S c i e nt i f ic Co ntri b u t i o n 31 s t Co ngr e ss o f t h e Eu ro p ea n Ac a d e m y o f Al l e rg y a n d C l inic a l Im mu n o l o g y 2012, E A AC I , 16 - 20 Ju n e 2012, G e n eva

D ear Co ngr e ss d e l e ga t e,

On behalf of HAL Allergy we would like to welcome you to the 31st EAACI congress in Geneva. The theme of this year’s congress is “At the crossroads of research, practice and education”. HAL Allergy is one of Europe’s top players in the field of allergen vaccination and devotes a major part of its resources and investments to the development of allergen specific immunotherapy. We are proud to present our ongoing research and clinical practice work during the biggest European congress on allergy and clinical immunology.

In this abstract book you will find fifteen abstracts dealing with clinical, pre-clinical and development work, including information on specific dates and times of presentations.

HAL Allergy’s clinical development program is committed to meeting today’s requirements for gaining registration for specific immunotherapy products. The clinical development program is represented with two abstracts related to the dose range finding study with PURETHAL® Mites which was initiated in the fall of 2011 and will be completed in spring 2013. In addition we also present you with clinical data on the use of PURETHAL®, SUBLIVAC® and VENOMENHAL® in daily practice.

At this year’s meeting there will be two oral presentations dealing with our latest pre-clinical work on a novel vaccine for peanut allergy. We are on track with our research program to develop a subcutaneous immunotherapy product to safely treat peanut-allergic patients and intend to complete all preclinical studies this year.

Furthermore, we present various abstracts on the characterisation and quantification of our allergen products, in particular our PURETHAL® range which involve chemically modified allergens, so-called allergoids. HAL Allergy is progressing with the development and implementation of new antibody based and physicochemical methodologies to characterise pharmaceutical allergoid preparations and to assure their quality.

We hope you will have a successful congress. Please visit our booth in the exhibition area should you require further information on our R&D program or on our products.

Kind regards,

Diderik Boot, PhD Dirk-Jan Opstelten, PhDMedical Director Research & Development Director

Diderik Boot, PhD

Medical Director

Dirk-Jan Opstelten, PhD

Research & DevelopmentDirector

Co nt e nt s

Scientific Contribution

ClinicalEvaluation of the cytokine response induced by allergen-specific immunotherapy with a modified allergoid in patients with allergic rhinits with or without asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Application of a standardised titrated nasal provocation test with HDM extract in a multicentre clinical study in allergic rhinitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Patient knowledge, perceptions, expectations, and satisfaction, on subcutaneous and sublingual allergen-specific immunotherapy: a real life survey . . . . . . . . . . . . . . . . . . . . . . . . . . 10Influence of co-morbidities and co-medications on safety and tolerability in the buildup phase of venom immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Design of dose range finding study with allergen specific immunotherapy in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Safety of pre- and coseasonal start of sublingual immunotherapy treatment in patients with pollen allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Pre-clinicalChemical modification of a peanut extract decreases IgE binding while the immunogenicity is maintained . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Allergenic potency of chemically modified Ara h2/h6 evaluated with basophil activation, RBL-SX38 activation, and solid phase IgE-immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20The content of allergens Ara h1, Ara h2, Ara h3 and Ara h6 in different peanut cultivars commonly consumed in Europe and the USA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

DevelopmentComparison of antibody-based assay with physicochemical assays for monitoring the stability of alum-adsorbed mite allergoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Characterisation of pollen allergoids with physicochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Characterisation of alum-adsorbed pollen allergoids with physicochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . 28An inhibition ELISA method for the determination of relative potency of mite preparations . . . . . . . . . . . . . . . . . . . . 30An antibody-based technique for stability studies on alum adsorbed birch pollen allergoid preparations . . . . . . . 32Biochemical characterisation of chemically modified Ara h2 isoforms; structure-function relationships . . . . . . . . . 34

Eva l u a t i o n o f t h e c y t o kin e r e s p o n s e in d u ce d by a l l e rge n - s p e c i f ic im mu n o t h e rap y w i t h a m o d i f i e d a l l e rg o i d in p a t i e nt s w i t h a l l e rgic r h ini t is w i t h o r w i t h o u t a s t h m a

A. Malet-Casajuana (1), A. Roger (2), P. Amat-Par (1), N. Depreux (2), T. Gonzalez-Quevedo (3), M.J. Cruz (4).

(1) Alergocentre, Barcelona, Spain, (2) Allergy Unit. Germans Trias Pujol University Hospital. Badalona, Spain. (3) Allergy Unit. Virgen del Rocío

University Hospital, Sevilla, Spain. (4) HAL Allergy, Barcelona, Spain.

BackgroundAllergy is characterized by a T helper type 2 (Th2) cell-mediated immune response. It has been demonstrated that after allergen-specific immunotherapy (SIT) there is a change in the balance between Th1/Th2 responses and the induction of a more allergen tolerant state. The aim of the present study was to investigate in patients with house dust mite (HDM) indu-ced allergic rhinitis/rhinoconjunctivitis the changes in Th1, Th2 and T regulatory (Treg) cytokines following SIT treatment

MethodsTwenty-nine adult patients (14F/15M, 33 yrs range 16-65 years) with a history of HDM induced allergic rhinitis/rhinocon-junctivitis with or without clinically stable mild persistent asthma were treated with subcutaneous SIT, containing 20,000 AUeq/ml of modified mites allergen extract adsorbed onto aluminium hydroxide. Blood samples were collected before and 3 and 6 months after SIT treatment. In serum samples IFN-γ, IL-10, IL-4, IL-5, and Il-13 levels were determined by flow cytometry using a commercial multiplexing kit (Milliplex Human Cytokine Kit, Millipore Corporation) according to the in-structions provided by the manufacturer. The samples were analyzed using a Fortessa flow cytometer.

Results Following SIT, a significant increase in the level of IFN-γ was found after 3 and 6 months of treatment (p = 0.004 and 0.005 respectively). This increase was also observed in the levels of IL-10 after 6 months of treatment (p = 0.019). Moreover, a tendency towards significant decrease of the levels of IL-5 were observed after 6 months, compared with the basal values (p = 0.088). No significant differences were found in the levels of IL-4 and IL13 after SIT.

ConclusionsThe results obtained in the present study suggest that SIT triggers a Th2 to Th1 shift and an induction of IL-10 secreting Treg cells. These events occur early and suggest a SIT tolerance induction in these patients.

6

EAACI, 16-20 June 2012, GenevaAbstract number: 2, Session date and time: Sunday 17 June; 10:30 - 12:00 Session title: OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment.

OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment.

Eva l u a t i o n o f t h e c y t o kin e r e s p o n s e in d u ce d by a l l e rge n - s p e c i f ic im mu n o t h e rap y w i t h a m o d i f i e d a l l e rg o i d in p a t i e nt s w i t h a l l e rgic r h ini t is w i t h o r w i t h o u t a s t h m a .

7

Bac

kgro

und

& Ai

m:

Alle

rgy

is c

hara

cter

ized

by

a T

help

er t

ype

2 (T

h2)

cell-

med

iate

d im

mun

e re

spon

se.

It ha

s be

en d

emon

stra

ted

that

afte

r al

lerg

en-s

peci

fic

imm

unot

hera

py (S

IT) t

here

is a

mod

ulat

ion

of a

llerg

en-s

peci

fic a

ntib

ody

resp

onse

s, a

cha

nge

in th

e ba

lanc

e be

twee

n Th

1/Th

2 re

spon

ses

and

the

indu

ctio

n of

a m

ore

alle

rgen

tole

rant

sta

te.T

he in

duct

ion

of a

llerg

en s

peci

fic s

erum

IgG

antib

odie

s ha

s pr

evio

usly

bee

n de

mon

stra

ted.

The

ai

m o

f the

pre

sent

stu

dy w

as to

inve

stig

ate

the

chan

ges

in T

h1, T

h2 a

nd T

regu

lato

ry (T

reg)

cyt

okin

es fo

llow

ing

the

initi

atio

n of

SIT

trea

tmen

t in

patie

nts

with

hou

se d

ust m

ite (H

DM

) ind

uced

alle

rgic

rhin

itis/

rhin

ocon

junc

tiviti

s

1 Ale

rgoc

entre

, Bar

celo

na, S

pain

, 2 Alle

rgy

Unit.

Ger

man

s Tr

ias

Pujo

lUni

vers

ity H

ospi

tal.

Bad

alon

a, S

pain

. 3 A

llerg

y Un

it. V

irgen

del R

ocío

Univ

ersi

ty H

ospi

tal,

Sevi

lla, S

pain

, 4 HAL

Alle

rgy,

Bar

celo

na, S

pain

.

2 -E

valu

atio

n of

the

cyto

kine

resp

onse

indu

ced

by a

llerg

en-s

peci

fic

imm

unot

hera

py w

ith a

mod

ified

alle

rgoi

din

pat

ient

s w

ith a

llerg

ic

rhin

itis

with

or w

ithou

t ast

hma

Figu

re 2

: Cyt

okin

e co

ncen

trat

ion

befo

re a

nd a

fter t

reat

men

t with

imm

unot

hera

py: A

= b

efor

e tr

eatm

ent;

B =

3 m

onth

s af

ter t

reat

men

t; C

= 6

mon

ths

afte

r tr

eatm

ent

Tabl

e 1:

Dem

ogra

phic

char

acte

ristic

san

dva

lues

ofsp

ecifi

can

tibod

ies

in th

est

udy

popu

latio

n.

Con

clus

ion:

The

resu

lts o

btai

ned

in th

e pr

esen

t stu

dy s

ugge

st th

at S

IT tr

igge

rs a

Th2

to T

h1 s

hift

and

an in

duct

ion

of IL

-10

secr

etin

g Tr

egce

lls. T

hese

eve

nts

occu

r ear

ly a

nd s

ugge

st a

SIT

tole

ranc

e in

duct

ion

in th

ese

patie

nts.

The

se c

hang

es a

re p

art o

f the

mec

hani

sm o

f act

ion

of im

mun

othe

rapy

and

is

prob

ably

rela

ted

to th

e cl

inic

al e

ffica

cy o

f the

trea

tmen

t.

Met

hods

:Ad

ult p

atie

nts

with

a h

isto

ry o

f HD

M in

duce

d al

lerg

ic rh

initi

s/rh

inoc

onju

nctiv

itis

with

or w

ithou

t con

com

itant

clin

ical

ly s

tabl

e m

ild p

ersi

sten

t ast

hma

wer

e sc

reen

ed.

Elig

ible

pat

ient

s w

ere

treat

ed w

ith s

ubcu

tane

ous

SIT

(PU

RET

HAL

®,

HAL

Alle

rgy

BV,

Leid

en,

The

Net

herla

nds)

, co

ntai

ning

20

,000

AU

eq/m

l of m

odifi

ed m

ites

alle

rgen

ext

ract

ads

orbe

d on

to a

lum

iniu

mhy

drox

ide.

Blo

od s

ampl

es w

ere

colle

cted

bef

ore,

3 a

nd 6

mon

ths

afte

r SIT

trea

tmen

t. In

ser

um s

ampl

es IF

N-

, IL-

10, I

L-4,

IL-5

, and

Il-1

3 le

vels

wer

e de

term

ined

by

flow

cyt

omet

ryus

ing

a co

mm

erci

al m

ultip

lexi

ng

kit (

Milli

plex

Hum

an C

ytok

ine

Kit,

Milli

pore

Cor

pora

tion)

. The

det

ectio

n sy

stem

was

a s

andw

ich

imm

unoa

ssay

per

form

ed o

n un

ique

fluo

resc

ent

bead

s, w

hich

con

tain

ed a

pre

cise

dis

tribu

tion

of t

wo

fluor

esce

ntdy

es,

and

a sp

ecifi

c cy

toki

ne c

aptu

re a

ntib

ody

linke

d to

its

sur

face

. Th

e flu

ores

cent

bea

d se

ts w

ere

incu

bate

d w

ith a

bio

logi

cal s

ampl

e. A

biot

inyla

ted

cyto

kine

repo

rter a

ntib

ody

was

then

use

d to

iden

tify

the

capt

ured

cy

toki

ne b

ound

to

the

spec

ific

fluor

esce

nt b

ead.

The

rea

ctio

n w

as d

etec

ted

by a

ddin

g a

stre

ptav

idin

-phy

coer

ythr

inco

njug

ate

to t

he r

eact

ion

mix

ture

. The

sam

ples

wer

e an

alyz

ed u

sing

a F

orte

ssa

flow

cyt

omet

er.

Res

ults

:Tw

enty

nin

e pa

tient

s (T

able

1, F

igur

e 1)

wer

e in

clud

ed. S

IT in

duce

d a

sign

ifica

nt in

crea

se in

ser

um le

vels

of

and

IL-1

0 an

d a

sign

ifica

nt

decr

ease

in IL

-5 (F

igur

e 2)

. No

sign

ifica

nt d

iffer

ence

s w

ere

foun

d in

the

leve

ls o

f IL-

4 an

d IL

13 a

fter S

IT.

P =

0.00

4

P =

0.03

3

P =

0.00

5

IFNγ A

IFNγ B

IFNγ C

0.1110100

P =

0.02

5

P =

0.37

4

P =

0.01

9

IL10 A

IL10 B

IL10 C

0.1110100

IL10 (pg/ml)

P =

0.27

1

P =

0.00

1

P =

0.08

8

IL5 A

IL5 B

IL5 C

0.010.

1110100

IL5 (pg/ml)

Figu

re 1

: Dis

trib

utio

n of

resp

irato

ry d

isea

se.

4.3

(3.0

-6.7

L)M

edia

n FV

C 1(L

) (ra

nge)

3.7

(2.4

-5.3

L)M

edia

n FE

V 1(L

) (ra

nge)

Patie

nts

n =

29G

ende

r (M

(%) /

F (%

))15

(51.

7%)/

14(4

8.3%

)

Mea

n ag

e in

yea

rs (r

ange

)33

(16-

65)

Med

ian

seru

m s

peci

fic Ig

E(IU

/ml)

to m

ites

(rang

e)17

.3 (1

.3-5

7.5)

Asth

ma

23%

Asth

ma

+ Rh

initi

s23

%

Rhin

itis

54%

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

Bac

kgro

und

& Ai

m:

Alle

rgy

is c

hara

cter

ized

by

a T

help

er t

ype

2 (T

h2)

cell-

med

iate

d im

mun

e re

spon

se.

It ha

s be

en d

emon

stra

ted

that

afte

r al

lerg

en-s

peci

fic

imm

unot

hera

py (S

IT) t

here

is a

mod

ulat

ion

of a

llerg

en-s

peci

fic a

ntib

ody

resp

onse

s, a

cha

nge

in th

e ba

lanc

e be

twee

n Th

1/Th

2 re

spon

ses

and

the

indu

ctio

n of

a m

ore

alle

rgen

tole

rant

sta

te.T

he in

duct

ion

of a

llerg

en s

peci

fic s

erum

IgG

antib

odie

s ha

s pr

evio

usly

bee

n de

mon

stra

ted.

The

ai

m o

f the

pre

sent

stu

dy w

as to

inve

stig

ate

the

chan

ges

in T

h1, T

h2 a

nd T

regu

lato

ry (T

reg)

cyt

okin

es fo

llow

ing

the

initi

atio

n of

SIT

trea

tmen

t in

patie

nts

with

hou

se d

ust m

ite (H

DM

) ind

uced

alle

rgic

rhin

itis/

rhin

ocon

junc

tiviti

s

1 Ale

rgoc

entre

, Bar

celo

na, S

pain

, 2 Alle

rgy

Unit.

Ger

man

s Tr

ias

Pujo

lUni

vers

ity H

ospi

tal.

Bad

alon

a, S

pain

. 3 A

llerg

y Un

it. V

irgen

del R

ocío

Univ

ersi

ty H

ospi

tal,

Sevi

lla, S

pain

, 4 HAL

Alle

rgy,

Bar

celo

na, S

pain

.

2 -E

valu

atio

n of

the

cyto

kine

resp

onse

indu

ced

by a

llerg

en-s

peci

fic

imm

unot

hera

py w

ith a

mod

ified

alle

rgoi

din

pat

ient

s w

ith a

llerg

ic

rhin

itis

with

or w

ithou

t ast

hma

Figu

re 2

: Cyt

okin

e co

ncen

trat

ion

befo

re a

nd a

fter t

reat

men

t with

imm

unot

hera

py: A

= b

efor

e tr

eatm

ent;

B =

3 m

onth

s af

ter t

reat

men

t; C

= 6

mon

ths

afte

r tr

eatm

ent

Tabl

e 1:

Dem

ogra

phic

char

acte

ristic

san

dva

lues

ofsp

ecifi

can

tibod

ies

in th

est

udy

popu

latio

n.

Con

clus

ion:

The

resu

lts o

btai

ned

in th

e pr

esen

t stu

dy s

ugge

st th

at S

IT tr

igge

rs a

Th2

to T

h1 s

hift

and

an in

duct

ion

of IL

-10

secr

etin

g Tr

egce

lls. T

hese

eve

nts

occu

r ear

ly a

nd s

ugge

st a

SIT

tole

ranc

e in

duct

ion

in th

ese

patie

nts.

The

se c

hang

es a

re p

art o

f the

mec

hani

sm o

f act

ion

of im

mun

othe

rapy

and

is

prob

ably

rela

ted

to th

e cl

inic

al e

ffica

cy o

f the

trea

tmen

t.

Met

hods

:Ad

ult p

atie

nts

with

a h

isto

ry o

f HD

M in

duce

d al

lerg

ic rh

initi

s/rh

inoc

onju

nctiv

itis

with

or w

ithou

t con

com

itant

clin

ical

ly s

tabl

e m

ild p

ersi

sten

t ast

hma

wer

e sc

reen

ed.

Elig

ible

pat

ient

s w

ere

treat

ed w

ith s

ubcu

tane

ous

SIT

(PU

RET

HAL

®,

HAL

Alle

rgy

BV,

Leid

en,

The

Net

herla

nds)

, co

ntai

ning

20

,000

AU

eq/m

l of m

odifi

ed m

ites

alle

rgen

ext

ract

ads

orbe

d on

to a

lum

iniu

mhy

drox

ide.

Blo

od s

ampl

es w

ere

colle

cted

bef

ore,

3 a

nd 6

mon

ths

afte

r SIT

trea

tmen

t. In

ser

um s

ampl

es IF

N-

, IL-

10, I

L-4,

IL-5

, and

Il-1

3 le

vels

wer

e de

term

ined

by

flow

cyt

omet

ryus

ing

a co

mm

erci

al m

ultip

lexi

ng

kit (

Milli

plex

Hum

an C

ytok

ine

Kit,

Milli

pore

Cor

pora

tion)

. The

det

ectio

n sy

stem

was

a s

andw

ich

imm

unoa

ssay

per

form

ed o

n un

ique

fluo

resc

ent

bead

s, w

hich

con

tain

ed a

pre

cise

dis

tribu

tion

of t

wo

fluor

esce

ntdy

es,

and

a sp

ecifi

c cy

toki

ne c

aptu

re a

ntib

ody

linke

d to

its

sur

face

. Th

e flu

ores

cent

bea

d se

ts w

ere

incu

bate

d w

ith a

bio

logi

cal s

ampl

e. A

biot

inyla

ted

cyto

kine

repo

rter a

ntib

ody

was

then

use

d to

iden

tify

the

capt

ured

cy

toki

ne b

ound

to

the

spec

ific

fluor

esce

nt b

ead.

The

rea

ctio

n w

as d

etec

ted

by a

ddin

g a

stre

ptav

idin

-phy

coer

ythr

inco

njug

ate

to t

he r

eact

ion

mix

ture

. The

sam

ples

wer

e an

alyz

ed u

sing

a F

orte

ssa

flow

cyt

omet

er.

Res

ults

:Tw

enty

nin

e pa

tient

s (T

able

1, F

igur

e 1)

wer

e in

clud

ed. S

IT in

duce

d a

sign

ifica

nt in

crea

se in

ser

um le

vels

of

and

IL-1

0 an

d a

sign

ifica

nt

decr

ease

in IL

-5 (F

igur

e 2)

. No

sign

ifica

nt d

iffer

ence

s w

ere

foun

d in

the

leve

ls o

f IL-

4 an

d IL

13 a

fter S

IT.

P =

0.00

4

P =

0.03

3

P =

0.00

5IFNγ

A

IFNγ B

IFNγ C

0.1110100

P =

0.02

5

P =

0.37

4

P =

0.01

9

IL10 A

IL10 B

IL10 C

0.1110100

IL10 (pg/ml)

P =

0.27

1

P =

0.00

1

P =

0.08

8

IL5 A

IL5 B

IL5 C

0.010.

1110100

IL5 (pg/ml)

Figu

re 1

: Dis

trib

utio

n of

resp

irato

ry d

isea

se.

4.3

(3.0

-6.7

L)M

edia

n FV

C 1(L

) (ra

nge)

3.7

(2.4

-5.3

L)M

edia

n FE

V 1(L

) (ra

nge)

Patie

nts

n =

29G

ende

r (M

(%) /

F (%

))15

(51.

7%)/

14(4

8.3%

)

Mea

n ag

e in

yea

rs (r

ange

)33

(16-

65)

Med

ian

seru

m s

peci

fic Ig

E(IU

/ml)

to m

ites

(rang

e)17

.3 (1

.3-5

7.5)

Asth

ma

23%

Asth

ma

+ Rh

initi

s23

%

Rhin

itis

54%

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

Bac

kgro

und

& Ai

m:

Alle

rgy

is c

hara

cter

ized

by

a T

help

er t

ype

2 (T

h2)

cell-

med

iate

d im

mun

e re

spon

se.

It ha

s be

en d

emon

stra

ted

that

afte

r al

lerg

en-s

peci

fic

imm

unot

hera

py (S

IT) t

here

is a

mod

ulat

ion

of a

llerg

en-s

peci

fic a

ntib

ody

resp

onse

s, a

cha

nge

in th

e ba

lanc

e be

twee

n Th

1/Th

2 re

spon

ses

and

the

indu

ctio

n of

a m

ore

alle

rgen

tole

rant

sta

te.T

he in

duct

ion

of a

llerg

en s

peci

fic s

erum

IgG

antib

odie

s ha

s pr

evio

usly

bee

n de

mon

stra

ted.

The

ai

m o

f the

pre

sent

stu

dy w

as to

inve

stig

ate

the

chan

ges

in T

h1, T

h2 a

nd T

regu

lato

ry (T

reg)

cyt

okin

es fo

llow

ing

the

initi

atio

n of

SIT

trea

tmen

t in

patie

nts

with

hou

se d

ust m

ite (H

DM

) ind

uced

alle

rgic

rhin

itis/

rhin

ocon

junc

tiviti

s

1 Ale

rgoc

entre

, Bar

celo

na, S

pain

, 2 Alle

rgy

Unit.

Ger

man

s Tr

ias

Pujo

lUni

vers

ity H

ospi

tal.

Bad

alon

a, S

pain

. 3 A

llerg

y Un

it. V

irgen

del R

ocío

Univ

ersi

ty H

ospi

tal,

Sevi

lla, S

pain

, 4 HAL

Alle

rgy,

Bar

celo

na, S

pain

.

2 -E

valu

atio

n of

the

cyto

kine

resp

onse

indu

ced

by a

llerg

en-s

peci

fic

imm

unot

hera

py w

ith a

mod

ified

alle

rgoi

din

pat

ient

s w

ith a

llerg

ic

rhin

itis

with

or w

ithou

t ast

hma

Figu

re 2

: Cyt

okin

e co

ncen

trat

ion

befo

re a

nd a

fter t

reat

men

t with

imm

unot

hera

py: A

= b

efor

e tr

eatm

ent;

B =

3 m

onth

s af

ter t

reat

men

t; C

= 6

mon

ths

afte

r tr

eatm

ent

Tabl

e 1:

Dem

ogra

phic

char

acte

ristic

san

dva

lues

ofsp

ecifi

can

tibod

ies

in th

est

udy

popu

latio

n.

Con

clus

ion:

The

resu

lts o

btai

ned

in th

e pr

esen

t stu

dy s

ugge

st th

at S

IT tr

igge

rs a

Th2

to T

h1 s

hift

and

an in

duct

ion

of IL

-10

secr

etin

g Tr

egce

lls. T

hese

eve

nts

occu

r ear

ly a

nd s

ugge

st a

SIT

tole

ranc

e in

duct

ion

in th

ese

patie

nts.

The

se c

hang

es a

re p

art o

f the

mec

hani

sm o

f act

ion

of im

mun

othe

rapy

and

is

prob

ably

rela

ted

to th

e cl

inic

al e

ffica

cy o

f the

trea

tmen

t.

Met

hods

:Ad

ult p

atie

nts

with

a h

isto

ry o

f HD

M in

duce

d al

lerg

ic rh

initi

s/rh

inoc

onju

nctiv

itis

with

or w

ithou

t con

com

itant

clin

ical

ly s

tabl

e m

ild p

ersi

sten

t ast

hma

wer

e sc

reen

ed.

Elig

ible

pat

ient

s w

ere

treat

ed w

ith s

ubcu

tane

ous

SIT

(PU

RET

HAL

®,

HAL

Alle

rgy

BV,

Leid

en,

The

Net

herla

nds)

, co

ntai

ning

20

,000

AU

eq/m

l of m

odifi

ed m

ites

alle

rgen

ext

ract

ads

orbe

d on

to a

lum

iniu

mhy

drox

ide.

Blo

od s

ampl

es w

ere

colle

cted

bef

ore,

3 a

nd 6

mon

ths

afte

r SIT

trea

tmen

t. In

ser

um s

ampl

es IF

N-

, IL-

10, I

L-4,

IL-5

, and

Il-1

3 le

vels

wer

e de

term

ined

by

flow

cyt

omet

ryus

ing

a co

mm

erci

al m

ultip

lexi

ng

kit (

Milli

plex

Hum

an C

ytok

ine

Kit,

Milli

pore

Cor

pora

tion)

. The

det

ectio

n sy

stem

was

a s

andw

ich

imm

unoa

ssay

per

form

ed o

n un

ique

fluo

resc

ent

bead

s, w

hich

con

tain

ed a

pre

cise

dis

tribu

tion

of t

wo

fluor

esce

ntdy

es,

and

a sp

ecifi

c cy

toki

ne c

aptu

re a

ntib

ody

linke

d to

its

sur

face

. Th

e flu

ores

cent

bea

d se

ts w

ere

incu

bate

d w

ith a

bio

logi

cal s

ampl

e. A

biot

inyla

ted

cyto

kine

repo

rter a

ntib

ody

was

then

use

d to

iden

tify

the

capt

ured

cy

toki

ne b

ound

to

the

spec

ific

fluor

esce

nt b

ead.

The

rea

ctio

n w

as d

etec

ted

by a

ddin

g a

stre

ptav

idin

-phy

coer

ythr

inco

njug

ate

to t

he r

eact

ion

mix

ture

. The

sam

ples

wer

e an

alyz

ed u

sing

a F

orte

ssa

flow

cyt

omet

er.

Res

ults

:Tw

enty

nin

e pa

tient

s (T

able

1, F

igur

e 1)

wer

e in

clud

ed. S

IT in

duce

d a

sign

ifica

nt in

crea

se in

ser

um le

vels

of

and

IL-1

0 an

d a

sign

ifica

nt

decr

ease

in IL

-5 (F

igur

e 2)

. No

sign

ifica

nt d

iffer

ence

s w

ere

foun

d in

the

leve

ls o

f IL-

4 an

d IL

13 a

fter S

IT.

P =

0.00

4

P =

0.03

3

P =

0.00

5

IFNγ A

IFNγ B

IFNγ C

0.1110100

P =

0.02

5

P =

0.37

4

P =

0.01

9

IL10 A

IL10 B

IL10 C

0.1110100

IL10 (pg/ml)

P =

0.27

1

P =

0.00

1

P =

0.08

8

IL5 A

IL5 B

IL5 C

0.010.

1110100

IL5 (pg/ml)

Figu

re 1

: Dis

trib

utio

n of

resp

irato

ry d

isea

se.

4.3

(3.0

-6.7

L)M

edia

n FV

C 1(L

) (ra

nge)

3.7

(2.4

-5.3

L)M

edia

n FE

V 1(L

) (ra

nge)

Patie

nts

n =

29G

ende

r (M

(%) /

F (%

))15

(51.

7%)/

14(4

8.3%

)

Mea

n ag

e in

yea

rs (r

ange

)33

(16-

65)

Med

ian

seru

m s

peci

fic Ig

E(IU

/ml)

to m

ites

(rang

e)17

.3 (1

.3-5

7.5)

Asth

ma

23%

Asth

ma

+ Rh

initi

s23

%

Rhin

itis

54%

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

Ap p l ic a t i o n o f a s t a n d ard i z e d t i tra t e d n a s a l p ro v o c a t i o n t e s t w i t h H D M ex tra c t in a mu l t ice ntr e cl inic a l s tu d y in a l l e rgic r h ini t is

C. Bachert (1), O. Pfaar (2), M.J. Nell (3), J.D. Boot (3), H. Nienhuis (3), Z. Diamant (4,5).

(1) UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) HAL Allergy BV,

Leiden, The Netherlands, (4) Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and (5) QPS-NL, Groningen, The Netherlands.

BackgroundTitrated nasal provocation test (TNPT) can be used in the diagnosis of allergic rhinitis (AR) and as a disease model in clinical trials evaluating the efficacy of treatment. However, the techniques for provocation and readouts vary, requiring standardization to enable the use of TNPT as a tool in multicentre trials. We performed a standardized TNPT and documented the upper airway response by symptom scores and peak nasal inspiratory flow (PNIF) measurements. In addition, we compared the upper airway responses across the different allergen concentrations and investigated a possible correlation.

Methods Patients with suspected HDM-induced AR were screened prior to enrollment into a multicentre efficacy study with HDM immunotherapy. Patients with a positive medical history and a positive skin prick test to HDM underwent a standardized TNPT starting with the diluent and up to 3 serial concentrations of a standardized allergen extract (D. Pter 100, 1,000 and 10,000 AU/ml, 1 puff per nostril) at 20 minutes intervals. The upper airway response was quantified by a composite symptom score according to Lebel and a PNIF measurement 15 minutes after each administration. TNPT was considered positive once a score ≥6 was reached. Symptom scores and PNIF were repeated 1 hour after the provocative allergen concentration.

Results 150 patients were screened. 103 patients (54M/49F, mean age 32 years, mean wheal size HDM 6mm) had a positive TNPT, the most prominent symptoms being nasal blockage and rhinorrhoea, followed by pruritus and sneezing. A consistent dose-response relationship was found between serial allergen concentrations and increasing symptom scores, matching a gradual decrease in PNIF. One hour after the provocative allergen concentration, both measurements had not returned to baseline values (Table). There was a moderate though highly significant correlation between symptom scores and PNIF (r=0.54; p<0.01).

ConclusionWhen applied in a multicentre setting in patients with suspected HDM-induced AR, a standardized TNPT with a HDM extract induces a consistent gradual increase in allergic symptoms and signs that can be reliably quantified by well-defined composite symptom scores and PNIF.

8

EAACI, 16-20 June 2012, GenevaAbstract number: 37, Session date and time: Sunday 17 June; 13:30 - 15:00 Session title: OAS 7 - Diagnosis and treatment of allergic rhinoconjunctivitis: recent development

Table: Symptom scores and PNIF (mean±SD) following TNPT with HDM allergen.

Timepoint Pre Post Diluent 100 AU/ml 1,000 AU/ml 10,000 AU/ml 1 hour Diluent Diluent post TNPT

Lebel Score 0.8±0.8 0.9±1.0 3.7±2.1 5.5±2.2 6.5±2.9 3.2±1.7

PNIF (L/min) 135±45 132±47 113±48 93±44 66±42 109±43

9

OAS 7 - Diagnosis and treatment of allergic rhinoconjunctivitis: recent development

Ap p l ic a t i o n o f a s t a n d ard i z e d t i tra t e d n a s a l p ro v o c a t i o n t e s t w i t h H D M ex tra c t in a mu l t ice ntr e cl inic a l s tu d y in a l l e rgic r h ini t is

1 UZ

Gen

t, Ea

r-, N

ose

and

Thro

at D

epar

tmen

t, G

ent,

Belg

ium

; 2 Cen

ter f

or R

hino

logy

and

Alle

rgol

ogy

Wie

sbad

en, G

erm

any;

3 H

AL A

llerg

y BV

, Le

iden

, The

Net

herla

nds;

4 Ska

ne U

nive

rsity

Hos

pita

l, D

ept o

f Res

pir M

ed &

Alle

rgol

, Lun

d, S

wed

en

and

5 QPS

-NL,

Gro

ning

en, T

he N

ethe

rland

s

37 -

Appl

icat

ion

of a

sta

ndar

dize

d tit

rate

d na

sal

prov

ocat

ion

test

with

HD

M e

xtra

ct in

a

mul

ticen

tre

clin

ical

stu

dy in

alle

rgic

rhin

itis

Bac

kgro

und

& Ai

m:

Titra

ted

nasa

l pro

voca

tion

test

s (T

NPT

) with

a re

leva

nt a

llerg

enca

n be

use

d in

the

diag

nosi

s of

alle

rgic

rhin

itis

(AR

) and

as

a di

seas

e m

odel

in

clin

ical

tria

ls e

valu

atin

g th

e ef

ficac

y of

tre

atm

ent.

How

ever

, bo

th t

he t

echn

ique

s us

ed f

or p

rovo

catio

n an

d th

e re

adou

ts o

f th

e ev

oked

upp

erai

rway

resp

onse

vary

, req

uirin

g st

anda

rdiz

atio

n to

ena

ble

the

use

of T

NPT

as

a to

ol in

mul

ticen

tre tr

ials

. We

perfo

rmed

a s

tand

ardi

zed

TNPT

an

d do

cum

ente

d th

e up

per a

irway

resp

onse

by

sym

ptom

sco

res

and

peak

nas

al in

spira

tory

flow

(PN

IF) m

easu

rem

ents

. In

addi

tion,

we

com

pare

d th

e up

per a

irway

resp

onse

s ac

ross

the

diffe

rent

alle

rgen

con

cent

ratio

ns a

nd in

vest

igat

ed a

pos

sibl

e co

rrela

tion

betw

een

both

read

outs

.

Con

clus

ion:

Whe

n ap

plie

d in

a m

ultic

entre

set

ting

in p

atie

nts

with

sus

pect

ed H

DM

-indu

ced

AR, a

sta

ndar

dize

d TN

PT w

ith a

HD

M e

xtra

ct in

duce

sa

cons

iste

nt g

radu

al in

crea

se in

alle

rgic

sym

ptom

s an

d si

gns

that

can

be

relia

bly

quan

tifie

d by

a w

ell-d

efin

ed c

ompo

site

sym

ptom

sc

ore

and

PNIF

mea

sure

men

ts.

Met

hods

:Pa

tient

s: M

/F a

ged

18-6

0 ye

ars

with

HD

M-re

late

d AR

, w

ith o

r w

ithou

t co

ncom

itant

, cl

inic

ally

sta

ble

asth

ma

(FEV

1 >7

0% p

redi

cted

) w

ere

scre

ened

prio

r to

enro

llmen

t int

o a

mul

ticen

tre e

ffica

cy s

tudy

with

HD

M im

mun

othe

rapy

. Pat

ient

s w

ith a

pos

itive

AR

-his

tory

and

a p

ositi

ve s

kin

pric

k te

st to

HD

M, u

nder

went

a s

tand

ardi

zed

TNPT

. M

etho

ds:

The

TNPT

sta

rted

with

the

alle

rgen

’s d

iluen

t fo

llow

ed b

y m

axim

al3

seria

l co

ncen

tratio

ns o

f a

stan

dard

ized

alle

rgen

ext

ract

(D

. Pt

eron

yssi

nus

100,

1,0

00 a

nd 1

0,00

0 AU

/ml,

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s), a

dmin

iste

red

by a

nas

al s

pray

ing

devi

ce (

1 pu

ff pe

r no

stril

) at

20

min

utes

int

erva

ls.

The

uppe

r ai

rway

res

pons

e w

as q

uant

ified

by

a co

mpo

site

sym

ptom

sco

re a

ccor

ding

to

Lebe

l an

d PN

IF

mea

sure

men

ts (h

ighe

st v

alue

of 3

mea

sure

d by

In-c

heck

nas

al in

spira

tory

flow

met

er [C

lem

ent C

lark

e, H

arlo

w, U

K])a

t bas

elin

e (p

re-d

iluen

t), 1

5 m

inut

es a

fter e

ach

adm

inis

tratio

n an

d 60

min

utes

pos

t-cha

lleng

e. S

ympt

oms

wer

e re

cord

ed u

sing

the

follo

win

g sc

orin

g sy

stem

: sne

ezes

2

= 0,

sn

eeze

s 3-

4 =

1 po

int,

snee

zes

5 =

3 po

ints

, ant

erio

r rh

inor

rhoe

a =

1 po

int,

post

erio

r rh

inor

rhoe

a =

1 po

int,

diffi

cult

brea

thin

g =

1 po

int,

1 bl

ocke

d no

stril

= 2

poi

nts,

2 b

lock

ed n

ostri

ls =

3 p

oint

s, n

asal

prur

itus

= 1

poin

t, pr

uritu

s in

pal

ate

or e

ar =

1 p

oint

, con

junc

tiviti

s =

1 po

int (

tota

l sc

ore

rang

e: 0

-11

poin

ts).

The

pre-

dilu

ent w

as n

ot a

llow

ed to

be

3. T

he T

NPT

was

con

side

red

posi

tive

once

a to

tal s

core

6

was

reac

hed.

Res

ults

: 15

0 pa

tient

s w

ere

scre

ened

. 103

pat

ient

s (T

able

1)h

ad a

pos

itive

TN

PT. T

he T

NPT

was

wel

l tol

erat

ed a

nd n

one

of th

epa

tient

s ex

perie

nced

any

cl

inic

ally

sig

nific

ant a

dver

se e

vent

s. T

he m

ost p

rom

inen

t sym

ptom

s w

ere

nasa

l blo

ckag

e an

d rh

inor

rhoe

a, fo

llow

ed b

y pr

uritu

s an

dsn

eezi

ng. A

co

nsis

tent

con

cent

ratio

n-re

spon

se r

elat

ions

hip

was

foun

d be

twee

n se

rial a

llerg

en c

once

ntra

tions

and

incr

easi

ng s

ympt

om s

core

s, m

atch

ing

a gr

adua

l dec

reas

e in

PN

IF. O

ne h

our a

fter t

he p

rovo

cativ

e al

lerg

en c

once

ntra

tion,

bot

h pa

ram

eter

s ha

d no

t com

plet

ely

retu

rned

to b

asel

ine

valu

es

(Fig

ure

1). T

here

was

a m

oder

ate

thou

gh h

ighl

y si

gnifi

cant

cor

rela

tion

betw

een

sym

ptom

sco

res

and

PNIF

mea

sure

men

ts (r

=0.5

4; p

<0.0

1). T

he

maj

ority

of p

atie

nts

had

a po

sitiv

e TN

PT-re

spon

se fo

llow

ing

adm

inis

tratio

n of

the

10,0

00 A

U/m

l con

cent

ratio

n (T

able

2).

Patie

nts

with

a p

ositi

ve

TNPT

-resp

onse

at a

low

er a

llerg

en c

once

ntra

tion

tend

ed to

hav

e a

larg

er w

heal

siz

e al

thou

gh d

iffer

ence

s w

ere

not s

tatis

tical

ly s

igni

fican

t (Ta

ble

2).

Tabl

e 2:

Pos

itive

TNP

T-re

spon

se a

nd c

orre

spon

ding

SP

T-w

heal

siz

es

Con

cent

ratio

n10

0 A

U/m

l1,

000

AU/

ml

10,0

00

AU/

ml

Posi

tive

resp

onse

(n)

1034

59M

ean

SPT-

whea

lsi

ze (m

m)

76

5Fi

gure

1: C

once

ntra

tion-

resp

onse

cur

ves

with

mea

n (

95%

CI)

chan

ges

in c

ompo

site

sym

ptom

sc

ores

(red

circ

les)

and

PNI

F m

easu

rem

ents

(blu

e ci

rcle

s) fo

llow

ing

TNPT

with

HDM

alle

rgen

.

Tabl

e 1:

Pat

ient

s’de

mog

raph

ic c

hara

cter

istic

s

Patie

nts

n =

103

Gen

der (

M (%

) / F

(%))

54(5

2.4%

)/ 49

(47.

6%)

Mea

n ag

e ±

SD in

yea

rs (r

ange

)32

±11

(18-

57)

SPT

to H

DM e

xtra

ct:

Mea

n wh

eal s

ize

in m

m ±

SD

(rang

e)6

±2

(3-1

2)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C. B

ache

rt1 , O. P

faar

2 , M.J

. Nel

l3 , J.D

. Boo

t3 , H.E

. Nie

nhui

s3 , Z. D

iam

ant4,

5 1 U

Z G

ent,

Ear-,

Nos

e an

d Th

roat

Dep

artm

ent,

Gen

t, Be

lgiu

m; 2 C

ente

r for

Rhi

nolo

gy a

nd A

llerg

olog

y W

iesb

aden

, Ger

man

y;

3 HAL

Alle

rgy

BV,

Leid

en, T

he N

ethe

rland

s; 4 S

kane

Uni

vers

ity H

ospi

tal,

Dep

t of R

espi

r Med

& A

llerg

ol, L

und,

Sw

eden

an

d 5 Q

PS-N

L, G

roni

ngen

, The

Net

herla

nds

37 -

Appl

icat

ion

of a

sta

ndar

dize

d tit

rate

d na

sal

prov

ocat

ion

test

with

HD

M e

xtra

ct in

a

mul

ticen

tre

clin

ical

stu

dy in

alle

rgic

rhin

itis

Bac

kgro

und

& Ai

m:

Titra

ted

nasa

l pro

voca

tion

test

s (T

NPT

) with

a re

leva

nt a

llerg

enca

n be

use

d in

the

diag

nosi

s of

alle

rgic

rhin

itis

(AR

) and

as

a di

seas

e m

odel

in

clin

ical

tria

ls e

valu

atin

g th

e ef

ficac

y of

tre

atm

ent.

How

ever

, bo

th t

he t

echn

ique

s us

ed f

or p

rovo

catio

n an

d th

e re

adou

ts o

f th

e ev

oked

upp

erai

rway

resp

onse

vary

, req

uirin

g st

anda

rdiz

atio

n to

ena

ble

the

use

of T

NPT

as

a to

ol in

mul

ticen

tre tr

ials

. We

perfo

rmed

a s

tand

ardi

zed

TNPT

an

d do

cum

ente

d th

e up

per a

irway

resp

onse

by

sym

ptom

sco

res

and

peak

nas

al in

spira

tory

flow

(PN

IF) m

easu

rem

ents

. In

addi

tion,

we

com

pare

d th

e up

per a

irway

resp

onse

s ac

ross

the

diffe

rent

alle

rgen

con

cent

ratio

ns a

nd in

vest

igat

ed a

pos

sibl

e co

rrela

tion

betw

een

both

read

outs

.

Con

clus

ion:

Whe

n ap

plie

d in

a m

ultic

entre

set

ting

in p

atie

nts

with

sus

pect

ed H

DM

-indu

ced

AR, a

sta

ndar

dize

d TN

PT w

ith a

HD

M e

xtra

ct in

duce

sa

cons

iste

nt g

radu

al in

crea

se in

alle

rgic

sym

ptom

s an

d si

gns

that

can

be

relia

bly

quan

tifie

d by

a w

ell-d

efin

ed c

ompo

site

sym

ptom

sc

ore

and

PNIF

mea

sure

men

ts.

Met

hods

:Pa

tient

s: M

/F a

ged

18-6

0 ye

ars

with

HD

M-re

late

d AR

, w

ith o

r w

ithou

t co

ncom

itant

, cl

inic

ally

sta

ble

asth

ma

(FEV

1 >7

0% p

redi

cted

) w

ere

scre

ened

prio

r to

enro

llmen

t int

o a

mul

ticen

tre e

ffica

cy s

tudy

with

HD

M im

mun

othe

rapy

. Pat

ient

s w

ith a

pos

itive

AR

-his

tory

and

a p

ositi

ve s

kin

pric

k te

st to

HD

M, u

nder

went

a s

tand

ardi

zed

TNPT

. M

etho

ds:

The

TNPT

sta

rted

with

the

alle

rgen

’s d

iluen

t fo

llow

ed b

y m

axim

al3

seria

l co

ncen

tratio

ns o

f a

stan

dard

ized

alle

rgen

ext

ract

(D

. Pt

eron

yssi

nus

100,

1,0

00 a

nd 1

0,00

0 AU

/ml,

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s), a

dmin

iste

red

by a

nas

al s

pray

ing

devi

ce (

1 pu

ff pe

r no

stril

) at

20

min

utes

int

erva

ls.

The

uppe

r ai

rway

res

pons

e w

as q

uant

ified

by

a co

mpo

site

sym

ptom

sco

re a

ccor

ding

to

Lebe

l an

d PN

IF

mea

sure

men

ts (h

ighe

st v

alue

of 3

mea

sure

d by

In-c

heck

nas

al in

spira

tory

flow

met

er [C

lem

ent C

lark

e, H

arlo

w, U

K])a

t bas

elin

e (p

re-d

iluen

t), 1

5 m

inut

es a

fter e

ach

adm

inis

tratio

n an

d 60

min

utes

pos

t-cha

lleng

e. S

ympt

oms

wer

e re

cord

ed u

sing

the

follo

win

g sc

orin

g sy

stem

: sne

ezes

2

= 0,

sn

eeze

s 3-

4 =

1 po

int,

snee

zes

5 =

3 po

ints

, ant

erio

r rh

inor

rhoe

a =

1 po

int,

post

erio

r rh

inor

rhoe

a =

1 po

int,

diffi

cult

brea

thin

g =

1 po

int,

1 bl

ocke

d no

stril

= 2

poi

nts,

2 b

lock

ed n

ostri

ls =

3 p

oint

s, n

asal

prur

itus

= 1

poin

t, pr

uritu

s in

pal

ate

or e

ar =

1 p

oint

, con

junc

tiviti

s =

1 po

int (

tota

l sc

ore

rang

e: 0

-11

poin

ts).

The

pre-

dilu

ent w

as n

ot a

llow

ed to

be

3. T

he T

NPT

was

con

side

red

posi

tive

once

a to

tal s

core

6

was

reac

hed.

Res

ults

: 15

0 pa

tient

s w

ere

scre

ened

. 103

pat

ient

s (T

able

1)h

ad a

pos

itive

TN

PT. T

he T

NPT

was

wel

l tol

erat

ed a

nd n

one

of th

epa

tient

s ex

perie

nced

any

cl

inic

ally

sig

nific

ant a

dver

se e

vent

s. T

he m

ost p

rom

inen

t sym

ptom

s w

ere

nasa

l blo

ckag

e an

d rh

inor

rhoe

a, fo

llow

ed b

y pr

uritu

s an

dsn

eezi

ng. A

co

nsis

tent

con

cent

ratio

n-re

spon

se r

elat

ions

hip

was

foun

d be

twee

n se

rial a

llerg

en c

once

ntra

tions

and

incr

easi

ng s

ympt

om s

core

s, m

atch

ing

a gr

adua

l dec

reas

e in

PN

IF. O

ne h

our a

fter t

he p

rovo

cativ

e al

lerg

en c

once

ntra

tion,

bot

h pa

ram

eter

s ha

d no

t com

plet

ely

retu

rned

to b

asel

ine

valu

es

(Fig

ure

1). T

here

was

a m

oder

ate

thou

gh h

ighl

y si

gnifi

cant

cor

rela

tion

betw

een

sym

ptom

sco

res

and

PNIF

mea

sure

men

ts (r

=0.5

4; p

<0.0

1). T

he

maj

ority

of p

atie

nts

had

a po

sitiv

e TN

PT-re

spon

se fo

llow

ing

adm

inis

tratio

n of

the

10,0

00 A

U/m

l con

cent

ratio

n (T

able

2).

Patie

nts

with

a p

ositi

ve

TNPT

-resp

onse

at a

low

er a

llerg

en c

once

ntra

tion

tend

ed to

hav

e a

larg

er w

heal

siz

e al

thou

gh d

iffer

ence

s w

ere

not s

tatis

tical

ly s

igni

fican

t (Ta

ble

2).

Tabl

e 2:

Pos

itive

TNP

T-re

spon

se a

nd c

orre

spon

ding

SP

T-w

heal

siz

es

Con

cent

ratio

n10

0 A

U/m

l1,

000

AU/

ml

10,0

00

AU/

ml

Posi

tive

resp

onse

(n)

1034

59M

ean

SPT-

whea

lsi

ze (m

m)

76

5Fi

gure

1: C

once

ntra

tion-

resp

onse

cur

ves

with

mea

n (

95%

CI)

chan

ges

in c

ompo

site

sym

ptom

sc

ores

(red

circ

les)

and

PNI

F m

easu

rem

ents

(blu

e ci

rcle

s) fo

llow

ing

TNPT

with

HDM

alle

rgen

.

Tabl

e 1:

Pat

ient

s’de

mog

raph

ic c

hara

cter

istic

s

Patie

nts

n =

103

Gen

der (

M (%

) / F

(%))

54(5

2.4%

)/ 49

(47.

6%)

Mea

n ag

e ±

SD in

yea

rs (r

ange

)32

±11

(18-

57)

SPT

to H

DM e

xtra

ct:

Mea

n wh

eal s

ize

in m

m ±

SD

(rang

e)6

±2

(3-1

2)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C. B

ache

rt1 , O. P

faar

2 , M.J

. Nel

l3 , J.D

. Boo

t3 , H.E

. Nie

nhui

s3 , Z. D

iam

ant4,

5

1 UZ

Gen

t, Ea

r-, N

ose

and

Thro

at D

epar

tmen

t, G

ent,

Belg

ium

; 2 Cen

ter f

or R

hino

logy

and

Alle

rgol

ogy

Wie

sbad

en, G

erm

any;

3 H

AL A

llerg

y BV

, Le

iden

, The

Net

herla

nds;

4 Ska

ne U

nive

rsity

Hos

pita

l, D

ept o

f Res

pir M

ed &

Alle

rgol

, Lun

d, S

wed

en

and

5 QPS

-NL,

Gro

ning

en, T

he N

ethe

rland

s

37 -

Appl

icat

ion

of a

sta

ndar

dize

d tit

rate

d na

sal

prov

ocat

ion

test

with

HD

M e

xtra

ct in

a

mul

ticen

tre

clin

ical

stu

dy in

alle

rgic

rhin

itis

Bac

kgro

und

& Ai

m:

Titra

ted

nasa

l pro

voca

tion

test

s (T

NPT

) with

a re

leva

nt a

llerg

enca

n be

use

d in

the

diag

nosi

s of

alle

rgic

rhin

itis

(AR

) and

as

a di

seas

e m

odel

in

clin

ical

tria

ls e

valu

atin

g th

e ef

ficac

y of

tre

atm

ent.

How

ever

, bo

th t

he t

echn

ique

s us

ed f

or p

rovo

catio

n an

d th

e re

adou

ts o

f th

e ev

oked

upp

erai

rway

resp

onse

vary

, req

uirin

g st

anda

rdiz

atio

n to

ena

ble

the

use

of T

NPT

as

a to

ol in

mul

ticen

tre tr

ials

. We

perfo

rmed

a s

tand

ardi

zed

TNPT

an

d do

cum

ente

d th

e up

per a

irway

resp

onse

by

sym

ptom

sco

res

and

peak

nas

al in

spira

tory

flow

(PN

IF) m

easu

rem

ents

. In

addi

tion,

we

com

pare

d th

e up

per a

irway

resp

onse

s ac

ross

the

diffe

rent

alle

rgen

con

cent

ratio

ns a

nd in

vest

igat

ed a

pos

sibl

e co

rrela

tion

betw

een

both

read

outs

.

Con

clus

ion:

Whe

n ap

plie

d in

a m

ultic

entre

set

ting

in p

atie

nts

with

sus

pect

ed H

DM

-indu

ced

AR, a

sta

ndar

dize

d TN

PT w

ith a

HD

M e

xtra

ct in

duce

sa

cons

iste

nt g

radu

al in

crea

se in

alle

rgic

sym

ptom

s an

d si

gns

that

can

be

relia

bly

quan

tifie

d by

a w

ell-d

efin

ed c

ompo

site

sym

ptom

sc

ore

and

PNIF

mea

sure

men

ts.

Met

hods

:Pa

tient

s: M

/F a

ged

18-6

0 ye

ars

with

HD

M-re

late

d AR

, w

ith o

r w

ithou

t co

ncom

itant

, cl

inic

ally

sta

ble

asth

ma

(FEV

1 >7

0% p

redi

cted

) w

ere

scre

ened

prio

r to

enro

llmen

t int

o a

mul

ticen

tre e

ffica

cy s

tudy

with

HD

M im

mun

othe

rapy

. Pat

ient

s w

ith a

pos

itive

AR

-his

tory

and

a p

ositi

ve s

kin

pric

k te

st to

HD

M, u

nder

went

a s

tand

ardi

zed

TNPT

. M

etho

ds:

The

TNPT

sta

rted

with

the

alle

rgen

’s d

iluen

t fo

llow

ed b

y m

axim

al3

seria

l co

ncen

tratio

ns o

f a

stan

dard

ized

alle

rgen

ext

ract

(D

. Pt

eron

yssi

nus

100,

1,0

00 a

nd 1

0,00

0 AU

/ml,

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s), a

dmin

iste

red

by a

nas

al s

pray

ing

devi

ce (

1 pu

ff pe

r no

stril

) at

20

min

utes

int

erva

ls.

The

uppe

r ai

rway

res

pons

e w

as q

uant

ified

by

a co

mpo

site

sym

ptom

sco

re a

ccor

ding

to

Lebe

l an

d PN

IF

mea

sure

men

ts (h

ighe

st v

alue

of 3

mea

sure

d by

In-c

heck

nas

al in

spira

tory

flow

met

er [C

lem

ent C

lark

e, H

arlo

w, U

K])a

t bas

elin

e (p

re-d

iluen

t), 1

5 m

inut

es a

fter e

ach

adm

inis

tratio

n an

d 60

min

utes

pos

t-cha

lleng

e. S

ympt

oms

wer

e re

cord

ed u

sing

the

follo

win

g sc

orin

g sy

stem

: sne

ezes

2

= 0,

sn

eeze

s 3-

4 =

1 po

int,

snee

zes

5 =

3 po

ints

, ant

erio

r rh

inor

rhoe

a =

1 po

int,

post

erio

r rh

inor

rhoe

a =

1 po

int,

diffi

cult

brea

thin

g =

1 po

int,

1 bl

ocke

d no

stril

= 2

poi

nts,

2 b

lock

ed n

ostri

ls =

3 p

oint

s, n

asal

prur

itus

= 1

poin

t, pr

uritu

s in

pal

ate

or e

ar =

1 p

oint

, con

junc

tiviti

s =

1 po

int (

tota

l sc

ore

rang

e: 0

-11

poin

ts).

The

pre-

dilu

ent w

as n

ot a

llow

ed to

be

3. T

he T

NPT

was

con

side

red

posi

tive

once

a to

tal s

core

6

was

reac

hed.

Res

ults

: 15

0 pa

tient

s w

ere

scre

ened

. 103

pat

ient

s (T

able

1)h

ad a

pos

itive

TN

PT. T

he T

NPT

was

wel

l tol

erat

ed a

nd n

one

of th

epa

tient

s ex

perie

nced

any

cl

inic

ally

sig

nific

ant a

dver

se e

vent

s. T

he m

ost p

rom

inen

t sym

ptom

s w

ere

nasa

l blo

ckag

e an

d rh

inor

rhoe

a, fo

llow

ed b

y pr

uritu

s an

dsn

eezi

ng. A

co

nsis

tent

con

cent

ratio

n-re

spon

se r

elat

ions

hip

was

foun

d be

twee

n se

rial a

llerg

en c

once

ntra

tions

and

incr

easi

ng s

ympt

om s

core

s, m

atch

ing

a gr

adua

l dec

reas

e in

PN

IF. O

ne h

our a

fter t

he p

rovo

cativ

e al

lerg

en c

once

ntra

tion,

bot

h pa

ram

eter

s ha

d no

t com

plet

ely

retu

rned

to b

asel

ine

valu

es

(Fig

ure

1). T

here

was

a m

oder

ate

thou

gh h

ighl

y si

gnifi

cant

cor

rela

tion

betw

een

sym

ptom

sco

res

and

PNIF

mea

sure

men

ts (r

=0.5

4; p

<0.0

1). T

he

maj

ority

of p

atie

nts

had

a po

sitiv

e TN

PT-re

spon

se fo

llow

ing

adm

inis

tratio

n of

the

10,0

00 A

U/m

l con

cent

ratio

n (T

able

2).

Patie

nts

with

a p

ositi

ve

TNPT

-resp

onse

at a

low

er a

llerg

en c

once

ntra

tion

tend

ed to

hav

e a

larg

er w

heal

siz

e al

thou

gh d

iffer

ence

s w

ere

not s

tatis

tical

ly s

igni

fican

t (Ta

ble

2).

Tabl

e 2:

Pos

itive

TNP

T-re

spon

se a

nd c

orre

spon

ding

SP

T-w

heal

siz

es

Con

cent

ratio

n10

0 A

U/m

l1,

000

AU/

ml

10,0

00

AU/

ml

Posi

tive

resp

onse

(n)

1034

59M

ean

SPT-

whea

lsi

ze (m

m)

76

5Fi

gure

1: C

once

ntra

tion-

resp

onse

cur

ves

with

mea

n (

95%

CI)

chan

ges

in c

ompo

site

sym

ptom

sc

ores

(red

circ

les)

and

PNI

F m

easu

rem

ents

(blu

e ci

rcle

s) fo

llow

ing

TNPT

with

HDM

alle

rgen

.

Tabl

e 1:

Pat

ient

s’de

mog

raph

ic c

hara

cter

istic

s

Patie

nts

n =

103

Gen

der (

M (%

) / F

(%))

54(5

2.4%

)/ 49

(47.

6%)

Mea

n ag

e ±

SD in

yea

rs (r

ange

)32

±11

(18-

57)

SPT

to H

DM e

xtra

ct:

Mea

n wh

eal s

ize

in m

m ±

SD

(rang

e)6

±2

(3-1

2)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C. B

ache

rt1 , O. P

faar

2 , M.J

. Nel

l3 , J.D

. Boo

t3 , H.E

. Nie

nhui

s3 , Z. D

iam

ant4,

5

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.

Pa t i e nt Kn o w l e d ge, Pe rce p t i o n s , E xp e c t a t i o n s , a n d S a t isf a c t i o n , o n Su b cu t a n e o u s a n d Su b l ing u a l Al l e rge n Sp e c i f ic Im mu n o t h e rap y: a r ea l l i f e su r v e y

I. Baiardini (1), F. Puggioni (2), S. Menoni (3), J.D. Boot (4), Z. Diamant (5), F. Braido (6), G.W. Canonica (6).

(1) University of Genoa, Allergy and Respiratory Diseases, Department of Internal Medicine, Genoa, Italy, (2) IRCCS Istituto Clinico Humanitas,

Allergy and respiratory Disease Department, Milano, Italy, (3) University of Genoa, Biostatistic Unit, Department of Health Science, Genoa,

Italy, (4) HAL Allergy BV, Leiden, The Netherlands, (5) Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and (6) QPS-NL,

Groningen, The Netherlands.

BackgroundAssessing patient’s viewpoint on treatment provides useful information enabling customized therapeutic approach. Presently, only a few small studies addressing the patient’s perspective on allergen specific immunotherapy (SIT) are available. The aim of this cross-sectional multicentre survey was to evaluate subjective viewpoints on SIT in a large cohort of patients treated with subcutaneous (SCIT) or sublingual (SLIT) immunotherapy.

MethodsA novel survey of 28 questions assessing patient’s knowledge (6), perception (12), expectations (5) and satisfaction (5) was developed by an expert panel. Sixteen physicians from 15 allergology centres in North Italy asked 30 consecutive patients with allergic rhinitis with or without asthma treated with SIT to fill out the survey. The physicians were also asked to report for each patient their satisfaction level regarding SIT.

ResultsFully completed surveys from 434 patients (55.3% male; 66.7% polysensitized, 74% SLIT) were analyzed. SIT consisted of grass pollen (37.1%), mites (26.6%), tree pollen (17%), flower pollen (9.2%), moulds (5.6%), epithelia (2%) and other (2.5%); mean duration of treatment was 2.5 years. Most patients acquired their SIT knowledge through their physician (95%). Conversely, the physicians opinion in their choice to start with SIT was important. The majority of patients (70.6%) believed that SIT should be continued for more than two years. Most patients perceived SIT to be safe and easy to integrate in daily routine. The main motivation when starting SIT were its potential effect to alter the course of the disease (45.7%), less need of anti-allergy drugs (28.2%), and dissatisfaction with the current pharmacotherapy (19.3%). Both patient and physician satisfaction was high (VAS score 74/100 and 78/100, respectively) and a significant correlation between patients’ and physicians’ satisfaction scores was found in both groups (SCIT: r = 0.612, p <0.001; SLIT: r = 0.608, p <0.001). No major difference was found in patients answers based on the level of education. Compared to SCIT, SLIT was frequently considered easier to take (p=0.024) and without side effects (p=0.026)

ConclusionThis real life, cross-sectional survey evaluated in a large cohort different aspects of patient’s perspective on SIT. Despite some gaps and misconceptions, the majority of patients had an adequate level of knowledge, perception, expectations and satisfaction about SIT, which corresponded well with the physician’s satisfaction level.

10

EAACI, 16-20 June 2012, GenevaAbstract number: 874, Session date and time: Monday 18 June; 12:15 - 13:30 Session title: Poster 32 - Update in the treatment of allergic rhinitis

11

Poster Session 32 - Update in the treatment of allergic rhinitis

Pa t i e nt Kn o w l e d ge, Pe rce p t i o n s , E xp e c t a t i o n s , a n d S a t isf a c t i o n , o n Su b cu t a n e o u s a n d Su b l ing u a l Al l e rge n Sp e c i f ic Im mu n o t h e rap y: a r ea l l i f e su r v e y

1 Uni

vers

ity o

f Gen

oa, A

llerg

y an

d Re

spira

tory

Dis

ease

s, D

epar

tmen

t of I

nter

nal M

edic

ine,

Gen

oa, I

taly

, 2 IRCC

S Is

titut

oCl

inic

oHu

man

itas,

Alle

rgy

and

resp

irato

ry D

isea

se

Depa

rtmen

t, M

ilano

, Ita

ly,3 U

nive

rsity

of G

enoa

, Bio

stat

istic

Unit,

Dep

artm

ent o

f Hea

lth S

cien

ce, G

enoa

, Ita

ly,4 H

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds,

5 S

kane

Univ

ersi

ty H

ospi

tal,

Dep

t of R

espi

rMed

& A

llerg

ol, L

und,

Sw

eden

and

QPS

-NL,

Gro

ning

en, T

he N

ethe

rland

s

874

-Pat

ient

Kno

wle

dge,

Per

cept

ions

, Exp

ecta

tions

and

Sa

tisfa

ctio

n on

Sub

cuta

neou

s an

d Su

blin

gual

Al

lerg

en S

peci

fic Im

mun

othe

rapy

:a re

al li

fe s

urve

y

Tabl

e 1:

Pat

ient

dem

ogra

phic

s an

d cl

inic

al d

ata

Figu

re 1

: Cor

rela

tion

betw

een

patie

nts’

and

phys

icia

ns’s

atis

fact

ion

leve

l (V

AS

scor

e) o

n su

bcut

aneo

us (l

eft p

anel

; r =

0.6

12, p

<0.

001)

and

su

blin

gual

(rig

ht p

anel

r =

0.60

8, p

<0.

001)

SIT

Su

rvey

Patie

nts

n =

434

Gen

der

238

Mal

es/ 1

92 F

emal

es

Mea

n ag

e ±

SD in

yea

rs

31.5

12

Mea

n du

ratio

n of

trea

tmen

t ±SD

in y

ears

2.5

±1.

3

Rhi

nitis

–AR

IA c

lass

ificat

ion

Inte

rmitt

ent

40.6

%

Pers

isten

t59

.4%

Mild

11.3

%

Mod

erat

e88

.7%

Asth

ma

–G

INA

Cla

ssific

atio

n

129

.7%

254

.5%

314

.3%

41.

5%

Sens

itiza

tion

Mon

osen

sitiz

ed33

.3%

Poly

sens

itize

d66

.7%

SCIT

26.0

%

SLIT

74.0

%

Adm

inis

tere

d al

lerg

en

Gra

ss p

olle

n37

.1%

Mite

s26

.6%

Tree

pol

len

17.0

%

Flow

er p

olle

n9.

2%

Oth

er10

.1%

Tabl

e 2:

Pat

ient

s pe

rcep

tion

abou

t SIT

Que

stio

nsPa

tient

answ

ers

Com

plet

ely

True

Pa

rtly

True

Partl

yFa

lseC

ompl

etel

y Fa

lseD

on’t

know

SIT

is sa

fe54

.5%

37.4

%0.

2%0.

2%7.

6%SI

T is

easy

to ta

ke68

.6%

27.5

%0.

5%-

3.5%

SIT

is ha

ndy

to u

se in

da

ily ro

utin

e47

.1%

42.7

%1.

8%0.

2%8.

1%

It is

eas

y to

rem

embe

r to

take

SIT

50.8

%37

.0%

2.8%

0.2%

9.2%

SIT

allo

ws to

bet

ter

cont

rol y

our a

llerg

y56

.0%

35.2

%0.

7%5.

1%3.

0%

1.0%

Did

not

spe

cifiy

Poss

ible

ans

wer

st

Patie

nan

swer

sR

ecov

ery

from

alle

rgy

38.2

%Im

prov

emen

t in

heal

th re

late

d qu

ality

of l

ife21

.4%

Sym

ptom

redu

ctio

n22

.6%

Less

nee

d of

ant

i-alle

rgy

med

icatio

n10

.5%

Avoi

d wo

rsen

ing

of th

e di

seas

e6.

4%

Tabl

e 3:

Pat

ient

s ex

pect

atio

n ab

out S

IT

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st:

the

pres

ente

r has

rece

ived

a tr

avel

gra

nt fr

om H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

:

®®

know

ledg

e

perc

eive

d

expe

cted

satis

fact

ion

I. B

aiar

dini

1 , F.

Pug

gion

i2 , S.

Men

oni3 ,

J.D

. Boo

t4 , Z.

Dia

man

t5 , F.

Bra

ido1 ,

G.W

. Can

onic

a1

1 Uni

vers

ity o

f Gen

oa, A

llerg

y an

d Re

spira

tory

Dis

ease

s, D

epar

tmen

t of I

nter

nal M

edic

ine,

Gen

oa, I

taly

, 2 IRCC

S Is

titut

oCl

inic

oHu

man

itas,

Alle

rgy

and

resp

irato

ry D

isea

se

Depa

rtmen

t, M

ilano

, Ita

ly,3 U

nive

rsity

of G

enoa

, Bio

stat

istic

Unit,

Dep

artm

ent o

f Hea

lth S

cien

ce, G

enoa

, Ita

ly,4 H

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds,

5 S

kane

Univ

ersi

ty H

ospi

tal,

Dep

t of R

espi

rMed

& A

llerg

ol, L

und,

Sw

eden

and

QPS

-NL,

Gro

ning

en, T

he N

ethe

rland

s

874

-Pat

ient

Kno

wle

dge,

Per

cept

ions

, Exp

ecta

tions

and

Sa

tisfa

ctio

n on

Sub

cuta

neou

s an

d Su

blin

gual

Al

lerg

en S

peci

fic Im

mun

othe

rapy

:a re

al li

fe s

urve

y

Tabl

e 1:

Pat

ient

dem

ogra

phic

s an

d cl

inic

al d

ata

Figu

re 1

: Cor

rela

tion

betw

een

patie

nts’

and

phys

icia

ns’s

atis

fact

ion

leve

l (V

AS

scor

e) o

n su

bcut

aneo

us (l

eft p

anel

; r =

0.6

12, p

<0.

001)

and

su

blin

gual

(rig

ht p

anel

r =

0.60

8, p

<0.

001)

SIT

Su

rvey

Patie

nts

n =

434

Gen

der

238

Mal

es/ 1

92 F

emal

es

Mea

n ag

e ±

SD in

yea

rs

31.5

12

Mea

n du

ratio

n of

trea

tmen

t ±SD

in y

ears

2.5

±1.

3

Rhi

nitis

–AR

IA c

lass

ificat

ion

Inte

rmitt

ent

40.6

%

Pers

isten

t59

.4%

Mild

11.3

%

Mod

erat

e88

.7%

Asth

ma

–G

INA

Cla

ssific

atio

n

129

.7%

254

.5%

314

.3%

41.

5%

Sens

itiza

tion

Mon

osen

sitiz

ed33

.3%

Poly

sens

itize

d66

.7%

SCIT

26.0

%

SLIT

74.0

%

Adm

inis

tere

d al

lerg

en

Gra

ss p

olle

n37

.1%

Mite

s26

.6%

Tree

pol

len

17.0

%

Flow

er p

olle

n9.

2%

Oth

er10

.1%

Tabl

e 2:

Pat

ient

s pe

rcep

tion

abou

t SIT

Que

stio

nsPa

tient

answ

ers

Com

plet

ely

True

Pa

rtly

True

Partl

yFa

lseC

ompl

etel

y Fa

lseD

on’t

know

SIT

is sa

fe54

.5%

37.4

%0.

2%0.

2%7.

6%SI

T is

easy

to ta

ke68

.6%

27.5

%0.

5%-

3.5%

SIT

is ha

ndy

to u

se in

da

ily ro

utin

e47

.1%

42.7

%1.

8%0.

2%8.

1%

It is

eas

y to

rem

embe

r to

take

SIT

50.8

%37

.0%

2.8%

0.2%

9.2%

SIT

allo

ws to

bet

ter

cont

rol y

our a

llerg

y56

.0%

35.2

%0.

7%5.

1%3.

0%

1.0%

Did

not

spe

cifiy

Poss

ible

ans

wer

st

Patie

nan

swer

sR

ecov

ery

from

alle

rgy

38.2

%Im

prov

emen

t in

heal

th re

late

d qu

ality

of l

ife21

.4%

Sym

ptom

redu

ctio

n22

.6%

Less

nee

d of

ant

i-alle

rgy

med

icatio

n10

.5%

Avoi

d wo

rsen

ing

of th

e di

seas

e6.

4%

Tabl

e 3:

Pat

ient

s ex

pect

atio

n ab

out S

IT

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st:

the

pres

ente

r has

rece

ived

a tr

avel

gra

nt fr

om H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

:

®®

know

ledg

e

perc

eive

d

expe

cted

satis

fact

ion

I. B

aiar

dini

1 , F.

Pug

gion

i2 , S.

Men

oni3 ,

J.D

. Boo

t4 , Z.

Dia

man

t5 , F.

Bra

ido1 ,

G.W

. Can

onic

a1

1 Uni

vers

ity o

f Gen

oa, A

llerg

y an

d Re

spira

tory

Dis

ease

s, D

epar

tmen

t of I

nter

nal M

edic

ine,

Gen

oa, I

taly

, 2 IRCC

S Is

titut

oCl

inic

oHu

man

itas,

Alle

rgy

and

resp

irato

ry D

isea

se

Depa

rtmen

t, M

ilano

, Ita

ly,3 U

nive

rsity

of G

enoa

, Bio

stat

istic

Unit,

Dep

artm

ent o

f Hea

lth S

cien

ce, G

enoa

, Ita

ly,4 H

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds,

5 S

kane

Univ

ersi

ty H

ospi

tal,

Dep

t of R

espi

rMed

& A

llerg

ol, L

und,

Sw

eden

and

QPS

-NL,

Gro

ning

en, T

he N

ethe

rland

s

874

-Pat

ient

Kno

wle

dge,

Per

cept

ions

, Exp

ecta

tions

and

Sa

tisfa

ctio

n on

Sub

cuta

neou

s an

d Su

blin

gual

Al

lerg

en S

peci

fic Im

mun

othe

rapy

:a re

al li

fe s

urve

y

Tabl

e 1:

Pat

ient

dem

ogra

phic

s an

d cl

inic

al d

ata

Figu

re 1

: Cor

rela

tion

betw

een

patie

nts’

and

phys

icia

ns’s

atis

fact

ion

leve

l (V

AS

scor

e) o

n su

bcut

aneo

us (l

eft p

anel

; r =

0.6

12, p

<0.

001)

and

su

blin

gual

(rig

ht p

anel

r =

0.60

8, p

<0.

001)

SIT

Su

rvey

Patie

nts

n =

434

Gen

der

238

Mal

es/ 1

92 F

emal

es

Mea

n ag

e ±

SD in

yea

rs

31.5

12

Mea

n du

ratio

n of

trea

tmen

t ±SD

in y

ears

2.5

±1.

3

Rhi

nitis

–AR

IA c

lass

ificat

ion

Inte

rmitt

ent

40.6

%

Pers

isten

t59

.4%

Mild

11.3

%

Mod

erat

e88

.7%

Asth

ma

–G

INA

Cla

ssific

atio

n

129

.7%

254

.5%

314

.3%

41.

5%

Sens

itiza

tion

Mon

osen

sitiz

ed33

.3%

Poly

sens

itize

d66

.7%

SCIT

26.0

%

SLIT

74.0

%

Adm

inis

tere

d al

lerg

en

Gra

ss p

olle

n37

.1%

Mite

s26

.6%

Tree

pol

len

17.0

%

Flow

er p

olle

n9.

2%

Oth

er10

.1%

Tabl

e 2:

Pat

ient

s pe

rcep

tion

abou

t SIT

Que

stio

nsPa

tient

answ

ers

Com

plet

ely

True

Pa

rtly

True

Partl

yFa

lseC

ompl

etel

y Fa

lseD

on’t

know

SIT

is sa

fe54

.5%

37.4

%0.

2%0.

2%7.

6%SI

T is

easy

to ta

ke68

.6%

27.5

%0.

5%-

3.5%

SIT

is ha

ndy

to u

se in

da

ily ro

utin

e47

.1%

42.7

%1.

8%0.

2%8.

1%

It is

eas

y to

rem

embe

r to

take

SIT

50.8

%37

.0%

2.8%

0.2%

9.2%

SIT

allo

ws to

bet

ter

cont

rol y

our a

llerg

y56

.0%

35.2

%0.

7%5.

1%3.

0%

1.0%

Did

not

spe

cifiy

Poss

ible

ans

wer

st

Patie

nan

swer

sR

ecov

ery

from

alle

rgy

38.2

%Im

prov

emen

t in

heal

th re

late

d qu

ality

of l

ife21

.4%

Sym

ptom

redu

ctio

n22

.6%

Less

nee

d of

ant

i-alle

rgy

med

icatio

n10

.5%

Avoi

d wo

rsen

ing

of th

e di

seas

e6.

4%

Tabl

e 3:

Pat

ient

s ex

pect

atio

n ab

out S

IT

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st:

the

pres

ente

r has

rece

ived

a tr

avel

gra

nt fr

om H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

:

®®

know

ledg

e

perc

eive

d

expe

cted

satis

fact

ion

I. B

aiar

dini

1 , F.

Pug

gion

i2 , S.

Men

oni3 ,

J.D

. Boo

t4 , Z.

Dia

man

t5 , F.

Bra

ido1 ,

G.W

. Can

onic

a1

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st: t

he p

rese

nter

has

rece

ived

a tr

avel

gra

nt fr

om H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

In f l u e n ce o f c o - m o r b i d i t i e s a n d c o - m e d ic a t i o n s o n s a f e t y a n d t o l e ra b il i t y in t h e b u il d u p p h a s e o f v e n o m im mu n o t h e rap y

12

A. Bauer, K. Bernkopf, P. Spornraft-Ragaller.

Department of Dermatology, University Hospital Carl Gustav Carus, TU Dresden, Germany.

BackgroundSafety and tolerability of specific venom immunotherapy (VIT) may be influenced by co-morbidities and co-medications. In the current study safety and tolerability of a 3 day ultra-rush protocol in patients with co-morbidities and co-medications was assessed during the build-up phase.

MethodsSafety and tolerability of VIT (wasp venom n=114; bee venom n=11) was analysed in patients with co-morbidities and co-medications. Starting with 0.02 µg venom, the maintenance dose of 100µg was reached in 15 dose increments within 3 days. Local and systemic reactions were documented during the build-up phase.

Results124 patients (67 female/57 male, 51 years range 18-81 years) with an established history of venom allergy were included. 55 (44%) patients suffered from cardiovascular co-morbidities, 22 (17.7%) from metabolic disorders, 24 (19.3%) from skin or respiratory atopy and 18 (14.5%) showed increased baseline tryptase levels >11.4 µg/l. During the build-up phase of VIT local reactions were seen in 46 (37.1%) patients and large local reactions in 69 (55.6%) patients. No significant differences were seen between the groups. Systemic reactions (SR) grade I-III were seen in 10 (8,1%) patients (SR I: 5 (4%) patients, SR II: 4 (3.2%), SR III: 1 (0.8%)). No SR grade IV were seen. Multivariate logistic regression analysis revealed ACE-inhibitor medication (OR 16.14; 95%CI 2.5-104.27), female sex (OR 9.17; 95%CI 1.34-62.97), increased baseline serum tryptase levels (OR 6.68; 95%CI 1.04-43.01) and therapy with bee venom (OR 30.99; 95%CI 3.18-302.34) to be relevant risk factor for SR during the buildup phase of VIT.

ConclusionSafety and tolerability of ultra rush VIT was not influenced by cardiovascular, metabolic or atopic co-morbidities. However, female sex, bee venom sensitization, increased baseline serum tryptase levels and medication with ACE-inhibitors were significantly associated with an increased risk to develop SR during the build-up phase of VIT.

EAACI, 16-20 June 2012, GenevaAbstract number: 1087, Session date and time: Monday 18 June; 12:15 - 13:30 Session title: Poster 46 - Advances in immunotherapy: latest studies

13

Poster Session 46 - Advances in immunotherapy: latest studies

In f l u e n ce o f c o - m o r b i d i t i e s a n d c o - m e d ic a t i o n s o n s a f e t y a n d t o l e ra b il i t y in t h e b u il d u p p h a s e o f v e n o m im mu n o t h e rap y

D e sign o f a d o s e ra nge f in d ing s tu d y w i t h a l l e rge n s p e c i f ic im mu n o t h e rap y in p a t i e nt s w i t h h o u s e d u s t mi t e in d u ce d a l l e rgic r h ini t is / r h in o c o n j u n c t iv i t is

14

C. Bachert (1), O. Pfaar (2), A. Roger (3), H. Riechelmann (4), J.N.G. Oude Elberink (5), M.J. Nell (6), J.D. Boot (6).

(1) UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) Unitat

d’Allèrgia, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, (4) Universitätsklinik für Hals-, Nasen- und Ohrenheilkunde, Innsbruck,

Austria, (5) Dept of Allergology, University Medical Center Groningen, University of Groningen, The Netherlands, (6) HAL Allergy BV, Leiden,

The Netherlands.

BackgroundIn order to comply with the 2008 EMA guidelines on the development of specific immunotherapy (SIT) products, a clinical development program was started to obtain full marketing authorization for a subcutaneous SIT allergoid for the treatment of house dust mite (HDM) allergy. Safety and tolerability of increasing doses have previously been determined in an open-label clinical study. The next step is a dose range finding (DRF) study to identify the optimal, i.e. safe and effective dose in patients with HDM induced allergic rhinitis/rhinoconjunctivitis (AR). As there is a wide variety of study designs in terms of inclusion criteria, doses, study duration, end-points, analysis of data, and control of environmental variables in the evalu-ation of SIT products, this abstract highlights the design and rationale of the DRF study.

MethodThe current study is a 1 year, multicenter, randomized, double-blind, placebo-controlled, 5 arm parallel-group, DRF study (ClinicalTrials.gov NCT01438463). Approximately 250 patients (50 per arm) with persistent, symptomatic AR with or wit-hout concomitant asthma related to HDM will be randomized. Due to the need of at least 4 active study arms in a DRF study, the use of a classical endpoint for SIT (i.e. the symptom & medication score) as primary parameter was not feasible because of the necessity of a large sample size. We therefore selected the titrated nasal provocation test (TNPT) with HDM allergen as the primary parameter. The TNPT is a reproducible exacerbation model of allergic rhinitis often applied to evaluate the efficacy of anti-allergy medications. The allergen-induced upper airway response will be quantified by recording symptom scores (Lebel-Score) and nasal flow (Peak Nasal Inspiratory Flow) after each HDM challenge. To minimize seasonal influ-ences the absolute difference in mean symptom score in the TNPT after one year of treatment and baseline will be used as primary endpoint. Patients will only be subjected to a TNPT in the absence of baseline symptoms and patients with seasonal allergies will only be challenged outside the corresponding pollen season. The study is performed in 40 clinical study centers in 5 European countries.

ResultsThe first patient was recruited in October 2011 and results will be available in 2013.

ConclusionAccording to the EMA guidelines we designed a Phase II DRF study to identify the optimal dose of a subcutaneous SIT allergoid for patients with HDM induced AR.

EAACI, 16-20 June 2012, GenevaAbstract number: 1393, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 68 - New clinical studies in immunotherapy

15

Poster Session 68 - New clinical studies in immunotherapy

D e sign o f a d o s e ra nge f in d ing s tu d y w i t h a l l e rge n s p e c i f ic im mu n o t h e rap y in p a t i e nt s w i t h h o u s e d u s t mi t e in d u ce d a l l e rgic r h ini t is / r h in o c o n j u n c t iv i t is

1 UZ

Gen

t, Ea

r-, N

ose

and

Thro

at D

epar

tmen

t, G

ent,

Belg

ium

, 2Ce

nter

for R

hino

logy

and

Alle

rgol

ogy

Wie

sbad

en, G

erm

any,

3Un

itat d

’Allè

rgia

, Hos

pita

l Un

iver

sita

ri G

erm

ans

Tria

s i P

ujol

, Bad

alon

a, S

pain

, 4Un

iver

sitä

tskl

inik

für H

als-

, Nas

en-u

nd O

hren

heilk

unde

, Inn

sbru

ck, A

ustr

ia, 5

Dept

of A

llerg

olog

y,

Univ

ersi

ty M

edic

al C

ente

r Gro

ning

en, U

nive

rsity

of G

roni

ngen

, The

Net

herla

nds,

6 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

1393

-D

esig

n of

a D

ose

Ran

ge F

indi

ng s

tudy

with

alle

rgen

sp

ecifi

c im

mun

othe

rapy

in p

atie

nts

with

H

DM

indu

ced

alle

rgic

rhin

itis/

rhin

ocon

junc

tiviti

s

Bac

kgro

und

& A

im:

In o

rder

to

com

ply

with

the

200

8 EM

A gu

idel

ines

on

the

deve

lopm

ent

of s

peci

fic i

mm

unot

hera

py (

SIT)

pro

duct

s, a

cl

inic

al d

evel

opm

ent

prog

ram

was

sta

rted

to o

btai

n fu

ll m

arke

ting

auth

oriz

atio

n fo

r a s

ubcu

tane

ous

SIT

alle

rgoi

d fo

r th

e tre

atm

ent o

f hou

se d

ust m

ite (H

DM

) alle

rgy

1 . Sa

fety

and

to

lera

bility

of

in

crea

sing

do

ses

have

pr

evio

usly

be

en

dete

rmin

ed in

an

open

-labe

l clin

ical

stu

dy. T

he n

ext s

tep

is a

do

se r

ange

find

ing

(DR

F) s

tudy

to id

entif

y th

e op

timal

, i.e

. sa

fe a

nd e

ffect

ive

dose

in

patie

nts

with

HD

M i

nduc

ed

alle

rgic

rhi

nitis

/rhin

ocon

junc

tivitis

(AR

). As

the

re i

s a

wide

va

riety

of s

tudy

des

igns

in te

rms

of in

clus

ion

crite

ria, d

oses

, st

udy

dura

tion,

end

-poi

nts,

ana

lysis

of

data

, an

d co

ntro

l of

envi

ronm

enta

l var

iabl

es in

the

eva

luat

ion

of S

IT p

rodu

cts,

th

is a

bstra

ct h

ighl

ight

s th

e de

sign

and

ratio

nale

of t

he D

RF

stud

y.

Figu

re 2

: Leb

el a

nd P

NIF

scor

e sh

eet f

or th

e TN

PT o

utco

me.

Figu

re 1

: Des

ign

of th

e st

udy

Con

clus

ion:

Acco

rdin

g to

the

EM

A gu

idel

ines

we

desi

gned

a P

hase

II

DR

F st

udy

to id

entif

y th

e op

timal

dos

e of

a s

ubcu

tane

ous

SIT

alle

rgoi

d fo

r pat

ient

s wi

th H

DM

indu

ced

AR.

Met

hods

:Th

e cu

rrent

stu

dy

is a

1 y

ear,

mul

ticen

ter,

rand

omiz

ed,

doub

le-b

lind,

pla

cebo

-con

trolle

d, 5

arm

par

alle

l-gro

up, D

RF

stud

y (C

linic

alTr

ials

.gov

NC

T014

3846

3). A

ppro

ximat

ely

250

patie

nts

(50

per a

rm)

with

per

sist

ent,

sym

ptom

atic

AR

with

or

with

out

conc

omita

nt a

sthm

a re

late

d to

HD

M w

ill be

ra

ndom

ized

. In

clus

ion

crite

ria a

re p

rese

nted

in

Tabl

e 1.

Pa

tient

s we

re tr

eate

d fo

r one

yea

r with

mite

s al

lerg

oid

(HAL

Al

lerg

y, L

eide

n, T

he N

ethe

rland

s) s

ee F

igur

e 1.

Due

to th

e ne

ed o

f at l

east

4 a

ctiv

e st

udy

arm

s in

a D

RF

stud

y, th

e us

e of

a

clas

sica

l en

dpoi

nt

for

SIT

(i.e.

th

e sy

mpt

om

& m

edic

atio

n sc

ore)

as

prim

ary

para

met

er w

as n

ot f

easi

ble

beca

use

of th

e ne

cess

ity o

f a la

rge

sam

ple

size

. The

refo

re,

the

titra

ted

nasa

l pr

ovoc

atio

n te

st

(TN

PT)

with

H

DM

al

lerg

en a

s th

e pr

imar

y pa

ram

eter

was

sel

ecte

d. T

he T

NPT

is

a re

prod

ucib

le e

xace

rbat

ion

mod

el o

f alle

rgic

rhin

itis

ofte

n ap

plie

d to

eva

luat

e th

e ef

ficac

y of

ant

i-alle

rgy

med

icat

ions

. Th

e al

lerg

en-in

duce

d up

per

airw

ay

resp

onse

w

ill be

qu

antif

ied

by r

ecor

ding

sym

ptom

sco

res

(Leb

el s

core

) an

d na

sal f

low

(Pe

ak N

asal

Insp

irato

ry F

low

, PN

IF)

afte

r ea

ch

HD

M

chal

leng

e (s

ee

Figu

res

2 an

d 3)

.To

m

inim

ize

seas

onal

in

fluen

ces

the

abso

lute

di

ffere

nce

in

mea

n sy

mpt

om s

core

in th

e TN

PT a

fter o

ne y

ear o

f tre

atm

ent a

nd

base

line

will

be u

sed

as p

rimar

y en

dpoi

nt. P

atie

nts

will

only

be

su

bjec

ted

to

a TN

PT

in

the

abse

nce

of

base

line

sym

ptom

s an

d pa

tient

s wi

th s

easo

nal a

llerg

ies

will

only

be

chal

leng

ed o

utsi

de t

he c

orre

spon

ding

pol

len

seas

on.

The

stud

y is

pe

rform

ed

in

40

clin

ical

st

udy

cent

ers

in

5 Eu

rope

an c

ount

ries.

Res

ults

: Th

e fir

st p

atie

nt w

as re

crui

ted

in O

ctob

er 2

011

and

the

last

pa

tient

was

enr

olle

d in

Mar

ch 2

012.

Res

ults

will

be a

vaila

ble

in 2

013.

Posi

tive

TNPT

for H

DM D

. pte

rext

ract

at s

cree

ning

(Leb

el s

core

6

at o

r bel

ow

10,0

00 A

U/m

l)

Seru

m s

peci

fic Ig

E-te

st (s

sIgE

) lev

el fo

r HD

M D

. pte

ror D

. far

at s

cree

ning

(>

0.7

U/m

l)

Posi

tive

SPT

to H

DM D

. pte

rand

/or D

. far

(mea

n wh

eal d

iam

eter

3m

m,

com

pare

d to

neg

ativ

e co

ntro

l and

neg

ativ

e co

ntro

l sho

uld

be n

egat

ive)

Patie

nts

with

a h

isto

ry o

f con

com

itant

ast

hma

shou

ld h

ave

a FE

V1>

70%

at

incl

usio

n. P

atie

nts

with

out a

his

tory

of a

sthm

a sh

ould

hav

e a

FEV1

> 7

0% o

r a

PEF

> 80

%

Patie

nts

with

alle

rgic

rhin

itis

or rh

inoc

onju

nctiv

itis

for a

t lea

st 1

yea

r; al

lerg

ic

sym

ptom

s re

late

d to

HDM

, with

or w

ithou

t con

com

itant

clin

ical

ly s

tabl

e co

ntro

lled

mild

to m

oder

ate

asth

ma

(acc

ordi

ng to

GIN

A cl

assi

ficat

ion)

Patie

nts

(mal

e or

fem

ale)

mus

t be

18 a

nd

60 y

ears

at s

cree

ning

Sign

ed in

form

ed c

onse

ntIn

clus

ion

crite

ria

Tabl

e 1:

Incl

usio

n cr

iteria

of t

he D

RF

stud

y.

Ref

eren

ces:

1EM

A G

uide

line

on th

e de

velo

pmen

t of p

rodu

cts

for

spec

ific im

mun

othe

rapy

for

the

treat

men

t of

al

lerg

ic

dise

ases

. C

HM

P/EW

P/18

504/

2006

, Lo

ndon

.20

N

ovem

ber 2

008.

PNIF

sco

re(h

ighe

stof

3

mea

sure

men

ts)

Tota

l LEB

EL s

core

Prur

itus

(Itch

yno

se=1

,Itc

hypa

late

and

/ore

ar=1

, Irr

itate

dey

es=1

)

Nas

alob

stru

ctio

n(D

ifficu

ltna

salb

reat

hing

=1,

One

nost

rilbl

ocke

d=2,

Bot

h no

stril

sbl

ocke

d=3)

Run

nyN

ose

(Pos

terio

r=1,

An

terio

r=1)

Snee

zing

(1-2

=0, 3

-4=1

,5

=3)

Tim

e of

mea

sure

men

t

-Ac

tual

Tim

e of

app

licat

ion

1h a

fter

last

ch.

20m

in.

afte

rpr

evio

us

20m

in.

afte

rpr

evio

us

20m

in.

afte

rdi

luen

t

--

Sche

dule

dtim

e fo

rnex

tch

alle

nge

Post

ch.

10,0

00

AU/m

l1,

000

AU/m

l10

0 AU

/ml

Post

D

iluen

tPr

e D

iluen

t(B

asel

ine)

Alle

rgen

Figu

re 3

: Per

form

ance

of a

nas

al c

halle

nge

(left)

and

PNI

F (r

ight

).

TNPT

& R

ando

mis

atio

nTN

PT12

mon

ths

10,0

00 A

Ueq/

dose

25,0

00 A

Ueq/

dose

50,0

00 A

Ueq/

dose

Plac

ebo

Scre

enin

g

3,33

3 AU

eq/d

ose

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C. B

ache

rt1 , O

. Pfa

ar2 ,

A. R

oger

3 , H.

Rie

chel

man

n4 , J.

N.G

. Oud

e El

berin

k5 , M

.J. N

ell6 ,

J.D.

Boo

t6

1 UZ

Gen

t, Ea

r-, N

ose

and

Thro

at D

epar

tmen

t, G

ent,

Belg

ium

, 2Ce

nter

for R

hino

logy

and

Alle

rgol

ogy

Wie

sbad

en, G

erm

any,

3Un

itat d

’Allè

rgia

, Hos

pita

l Un

iver

sita

ri G

erm

ans

Tria

s i P

ujol

, Bad

alon

a, S

pain

, 4Un

iver

sitä

tskl

inik

für H

als-

, Nas

en-u

nd O

hren

heilk

unde

, Inn

sbru

ck, A

ustr

ia, 5

Dept

of A

llerg

olog

y,

Univ

ersi

ty M

edic

al C

ente

r Gro

ning

en, U

nive

rsity

of G

roni

ngen

, The

Net

herla

nds,

6 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

1393

-D

esig

n of

a D

ose

Ran

ge F

indi

ng s

tudy

with

alle

rgen

sp

ecifi

c im

mun

othe

rapy

in p

atie

nts

with

H

DM

indu

ced

alle

rgic

rhin

itis/

rhin

ocon

junc

tiviti

s

Bac

kgro

und

& A

im:

In o

rder

to

com

ply

with

the

200

8 EM

A gu

idel

ines

on

the

deve

lopm

ent

of s

peci

fic i

mm

unot

hera

py (

SIT)

pro

duct

s, a

cl

inic

al d

evel

opm

ent

prog

ram

was

sta

rted

to o

btai

n fu

ll m

arke

ting

auth

oriz

atio

n fo

r a s

ubcu

tane

ous

SIT

alle

rgoi

d fo

r th

e tre

atm

ent o

f hou

se d

ust m

ite (H

DM

) alle

rgy

1 . Sa

fety

and

to

lera

bility

of

in

crea

sing

do

ses

have

pr

evio

usly

be

en

dete

rmin

ed in

an

open

-labe

l clin

ical

stu

dy. T

he n

ext s

tep

is a

do

se r

ange

find

ing

(DR

F) s

tudy

to id

entif

y th

e op

timal

, i.e

. sa

fe a

nd e

ffect

ive

dose

in

patie

nts

with

HD

M i

nduc

ed

alle

rgic

rhi

nitis

/rhin

ocon

junc

tivitis

(AR

). As

the

re i

s a

wide

va

riety

of s

tudy

des

igns

in te

rms

of in

clus

ion

crite

ria, d

oses

, st

udy

dura

tion,

end

-poi

nts,

ana

lysis

of

data

, an

d co

ntro

l of

envi

ronm

enta

l var

iabl

es in

the

eva

luat

ion

of S

IT p

rodu

cts,

th

is a

bstra

ct h

ighl

ight

s th

e de

sign

and

ratio

nale

of t

he D

RF

stud

y.

Figu

re 2

: Leb

el a

nd P

NIF

scor

e sh

eet f

or th

e TN

PT o

utco

me.

Figu

re 1

: Des

ign

of th

e st

udy

Con

clus

ion:

Acco

rdin

g to

the

EM

A gu

idel

ines

we

desi

gned

a P

hase

II

DR

F st

udy

to id

entif

y th

e op

timal

dos

e of

a s

ubcu

tane

ous

SIT

alle

rgoi

d fo

r pat

ient

s wi

th H

DM

indu

ced

AR.

Met

hods

:Th

e cu

rrent

stu

dy

is a

1 y

ear,

mul

ticen

ter,

rand

omiz

ed,

doub

le-b

lind,

pla

cebo

-con

trolle

d, 5

arm

par

alle

l-gro

up, D

RF

stud

y (C

linic

alTr

ials

.gov

NC

T014

3846

3). A

ppro

ximat

ely

250

patie

nts

(50

per a

rm)

with

per

sist

ent,

sym

ptom

atic

AR

with

or

with

out

conc

omita

nt a

sthm

a re

late

d to

HD

M w

ill be

ra

ndom

ized

. In

clus

ion

crite

ria a

re p

rese

nted

in

Tabl

e 1.

Pa

tient

s we

re tr

eate

d fo

r one

yea

r with

mite

s al

lerg

oid

(HAL

Al

lerg

y, L

eide

n, T

he N

ethe

rland

s) s

ee F

igur

e 1.

Due

to th

e ne

ed o

f at l

east

4 a

ctiv

e st

udy

arm

s in

a D

RF

stud

y, th

e us

e of

a

clas

sica

l en

dpoi

nt

for

SIT

(i.e.

th

e sy

mpt

om

& m

edic

atio

n sc

ore)

as

prim

ary

para

met

er w

as n

ot f

easi

ble

beca

use

of th

e ne

cess

ity o

f a la

rge

sam

ple

size

. The

refo

re,

the

titra

ted

nasa

l pr

ovoc

atio

n te

st

(TN

PT)

with

H

DM

al

lerg

en a

s th

e pr

imar

y pa

ram

eter

was

sel

ecte

d. T

he T

NPT

is

a re

prod

ucib

le e

xace

rbat

ion

mod

el o

f alle

rgic

rhin

itis

ofte

n ap

plie

d to

eva

luat

e th

e ef

ficac

y of

ant

i-alle

rgy

med

icat

ions

. Th

e al

lerg

en-in

duce

d up

per

airw

ay

resp

onse

w

ill be

qu

antif

ied

by r

ecor

ding

sym

ptom

sco

res

(Leb

el s

core

) an

d na

sal f

low

(Pe

ak N

asal

Insp

irato

ry F

low

, PN

IF)

afte

r ea

ch

HD

M

chal

leng

e (s

ee

Figu

res

2 an

d 3)

.To

m

inim

ize

seas

onal

in

fluen

ces

the

abso

lute

di

ffere

nce

in

mea

n sy

mpt

om s

core

in th

e TN

PT a

fter o

ne y

ear o

f tre

atm

ent a

nd

base

line

will

be u

sed

as p

rimar

y en

dpoi

nt. P

atie

nts

will

only

be

su

bjec

ted

to

a TN

PT

in

the

abse

nce

of

base

line

sym

ptom

s an

d pa

tient

s wi

th s

easo

nal a

llerg

ies

will

only

be

chal

leng

ed o

utsi

de t

he c

orre

spon

ding

pol

len

seas

on.

The

stud

y is

pe

rform

ed

in

40

clin

ical

st

udy

cent

ers

in

5 Eu

rope

an c

ount

ries.

Res

ults

: Th

e fir

st p

atie

nt w

as re

crui

ted

in O

ctob

er 2

011

and

the

last

pa

tient

was

enr

olle

d in

Mar

ch 2

012.

Res

ults

will

be a

vaila

ble

in 2

013.

Posi

tive

TNPT

for H

DM D

. pte

rext

ract

at s

cree

ning

(Leb

el s

core

6

at o

r bel

ow

10,0

00 A

U/m

l)

Seru

m s

peci

fic Ig

E-te

st (s

sIgE

) lev

el fo

r HD

M D

. pte

ror D

. far

at s

cree

ning

(>

0.7

U/m

l)

Posi

tive

SPT

to H

DM D

. pte

rand

/or D

. far

(mea

n wh

eal d

iam

eter

3m

m,

com

pare

d to

neg

ativ

e co

ntro

l and

neg

ativ

e co

ntro

l sho

uld

be n

egat

ive)

Patie

nts

with

a h

isto

ry o

f con

com

itant

ast

hma

shou

ld h

ave

a FE

V1>

70%

at

incl

usio

n. P

atie

nts

with

out a

his

tory

of a

sthm

a sh

ould

hav

e a

FEV1

> 7

0% o

r a

PEF

> 80

%

Patie

nts

with

alle

rgic

rhin

itis

or rh

inoc

onju

nctiv

itis

for a

t lea

st 1

yea

r; al

lerg

ic

sym

ptom

s re

late

d to

HDM

, with

or w

ithou

t con

com

itant

clin

ical

ly s

tabl

e co

ntro

lled

mild

to m

oder

ate

asth

ma

(acc

ordi

ng to

GIN

A cl

assi

ficat

ion)

Patie

nts

(mal

e or

fem

ale)

mus

t be

18 a

nd

60 y

ears

at s

cree

ning

Sign

ed in

form

ed c

onse

ntIn

clus

ion

crite

ria

Tabl

e 1:

Incl

usio

n cr

iteria

of t

he D

RF

stud

y.

Ref

eren

ces:

1EM

A G

uide

line

on th

e de

velo

pmen

t of p

rodu

cts

for

spec

ific im

mun

othe

rapy

for

the

treat

men

t of

al

lerg

ic

dise

ases

. C

HM

P/EW

P/18

504/

2006

, Lo

ndon

.20

N

ovem

ber 2

008.

PNIF

sco

re(h

ighe

stof

3

mea

sure

men

ts)

Tota

l LEB

EL s

core

Prur

itus

(Itch

yno

se=1

,Itc

hypa

late

and

/ore

ar=1

, Irr

itate

dey

es=1

)

Nas

alob

stru

ctio

n(D

ifficu

ltna

salb

reat

hing

=1,

One

nost

rilbl

ocke

d=2,

Bot

h no

stril

sbl

ocke

d=3)

Run

nyN

ose

(Pos

terio

r=1,

An

terio

r=1)

Snee

zing

(1-2

=0, 3

-4=1

,5

=3)

Tim

e of

mea

sure

men

t

-Ac

tual

Tim

e of

app

licat

ion

1h a

fter

last

ch.

20m

in.

afte

rpr

evio

us

20m

in.

afte

rpr

evio

us

20m

in.

afte

rdi

luen

t

--

Sche

dule

dtim

e fo

rnex

tch

alle

nge

Post

ch.

10,0

00

AU/m

l1,

000

AU/m

l10

0 AU

/ml

Post

D

iluen

tPr

e D

iluen

t(B

asel

ine)

Alle

rgen

Figu

re 3

: Per

form

ance

of a

nas

al c

halle

nge

(left)

and

PNI

F (r

ight

).

TNPT

& R

ando

mis

atio

nTN

PT12

mon

ths

10,0

00 A

Ueq/

dose

25,0

00 A

Ueq/

dose

50,0

00 A

Ueq/

dose

Plac

ebo

Scre

enin

g

3,33

3 AU

eq/d

ose

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C. B

ache

rt1 , O

. Pfa

ar2 ,

A. R

oger

3 , H.

Rie

chel

man

n4 , J.

N.G

. Oud

e El

berin

k5 , M

.J. N

ell6 ,

J.D.

Boo

t6

1 UZ

Gen

t, Ea

r-, N

ose

and

Thro

at D

epar

tmen

t, G

ent,

Belg

ium

, 2Ce

nter

for R

hino

logy

and

Alle

rgol

ogy

Wie

sbad

en, G

erm

any,

3Un

itat d

’Allè

rgia

, Hos

pita

l Un

iver

sita

ri G

erm

ans

Tria

s i P

ujol

, Bad

alon

a, S

pain

, 4Un

iver

sitä

tskl

inik

für H

als-

, Nas

en-u

nd O

hren

heilk

unde

, Inn

sbru

ck, A

ustr

ia, 5

Dept

of A

llerg

olog

y,

Univ

ersi

ty M

edic

al C

ente

r Gro

ning

en, U

nive

rsity

of G

roni

ngen

, The

Net

herla

nds,

6 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

1393

-D

esig

n of

a D

ose

Ran

ge F

indi

ng s

tudy

with

alle

rgen

sp

ecifi

c im

mun

othe

rapy

in p

atie

nts

with

H

DM

indu

ced

alle

rgic

rhin

itis/

rhin

ocon

junc

tiviti

s

Bac

kgro

und

& A

im:

In o

rder

to

com

ply

with

the

200

8 EM

A gu

idel

ines

on

the

deve

lopm

ent

of s

peci

fic i

mm

unot

hera

py (

SIT)

pro

duct

s, a

cl

inic

al d

evel

opm

ent

prog

ram

was

sta

rted

to o

btai

n fu

ll m

arke

ting

auth

oriz

atio

n fo

r a s

ubcu

tane

ous

SIT

alle

rgoi

d fo

r th

e tre

atm

ent o

f hou

se d

ust m

ite (H

DM

) alle

rgy

1 . Sa

fety

and

to

lera

bility

of

in

crea

sing

do

ses

have

pr

evio

usly

be

en

dete

rmin

ed in

an

open

-labe

l clin

ical

stu

dy. T

he n

ext s

tep

is a

do

se r

ange

find

ing

(DR

F) s

tudy

to id

entif

y th

e op

timal

, i.e

. sa

fe a

nd e

ffect

ive

dose

in

patie

nts

with

HD

M i

nduc

ed

alle

rgic

rhi

nitis

/rhin

ocon

junc

tivitis

(AR

). As

the

re i

s a

wide

va

riety

of s

tudy

des

igns

in te

rms

of in

clus

ion

crite

ria, d

oses

, st

udy

dura

tion,

end

-poi

nts,

ana

lysis

of

data

, an

d co

ntro

l of

envi

ronm

enta

l var

iabl

es in

the

eva

luat

ion

of S

IT p

rodu

cts,

th

is a

bstra

ct h

ighl

ight

s th

e de

sign

and

ratio

nale

of t

he D

RF

stud

y.

Figu

re 2

: Leb

el a

nd P

NIF

scor

e sh

eet f

or th

e TN

PT o

utco

me.

Figu

re 1

: Des

ign

of th

e st

udy

Con

clus

ion:

Acco

rdin

g to

the

EM

A gu

idel

ines

we

desi

gned

a P

hase

II

DR

F st

udy

to id

entif

y th

e op

timal

dos

e of

a s

ubcu

tane

ous

SIT

alle

rgoi

d fo

r pat

ient

s wi

th H

DM

indu

ced

AR.

Met

hods

:Th

e cu

rrent

stu

dy

is a

1 y

ear,

mul

ticen

ter,

rand

omiz

ed,

doub

le-b

lind,

pla

cebo

-con

trolle

d, 5

arm

par

alle

l-gro

up, D

RF

stud

y (C

linic

alTr

ials

.gov

NC

T014

3846

3). A

ppro

ximat

ely

250

patie

nts

(50

per a

rm)

with

per

sist

ent,

sym

ptom

atic

AR

with

or

with

out

conc

omita

nt a

sthm

a re

late

d to

HD

M w

ill be

ra

ndom

ized

. In

clus

ion

crite

ria a

re p

rese

nted

in

Tabl

e 1.

Pa

tient

s we

re tr

eate

d fo

r one

yea

r with

mite

s al

lerg

oid

(HAL

Al

lerg

y, L

eide

n, T

he N

ethe

rland

s) s

ee F

igur

e 1.

Due

to th

e ne

ed o

f at l

east

4 a

ctiv

e st

udy

arm

s in

a D

RF

stud

y, th

e us

e of

a

clas

sica

l en

dpoi

nt

for

SIT

(i.e.

th

e sy

mpt

om

& m

edic

atio

n sc

ore)

as

prim

ary

para

met

er w

as n

ot f

easi

ble

beca

use

of th

e ne

cess

ity o

f a la

rge

sam

ple

size

. The

refo

re,

the

titra

ted

nasa

l pr

ovoc

atio

n te

st

(TN

PT)

with

H

DM

al

lerg

en a

s th

e pr

imar

y pa

ram

eter

was

sel

ecte

d. T

he T

NPT

is

a re

prod

ucib

le e

xace

rbat

ion

mod

el o

f alle

rgic

rhin

itis

ofte

n ap

plie

d to

eva

luat

e th

e ef

ficac

y of

ant

i-alle

rgy

med

icat

ions

. Th

e al

lerg

en-in

duce

d up

per

airw

ay

resp

onse

w

ill be

qu

antif

ied

by r

ecor

ding

sym

ptom

sco

res

(Leb

el s

core

) an

d na

sal f

low

(Pe

ak N

asal

Insp

irato

ry F

low

, PN

IF)

afte

r ea

ch

HD

M

chal

leng

e (s

ee

Figu

res

2 an

d 3)

.To

m

inim

ize

seas

onal

in

fluen

ces

the

abso

lute

di

ffere

nce

in

mea

n sy

mpt

om s

core

in th

e TN

PT a

fter o

ne y

ear o

f tre

atm

ent a

nd

base

line

will

be u

sed

as p

rimar

y en

dpoi

nt. P

atie

nts

will

only

be

su

bjec

ted

to

a TN

PT

in

the

abse

nce

of

base

line

sym

ptom

s an

d pa

tient

s wi

th s

easo

nal a

llerg

ies

will

only

be

chal

leng

ed o

utsi

de t

he c

orre

spon

ding

pol

len

seas

on.

The

stud

y is

pe

rform

ed

in

40

clin

ical

st

udy

cent

ers

in

5 Eu

rope

an c

ount

ries.

Res

ults

: Th

e fir

st p

atie

nt w

as re

crui

ted

in O

ctob

er 2

011

and

the

last

pa

tient

was

enr

olle

d in

Mar

ch 2

012.

Res

ults

will

be a

vaila

ble

in 2

013.

Posi

tive

TNPT

for H

DM D

. pte

rext

ract

at s

cree

ning

(Leb

el s

core

6

at o

r bel

ow

10,0

00 A

U/m

l)

Seru

m s

peci

fic Ig

E-te

st (s

sIgE

) lev

el fo

r HD

M D

. pte

ror D

. far

at s

cree

ning

(>

0.7

U/m

l)

Posi

tive

SPT

to H

DM D

. pte

rand

/or D

. far

(mea

n wh

eal d

iam

eter

3m

m,

com

pare

d to

neg

ativ

e co

ntro

l and

neg

ativ

e co

ntro

l sho

uld

be n

egat

ive)

Patie

nts

with

a h

isto

ry o

f con

com

itant

ast

hma

shou

ld h

ave

a FE

V1>

70%

at

incl

usio

n. P

atie

nts

with

out a

his

tory

of a

sthm

a sh

ould

hav

e a

FEV1

> 7

0% o

r a

PEF

> 80

%

Patie

nts

with

alle

rgic

rhin

itis

or rh

inoc

onju

nctiv

itis

for a

t lea

st 1

yea

r; al

lerg

ic

sym

ptom

s re

late

d to

HDM

, with

or w

ithou

t con

com

itant

clin

ical

ly s

tabl

e co

ntro

lled

mild

to m

oder

ate

asth

ma

(acc

ordi

ng to

GIN

A cl

assi

ficat

ion)

Patie

nts

(mal

e or

fem

ale)

mus

t be

18 a

nd

60 y

ears

at s

cree

ning

Sign

ed in

form

ed c

onse

ntIn

clus

ion

crite

ria

Tabl

e 1:

Incl

usio

n cr

iteria

of t

he D

RF

stud

y.

Ref

eren

ces:

1EM

A G

uide

line

on th

e de

velo

pmen

t of p

rodu

cts

for

spec

ific im

mun

othe

rapy

for

the

treat

men

t of

al

lerg

ic

dise

ases

. C

HM

P/EW

P/18

504/

2006

, Lo

ndon

.20

N

ovem

ber 2

008.

PNIF

sco

re(h

ighe

stof

3

mea

sure

men

ts)

Tota

l LEB

EL s

core

Prur

itus

(Itch

yno

se=1

,Itc

hypa

late

and

/ore

ar=1

, Irr

itate

dey

es=1

)

Nas

alob

stru

ctio

n(D

ifficu

ltna

salb

reat

hing

=1,

One

nost

rilbl

ocke

d=2,

Bot

h no

stril

sbl

ocke

d=3)

Run

nyN

ose

(Pos

terio

r=1,

An

terio

r=1)

Snee

zing

(1-2

=0, 3

-4=1

,5

=3)

Tim

e of

mea

sure

men

t

-Ac

tual

Tim

e of

app

licat

ion

1h a

fter

last

ch.

20m

in.

afte

rpr

evio

us

20m

in.

afte

rpr

evio

us

20m

in.

afte

rdi

luen

t

--

Sche

dule

dtim

e fo

rnex

tch

alle

nge

Post

ch.

10,0

00

AU/m

l1,

000

AU/m

l10

0 AU

/ml

Post

D

iluen

tPr

e D

iluen

t(B

asel

ine)

Alle

rgen

Figu

re 3

: Per

form

ance

of a

nas

al c

halle

nge

(left)

and

PNI

F (r

ight

).

TNPT

& R

ando

mis

atio

nTN

PT12

mon

ths

10,0

00 A

Ueq/

dose

25,0

00 A

Ueq/

dose

50,0

00 A

Ueq/

dose

Plac

ebo

Scre

enin

g

3,33

3 AU

eq/d

ose

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C. B

ache

rt1 , O

. Pfa

ar2 ,

A. R

oger

3 , H.

Rie

chel

man

n4 , J.

N.G

. Oud

e El

berin

k5 , M

.J. N

ell6 ,

J.D.

Boo

t6

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.

S a f e t y o f p r e - a n d c o s ea s o n a l s t ar t o f su b l ing u a l im mu n o t h e rap y tr ea t m e nt in p a t i e nt s w i t h p o l l e n a l l e rg y

16

A. Distler (1), H. Kietzmann (2), H. Brüning (2), A. Roger (3), N. Angelova (4), J.D. Boot (4).

(1) HAL Allergie GmbH, Medical Department, Düsseldorf, Germany, (2) CutiCon, Kiel, Germany, (3) Allergy Unit University Hospital Germans

Trias Pujol. Badalona, Spain, (4) HAL Allergy BV, Leiden, Netherlands.

BackgroundStart of treatment with sublingual allergen immunotherapy (SLIT) during the relevant pollen season (co-seasonal) may result in more and/or worse adverse events (AEs) compared to the start outside the pollen season (pre-seasonal). A post marketing surveillance study in adults and children treated with SLIT pollen was performed to evaluate safety and clinical efficacy during routine application at home.

MethodsSubjects with allergic rhinoconjunctivitis with or without mild asthma due to pollen were treated with SLIT Pollen for approximately 1 year, according to onsite routine. Patients started with one sublingual drop and add one drop each consecutive day until on day 5 the maintenance dose is reached. At each visit, the number of daily drops and AEs were recorded. The patients filled in a dairy during the first 30 treatment days to monitor AEs. The treatment’s efficacy was evaluated after 1 year. The start of the respective pollen season was based on local pollen counts in Germany and Spain.

Results129 pts (88 female, mean age 38 years) were included in the study: 43.4% received grasses, 8.5% birch, and 47.3% received a combination or other pollen antigens. In total 40.3% of the patients started SLIT co- and 59.7% pre-seasonally. 89.1% of patients reached maintenance dose within 5 days (88.5% co- vs 89.6% pre-seasonal). During up-dosing 37.9% of the patients (38.4% co- vs 37.6% pre-seasonal) experienced a total of 82 adverse drug reactions. 60 local reactions (most common were allergic reactions in the mouth and abdominal pain) and 22 systemic reactions (most common were pruritus of eyes and ears). No difference in the number and severity of adverse drug reactions was observed between the two groups. Patients’ assessment of their own situation improved in 74.4%, remained equal in 11.6% and was unknown in 13.9% of the patients. No patient reported a worsening of their situation. The satisfaction of patients about the use of SLIT pollen was (very) satisfied in 75.2%, (very) unsatisfied in 11.6% and unknown in 12.4% of the patients.

ConclusionCo-seasonal start of treatment was found to be as safe as the pre-seasonal start of treatment with SLIT pollen. This is practical since it is difficult to up-dose before the season due to lengthening of the pollen season, new pollen allergens and a high number of patients with multiple pollen allergies. Subjects were generally satisfied with their treatment.

EAACI, 16-20 June 2012, GenevaAbstract number: 1394, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 68 - New clinical studies in immunotherapy

17

Poster Session 68 - New clinical studies in immunotherapy

S a f e t y o f p r e - a n d c o s ea s o n a l s t ar t o f su b l ing u a l im mu n o t h e rap y tr ea t m e nt in p a t i e nt s w i t h p o l l e n a l l e rg y

1394

-Sa

fety

of p

re-a

nd c

o-se

ason

al s

tart

of s

ublin

gual

im

mun

othe

rapy

trea

tmen

t in

patie

nts

with

pol

len

alle

rgy

, B

adal

ona,

Spa

in; 4 H

AL A

llerg

y B

VLe

iden

, Net

herla

nds

Bac

kgro

und

& A

im:

Diff

eren

t up

dosi

ngsc

hedu

les

for

subl

ingu

al

alle

rgen

im

mun

othe

rapy

(SL

IT)

exis

t. St

art

of t

reat

men

t du

ring

the

rele

vant

pol

len

seas

on (

co-s

easo

nal)

may

res

ult

in m

ore

and/

or w

orse

adv

erse

reac

tions

com

pare

d to

a s

tart

outs

ide

the

polle

n se

ason

(pre

-sea

sona

l). T

o in

vest

igat

e th

is, a

non

in

terv

entio

nal s

tudy

in a

dults

and

chi

ldre

n tre

ated

with

SLI

T po

llen

was

per

form

ed to

eva

luat

e sa

fety

and

clin

ical

effi

cacy

du

ring

rout

ine

appl

icat

ion.

Met

hods

:Th

is s

tudy

was

per

form

ed in

18

clin

ical

cen

tres

in G

erm

any

and

Spai

n.

Subj

ects

w

ith

a hi

stor

y of

al

lerg

ic

rhin

ocon

junc

tiviti

sw

ith o

r w

ithou

t mild

con

com

itant

ast

hma

due

to p

olle

n w

ere

treat

ed w

ith S

LIT

polle

n (S

UBL

IVAC

®

HAL

Al

lerg

y BV

, Le

iden

, Th

e N

ethe

rland

s)fo

r ap

prox

imat

ely

1 ye

ar, a

ccor

ding

to o

nsite

rou

tine.

Pat

ient

s (p

ts)

star

ted

with

one

sub

lingu

al d

rop

and

adde

d on

e dr

op

each

con

secu

tive

day

until

on

day

5 th

e m

aint

enan

ce d

ose

was

rea

ched

. Tr

eatm

ent

was

con

tinue

d du

ring

the

polle

n se

ason

. At

eac

h vi

sit,

the

num

ber

of d

aily

dro

ps a

nd A

Esw

ere

reco

rded

. The

pat

ient

s fil

led

in a

dia

ry d

urin

g th

e fir

st

30 tr

eatm

ent d

ays

to m

onito

r AEs

. Afte

r 1 y

ear o

f tre

atm

ent

patie

nts

scor

ed th

eir a

llerg

y co

mpl

aint

s co

mpa

red

to b

efor

e tre

atm

ent.

The

star

t of

the

res

pect

ive

polle

n se

ason

was

ba

sed

on lo

cal p

olle

n co

unts

in G

erm

any

and

Spai

n. B

ased

on

this

dat

a pa

tient

s w

ere

clas

sifie

d as

co-

or p

re-s

easo

nal.

Con

clus

ion:

Co-

seas

onal

sta

rt of

trea

tmen

t was

foun

d to

be

as s

afe

as

the

pre-

seas

onal

sta

rt of

trea

tmen

t with

SLI

T po

llen.

Thi

s is

pr

actic

al s

ince

it is

diff

icul

t to

up-

dose

bef

ore

the

seas

on

due

to

leng

then

ing

of

the

polle

n se

ason

, ne

w

polle

n al

lerg

ens

and

a hi

gh n

umbe

r of p

atie

nts

with

mul

tiple

pol

len

alle

rgie

s.

Subj

ects

w

ere

gene

rally

sa

tisfie

d w

ith

thei

r tre

atm

ent.

Res

ults

: 12

9 pt

s w

ere

incl

uded

in th

e st

udy

(Tab

le 1

). At

the

time

of

the

stud

y al

l pa

tient

s w

ere

only

pre

scrib

ed o

ne t

ype

of

polle

n SL

IT. T

he a

llerg

ens

are

liste

d in

Tab

le 1

.In

tot

al 4

0.3%

of

the

patie

nts

star

ted

SLIT

in t

he s

easo

n (c

o-se

ason

al)

and

59.7

%

outs

ide

(pre

-sea

sona

l) th

e se

ason

. 89

.1%

of

pa

tient

s re

ache

d m

aint

enan

ce d

ose

with

in 5

day

s (8

8.5%

co-

vs. 8

9.6%

pre

-sea

sona

l).

Dur

ing

up-d

osin

g a

tota

l of 8

2 ad

vers

e re

actio

ns in

49

pts

wer

e re

porte

d; 6

0 lo

cal

reac

tions

(m

ost

com

mon

wer

e al

lerg

ic re

actio

ns in

the

mou

th a

nd a

bdom

inal

pai

n) a

nd 2

2 sy

stem

ic r

eact

ions

(m

ost

com

mon

wer

e pr

uritu

sof

eye

s an

d ea

rs).

No

diffe

renc

es i

n th

e nu

mbe

r an

d se

verit

y of

ad

vers

e dr

ug r

eact

ions

wer

e ob

serv

ed b

etw

een

the

two

grou

ps (T

able

2 a

nd 3

). Al

mos

t all

patie

nts

repo

rted

an im

prov

emen

t and

no

patie

nt

repo

rted

a w

orse

ning

of t

heir

alle

rgic

com

plai

nts

(Fig

ure

1).

No

diffe

renc

e w

as s

een

betw

een

the

co-a

nd p

re-s

easo

nal

grou

ps. P

atie

nts

wer

e ge

nera

lly s

atis

fied

abou

t the

use

of

SLIT

po

llen:

34

.1%

w

ere

very

sa

tisfie

d,

41.1

%

wer

e sa

tisfie

d,

9.3%

w

ere

unsa

tisfie

d,

2.3%

w

ere

very

un

satis

fied

and

13.2

% w

ere

unkn

own.

Co-

seas

onal

Pre-

seas

onal

Tota

l(n

= 1

29)

Gen

der (

M /

F)20

/ 33

20 /

5640

/ 89

Mea

n ag

e ±

SD in

yea

rs36

.3 ±

15.3

38.3

±15

.337

.8 ±

15.2

Adol

esce

nts

75

12

Pres

crib

ed a

llerg

ens

-Gra

sses

-Tre

es-P

arie

taria

sp.

-Ole

aeu

rope

a

53 20 21 11 1

76 37 33 5 1

129 57

54 16

2

Tabl

e 1:

Pat

ient

dem

ogra

phic

s an

d ad

min

iste

red

alle

rgen

s

Tabl

e 3:

Ove

rvie

w o

f mos

t fre

quen

t adv

erse

reac

tions

Figu

re 1

: Pat

ient

s’as

sess

men

t of i

mpr

ovem

ent i

n th

e co

-(le

ft) a

nd p

re-

seas

onal

(rig

ht) g

roup

.

Tabl

e 2:

Occ

urre

nce

of a

dver

se re

actio

ns

Co-

seas

onal

Pre-

seas

onal

Tota

l

Patie

nts

with

adv

erse

reac

tions

38.4

%37

.6%

37.9

%

Patie

nts

with

loca

l rea

ctio

ns34

%34

.2%

34.1

%

Patie

nts

with

sys

tem

ic re

actio

ns9.

4%13

.2%

11.6

%

33%

53%14

%0%

35%

53%12

%0%

Muc

h im

prov

edIm

prov

edEq

ual

Wor

sene

d

Med

DR

APr

efer

red

Term

Num

ber o

f adv

erse

re

actio

nsN

umbe

r of

patie

nts

[%]

Swol

len

tong

ue

Ora

lpru

ritus

Abdo

min

alpa

in

Burn

ing

sens

atio

n

Para

esth

esia

oral

Lip

swel

ling

Ear p

rurit

us

Glo

ssod

ynia

Ora

ldisc

omfo

rt

Thro

atirr

itatio

n

Dys

peps

ia

Gin

giva

lpru

ritus

Lip

prur

itus

Prur

itus

Swel

ling

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

yEA

ACI C

ongr

ess

2012

A. D

istle

r1 , H.

Kie

tzm

ann2 ,

H. B

rüni

ng2 ,

A. R

oger

3 , N.

Ang

elov

a4 , J.

D. B

oot4

Düs

seld

orf,

Ger

man

y; 2 C

utiC

on, K

iel,

Ger

man

y; 3 A

llerg

y Un

it Un

iver

sity

Hos

pita

l Ger

man

s Tr

ias

Pujo

l. 1 H

AL A

llerg

ieG

mbH

,

1394

-Sa

fety

of p

re-a

nd c

o-se

ason

al s

tart

of s

ublin

gual

im

mun

othe

rapy

trea

tmen

t in

patie

nts

with

pol

len

alle

rgy

, Ba

dalo

na, S

pain

; 4 HAL

Alle

rgy

BVLe

iden

, Net

herla

nds

Bac

kgro

und

& A

im:

Diff

eren

t up

dosi

ngsc

hedu

les

for

subl

ingu

al

alle

rgen

im

mun

othe

rapy

(SL

IT)

exis

t. St

art

of t

reat

men

t du

ring

the

rele

vant

pol

len

seas

on (

co-s

easo

nal)

may

res

ult

in m

ore

and/

or w

orse

adv

erse

reac

tions

com

pare

d to

a s

tart

outs

ide

the

polle

n se

ason

(pre

-sea

sona

l). T

o in

vest

igat

e th

is, a

non

in

terv

entio

nal s

tudy

in a

dults

and

chi

ldre

n tre

ated

with

SLI

T po

llen

was

perfo

rmed

to e

valu

ate

safe

ty a

nd c

linic

al e

ffica

cy

durin

g ro

utin

e ap

plic

atio

n.

Met

hods

:Th

is s

tudy

was

per

form

ed in

18

clin

ical

cen

tres

in G

erm

any

and

Spai

n.

Subj

ects

w

ith

a hi

stor

y of

al

lerg

ic

rhin

ocon

junc

tivitis

with

or

with

out m

ild c

onco

mita

nt a

sthm

a du

e to

pol

len

were

tre

ated

with

SLI

T po

llen

(SU

BLIV

AC®

HAL

Al

lerg

y BV

, Le

iden

, Th

e N

ethe

rland

s)fo

r ap

prox

imat

ely

1 ye

ar, a

ccor

ding

to o

nsite

rou

tine.

Pat

ient

s (p

ts) s

tarte

d w

ith o

ne s

ublin

gual

dro

p an

d ad

ded

one

drop

ea

ch c

onse

cutiv

e da

y un

til o

n da

y 5

the

mai

nten

ance

dos

e wa

s re

ache

d. T

reat

men

t wa

s co

ntin

ued

durin

g th

e po

llen

seas

on.

At e

ach

visi

t, th

e nu

mbe

r of

dai

ly d

rops

and

AEs

were

reco

rded

. The

pat

ient

s fil

led

in a

dia

ry d

urin

g th

e fir

st

30 tr

eatm

ent d

ays

to m

onito

r AEs

. Afte

r 1 y

ear o

f tre

atm

ent

patie

nts

scor

ed th

eir a

llerg

y co

mpl

aint

s co

mpa

red

to b

efor

e tre

atm

ent.

The

star

t of

the

res

pect

ive

polle

n se

ason

was

ba

sed

on lo

cal p

olle

n co

unts

in G

erm

any

and

Spai

n. B

ased

on

this

dat

a pa

tient

s w

ere

clas

sifie

d as

co-

or p

re-s

easo

nal.

Con

clus

ion:

Co-

seas

onal

sta

rt of

trea

tmen

t was

foun

d to

be

as s

afe

as

the

pre-

seas

onal

sta

rt of

trea

tmen

t with

SLI

T po

llen.

Thi

s is

pr

actic

al s

ince

it is

diff

icul

t to

up-

dose

bef

ore

the

seas

on

due

to

leng

then

ing

of

the

polle

n se

ason

, ne

w po

llen

alle

rgen

s an

d a

high

num

ber o

f pat

ient

s wi

th m

ultip

le p

olle

n al

lerg

ies.

Su

bjec

ts

were

ge

nera

lly

satis

fied

with

th

eir

treat

men

t.

Res

ults

: 12

9 pt

s we

re in

clud

ed in

the

stud

y (T

able

1).

At th

e tim

e of

th

e st

udy

all

patie

nts

wer

e on

ly p

resc

ribed

one

typ

e of

po

llen

SLIT

. The

alle

rgen

s ar

e lis

ted

in T

able

1.

In t

otal

40.

3% o

f th

e pa

tient

s st

arte

d SL

IT in

the

sea

son

(co-

seas

onal

) an

d 59

.7%

ou

tsid

e (p

re-s

easo

nal)

the

seas

on.

89.1

% o

f pa

tient

s re

ache

d m

aint

enan

ce d

ose

with

in 5

day

s (8

8.5%

co-

vs. 8

9.6%

pre

-sea

sona

l).

Dur

ing

up-d

osin

g a

tota

l of 8

2 ad

vers

e re

actio

ns in

49

pts

were

rep

orte

d; 6

0 lo

cal

reac

tions

(m

ost

com

mon

wer

e al

lerg

ic re

actio

ns in

the

mou

th a

nd a

bdom

inal

pai

n) a

nd 2

2 sy

stem

ic r

eact

ions

(m

ost

com

mon

wer

e pr

uritu

sof

eye

s an

d ea

rs).

No

diffe

renc

es i

n th

e nu

mbe

r an

d se

verit

y of

ad

vers

e dr

ug r

eact

ions

wer

e ob

serv

ed b

etw

een

the

two

grou

ps (T

able

2 a

nd 3

). Al

mos

t all

patie

nts

repo

rted

an im

prov

emen

t and

no

patie

nt

repo

rted

a w

orse

ning

of t

heir

alle

rgic

com

plai

nts

(Fig

ure

1).

No

diffe

renc

e w

as s

een

betw

een

the

co-a

nd p

re-s

easo

nal

grou

ps. P

atie

nts

were

gen

eral

ly s

atis

fied

abou

t the

use

of

SLIT

po

llen:

34

.1%

we

re

very

sa

tisfie

d,

41.1

%

were

sa

tisfie

d,

9.3%

we

re

unsa

tisfie

d,

2.3%

we

re

very

un

satis

fied

and

13.2

% w

ere

unkn

own.

Co-

seas

onal

Pre-

seas

onal

Tota

l(n

= 1

29)

Gen

der (

M /

F)20

/ 33

20 /

5640

/ 89

Mea

n ag

e ±

SD in

yea

rs36

.3 ±

15.3

38.3

±15

.337

.8 ±

15.2

Adol

esce

nts

75

12

Pres

crib

ed a

llerg

ens

-Gra

sses

-Tre

es-P

arie

taria

sp.

-Ole

aeu

rope

a

53 20 21 11 1

76 37 33 5 1

129 57

54 16

2

Tabl

e 1:

Pat

ient

dem

ogra

phic

s an

d ad

min

iste

red

alle

rgen

s

Tabl

e 3:

Ove

rvie

w o

f mos

t fre

quen

t adv

erse

reac

tions

Figu

re 1

: Pat

ient

s’as

sess

men

t of i

mpr

ovem

ent i

n th

e co

-(le

ft) a

nd p

re-

seas

onal

(rig

ht) g

roup

.

Tabl

e 2:

Occ

urre

nce

of a

dver

se re

actio

ns

Co-

seas

onal

Pre-

seas

onal

Tota

l

Patie

nts

with

adv

erse

reac

tions

38.4

%37

.6%

37.9

%

Patie

nts

with

loca

l rea

ctio

ns34

%34

.2%

34.1

%

Patie

nts

with

sys

tem

ic re

actio

ns9.

4%13

.2%

11.6

%

33%

53%14

%0%

35%

53%12

%0%

Muc

h im

prov

edIm

prov

edEq

ual

Wor

sene

d

Med

DR

APr

efer

red

Term

Num

ber o

f adv

erse

re

actio

nsN

umbe

r of

patie

nts

[%]

Swol

len

tong

ue

Ora

lpru

ritus

Abdo

min

alpa

in

Burn

ing

sens

atio

n

Para

esth

esia

oral

Lip

swel

ling

Ear p

rurit

us

Glo

ssod

ynia

Ora

ldisc

omfo

rt

Thro

atirr

itatio

n

Dys

peps

ia

Gin

giva

lpru

ritus

Lip

prur

itus

Prur

itus

Swel

ling

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

wing

, rea

lorp

erce

ived

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy

EAAC

I Con

gres

s 20

12

A. D

istle

r1 , H.

Kie

tzm

ann2 ,

H. B

rüni

ng2 ,

A. R

oger

3 , N.

Ang

elov

a4 , J.

D. B

oot4

Düs

seld

orf,

Ger

man

y; 2 C

utiC

on, K

iel,

Ger

man

y; 3 A

llerg

y Un

it Un

iver

sity

Hos

pita

l Ger

man

s Tr

ias

Pujo

l. 1 H

AL A

llerg

ieG

mbH

,

1394

-Sa

fety

of p

re-a

nd c

o-se

ason

al s

tart

of s

ublin

gual

im

mun

othe

rapy

trea

tmen

t in

patie

nts

with

pol

len

alle

rgy

, B

adal

ona,

Spa

in; 4 H

AL A

llerg

y B

VLe

iden

, Net

herla

nds

Bac

kgro

und

& A

im:

Diff

eren

t up

dosi

ngsc

hedu

les

for

subl

ingu

al

alle

rgen

im

mun

othe

rapy

(SL

IT)

exis

t. St

art

of t

reat

men

t du

ring

the

rele

vant

pol

len

seas

on (

co-s

easo

nal)

may

res

ult

in m

ore

and/

or w

orse

adv

erse

reac

tions

com

pare

d to

a s

tart

outs

ide

the

polle

n se

ason

(pre

-sea

sona

l). T

o in

vest

igat

e th

is, a

non

in

terv

entio

nal s

tudy

in a

dults

and

chi

ldre

n tre

ated

with

SLI

T po

llen

was

per

form

ed to

eva

luat

e sa

fety

and

clin

ical

effi

cacy

du

ring

rout

ine

appl

icat

ion.

Met

hods

:Th

is s

tudy

was

per

form

ed in

18

clin

ical

cen

tres

in G

erm

any

and

Spai

n.

Subj

ects

w

ith

a hi

stor

y of

al

lerg

ic

rhin

ocon

junc

tiviti

sw

ith o

r w

ithou

t mild

con

com

itant

ast

hma

due

to p

olle

n w

ere

treat

ed w

ith S

LIT

polle

n (S

UBL

IVAC

®

HAL

Al

lerg

y BV

, Le

iden

, Th

e N

ethe

rland

s)fo

r ap

prox

imat

ely

1 ye

ar, a

ccor

ding

to o

nsite

rou

tine.

Pat

ient

s (p

ts)

star

ted

with

one

sub

lingu

al d

rop

and

adde

d on

e dr

op

each

con

secu

tive

day

until

on

day

5 th

e m

aint

enan

ce d

ose

was

rea

ched

. Tr

eatm

ent

was

con

tinue

d du

ring

the

polle

n se

ason

. At

eac

h vi

sit,

the

num

ber

of d

aily

dro

ps a

nd A

Esw

ere

reco

rded

. The

pat

ient

s fil

led

in a

dia

ry d

urin

g th

e fir

st

30 tr

eatm

ent d

ays

to m

onito

r AEs

. Afte

r 1 y

ear o

f tre

atm

ent

patie

nts

scor

ed th

eir a

llerg

y co

mpl

aint

s co

mpa

red

to b

efor

e tre

atm

ent.

The

star

t of

the

res

pect

ive

polle

n se

ason

was

ba

sed

on lo

cal p

olle

n co

unts

in G

erm

any

and

Spai

n. B

ased

on

this

dat

a pa

tient

s w

ere

clas

sifie

d as

co-

or p

re-s

easo

nal.

Con

clus

ion:

Co-

seas

onal

sta

rt of

trea

tmen

t was

foun

d to

be

as s

afe

as

the

pre-

seas

onal

sta

rt of

trea

tmen

t with

SLI

T po

llen.

Thi

s is

pr

actic

al s

ince

it is

diff

icul

t to

up-

dose

bef

ore

the

seas

on

due

to

leng

then

ing

of

the

polle

n se

ason

, ne

w

polle

n al

lerg

ens

and

a hi

gh n

umbe

r of p

atie

nts

with

mul

tiple

pol

len

alle

rgie

s.

Subj

ects

w

ere

gene

rally

sa

tisfie

d w

ith

thei

r tre

atm

ent.

Res

ults

: 12

9 pt

s w

ere

incl

uded

in th

e st

udy

(Tab

le 1

). At

the

time

of

the

stud

y al

l pa

tient

s w

ere

only

pre

scrib

ed o

ne t

ype

of

polle

n SL

IT. T

he a

llerg

ens

are

liste

d in

Tab

le 1

.In

tot

al 4

0.3%

of

the

patie

nts

star

ted

SLIT

in t

he s

easo

n (c

o-se

ason

al)

and

59.7

%

outs

ide

(pre

-sea

sona

l) th

e se

ason

. 89

.1%

of

pa

tient

s re

ache

d m

aint

enan

ce d

ose

with

in 5

day

s (8

8.5%

co-

vs. 8

9.6%

pre

-sea

sona

l).

Dur

ing

up-d

osin

g a

tota

l of 8

2 ad

vers

e re

actio

ns in

49

pts

wer

e re

porte

d; 6

0 lo

cal

reac

tions

(m

ost

com

mon

wer

e al

lerg

ic re

actio

ns in

the

mou

th a

nd a

bdom

inal

pai

n) a

nd 2

2 sy

stem

ic r

eact

ions

(m

ost

com

mon

wer

e pr

uritu

sof

eye

s an

d ea

rs).

No

diffe

renc

es i

n th

e nu

mbe

r an

d se

verit

y of

ad

vers

e dr

ug r

eact

ions

wer

e ob

serv

ed b

etw

een

the

two

grou

ps (T

able

2 a

nd 3

). Al

mos

t all

patie

nts

repo

rted

an im

prov

emen

t and

no

patie

nt

repo

rted

a w

orse

ning

of t

heir

alle

rgic

com

plai

nts

(Fig

ure

1).

No

diffe

renc

e w

as s

een

betw

een

the

co-a

nd p

re-s

easo

nal

grou

ps. P

atie

nts

wer

e ge

nera

lly s

atis

fied

abou

t the

use

of

SLIT

po

llen:

34

.1%

w

ere

very

sa

tisfie

d,

41.1

%

wer

e sa

tisfie

d,

9.3%

w

ere

unsa

tisfie

d,

2.3%

w

ere

very

un

satis

fied

and

13.2

% w

ere

unkn

own.

Co-

seas

onal

Pre-

seas

onal

Tota

l(n

= 1

29)

Gen

der (

M /

F)20

/ 33

20 /

5640

/ 89

Mea

n ag

e ±

SD in

yea

rs36

.3 ±

15.3

38.3

±15

.337

.8 ±

15.2

Adol

esce

nts

75

12

Pres

crib

ed a

llerg

ens

-Gra

sses

-Tre

es-P

arie

taria

sp.

-Ole

aeu

rope

a

53 20 21 11 1

76 37 33 5 1

129 57

54 16

2

Tabl

e 1:

Pat

ient

dem

ogra

phic

s an

d ad

min

iste

red

alle

rgen

s

Tabl

e 3:

Ove

rvie

w o

f mos

t fre

quen

t adv

erse

reac

tions

Figu

re 1

: Pat

ient

s’as

sess

men

t of i

mpr

ovem

ent i

n th

e co

-(le

ft) a

nd p

re-

seas

onal

(rig

ht) g

roup

.

Tabl

e 2:

Occ

urre

nce

of a

dver

se re

actio

ns

Co-

seas

onal

Pre-

seas

onal

Tota

l

Patie

nts

with

adv

erse

reac

tions

38.4

%37

.6%

37.9

%

Patie

nts

with

loca

l rea

ctio

ns34

%34

.2%

34.1

%

Patie

nts

with

sys

tem

ic re

actio

ns9.

4%13

.2%

11.6

%

33%

53%14

%0%

35%

53%12

%0%

Muc

h im

prov

edIm

prov

edEq

ual

Wor

sene

d

Med

DR

APr

efer

red

Term

Num

ber o

f adv

erse

re

actio

nsN

umbe

r of

patie

nts

[%]

Swol

len

tong

ue

Ora

lpru

ritus

Abdo

min

alpa

in

Burn

ing

sens

atio

n

Para

esth

esia

oral

Lip

swel

ling

Ear p

rurit

us

Glo

ssod

ynia

Ora

ldisc

omfo

rt

Thro

atirr

itatio

n

Dys

peps

ia

Gin

giva

lpru

ritus

Lip

prur

itus

Prur

itus

Swel

ling

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

yEA

ACI C

ongr

ess

2012

A. D

istle

r1 , H.

Kie

tzm

ann2 ,

H. B

rüni

ng2 ,

A. R

oger

3 , N.

Ang

elov

a4 , J.

D. B

oot4

Düs

seld

orf,

Ger

man

y; 2 C

utiC

on, K

iel,

Ger

man

y; 3 A

llerg

y Un

it Un

iver

sity

Hos

pita

l Ger

man

s Tr

ias

Pujo

l. 1 H

AL A

llerg

ieG

mbH

,

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.

C h e mic a l m o d i f ic a t i o n o f a p ea n u t ex tra c t d e cr ea s e s Ig E b in d ing w h il e t h e im mu n o ge nic i t y is m a int a in e d

18

H. van der Kleij (1), J. Smit (2), S.C. Dreskin (3), H. Warmenhoven (1), D. Verbart (1), E. Knol (4), A. Knulst (4), E. Kerkvliet (1),

R. van den Hout (1), R. van Ree (5), R. Pieters (2), E. van Hoffen (4), S.J. Koppelman (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Institute for Risk Assessment Sciences, Immunotoxicology, Utrecht University, Utrecht,

The Netherlands, (3) University of Colorado Denver School of Medicine, Aurora, CO, USA, (4) Dermatology/Allergology, University Medical

Center Utrecht, Utrecht, The Netherlands, (5) Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands.

RationalePeanuts are one of the most common foods responsible for food-induced anaphylaxis in adults. Until now, a curative treatment is not available for peanut-allergic patients. Peanut sIT could be an option but earlier attempts with aqueous peanut extract were hampered by safety issues. Therefore, a chemically modified peanut extract with improved safety characteristics is being investigated for its potential use in immunotherapy.

MethodsA peanut extract (PE) was prepared and modified by reduction of the disulfide bonds and subsequent alkylation of the free Cys residues. The modified extract (mPE) was analysed for biochemical parameters using electrophoreses and far UV CD spectroscopy. Using a set of sera obtained from 20 US and 9 Dutch peanut allergic patients, solid-state IgE-binding assays and mediator release assays (MRA) using RBL SX38 cells and human basophils were performed. The potency ofPE and mPE to activate T-cells was evaluated using PE-specific T-cell lines from 9 peanut-allergic patients. The potency of PE and mPE to induce PE-specific IgG was evaluated in mice too. Furthermore, the in vivo safety of PE and mPE was tested by measuring body temperature upon subcutaneous challenges in mice allergic to PE.

ResultsThe IgE-binding of mPE was for all patients tested lower than for PE (mean remaining potency: 7.2% ± 5%), which was confirmed by IgE-immunoblotting. The potency of mPE in MRA was for all patients tested lower than for PE (mean reduction: 10 to100 fold, range 3 to >10,000 fold). PE-specific T-cell lines were activated equally well with mPE and PE. Mice sensitized with PE experienced a severe anaphylactic reaction upon subcutaneous challenge starting at 0.01 mg PE, while 0.3 mg mPE was tolerated without signs of an allergic reaction. PE and mPE were equally potent in inducing PE-specific IgG antibodies in mice.

ConclusionsUsing in vitro and in vivo models, we have shown that an allergoid preparation of peanut extract has a reduced allergenicity compared to its native counterpart, while the immunogenicity is maintained.

EAACI, 16-20 June 2012, GenevaAbstract number: 4, Session date and time: Sunday 17 June; 10:30 - 12:00 Session title: OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment

19

OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment

C h e mic a l m o d i f ic a t i o n o f a p ea n u t ex tra c t d e cr ea s e s Ig E b in d ing w h il e t h e im mu n o ge nic i t y is m a int a in e d

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Ins

titut

e fo

r Ris

k A

sses

smen

t Sci

ence

s an

d U

trec

ht C

entr

e fo

r Foo

d Al

lerg

y, U

trec

ht,

The

Net

herla

nds,

3 Uni

vers

ity o

f Col

orad

o D

enve

r Sch

ool o

f Med

icin

e, A

uror

a, C

O, U

SA,

4 Dep

. Der

mat

olog

y/Al

lerg

olog

y, U

MC

Utr

echt

, Utr

echt

, Th

e N

ethe

rland

s, 5 E

xper

imen

tal I

mm

unol

ogy,

Aca

dem

ic M

edic

al C

entr

e, A

mst

erda

m, T

heN

ethe

rland

s

4 -C

hem

ical

mod

ifica

tion

of a

pea

nut e

xtra

ct d

ecre

ases

Ig

Ebi

ndin

g w

hile

the

imm

unog

enic

ity is

mai

ntai

ned

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•A

cur

ativ

e tre

atm

ent i

s no

t ava

ilabl

e.

•A m

odifi

ed p

eanu

t ext

ract

(alle

rgoi

d) is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

.

Con

clus

ion:

An

alle

rgoi

dpr

epar

atio

n of

pe

anut

ex

tract

ha

s a

redu

ced

alle

rgen

icity

com

pare

d to

its

nativ

e co

unte

rpar

t, w

hile

th

e im

mun

ogen

icity

is m

aint

aine

d.

Met

hods

:Th

e Pe

anut

ext

ract

(PE)

pre

para

tion

isch

emic

ally

mod

ified

by

redu

ctio

n of

the

intra

mol

ecul

ardi

sulfi

de b

onds

of P

E,an

d al

kyla

tion

of

the

resu

lting

fre

e su

lfhyd

rylg

roup

s (R

A).

For i

mm

unog

enic

ity, t

he a

llerg

oid

was

adso

rbed

to a

lum

iniu

m h

ydro

xide

.

Res

ults

:Ef

ficac

y:•P

E-sp

ecifi

c hu

man

T-c

ell l

ines

wer

eac

tivat

ed e

qual

ly w

ell w

ith P

E an

d R

A-PE

.•I

n m

ice,

PE

and

RA-

PE w

ere

equa

llypo

tent

in in

duci

ng P

E-sp

ecifi

c Ig

Gan

tibod

ies

in m

ice.

Safe

ty:

•Mod

ifica

tion

low

ered

the

IgE-

bind

ing

inal

l pat

ient

s to

a m

ean

rem

aini

ng p

oten

cyof

7.2

%5%

. •T

he m

edia

tor r

elea

se a

ssay

(MR

A)sh

owed

a re

duce

d po

tenc

y of

10-

100

fold

(ran

ge 3

to >

10,0

00 fo

ld).

•Mic

e se

nsiti

zed

with

PE

expe

rienc

ed a

seve

re a

naph

ylac

tic re

actio

n up

onsu

bcut

aneo

us c

halle

nge

with

0.1

mg

PE, w

hile

3 m

g R

A-PE

was

stil

lto

lera

ted

with

out p

robl

ems.

Figu

re 1

: Res

pons

e of

PE-

spec

ific

TCLs

(T c

ell l

ines

) to

nativ

e PE

and

RA

-PE.

Prol

ifera

tion

was

expr

esse

d as

the

stim

ulat

ion

inde

x (S

I, i.e

., pr

olife

ratio

n to

alle

rgen

stim

ulat

ion

divi

ded

by b

lanc

).A.

Rep

rese

ntat

ive

exam

ples

of a

TC

L of

an

alle

rgic

pat

ient

. B. I

mm

unog

enic

ity o

f PE

and

RA-

PE in

9 T

CLs

.

A. Fi

gure

3: I

nhib

ition

ELI

SA

. A

. Inh

ibiti

on c

urve

s of

the

nativ

e an

d m

odifie

d PE

of a

US

seru

m

pool

of 2

0 pa

tient

s. T

est s

ampl

es c

ompe

te w

ith P

E co

ated

to E

LISA

pl

ates

for b

indi

ng to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sa

mpl

e re

sult

in in

hibi

tion

of b

indi

ng. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of P

E co

mpa

red

to th

at in

abs

ence

of P

E is

cal

cula

ted

as

perc

enta

ge in

hibi

tion.

B. Ig

Epo

tenc

y of

RA-

CPE

rela

tive

to n

ativ

e C

PE d

eter

min

ed in

an

IgE

inhi

bitio

n EL

ISA

usin

g se

ra o

f 20

pean

ut a

llerg

ic p

atie

nts

from

the

US.

An

in-h

ouse

refe

renc

e (IH

R) a

llerg

en p

repa

ratio

n is

test

ed to

com

pare

the

inhi

bitio

n cu

rves

by

para

llel l

ine

anal

yses

. The

IgE

bind

ing

effic

ienc

y is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to th

e IH

R.

1,3

pool

5,9

D11

48,

8D

107

6,2

D10

512

,2D

103

8,2

D98

8,2

D81

2,8

D80

11,8

D77

4,5

D74

9,1

D70

11,3

D69

4,3

D68

0,3

D65

17,8

D64

4,1

D62

5,3

D60

1,4

D50

16,9

D48

0,4

D44

1,3

D19

Rea

lativ

epo

tenc

y (%

) Sa

mpl

eB

.

Figu

re4:

The

pot

ency

of n

ativ

ePE

and

RA

-PE

in th

e in

dire

ct M

RA

. A. R

epre

sent

ativ

eex

ampl

e.

B.R

elat

ive

pote

ncy

of n

ativ

ePE

and

RA-

PE to

act

ivat

eba

soph

ilsFigu

re 2

: Spe

cific

IgG

resp

onse

s to

nat

ive

vers

us m

odifi

ed P

E.Sp

ecific

IgG

titre

s at

day

63

afte

r 5 im

mun

izat

ions

(i.p

.) wi

th 1

0 or

100

µg

prod

uct.

Con

trol m

ice

were

inje

cted

with

mat

rix o

nly.

Dot

s re

pres

ent t

he

IgG

titre

of i

ndiv

idua

l mic

e in

arb

itrar

y un

its (A

U) a

nd b

ars

repr

esen

t the

m

edia

n tit

re o

f eac

h gr

oup.

010

2030

4050

6070

8090

100

28303234363840

0.6

mg

PE0.

1 m

g PE

3 m

g PE

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celcius)

010

2030

4050

6070

8090

100

283032343638400.

1 m

g PE

-RA

0.6

mg

PE-R

A

3 m

g PE

-RA

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celsius)

Cha

lleng

eby

subc

utan

eous

inje

ctio

n

6 m

g PE

+ 1

5 g

Cho

lera

Tox

inby

oral

gava

ge

Day

: 0

1

2

7

14

21

2

8

42

Figu

re 5

: Ani

mal

mod

el.M

ice

were

sen

sitiz

ed w

ith 6

mg

PE

and

15 µ

g C

hole

ra T

oxin

via

ora

l gav

age

and

chal

leng

ed (i

.p.)

with

PE,

PE

adso

rbed

to a

lum

, RA-

PE p

repa

ratio

n an

d R

A-PE

ad

sorb

ed to

alu

m (n

=6 p

er g

roup

).

Figu

re 6

: Bod

y te

mpe

ratu

re fo

llow

ing

i.p. c

halle

nge;

Ana

phyl

axis

to th

e na

tive

prep

arat

ion.

An i.

p. c

halle

nge

with

0.6

mg

PE p

er

mou

se re

sulte

d in

an

anap

hyla

ctic

sho

ck re

spon

se. T

he m

ice

in th

e gr

oups

chal

leng

edwi

thm

odifie

dPE

sho

wed

smal

ldec

reas

esin

te

mpe

ratu

reat

the

high

estd

ose

(3 m

g R

A-PE

). C

ontro

lmic

edi

dno

tres

pond

to th

e ch

alle

nge

(with

3 m

g of

PE

orPE

-RA)

.

blan

c50

2512

.56.

253.

131.

560255075100

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)Proliferation (SI)

blan

c50

2512

.56.

253.

131.

56050100

150

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

0.010.

1110100

p<0.

01

PE

R

A-PE

relative basophil activation (%)

02550PE PE

-RA

con

0.0

3 0

.01

0.3

1

3

10

100

10

00

conc

entr

atio

n (n

g/m

l)

% CD63+ basophils

0.01

0.1

110

100

0255075100

PE PE-R

A

inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)110100

p=0.

25

PE

PE-

RA

stimulation index

con

0

100

200

300

10μ μμμ

g

100

μ μμμg

10μ μμμg

100μ μμμg

PEPE

-RA

IgG (AU)

A.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

J. S

mit2 ,

S.C

. Dre

skin

3 , H

. War

men

hove

n1 , D

. Ver

bart

1 , E.

Kno

l4 , A

. Knu

lst4 ,

E. K

erkv

liet1 ,

R. v

an d

en H

out1 ,

R. v

an R

ee5 ,

R. P

iete

rs2 ,

E. v

an H

offe

n4 , S.

J. K

oppe

lman

1

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Ins

titut

e fo

r Ris

k As

sess

men

t Sci

ence

s an

d U

trec

ht C

entr

e fo

r Foo

d Al

lerg

y, U

trec

ht,

The

Net

herla

nds,

3 Uni

vers

ity o

f Col

orad

o D

enve

r Sch

ool o

f Med

icin

e, A

uror

a, C

O, U

SA,

4 Dep

. Der

mat

olog

y/Al

lerg

olog

y, U

MC

Utr

echt

, Utr

echt

, Th

e N

ethe

rland

s, 5 E

xper

imen

tal I

mm

unol

ogy,

Aca

dem

ic M

edic

al C

entr

e, A

mst

erda

m, T

heN

ethe

rland

s

4 -C

hem

ical

mod

ifica

tion

of a

pea

nut e

xtra

ct d

ecre

ases

Ig

Ebi

ndin

g w

hile

the

imm

unog

enic

ity is

mai

ntai

ned

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•A

cur

ativ

e tre

atm

ent i

s no

t ava

ilabl

e.

•A m

odifi

ed p

eanu

t ext

ract

(alle

rgoi

d) is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

.

Con

clus

ion:

An

alle

rgoi

dpr

epar

atio

n of

pe

anut

ex

tract

ha

s a

redu

ced

alle

rgen

icity

com

pare

d to

its

nativ

e co

unte

rpar

t, w

hile

th

e im

mun

ogen

icity

is m

aint

aine

d.

Met

hods

:Th

e Pe

anut

ext

ract

(PE)

pre

para

tion

isch

emic

ally

mod

ified

by

redu

ctio

n of

the

intra

mol

ecul

ardi

sulfi

de b

onds

of P

E,an

d al

kyla

tion

of

the

resu

lting

fre

e su

lfhyd

rylg

roup

s (R

A).

For i

mm

unog

enic

ity, t

he a

llerg

oid

was

adso

rbed

to a

lum

iniu

m h

ydro

xide

.

Res

ults

:Ef

ficac

y:•P

E-sp

ecifi

c hu

man

T-c

ell l

ines

wer

eac

tivat

ed e

qual

ly w

ell w

ith P

E an

d R

A-PE

.•I

n m

ice,

PE

and

RA-

PE w

ere

equa

llypo

tent

in in

duci

ng P

E-sp

ecifi

c Ig

Gan

tibod

ies

in m

ice.

Safe

ty:

•Mod

ifica

tion

low

ered

the

IgE-

bind

ing

inal

l pat

ient

s to

a m

ean

rem

aini

ng p

oten

cyof

7.2

%5%

. •T

he m

edia

tor r

elea

se a

ssay

(MR

A)sh

owed

a re

duce

d po

tenc

y of

10-

100

fold

(ran

ge 3

to >

10,0

00 fo

ld).

•Mic

e se

nsiti

zed

with

PE

expe

rienc

ed a

seve

re a

naph

ylac

tic re

actio

n up

onsu

bcut

aneo

us c

halle

nge

with

0.1

mg

PE, w

hile

3 m

g R

A-PE

was

stil

lto

lera

ted

with

out p

robl

ems.

Figu

re 1

: Res

pons

e of

PE-

spec

ific

TCLs

(T c

ell l

ines

) to

nativ

e PE

and

RA

-PE.

Prol

ifera

tion

was

expr

esse

d as

the

stim

ulat

ion

inde

x (S

I, i.e

., pr

olife

ratio

n to

alle

rgen

stim

ulat

ion

divi

ded

by b

lanc

).A.

Rep

rese

ntat

ive

exam

ples

of a

TC

L of

an

alle

rgic

pat

ient

. B. I

mm

unog

enic

ity o

f PE

and

RA-

PE in

9 T

CLs

.

A. Fi

gure

3: I

nhib

ition

ELI

SA

. A

. Inh

ibiti

on c

urve

s of

the

nativ

e an

d m

odifie

d PE

of a

US

seru

m

pool

of 2

0 pa

tient

s. T

est s

ampl

es c

ompe

te w

ith P

E co

ated

to E

LISA

pl

ates

for b

indi

ng to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sa

mpl

e re

sult

in in

hibi

tion

of b

indi

ng. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of P

E co

mpa

red

to th

at in

abs

ence

of P

E is

cal

cula

ted

as

perc

enta

ge in

hibi

tion.

B. Ig

Epo

tenc

y of

RA-

CPE

rela

tive

to n

ativ

e C

PE d

eter

min

ed in

an

IgE

inhi

bitio

n EL

ISA

usin

g se

ra o

f 20

pean

ut a

llerg

ic p

atie

nts

from

the

US.

An

in-h

ouse

refe

renc

e (IH

R) a

llerg

en p

repa

ratio

n is

test

ed to

com

pare

the

inhi

bitio

n cu

rves

by

para

llel l

ine

anal

yses

. The

IgE

bind

ing

effic

ienc

y is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to th

e IH

R.

1,3

pool

5,9

D11

48,

8D

107

6,2

D10

512

,2D

103

8,2

D98

8,2

D81

2,8

D80

11,8

D77

4,5

D74

9,1

D70

11,3

D69

4,3

D68

0,3

D65

17,8

D64

4,1

D62

5,3

D60

1,4

D50

16,9

D48

0,4

D44

1,3

D19

Rea

lativ

epo

tenc

y (%

) Sa

mpl

eB

.

Figu

re4:

The

pot

ency

of n

ativ

ePE

and

RA

-PE

in th

e in

dire

ct M

RA

. A. R

epre

sent

ativ

eex

ampl

e.

B.R

elat

ive

pote

ncy

of n

ativ

ePE

and

RA-

PE to

act

ivat

eba

soph

ilsFigu

re 2

: Spe

cific

IgG

resp

onse

s to

nat

ive

vers

us m

odifi

ed P

E.Sp

ecific

IgG

titre

s at

day

63

afte

r 5 im

mun

izat

ions

(i.p

.) wi

th 1

0 or

100

µg

prod

uct.

Con

trol m

ice

were

inje

cted

with

mat

rix o

nly.

Dot

s re

pres

ent t

he

IgG

titre

of i

ndiv

idua

l mic

e in

arb

itrar

y un

its (A

U) a

nd b

ars

repr

esen

t the

m

edia

n tit

re o

f eac

h gr

oup.

010

2030

4050

6070

8090

100

28303234363840

0.6

mg

PE0.

1 m

g PE

3 m

g PE

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celcius)

010

2030

4050

6070

8090

100

283032343638400.

1 m

g PE

-RA

0.6

mg

PE-R

A

3 m

g PE

-RA

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celsius)

Cha

lleng

eby

subc

utan

eous

inje

ctio

n

6 m

g PE

+ 1

5 g

Cho

lera

Tox

inby

oral

gava

ge

Day

: 0

1

2

7

14

21

2

8

42

Figu

re 5

: Ani

mal

mod

el.M

ice

were

sen

sitiz

ed w

ith 6

mg

PE

and

15 µ

g C

hole

ra T

oxin

via

ora

l gav

age

and

chal

leng

ed (i

.p.)

with

PE,

PE

adso

rbed

to a

lum

, RA-

PE p

repa

ratio

n an

d R

A-PE

ad

sorb

ed to

alu

m (n

=6 p

er g

roup

).

Figu

re 6

: Bod

y te

mpe

ratu

re fo

llow

ing

i.p. c

halle

nge;

Ana

phyl

axis

to th

e na

tive

prep

arat

ion.

An i.

p. c

halle

nge

with

0.6

mg

PE p

er

mou

se re

sulte

d in

an

anap

hyla

ctic

sho

ck re

spon

se. T

he m

ice

in th

e gr

oups

chal

leng

edwi

thm

odifie

dPE

sho

wed

smal

ldec

reas

esin

te

mpe

ratu

reat

the

high

estd

ose

(3 m

g R

A-PE

). C

ontro

lmic

edi

dno

tres

pond

to th

e ch

alle

nge

(with

3 m

g of

PE

orPE

-RA)

.

blan

c50

2512

.56.

253.

131.

560255075100

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)Proliferation (SI)

blan

c50

2512

.56.

253.

131.

56050100

150

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

0.010.

1110100

p<0.

01

PE

R

A-PE

relative basophil activation (%)

02550PE PE

-RA

con

0.0

3 0

.01

0.3

1

3

10

100

10

00

conc

entr

atio

n (n

g/m

l)

% CD63+ basophils

0.01

0.1

110

100

0255075100

PE PE-R

A

inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)110100

p=0.

25

PE

PE-

RA

stimulation index

con

0

100

200

300

10μ μμμ

g

100

μ μμμg

10μ μμμg

100μ μμμg

PEPE

-RA

IgG (AU)

A.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

J. S

mit2 ,

S.C

. Dre

skin

3 , H

. War

men

hove

n1 , D

. Ver

bart

1 , E.

Kno

l4 , A

. Knu

lst4 ,

E. K

erkv

liet1 ,

R. v

an d

en H

out1 ,

R. v

an R

ee5 ,

R. P

iete

rs2 ,

E. v

an H

offe

n4 , S.

J. K

oppe

lman

1

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Inst

itute

for R

isk

Asse

ssm

ent S

cien

ces

and

Utr

echt

Cen

tre

for F

ood

Alle

rgy,

Utr

echt

, Th

e N

ethe

rland

s, 3 U

nive

rsity

of C

olor

ado

Den

ver S

choo

l of M

edic

ine,

Aur

ora,

CO

, USA

,4 D

ep. D

erm

atol

ogy/

Alle

rgol

ogy,

UM

C U

trec

ht, U

trec

ht,

The

Net

herla

nds,

5 Exp

erim

enta

l Im

mun

olog

y, A

cade

mic

Med

ical

Cen

tre,

Am

ster

dam

, The

Net

herla

nds

4 -C

hem

ical

mod

ifica

tion

of a

pea

nut e

xtra

ct d

ecre

ases

Ig

Ebi

ndin

g w

hile

the

imm

unog

enic

ity is

mai

ntai

ned

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•A

cur

ativ

e tre

atm

ent i

s no

t ava

ilabl

e.

•A m

odifi

ed p

eanu

t ext

ract

(alle

rgoi

d) is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

.

Con

clus

ion:

An

alle

rgoi

dpr

epar

atio

n of

pe

anut

ex

tract

ha

s a

redu

ced

alle

rgen

icity

com

pare

d to

its

nativ

e co

unte

rpar

t, w

hile

th

e im

mun

ogen

icity

is m

aint

aine

d.

Met

hods

:Th

e Pe

anut

ext

ract

(PE)

pre

para

tion

isch

emic

ally

mod

ified

by

redu

ctio

n of

the

intra

mol

ecul

ardi

sulfi

de b

onds

of P

E,an

d al

kyla

tion

of

the

resu

lting

fre

e su

lfhyd

rylg

roup

s (R

A).

For i

mm

unog

enic

ity, t

he a

llerg

oid

was

adso

rbed

to a

lum

iniu

m h

ydro

xide

.

Res

ults

:Ef

ficac

y:•P

E-sp

ecifi

c hu

man

T-c

ell l

ines

wer

eac

tivat

ed e

qual

ly w

ell w

ith P

E an

d R

A-PE

.•I

n m

ice, P

E an

d R

A-PE

wer

e eq

ually

pote

nt in

indu

cing

PE-

spec

ific

IgG

antib

odie

s in

mic

e.Sa

fety

:•M

odifi

catio

n lo

wer

ed th

e Ig

E-bi

ndin

g in

all p

atie

nts

to a

mea

n re

mai

ning

pot

ency

of 7

.2%

5%.

•The

med

iato

r rel

ease

ass

ay (M

RA)

show

ed a

redu

ced

pote

ncy

of 1

0-10

0fo

ld (r

ange

3 to

>10

,000

fold

). •M

ice

sens

itize

d w

ith P

E ex

perie

nced

ase

vere

ana

phyl

actic

reac

tion

upon

subc

utan

eous

cha

lleng

e w

ith 0

.1 m

gPE

, whi

le 3

mg

RA-

PE w

as s

till

tole

rate

d w

ithou

t pro

blem

s.

Figu

re 1

: Res

pons

e of

PE-

spec

ific

TCLs

(T c

ell l

ines

) to

nativ

e PE

and

RA-

PE.

Prol

ifera

tion

was

expr

esse

d as

the

stim

ulat

ion

inde

x (S

I, i.e

., pr

olife

ratio

n to

alle

rgen

stim

ulat

ion

divi

ded

by b

lanc

).A.

Rep

rese

ntat

ive

exam

ples

of a

TC

L of

an

alle

rgic

pat

ient

. B. I

mm

unog

enic

ity o

f PE

and

RA-

PE in

9 T

CLs

.

A.

Figu

re 3

: Inh

ibiti

on E

LISA

. A.

Inhi

bitio

n cu

rves

of t

he n

ativ

e an

d m

odifie

d PE

of a

US

seru

m

pool

of 2

0 pa

tient

s. T

est s

ampl

es c

ompe

te w

ith P

E co

ated

to E

LISA

pl

ates

for b

indi

ng to

ser

um Ig

E. In

crea

sing

conc

entra

tions

of t

est

sam

ple

resu

lt in

inhi

bitio

n of

bin

ding

. The

am

ount

of t

he Ig

Ebi

ndin

g in

pre

senc

e of

PE

com

pare

d to

that

in a

bsen

ce o

f PE

is c

alcu

late

d as

pe

rcen

tage

inhi

bitio

n.

B. Ig

Epo

tenc

y of

RA-

CPE

rela

tive

to n

ativ

e C

PE d

eter

min

ed in

an

IgE

inhi

bitio

n EL

ISA

usin

g se

ra o

f 20

pean

ut a

llerg

ic p

atie

nts

from

the

US.

An

in-h

ouse

refe

renc

e (IH

R) a

llerg

en p

repa

ratio

n is

test

ed to

com

pare

the

inhi

bitio

n cu

rves

by

para

llel l

ine

anal

yses

. The

IgE

bind

ing

effic

ienc

y is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to th

e IH

R.

1,3

pool

5,9

D11

48,

8D

107

6,2

D10

512

,2D

103

8,2

D98

8,2

D81

2,8

D80

11,8

D77

4,5

D74

9,1

D70

11,3

D69

4,3

D68

0,3

D65

17,8

D64

4,1

D62

5,3

D60

1,4

D50

16,9

D48

0,4

D44

1,3

D19

Rea

lativ

epo

tenc

y (%

) Sa

mpl

eB

.

Figu

re4:

The

pot

ency

of n

ativ

ePE

and

RA-

PE

in th

e in

dire

ct M

RA.

A. R

epre

sent

ativ

eex

ampl

e.

B.R

elat

ive

pote

ncy

of n

ativ

ePE

and

RA-

PE to

act

ivat

eba

soph

ilsFigu

re 2

: Spe

cific

IgG

resp

onse

s to

nat

ive

vers

us m

odifi

ed P

E.Sp

ecific

IgG

titre

s at

day

63

afte

r 5 im

mun

izatio

ns (i

.p.)

with

10

or 1

00 µ

g pr

oduc

t. C

ontro

l mic

e we

re in

ject

ed w

ith m

atrix

onl

y. D

ots

repr

esen

t the

Ig

Gtit

re o

f ind

ivid

ual m

ice

in a

rbitr

ary

units

(AU

) and

bar

s re

pres

ent t

he

med

ian

titre

of e

ach

grou

p.

010

2030

4050

6070

8090

100

28303234363840

0.6

mg

PE0.

1 m

g PE

3 m

g PE

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celcius)

010

2030

4050

6070

8090

100

283032343638400.

1 m

g PE

-RA

0.6

mg

PE-R

A

3 m

g PE

-RA

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celsius)

Cha

lleng

eby

subc

utan

eous

inje

ctio

n

6 m

g PE

+ 1

5 g

Cho

lera

Tox

inby

oral

gava

ge

Day

: 0

1

2

7

14

21

2

8

42

Figu

re 5

: Ani

mal

mod

el.M

ice

were

sen

sitiz

ed w

ith 6

mg

PE

and

15 µ

g C

hole

ra T

oxin

via

ora

l gav

age

and

chal

leng

ed (i

.p.)

with

PE,

PE

adso

rbed

to a

lum

, RA-

PE p

repa

ratio

n an

d R

A-PE

ad

sorb

ed to

alu

m (n

=6 p

er g

roup

).

Figu

re 6

: Bod

y te

mpe

ratu

re fo

llow

ing

i.p. c

halle

nge;

Ana

phyl

axis

to th

e na

tive

prep

arat

ion.

An i.

p. c

halle

nge

with

0.6

mg

PE p

er

mou

se re

sulte

d in

an

anap

hyla

ctic

sho

ck re

spon

se. T

he m

ice

in th

e gr

oups

chal

leng

edwi

thm

odifie

dPE

sho

wed

smal

ldec

reas

esin

te

mpe

ratu

reat

the

high

estd

ose

(3 m

g R

A-PE

). C

ontro

lmic

edi

dno

tres

pond

to th

e ch

alle

nge

(with

3 m

g of

PE

orPE

-RA)

.

blan

c50

2512

.56.

253.

131.

560255075100

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)bl

anc

5025

12.5

6.25

3.13

1.56

050100

150

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

0.010.

1110100

p<0.

01

PE

R

A-PE

relative basophil activation (%)

02550PE PE

-RA

con

0.0

3 0

.01

0.3

1

3

10

100

10

00

conc

entr

atio

n (n

g/m

l)

% CD63+ basophils

0.01

0.1

110

100

0255075100

PE PE-R

A

inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

110100

p=0.

25

PE

PE-

RA

stimulation index

con

0

100

200

300

10μ μμμ

g

100

μ μμμg

10μ μμμg

100μ μμμg

PEPE

-RA

IgG (AU)

A.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

J. S

mit2 ,

S.C

. Dre

skin

3 , H

. War

men

hove

n1 , D

. Ver

bart

1 , E.

Kno

l4 , A

. Knu

lst4 ,

E. K

erkv

liet1 ,

R. v

an d

en H

out1 ,

R. v

an R

ee5 ,

R. P

iete

rs2 ,

E. v

an H

offe

n4 , S.

J. K

oppe

lman

1

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Ins

titut

e fo

r Ris

k A

sses

smen

t Sci

ence

s an

d U

trec

ht C

entr

e fo

r Foo

d Al

lerg

y, U

trec

ht,

The

Net

herla

nds,

3 Uni

vers

ity o

f Col

orad

o D

enve

r Sch

ool o

f Med

icin

e, A

uror

a, C

O, U

SA,

4 Dep

. Der

mat

olog

y/Al

lerg

olog

y, U

MC

Utr

echt

, Utr

echt

, Th

e N

ethe

rland

s, 5 E

xper

imen

tal I

mm

unol

ogy,

Aca

dem

ic M

edic

al C

entr

e, A

mst

erda

m, T

heN

ethe

rland

s

4 -C

hem

ical

mod

ifica

tion

of a

pea

nut e

xtra

ct d

ecre

ases

Ig

Ebi

ndin

g w

hile

the

imm

unog

enic

ity is

mai

ntai

ned

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•A

cur

ativ

e tre

atm

ent i

s no

t ava

ilabl

e.

•A m

odifi

ed p

eanu

t ext

ract

(alle

rgoi

d) is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

.

Con

clus

ion:

An

alle

rgoi

dpr

epar

atio

n of

pe

anut

ex

tract

ha

s a

redu

ced

alle

rgen

icity

com

pare

d to

its

nativ

e co

unte

rpar

t, w

hile

th

e im

mun

ogen

icity

is m

aint

aine

d.

Met

hods

:Th

e Pe

anut

ext

ract

(PE)

pre

para

tion

isch

emic

ally

mod

ified

by

redu

ctio

n of

the

intra

mol

ecul

ardi

sulfi

de b

onds

of P

E,an

d al

kyla

tion

of

the

resu

lting

fre

e su

lfhyd

rylg

roup

s (R

A).

For i

mm

unog

enic

ity, t

he a

llerg

oid

was

adso

rbed

to a

lum

iniu

m h

ydro

xide

.

Res

ults

:Ef

ficac

y:•P

E-sp

ecifi

c hu

man

T-c

ell l

ines

wer

eac

tivat

ed e

qual

ly w

ell w

ith P

E an

d R

A-PE

.•I

n m

ice,

PE

and

RA-

PE w

ere

equa

llypo

tent

in in

duci

ng P

E-sp

ecifi

c Ig

Gan

tibod

ies

in m

ice.

Safe

ty:

•Mod

ifica

tion

low

ered

the

IgE-

bind

ing

inal

l pat

ient

s to

a m

ean

rem

aini

ng p

oten

cyof

7.2

%5%

. •T

he m

edia

tor r

elea

se a

ssay

(MR

A)sh

owed

a re

duce

d po

tenc

y of

10-

100

fold

(ran

ge 3

to >

10,0

00 fo

ld).

•Mic

e se

nsiti

zed

with

PE

expe

rienc

ed a

seve

re a

naph

ylac

tic re

actio

n up

onsu

bcut

aneo

us c

halle

nge

with

0.1

mg

PE, w

hile

3 m

g R

A-PE

was

stil

lto

lera

ted

with

out p

robl

ems.

Figu

re 1

: Res

pons

e of

PE-

spec

ific

TCLs

(T c

ell l

ines

) to

nativ

e PE

and

RA

-PE.

Prol

ifera

tion

was

expr

esse

d as

the

stim

ulat

ion

inde

x (S

I, i.e

., pr

olife

ratio

n to

alle

rgen

stim

ulat

ion

divi

ded

by b

lanc

).A.

Rep

rese

ntat

ive

exam

ples

of a

TC

L of

an

alle

rgic

pat

ient

. B. I

mm

unog

enic

ity o

f PE

and

RA-

PE in

9 T

CLs

.

A. Fi

gure

3: I

nhib

ition

ELI

SA

. A

. Inh

ibiti

on c

urve

s of

the

nativ

e an

d m

odifie

d PE

of a

US

seru

m

pool

of 2

0 pa

tient

s. T

est s

ampl

es c

ompe

te w

ith P

E co

ated

to E

LISA

pl

ates

for b

indi

ng to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sa

mpl

e re

sult

in in

hibi

tion

of b

indi

ng. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of P

E co

mpa

red

to th

at in

abs

ence

of P

E is

cal

cula

ted

as

perc

enta

ge in

hibi

tion.

B. Ig

Epo

tenc

y of

RA-

CPE

rela

tive

to n

ativ

e C

PE d

eter

min

ed in

an

IgE

inhi

bitio

n EL

ISA

usin

g se

ra o

f 20

pean

ut a

llerg

ic p

atie

nts

from

the

US.

An

in-h

ouse

refe

renc

e (IH

R) a

llerg

en p

repa

ratio

n is

test

ed to

com

pare

the

inhi

bitio

n cu

rves

by

para

llel l

ine

anal

yses

. The

IgE

bind

ing

effic

ienc

y is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to th

e IH

R.

1,3

pool

5,9

D11

48,

8D

107

6,2

D10

512

,2D

103

8,2

D98

8,2

D81

2,8

D80

11,8

D77

4,5

D74

9,1

D70

11,3

D69

4,3

D68

0,3

D65

17,8

D64

4,1

D62

5,3

D60

1,4

D50

16,9

D48

0,4

D44

1,3

D19

Rea

lativ

epo

tenc

y (%

) Sa

mpl

eB

.

Figu

re4:

The

pot

ency

of n

ativ

ePE

and

RA

-PE

in th

e in

dire

ct M

RA

. A. R

epre

sent

ativ

eex

ampl

e.

B.R

elat

ive

pote

ncy

of n

ativ

ePE

and

RA-

PE to

act

ivat

eba

soph

ilsFigu

re 2

: Spe

cific

IgG

resp

onse

s to

nat

ive

vers

us m

odifi

ed P

E.Sp

ecific

IgG

titre

s at

day

63

afte

r 5 im

mun

izat

ions

(i.p

.) wi

th 1

0 or

100

µg

prod

uct.

Con

trol m

ice

were

inje

cted

with

mat

rix o

nly.

Dot

s re

pres

ent t

he

IgG

titre

of i

ndiv

idua

l mic

e in

arb

itrar

y un

its (A

U) a

nd b

ars

repr

esen

t the

m

edia

n tit

re o

f eac

h gr

oup.

010

2030

4050

6070

8090

100

28303234363840

0.6

mg

PE0.

1 m

g PE

3 m

g PE

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celcius)

010

2030

4050

6070

8090

100

283032343638400.

1 m

g PE

-RA

0.6

mg

PE-R

A

3 m

g PE

-RA

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celsius)

Cha

lleng

eby

subc

utan

eous

inje

ctio

n

6 m

g PE

+ 1

5 g

Cho

lera

Tox

inby

oral

gava

ge

Day

: 0

1

2

7

14

21

2

8

42

Figu

re 5

: Ani

mal

mod

el.M

ice

were

sen

sitiz

ed w

ith 6

mg

PE

and

15 µ

g C

hole

ra T

oxin

via

ora

l gav

age

and

chal

leng

ed (i

.p.)

with

PE,

PE

adso

rbed

to a

lum

, RA-

PE p

repa

ratio

n an

d R

A-PE

ad

sorb

ed to

alu

m (n

=6 p

er g

roup

).

Figu

re 6

: Bod

y te

mpe

ratu

re fo

llow

ing

i.p. c

halle

nge;

Ana

phyl

axis

to th

e na

tive

prep

arat

ion.

An i.

p. c

halle

nge

with

0.6

mg

PE p

er

mou

se re

sulte

d in

an

anap

hyla

ctic

sho

ck re

spon

se. T

he m

ice

in th

e gr

oups

chal

leng

edwi

thm

odifie

dPE

sho

wed

smal

ldec

reas

esin

te

mpe

ratu

reat

the

high

estd

ose

(3 m

g R

A-PE

). C

ontro

lmic

edi

dno

tres

pond

to th

e ch

alle

nge

(with

3 m

g of

PE

orPE

-RA)

.

blan

c50

2512

.56.

253.

131.

560255075100

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

blan

c50

2512

.56.

253.

131.

56050100

150

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

0.010.

1110100

p<0.

01

PE

R

A-PE

relative basophil activation (%)

02550PE PE

-RA

con

0.0

3 0

.01

0.3

1

3

10

100

10

00

conc

entr

atio

n (n

g/m

l)

% CD63+ basophils

0.01

0.1

110

100

0255075100

PE PE-R

A

inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

110100

p=0.

25

PE

PE-

RA

stimulation index

con

0

100

200

300

10μ μμμ

g

100

μ μμμg

10μ μμμg

100μ μμμg

PEPE

-RA

IgG (AU)

A.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

J. S

mit2 ,

S.C

. Dre

skin

3 , H

. War

men

hove

n1 , D

. Ver

bart

1 , E.

Kno

l4 , A

. Knu

lst4 ,

E. K

erkv

liet1 ,

R. v

an d

en H

out1 ,

R. v

an R

ee5 ,

R. P

iete

rs2 ,

E. v

an H

offe

n4 , S.

J. K

oppe

lman

1

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Ins

titut

e fo

r Ris

k As

sess

men

t Sci

ence

s an

d U

trec

ht C

entr

e fo

r Foo

d Al

lerg

y, U

trec

ht,

The

Net

herla

nds,

3 Uni

vers

ity o

f Col

orad

o D

enve

r Sch

ool o

f Med

icin

e, A

uror

a, C

O, U

SA,

4 Dep

. Der

mat

olog

y/Al

lerg

olog

y, U

MC

Utr

echt

, Utr

echt

, Th

e N

ethe

rland

s, 5 E

xper

imen

tal I

mm

unol

ogy,

Aca

dem

ic M

edic

al C

entr

e, A

mst

erda

m, T

heN

ethe

rland

s

4 -C

hem

ical

mod

ifica

tion

of a

pea

nut e

xtra

ct d

ecre

ases

Ig

Ebi

ndin

g w

hile

the

imm

unog

enic

ity is

mai

ntai

ned

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•A

cur

ativ

e tre

atm

ent i

s no

t ava

ilabl

e.

•A m

odifi

ed p

eanu

t ext

ract

(alle

rgoi

d) is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

.

Con

clus

ion:

An

alle

rgoi

dpr

epar

atio

n of

pe

anut

ex

tract

ha

s a

redu

ced

alle

rgen

icity

com

pare

d to

its

nativ

e co

unte

rpar

t, w

hile

th

e im

mun

ogen

icity

is m

aint

aine

d.

Met

hods

:Th

e Pe

anut

ext

ract

(PE)

pre

para

tion

isch

emic

ally

mod

ified

by

redu

ctio

n of

the

intra

mol

ecul

ardi

sulfi

de b

onds

of P

E,an

d al

kyla

tion

of

the

resu

lting

fre

e su

lfhyd

rylg

roup

s (R

A).

For i

mm

unog

enic

ity, t

he a

llerg

oid

was

adso

rbed

to a

lum

iniu

m h

ydro

xide

.

Res

ults

:Ef

ficac

y:•P

E-sp

ecifi

c hu

man

T-c

ell l

ines

wer

eac

tivat

ed e

qual

ly w

ell w

ith P

E an

d R

A-PE

.•I

n m

ice,

PE

and

RA-

PE w

ere

equa

llypo

tent

in in

duci

ng P

E-sp

ecifi

c Ig

Gan

tibod

ies

in m

ice.

Safe

ty:

•Mod

ifica

tion

low

ered

the

IgE-

bind

ing

inal

l pat

ient

s to

a m

ean

rem

aini

ng p

oten

cyof

7.2

%5%

. •T

he m

edia

tor r

elea

se a

ssay

(MR

A)sh

owed

a re

duce

d po

tenc

y of

10-

100

fold

(ran

ge 3

to >

10,0

00 fo

ld).

•Mic

e se

nsiti

zed

with

PE

expe

rienc

ed a

seve

re a

naph

ylac

tic re

actio

n up

onsu

bcut

aneo

us c

halle

nge

with

0.1

mg

PE, w

hile

3 m

g R

A-PE

was

stil

lto

lera

ted

with

out p

robl

ems.

Figu

re 1

: Res

pons

e of

PE-

spec

ific

TCLs

(T c

ell l

ines

) to

nativ

e PE

and

RA

-PE.

Prol

ifera

tion

was

expr

esse

d as

the

stim

ulat

ion

inde

x (S

I, i.e

., pr

olife

ratio

n to

alle

rgen

stim

ulat

ion

divi

ded

by b

lanc

).A.

Rep

rese

ntat

ive

exam

ples

of a

TC

L of

an

alle

rgic

pat

ient

. B. I

mm

unog

enic

ity o

f PE

and

RA-

PE in

9 T

CLs

.

A. Fi

gure

3: I

nhib

ition

ELI

SA

. A

. Inh

ibiti

on c

urve

s of

the

nativ

e an

d m

odifie

d PE

of a

US

seru

m

pool

of 2

0 pa

tient

s. T

est s

ampl

es c

ompe

te w

ith P

E co

ated

to E

LISA

pl

ates

for b

indi

ng to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sa

mpl

e re

sult

in in

hibi

tion

of b

indi

ng. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of P

E co

mpa

red

to th

at in

abs

ence

of P

E is

cal

cula

ted

as

perc

enta

ge in

hibi

tion.

B. Ig

Epo

tenc

y of

RA-

CPE

rela

tive

to n

ativ

e C

PE d

eter

min

ed in

an

IgE

inhi

bitio

n EL

ISA

usin

g se

ra o

f 20

pean

ut a

llerg

ic p

atie

nts

from

the

US.

An

in-h

ouse

refe

renc

e (IH

R) a

llerg

en p

repa

ratio

n is

test

ed to

com

pare

the

inhi

bitio

n cu

rves

by

para

llel l

ine

anal

yses

. The

IgE

bind

ing

effic

ienc

y is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to th

e IH

R.

1,3

pool

5,9

D11

48,

8D

107

6,2

D10

512

,2D

103

8,2

D98

8,2

D81

2,8

D80

11,8

D77

4,5

D74

9,1

D70

11,3

D69

4,3

D68

0,3

D65

17,8

D64

4,1

D62

5,3

D60

1,4

D50

16,9

D48

0,4

D44

1,3

D19

Rea

lativ

epo

tenc

y (%

) Sa

mpl

eB

.

Figu

re4:

The

pot

ency

of n

ativ

ePE

and

RA

-PE

in th

e in

dire

ct M

RA

. A. R

epre

sent

ativ

eex

ampl

e.

B.R

elat

ive

pote

ncy

of n

ativ

ePE

and

RA-

PE to

act

ivat

eba

soph

ilsFigu

re 2

: Spe

cific

IgG

resp

onse

s to

nat

ive

vers

us m

odifi

ed P

E.Sp

ecific

IgG

titre

s at

day

63

afte

r 5 im

mun

izat

ions

(i.p

.) wi

th 1

0 or

100

µg

prod

uct.

Con

trol m

ice

were

inje

cted

with

mat

rix o

nly.

Dot

s re

pres

ent t

he

IgG

titre

of i

ndiv

idua

l mic

e in

arb

itrar

y un

its (A

U) a

nd b

ars

repr

esen

t the

m

edia

n tit

re o

f eac

h gr

oup.

010

2030

4050

6070

8090

100

28303234363840

0.6

mg

PE0.

1 m

g PE

3 m

g PE

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celcius)

010

2030

4050

6070

8090

100

283032343638400.

1 m

g PE

-RA

0.6

mg

PE-R

A

3 m

g PE

-RA

Tim

e af

ter c

halle

nge

(min

utes

)

Temperature (Celsius)

Cha

lleng

eby

subc

utan

eous

inje

ctio

n

6 m

g PE

+ 1

5 g

Cho

lera

Tox

inby

oral

gava

ge

Day

: 0

1

2

7

14

21

2

8

42

Figu

re 5

: Ani

mal

mod

el.M

ice

were

sen

sitiz

ed w

ith 6

mg

PE

and

15 µ

g C

hole

ra T

oxin

via

ora

l gav

age

and

chal

leng

ed (i

.p.)

with

PE,

PE

adso

rbed

to a

lum

, RA-

PE p

repa

ratio

n an

d R

A-PE

ad

sorb

ed to

alu

m (n

=6 p

er g

roup

).

Figu

re 6

: Bod

y te

mpe

ratu

re fo

llow

ing

i.p. c

halle

nge;

Ana

phyl

axis

to th

e na

tive

prep

arat

ion.

An i.

p. c

halle

nge

with

0.6

mg

PE p

er

mou

se re

sulte

d in

an

anap

hyla

ctic

sho

ck re

spon

se. T

he m

ice

in th

e gr

oups

chal

leng

edwi

thm

odifie

dPE

sho

wed

smal

ldec

reas

esin

te

mpe

ratu

reat

the

high

estd

ose

(3 m

g R

A-PE

). C

ontro

lmic

edi

dno

tres

pond

to th

e ch

alle

nge

(with

3 m

g of

PE

orPE

-RA)

.

blan

c50

2512

.56.

253.

131.

560255075100

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

blan

c50

2512

.56.

253.

131.

56050100

150

PE RA-

PE

conc

entr

atio

n (μ μμμ

g/m

l)

Proliferation (SI)

0.010.

1110100

p<0.

01

PE

R

A-PE

relative basophil activation (%)

02550PE PE

-RA

con

0.0

3 0

.01

0.3

1

3

10

100

10

00

conc

entr

atio

n (n

g/m

l)

% CD63+ basophils

0.01

0.1

110

100

0255075100

PE PE-R

A

inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

110100

p=0.

25

PE

PE-

RA

stimulation index

con

0

100

200

300

10μ μμμ

g

100

μ μμμg

10μ μμμg

100μ μμμg

PEPE

-RA

IgG (AU)

A.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

J. S

mit2 ,

S.C

. Dre

skin

3 , H

. War

men

hove

n1 , D

. Ver

bart

1 , E.

Kno

l4 , A

. Knu

lst4 ,

E. K

erkv

liet1 ,

R. v

an d

en H

out1 ,

R. v

an R

ee5 ,

R. P

iete

rs2 ,

E. v

an H

offe

n4 , S.

J. K

oppe

lman

1

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.

Al l e rge nic p o t e n c y o f c h e mic a l l y m o d i f i e d Ara h 2 / h 6 eva l u a t e d w i t h b a s o p h il a c t iva t i o n , R B L- S X 3 8 a c t iva t i o n , a n d s o l i d p h a s e Ig E - im mu n o a ss ay

20

H. van der Kleij (1), H. Warmenhoven (1), D. Verbart (1), Q. Wang (2), R. van Ree (3), S.C. Dreskin (2), S.J. Koppelman (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) University of Colorado Denver School of Medicine, Aurora, CO, USA, (3) Experimental

Immunology, Academic Medical Centre, Amsterdam, The Netherlands.

RationalePeanuts are one of the most common foods responsible for food-induced anaphylaxis and there is no curative treatment for peanut allergy. Peanut sIT could be an option but earlier attempts with aqueous peanut extract were hampered by safety issues. Major peanut allergens Ara h2 and Ara h6 have been modified to make them suitable for immunotherapy. This study evaluates the potency of modified Ara h2 and Ara h6 in mediator release assays in comparison with native, not modified Ara h2 and Ara h6.

MethodsAra h2 and Ara h6 were purified and chemically modified by means of reduction of disulfide bonds and subsequent alkylation of free Cys. Twenty patients from the greater Denver area were recruited and serum was collected. Total- and peanut-specific IgE was determined as well as IgE to individual peanut allergens. RBL SX38 cell expressing the human IgE receptor and basophils from healthy donors were loaded with the serum samples and were subsequently exposed to different concentrations of either native or modified Ara h2 / Ara h6. Mediator release was determine and expressed as percentage of maximum release. Potency of native and modified Ara h2 / Ara h6 was also evaluated applying a solid phase IgE- binding assay performed with individual patient sera.

ResultsFor all tested patient sera it was observed that the potency of modified Ara h2 /Ara h 6 was reduced compared to native Ara h2 / Ara h6. Using the RBL-SX38 assay a mean reduction of 100- fold was found (range: 3 to <10,000 fold). Similar data were found with the basophil assay and the fold reduction observed per patient correlated between the two mediator release tests. For modified Ara h2 / Ara h6, IgE-binding as determined using the solid phase IgE-binding assay was for all patient sera reduced compared to native Ara h2 / Ara h6. Sera for which the modification had the largest effect on the activity in mediator release assays also showed the largest reduction in potency using the solid-phase IgE binding test.

ConclusionsThe allergenic potency of modified Ara h2/Ara h6 is about 100-fold lower that that of native Ara h2/Ara h6, making them more suitable candidates for subcutaneous immunotherapy.

EAACI, 16-20 June 2012, GenevaAbstract number: 62, Session date and time: Sunday 17 June; 15:30 - 17:00 Session title: OAS 11 - Molecules and multiplexing in allergy diagnosis

21

OAS 11 - Molecules and multiplexing in allergy diagnosis

Al l e rge nic p o t e n c y o f c h e mic a l l y m o d i f i e d Ara h 2 / h 6 eva l u a t e d w i t h b a s o p h il a c t iva t i o n , R B L- S X 3 8 a c t iva t i o n , a n d s o l i d p h a s e Ig E - im mu n o a ss ay

1H

AL A

llerg

y B

V, L

eide

n, T

he N

ethe

rland

s, 2

Uni

vers

ity o

f Col

orad

o D

enve

r Sch

ool o

f Med

icin

e, A

uror

a, C

O, U

SA3 Ex

perim

enta

l Im

mun

olog

y, A

cade

mic

Med

ical

Cen

tre,

Am

ster

dam

, The

Net

herla

nds

62 -

Alle

rgen

ic p

oten

cy o

f che

mic

ally

mod

ified

Ara

h2/h

6 ev

alua

ted

with

bas

ophi

lact

ivat

ion,

RB

L-SX

38 a

ctiv

atio

n, a

nd

solid

pha

se Ig

E-im

mun

oass

ay

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•P

eanu

t sIT

with

aqu

eous

pea

nut e

xtra

ctha

s fa

iled

beca

use

of s

afet

y is

sues

.•A

mod

ified

pre

para

tion

of th

e 2

maj

orpe

anut

alle

rgen

s Ar

ah2

and

Ara

h6 is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

IgE

inhi

bitio

n cu

rves

of t

he n

ativ

e an

d m

odifie

d Ar

ah2

/6 o

f ind

ivid

ual

patie

nts.

Te

st s

ampl

es c

ompe

te w

ith n

ativ

e Ar

ah2

/6 c

oate

d to

ELI

SA p

late

s fo

r bin

ding

to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sam

ple

resu

lt in

inhi

bitio

n of

bi

ndin

g. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of A

rah2

/6 c

ompa

red

to

that

in a

bsen

ce o

f Ara

h2/6

is c

alcu

late

d as

per

cent

age

inhi

bitio

n.

Tabl

e1:

Ser

olog

yof

recr

uite

dpa

tient

s

Con

clus

ion:

The

alle

rgen

ic p

oten

cy o

f mod

ified

Ara

h2/6

is a

bout

100

-fold

low

er th

an th

at o

fna

tive

Ara

h2/A

ra h

6, m

akin

g th

em m

ore

suita

ble

cand

idat

es fo

r sub

cuta

neou

sim

mun

othe

rapy

.

Met

hods

:•A

rah2

and

Ara

h6 w

ere

purif

ied

and

chem

ical

ly m

odifi

ed b

y m

eans

of

redu

ctio

n (R

) of d

isul

fide

bond

s an

dsu

bseq

uent

alk

ylat

ion

(A) o

f fre

e C

ys.

Prep

arat

ions

wer

e ch

arac

teriz

ed b

ySD

S-PA

GE

and

by fa

r-UV

CD

spec

tosc

ropy

.•T

wen

ty p

atie

nts

from

the

grea

ter

Den

ver a

rea

wer

e re

crui

ted

and

seru

mw

as c

olle

cted

. •R

BL S

X38

cells

and

bas

ophi

lsfro

mhe

alth

y do

nors

wer

e lo

aded

with

the

seru

m s

ampl

es a

nd s

ubse

quen

tlyex

pose

d to

eith

er n

ativ

e Ar

ah2

/6 o

rm

odifi

ed A

rah2

/6 (R

A-Ar

ah2

/6).

•Pot

ency

was

als

o ev

alua

ted

appl

ying

aso

lid p

hase

IgE-

bind

ing

assa

ype

rform

ed w

ith in

divi

dual

pat

ient

ser

a.

Res

ults

: Al

l tes

ted

patie

nt s

era

disp

laye

d a

redu

ced

resp

onse

to R

A-Ar

ah2

/6:

•A m

ean

redu

ctio

n of

100

-fol

d w

asfo

und

(rang

e: 3

to <

10,0

00 fo

ld) u

sing

both

the

RBL

SX3

8 an

d th

e in

dire

ctba

soph

ilas

say.

•T

he fo

ld re

duct

ion

obse

rved

per

pat

ient

corre

late

d be

twee

n th

e tw

o m

edia

tor

rele

ase

test

s.

•Ser

a fo

r whi

ch th

e m

odifi

catio

n ha

d th

ela

rges

t effe

ct o

n th

e ac

tivity

in m

edia

tor

rele

ase

assa

ys a

lso

show

ed th

e la

rges

t re

duct

ion

in p

oten

cy u

sing

the

solid

-ph

ase

IgE

bind

ing

test

.

A.

Mw

1

2

B.N

ativ

e co

nglu

tinsh

ows

a ty

pica

l sp

ectru

m fo

r pro

tein

s co

nsis

ting

mai

nly

of a

lpha

-hel

ices

(min

imal

ellip

ticity

valu

es a

t 208

and

222

nm

) but

als

o

beta

-stru

ctur

e. In

con

trast

, RA

show

s a

typi

cal s

pect

rum

for u

nfol

ded

prot

ein.

St

rong

dec

reas

e of

ellip

ticity

at 2

22 n

m

indi

cate

s lo

ss o

f alp

ha-h

elic

es a

nd

incr

ease

of r

ando

m c

oil s

truct

ure.

Thi

s ca

n be

exp

lain

ed b

y th

e fa

ct th

at th

e di

sulp

hide

brid

ges

have

bee

n cl

eave

d.

200

210

220

230

240

250

260

-15

-10-5051015

Nat

ive

RA

Wav

elen

gth

(nm

)

CD (mDeg)

The

far-U

V C

D s

pect

ra o

f nat

ive

and

mod

ified

con

glut

in.

117

199

88179780219

545

505

194

1112

290

293

2421

280337306

533

138

101573

Tota

l IgE

226514787

1681285

4929500

1352180

591

135638716539

Pean

ut-

Spec

ific

IgE

2,0

D11

40,

7D

107

0D

105

6,4

D10

30,

4D

98

9,4

D81

9,7

D80

1,5

D77

0,5

D74

0D

70

3,4

D69

0D

68

0D

650,

8D

640,

3D

620,

5D

60

0,3

D50

0,4

D48

0,2

D44

0D

19

Rel

ativ

e po

tenc

y (%

) Sa

mpl

e

0.01

0.1

110

100

1000

0255075100

D10

3

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.1

110

100

1000

0102030Se

rum

poo

l

nativ

e

RA

ng/m

l

net % degranulationFigu

re 3

: Deg

ranu

latio

nof

RB

L SX

-38

cells

to n

ativ

e or

mod

ified

Ara

h2/6

1010

010

000102030

D65

nativ

e

RA

ng/m

l

net degranulation

110

100

1000

0102030D8

0

nativ

e

RA

ng/m

l

net degranulation

A.T

he n

ativ

e (1

) and

RA

(2)

Ara

h2/h

6 pr

epar

atio

ns

were

ana

lyzed

by

SDS-

PAG

E. T

he p

rote

ins

were

vi

sual

ized

usi

ng C

oom

assi

e-st

aini

ng.

0.01

110

010

000

0102030405060D

65

nativ

e

RA

ng/m

l

release (%)

0.01

110

010

000

0102030405060D

68

nativ

e

RA

ng/m

l

release (%)

0.01

110

010

000

0102030405060D

64 nativ

e

RA

ng/m

l

release (%)

Figu

re 4

: Deg

ranu

latio

nof

hum

an b

asop

hils

to n

ativ

e or

mod

ified

Ara

h2/6

RBL

SX-

38 c

ells

wer

e se

nsitiz

ed e

ither

with

a s

erum

poo

l (A)

or i

ndiv

idua

l ser

a (B

and

C) a

nd tr

igge

red

with

nat

ive

Ara

h 2/

6 or

RA-

Ara

h 2/

6.

Dat

a ar

e sh

own

as “n

et”d

egra

nula

tion

(tota

l deg

ranu

latio

nm

inus

bac

kgro

und)

.

In th

e in

dire

ct B

AT, h

uman

bas

ophi

lswe

re s

ensit

ized

with

indi

vidu

al s

era

and

trig

gere

d wi

th n

ativ

e Ar

ah

2/6

or R

A-Ar

ah

2/6.

Dat

a ar

e sh

own

as %

rele

ase

(in re

latio

n to

tota

l deg

ranu

latio

nm

inus

bac

kgro

und)

.

B.

Figu

re 1

. Cha

ract

eris

tics

of p

eanu

t pre

para

tions

Figu

re 2

: Sol

id p

hase

IgE-

bind

ing

test

0.01

0.1

110

100

1000

0255075100

D64

nativ

eR

A

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.01

0.1

110

100

1000

0255075100

D10

5

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.01

0.1

110

100

1000

0255075100

D11

4

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

IgE

bind

ing

pote

ncy

of th

e na

tive

and

mod

ified

prep

arat

ions

wer

e de

term

ined

in 2

0 U

S pa

tient

s wi

th

esta

blis

hed

clin

ical

alle

rgy

for p

eanu

t.Th

e Ig

Ebi

ndin

g is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to a

n in

-hou

se re

fere

nce

alle

rgen

pre

para

tion.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

H. W

arm

enho

ven1 ,

D. V

erba

rt1 ,

Q. W

ang2 ,

R. v

an R

ee3 ,

S.C

. Dre

skin

2 , S.

J. K

oppe

lman

11

HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2U

nive

rsity

of C

olor

ado

Den

ver S

choo

l of M

edic

ine,

Aur

ora,

CO

, USA

3 Ex

perim

enta

l Im

mun

olog

y, A

cade

mic

Med

ical

Cen

tre,

Am

ster

dam

, The

Net

herla

nds

62 -

Alle

rgen

ic p

oten

cy o

f che

mic

ally

mod

ified

Ara

h2/h

6 ev

alua

ted

with

bas

ophi

lact

ivat

ion,

RB

L-SX

38 a

ctiv

atio

n, a

nd

solid

pha

se Ig

E-im

mun

oass

ay

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•P

eanu

t sIT

with

aqu

eous

pea

nut e

xtra

ctha

s fa

iled

beca

use

of s

afet

y is

sues

.•A

mod

ified

pre

para

tion

of th

e 2

maj

orpe

anut

alle

rgen

s Ar

ah2

and

Ara

h6 is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

IgE

inhi

bitio

n cu

rves

of t

he n

ativ

e an

d m

odifie

d Ar

ah2

/6 o

f ind

ivid

ual

patie

nts.

Te

st s

ampl

es c

ompe

te w

ith n

ativ

e Ar

ah2

/6 c

oate

d to

ELI

SA p

late

s fo

r bin

ding

to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sam

ple

resu

lt in

inhi

bitio

n of

bi

ndin

g. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of A

rah2

/6 c

ompa

red

to

that

in a

bsen

ce o

f Ara

h2/6

is c

alcu

late

d as

per

cent

age

inhi

bitio

n.

Tabl

e1:

Ser

olog

yof

recr

uite

dpa

tient

s

Con

clus

ion:

The

alle

rgen

ic p

oten

cy o

f mod

ified

Ara

h2/6

is a

bout

100

-fold

low

er th

an th

at o

fna

tive

Ara

h2/A

ra h

6, m

akin

g th

em m

ore

suita

ble

cand

idat

es fo

r sub

cuta

neou

sim

mun

othe

rapy

.

Met

hods

:•A

rah2

and

Ara

h6 w

ere

purif

ied

and

chem

ical

ly m

odifi

ed b

y m

eans

of

redu

ctio

n (R

) of d

isul

fide

bond

s an

dsu

bseq

uent

alk

ylat

ion

(A) o

f fre

e C

ys.

Prep

arat

ions

wer

e ch

arac

teriz

ed b

ySD

S-PA

GE

and

by fa

r-UV

CD

spec

tosc

ropy

.•T

wen

ty p

atie

nts

from

the

grea

ter

Den

ver a

rea

wer

e re

crui

ted

and

seru

mw

as c

olle

cted

. •R

BL S

X38

cells

and

bas

ophi

lsfro

mhe

alth

y do

nors

wer

e lo

aded

with

the

seru

m s

ampl

es a

nd s

ubse

quen

tlyex

pose

d to

eith

er n

ativ

e Ar

ah2

/6 o

rm

odifi

ed A

rah2

/6 (R

A-Ar

ah2

/6).

•Pot

ency

was

als

o ev

alua

ted

appl

ying

aso

lid p

hase

IgE-

bind

ing

assa

ype

rform

ed w

ith in

divi

dual

pat

ient

ser

a.

Res

ults

: Al

l tes

ted

patie

nt s

era

disp

laye

d a

redu

ced

resp

onse

to R

A-Ar

ah2

/6:

•A m

ean

redu

ctio

n of

100

-fol

d w

asfo

und

(rang

e: 3

to <

10,0

00 fo

ld) u

sing

both

the

RBL

SX3

8 an

d th

e in

dire

ctba

soph

ilas

say.

•T

he fo

ld re

duct

ion

obse

rved

per

pat

ient

corre

late

d be

twee

n th

e tw

o m

edia

tor

rele

ase

test

s.

•Ser

a fo

r whi

ch th

e m

odifi

catio

n ha

d th

ela

rges

t effe

ct o

n th

e ac

tivity

in m

edia

tor

rele

ase

assa

ys a

lso

show

ed th

e la

rges

t re

duct

ion

in p

oten

cy u

sing

the

solid

-ph

ase

IgE

bind

ing

test

.

A.

Mw

1

2

B.N

ativ

e co

nglu

tinsh

ows

a ty

pica

l sp

ectru

m fo

r pro

tein

s co

nsis

ting

mai

nly

of a

lpha

-hel

ices

(min

imal

ellip

ticity

valu

es a

t 208

and

222

nm

) but

als

o

beta

-stru

ctur

e. In

con

trast

, RA

show

s a

typi

cal s

pect

rum

for u

nfol

ded

prot

ein.

St

rong

dec

reas

e of

ellip

ticity

at 2

22 n

m

indi

cate

s lo

ss o

f alp

ha-h

elic

es a

nd

incr

ease

of r

ando

m c

oil s

truct

ure.

Thi

s ca

n be

exp

lain

ed b

y th

e fa

ct th

at th

e di

sulp

hide

brid

ges

have

bee

n cl

eave

d.

200

210

220

230

240

250

260

-15

-10-5051015

Nat

ive

RA

Wav

elen

gth

(nm

)

CD (mDeg)

The

far-U

V C

D s

pect

ra o

f nat

ive

and

mod

ified

con

glut

in.

117

199

88179780219

545

505

194

1112

290

293

2421

280337306

533

138

101573

Tota

l IgE

226514787

1681285

4929500

1352180

591

135638716539

Pean

ut-

Spec

ific

IgE

2,0

D11

40,

7D

107

0D

105

6,4

D10

30,

4D

98

9,4

D81

9,7

D80

1,5

D77

0,5

D74

0D

70

3,4

D69

0D

68

0D

650,

8D

640,

3D

620,

5D

60

0,3

D50

0,4

D48

0,2

D44

0D

19

Rel

ativ

e po

tenc

y (%

) Sa

mpl

e

0.01

0.1

110

100

1000

0255075100

D10

3

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.1

110

100

1000

0102030Se

rum

poo

l

nativ

e

RA

ng/m

l

net % degranulationFigu

re 3

: Deg

ranu

latio

nof

RB

L SX

-38

cells

to n

ativ

e or

mod

ified

Ara

h2/6

1010

010

000102030

D65

nativ

e

RA

ng/m

l

net degranulation

110

100

1000

0102030D8

0

nativ

e

RA

ng/m

l

net degranulation

A.T

he n

ativ

e (1

) and

RA

(2)

Ara

h2/h

6 pr

epar

atio

ns

were

ana

lyzed

by

SDS-

PAG

E. T

he p

rote

ins

were

vi

sual

ized

usi

ng C

oom

assi

e-st

aini

ng.

0.01

110

010

000

0102030405060D

65

nativ

e

RA

ng/m

l

release (%)

0.01

110

010

000

0102030405060D

68

nativ

e

RA

ng/m

l

release (%)

0.01

110

010

000

0102030405060D

64 nativ

e

RA

ng/m

l

release (%)

Figu

re 4

: Deg

ranu

latio

nof

hum

an b

asop

hils

to n

ativ

e or

mod

ified

Ara

h2/6

RBL

SX-

38 c

ells

wer

e se

nsitiz

ed e

ither

with

a s

erum

poo

l (A)

or i

ndiv

idua

l ser

a (B

and

C) a

nd tr

igge

red

with

nat

ive

Ara

h 2/

6 or

RA-

Ara

h 2/

6.

Dat

a ar

e sh

own

as “n

et”d

egra

nula

tion

(tota

l deg

ranu

latio

nm

inus

bac

kgro

und)

.

In th

e in

dire

ct B

AT, h

uman

bas

ophi

lswe

re s

ensit

ized

with

indi

vidu

al s

era

and

trig

gere

d wi

th n

ativ

e Ar

ah

2/6

or R

A-Ar

ah

2/6.

Dat

a ar

e sh

own

as %

rele

ase

(in re

latio

n to

tota

l deg

ranu

latio

nm

inus

bac

kgro

und)

.

B.

Figu

re 1

. Cha

ract

eris

tics

of p

eanu

t pre

para

tions

Figu

re 2

: Sol

id p

hase

IgE-

bind

ing

test

0.01

0.1

110

100

1000

0255075100

D64

nativ

eR

A

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.01

0.1

110

100

1000

0255075100

D10

5

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.01

0.1

110

100

1000

0255075100

D11

4

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

IgE

bind

ing

pote

ncy

of th

e na

tive

and

mod

ified

prep

arat

ions

wer

e de

term

ined

in 2

0 U

S pa

tient

s wi

th

esta

blis

hed

clin

ical

alle

rgy

for p

eanu

t.Th

e Ig

Ebi

ndin

g is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to a

n in

-hou

se re

fere

nce

alle

rgen

pre

para

tion.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

H. W

arm

enho

ven1 ,

D. V

erba

rt1 ,

Q. W

ang2 ,

R. v

an R

ee3 ,

S.C

. Dre

skin

2 , S.

J. K

oppe

lman

1

1H

AL A

llerg

y B

V, L

eide

n, T

he N

ethe

rland

s, 2

Uni

vers

ity o

f Col

orad

o D

enve

r Sch

ool o

f Med

icin

e, A

uror

a, C

O, U

SA3 Ex

perim

enta

l Im

mun

olog

y, A

cade

mic

Med

ical

Cen

tre,

Am

ster

dam

, The

Net

herla

nds

62 -

Alle

rgen

ic p

oten

cy o

f che

mic

ally

mod

ified

Ara

h2/h

6 ev

alua

ted

with

bas

ophi

lact

ivat

ion,

RB

L-SX

38 a

ctiv

atio

n, a

nd

solid

pha

se Ig

E-im

mun

oass

ay

Bac

kgro

und

& A

im:

•Pea

nuts

are

one

of t

he m

ost c

omm

onfo

ods

resp

onsi

ble

for f

ood-

indu

ced

anap

hyla

xis

in a

dults

.•P

eanu

t sIT

with

aqu

eous

pea

nut e

xtra

ctha

s fa

iled

beca

use

of s

afet

y is

sues

.•A

mod

ified

pre

para

tion

of th

e 2

maj

orpe

anut

alle

rgen

s Ar

ah2

and

Ara

h6 is

bein

g in

vest

igat

ed fo

r its

pot

entia

l use

in im

mun

othe

rapy

IgE

inhi

bitio

n cu

rves

of t

he n

ativ

e an

d m

odifie

d Ar

ah2

/6 o

f ind

ivid

ual

patie

nts.

Te

st s

ampl

es c

ompe

te w

ith n

ativ

e Ar

ah2

/6 c

oate

d to

ELI

SA p

late

s fo

r bin

ding

to

ser

um Ig

E. In

crea

sing

con

cent

ratio

ns o

f tes

t sam

ple

resu

lt in

inhi

bitio

n of

bi

ndin

g. T

he a

mou

nt o

f the

IgE

bind

ing

in p

rese

nce

of A

rah2

/6 c

ompa

red

to

that

in a

bsen

ce o

f Ara

h2/6

is c

alcu

late

d as

per

cent

age

inhi

bitio

n.

Tabl

e1:

Ser

olog

yof

recr

uite

dpa

tient

s

Con

clus

ion:

The

alle

rgen

ic p

oten

cy o

f mod

ified

Ara

h2/6

is a

bout

100

-fold

low

er th

an th

at o

fna

tive

Ara

h2/A

ra h

6, m

akin

g th

em m

ore

suita

ble

cand

idat

es fo

r sub

cuta

neou

sim

mun

othe

rapy

.

Met

hods

:•A

rah2

and

Ara

h6 w

ere

purif

ied

and

chem

ical

ly m

odifi

ed b

y m

eans

of

redu

ctio

n (R

) of d

isul

fide

bond

s an

dsu

bseq

uent

alk

ylat

ion

(A) o

f fre

e C

ys.

Prep

arat

ions

wer

e ch

arac

teriz

ed b

ySD

S-PA

GE

and

by fa

r-UV

CD

spec

tosc

ropy

.•T

wen

ty p

atie

nts

from

the

grea

ter

Den

ver a

rea

wer

e re

crui

ted

and

seru

mw

as c

olle

cted

. •R

BL S

X38

cells

and

bas

ophi

lsfro

mhe

alth

y do

nors

wer

e lo

aded

with

the

seru

m s

ampl

es a

nd s

ubse

quen

tlyex

pose

d to

eith

er n

ativ

e Ar

ah2

/6 o

rm

odifi

ed A

rah2

/6 (R

A-Ar

ah2

/6).

•Pot

ency

was

als

o ev

alua

ted

appl

ying

aso

lid p

hase

IgE-

bind

ing

assa

ype

rform

ed w

ith in

divi

dual

pat

ient

ser

a.

Res

ults

: Al

l tes

ted

patie

nt s

era

disp

laye

d a

redu

ced

resp

onse

to R

A-Ar

ah2

/6:

•A m

ean

redu

ctio

n of

100

-fol

d w

asfo

und

(rang

e: 3

to <

10,0

00 fo

ld) u

sing

both

the

RBL

SX3

8 an

d th

e in

dire

ctba

soph

ilas

say.

•T

he fo

ld re

duct

ion

obse

rved

per

pat

ient

corre

late

d be

twee

n th

e tw

o m

edia

tor

rele

ase

test

s.

•Ser

a fo

r whi

ch th

e m

odifi

catio

n ha

d th

ela

rges

t effe

ct o

n th

e ac

tivity

in m

edia

tor

rele

ase

assa

ys a

lso

show

ed th

e la

rges

t re

duct

ion

in p

oten

cy u

sing

the

solid

-ph

ase

IgE

bind

ing

test

.

A.

Mw

1

2

B.N

ativ

e co

nglu

tinsh

ows

a ty

pica

l sp

ectru

m fo

r pro

tein

s co

nsis

ting

mai

nly

of a

lpha

-hel

ices

(min

imal

ellip

ticity

valu

es a

t 208

and

222

nm

) but

als

o

beta

-stru

ctur

e. In

con

trast

, RA

show

s a

typi

cal s

pect

rum

for u

nfol

ded

prot

ein.

St

rong

dec

reas

e of

ellip

ticity

at 2

22 n

m

indi

cate

s lo

ss o

f alp

ha-h

elic

es a

nd

incr

ease

of r

ando

m c

oil s

truct

ure.

Thi

s ca

n be

exp

lain

ed b

y th

e fa

ct th

at th

e di

sulp

hide

brid

ges

have

bee

n cl

eave

d.

200

210

220

230

240

250

260

-15

-10-5051015

Nat

ive

RA

Wav

elen

gth

(nm

)

CD (mDeg)

The

far-U

V C

D s

pect

ra o

f nat

ive

and

mod

ified

con

glut

in.

117

199

88179780219

545

505

194

1112

290

293

2421

280337306

533

138

101573

Tota

l IgE

226514787

1681285

4929500

1352180

591

135638716539

Pean

ut-

Spec

ific

IgE

2,0

D11

40,

7D

107

0D

105

6,4

D10

30,

4D

98

9,4

D81

9,7

D80

1,5

D77

0,5

D74

0D

70

3,4

D69

0D

68

0D

650,

8D

640,

3D

620,

5D

60

0,3

D50

0,4

D48

0,2

D44

0D

19

Rel

ativ

e po

tenc

y (%

) Sa

mpl

e

0.01

0.1

110

100

1000

0255075100

D10

3

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.1

110

100

1000

0102030Se

rum

poo

l

nativ

e

RA

ng/m

l

net % degranulationFigu

re 3

: Deg

ranu

latio

nof

RB

L SX

-38

cells

to n

ativ

e or

mod

ified

Ara

h2/6

1010

010

000102030

D65

nativ

e

RA

ng/m

l

net degranulation

110

100

1000

0102030D8

0

nativ

e

RA

ng/m

l

net degranulation

A.T

he n

ativ

e (1

) and

RA

(2)

Ara

h2/h

6 pr

epar

atio

ns

were

ana

lyzed

by

SDS-

PAG

E. T

he p

rote

ins

were

vi

sual

ized

usi

ng C

oom

assi

e-st

aini

ng.

0.01

110

010

000

0102030405060D

65

nativ

e

RA

ng/m

l

release (%)

0.01

110

010

000

0102030405060D

68

nativ

e

RA

ng/m

l

release (%)

0.01

110

010

000

0102030405060D

64 nativ

e

RA

ng/m

l

release (%)

Figu

re 4

: Deg

ranu

latio

nof

hum

an b

asop

hils

to n

ativ

e or

mod

ified

Ara

h2/6

RBL

SX-

38 c

ells

wer

e se

nsitiz

ed e

ither

with

a s

erum

poo

l (A)

or i

ndiv

idua

l ser

a (B

and

C) a

nd tr

igge

red

with

nat

ive

Ara

h 2/

6 or

RA-

Ara

h 2/

6.

Dat

a ar

e sh

own

as “n

et”d

egra

nula

tion

(tota

l deg

ranu

latio

nm

inus

bac

kgro

und)

.

In th

e in

dire

ct B

AT, h

uman

bas

ophi

lswe

re s

ensit

ized

with

indi

vidu

al s

era

and

trig

gere

d wi

th n

ativ

e Ar

ah

2/6

or R

A-Ar

ah

2/6.

Dat

a ar

e sh

own

as %

rele

ase

(in re

latio

n to

tota

l deg

ranu

latio

nm

inus

bac

kgro

und)

.

B.

Figu

re 1

. Cha

ract

eris

tics

of p

eanu

t pre

para

tions

Figu

re 2

: Sol

id p

hase

IgE-

bind

ing

test

0.01

0.1

110

100

1000

0255075100

D64

nativ

eR

A

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.01

0.1

110

100

1000

0255075100

D10

5

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

0.01

0.1

110

100

1000

0255075100

D11

4

nativ

e

RA

Inhi

bito

r con

cent

ratio

n (μ μμμ

g/m

l)

Inhibition (%)

IgE

bind

ing

pote

ncy

of th

e na

tive

and

mod

ified

prep

arat

ions

wer

e de

term

ined

in 2

0 U

S pa

tient

s wi

th

esta

blis

hed

clin

ical

alle

rgy

for p

eanu

t.Th

e Ig

Ebi

ndin

g is

exp

ress

ed a

s re

lativ

e po

tenc

y (R

P) c

ompa

red

to a

n in

-hou

se re

fere

nce

alle

rgen

pre

para

tion.

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

H. v

an d

er K

leij1 ,

H. W

arm

enho

ven1 ,

D. V

erba

rt1 ,

Q. W

ang2 ,

R. v

an R

ee3 ,

S.C

. Dre

skin

2 , S.

J. K

oppe

lman

1

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.

T h e c o nt e nt o f a l l e rge n s Ara h 1, Ara h 2, Ara h 3 a n d Ara h 6 in d i f f e r e nt p ea n u t cu l t ivars c o m m o n l y c o n su m e d in Eu ro p e a n d t h e U SA

22

S.J. Koppelman(1,2), D. Apostolovic (1), H. Warmenhoven (1), D. Verbart (1), S. L. Taylor (2), T. Isleib (3), S. Maleki (3).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Food Allergy Research and Resources Programme, University of Nebraska, Lincoln NE, USA,

(3) US Dept of Agriculture, New Orleans LA, USA.

RationalePeanut allergens Ara h2 and Ara h6 are considered to be the most relevant peanut allergens, while Ara h1 and to a lesser extent Ara h3, may play an important role as well. There are four main peanut cultivars commonly consumed in the Western world and their major allergen content is only partially known. Also, the effect of roasting on the extractability of major allergens is not well known.

MethodsTwelve peanuts samples comprising the four main cultivars Runner, Spanish, Valencia, and Virginia, were obtained and part of these sample were roasted (140 °C for 15 minutes in a preheated circulating hot air oven). Extracts were made and total protein content was measured using Bradford analysis. SDS-PAGE was used to visualize the protein bands patterns and the content of Ara h1, Ara h2, Ara h3, and Ara h6 was quantified using rp-HPLC. A solid phase IgE binding test was applied for evaluation of allergenic potency.

ResultsProtein concentration in varietal extracts varied from 13.9 to 21.2 mg/ml, and roasting reduced the extractability by 22 to 50%. Band patterns indicated that the four allergens of interest were present in samples of all varieties. The ranges of individual allergens in the tested samples were as follows: Ara h1: 11-32%; Ara h2: 7-16%; Ara h3: 38-76%; and Ara h6: 4-14%. In extracts of roasted peanuts, the relative content of Ara h1 was lower while that of Ara h3 was higher compared to non-roasted. The relative content of Ara h2 and Ara h6 did not vary between raw and roasted. The allergenic potencies compared to in-house reference extracts varied from 99 to 139% for the raw samples, and roasting increased the potency only marginally with an increase of 14 to 30% relative to non-roasted counterparts.

ConclusionsThe main peanut cultivars consumed in the Western world all contain the main peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 with a slight variation of the relative content of these allergens between the different cultivars. Roasting had minimal effects on allergenic potency with the sera used in this study.

EAACI, 16-20 June 2012, GenevaAbstract number: 1463, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 73 - Molecular components in allergy diagnosis

23

Poster Session 73 - Molecular components in allergy diagnosis

T h e c o nt e nt o f a l l e rge n s Ara h 1, Ara h 2, Ara h 3 a n d Ara h 6 in d i f f e r e nt p ea n u t cu l t ivars c o m m o n l y c o n su m e d in Eu ro p e a n d t h e U SA

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Foo

d Al

lerg

y R

esea

rch

and

Reso

urce

s Pr

ogra

m, U

nive

rsity

of

Nebr

aska

, Lin

coln

, NE,

USA

, 3 US

Dep

t of A

gric

ultu

re, N

ew O

rlean

s LA

, USA

Figu

re 3

: Pot

ency

det

erm

inat

ion

of th

e va

rious

ext

ract

s us

ing

hum

an Ig

EEL

ISA.

Extra

cts

of th

e pe

anut

ext

ract

s we

re d

ilute

d in

a

rang

e of

0.0

1 to

10

g/m

l. Ex

ampl

es o

f of V

irgin

ia

pean

ut (G

reen

: Bra

ntle

y ra

w; Y

ello

w: G

rego

ry

raw;

Red

: NC

-v11

roas

ted;

Blu

e: N

C-v

11 ra

w) a

re

show

n in

com

paris

on to

the

IHR

.Th

e pl

asm

a po

ol u

sed

cons

iste

d of

14

plas

mas

fro

m p

atie

nts,

all c

linic

ally

alle

rgic

to p

eanu

t, m

ean

IgE

to p

eanu

36,9

kU

/L.

1463

-Th

e co

nten

t of a

llerg

ens

Ara

h1, A

rah2

, Ar

ah3

and

Ara

h6 in

diff

eren

t pea

nut c

ultiv

ars

com

mon

ly c

onsu

med

in E

urop

e an

d th

e U

SA

Bac

kgro

und

& A

im:

Pean

utal

lerg

ens

Ara

h2 a

nd A

ra h

6 ar

e co

nsid

ered

to b

eth

e m

ost

rele

vant

pea

nut

alle

rgen

s, w

hile

Ara

h1 a

nd t

o a

less

erex

tent

Ara

h3, m

aypl

ayan

impo

rtant

role

as

wel

l. Th

ere

are

four

mai

npe

anut

culti

vars

co

mm

only

cons

umed

in t

he W

este

rn w

orld

and

thei

rm

ajor

al

lerg

enco

nten

t is

on

lypa

rtial

lykn

own.

Als

o, t

he e

ffect

of

roas

ting

onth

e ex

tract

abilit

yof

maj

or a

llerg

ens

is n

otw

ellk

now

n.

Tabl

e 1:

Ext

ract

able

pro

tein

, con

tent

of i

ndiv

idua

l alle

rgen

s an

d Ig

E-bi

ndin

g po

tenc

y of

the

vario

us p

eanu

t sam

ples

Figu

re 2

: Det

erm

inat

ion

of p

eanu

t alle

rgen

s by

rp-H

PLC

. A.

Exa

mpl

e of

chr

omat

ogra

m o

f sam

ples

of V

irgin

ia (G

reen

: Bra

ntle

y ra

w; Y

ello

w: G

rego

ry ra

w; R

ed: N

C-v

11 ro

aste

d; B

lue:

NC

-v11

raw)

. In

divi

dual

cur

ves

were

shi

fted

verti

cally

, ran

ge fo

r eac

h sa

mpl

eis

0-8

00 m

AU.

B. C

hrom

atog

ram

of p

urifie

d pe

anut

alle

rgen

refe

renc

es A

rah1

: blu

e; A

rah2

: red

; Ara

h3: y

ello

w; A

rah6

: gre

en.

Col

umn:

X-B

ridge

BEH

Phe

nyl;

3.5

m; 1

35Å;

[2.1

x 1

50 m

m].

Tem

pera

ture

: 30

1 C

. Sam

ple

volu

me:

0.0

2 m

l, flo

w ra

te: 0

.3 m

l/min

. El

uent

A: 0

.1 %

TFA

in M

Q W

ater

; elu

ent B

: 0.0

85 %

TFA

in M

eOH

. Abs

orba

nces

were

mea

sure

d by

dio

de a

rray

and

reco

rded

for 2

15

and

280

nm. S

ampl

e si

ze fo

r ext

ract

s: 2

0 lo

f 1 m

g/m

l sol

utio

n.

Con

clus

ion:

The

mai

n pe

anut

cul

tivar

s co

nsum

ed in

the

Wes

tern

wor

ld a

ll co

ntai

n th

e m

ain

pean

ut

alle

rgen

s Ar

ah1

, Ara

h2, A

rah3

, and

Ara

h6

with

a s

light

var

iatio

n of

the

rela

tive

cont

ent

of

thes

e al

lerg

ens

betw

een

the

diffe

rent

cu

ltiva

rs.

Roa

stin

g ha

d m

inim

al e

ffect

s on

al

lerg

enic

pot

ency

with

the

sera

use

d in

this

st

udy.

Met

hods

:Tw

elve

pea

nuts

sam

ples

com

pris

ing

the

four

m

ain

culti

vars

Run

ner,

Span

ish,

Val

enci

a,

and

Virg

inia

, w

ere

obta

ined

an

d pa

rt of

th

ese

sam

ple

wer

e ro

aste

d (1

40

Cfo

r 15

m

inut

es i

n a

preh

eate

d ci

rcul

atin

g ho

t ai

r ov

en).

Extra

cts

wer

e m

ade

and

tota

l pro

tein

co

nten

t w

as

mea

sure

d us

ing

Brad

ford

an

alys

is.

SDS-

PAG

E w

as u

sed

to v

isua

lize

the

prot

ein

band

s pa

ttern

s an

d th

e co

nten

t of

Ara

h1, A

rah2

, Ara

h3, a

nd A

rah6

was

qu

antif

ied

usin

g rp

-HPL

C. A

sol

id p

hase

IgE

bind

ing

test

was

app

lied

for

eval

uatio

n of

al

lerg

enic

pot

ency

.An

extra

ct f

rom

Virg

inia

pe

anut

s(ra

w, G

reer

, Len

oir

NC

, USA

) w

as

used

as in

-hou

sere

fere

nce

(IHR

).

Res

ults

: Pr

otei

n co

ncen

tratio

n in

va

rieta

l ex

tract

s va

ried

from

13.

9 to

21.

2 m

g/m

l, an

d ro

astin

g re

duce

d th

e ex

tract

abilit

y by

22

to 5

0%.

Band

pa

ttern

s in

dica

ted

that

th

e fo

ur

alle

rgen

s of

in

tere

st

wer

e pr

esen

t in

sa

mpl

es

of

all

varie

ties.

Th

e ra

nges

of

in

divi

dual

alle

rgen

s in

the

tes

ted

sam

ples

w

ere

as fo

llow

s: A

rah1

: 11-

32%

; Ara

h2: 7

-16

%; A

rah3

: 38-

76%

; and

Ara

h6: 4

-14%

. In

extra

cts

of

roas

ted

pean

uts,

th

e re

lativ

e co

nten

t of A

rah1

was

low

er w

hile

that

of A

rah3

was

hig

her

com

pare

d to

non

-roas

ted.

Th

e re

lativ

e co

nten

t of

Ara

h2 a

nd A

rah6

di

d no

t var

y be

twee

n ra

w a

nd r

oast

ed. T

he

alle

rgen

ic

pote

ncie

s co

mpa

red

to in

-hou

se

refe

renc

e ex

tract

s va

ried

from

99

to 1

39%

fo

r the

raw

sam

ples

, and

roas

ting

incr

ease

d th

e po

tenc

y on

ly m

argi

nally

with

an

incr

ease

of

14

to

30

%

rela

tive

to

non-

roas

ted

coun

terp

arts

.

Figu

re 1

: SD

S-PA

GE

of th

e va

rious

pea

nut s

ampl

es

and

purif

ied

pean

ut a

llerg

en re

fere

nces

A. P

eanu

t sam

ple

anal

ysed

und

er re

duci

ng c

ondi

tions

. B

. Pea

nut s

ampl

e an

alys

ed u

nder

non

-redu

cing

con

ditio

ns.

1. B

io-R

ad M

olec

ular

wei

ght m

arke

r (kD

a)

2. L

aem

mlis

ampl

e bu

ffer

3. R

unne

r; G

AO6G

Run

ner (

Raw

) 4.

Run

ner;

Tiftg

uard

Run

ner (

Raw

)5.

Run

ner;

Flor

unne

r4/1

4 (R

aw)

6. R

unne

r; Fl

orun

ner4

/14

(Roa

sted

) 7.

Spa

nish

; Olin

Spa

nish

(Raw

) 8.

Spa

nish

; Olin

Spa

nish

(Roa

sted

) 9.

Val

encia

; Val

enci

a A

(Raw

) 10

. Val

enci

a; V

alen

cia

A (R

oast

ed)

11. V

irgin

ia; N

c-v1

1 Vi

rgin

ia (R

aw)

12. V

irgin

ia; N

c-v1

1 Vi

rgin

ia (R

oast

ed)

13. V

irgin

ia; G

rego

ry V

irgin

ia (R

aw)

14. V

irgin

ia; B

rant

ley

Virg

inia

(Raw

)

Purif

ied

alle

rgen

s Ar

ah1

, Ara

h2, A

rah3

, and

Ara

h6 w

ere

used

for b

and

assi

gnm

ent (

right

mar

gin)

.

A. B.

5 10

15

A215 (mAU)

Tim

e (m

in)

0

300

600

900

1200

5 10

15

A215 (mAU)

Tim

e (m

in)

B.

A. 800 0

0,0

25,0

50,0

75,0

100,

0 0,01

0,

10

1,00

10

,00

Inhibition (%)

Inhi

bito

r con

cent

ratio

n (µ

g/m

l)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

or p

erce

ived

conf

licts

of in

tere

st:

the

pres

ente

r was

an

empl

oyee

of H

AL A

llerg

ydu

ring

whe

nth

isre

sear

ch w

as c

ondu

cted

EAAC

I Con

gres

s 20

12

S.J.

Kop

pelm

an1,

2 , D

. Apo

stol

ovic

1 , H

. War

men

hove

n1, D

. Ver

bart

1 , S.

L. T

aylo

r2, T

. Isl

eib3

and

S. M

alek

i3

1 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s, 2 F

ood

Alle

rgy

Rese

arch

and

Res

ourc

es P

rogr

am, U

nive

rsity

of

Nebr

aska

, Lin

coln

, NE,

USA

, 3 US

Dept

of A

gric

ultu

re, N

ew O

rlean

s LA

, USA

Figu

re 3

: Pot

ency

det

erm

inat

ion

of th

e va

rious

ext

ract

s us

ing

hum

an Ig

EEL

ISA.

Extra

cts

of th

e pe

anut

ext

ract

s we

re d

ilute

d in

a

rang

e of

0.0

1 to

10

g/m

l. Ex

ampl

es o

f of V

irgin

ia

pean

ut (G

reen

: Bra

ntle

y ra

w; Y

ello

w: G

rego

ry

raw;

Red

: NC

-v11

roas

ted;

Blu

e: N

C-v

11 ra

w) a

re

show

n in

com

paris

on to

the

IHR

.Th

e pl

asm

a po

ol u

sed

cons

isted

of 1

4 pl

asm

as

from

pat

ient

s, a

ll clin

ically

alle

rgic

to p

eanu

t, m

ean

IgE

to p

eanu

36,9

kU

/L.

1463

-Th

e co

nten

t of a

llerg

ens

Ara

h1, A

rah2

, Ar

ah3

and

Ara

h6 in

diff

eren

t pea

nut c

ultiv

ars

com

mon

ly c

onsu

med

in E

urop

e an

d th

e U

SA

Bac

kgro

und

& A

im:

Pean

utal

lerg

ens

Ara

h2 a

nd A

ra h

6 ar

e co

nsid

ered

to b

eth

e m

ost

rele

vant

pea

nut

alle

rgen

s, w

hile

Ara

h1 a

nd t

o a

less

erex

tent

Ara

h3, m

aypl

ayan

impo

rtant

role

as

wel

l. Th

ere

are

four

mai

npe

anut

culti

vars

co

mm

only

cons

umed

in t

he W

este

rn w

orld

and

thei

rm

ajor

al

lerg

enco

nten

t is

on

lypa

rtial

lykn

own.

Als

o, t

he e

ffect

of

roas

ting

onth

e ex

tract

abilit

yof

maj

or a

llerg

ens

is n

otw

ellk

now

n.

Tabl

e 1:

Ext

ract

able

pro

tein

, con

tent

of i

ndiv

idua

l alle

rgen

s an

d Ig

E-bi

ndin

g po

tenc

y of

the

vario

us p

eanu

t sam

ples

Figu

re 2

: Det

erm

inat

ion

of p

eanu

t alle

rgen

s by

rp-H

PLC

. A.

Exa

mpl

e of

chr

omat

ogra

m o

f sam

ples

of V

irgin

ia (G

reen

: Bra

ntle

y ra

w; Y

ello

w: G

rego

ry ra

w; R

ed: N

C-v

11 ro

aste

d; B

lue:

NC

-v11

raw)

. In

divi

dual

cur

ves

were

shi

fted

verti

cally

, ran

ge fo

r eac

h sa

mpl

eis

0-80

0 m

AU.

B. C

hrom

atog

ram

of p

urifie

d pe

anut

alle

rgen

refe

renc

es A

rah1

: blu

e; A

rah2

: red

; Ara

h3: y

ello

w; A

rah6

: gre

en.

Col

umn:

X-B

ridge

BEH

Phe

nyl;

3.5

m; 1

35Å;

[2.1

x 1

50 m

m].

Tem

pera

ture

: 30

1 C

. Sam

ple

volu

me:

0.0

2 m

l, flo

w ra

te: 0

.3 m

l/min

. El

uent

A: 0

.1 %

TFA

in M

Q W

ater

; elu

ent B

: 0.0

85 %

TFA

in M

eOH

. Abs

orba

nces

were

mea

sure

d by

dio

de a

rray

and

reco

rded

for 2

15

and

280

nm. S

ampl

e si

ze fo

r ext

ract

s: 2

0 lo

f 1 m

g/m

l sol

utio

n.

Con

clus

ion:

The

mai

n pe

anut

cul

tivar

s co

nsum

ed in

the

Wes

tern

wor

ld a

ll co

ntai

n th

e m

ain

pean

ut

alle

rgen

s Ar

ah1

, Ara

h2, A

rah3

, and

Ara

h6

with

a s

light

var

iatio

n of

the

rela

tive

cont

ent

of

thes

e al

lerg

ens

betw

een

the

diffe

rent

cu

ltiva

rs.

Roa

stin

g ha

d m

inim

al e

ffect

s on

al

lerg

enic

pot

ency

with

the

sera

use

d in

this

st

udy.

Met

hods

:Tw

elve

pea

nuts

sam

ples

com

pris

ing

the

four

m

ain

culti

vars

Run

ner,

Span

ish,

Val

enci

a,

and

Virg

inia

, w

ere

obta

ined

an

d pa

rt of

th

ese

sam

ple

wer

e ro

aste

d (1

40

Cfo

r 15

m

inut

es i

n a

preh

eate

d ci

rcul

atin

g ho

t ai

r ov

en).

Extra

cts

wer

e m

ade

and

tota

l pro

tein

co

nten

t w

as

mea

sure

d us

ing

Brad

ford

an

alys

is.

SDS-

PAG

E w

as u

sed

to v

isua

lize

the

prot

ein

band

s pa

ttern

s an

d th

e co

nten

t of

Ara

h1, A

rah2

, Ara

h3, a

nd A

rah6

was

qu

antif

ied

usin

g rp

-HPL

C. A

sol

id p

hase

IgE

bind

ing

test

was

app

lied

for

eval

uatio

n of

al

lerg

enic

pot

ency

.An

extra

ct f

rom

Virg

inia

pe

anut

s(ra

w, G

reer

, Len

oirN

C, U

SA )

was

us

edas

in-h

ouse

refe

renc

e(IH

R).

Res

ults

: Pr

otei

n co

ncen

tratio

n in

va

rieta

l ex

tract

s va

ried

from

13.

9 to

21.

2 m

g/m

l, an

d ro

astin

g re

duce

d th

e ex

tract

abilit

y by

22

to 5

0%.

Band

pa

ttern

s in

dica

ted

that

th

e fo

ur

alle

rgen

s of

in

tere

st

wer

e pr

esen

t in

sa

mpl

es

of

all

varie

ties.

Th

e ra

nges

of

in

divi

dual

alle

rgen

s in

the

tes

ted

sam

ples

w

ere

as fo

llow

s: A

rah1

: 11-

32%

; Ara

h2: 7

-16

%; A

rah3

: 38-

76%

; and

Ara

h6: 4

-14%

. In

extra

cts

of

roas

ted

pean

uts,

th

e re

lativ

e co

nten

t of A

rah1

was

low

er w

hile

that

of A

rah3

was

hig

her

com

pare

d to

non

-roas

ted.

Th

e re

lativ

e co

nten

t of

Ara

h2 a

nd A

rah6

di

d no

t var

y be

twee

n ra

w a

nd r

oast

ed. T

he

alle

rgen

ic

pote

ncie

s co

mpa

red

to in

-hou

se

refe

renc

e ex

tract

s va

ried

from

99

to 1

39%

fo

r the

raw

sam

ples

, and

roas

ting

incr

ease

d th

e po

tenc

y on

ly m

argi

nally

with

an

incr

ease

of

14

to

30

%

rela

tive

to

non-

roas

ted

coun

terp

arts

.

Figu

re 1

: SD

S-PA

GE

of th

e va

rious

pea

nut s

ampl

es

and

purif

ied

pean

ut a

llerg

en re

fere

nces

A. P

eanu

t sam

ple

anal

ysed

und

er re

ducin

g co

nditio

ns.

B. P

eanu

t sam

ple

anal

ysed

und

er n

on-re

ducin

g co

nditio

ns.

1. B

io-R

ad M

olec

ular

wei

ght m

arke

r (kD

a)

2. L

aem

mlis

ampl

e bu

ffer

3. R

unne

r; G

AO6G

Run

ner (

Raw)

4.

Run

ner;

Tiftg

uard

Runn

er (R

aw)

5. R

unne

r; Fl

orun

ner4

/14

(Raw

) 6.

Run

ner;

Flor

unne

r4/1

4 (R

oast

ed)

7. S

pani

sh; O

lin S

pani

sh (R

aw)

8. S

pani

sh; O

lin S

pani

sh (R

oast

ed)

9. V

alen

cia; V

alen

cia A

(Raw

) 10

. Val

encia

; Val

encia

A (R

oast

ed)

11. V

irgin

ia; N

c-v1

1 Vi

rgin

ia (R

aw)

12. V

irgin

ia; N

c-v1

1 Vi

rgin

ia (R

oast

ed)

13. V

irgin

ia; G

rego

ry V

irgin

ia (R

aw)

14. V

irgin

ia; B

rant

ley

Virg

inia

(Raw

)

Purif

ied

alle

rgen

s Ar

ah1

, Ara

h2, A

rah3

, and

Ara

h6 w

ere

used

for b

and

assig

nmen

t (rig

ht m

argi

n).

A. B.

5 10

15

A215 (mAU)

Tim

e (m

in)

0

300

600

900

1200

5 10

15

A215 (mAU)

Tim

e (m

in)

B.A. 80

0 0

0,0

25,0

50,0

75,0

100,

0 0,01

0,

10

1,00

10

,00

Inhibition (%)

Inhi

bito

r con

cent

ratio

n (µ

g/m

l)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

or p

erce

ived

conf

licts

of in

tere

st:

the

pres

ente

r was

an

empl

oyee

of H

AL A

llerg

ydu

ring

whe

nth

isre

sear

ch w

as c

ondu

cted

EAAC

I Con

gres

s 20

12

S.J.

Kop

pelm

an1,

2 , D

. Apo

stol

ovic

1 , H

. War

men

hove

n1, D

. Ver

bart

1 , S.

L. T

aylo

r2, T

. Isl

eib3

and

S. M

alek

i3

1 HAL

Alle

rgy

BV,

Lei

den,

The

Net

herla

nds,

2 Foo

d Al

lerg

y R

esea

rch

and

Reso

urce

s Pr

ogra

m, U

nive

rsity

of

Nebr

aska

, Lin

coln

, NE,

USA

, 3 US

Dep

t of A

gric

ultu

re, N

ew O

rlean

s LA

, USA

Figu

re 3

: Pot

ency

det

erm

inat

ion

of th

e va

rious

ext

ract

s us

ing

hum

an Ig

EEL

ISA.

Extra

cts

of th

e pe

anut

ext

ract

s we

re d

ilute

d in

a

rang

e of

0.0

1 to

10

g/m

l. Ex

ampl

es o

f of V

irgin

ia

pean

ut (G

reen

: Bra

ntle

y ra

w; Y

ello

w: G

rego

ry

raw;

Red

: NC

-v11

roas

ted;

Blu

e: N

C-v

11 ra

w) a

re

show

n in

com

paris

on to

the

IHR

.Th

e pl

asm

a po

ol u

sed

cons

iste

d of

14

plas

mas

fro

m p

atie

nts,

all c

linic

ally

alle

rgic

to p

eanu

t, m

ean

IgE

to p

eanu

36,9

kU

/L.

1463

-Th

e co

nten

t of a

llerg

ens

Ara

h1, A

rah2

, Ar

ah3

and

Ara

h6 in

diff

eren

t pea

nut c

ultiv

ars

com

mon

ly c

onsu

med

in E

urop

e an

d th

e U

SA

Bac

kgro

und

& A

im:

Pean

utal

lerg

ens

Ara

h2 a

nd A

ra h

6 ar

e co

nsid

ered

to b

eth

e m

ost

rele

vant

pea

nut

alle

rgen

s, w

hile

Ara

h1 a

nd t

o a

less

erex

tent

Ara

h3, m

aypl

ayan

impo

rtant

role

as

wel

l. Th

ere

are

four

mai

npe

anut

culti

vars

co

mm

only

cons

umed

in t

he W

este

rn w

orld

and

thei

rm

ajor

al

lerg

enco

nten

t is

on

lypa

rtial

lykn

own.

Als

o, t

he e

ffect

of

roas

ting

onth

e ex

tract

abilit

yof

maj

or a

llerg

ens

is n

otw

ellk

now

n.

Tabl

e 1:

Ext

ract

able

pro

tein

, con

tent

of i

ndiv

idua

l alle

rgen

s an

d Ig

E-bi

ndin

g po

tenc

y of

the

vario

us p

eanu

t sam

ples

Figu

re 2

: Det

erm

inat

ion

of p

eanu

t alle

rgen

s by

rp-H

PLC

. A.

Exa

mpl

e of

chr

omat

ogra

m o

f sam

ples

of V

irgin

ia (G

reen

: Bra

ntle

y ra

w; Y

ello

w: G

rego

ry ra

w; R

ed: N

C-v

11 ro

aste

d; B

lue:

NC

-v11

raw)

. In

divi

dual

cur

ves

were

shi

fted

verti

cally

, ran

ge fo

r eac

h sa

mpl

eis

0-8

00 m

AU.

B. C

hrom

atog

ram

of p

urifie

d pe

anut

alle

rgen

refe

renc

es A

rah1

: blu

e; A

rah2

: red

; Ara

h3: y

ello

w; A

rah6

: gre

en.

Col

umn:

X-B

ridge

BEH

Phe

nyl;

3.5

m; 1

35Å;

[2.1

x 1

50 m

m].

Tem

pera

ture

: 30

1 C

. Sam

ple

volu

me:

0.0

2 m

l, flo

w ra

te: 0

.3 m

l/min

. El

uent

A: 0

.1 %

TFA

in M

Q W

ater

; elu

ent B

: 0.0

85 %

TFA

in M

eOH

. Abs

orba

nces

were

mea

sure

d by

dio

de a

rray

and

reco

rded

for 2

15

and

280

nm. S

ampl

e si

ze fo

r ext

ract

s: 2

0 lo

f 1 m

g/m

l sol

utio

n.

Con

clus

ion:

The

mai

n pe

anut

cul

tivar

s co

nsum

ed in

the

Wes

tern

wor

ld a

ll co

ntai

n th

e m

ain

pean

ut

alle

rgen

s Ar

ah1

, Ara

h2, A

rah3

, and

Ara

h6

with

a s

light

var

iatio

n of

the

rela

tive

cont

ent

of

thes

e al

lerg

ens

betw

een

the

diffe

rent

cu

ltiva

rs.

Roa

stin

g ha

d m

inim

al e

ffect

s on

al

lerg

enic

pot

ency

with

the

sera

use

d in

this

st

udy.

Met

hods

:Tw

elve

pea

nuts

sam

ples

com

pris

ing

the

four

m

ain

culti

vars

Run

ner,

Span

ish,

Val

enci

a,

and

Virg

inia

, w

ere

obta

ined

an

d pa

rt of

th

ese

sam

ple

wer

e ro

aste

d (1

40

Cfo

r 15

m

inut

es i

n a

preh

eate

d ci

rcul

atin

g ho

t ai

r ov

en).

Extra

cts

wer

e m

ade

and

tota

l pro

tein

co

nten

t w

as

mea

sure

d us

ing

Brad

ford

an

alys

is.

SDS-

PAG

E w

as u

sed

to v

isua

lize

the

prot

ein

band

s pa

ttern

s an

d th

e co

nten

t of

Ara

h1, A

rah2

, Ara

h3, a

nd A

rah6

was

qu

antif

ied

usin

g rp

-HPL

C. A

sol

id p

hase

IgE

bind

ing

test

was

app

lied

for

eval

uatio

n of

al

lerg

enic

pot

ency

.An

extra

ct f

rom

Virg

inia

pe

anut

s(ra

w, G

reer

, Len

oir

NC

, USA

) w

as

used

as in

-hou

sere

fere

nce

(IHR

).

Res

ults

: Pr

otei

n co

ncen

tratio

n in

va

rieta

l ex

tract

s va

ried

from

13.

9 to

21.

2 m

g/m

l, an

d ro

astin

g re

duce

d th

e ex

tract

abilit

y by

22

to 5

0%.

Band

pa

ttern

s in

dica

ted

that

th

e fo

ur

alle

rgen

s of

in

tere

st

wer

e pr

esen

t in

sa

mpl

es

of

all

varie

ties.

Th

e ra

nges

of

in

divi

dual

alle

rgen

s in

the

tes

ted

sam

ples

w

ere

as fo

llow

s: A

rah1

: 11-

32%

; Ara

h2: 7

-16

%; A

rah3

: 38-

76%

; and

Ara

h6: 4

-14%

. In

extra

cts

of

roas

ted

pean

uts,

th

e re

lativ

e co

nten

t of A

rah1

was

low

er w

hile

that

of A

rah3

was

hig

her

com

pare

d to

non

-roas

ted.

Th

e re

lativ

e co

nten

t of

Ara

h2 a

nd A

rah6

di

d no

t var

y be

twee

n ra

w a

nd r

oast

ed. T

he

alle

rgen

ic

pote

ncie

s co

mpa

red

to in

-hou

se

refe

renc

e ex

tract

s va

ried

from

99

to 1

39%

fo

r the

raw

sam

ples

, and

roas

ting

incr

ease

d th

e po

tenc

y on

ly m

argi

nally

with

an

incr

ease

of

14

to

30

%

rela

tive

to

non-

roas

ted

coun

terp

arts

.

Figu

re 1

: SD

S-PA

GE

of th

e va

rious

pea

nut s

ampl

es

and

purif

ied

pean

ut a

llerg

en re

fere

nces

A. P

eanu

t sam

ple

anal

ysed

und

er re

duci

ng c

ondi

tions

. B

. Pea

nut s

ampl

e an

alys

ed u

nder

non

-redu

cing

con

ditio

ns.

1. B

io-R

ad M

olec

ular

wei

ght m

arke

r (kD

a)

2. L

aem

mlis

ampl

e bu

ffer

3. R

unne

r; G

AO6G

Run

ner (

Raw

) 4.

Run

ner;

Tiftg

uard

Run

ner (

Raw

)5.

Run

ner;

Flor

unne

r4/1

4 (R

aw)

6. R

unne

r; Fl

orun

ner4

/14

(Roa

sted

) 7.

Spa

nish

; Olin

Spa

nish

(Raw

) 8.

Spa

nish

; Olin

Spa

nish

(Roa

sted

) 9.

Val

encia

; Val

enci

a A

(Raw

) 10

. Val

enci

a; V

alen

cia

A (R

oast

ed)

11. V

irgin

ia; N

c-v1

1 Vi

rgin

ia (R

aw)

12. V

irgin

ia; N

c-v1

1 Vi

rgin

ia (R

oast

ed)

13. V

irgin

ia; G

rego

ry V

irgin

ia (R

aw)

14. V

irgin

ia; B

rant

ley

Virg

inia

(Raw

)

Purif

ied

alle

rgen

s Ar

ah1

, Ara

h2, A

rah3

, and

Ara

h6 w

ere

used

for b

and

assi

gnm

ent (

right

mar

gin)

.

A. B.

5 10

15

A215 (mAU)

Tim

e (m

in)

0

300

600

900

1200

5 10

15

A215 (mAU)

Tim

e (m

in)

B.

A. 800 0

0,0

25,0

50,0

75,0

100,

0 0,01

0,

10

1,00

10

,00

Inhibition (%)

Inhi

bito

r con

cent

ratio

n (µ

g/m

l)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

or p

erce

ived

conf

licts

of in

tere

st:

the

pres

ente

r was

an

empl

oyee

of H

AL A

llerg

ydu

ring

whe

nth

isre

sear

ch w

as c

ondu

cted

EAAC

I Con

gres

s 20

12

S.J.

Kop

pelm

an1,

2 , D

. Apo

stol

ovic

1 , H

. War

men

hove

n1, D

. Ver

bart

1 , S.

L. T

aylo

r2, T

. Isl

eib3

and

S. M

alek

i3

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er w

as a

n em

ploy

ee o

f HAL

Alle

rgy

whe

n th

is re

sear

ch w

as c

ondu

cted

.

Co m p aris o n o f a nt i b o d y - b a s e d a ss ay w i t h p h y sic o c h e mic a l a ss ay s f o r m o ni t o ring t h e s t a b il i t y o f a l u m - a ds o r b e d mi t e a l l e rg o i d

24

D. Luykx, E. Kerkvliet, J. de Bruijn, N. Sinnige, R. van den Hout.

HAL Allergy BV, Leiden, The Netherlands.

BackgroundAn IgG inhibition assay has been developed previously to monitor the stability of alum-adsorbed mite allergoids in a mite allergy vaccine (ALU-M). In addition, two physicochemical methods have been developed to monitor the structural stability of the mite vaccine. These methods concern circular dichroism (CD) and front-face fluorescence spectroscopy (FF-fluorescence). In order to find out whether there is a relation between the antibody-based assay and physicochemical methods, temperature-stressed samples were analysed with both types of methods and the results were compared.

MethodsALU-M: D. pteronyssinus and D. farinae mite extracts were modified with glutaraldehyde and adsorbed onto aluminium hydroxide. After adsorption the species were mixed. IgG inhibition assay: Mite allergoid specific IgG antibodies were used to determine the 50% IgG inhibition value. CD: Far-UV CD spectra were recorded from 195 to 260 nm while stirring the sample suspension. FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm (excitation at 280 nm).

ResultsStorage of the ALU-M vaccine at various elevated temperatures resulted in increased IgG inhibition values and spectral alterations with CD and FF-fluorescence, meaning structural alterations of the adsorbed protein. The increase of the 50% IgG inhibition value indicates a reduction of IgG binding to the epitopes. The spectral alterations observed with CD indicate partial loss of α-helices and increase of β-structure at higher temperatures. The spectral alterations observed with FF-fluorescence indicate formation of a more rigid protein structure at higher temperatures. When the temperature stress stability data obtained with the three methods were compared, a relation was observed between the IgG inhibition assay and CD assay by means of the CD-ratio 207/222 nm. Samples stored at elevated temperatures showed a similar trend with respect to IgG inhibition and CD ratio; both values increased in time.

ConclusionBy performing stress stability studies on ALU-M it was shown that the IgG inhibition assay and physicochemical assays were stability indicating. The results obtained with the IgG inhibition assay and CD assay relate well suggesting a relation between the IgG binding and secondary protein structure of the adsorbed mite allergoid.

EAACI, 16-20 June 2012, GenevaAbstract number: 278, Session date and time: Monday 18 June; 10:45 - 12:15 Session title: PDS 5 - Immunotherapy: vaccines and mechanisms

25

PDS 5 - Immunotherapy: vaccines and mechanisms

Co m p aris o n o f a nt i b o d y - b a s e d a ss ay w i t h p h y sic o c h e mic a l a ss ay s f o r m o ni t o ring t h e s t a b il i t y o f a l u m - a ds o r b e d mi t e a l l e rg o i d

278

-Com

paris

on o

f ant

ibod

y-ba

sed

assa

y w

ith p

hysi

coch

emic

al a

ssay

s fo

r mon

itorin

g th

e st

abili

ty o

f alu

m-a

dsor

bed

mite

alle

rgoi

dD.

Luy

kx, E

. Ker

kvlie

t, J.

de

Brui

jn, N

. Sin

nige

and

R. v

an d

en H

out

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Back

grou

nd &

Aim

:An

IgG

inhi

bitio

n as

say

has

been

dev

elop

ed p

revi

ousl

y to

mon

itor

the

stab

ility

of a

lum

-ads

orbe

d m

ite a

llerg

oids

in a

mite

alle

rgy

vacc

ine

(ALU

-M

). In

add

ition,

two

phys

icoc

hem

ical

met

hods

hav

e be

en d

evel

oped

to

mon

itor

the

stru

ctur

al s

tabi

lity o

f th

e m

ite v

acci

ne:

circ

ular

dich

roism

(CD)

and

fro

nt-fa

ce f

luor

esce

nce

spec

trosc

opy

(FF-

fluor

esce

nce)

. In

or

der t

o fin

d ou

t whe

ther

ther

e is

a re

latio

n be

twee

n th

e an

tibod

y-ba

sed

assa

y an

d ph

ysic

oche

mica

l m

etho

ds,

tem

pera

ture

-stre

ssed

sam

ples

we

re a

nalys

ed

with

bot

h ty

pes

of m

etho

ds a

nd t

he r

esul

ts w

ere

com

pare

d.

Met

hods

:AL

U-M

: D. p

tero

nyss

inus

and

D. fa

rinae

mite

ext

ract

s we

re s

epar

atel

y m

odifi

ed w

ith g

luta

rald

ehyd

ean

d ad

sorb

ed o

nto

alum

iniu

m h

ydro

xide

. Af

terw

ards

the

two

spec

ies

were

mix

ed.

Stre

ss c

ondi

tions

: ALU

-M w

as s

tore

d at

5 o

r 37º

C fo

r sev

eral

mon

ths,

at

50ºC

for s

ever

al d

ays,

or a

t 90º

C fo

r 6 h

ours

.

IgG

inhi

bitio

n as

say :

Mite

alle

rgoi

dsp

ecifi

c Ig

Gan

tibod

ies

were

use

d to

de

term

ine

the

50%

IgG

inhi

bitio

n va

lue.

CD: F

ar-U

V CD

spe

ctra

wer

e re

cord

ed fr

om 1

95 to

260

nm

and

und

er

stirr

ing

cond

itions

of

the

sam

ple.

The

CD

ratio

207

/222

nm

wa

s m

onito

red.

FF-fl

uore

scen

ce:

Fluo

resc

ence

em

issio

n sp

ectra

wer

e re

cord

ed f

rom

29

0 to

400

nm

with

exc

itatio

n at

280

nm

. The

fluo

resc

ence

inte

nsity

at

326

nm w

as m

onito

red.

Resu

lts:

The

stab

ility

of A

LU-M

was

stu

died

by

mon

itorin

g th

e 50

% Ig

Gin

hibi

tion

beha

viou

r, CD

-ratio

207

/222

nm

and

FF-

fluor

esce

nce

inte

nsity

at

326

nm a

t var

ious

tem

pera

ture

s fo

r diff

eren

t per

iods

of t

ime

(Fig

. 1an

d 2)

. Th

e an

tibod

y-ba

sed

assa

y an

d ph

ysic

oche

mica

l ass

ays

did

not

show

an

y ch

ange

s wh

en A

LU-M

was

sto

red

at 5

ºC fo

r a p

erio

d of

1 y

ear (

Fig.

1)

. St

orag

e of

the

ALU

-M v

acci

ne a

t 37

ºC r

esul

ted

in in

crea

sed

IgG

inhi

bitio

n va

lues

and

spe

ctra

l alte

ratio

ns w

ith C

D an

d FF

-fluo

resc

ence

(F

ig.

1),

mea

ning

stru

ctur

al a

ltera

tions

of

the

adso

rbed

pro

tein

. Th

e in

crea

se o

f th

e 50

% I

gGin

hibi

tion

valu

e in

dica

tes

a re

duct

ion

of I

gGbi

ndin

g to

the

epi

tope

s. T

he s

pect

ral

alte

ratio

ns o

bser

ved

with

CD

in

dica

te p

artia

l los

s of

-h

elic

es a

nd in

crea

se o

f -s

truct

ure

at h

ighe

r te

mpe

ratu

res

(incr

ease

of

CD

ra

tio

207/

222

nm).

The

spec

tral

alte

ratio

ns o

bser

ved

with

FF-

fluor

esce

nce

indi

cate

form

atio

n of

a m

ore

rigid

pro

tein

stru

ctur

e at

hig

her t

empe

ratu

res

(incr

ease

of f

luor

esce

nce

at 3

26 n

m).

Com

paris

on o

f th

e re

sults

of

the

thre

e di

ffere

nt a

ssay

s ob

tain

ed a

t 37

ºC in

dica

te t

hat

the

antib

ody-

base

d an

d CD

ass

ay a

re

mor

e se

nsitiv

e to

de

tect

st

ruct

ural

ch

ange

s fo

r AL

U-M

th

an

FF-

fluor

esce

nce

(i.e.

, afte

r 1 m

onth

). Th

e sa

me

hold

s wh

en th

e va

ccin

e is

stor

ed a

t 50

ºC f

or a

few

day

s (F

ig.

2).

In t

his

case

the

flu

ores

cenc

e in

tens

ity f

irst

decr

ease

s in

tim

e be

fore

it in

crea

ses

agai

n. A

ppar

ently

, th

e co

nditi

ons

at 5

0ºC

quen

ch th

e flu

ores

cenc

e si

gnal

.

Whe

n th

e te

mpe

ratu

re s

tress

sta

bility

dat

a ob

tain

ed w

ith t

he t

hree

m

etho

ds w

ere

com

pare

d, a

rel

atio

n is

obs

erve

d be

twee

n th

e Ig

Gin

hibi

tion

assa

y an

d CD

ass

ay b

y m

eans

of t

he C

D-ra

tio 2

07/2

22 n

m

(Fig

. 3).

Sam

ples

sto

red

at e

leva

ted

tem

pera

ture

s (i.

e., 3

7, 5

0 an

d 90

ºC)

show

ed a

sim

ilar t

rend

with

resp

ect t

o Ig

Gin

hibi

tion

and

CD ra

tio. T

he

data

of t

hese

ass

ays

supp

ort e

ach

othe

r ind

icat

ing

that

bot

h as

says

can

be

use

d as

sta

bilit

y in

dica

ting

assa

y fo

r ALU

-M.

Conc

lusi

on:

By p

erfo

rmin

g st

ress

sta

bility

stu

dies

on

ALU

-M it

was

sho

wn th

at th

e Ig

Gin

hibi

tion

assa

y an

d ph

ysico

chem

ical

assa

ys

were

st

abilit

y in

dica

ting.

The

res

ults

obt

aine

d wi

th t

he I

gGin

hibi

tion

assa

y an

d CD

as

say

rela

te w

ell s

ugge

stin

g a

rela

tion

betw

een

the

IgG

bind

ing

and

seco

ndar

y pr

otei

n st

ruct

ure

of th

e ad

sorb

ed m

ite a

llerg

oid.

Bot

h as

says

we

re s

hown

to b

e m

ore

sens

itive

than

the

FF-fl

uore

scen

ce a

ssay

.

Figu

re

1:

(A)

50%

Ig

Gin

hibi

tion

valu

es,

(B)

CD

ra

tio 2

07/2

22 n

m a

nd (C

) FF-

fluor

esce

nce

inte

nsity

of

AL

U-M

sto

red

at 5

ºC (

blue

) or

37º

C(g

reen

) fo

r se

vera

l m

onth

s.

Figu

re 2

: (A

) 50

% I

gGin

hibi

tion

valu

es,

(B)

CD

rat

io 2

07/2

22 n

m a

nd (

C)

FF-

fluor

esce

nce

inte

nsity

of A

LU-M

sto

red

at 5

0ºC

(red

) for

sev

eral

day

s.

Figu

re 3

: 50%

IgG

inhi

bitio

n da

ta v

sC

D r

atio

207

/222

nm

da

ta o

btai

ned

afte

r st

orag

e of

ALU

-M a

t 5º

C(b

lue)

37º

C

(gre

en),

50ºC

(red

) and

90º

C

(yel

low

).

0

500

1000

1500

2000

2500

3000

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

50% IgG inhibition (AUeq/ml)

5°C

37°C

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

CD ratio

5°C

37°C

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

FF fluorescence (x1E7)

5°C

37°C

50°C

0

500

1000

1500

2000

2500

3000

02

46

810

Stor

age

time

in d

ays

50 % IgG inhibition(AUeq/ml)

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

02

46

810

Stor

age

time

in d

ays

CD ratio

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

02

46

810

Stor

age

time

in d

ays

FF fluoresence (x1E7)

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

050

010

0015

0020

0025

0030

0035

0040

00

50%

IgG

Inhi

bitio

n (A

Ueq/

ml)

CD ratio

5°C

37°C

50°C

1000

0

1.50

90° °°°C

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

050

010

0015

0020

0025

0030

0035

0040

00

50%

IgG

Inhi

bitio

n (A

Ueq/

ml)

CD ratio

5°C

37°C

50°C

1000

0

1.50

90° °°°C

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

278

-Com

paris

on o

f ant

ibod

y-ba

sed

assa

y w

ith p

hysi

coch

emic

al a

ssay

s fo

r mon

itorin

g th

e st

abili

ty o

f alu

m-a

dsor

bed

mite

alle

rgoi

dD.

Luy

kx, E

. Ker

kvlie

t, J.

de

Brui

jn, N

. Sin

nige

and

R. v

an d

en H

out

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Back

grou

nd &

Aim

:An

IgG

inhi

bitio

n as

say

has

been

dev

elop

ed p

revi

ousl

y to

mon

itor

the

stab

ility

of a

lum

-ads

orbe

d m

ite a

llerg

oids

in a

mite

alle

rgy

vacc

ine

(ALU

-M

). In

add

ition,

two

phys

icoc

hem

ical

met

hods

hav

e be

en d

evel

oped

to

mon

itor

the

stru

ctur

al s

tabi

lity o

f th

e m

ite v

acci

ne:

circ

ular

dich

roism

(CD)

and

fro

nt-fa

ce f

luor

esce

nce

spec

trosc

opy

(FF-

fluor

esce

nce)

. In

or

der t

o fin

d ou

t whe

ther

ther

e is

a re

latio

n be

twee

n th

e an

tibod

y-ba

sed

assa

y an

d ph

ysic

oche

mica

l m

etho

ds,

tem

pera

ture

-stre

ssed

sam

ples

we

re a

naly

sed

with

bot

h ty

pes

of m

etho

ds a

nd t

he r

esul

ts w

ere

com

pare

d.

Met

hods

:AL

U-M

: D. p

tero

nyss

inus

and

D. fa

rinae

mite

ext

ract

s we

re s

epar

atel

y m

odifi

ed w

ith g

luta

rald

ehyd

ean

d ad

sorb

ed o

nto

alum

iniu

m h

ydro

xide

. Af

terw

ards

the

two

spec

ies

were

mix

ed.

Stre

ss c

ondi

tions

: ALU

-M w

as s

tore

d at

5 o

r 37º

C fo

r sev

eral

mon

ths,

at

50ºC

for s

ever

al d

ays,

or a

t 90º

C fo

r 6 h

ours

.

IgG

inhi

bitio

n as

say :

Mite

alle

rgoi

dsp

ecifi

c Ig

Gan

tibod

ies

were

use

d to

de

term

ine

the

50%

IgG

inhi

bitio

n va

lue.

CD: F

ar-U

V CD

spe

ctra

wer

e re

cord

ed fr

om 1

95 to

260

nm

and

und

er

stirr

ing

cond

itions

of

the

sam

ple.

The

CD

ratio

207

/222

nm

wa

s m

onito

red.

FF-fl

uore

scen

ce:

Fluo

resc

ence

em

issio

n sp

ectra

wer

e re

cord

ed f

rom

29

0 to

400

nm

with

exc

itatio

n at

280

nm

. The

fluo

resc

ence

inte

nsity

at

326

nm w

as m

onito

red.

Resu

lts:

The

stab

ility

of A

LU-M

was

stu

died

by

mon

itorin

g th

e 50

% Ig

Gin

hibi

tion

beha

viou

r, C

D-ra

tio 2

07/2

22 n

m a

nd F

F-flu

ores

cenc

e in

tens

ity a

t 32

6 nm

at v

ario

us te

mpe

ratu

res

for d

iffer

ent p

erio

ds o

f tim

e (F

ig. 1

and

2).

The

antib

ody-

base

d as

say

and

phys

icoc

hem

ical a

ssay

s di

d no

t sh

ow

any

chan

ges

when

ALU

-M w

as s

tore

d at

5ºC

for a

per

iod

of 1

yea

r (Fi

g.

1).

Stor

age

of t

he A

LU-M

vac

cine

at

37ºC

res

ulte

d in

incr

ease

d Ig

Gin

hibi

tion

valu

es a

nd s

pect

ral a

ltera

tions

with

CD

and

FF-fl

uore

scen

ce

(Fig

. 1)

, m

eani

ng s

truct

ural

alte

ratio

ns o

f th

e ad

sorb

ed p

rote

in.

The

incr

ease

of

the

50%

IgG

inhi

bitio

n va

lue

indi

cate

s a

redu

ctio

n of

IgG

bind

ing

to t

he e

pito

pes.

The

spe

ctra

l al

tera

tions

obs

erve

d wi

th C

D

indi

cate

par

tial l

oss

of

-hel

ices

and

incr

ease

of

-stru

ctur

e at

hig

her

tem

pera

ture

s (in

crea

se

of

CD

ratio

20

7/22

2 nm

). Th

e sp

ectra

l al

tera

tions

obs

erve

d wi

th F

F-flu

ores

cenc

e in

dica

te fo

rmat

ion

of a

mor

e rig

id p

rote

in s

truct

ure

at h

ighe

r tem

pera

ture

s (in

crea

se o

f flu

ores

cenc

e at

326

nm

). Co

mpa

rison

of

the

resu

lts o

f th

e th

ree

diffe

rent

ass

ays

obta

ined

at

37ºC

indi

cate

tha

t th

e an

tibod

y-ba

sed

and

CD a

ssay

are

m

ore

sens

itive

to

dete

ct

stru

ctur

al

chan

ges

for

ALU

-M

than

FF

-flu

ores

cenc

e (i.

e., a

fter 1

mon

th).

The

sam

e ho

lds

when

the

vacc

ine

is st

ored

at

50ºC

for

a f

ew d

ays

(Fig

. 2)

. In

thi

s ca

se t

he f

luor

esce

nce

inte

nsity

firs

t de

crea

ses

in t

ime

befo

re it

incr

ease

s ag

ain.

App

aren

tly,

the

cond

ition

s at

50º

C qu

ench

the

fluor

esce

nce

sign

al.

Whe

n th

e te

mpe

ratu

re s

tress

sta

bility

dat

a ob

tain

ed w

ith t

he t

hree

m

etho

ds w

ere

com

pare

d, a

rel

atio

n is

obs

erve

d be

twee

n th

e Ig

Gin

hibi

tion

assa

y an

d CD

ass

ay b

y m

eans

of t

he C

D-ra

tio 2

07/2

22 n

m

(Fig

. 3).

Sam

ples

sto

red

at e

leva

ted

tem

pera

ture

s (i.

e., 3

7, 5

0 an

d 90

ºC)

show

ed a

sim

ilar t

rend

with

resp

ect t

o Ig

Gin

hibi

tion

and

CD ra

tio. T

he

data

of t

hese

ass

ays

supp

ort e

ach

othe

r ind

icat

ing

that

bot

h as

says

can

be

use

d as

sta

bilit

y in

dica

ting

assa

y fo

r ALU

-M.

Conc

lusi

on:

By p

erfo

rmin

g st

ress

sta

bility

stu

dies

on

ALU

-M it

was

sho

wn th

at th

e Ig

Gin

hibi

tion

assa

y an

d ph

ysico

chem

ical

assa

ys

were

st

abilit

y in

dica

ting.

The

res

ults

obt

aine

d wi

th t

he I

gGin

hibi

tion

assa

y an

d CD

as

say

rela

te w

ell s

ugge

stin

g a

rela

tion

betw

een

the

IgG

bind

ing

and

seco

ndar

y pr

otei

n st

ruct

ure

of th

e ad

sorb

ed m

ite a

llerg

oid.

Bot

h as

says

we

re s

hown

to b

e m

ore

sens

itive

than

the

FF-fl

uore

scen

ce a

ssay

.

Figu

re

1:

(A)

50%

Ig

Gin

hibi

tion

valu

es,

(B)

CD

ra

tio 2

07/2

22 n

m a

nd (C

) FF-

fluor

esce

nce

inte

nsity

of

AL

U-M

sto

red

at 5

ºC (

blue

) or

37º

C(g

reen

) fo

r se

vera

l m

onth

s.

Figu

re 2

: (A

) 50

% I

gGin

hibi

tion

valu

es,

(B)

CD

rat

io 2

07/2

22 n

m a

nd (

C)

FF-

fluor

esce

nce

inte

nsity

of A

LU-M

sto

red

at 5

0ºC

(red

) for

sev

eral

day

s.

Figu

re 3

: 50%

IgG

inhi

bitio

n da

ta v

sC

D r

atio

207

/222

nm

da

ta o

btai

ned

afte

r st

orag

e of

ALU

-M a

t 5º

C(b

lue)

37º

C

(gre

en),

50ºC

(red

) and

90º

C

(yel

low

).

0

500

1000

1500

2000

2500

3000

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

50% IgG inhibition (AUeq/ml)

5°C

37°C

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

CD ratio

5°C

37°C

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

FF fluorescence (x1E7)

5°C

37°C

50°C

0

500

1000

1500

2000

2500

3000

02

46

810

Stor

age

time

in d

ays

50 % IgG inhibition(AUeq/ml)

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

02

46

810

Stor

age

time

in d

ays

CD ratio

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

02

46

810

Stor

age

time

in d

ays

FF fluoresence (x1E7)

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

050

010

0015

0020

0025

0030

0035

0040

00

50%

IgG

Inhi

bitio

n (A

Ueq/

ml)

CD ratio

5°C

37°C

50°C

1000

0

1.50

90° °°°C

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

050

010

0015

0020

0025

0030

0035

0040

00

50%

IgG

Inhi

bitio

n (A

Ueq/

ml)

CD ratio

5°C

37°C

50°C

1000

0

1.50

90° °°°C

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

278

-Com

paris

on o

f ant

ibod

y-ba

sed

assa

y w

ith p

hysi

coch

emic

al a

ssay

s fo

r mon

itorin

g th

e st

abili

ty o

f alu

m-a

dsor

bed

mite

alle

rgoi

dD.

Luy

kx, E

. Ker

kvlie

t, J.

de

Brui

jn, N

. Sin

nige

and

R. v

an d

en H

out

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Back

grou

nd &

Aim

:An

IgG

inhi

bitio

n as

say

has

been

dev

elop

ed p

revi

ousl

y to

mon

itor

the

stab

ility

of a

lum

-ads

orbe

d m

ite a

llerg

oids

in a

mite

alle

rgy

vacc

ine

(ALU

-M

). In

add

ition,

two

phys

icoc

hem

ical

met

hods

hav

e be

en d

evel

oped

to

mon

itor

the

stru

ctur

al s

tabi

lity o

f th

e m

ite v

acci

ne:

circ

ular

dich

roism

(CD)

and

fro

nt-fa

ce f

luor

esce

nce

spec

trosc

opy

(FF-

fluor

esce

nce)

. In

or

der t

o fin

d ou

t whe

ther

ther

e is

a re

latio

n be

twee

n th

e an

tibod

y-ba

sed

assa

y an

d ph

ysic

oche

mica

l m

etho

ds,

tem

pera

ture

-stre

ssed

sam

ples

we

re a

nalys

ed

with

bot

h ty

pes

of m

etho

ds a

nd t

he r

esul

ts w

ere

com

pare

d.

Met

hods

:AL

U-M

: D. p

tero

nyss

inus

and

D. fa

rinae

mite

ext

ract

s we

re s

epar

atel

y m

odifi

ed w

ith g

luta

rald

ehyd

ean

d ad

sorb

ed o

nto

alum

iniu

m h

ydro

xide

. Af

terw

ards

the

two

spec

ies

were

mix

ed.

Stre

ss c

ondi

tions

: ALU

-M w

as s

tore

d at

5 o

r 37º

C fo

r sev

eral

mon

ths,

at

50ºC

for s

ever

al d

ays,

or a

t 90º

C fo

r 6 h

ours

.

IgG

inhi

bitio

n as

say :

Mite

alle

rgoi

dsp

ecifi

c Ig

Gan

tibod

ies

were

use

d to

de

term

ine

the

50%

IgG

inhi

bitio

n va

lue.

CD: F

ar-U

V CD

spe

ctra

wer

e re

cord

ed fr

om 1

95 to

260

nm

and

und

er

stirr

ing

cond

itions

of

the

sam

ple.

The

CD

ratio

207

/222

nm

wa

s m

onito

red.

FF-fl

uore

scen

ce:

Fluo

resc

ence

em

issio

n sp

ectra

wer

e re

cord

ed f

rom

29

0 to

400

nm

with

exc

itatio

n at

280

nm

. The

fluo

resc

ence

inte

nsity

at

326

nm w

as m

onito

red.

Resu

lts:

The

stab

ility

of A

LU-M

was

stu

died

by

mon

itorin

g th

e 50

% Ig

Gin

hibi

tion

beha

viou

r, CD

-ratio

207

/222

nm

and

FF-

fluor

esce

nce

inte

nsity

at

326

nm a

t var

ious

tem

pera

ture

s fo

r diff

eren

t per

iods

of t

ime

(Fig

. 1an

d 2)

. Th

e an

tibod

y-ba

sed

assa

y an

d ph

ysic

oche

mica

l ass

ays

did

not

show

an

y ch

ange

s wh

en A

LU-M

was

sto

red

at 5

ºC fo

r a p

erio

d of

1 y

ear (

Fig.

1)

. St

orag

e of

the

ALU

-M v

acci

ne a

t 37

ºC r

esul

ted

in in

crea

sed

IgG

inhi

bitio

n va

lues

and

spe

ctra

l alte

ratio

ns w

ith C

D an

d FF

-fluo

resc

ence

(F

ig.

1),

mea

ning

stru

ctur

al a

ltera

tions

of

the

adso

rbed

pro

tein

. Th

e in

crea

se o

f th

e 50

% I

gGin

hibi

tion

valu

e in

dica

tes

a re

duct

ion

of I

gGbi

ndin

g to

the

epi

tope

s. T

he s

pect

ral

alte

ratio

ns o

bser

ved

with

CD

in

dica

te p

artia

l los

s of

-h

elic

es a

nd in

crea

se o

f -s

truct

ure

at h

ighe

r te

mpe

ratu

res

(incr

ease

of

CD

ra

tio

207/

222

nm).

The

spec

tral

alte

ratio

ns o

bser

ved

with

FF-

fluor

esce

nce

indi

cate

form

atio

n of

a m

ore

rigid

pro

tein

stru

ctur

e at

hig

her t

empe

ratu

res

(incr

ease

of f

luor

esce

nce

at 3

26 n

m).

Com

paris

on o

f th

e re

sults

of

the

thre

e di

ffere

nt a

ssay

s ob

tain

ed a

t 37

ºC in

dica

te t

hat

the

antib

ody-

base

d an

d CD

ass

ay a

re

mor

e se

nsitiv

e to

de

tect

st

ruct

ural

ch

ange

s fo

r AL

U-M

th

an

FF-

fluor

esce

nce

(i.e.

, afte

r 1 m

onth

). Th

e sa

me

hold

s wh

en th

e va

ccin

e is

stor

ed a

t 50

ºC f

or a

few

day

s (F

ig.

2).

In t

his

case

the

flu

ores

cenc

e in

tens

ity f

irst

decr

ease

s in

tim

e be

fore

it in

crea

ses

agai

n. A

ppar

ently

, th

e co

nditi

ons

at 5

0ºC

quen

ch th

e flu

ores

cenc

e si

gnal

.

Whe

n th

e te

mpe

ratu

re s

tress

sta

bility

dat

a ob

tain

ed w

ith t

he t

hree

m

etho

ds w

ere

com

pare

d, a

rel

atio

n is

obs

erve

d be

twee

n th

e Ig

Gin

hibi

tion

assa

y an

d CD

ass

ay b

y m

eans

of t

he C

D-ra

tio 2

07/2

22 n

m

(Fig

. 3).

Sam

ples

sto

red

at e

leva

ted

tem

pera

ture

s (i.

e., 3

7, 5

0 an

d 90

ºC)

show

ed a

sim

ilar t

rend

with

resp

ect t

o Ig

Gin

hibi

tion

and

CD ra

tio. T

he

data

of t

hese

ass

ays

supp

ort e

ach

othe

r ind

icat

ing

that

bot

h as

says

can

be

use

d as

sta

bilit

y in

dica

ting

assa

y fo

r ALU

-M.

Conc

lusi

on:

By p

erfo

rmin

g st

ress

sta

bility

stu

dies

on

ALU

-M it

was

sho

wn th

at th

e Ig

Gin

hibi

tion

assa

y an

d ph

ysico

chem

ical

assa

ys

were

st

abilit

y in

dica

ting.

The

res

ults

obt

aine

d wi

th t

he I

gGin

hibi

tion

assa

y an

d CD

as

say

rela

te w

ell s

ugge

stin

g a

rela

tion

betw

een

the

IgG

bind

ing

and

seco

ndar

y pr

otei

n st

ruct

ure

of th

e ad

sorb

ed m

ite a

llerg

oid.

Bot

h as

says

we

re s

hown

to b

e m

ore

sens

itive

than

the

FF-fl

uore

scen

ce a

ssay

.

Figu

re

1:

(A)

50%

Ig

Gin

hibi

tion

valu

es,

(B)

CD

ra

tio 2

07/2

22 n

m a

nd (C

) FF-

fluor

esce

nce

inte

nsity

of

AL

U-M

sto

red

at 5

ºC (

blue

) or

37º

C(g

reen

) fo

r se

vera

l m

onth

s.

Figu

re 2

: (A

) 50

% I

gGin

hibi

tion

valu

es,

(B)

CD

rat

io 2

07/2

22 n

m a

nd (

C)

FF-

fluor

esce

nce

inte

nsity

of A

LU-M

sto

red

at 5

0ºC

(red

) for

sev

eral

day

s.

Figu

re 3

: 50%

IgG

inhi

bitio

n da

ta v

sC

D r

atio

207

/222

nm

da

ta o

btai

ned

afte

r st

orag

e of

ALU

-M a

t 5º

C(b

lue)

37º

C

(gre

en),

50ºC

(red

) and

90º

C

(yel

low

).

0

500

1000

1500

2000

2500

3000

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

50% IgG inhibition (AUeq/ml)

5°C

37°C

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

CD ratio

5°C

37°C

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

01

23

45

67

89

1011

12St

orag

e tim

e in

mon

ths

FF fluorescence (x1E7)

5°C

37°C

50°C

0

500

1000

1500

2000

2500

3000

02

46

810

Stor

age

time

in d

ays

50 % IgG inhibition(AUeq/ml)

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

02

46

810

Stor

age

time

in d

ays

CD ratio

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

02

46

810

Stor

age

time

in d

ays

FF fluoresence (x1E7)

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

050

010

0015

0020

0025

0030

0035

0040

00

50%

IgG

Inhi

bitio

n (A

Ueq/

ml)

CD ratio

5°C

37°C

50°C

1000

0

1.50

90° °°°C

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

050

010

0015

0020

0025

0030

0035

0040

00

50%

IgG

Inhi

bitio

n (A

Ueq/

ml)

CD ratio

5°C

37°C

50°C

1000

0

1.50

90° °°°C

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12In

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

C h ara c t e ris a t i o n o f p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s

26

D. Luykx (1), J. de Bruijn (1), J. Cordewener (2), T. America (2), R. van den Hout (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Plant Research International, Wageningen, The Netherlands.

BackgroundBirch and grass allergoids are drug substances for birch and grass allergy vaccines, respectively. Until now these allergoids have been poorly characterised due to their complexity. Nevertheless, a better characterisation is needed. This includes identification of the relevant allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures. Several physicochemical techniques were tested for their suitability to analyse these characteristics in the allergoid and corresponding allergen extract: Mass spectrometry (MS), SDS-PAGE, HPLC-SEC, lysine determination and fluorescence spectroscopy.

MethodsAllergoids: Allergen extracts from birch and grasses were treated with glutaraldehyde to obtain birch and grass allergoids, respectively. MS: Preparation of tryptic digests and peptide separation via nano-LC before electrospray ionisation. Ionised peptides were fragmented revealing sequences. SDS-PAGE: 10-20% gels with Coomassie Blue staining and reduced samples. HPLC: GF250 and GF450 SEC columns were used combined with UV-detection. Lysine determination: Samples were hydrolysed into amino acids followed by free lysine determination with HPLC. Fluorescence: Emission spectra were recorded from 290-400 nm, with excitation at 280 nm.

ResultsSeveral isoforms of relevant birch allergen Bet v1 were identified in the birch allergoid with MS. In the grass allergoid relevant grass allergens group 1 and 5 were identified. SDS-PAGE showed for both allergoids the formation of various high molecular weight molecules including masses ≥250kDa. HPLC-SEC showed for both allergoids the formation of molecules ≥670 kDa. For both pollen extracts a minority of HPLC peaks (6 and 9%) was corresponding to proteins with a molecular mass >44 kDa. For the allergoids the majority of peaks (76 and 75%) was corresponding to proteins with a molecular mass (far) >44 kDa. Determination of free lysines showed that the majority of the lysines were modified in both pollen allergoids (73 and 74%). Fluorescence indicated hydrophobic tertiary protein structures after modification of the pollen extracts.

ConclusionApplying a combination of physicochemical techniques was shown to be a suitable approach to characterise the pollen allergoids well: Identification of the relevant allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures were accomplished.

EAACI, 16-20 June 2012, GenevaAbstract number: 287, Session date and time: Monday 18 June; 10:45 - 12:15 Session title: PDS 5 - Immunotherapy: vaccines and mechanisms

27

PDS 5 - Immunotherapy: vaccines and mechanisms

C h ara c t e ris a t i o n o f p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s

287

-Cha

ract

eris

atio

n of

pol

len

alle

rgoi

dsw

ith p

hysi

coch

emic

al te

chni

ques

D. L

uykx

1 , J.

de

Brui

jn1 1, J

. Cor

dew

ener

2

2

, T. A

mer

ica2

and

R. v

an d

en H

out1

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Plan

t Res

earc

h In

tern

atio

nal,

Wag

enin

gen,

The

Net

herla

nds

Back

grou

nd &

Aim

:Bi

rch

and

gras

s al

lerg

oids

are

used

as

drug

sub

stan

ces

for b

irch

and

gras

s al

lerg

y va

ccin

es, r

espe

ctive

ly. U

ntil

now

thes

e al

lerg

oids

have

bee

n po

orly

ch

arac

teris

ed d

ue to

thei

r co

mpl

exity

. A b

ette

r ch

arac

teris

atio

nis

need

ed.

This

inc

lude

s id

entif

icat

ion

of t

he m

ajor

alle

rgen

s, d

eter

min

atio

n of

the

de

gree

of p

olym

eris

atio

n an

d cr

oss-

linki

ng, a

nd in

vest

igat

ion

of th

e pr

otei

n st

ruct

ures

. Se

vera

l ph

ysic

oche

mica

l te

chni

ques

wer

e ap

plie

d to

ana

lyse

th

ese

char

acte

ristic

s in

thr

ee s

ucce

ssiv

e, s

epar

atel

y pr

oduc

ed b

irch

and

gras

s al

lerg

oids

and

corre

spon

ding

alle

rgen

ext

ract

s: M

ass

spec

trom

etry

(M

S),

SDS-

PAG

E,

HPL

C-S

EC,

lysin

e de

term

inat

ion

and

fluor

esce

nce

spec

trosc

opy.

Met

hods

:Al

lerg

oids

: Alle

rgen

ext

ract

s fro

m b

irch

and

mixe

d gr

ass

polle

n we

re tr

eate

d wi

th g

luta

rald

ehyd

eto

obt

ain

birc

h an

d gr

ass

alle

rgoi

ds, r

espe

ctiv

ely.

MS:

Firs

t try

ptic

dige

sts

were

pre

pare

d fro

m th

e sa

mpl

es. S

ubse

quen

tly, t

he

pept

ides

wer

e se

para

ted

with

nan

o-liq

uid

chro

mat

ogra

phy

befo

re e

nter

ing

the

MS

inst

rum

ent

via

elec

trosp

ray

ioni

satio

n.

Ioni

sed

pept

ides

we

re

fragm

ente

d re

veal

ing

amin

o ac

id s

eque

nces

(i.e

., id

entif

icatio

n).

SDS-

PAG

E: R

educ

ed s

ampl

es w

ere

appl

ied

onto

10-

20%

pol

yacr

ylam

ide

gels

and

sta

ined

with

Coo

mas

sie

Blue

.

HPL

C-S

EC:

A G

F250

or

GF4

50 s

ize e

xclu

sion

chr

omat

ogra

phy

(SEC

) co

lum

n wa

s us

ed c

ombi

ned

with

UV-

dete

ctio

n (2

15 n

m).

Lysi

ne d

eter

min

atio

n : S

ampl

es w

ere

hydr

olys

ed in

to a

min

o ac

ids

follo

wed

by l

ysin

e de

term

inat

ion

with

HPL

C. B

y de

term

inin

g th

e am

ount

of

free

lysi

nes

in t

he e

xtra

ct a

nd a

llerg

oid,

the

am

ount

of m

odifi

ed ly

sine

sin

the

al

lerg

oid

can

be c

alcu

late

d.

Fluo

resc

ence

spe

ctro

scop

y : E

mis

sion

spec

tra w

ere

reco

rded

from

290

-400

nm

with

exc

itatio

n at

280

nm

.

Resu

lts:

MS:

Sev

eral

isof

orm

sof

the

maj

or b

irch

alle

rgen

Bet

v1

were

iden

tifie

d in

th

e th

ree

birc

h al

lerg

oids

(a, b

, c, d

/h, f

/i). N

ext t

o th

e m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5, a

lso

grou

p 2,

3, 4

and

6 a

llerg

ens

were

iden

tifie

d in

the

thre

e gr

ass

alle

rgoi

ds(d

ata

not s

hown

).

SDS-

PAG

E: F

or t

he t

hree

birc

h ex

tract

s, s

imila

r pr

otei

n pr

ofile

s we

re

obse

rved

(Fi

g. 1

). Th

e ba

nd c

orre

spon

ding

to

15-1

8 kD

ain

dica

tes

the

pres

ence

of m

ajor

alle

rgen

Bet

v1.

For

the

thre

e gr

ass

extra

cts

also

sim

ilar

prot

ein

prof

iles

were

obs

erve

d. H

ere,

the

band

s co

rresp

ondi

ng to

27-3

4 kD

ain

dica

te th

e pr

esen

ce o

f maj

or a

llerg

ens

grou

p 1

and

5. F

or a

ll al

lerg

oids

the

form

atio

n of

var

ious

hig

h m

olec

ular

wei

ght m

olec

ules

is s

hown

via

a s

mea

r an

d a

band

aro

und

250

kDa

(mol

ecul

es

250

kDa)

on

gel.

HPL

C-S

EC:

For

the

thre

e bi

rch

and

gras

s ex

tract

s a

min

ority

of

HPL

C

peak

s is

cor

resp

ondi

ng t

o pr

otei

ns w

ith a

mol

ecul

ar m

ass

abov

e 44

kDa

(Fig

. 2)

. Th

e ch

rom

atog

ram

s of

the

birc

h an

d gr

ass

alle

rgoi

dba

tche

s sh

owed

the

pre

senc

e of

a v

arie

ty o

f hi

gh m

olec

ular

wei

ght

mol

ecul

es

incl

udin

g m

olec

ules

with

mol

ecul

ar m

asse

s 67

0 kD

a. H

ere,

a c

onsi

sten

t m

ajor

ity

of

peak

s (7

4-77

%

for

birc

h an

d 68

-79%

fo

r gr

asse

s)

is co

rresp

ondi

ng t

o pr

otei

ns w

ith a

mol

ecul

ar m

ass

(far)

abov

e 44

kDa

show

ing

a co

nsis

tent

deg

ree

of p

rote

in p

olym

eris

atio

n (T

able

1-2

).

Lysi

ne d

eter

min

atio

n: F

or t

he b

irch

alle

rgoi

ds74

-79%

of t

he ly

sine

swe

re

mod

ified

and

for

the

gras

s al

lerg

oids

73-7

6% o

f the

lysi

nes

were

mod

ified

(Tab

le 1

-2).

This

sho

ws a

con

sist

ent d

egre

e of

pro

tein

cro

ss-li

nkin

g.

Fluo

resc

ence

spe

ctro

scop

y : M

odific

atio

n of

the

birc

h an

d gr

ass

extra

cts

resu

lted

in a

shi

ft of

the

emis

sion

max

imum

to a

lowe

r wa

vele

ngth

and

a

decr

ease

of f

luor

esce

nce

inte

nsity

(dat

a no

t sho

wn).

This

indi

cate

s th

at th

e su

rroun

ding

s of

the

arom

atic

am

ino

acid

s in

the

prot

eins

cha

nge

to a

mor

e hy

drop

hobi

c en

viro

nmen

t whi

ch is

indi

cativ

e fo

r pro

tein

pol

ymer

isat

ion.

The

em

issi

on

max

imum

va

lues

ob

tain

ed

for

the

polle

n al

lerg

oids

show

co

nsis

tenc

y as

wel

l (Ta

ble

1-2)

.

Conc

lusi

on:

Appl

ying

a c

ombi

natio

n of

phy

sicoc

hem

ical

tech

niqu

es w

as s

hown

to b

e a

suita

ble

appr

oach

to c

hara

cter

ise th

e po

llen

alle

rgoi

dswe

ll: Id

entif

icat

ion

of

the

maj

or a

llerg

ens,

det

erm

inat

ion

of t

he d

egre

e of

pol

ymer

isat

ion

and

cros

s-lin

king

, and

inve

stig

atio

n of

the

prot

ein

stru

ctur

es w

ere

acco

mpl

ishe

d.

This

stu

dy s

hows

con

sist

ency

for t

he m

odifi

catio

n of

the

polle

n ex

tract

s.

Figu

re 2

: H

PLC

-SEC

pat

tern

s (A

215

nm)

of c

alib

ratio

n st

anda

rd (

A1 a

nd B

1), t

hree

birc

h ex

trac

ts (

blue

lin

e) a

nd c

orre

spon

ding

alle

rgoi

ds(r

ed l

ine)

(A2

-A4)

, an

d th

ree

gras

s ex

trac

ts (b

lue

line)

and

cor

resp

ondi

ng a

llerg

oids

(red

line)

(B2-

B4)

. The

bla

ck v

ertic

al li

ne

in th

e ch

rom

atog

ram

spl

its p

eaks

cor

resp

ondi

ng to

mol

ecul

ar m

asse

s ab

ove

and

belo

w

44 k

Da.

A G

F250

SEC

col

umn

was

app

lied

for b

irch,

a G

F450

SEC

col

umn

for g

rass

es.

Tabl

e 1:

Phy

sico

chem

ical

cha

ract

eris

tics

of th

ree

batc

hes

of b

irch

alle

rgoi

ds

670

kDa

158

kDa44

kDa 17

kDa 1.35

kDa

Absorbance 215 nm

Rete

ntio

ntim

e (m

in)

670

kDa 15

8 kD

a44 k

Da 17 k

Da 1.35

kDa

Stan

dard

Stan

dard

Birc

h1

Birc

h2

Birc

h3

Gra

ss1

Gra

ss2

Gra

ss3

A1 A2 A3 A4

B1 B2 B3 B4

Tabl

e 2:

Phy

sico

chem

ical

cha

ract

eris

tics

of th

ree

batc

hes

of g

rass

alle

rgoi

ds

Ch

ara

cte

ris

tic

Bir

ch

1

Bir

ch

2

B

irc

h 3

HP

LC

: P

eak

are

a a

bo

ve 4

4 k

Da

(%

)

75

7

4

77

Mo

difi

ed

lysi

ne

s (%

)

74

7

9

78

Flu

ore

scen

ce e

mis

sion m

axi

mum

(nm

) 3

11

3

11

3

11

C

ha

rac

teri

sti

c

G

ras

s 1

G

ras

s 2

Gra

ss 3

HP

LC

: P

eak

are

a a

bo

ve 4

4 k

Da

(%

)

76

6

8

79

Mo

difi

ed

lysi

ne

s (%

)

73

7

3

76

Flu

ore

sce

nce

em

issi

on m

axi

mu

m (

nm

) 3

29

3

29

3

30

Figu

re 1

: SD

S-PA

GE

patte

rns

of m

olec

ular

wei

ght

mar

ker

(M),

thre

e bi

rch

(1-3

) an

d th

ree

gras

s (4

-6)

extr

acts

and

cor

resp

ondi

ng a

llerg

oids

(1-6

). M

ajor

birc

h al

lerg

en B

et v

1 an

d m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5 ar

e bo

xed.

M1

23

54

61

24

35

6

Extr

acts

Alle

rgoi

dsG

rass

Birc

hB

irch

Birc

hG

rass

Birc

hB

irch

Gra

ssG

rass

Birc

hG

rass

Birc

hG

rass

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

287

-Cha

ract

eris

atio

n of

pol

len

alle

rgoi

dsw

ith p

hysi

coch

emic

al te

chni

ques

D. L

uykx

1 , J.

de

Brui

jn1 1, J

. Cor

dew

ener

2

2

, T. A

mer

ica2

and

R. v

an d

en H

out1

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Plan

t Res

earc

h In

tern

atio

nal,

Wag

enin

gen,

The

Net

herla

nds

Back

grou

nd &

Aim

:Bi

rch

and

gras

s al

lerg

oids

are

used

as

drug

sub

stan

ces

for b

irch

and

gras

s al

lerg

y va

ccin

es, r

espe

ctive

ly. U

ntil

now

thes

e al

lerg

oids

have

bee

n po

orly

ch

arac

teris

ed d

ue to

thei

r co

mpl

exity

. A b

ette

r ch

arac

teris

atio

nis

need

ed.

This

inc

lude

s id

entif

icat

ion

of t

he m

ajor

alle

rgen

s, d

eter

min

atio

n of

the

de

gree

of p

olym

eris

atio

n an

d cr

oss-

linki

ng, a

nd in

vest

igat

ion

of th

e pr

otei

n st

ruct

ures

. Se

vera

l ph

ysic

oche

mica

l te

chni

ques

wer

e ap

plie

d to

ana

lyse

th

ese

char

acte

ristic

s in

thr

ee s

ucce

ssiv

e, s

epar

atel

y pr

oduc

ed b

irch

and

gras

s al

lerg

oids

and

corre

spon

ding

alle

rgen

ext

ract

s: M

ass

spec

trom

etry

(M

S),

SDS-

PAG

E,

HPL

C-S

EC,

lysin

e de

term

inat

ion

and

fluor

esce

nce

spec

trosc

opy.

Met

hods

:Al

lerg

oids

: Alle

rgen

ext

ract

s fro

m b

irch

and

mixe

d gr

ass

polle

n we

re tr

eate

d wi

th g

luta

rald

ehyd

eto

obt

ain

birc

h an

d gr

ass

alle

rgoi

ds, r

espe

ctiv

ely.

MS:

Firs

t try

ptic

dige

sts

were

pre

pare

d fro

m th

e sa

mpl

es. S

ubse

quen

tly, t

he

pept

ides

wer

e se

para

ted

with

nan

o-liq

uid

chro

mat

ogra

phy

befo

re e

nter

ing

the

MS

inst

rum

ent

via

elec

trosp

ray

ioni

satio

n.

Ioni

sed

pept

ides

we

re

fragm

ente

d re

veal

ing

amin

o ac

id s

eque

nces

(i.e

., id

entif

icatio

n).

SDS-

PAG

E: R

educ

ed s

ampl

es w

ere

appl

ied

onto

10-

20%

pol

yacr

ylam

ide

gels

and

sta

ined

with

Coo

mas

sie

Blue

.

HPL

C-S

EC:

A G

F250

or

GF4

50 s

ize e

xclu

sion

chr

omat

ogra

phy

(SEC

) co

lum

n wa

s us

ed c

ombi

ned

with

UV-

dete

ctio

n (2

15 n

m).

Lysi

ne d

eter

min

atio

n : S

ampl

es w

ere

hydr

olys

ed in

to a

min

o ac

ids

follo

wed

by l

ysin

e de

term

inat

ion

with

HPL

C. B

y de

term

inin

g th

e am

ount

of

free

lysi

nes

in t

he e

xtra

ct a

nd a

llerg

oid,

the

am

ount

of m

odifi

ed ly

sine

sin

the

al

lerg

oid

can

be c

alcu

late

d.

Fluo

resc

ence

spe

ctro

scop

y : E

mis

sion

spec

tra w

ere

reco

rded

from

290

-400

nm

with

exc

itatio

n at

280

nm

.

Resu

lts:

MS:

Sev

eral

isof

orm

sof

the

maj

or b

irch

alle

rgen

Bet

v1

were

iden

tifie

d in

th

e th

ree

birc

h al

lerg

oids

(a, b

, c, d

/h, f

/i). N

ext t

o th

e m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5, a

lso

grou

p 2,

3, 4

and

6 a

llerg

ens

were

iden

tifie

d in

the

thre

e gr

ass

alle

rgoi

ds(d

ata

not s

hown

).

SDS-

PAG

E: F

or t

he t

hree

birc

h ex

tract

s, s

imila

r pr

otei

n pr

ofile

s we

re

obse

rved

(Fi

g. 1

). Th

e ba

nd c

orre

spon

ding

to

15-1

8 kD

ain

dica

tes

the

pres

ence

of m

ajor

alle

rgen

Bet

v1.

For

the

thre

e gr

ass

extra

cts

also

sim

ilar

prot

ein

prof

iles

were

obs

erve

d. H

ere,

the

band

s co

rresp

ondi

ng to

27-3

4 kD

ain

dica

te th

e pr

esen

ce o

f maj

or a

llerg

ens

grou

p 1

and

5. F

or a

ll al

lerg

oids

the

form

atio

n of

var

ious

hig

h m

olec

ular

wei

ght m

olec

ules

is s

hown

via

a s

mea

r an

d a

band

aro

und

250

kDa

(mol

ecul

es

250

kDa)

on

gel.

HPL

C-S

EC:

For

the

thre

e bi

rch

and

gras

s ex

tract

s a

min

ority

of

HPL

C

peak

s is

cor

resp

ondi

ng t

o pr

otei

ns w

ith a

mol

ecul

ar m

ass

abov

e 44

kDa

(Fig

. 2)

. Th

e ch

rom

atog

ram

s of

the

birc

h an

d gr

ass

alle

rgoi

dba

tche

s sh

owed

the

pre

senc

e of

a v

arie

ty o

f hi

gh m

olec

ular

wei

ght

mol

ecul

es

incl

udin

g m

olec

ules

with

mol

ecul

ar m

asse

s 67

0 kD

a. H

ere,

a c

onsi

sten

t m

ajor

ity

of

peak

s (7

4-77

%

for

birc

h an

d 68

-79%

fo

r gr

asse

s)

is co

rresp

ondi

ng t

o pr

otei

ns w

ith a

mol

ecul

ar m

ass

(far)

abov

e 44

kDa

show

ing

a co

nsis

tent

deg

ree

of p

rote

in p

olym

eris

atio

n (T

able

1-2

).

Lysi

ne d

eter

min

atio

n: F

or t

he b

irch

alle

rgoi

ds74

-79%

of t

he ly

sine

swe

re

mod

ified

and

for

the

gras

s al

lerg

oids

73-7

6% o

f the

lysi

nes

were

mod

ified

(Tab

le 1

-2).

This

sho

ws a

con

sist

ent d

egre

e of

pro

tein

cro

ss-li

nkin

g.

Fluo

resc

ence

spe

ctro

scop

y : M

odific

atio

n of

the

birc

h an

d gr

ass

extra

cts

resu

lted

in a

shi

ft of

the

emis

sion

max

imum

to a

lowe

r wa

vele

ngth

and

a

decr

ease

of f

luor

esce

nce

inte

nsity

(dat

a no

t sho

wn).

This

indi

cate

s th

at th

e su

rroun

ding

s of

the

arom

atic

am

ino

acid

s in

the

prot

eins

cha

nge

to a

mor

e hy

drop

hobi

c en

viro

nmen

t whi

ch is

indi

cativ

e fo

r pro

tein

pol

ymer

isat

ion.

The

em

issi

on

max

imum

va

lues

ob

tain

ed

for

the

polle

n al

lerg

oids

show

co

nsis

tenc

y as

wel

l (Ta

ble

1-2)

.

Conc

lusi

on:

Appl

ying

a c

ombi

natio

n of

phy

sicoc

hem

ical

tech

niqu

es w

as s

hown

to b

e a

suita

ble

appr

oach

to c

hara

cter

ise th

e po

llen

alle

rgoi

dswe

ll: Id

entif

icat

ion

of

the

maj

or a

llerg

ens,

det

erm

inat

ion

of t

he d

egre

e of

pol

ymer

isat

ion

and

cros

s-lin

king

, and

inve

stig

atio

n of

the

prot

ein

stru

ctur

es w

ere

acco

mpl

ishe

d.

This

stu

dy s

hows

con

sist

ency

for t

he m

odifi

catio

n of

the

polle

n ex

tract

s.

Figu

re 2

: H

PLC

-SEC

pat

tern

s (A

215

nm)

of c

alib

ratio

n st

anda

rd (

A1 a

nd B

1), t

hree

birc

h ex

trac

ts (

blue

lin

e) a

nd c

orre

spon

ding

alle

rgoi

ds(r

ed l

ine)

(A2

-A4)

, an

d th

ree

gras

s ex

trac

ts (b

lue

line)

and

cor

resp

ondi

ng a

llerg

oids

(red

line)

(B2-

B4)

. The

bla

ck v

ertic

al li

ne

in th

e ch

rom

atog

ram

spl

its p

eaks

cor

resp

ondi

ng to

mol

ecul

ar m

asse

s ab

ove

and

belo

w

44 k

Da.

A G

F250

SEC

col

umn

was

app

lied

for b

irch,

a G

F450

SEC

col

umn

for g

rass

es.

Tabl

e 1:

Phy

sico

chem

ical

cha

ract

eris

tics

of th

ree

batc

hes

of b

irch

alle

rgoi

ds

670

kDa

158

kDa44

kDa 17

kDa 1.35

kDa

Absorbance 215 nm

Rete

ntio

ntim

e (m

in)

670

kDa 15

8 kD

a44 k

Da 17 k

Da 1.35

kDa

Stan

dard

Stan

dard

Birc

h1

Birc

h2

Birc

h3

Gra

ss1

Gra

ss2

Gra

ss3

A1 A2 A3 A4

B1 B2 B3 B4

Tabl

e 2:

Phy

sico

chem

ical

cha

ract

eris

tics

of th

ree

batc

hes

of g

rass

alle

rgoi

ds

Ch

ara

cte

ris

tic

Bir

ch

1

Bir

ch

2

B

irc

h 3

HP

LC

: P

eak

are

a a

bo

ve 4

4 k

Da

(%

)

75

7

4

77

Mo

difi

ed

lysi

ne

s (%

)

74

7

9

78

Flu

ore

scen

ce e

mis

sion m

axi

mum

(nm

) 3

11

3

11

3

11

C

ha

rac

teri

sti

c

G

ras

s 1

G

ras

s 2

Gra

ss 3

HP

LC

: P

eak

are

a a

bo

ve 4

4 k

Da

(%

)

76

6

8

79

Mo

difi

ed

lysi

ne

s (%

)

73

7

3

76

Flu

ore

sce

nce

em

issi

on m

axi

mu

m (

nm

) 3

29

3

29

3

30

Figu

re 1

: SD

S-PA

GE

patte

rns

of m

olec

ular

wei

ght

mar

ker

(M),

thre

e bi

rch

(1-3

) an

d th

ree

gras

s (4

-6)

extr

acts

and

cor

resp

ondi

ng a

llerg

oids

(1-6

). M

ajor

birc

h al

lerg

en B

et v

1 an

d m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5 ar

e bo

xed.

M1

23

54

61

24

35

6

Extr

acts

Alle

rgoi

dsG

rass

Birc

hB

irch

Birc

hG

rass

Birc

hB

irch

Gra

ssG

rass

Birc

hG

rass

Birc

hG

rass

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

287

-Cha

ract

eris

atio

n of

pol

len

alle

rgoi

dsw

ith p

hysi

coch

emic

al te

chni

ques

D. L

uykx

1 , J.

de

Brui

jn1 1, J

. Cor

dew

ener

2

2

, T. A

mer

ica2

and

R. v

an d

en H

out1

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Plan

t Res

earc

h In

tern

atio

nal,

Wag

enin

gen,

The

Net

herla

nds

Back

grou

nd &

Aim

:Bi

rch

and

gras

s al

lerg

oids

are

used

as

drug

sub

stan

ces

for b

irch

and

gras

s al

lerg

y va

ccin

es, r

espe

ctive

ly. U

ntil

now

thes

e al

lerg

oids

have

bee

n po

orly

ch

arac

teris

ed d

ue to

thei

r co

mpl

exity

. A b

ette

r ch

arac

teris

atio

nis

need

ed.

This

inc

lude

s id

entif

icat

ion

of t

he m

ajor

alle

rgen

s, d

eter

min

atio

n of

the

de

gree

of p

olym

eris

atio

n an

d cr

oss-

linki

ng, a

nd in

vest

igat

ion

of th

e pr

otei

n st

ruct

ures

. Se

vera

l ph

ysic

oche

mica

l te

chni

ques

wer

e ap

plie

d to

ana

lyse

th

ese

char

acte

ristic

s in

thr

ee s

ucce

ssiv

e, s

epar

atel

y pr

oduc

ed b

irch

and

gras

s al

lerg

oids

and

corre

spon

ding

alle

rgen

ext

ract

s: M

ass

spec

trom

etry

(M

S),

SDS-

PAG

E,

HPL

C-S

EC,

lysin

e de

term

inat

ion

and

fluor

esce

nce

spec

trosc

opy.

Met

hods

:Al

lerg

oids

: Alle

rgen

ext

ract

s fro

m b

irch

and

mixe

d gr

ass

polle

n we

re tr

eate

d wi

th g

luta

rald

ehyd

eto

obt

ain

birc

h an

d gr

ass

alle

rgoi

ds, r

espe

ctiv

ely.

MS:

Firs

t try

ptic

dige

sts

were

pre

pare

d fro

m th

e sa

mpl

es. S

ubse

quen

tly, t

he

pept

ides

wer

e se

para

ted

with

nan

o-liq

uid

chro

mat

ogra

phy

befo

re e

nter

ing

the

MS

inst

rum

ent

via

elec

trosp

ray

ioni

satio

n.

Ioni

sed

pept

ides

we

re

fragm

ente

d re

veal

ing

amin

o ac

id s

eque

nces

(i.e

., id

entif

icatio

n).

SDS-

PAG

E: R

educ

ed s

ampl

es w

ere

appl

ied

onto

10-

20%

pol

yacr

ylam

ide

gels

and

sta

ined

with

Coo

mas

sie

Blue

.

HPL

C-S

EC:

A G

F250

or

GF4

50 s

ize e

xclu

sion

chr

omat

ogra

phy

(SEC

) co

lum

n wa

s us

ed c

ombi

ned

with

UV-

dete

ctio

n (2

15 n

m).

Lysi

ne d

eter

min

atio

n : S

ampl

es w

ere

hydr

olys

ed in

to a

min

o ac

ids

follo

wed

by l

ysin

e de

term

inat

ion

with

HPL

C. B

y de

term

inin

g th

e am

ount

of

free

lysi

nes

in t

he e

xtra

ct a

nd a

llerg

oid,

the

am

ount

of m

odifi

ed ly

sine

sin

the

al

lerg

oid

can

be c

alcu

late

d.

Fluo

resc

ence

spe

ctro

scop

y : E

mis

sion

spec

tra w

ere

reco

rded

from

290

-400

nm

with

exc

itatio

n at

280

nm

.

Resu

lts:

MS:

Sev

eral

isof

orm

sof

the

maj

or b

irch

alle

rgen

Bet

v1

were

iden

tifie

d in

th

e th

ree

birc

h al

lerg

oids

(a, b

, c, d

/h, f

/i). N

ext t

o th

e m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5, a

lso

grou

p 2,

3, 4

and

6 a

llerg

ens

were

iden

tifie

d in

the

thre

e gr

ass

alle

rgoi

ds(d

ata

not s

hown

).

SDS-

PAG

E: F

or t

he t

hree

birc

h ex

tract

s, s

imila

r pr

otei

n pr

ofile

s we

re

obse

rved

(Fi

g. 1

). Th

e ba

nd c

orre

spon

ding

to

15-1

8 kD

ain

dica

tes

the

pres

ence

of m

ajor

alle

rgen

Bet

v1.

For

the

thre

e gr

ass

extra

cts

also

sim

ilar

prot

ein

prof

iles

were

obs

erve

d. H

ere,

the

band

s co

rresp

ondi

ng to

27-3

4 kD

ain

dica

te th

e pr

esen

ce o

f maj

or a

llerg

ens

grou

p 1

and

5. F

or a

ll al

lerg

oids

the

form

atio

n of

var

ious

hig

h m

olec

ular

wei

ght m

olec

ules

is s

hown

via

a s

mea

r an

d a

band

aro

und

250

kDa

(mol

ecul

es

250

kDa)

on

gel.

HPL

C-S

EC:

For

the

thre

e bi

rch

and

gras

s ex

tract

s a

min

ority

of

HPL

C

peak

s is

cor

resp

ondi

ng t

o pr

otei

ns w

ith a

mol

ecul

ar m

ass

abov

e 44

kDa

(Fig

. 2)

. Th

e ch

rom

atog

ram

s of

the

birc

h an

d gr

ass

alle

rgoi

dba

tche

s sh

owed

the

pre

senc

e of

a v

arie

ty o

f hi

gh m

olec

ular

wei

ght

mol

ecul

es

incl

udin

g m

olec

ules

with

mol

ecul

ar m

asse

s 67

0 kD

a. H

ere,

a c

onsi

sten

t m

ajor

ity

of

peak

s (7

4-77

%

for

birc

h an

d 68

-79%

fo

r gr

asse

s)

is co

rresp

ondi

ng t

o pr

otei

ns w

ith a

mol

ecul

ar m

ass

(far)

abov

e 44

kDa

show

ing

a co

nsis

tent

deg

ree

of p

rote

in p

olym

eris

atio

n (T

able

1-2

).

Lysi

ne d

eter

min

atio

n: F

or t

he b

irch

alle

rgoi

ds74

-79%

of t

he ly

sine

swe

re

mod

ified

and

for

the

gras

s al

lerg

oids

73-7

6% o

f the

lysi

nes

were

mod

ified

(Tab

le 1

-2).

This

sho

ws a

con

sist

ent d

egre

e of

pro

tein

cro

ss-li

nkin

g.

Fluo

resc

ence

spe

ctro

scop

y : M

odific

atio

n of

the

birc

h an

d gr

ass

extra

cts

resu

lted

in a

shi

ft of

the

emis

sion

max

imum

to a

lowe

r wa

vele

ngth

and

a

decr

ease

of f

luor

esce

nce

inte

nsity

(dat

a no

t sho

wn).

This

indi

cate

s th

at th

e su

rroun

ding

s of

the

arom

atic

am

ino

acid

s in

the

prot

eins

cha

nge

to a

mor

e hy

drop

hobi

c en

viro

nmen

t whi

ch is

indi

cativ

e fo

r pro

tein

pol

ymer

isat

ion.

The

em

issi

on

max

imum

va

lues

ob

tain

ed

for

the

polle

n al

lerg

oids

show

co

nsis

tenc

y as

wel

l (Ta

ble

1-2)

.

Conc

lusi

on:

Appl

ying

a c

ombi

natio

n of

phy

sicoc

hem

ical

tech

niqu

es w

as s

hown

to b

e a

suita

ble

appr

oach

to c

hara

cter

ise th

e po

llen

alle

rgoi

dswe

ll: Id

entif

icat

ion

of

the

maj

or a

llerg

ens,

det

erm

inat

ion

of t

he d

egre

e of

pol

ymer

isat

ion

and

cros

s-lin

king

, and

inve

stig

atio

n of

the

prot

ein

stru

ctur

es w

ere

acco

mpl

ishe

d.

This

stu

dy s

hows

con

sist

ency

for t

he m

odifi

catio

n of

the

polle

n ex

tract

s.

Figu

re 2

: H

PLC

-SEC

pat

tern

s (A

215

nm)

of c

alib

ratio

n st

anda

rd (

A1 a

nd B

1), t

hree

birc

h ex

trac

ts (

blue

lin

e) a

nd c

orre

spon

ding

alle

rgoi

ds(r

ed l

ine)

(A2

-A4)

, an

d th

ree

gras

s ex

trac

ts (b

lue

line)

and

cor

resp

ondi

ng a

llerg

oids

(red

line)

(B2-

B4)

. The

bla

ck v

ertic

al li

ne

in th

e ch

rom

atog

ram

spl

its p

eaks

cor

resp

ondi

ng to

mol

ecul

ar m

asse

s ab

ove

and

belo

w

44 k

Da.

A G

F250

SEC

col

umn

was

app

lied

for b

irch,

a G

F450

SEC

col

umn

for g

rass

es.

Tabl

e 1:

Phy

sico

chem

ical

cha

ract

eris

tics

of th

ree

batc

hes

of b

irch

alle

rgoi

ds

670

kDa

158

kDa44

kDa 17

kDa 1.35

kDa

Absorbance 215 nm

Rete

ntio

ntim

e (m

in)

670

kDa 15

8 kD

a44 k

Da 17 k

Da 1.35

kDa

Stan

dard

Stan

dard

Birc

h1

Birc

h2

Birc

h3

Gra

ss1

Gra

ss2

Gra

ss3

A1 A2 A3 A4

B1 B2 B3 B4

Tabl

e 2:

Phy

sico

chem

ical

cha

ract

eris

tics

of th

ree

batc

hes

of g

rass

alle

rgoi

ds

Ch

ara

cte

ris

tic

Bir

ch

1

Bir

ch

2

B

irc

h 3

HP

LC

: P

eak

are

a a

bo

ve 4

4 k

Da

(%

)

75

7

4

77

Mo

difi

ed

lysi

ne

s (%

)

74

7

9

78

Flu

ore

scen

ce e

mis

sion m

axi

mum

(nm

) 3

11

3

11

3

11

C

ha

rac

teri

sti

c

G

ras

s 1

G

ras

s 2

Gra

ss 3

HP

LC

: P

eak

are

a a

bo

ve 4

4 k

Da

(%

)

76

6

8

79

Mo

difi

ed

lysi

ne

s (%

)

73

7

3

76

Flu

ore

sce

nce

em

issi

on m

axi

mu

m (

nm

) 3

29

3

29

3

30

Figu

re 1

: SD

S-PA

GE

patte

rns

of m

olec

ular

wei

ght

mar

ker

(M),

thre

e bi

rch

(1-3

) an

d th

ree

gras

s (4

-6)

extr

acts

and

cor

resp

ondi

ng a

llerg

oids

(1-6

). M

ajor

birc

h al

lerg

en B

et v

1 an

d m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5 ar

e bo

xed.

M1

23

54

61

24

35

6

Extr

acts

Alle

rgoi

dsG

rass

Birc

hB

irch

Birc

hG

rass

Birc

hB

irch

Gra

ssG

rass

Birc

hG

rass

Birc

hG

rass

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st: t

he p

rese

nter

is a

n em

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

C h ara c t e ris a t i o n o f a l u m - a ds o r b e d p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s

28

D. Luykx (1), J. de Bruijn (1), J. Cordewener (2), T. America (2), R. van den Hout (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) Plant Research International, Wageningen, The Netherlands.

BackgroundSeveral physicochemical techniques have been selected to investigate their suitability to characterise a birch allergy vaccine (ALU-B) and grass allergy vaccine (ALU-G). These techniques are mass spectrometry (MS), circular dichroism (CD), and front-face fluorescence (FF-fluorescence). They should be able to investigate the alum-adsorbed and modified allergens on all structural levels, i.e. primary structure (for protein identification), secondary and tertiary structure (for structural stability). Samples were temperature stressed to find out if these techniques are stability indicating.

MethodAlum-adsorbed pollen allergoids: Allergen extracts from birch and grasses were modified with glutaraldehyde and adsorbed onto aluminium hydroxide to obtain ALU-B and ALU-G, respectively. MS: Preparation of tryptic digests and peptide separation via nano-LC before electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences. CD: Far-UV CD spectra were recorded from 195 to 260 nm while stirring the sample suspension. FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm, with excitation at 280 nm.

ResultsVia MS several isoforms of relevant birch allergen Bet v1 were identified in ALU-B. In ALU-G relevant grass allergens group 1 and 5 were identified. The CD-spectrum of ALU-B and ALU-G represented protein structures consisting of mainly α-helices and ß-structure. The FF-fluorescence spectrum of both products showed an emission maximum at 326 nm suggesting hydrophobic surroundings for the aromatic amino acids present in both protein samples. Storage of ALU-B and ALU-G at several elevated temperatures resulted in spectral alterations meaning structural alterations of protein. CD spectra indicated partially loss of α-helices (increase of ratio 207/222 nm) while fluorescence spectra indicated formation of a less rigid structure (decrease of fluorescence at 326 nm).

ConclusionMS, CD and FF-fluorescence were able to characterise the complex birch and grass allergy vaccines on all structural levels, i.e. primary, secondary and tertiary structure. Via MS the relevant birch and grass allergens could be identified while via CD and FF-fluorescence the protein structures could be investigated. Moreover, CD and FF-fluorescence were shown to be promising stability indicating assays.

EAACI, 16-20 June 2012, GenevaAbstract number: 560, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations

29

Poster Session 13 - Immunotherapy: allergens and allergen-preparations

C h ara c t e ris a t i o n o f a l u m - a ds o r b e d p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

Time (

mont

hs)

CD ratio 207/222 nm

5°C

37°C

0510152025

01

23

Time (

month

s)

Fluorescence intensity (106 x LU)

5°C

37°C

0510152025

01

23

Time (

mont

hs)

Fluorescence intensity (106 x LU)

5°C

37°C

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

Time (

mont

hs)

CD ratio 207/222 nm

5°C

37°C

0510152025

290

300

310

320

330

340

350

360

370

380

390

400

Wave

length

(nm)

Fluorescence intensity (106 x LU)

0510152025

290

300

310

320

330

340

350

360

370

380

390

400

Wave

length

(nm)

Fluorescence intensity (106 x LU)

560

-Cha

ract

eris

atio

nof

alu

m-a

dsor

bed

polle

n al

lerg

oids

with

ph

ysic

oche

mic

al te

chni

ques

D. L

uykx

1

1, J

. de

Brui

jn1

2

, J. C

orde

wen

er2 ,

T. A

mer

ica2

and

R. v

an d

en H

out1

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Plan

t Res

earc

h In

tern

atio

nal,

Wag

enin

gen,

The

Net

herla

nds

Back

grou

nd &

Aim

:Se

vera

l phy

sico

chem

ical t

echn

ique

s ha

ve b

een

sele

cted

to in

vest

igat

e th

eir s

uita

bilit

y to

cha

ract

eris

e a

birc

h al

lerg

y va

ccin

e (A

LU-B

) and

gra

ss

alle

rgy

vacc

ine

(ALU

-G).

Thes

e te

chni

ques

are

mas

s sp

ectro

met

ry

(MS)

, ci

rcul

ar

dich

roism

(CD)

, an

d fro

nt-fa

ce

fluor

esce

nce

(FF-

fluor

esce

nce)

. Th

ey s

houl

d be

abl

e to

inv

estig

ate

the

alum

-ads

orbe

d m

odifi

ed a

llerg

ens

on a

ll st

ruct

ural

lev

els,

i.e

. pr

imar

y st

ruct

ure

(for

prot

ein

iden

tific

atio

n),

seco

ndar

y an

d te

rtiar

y st

ruct

ure

(for

stru

ctur

al

stab

ility)

. Sa

mpl

es w

ere

tem

pera

ture

stre

ssed

to

find

out

if th

ese

tech

niqu

es a

re s

tabi

lity

indi

catin

g.

Met

hods

:Al

um-a

dsor

bed

polle

n al

lerg

oids

: Alle

rgen

ext

ract

s fro

m b

irch

and

mixe

d gr

ass

polle

n we

re m

odifi

ed w

ith g

luta

rald

ehyd

e, a

nd a

dsor

bed

onto

al

umin

ium

hyd

roxi

de to

obt

ain

ALU

-B a

nd A

LU-G

, res

pect

ivel

y.

Stre

ss c

ondi

tions

: ALU

-B a

nd A

LU-G

wer

e st

ored

at 5

and

37º

C fo

r 1-3

m

onth

s, a

t 50º

Cfo

r 12

hour

s, a

nd a

t 90º

C fo

r 1 h

our.

MS:

The

app

lied

proc

edur

e fir

stly

invo

lved

prep

arat

ion

of tr

yptic

dige

sts

from

the

sam

ple.

Nex

t, th

e pe

ptid

es w

ere

sepa

rate

d vi

a 2D

nan

o-liq

uid

chro

mat

ogra

phy

befo

re e

lect

rosp

ray

ioni

satio

n an

d en

terin

g th

e M

S in

stru

men

t. Fo

r pr

otei

n id

entif

icatio

n,

the

ioni

sed

pept

ides

we

re

fragm

ente

d re

veal

ing

the

amin

o ac

id s

eque

nce.

CD: F

ar-U

V CD

spe

ctra

wer

e re

cord

ed fr

om 1

95 to

260

nm

and

und

er

stirr

ing

cond

itions

of

the

sam

ple.

The

CD

ratio

207

/222

nm

wa

s m

onito

red.

FF-fl

uore

scen

ce:

Fluo

resc

ence

em

issi

on s

pect

ra w

ere

reco

rded

fro

m

290

to 4

00 n

m w

ith e

xcita

tion

at 2

80 n

m. T

he fl

uore

scen

ce in

tens

ity a

t 32

6 nm

was

mon

itore

d.

Resu

lts:

Via

MS

seve

ral

isofo

rms

of t

he m

ajor

birc

h al

lerg

en B

et v

1 we

re

iden

tifie

d in

ALU

-B (a

, b, c

, d/h

, f/i)

. In

ALU

-G th

e m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5 we

re id

entif

ied

(dat

a no

t sho

wn).

The

CD-

spec

trum

of A

LU-B

and

ALU

-G r

epre

sent

ed p

rote

in s

truct

ures

co

nsis

ting

of m

ainl

y -h

elic

es (i

.e.,

ellip

ticity

at 2

08 a

nd 2

22 n

m) a

nd

-st

ruct

ure

(Fig

. 1)

. Th

e FF

-fluo

resc

ence

spe

ctru

m o

f bo

th v

acci

nes

show

ed a

n em

issi

on m

axim

um a

t 32

6 nm

sug

gest

ing

hydr

opho

bic

surro

undi

ngs

for

the

arom

atic

am

ino

acid

s pr

esen

t in

bot

h pr

otei

n sa

mpl

es (F

ig. 2

).

Stor

age

of A

LU-B

and

ALU

-G a

t sev

eral

ele

vate

d te

mpe

ratu

res

resu

lted

in s

pect

ral

alte

ratio

ns m

eani

ng s

truct

ural

alte

ratio

ns o

f pr

otei

n. C

D sp

ectra

indi

cate

d pa

rtial

ly lo

ss o

f -h

elic

es (

incr

ease

of

ratio

207

/222

nm

) wh

ile f

luor

esce

nce

spec

tra i

ndic

ated

for

mat

ion

of a

les

s rig

id

stru

ctur

e (d

ecre

ase

of fl

uore

scen

ce a

t 326

nm

).By

mon

itorin

g th

e C

D-ra

tio 2

07/2

22 n

m (

i.e.,

loss

of

-hel

ices

) an

d flu

ores

cenc

e in

tens

ity a

t 326

nm

(i.e

., rig

idity

), th

e st

ruct

ural

sta

bilit

y of

AL

U-B

and

ALU

-G w

as fo

llowe

d at

5 a

nd 3

7ºC

for 1

-3 m

onth

s (F

ig. 3

-4).

Stor

age

at

5ºC

di

d no

t af

fect

the

se

cond

ary

and

terti

ary

prot

ein

stru

ctur

es o

f bot

h va

ccin

es. H

owev

er, s

truct

ural

cha

nges

wer

e ob

serv

ed

for

the

polle

n va

ccin

es w

hen

stor

ed a

t 37

ºC.

Stor

age

at 5

0ºC

for

12

hour

s di

d re

sult

in s

imila

r st

ruct

ural

cha

nges

as

was

foun

d af

ter

1-2

mon

ths

at 3

7ºC

(dat

a no

t sho

wn).

The

prot

ein

stru

ctur

es o

f ALU

-B a

nd

ALU

-G w

ere

mos

tly e

ffect

ed w

hen

stor

ed a

t 90º

C fo

r 1

hour

(da

ta n

ot

show

n). I

n th

is ca

se th

e C

D-ra

tio in

crea

sed

to 1

.20

and

1.56

for A

LU-B

an

d AL

U-G

, re

spec

tivel

y.

With

re

spec

t to

th

e flu

ores

cenc

e,

the

inte

nsiti

es d

ecre

ased

for b

oth

vacc

ines

to 9

.5 x

106 .

Conc

lusi

on:

MS,

CD

and

FF-fl

uore

scen

ce w

ere

able

to

char

acte

rise

the

com

plex

bi

rch

and

gras

s al

lerg

y va

ccin

es o

n al

l stru

ctur

al l

evel

s, i

.e.

prim

ary,

se

cond

ary

and

terti

ary

stru

ctur

e. V

ia M

S th

e m

ajor

birc

h an

d gr

ass

alle

rgen

s we

re id

entif

ied

while

via

CD

and

FF-fl

uore

scen

ce th

e pr

otei

n st

ruct

ures

wer

e el

ucid

ated

. CD

and

FF-fl

uore

scen

ce w

ere

show

n to

be

prom

isin

g st

abilit

y in

dica

ting

assa

ys.

Figu

re 4

: Flu

ores

cenc

e in

tens

ity a

t 326

nm

of A

LU-B

(A) a

nd A

LU-G

(B) s

tore

d at

5 a

nd

37ºC

for 1

-3 m

onth

s.

Figu

re 1

: Fa

r-UV

CD

-spe

ctru

m o

f AL

U-B

(A)

and

ALU

-G (

B).

The

arro

ws

indi

cate

the

el

liptic

ityat

207

and

222

nm

.

Figu

re 3

: CD

-ratio

207

/222

nm

of

ALU

-B (

A) a

nd A

LU-G

(B) s

tore

d at

5 a

nd 3

7ºC

for

1-3

mon

ths.

Figu

re 2

: FF-

fluor

esce

nce

spec

trum

of A

LU-B

(A) a

nd A

LU-G

(B).

The

arro

w in

dica

tes

the

fluor

esce

nce

inte

nsity

at 3

26 n

m. T

he p

eak

at 3

10 n

m re

pres

ents

the

wat

er ra

man

peak

.

-20-15-10-50510

190

200

210

220

230

240

250

260

Wav

eleng

th (n

m)

Ellipticity (mDeg)

-10-50510

190

200

210

220

230

240

250

260

Wav

eleng

th (nm

)

Ellipticity (mDeg)

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

Time (

mont

hs)

CD ratio 207/222 nm

5°C

37°C

0510152025

01

23

Time (

month

s)

Fluorescence intensity (106 x LU)

5°C

37°C

0510152025

01

23

Time (

mont

hs)

Fluorescence intensity (106 x LU)

5°C

37°C

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

Time (

mont

hs)

CD ratio 207/222 nm

5°C

37°C

0510152025

290

300

310

320

330

340

350

360

370

380

390

400

Wave

length

(nm)

Fluorescence intensity (106 x LU)

0510152025

290

300

310

320

330

340

350

360

370

380

390

400

Wave

length

(nm)

Fluorescence intensity (106 x LU)

560

-Cha

ract

eris

atio

nof

alu

m-a

dsor

bed

polle

n al

lerg

oids

with

ph

ysic

oche

mic

al te

chni

ques

D. L

uykx

1

1, J

. de

Brui

jn1

2

, J. C

orde

wen

er2 ,

T. A

mer

ica2

and

R. v

an d

en H

out1

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Plan

t Res

earc

h In

tern

atio

nal,

Wag

enin

gen,

The

Net

herla

nds

Back

grou

nd &

Aim

:Se

vera

l phy

sico

chem

ical t

echn

ique

s ha

ve b

een

sele

cted

to in

vest

igat

e th

eir s

uita

bilit

y to

cha

ract

eris

e a

birc

h al

lerg

y va

ccin

e (A

LU-B

) and

gra

ss

alle

rgy

vacc

ine

(ALU

-G).

Thes

e te

chni

ques

are

mas

s sp

ectro

met

ry

(MS)

, ci

rcul

ar

dich

roism

(CD)

, an

d fro

nt-fa

ce

fluor

esce

nce

(FF-

fluor

esce

nce)

. Th

ey s

houl

d be

abl

e to

inv

estig

ate

the

alum

-ads

orbe

d m

odifi

ed a

llerg

ens

on a

ll st

ruct

ural

lev

els,

i.e

. pr

imar

y st

ruct

ure

(for

prot

ein

iden

tific

atio

n),

seco

ndar

y an

d te

rtiar

y st

ruct

ure

(for

stru

ctur

al

stab

ility)

. Sa

mpl

es w

ere

tem

pera

ture

stre

ssed

to

find

out

if th

ese

tech

niqu

es a

re s

tabi

lity

indi

catin

g.

Met

hods

:Al

um-a

dsor

bed

polle

n al

lerg

oids

: Alle

rgen

ext

ract

s fro

m b

irch

and

mixe

d gr

ass

polle

n we

re m

odifi

ed w

ith g

luta

rald

ehyd

e, a

nd a

dsor

bed

onto

al

umin

ium

hyd

roxi

de to

obt

ain

ALU

-B a

nd A

LU-G

, res

pect

ivel

y.

Stre

ss c

ondi

tions

: ALU

-B a

nd A

LU-G

wer

e st

ored

at 5

and

37º

C fo

r 1-3

m

onth

s, a

t 50º

Cfo

r 12

hour

s, a

nd a

t 90º

C fo

r 1 h

our.

MS:

The

app

lied

proc

edur

e fir

stly

invo

lved

prep

arat

ion

of tr

yptic

dige

sts

from

the

sam

ple.

Nex

t, th

e pe

ptid

es w

ere

sepa

rate

d vi

a 2D

nan

o-liq

uid

chro

mat

ogra

phy

befo

re e

lect

rosp

ray

ioni

satio

n an

d en

terin

g th

e M

S in

stru

men

t. Fo

r pr

otei

n id

entif

icatio

n,

the

ioni

sed

pept

ides

we

re

fragm

ente

d re

veal

ing

the

amin

o ac

id s

eque

nce.

CD: F

ar-U

V CD

spe

ctra

wer

e re

cord

ed fr

om 1

95 to

260

nm

and

und

er

stirr

ing

cond

itions

of

the

sam

ple.

The

CD

ratio

207

/222

nm

wa

s m

onito

red.

FF-fl

uore

scen

ce:

Fluo

resc

ence

em

issi

on s

pect

ra w

ere

reco

rded

fro

m

290

to 4

00 n

m w

ith e

xcita

tion

at 2

80 n

m. T

he fl

uore

scen

ce in

tens

ity a

t 32

6 nm

was

mon

itore

d.

Resu

lts:

Via

MS

seve

ral

isofo

rms

of t

he m

ajor

birc

h al

lerg

en B

et v

1 we

re

iden

tifie

d in

ALU

-B (a

, b, c

, d/h

, f/i)

. In

ALU

-G th

e m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5 we

re id

entif

ied

(dat

a no

t sho

wn).

The

CD-

spec

trum

of A

LU-B

and

ALU

-G r

epre

sent

ed p

rote

in s

truct

ures

co

nsis

ting

of m

ainl

y -h

elic

es (i

.e.,

ellip

ticity

at 2

08 a

nd 2

22 n

m) a

nd

-st

ruct

ure

(Fig

. 1)

. Th

e FF

-fluo

resc

ence

spe

ctru

m o

f bo

th v

acci

nes

show

ed a

n em

issi

on m

axim

um a

t 32

6 nm

sug

gest

ing

hydr

opho

bic

surro

undi

ngs

for

the

arom

atic

am

ino

acid

s pr

esen

t in

bot

h pr

otei

n sa

mpl

es (F

ig. 2

).

Stor

age

of A

LU-B

and

ALU

-G a

t sev

eral

ele

vate

d te

mpe

ratu

res

resu

lted

in s

pect

ral

alte

ratio

ns m

eani

ng s

truct

ural

alte

ratio

ns o

f pr

otei

n. C

D sp

ectra

indi

cate

d pa

rtial

ly lo

ss o

f -h

elic

es (

incr

ease

of

ratio

207

/222

nm

) wh

ile f

luor

esce

nce

spec

tra i

ndic

ated

for

mat

ion

of a

les

s rig

id

stru

ctur

e (d

ecre

ase

of fl

uore

scen

ce a

t 326

nm

).By

mon

itorin

g th

e C

D-ra

tio 2

07/2

22 n

m (

i.e.,

loss

of

-hel

ices

) an

d flu

ores

cenc

e in

tens

ity a

t 326

nm

(i.e

., rig

idity

), th

e st

ruct

ural

sta

bilit

y of

AL

U-B

and

ALU

-G w

as fo

llowe

d at

5 a

nd 3

7ºC

for 1

-3 m

onth

s (F

ig. 3

-4).

Stor

age

at

5ºC

di

d no

t af

fect

the

se

cond

ary

and

terti

ary

prot

ein

stru

ctur

es o

f bot

h va

ccin

es. H

owev

er, s

truct

ural

cha

nges

wer

e ob

serv

ed

for

the

polle

n va

ccin

es w

hen

stor

ed a

t 37

ºC.

Stor

age

at 5

0ºC

for

12

hour

s di

d re

sult

in s

imila

r st

ruct

ural

cha

nges

as

was

foun

d af

ter

1-2

mon

ths

at 3

7ºC

(dat

a no

t sho

wn).

The

prot

ein

stru

ctur

es o

f ALU

-B a

nd

ALU

-G w

ere

mos

tly e

ffect

ed w

hen

stor

ed a

t 90º

C fo

r 1

hour

(da

ta n

ot

show

n). I

n th

is ca

se th

e C

D-ra

tio in

crea

sed

to 1

.20

and

1.56

for A

LU-B

an

d AL

U-G

, re

spec

tivel

y.

With

re

spec

t to

th

e flu

ores

cenc

e,

the

inte

nsiti

es d

ecre

ased

for b

oth

vacc

ines

to 9

.5 x

106 .

Conc

lusi

on:

MS,

CD

and

FF-fl

uore

scen

ce w

ere

able

to

char

acte

rise

the

com

plex

bi

rch

and

gras

s al

lerg

y va

ccin

es o

n al

l stru

ctur

al l

evel

s, i

.e.

prim

ary,

se

cond

ary

and

terti

ary

stru

ctur

e. V

ia M

S th

e m

ajor

birc

h an

d gr

ass

alle

rgen

s we

re id

entif

ied

while

via

CD

and

FF-fl

uore

scen

ce th

e pr

otei

n st

ruct

ures

wer

e el

ucid

ated

. CD

and

FF-fl

uore

scen

ce w

ere

show

n to

be

prom

isin

g st

abilit

y in

dica

ting

assa

ys.

Figu

re 4

: Flu

ores

cenc

e in

tens

ity a

t 326

nm

of A

LU-B

(A) a

nd A

LU-G

(B) s

tore

d at

5 a

nd

37ºC

for 1

-3 m

onth

s.

Figu

re 1

: Fa

r-UV

CD

-spe

ctru

m o

f AL

U-B

(A)

and

ALU

-G (

B).

The

arro

ws

indi

cate

the

el

liptic

ityat

207

and

222

nm

.

Figu

re 3

: CD

-ratio

207

/222

nm

of

ALU

-B (

A) a

nd A

LU-G

(B) s

tore

d at

5 a

nd 3

7ºC

for

1-3

mon

ths.

Figu

re 2

: FF-

fluor

esce

nce

spec

trum

of A

LU-B

(A) a

nd A

LU-G

(B).

The

arro

w in

dica

tes

the

fluor

esce

nce

inte

nsity

at 3

26 n

m. T

he p

eak

at 3

10 n

m re

pres

ents

the

wat

er ra

man

peak

.

-20-15-10-50510

190

200

210

220

230

240

250

260

Wav

eleng

th (n

m)

Ellipticity (mDeg)

-10-50510

190

200

210

220

230

240

250

260

Wav

eleng

th (nm

)

Ellipticity (mDeg)

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

Time (

mont

hs)

CD ratio 207/222 nm

5°C

37°C

0510152025

01

23

Time (

month

s)

Fluorescence intensity (106 x LU)

5°C

37°C

0510152025

01

23

Time (

mont

hs)

Fluorescence intensity (106 x LU)

5°C

37°C

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

01

23

Time (

mont

hs)

CD ratio 207/222 nm

5°C

37°C

0510152025

290

300

310

320

330

340

350

360

370

380

390

400

Wave

length

(nm)

Fluorescence intensity (106 x LU)

0510152025

290

300

310

320

330

340

350

360

370

380

390

400

Wave

length

(nm)

Fluorescence intensity (106 x LU)

560

-Cha

ract

eris

atio

nof

alu

m-a

dsor

bed

polle

n al

lerg

oids

with

ph

ysic

oche

mic

al te

chni

ques

D. L

uykx

1

1, J

. de

Brui

jn1

2

, J. C

orde

wen

er2 ,

T. A

mer

ica2

and

R. v

an d

en H

out1

HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s

Plan

t Res

earc

h In

tern

atio

nal,

Wag

enin

gen,

The

Net

herla

nds

Back

grou

nd &

Aim

:Se

vera

l phy

sico

chem

ical t

echn

ique

s ha

ve b

een

sele

cted

to in

vest

igat

e th

eir s

uita

bilit

y to

cha

ract

eris

e a

birc

h al

lerg

y va

ccin

e (A

LU-B

) and

gra

ss

alle

rgy

vacc

ine

(ALU

-G).

Thes

e te

chni

ques

are

mas

s sp

ectro

met

ry

(MS)

, ci

rcul

ar

dich

roism

(CD)

, an

d fro

nt-fa

ce

fluor

esce

nce

(FF-

fluor

esce

nce)

. Th

ey s

houl

d be

abl

e to

inv

estig

ate

the

alum

-ads

orbe

d m

odifi

ed a

llerg

ens

on a

ll st

ruct

ural

lev

els,

i.e

. pr

imar

y st

ruct

ure

(for

prot

ein

iden

tific

atio

n),

seco

ndar

y an

d te

rtiar

y st

ruct

ure

(for

stru

ctur

al

stab

ility)

. Sa

mpl

es w

ere

tem

pera

ture

stre

ssed

to

find

out

if th

ese

tech

niqu

es a

re s

tabi

lity

indi

catin

g.

Met

hods

:Al

um-a

dsor

bed

polle

n al

lerg

oids

: Alle

rgen

ext

ract

s fro

m b

irch

and

mixe

d gr

ass

polle

n we

re m

odifi

ed w

ith g

luta

rald

ehyd

e, a

nd a

dsor

bed

onto

al

umin

ium

hyd

roxi

de to

obt

ain

ALU

-B a

nd A

LU-G

, res

pect

ivel

y.

Stre

ss c

ondi

tions

: ALU

-B a

nd A

LU-G

wer

e st

ored

at 5

and

37º

C fo

r 1-3

m

onth

s, a

t 50º

Cfo

r 12

hour

s, a

nd a

t 90º

C fo

r 1 h

our.

MS:

The

app

lied

proc

edur

e fir

stly

invo

lved

prep

arat

ion

of tr

yptic

dige

sts

from

the

sam

ple.

Nex

t, th

e pe

ptid

es w

ere

sepa

rate

d vi

a 2D

nan

o-liq

uid

chro

mat

ogra

phy

befo

re e

lect

rosp

ray

ioni

satio

n an

d en

terin

g th

e M

S in

stru

men

t. Fo

r pr

otei

n id

entif

icatio

n,

the

ioni

sed

pept

ides

we

re

fragm

ente

d re

veal

ing

the

amin

o ac

id s

eque

nce.

CD: F

ar-U

V CD

spe

ctra

wer

e re

cord

ed fr

om 1

95 to

260

nm

and

und

er

stirr

ing

cond

itions

of

the

sam

ple.

The

CD

ratio

207

/222

nm

wa

s m

onito

red.

FF-fl

uore

scen

ce:

Fluo

resc

ence

em

issi

on s

pect

ra w

ere

reco

rded

fro

m

290

to 4

00 n

m w

ith e

xcita

tion

at 2

80 n

m. T

he fl

uore

scen

ce in

tens

ity a

t 32

6 nm

was

mon

itore

d.

Resu

lts:

Via

MS

seve

ral

isofo

rms

of t

he m

ajor

birc

h al

lerg

en B

et v

1 we

re

iden

tifie

d in

ALU

-B (a

, b, c

, d/h

, f/i)

. In

ALU

-G th

e m

ajor

gra

ss a

llerg

ens

grou

p 1

and

5 we

re id

entif

ied

(dat

a no

t sho

wn).

The

CD-

spec

trum

of A

LU-B

and

ALU

-G r

epre

sent

ed p

rote

in s

truct

ures

co

nsis

ting

of m

ainl

y -h

elic

es (i

.e.,

ellip

ticity

at 2

08 a

nd 2

22 n

m) a

nd

-st

ruct

ure

(Fig

. 1)

. Th

e FF

-fluo

resc

ence

spe

ctru

m o

f bo

th v

acci

nes

show

ed a

n em

issi

on m

axim

um a

t 32

6 nm

sug

gest

ing

hydr

opho

bic

surro

undi

ngs

for

the

arom

atic

am

ino

acid

s pr

esen

t in

bot

h pr

otei

n sa

mpl

es (F

ig. 2

).

Stor

age

of A

LU-B

and

ALU

-G a

t sev

eral

ele

vate

d te

mpe

ratu

res

resu

lted

in s

pect

ral

alte

ratio

ns m

eani

ng s

truct

ural

alte

ratio

ns o

f pr

otei

n. C

D sp

ectra

indi

cate

d pa

rtial

ly lo

ss o

f -h

elic

es (

incr

ease

of

ratio

207

/222

nm

) wh

ile f

luor

esce

nce

spec

tra i

ndic

ated

for

mat

ion

of a

les

s rig

id

stru

ctur

e (d

ecre

ase

of fl

uore

scen

ce a

t 326

nm

).By

mon

itorin

g th

e C

D-ra

tio 2

07/2

22 n

m (

i.e.,

loss

of

-hel

ices

) an

d flu

ores

cenc

e in

tens

ity a

t 326

nm

(i.e

., rig

idity

), th

e st

ruct

ural

sta

bilit

y of

AL

U-B

and

ALU

-G w

as fo

llowe

d at

5 a

nd 3

7ºC

for 1

-3 m

onth

s (F

ig. 3

-4).

Stor

age

at

5ºC

di

d no

t af

fect

the

se

cond

ary

and

terti

ary

prot

ein

stru

ctur

es o

f bot

h va

ccin

es. H

owev

er, s

truct

ural

cha

nges

wer

e ob

serv

ed

for

the

polle

n va

ccin

es w

hen

stor

ed a

t 37

ºC.

Stor

age

at 5

0ºC

for

12

hour

s di

d re

sult

in s

imila

r st

ruct

ural

cha

nges

as

was

foun

d af

ter

1-2

mon

ths

at 3

7ºC

(dat

a no

t sho

wn).

The

prot

ein

stru

ctur

es o

f ALU

-B a

nd

ALU

-G w

ere

mos

tly e

ffect

ed w

hen

stor

ed a

t 90º

C fo

r 1

hour

(da

ta n

ot

show

n). I

n th

is ca

se th

e C

D-ra

tio in

crea

sed

to 1

.20

and

1.56

for A

LU-B

an

d AL

U-G

, re

spec

tivel

y.

With

re

spec

t to

th

e flu

ores

cenc

e,

the

inte

nsiti

es d

ecre

ased

for b

oth

vacc

ines

to 9

.5 x

106 .

Conc

lusi

on:

MS,

CD

and

FF-fl

uore

scen

ce w

ere

able

to

char

acte

rise

the

com

plex

bi

rch

and

gras

s al

lerg

y va

ccin

es o

n al

l stru

ctur

al l

evel

s, i

.e.

prim

ary,

se

cond

ary

and

terti

ary

stru

ctur

e. V

ia M

S th

e m

ajor

birc

h an

d gr

ass

alle

rgen

s we

re id

entif

ied

while

via

CD

and

FF-fl

uore

scen

ce th

e pr

otei

n st

ruct

ures

wer

e el

ucid

ated

. CD

and

FF-fl

uore

scen

ce w

ere

show

n to

be

prom

isin

g st

abilit

y in

dica

ting

assa

ys.

Figu

re 4

: Flu

ores

cenc

e in

tens

ity a

t 326

nm

of A

LU-B

(A) a

nd A

LU-G

(B) s

tore

d at

5 a

nd

37ºC

for 1

-3 m

onth

s.

Figu

re 1

: Fa

r-UV

CD

-spe

ctru

m o

f AL

U-B

(A)

and

ALU

-G (

B).

The

arro

ws

indi

cate

the

el

liptic

ityat

207

and

222

nm

.

Figu

re 3

: CD

-ratio

207

/222

nm

of

ALU

-B (

A) a

nd A

LU-G

(B) s

tore

d at

5 a

nd 3

7ºC

for

1-3

mon

ths.

Figu

re 2

: FF-

fluor

esce

nce

spec

trum

of A

LU-B

(A) a

nd A

LU-G

(B).

The

arro

w in

dica

tes

the

fluor

esce

nce

inte

nsity

at 3

26 n

m. T

he p

eak

at 3

10 n

m re

pres

ents

the

wat

er ra

man

peak

.

-20-15-10-50510

190

200

210

220

230

240

250

260

Wav

eleng

th (n

m)

Ellipticity (mDeg)

-10-50510

190

200

210

220

230

240

250

260

Wav

eleng

th (nm

)

Ellipticity (mDeg)

In re

latio

n to

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

al o

r per

ceiv

ed c

onflic

ts o

f int

eres

t: th

e pr

esen

ter i

s an

em

ploy

ee o

f HAL

EAAC

I Con

gres

s 20

12

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st: t

he p

rese

nter

is a

n em

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

An In h i b i t i o n E LI SA m e t h o d f o r t h e d e t e r min a t i o n o f R e l a t iv e Po t e n c y o f M i t e p r e p ara t i o n s

30

E. Kerkvliet, I. Peekel, R. van den Hout.

HAL Allergy BV, Leiden, The Netherlands.

BackgroundWe have developed and (pre-)validated a new method for the determination of IgE potency of mites. Advantages of the new method are i) all components are well characterised and verifiable, ii) the method is specific for different mite cultures and has a high precision, and iii) the costs of reagents are relatively low.

Method The assay starts with a pre-incubation of mite preparations with a sera pool obtained from patients with established clinical allergy for mite allergens. Thereafter, the mixture is transferred to 96-well plates coated with mite allergens from the mite species and mite source of interest. Unbound specific IgE binds to the coated wells and the total amount of IgE is quantitatively stained by using an anti-IgE streptavidine conjugate. The extent to which the IgE binds to the coat is compared with the maximum amount of IgE that was bound if the sera pool was not pre-incubated (E-max). The difference of the pre-incubated samples and E-max is indicated as percentage inhibition of the IgE preparation. As control, an in-house reference (IHR) is included with the assay to compare the inhibition curves by parallel line analyses. The IgE binding efficiency of an allergen preparation is expressed by relative potency (RP) compared to the IHR. The allergen preparations that were tested with the new method are different mite drug substances from Dermatophagoides pteronyssinus and Dermatophagoides farinae. The results were compared with results obtained with the ImmunoCap assay.

Results All mite preparations tested showed linear inhibition curves with high correlations (R2≥0,99) and reproducible RP data (intermediate precision (CV) of 10%). Furthermore, RP data measured with the new method were comparable with the RP data measured with the ImmunoCap.

Conclusion The newly developed IgE inhibition ELISA is well suited for use of potency determination of different mite species from bodies and cultures. In comparison with the ImmunoCap assay, the newly developed assay has an improved precision and all reagents are well characterised.

EAACI, 16-20 June 2012, GenevaAbstract number: 571, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations

31

Poster Session 13 - Immunotherapy: allergens and allergen-preparations

An In h i b i t i o n E LI SA m e t h o d f o r t h e d e t e r min a t i o n o f R e l a t iv e Po t e n c y o f M i t e p r e p ara t i o n s

571

-An

Inhi

bitio

n EL

ISA

met

hod

for t

he d

eter

min

atio

n of

R

elat

ive

Pote

ncy

of M

ite p

repa

ratio

nsE.

Ker

kvlie

t, I.

Peek

el a

nd R

. van

den

Hou

tH

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds

Back

grou

nd a

nd a

im:

IgE

pote

ncy

assa

ys w

ith s

peci

fic re

agen

ts s

uch

as b

iotin

ylat

edal

lerg

en e

xtra

cts

or a

llerg

en c

aps

used

in th

e Im

mun

oCap

syst

em a

re re

lativ

ely

expe

nsiv

e an

d co

ntai

n re

agen

ts th

at c

an n

ot b

e ch

arac

teriz

ed.I

n ad

ditio

n, th

e Im

mun

oCap

assa

y fo

r mite

s wa

s no

t dev

elop

ed fo

r pot

ency

test

ing

of d

iffer

ent m

ite c

ultu

res.

Ther

efor

e, a

new

met

hod

for t

he d

eter

min

atio

n of

IgE

pote

ncy

of m

ites

was

deve

lope

d an

d pr

e-va

lidat

ed

Conc

lusi

on:

The

newl

y de

velo

ped

IgE

inhi

bitio

n EL

ISA

is w

ell s

uite

d fo

r use

of p

oten

cy d

eter

min

atio

n of

diff

eren

t mite

spe

cies

from

bod

ies

and

cultu

res.

In c

ompa

rison

with

the

Imm

unoC

apas

say,

the

newl

y de

velo

ped

assa

y ha

s an

impr

oved

pre

cisi

onan

d al

l rea

gent

s ar

e we

ll ch

arac

teris

ed.

Tabl

e 1.

RP

data

and

var

iatio

n (C

V%) f

rom

4 d

iffer

ent

sam

ples

test

ed in

two

conc

entra

tions

. The

RP

valu

e is

an

ave

rage

of

3 di

ffere

nt r

uns.

Eac

h in

divi

dual

RP

valu

e is

an a

vera

ge o

f an

inde

pend

ent d

uplic

ate.

Met

hods

:iE

LISA

: The

ass

ay s

tarts

with

coa

ting

of m

icot

iterp

late

s us

ing

mite

ext

ract

s of

the

mite

sou

rce

of in

tere

st (F

ig 1

, A),

follo

wed

by a

n in

cuba

tion

in th

e co

ated

we

lls o

f mite

pre

para

tions

with

a s

era

pool

obt

aine

d fro

m p

atie

nts

with

est

ablis

hed

clin

ical

alle

rgy

for m

ite a

llerg

ens

(Fig

1, B

). U

nbou

nd s

peci

fic Ig

Ebi

nds

to th

e co

ated

wel

ls a

nd th

e to

tal a

mou

nt o

f IgE

is qu

antit

ative

ly st

aine

d by

usin

g an

HRP

ant

i-IgE

conj

ugat

e (F

ig 1

, C).

The

exte

nt to

whi

ch Ig

Ebi

nds

to th

e co

at is

co

mpa

red

with

the

max

imum

am

ount

of I

gEth

at w

as b

ound

if o

nly

seru

m w

as in

cuba

ted

(E-m

ax).

The

diffe

renc

e of

the

pre-

incu

bate

d sa

mpl

es a

nd E

-max

is

indi

cate

d as

per

cent

age

inhi

bitio

n of

the

IgE

prep

arat

ion.

As

cont

rol,

an in

-hou

se re

fere

nce

(IHR

) is

incl

uded

to c

ompa

re th

e in

hibi

tion

curv

es b

y pa

ralle

l lin

e an

alys

es (

PLA)

(Fi

g 1,

D).

The

IgE

bind

ing

effic

ienc

y of

an

alle

rgen

pre

para

tion

is e

xpre

ssed

by

rela

tive

pote

ncy

(RP)

com

pare

d to

the

IHR.

The

alle

rgen

pr

epar

atio

ns th

at w

ere

test

ed w

ith th

e ne

w m

etho

d ar

e di

ffere

nt m

ite e

xtra

cts

from

Der

mat

opha

goid

espt

eron

yssi

nus

and

Der

mat

opha

goid

esfa

rinae

.The

re

sults

of t

he iE

LISA

were

com

pare

d wi

th re

sults

obt

aine

d wi

th th

e Im

mun

oCap

assa

y

Res

ults

:Al

l mite

pre

para

tions

test

ed s

howe

d lin

ear i

nhib

ition

curv

es w

ith h

igh

corre

latio

ns (R

20,

99).

Diffe

rent

mite

spe

cies

from

bot

h bo

dies

and

cul

ture

s we

rete

sted

in

two

conc

entra

tions

in tr

iplic

ate

(Tab

le 1

). Fu

rther

mor

e, in

tra-a

ssay

(Tab

le 2

) and

inte

r-ass

ay (T

able

3) p

reci

sion

was

det

erm

ined

for 4

diff

eren

t mite

pro

duct

s an

d sh

ow a

max

imal

day

-to-d

ay v

aria

tion

of 1

0% (n

=6).

No s

igni

fican

t diff

eren

ces

were

foun

d wh

en R

P da

ta m

easu

red

with

the

new

met

hod

were

com

pare

d wi

th R

P da

ta m

easu

red

with

the

Imm

unoC

ap.

80.

550.

5

31.

051

Mite

mix

cultu

res

90.

680.

5

11.

191

D. f

arin

ae c

ultu

res

170.

400.

5

120.

741

D. p

terb

odie

s

70.

470.

5

30.

851

D. f

arin

aebo

dies

CV%

Mea

nR

PTe

sted

dose

Test

sam

ple

104106

CV%

1.20

1.60

0.60

0.61

Mea

nR

P

Imm

unoC

apiE

LISA

201.

02M

item

ix cu

lture

s

461.

15D

. far

inae

cul

ture

s

170.

75D

. pte

rbod

ies

230.

84D

. far

inae

bodi

es

CV%

Mea

nR

PTe

st s

ampl

e

Tabl

e 3.

RP

data

of 4

diff

eren

t sam

ples

test

ed w

ith th

e iE

LISA

and

the

Imm

unoC

ap.

The

resu

lts o

f th

e iE

LISA

are

from

the

int

er-a

ssay

pr

ecis

ion

dete

rmin

atio

n. iE

LISA

: ave

rage

and

CV%

of 6

inde

pend

ent

runs

; Im

mun

oCap

: ave

rage

and

CV%

of 3

inde

pend

ent r

uns.

0.93

1.19

0.69

0.91

Mea

nR

P

11M

item

ix cu

lture

s

12D

. far

inae

cu

lture

s

4D

. pte

rbo

dies

3D

. far

inae

bodi

es

CV%

Test

sam

ple

Tabl

e 2.

Int

ra-m

edia

te p

reci

sion

of

RP

data

and

var

iatio

n (C

V%)

of 4

diff

eren

t sa

mpl

es.

Intra

med

iate

prec

isio

n is

de

term

ined

in

th

ree

runs

; th

ese

data

re

pres

ent

the

data

fro

m t

he r

un w

ith t

he

high

est

varia

tion.

The

mea

n R

P va

lue

is

an a

vera

ge o

f 3 d

iffer

ent s

ampl

e di

lutio

ns

anal

ysed

in o

ne ru

n.

Imm

unoC

ap: T

he Im

mun

oCap

assa

y in

brie

f: th

e as

say

star

ts w

ith a

pre

-incu

batio

n of

mite

ext

ract

and

ser

um, a

fter

whic

h th

e m

ixtur

e is

trans

ferre

d to

the

Imm

unoC

APsy

stem

(Ph

adia

AB, S

wede

n) lo

aded

with

CAP

sco

ated

with

mite

alle

rgen

s. U

nbou

nd Ig

E, w

hich

was

una

ble

to b

ind

the

prod

uct d

urin

g pr

e-in

cuba

tion,

bin

ds to

the

CAP

and

is d

etec

ted

by a

n an

ti-Ig

Eco

njug

ate.

A fl

uoro

geni

csu

bstra

te is

add

ed a

nd th

e am

ount

of I

gEon

the

CAP

is m

easu

red

quan

titat

ively

by

fluor

esce

nce.

RP

is de

term

ined

by

usin

g PL

A.

020406080100

110

100

1000

mite

con

cent

ratio

n in

AU

/ml

IgE inhibition (IgE)

IHR

mite

ext

ract

Coa

ting

of m

ite e

xtra

ct

ME-

BM

E-C

Mixt

ure

of m

ite p

rote

ins

and

patie

nt s

erum

is

adde

d

Com

petit

ion

betw

een

co

at &

mite

sam

ple

for

bind

ing

of Ig

Ein

the

seru

m

Wash

Wash

Boun

d Ig

Eis

det

ecte

d wi

th H

RP la

bele

dα

-IgE

HRP

is s

tain

ed w

ith T

MB

Col

our i

s de

tect

ed a

t 45

0nm

Line

ar c

urve

s ar

e co

mpu

ted

by u

sing

ratio

(%) b

ound

IgE

vsE-

max

. Pot

ency

is

dete

rmin

ed b

y us

ing

PLA.

AB

CD

= m

iteex

tract

= Ig

E=

TMB

= H

RP-la

bele

d co

njug

ate

020406080100

110

100

1000

mite

con

cent

ratio

n in

AU

/ml

IgE inhibition (IgE)

IHR

mite

ext

ract

Coa

ting

of m

ite e

xtra

ct

ME-

BM

E-C

Mixt

ure

of m

ite p

rote

ins

and

patie

nt s

erum

is

adde

d

Com

petit

ion

betw

een

co

at &

mite

sam

ple

for

bind

ing

of Ig

Ein

the

seru

m

Wash

Wash

Boun

d Ig

Eis

det

ecte

d wi

th H

RP la

bele

dα

-IgE

HRP

is s

tain

ed w

ith T

MB

Col

our i

s de

tect

ed a

t 45

0nm

Line

ar c

urve

s ar

e co

mpu

ted

by u

sing

ratio

(%) b

ound

IgE

vsE-

max

. Pot

ency

is

dete

rmin

ed b

y us

ing

PLA.

AB

CD

= m

iteex

tract

= Ig

E=

TMB

= H

RP-la

bele

d co

njug

ate

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

571

-An

Inhi

bitio

n EL

ISA

met

hod

for t

he d

eter

min

atio

n of

R

elat

ive

Pote

ncy

of M

ite p

repa

ratio

nsE.

Ker

kvlie

t, I.

Peek

el a

nd R

. van

den

Hou

tH

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds

Back

grou

nd a

nd a

im:

IgE

pote

ncy

assa

ys w

ith s

peci

fic re

agen

ts s

uch

as b

iotin

ylat

edal

lerg

en e

xtra

cts

or a

llerg

en c

aps

used

in th

e Im

mun

oCap

syst

em a

re re

lativ

ely

expe

nsiv

e an

d co

ntai

n re

agen

ts th

at c

an n

ot b

e ch

arac

teriz

ed.I

n ad

ditio

n, th

e Im

mun

oCap

assa

y fo

r mite

s wa

s no

t dev

elop

ed fo

r pot

ency

test

ing

of d

iffer

ent m

ite c

ultu

res.

Ther

efor

e, a

new

met

hod

for t

he d

eter

min

atio

n of

IgE

pote

ncy

of m

ites

was

deve

lope

d an

d pr

e-va

lidat

ed

Conc

lusi

on:

The

newl

y de

velo

ped

IgE

inhi

bitio

n EL

ISA

is w

ell s

uite

d fo

r use

of p

oten

cy d

eter

min

atio

n of

diff

eren

t mite

spe

cies

from

bod

ies

and

cultu

res.

In c

ompa

rison

with

the

Imm

unoC

apas

say,

the

newl

y de

velo

ped

assa

y ha

s an

impr

oved

pre

cisi

onan

d al

l rea

gent

s ar

e we

ll ch

arac

teris

ed.

Tabl

e 1.

RP

data

and

var

iatio

n (C

V%) f

rom

4 d

iffer

ent

sam

ples

test

ed in

two

conc

entra

tions

. The

RP

valu

e is

an

ave

rage

of

3 di

ffere

nt r

uns.

Eac

h in

divi

dual

RP

valu

e is

an a

vera

ge o

f an

inde

pend

ent d

uplic

ate.

Met

hods

:iE

LISA

: The

ass

ay s

tarts

with

coa

ting

of m

icot

iterp

late

s us

ing

mite

ext

ract

s of

the

mite

sou

rce

of in

tere

st (F

ig 1

, A),

follo

wed

by a

n in

cuba

tion

in th

e co

ated

we

lls o

f mite

pre

para

tions

with

a s

era

pool

obt

aine

d fro

m p

atie

nts

with

est

ablis

hed

clin

ical

alle

rgy

for m

ite a

llerg

ens

(Fig

1, B

). U

nbou

nd s

peci

fic Ig

Ebi

nds

to th

e co

ated

wel

ls a

nd th

e to

tal a

mou

nt o

f IgE

is qu

antit

ative

ly st

aine

d by

usin

g an

HRP

ant

i-IgE

conj

ugat

e (F

ig 1

, C).

The

exte

nt to

whi

ch Ig

Ebi

nds

to th

e co

at is

co

mpa

red

with

the

max

imum

am

ount

of I

gEth

at w

as b

ound

if o

nly

seru

m w

as in

cuba

ted

(E-m

ax).

The

diffe

renc

e of

the

pre-

incu

bate

d sa

mpl

es a

nd E

-max

is

indi

cate

d as

per

cent

age

inhi

bitio

n of

the

IgE

prep

arat

ion.

As

cont

rol,

an in

-hou

se re

fere

nce

(IHR

) is

incl

uded

to c

ompa

re th

e in

hibi

tion

curv

es b

y pa

ralle

l lin

e an

alys

es (

PLA)

(Fi

g 1,

D).

The

IgE

bind

ing

effic

ienc

y of

an

alle

rgen

pre

para

tion

is e

xpre

ssed

by

rela

tive

pote

ncy

(RP)

com

pare

d to

the

IHR.

The

alle

rgen

pr

epar

atio

ns th

at w

ere

test

ed w

ith th

e ne

w m

etho

d ar

e di

ffere

nt m

ite e

xtra

cts

from

Der

mat

opha

goid

espt

eron

yssi

nus

and

Der

mat

opha

goid

esfa

rinae

.The

re

sults

of t

he iE

LISA

were

com

pare

d wi

th re

sults

obt

aine

d wi

th th

e Im

mun

oCap

assa

y

Res

ults

:Al

l mite

pre

para

tions

test

ed s

howe

d lin

ear i

nhib

ition

curv

es w

ith h

igh

corre

latio

ns (R

20,

99).

Diffe

rent

mite

spe

cies

from

bot

h bo

dies

and

cul

ture

s we

rete

sted

in

two

conc

entra

tions

in tr

iplic

ate

(Tab

le 1

). Fu

rther

mor

e, in

tra-a

ssay

(Tab

le 2

) and

inte

r-ass

ay (T

able

3) p

reci

sion

was

det

erm

ined

for 4

diff

eren

t mite

pro

duct

s an

d sh

ow a

max

imal

day

-to-d

ay v

aria

tion

of 1

0% (n

=6).

No s

igni

fican

t diff

eren

ces

were

foun

d wh

en R

P da

ta m

easu

red

with

the

new

met

hod

were

com

pare

d wi

th R

P da

ta m

easu

red

with

the

Imm

unoC

ap.

80.

550.

5

31.

051

Mite

mix

cultu

res

90.

680.

5

11.

191

D. f

arin

ae c

ultu

res

170.

400.

5

120.

741

D. p

terb

odie

s

70.

470.

5

30.

851

D. f

arin

aebo

dies

CV%

Mea

nR

PTe

sted

dose

Test

sam

ple

104106

CV%

1.20

1.60

0.60

0.61

Mea

nR

P

Imm

unoC

apiE

LISA

201.

02M

item

ix cu

lture

s

461.

15D

. far

inae

cul

ture

s

170.

75D

. pte

rbod

ies

230.

84D

. far

inae

bodi

es

CV%

Mea

nR

PTe

st s

ampl

e

Tabl

e 3.

RP

data

of 4

diff

eren

t sam

ples

test

ed w

ith th

e iE

LISA

and

the

Imm

unoC

ap.

The

resu

lts o

f th

e iE

LISA

are

from

the

int

er-a

ssay

pr

ecis

ion

dete

rmin

atio

n. iE

LISA

: ave

rage

and

CV%

of 6

inde

pend

ent

runs

; Im

mun

oCap

: ave

rage

and

CV%

of 3

inde

pend

ent r

uns.

0.93

1.19

0.69

0.91

Mea

nR

P

11M

item

ix cu

lture

s

12D

. far

inae

cu

lture

s

4D

. pte

rbo

dies

3D

. far

inae

bodi

es

CV%

Test

sam

ple

Tabl

e 2.

Int

ra-m

edia

te p

reci

sion

of

RP

data

and

var

iatio

n (C

V%)

of 4

diff

eren

t sa

mpl

es.

Intra

med

iate

prec

isio

n is

de

term

ined

in

th

ree

runs

; th

ese

data

re

pres

ent

the

data

fro

m t

he r

un w

ith t

he

high

est

varia

tion.

The

mea

n R

P va

lue

is

an a

vera

ge o

f 3 d

iffer

ent s

ampl

e di

lutio

ns

anal

ysed

in o

ne ru

n.

Imm

unoC

ap: T

he Im

mun

oCap

assa

y in

brie

f: th

e as

say

star

ts w

ith a

pre

-incu

batio

n of

mite

ext

ract

and

ser

um, a

fter

whic

h th

e m

ixtur

e is

trans

ferre

d to

the

Imm

unoC

APsy

stem

(Ph

adia

AB, S

wede

n) lo

aded

with

CAP

sco

ated

with

mite

alle

rgen

s. U

nbou

nd Ig

E, w

hich

was

una

ble

to b

ind

the

prod

uct d

urin

g pr

e-in

cuba

tion,

bin

ds to

the

CAP

and

is d

etec

ted

by a

n an

ti-Ig

Eco

njug

ate.

A fl

uoro

geni

csu

bstra

te is

add

ed a

nd th

e am

ount

of I

gEon

the

CAP

is m

easu

red

quan

titat

ively

by

fluor

esce

nce.

RP

is de

term

ined

by

usin

g PL

A.

020406080100

110

100

1000

mite

con

cent

ratio

n in

AU

/ml

IgE inhibition (IgE)

IHR

mite

ext

ract

Coa

ting

of m

ite e

xtra

ct

ME-

BM

E-C

Mixt

ure

of m

ite p

rote

ins

and

patie

nt s

erum

is

adde

d

Com

petit

ion

betw

een

co

at &

mite

sam

ple

for

bind

ing

of Ig

Ein

the

seru

m

Wash

Wash

Boun

d Ig

Eis

det

ecte

d wi

th H

RP la

bele

dα

-IgE

HRP

is s

tain

ed w

ith T

MB

Col

our i

s de

tect

ed a

t 45

0nm

Line

ar c

urve

s ar

e co

mpu

ted

by u

sing

ratio

(%) b

ound

IgE

vsE-

max

. Pot

ency

is

dete

rmin

ed b

y us

ing

PLA.

AB

CD

= m

iteex

tract

= Ig

E=

TMB

= H

RP-la

bele

d co

njug

ate

020406080100

110

100

1000

mite

con

cent

ratio

n in

AU

/ml

IgE inhibition (IgE)

IHR

mite

ext

ract

Coa

ting

of m

ite e

xtra

ct

ME-

BM

E-C

Mixt

ure

of m

ite p

rote

ins

and

patie

nt s

erum

is

adde

d

Com

petit

ion

betw

een

co

at &

mite

sam

ple

for

bind

ing

of Ig

Ein

the

seru

m

Wash

Wash

Boun

d Ig

Eis

det

ecte

d wi

th H

RP la

bele

dα

-IgE

HRP

is s

tain

ed w

ith T

MB

Col

our i

s de

tect

ed a

t 45

0nm

Line

ar c

urve

s ar

e co

mpu

ted

by u

sing

ratio

(%) b

ound

IgE

vsE-

max

. Pot

ency

is

dete

rmin

ed b

y us

ing

PLA.

AB

CD

= m

iteex

tract

= Ig

E=

TMB

= H

RP-la

bele

d co

njug

ate

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

571

-An

Inhi

bitio

n EL

ISA

met

hod

for t

he d

eter

min

atio

n of

R

elat

ive

Pote

ncy

of M

ite p

repa

ratio

nsE.

Ker

kvlie

t, I.

Peek

el a

nd R

. van

den

Hou

tH

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds

Back

grou

nd a

nd a

im:

IgE

pote

ncy

assa

ys w

ith s

peci

fic re

agen

ts s

uch

as b

iotin

ylat

edal

lerg

en e

xtra

cts

or a

llerg

en c

aps

used

in th

e Im

mun

oCap

syst

em a

re re

lativ

ely

expe

nsiv

e an

d co

ntai

n re

agen

ts th

at c

an n

ot b

e ch

arac

teriz

ed.I

n ad

ditio

n, th

e Im

mun

oCap

assa

y fo

r mite

s wa

s no

t dev

elop

ed fo

r pot

ency

test

ing

of d

iffer

ent m

ite c

ultu

res.

Ther

efor

e, a

new

met

hod

for t

he d

eter

min

atio

n of

IgE

pote

ncy

of m

ites

was

deve

lope

d an

d pr

e-va

lidat

ed

Conc

lusi

on:

The

newl

y de

velo

ped

IgE

inhi

bitio

n EL

ISA

is w

ell s

uite

d fo

r use

of p

oten

cy d

eter

min

atio

n of

diff

eren

t mite

spe

cies

from

bod

ies

and

cultu

res.

In c

ompa

rison

with

the

Imm

unoC

apas

say,

the

newl

y de

velo

ped

assa

y ha

s an

impr

oved

pre

cisi

onan

d al

l rea

gent

s ar

e we

ll ch

arac

teris

ed.

Tabl

e 1.

RP

data

and

var

iatio

n (C

V%) f

rom

4 d

iffer

ent

sam

ples

test

ed in

two

conc

entra

tions

. The

RP

valu

e is

an

ave

rage

of

3 di

ffere

nt r

uns.

Eac

h in

divi

dual

RP

valu

e is

an a

vera

ge o

f an

inde

pend

ent d

uplic

ate.

Met

hods

:iE

LISA

: The

ass

ay s

tarts

with

coa

ting

of m

icot

iterp

late

s us

ing

mite

ext

ract

s of

the

mite

sou

rce

of in

tere

st (F

ig 1

, A),

follo

wed

by a

n in

cuba

tion

in th

e co

ated

we

lls o

f mite

pre

para

tions

with

a s

era

pool

obt

aine

d fro

m p

atie

nts

with

est

ablis

hed

clin

ical

alle

rgy

for m

ite a

llerg

ens

(Fig

1, B

). U

nbou

nd s

peci

fic Ig

Ebi

nds

to th

e co

ated

wel

ls a

nd th

e to

tal a

mou

nt o

f IgE

is qu

antit

ative

ly st

aine

d by

usin

g an

HRP

ant

i-IgE

conj

ugat

e (F

ig 1

, C).

The

exte

nt to

whi

ch Ig

Ebi

nds

to th

e co

at is

co

mpa

red

with

the

max

imum

am

ount

of I

gEth

at w

as b

ound

if o

nly

seru

m w

as in

cuba

ted

(E-m

ax).

The

diffe

renc

e of

the

pre-

incu

bate

d sa

mpl

es a

nd E

-max

is

indi

cate

d as

per

cent

age

inhi

bitio

n of

the

IgE

prep

arat

ion.

As

cont

rol,

an in

-hou

se re

fere

nce

(IHR

) is

incl

uded

to c

ompa

re th

e in

hibi

tion

curv

es b

y pa

ralle

l lin

e an

alys

es (

PLA)

(Fi

g 1,

D).

The

IgE

bind

ing

effic

ienc

y of

an

alle

rgen

pre

para

tion

is e

xpre

ssed

by

rela

tive

pote

ncy

(RP)

com

pare

d to

the

IHR.

The

alle

rgen

pr

epar

atio

ns th

at w

ere

test

ed w

ith th

e ne

w m

etho

d ar

e di

ffere

nt m

ite e

xtra

cts

from

Der

mat

opha

goid

espt

eron

yssi

nus

and

Der

mat

opha

goid

esfa

rinae

.The

re

sults

of t

he iE

LISA

were

com

pare

d wi

th re

sults

obt

aine

d wi

th th

e Im

mun

oCap

assa

y

Res

ults

:Al

l mite

pre

para

tions

test

ed s

howe

d lin

ear i

nhib

ition

curv

es w

ith h

igh

corre

latio

ns (R

20,

99).

Diffe

rent

mite

spe

cies

from

bot

h bo

dies

and

cul

ture

s we

rete

sted

in

two

conc

entra

tions

in tr

iplic

ate

(Tab

le 1

). Fu

rther

mor

e, in

tra-a

ssay

(Tab

le 2

) and

inte

r-ass

ay (T

able

3) p

reci

sion

was

det

erm

ined

for 4

diff

eren

t mite

pro

duct

s an

d sh

ow a

max

imal

day

-to-d

ay v

aria

tion

of 1

0% (n

=6).

No s

igni

fican

t diff

eren

ces

were

foun

d wh

en R

P da

ta m

easu

red

with

the

new

met

hod

were

com

pare

d wi

th R

P da

ta m

easu

red

with

the

Imm

unoC

ap.

80.

550.

5

31.

051

Mite

mix

cultu

res

90.

680.

5

11.

191

D. f

arin

ae c

ultu

res

170.

400.

5

120.

741

D. p

terb

odie

s

70.

470.

5

30.

851

D. f

arin

aebo

dies

CV%

Mea

nR

PTe

sted

dose

Test

sam

ple

104106

CV%

1.20

1.60

0.60

0.61

Mea

nR

P

Imm

unoC

apiE

LISA

201.

02M

item

ix cu

lture

s

461.

15D

. far

inae

cul

ture

s

170.

75D

. pte

rbod

ies

230.

84D

. far

inae

bodi

es

CV%

Mea

nR

PTe

st s

ampl

e

Tabl

e 3.

RP

data

of 4

diff

eren

t sam

ples

test

ed w

ith th

e iE

LISA

and

the

Imm

unoC

ap.

The

resu

lts o

f th

e iE

LISA

are

from

the

int

er-a

ssay

pr

ecis

ion

dete

rmin

atio

n. iE

LISA

: ave

rage

and

CV%

of 6

inde

pend

ent

runs

; Im

mun

oCap

: ave

rage

and

CV%

of 3

inde

pend

ent r

uns.

0.93

1.19

0.69

0.91

Mea

nR

P

11M

item

ix cu

lture

s

12D

. far

inae

cu

lture

s

4D

. pte

rbo

dies

3D

. far

inae

bodi

es

CV%

Test

sam

ple

Tabl

e 2.

Int

ra-m

edia

te p

reci

sion

of

RP

data

and

var

iatio

n (C

V%)

of 4

diff

eren

t sa

mpl

es.

Intra

med

iate

prec

isio

n is

de

term

ined

in

th

ree

runs

; th

ese

data

re

pres

ent

the

data

fro

m t

he r

un w

ith t

he

high

est

varia

tion.

The

mea

n R

P va

lue

is

an a

vera

ge o

f 3 d

iffer

ent s

ampl

e di

lutio

ns

anal

ysed

in o

ne ru

n.

Imm

unoC

ap: T

he Im

mun

oCap

assa

y in

brie

f: th

e as

say

star

ts w

ith a

pre

-incu

batio

n of

mite

ext

ract

and

ser

um, a

fter

whic

h th

e m

ixtur

e is

trans

ferre

d to

the

Imm

unoC

APsy

stem

(Ph

adia

AB, S

wede

n) lo

aded

with

CAP

sco

ated

with

mite

alle

rgen

s. U

nbou

nd Ig

E, w

hich

was

una

ble

to b

ind

the

prod

uct d

urin

g pr

e-in

cuba

tion,

bin

ds to

the

CAP

and

is d

etec

ted

by a

n an

ti-Ig

Eco

njug

ate.

A fl

uoro

geni

csu

bstra

te is

add

ed a

nd th

e am

ount

of I

gEon

the

CAP

is m

easu

red

quan

titat

ively

by

fluor

esce

nce.

RP

is de

term

ined

by

usin

g PL

A.

020406080100

110

100

1000

mite

con

cent

ratio

n in

AU

/ml

IgE inhibition (IgE)

IHR

mite

ext

ract

Coa

ting

of m

ite e

xtra

ct

ME-

BM

E-C

Mixt

ure

of m

ite p

rote

ins

and

patie

nt s

erum

is

adde

d

Com

petit

ion

betw

een

co

at &

mite

sam

ple

for

bind

ing

of Ig

Ein

the

seru

m

Wash

Wash

Boun

d Ig

Eis

det

ecte

d wi

th H

RP la

bele

dα

-IgE

HRP

is s

tain

ed w

ith T

MB

Col

our i

s de

tect

ed a

t 45

0nm

Line

ar c

urve

s ar

e co

mpu

ted

by u

sing

ratio

(%) b

ound

IgE

vsE-

max

. Pot

ency

is

dete

rmin

ed b

y us

ing

PLA.

AB

CD

= m

iteex

tract

= Ig

E=

TMB

= H

RP-la

bele

d co

njug

ate

020406080100

110

100

1000

mite

con

cent

ratio

n in

AU

/ml

IgE inhibition (IgE)

IHR

mite

ext

ract

Coa

ting

of m

ite e

xtra

ct

ME-

BM

E-C

Mixt

ure

of m

ite p

rote

ins

and

patie

nt s

erum

is

adde

d

Com

petit

ion

betw

een

co

at &

mite

sam

ple

for

bind

ing

of Ig

Ein

the

seru

m

Wash

Wash

Boun

d Ig

Eis

det

ecte

d wi

th H

RP la

bele

dα

-IgE

HRP

is s

tain

ed w

ith T

MB

Col

our i

s de

tect

ed a

t 45

0nm

Line

ar c

urve

s ar

e co

mpu

ted

by u

sing

ratio

(%) b

ound

IgE

vsE-

max

. Pot

ency

is

dete

rmin

ed b

y us

ing

PLA.

AB

CD

= m

iteex

tract

= Ig

E=

TMB

= H

RP-la

bele

d co

njug

ate

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st: t

he p

rese

nter

is a

n em

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

An a nt i b o d y - b a s e d t e c h ni q u e f o r s t a b il i t y s tu d i e s o n a l u m a ds o r b e d b irc h p o l l e n a l l e rg o i d p r e p ara t i o n s

32

E. Kerkvliet, C. Franso, A. Segaar, R. van den Hout.

HAL Allergy BV, Leiden, The Netherlands.

Background IgE based potency assays for allergens are essential tests for monitoring stability in extracts. However, these IgE potency assays can not be used for potency determination of allergoid-based products, since they essentially lack IgE reactivity. Furthermore, aluminium can deteriorate binding of antibodies to proteins attached to a solid phase. We have developed and validated an IgG based inhibition assay that is suitable for determining IgG inhibition of an alum-adsorbed birch pollen allergoid (ALU-B). In order to find out whether the assay is stability indicating, temperature-stressed samples were analysed with the new method.

Method Rabbit IgG antibodies specific for birch allergoid are pre-incubated with various concentrations of ALU-B. The mix is then added to a birch allergoid coated microtiter plate to bind free allergoid-specific IgG, which is quantitatively measured by a donkey anti-rabbit HRP conjugate and stained with TMB. The extent to which the IgG antibodies bind the plate in presence of added inhibitor (ALU-B) is compared with the maximum amount of IgG antibodies that binds in absence of the inhibitor. Results are finally expressed as percentage inhibition relative to the uninhibited value and the concentration at 50% inhibi-tion is used as read out. An in house reference of birch allergoid is included as assay control and is used for factorizing the IgG inhibition values to reduce day to day variation.

Results Validation of the IgG inhibition assay showed that the assay is specific for birch preparations, linear, precise (CV=16%) and robust. Furthermore, the assay detected a two-fold decrease in IgG inhibition when two doses of ALU-B with a two-fold difference in strength were analysed. Storage of the ALU-B vaccine at various elevated temperatures resulted in increased 50% IgG inhibition values indicating a reduction of IgG binding to the epitopes.

Conclusion We have successfully developed and validated an assay that determines IgG inhibition of the alum-adsorbed birch pol-len vaccine. Moreover, the sensitivity of the assay is sufficient to at least detect a two-fold reduction in potency. Although the assay is still under evaluation whether the IgG inhibition assay can be used for stability studies of ALU-B, preliminary results showed reduced IgG binding to temperature stressed ALU-B vaccine.

EAACI, 16-20 June 2012, GenevaAbstract number: 572, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations

33

Poster Session 13 - Immunotherapy: allergens and allergen-preparations

An a nt i b o d y - b a s e d t e c h ni q u e f o r s t a b il i t y s tu d i e s o n a l u m a ds o r b e d b irc h p o l l e n a l l e rg o i d p r e p ara t i o n s

572

-An

antib

ody-

base

d Te

chni

que

for S

tabi

lity

Stud

ies

on

Alum

Ads

orbe

d B

irch

polle

n al

lerg

oid

prep

arat

ions

E. K

erkv

liet,

C. F

rans

o, A

. Seg

aar a

nd R

. van

den

Hou

tH

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds

Back

grou

nd a

nd a

im:

IgE

base

d po

tenc

y as

says

for

alle

rgen

s ar

e es

sent

ial t

ests

for

mon

itorin

g st

abilit

y in

ext

ract

s. H

owev

er,

thes

e Ig

Epo

tenc

y as

says

can

not

be

used

for p

oten

cy d

eter

min

atio

n of

alle

rgoi

d-ba

sed

prod

ucts

, si

nce

they

es

sent

ially

la

ck

IgE

reac

tivity

. Fu

rther

mor

e, a

lum

iniu

m c

an d

eter

iora

te b

indi

ng o

f ant

ibod

ies

to

prot

eins

atta

ched

to a

sol

id p

hase

. We

prev

ious

ly d

evel

oped

an

IgG

base

d in

hibi

tion

assa

y fo

r m

onito

ring

stab

ility

of a

lum

ad

sorb

ed m

ite p

repa

ratio

ns.

A sim

ilar

assa

y ha

s no

w be

en

deve

lope

d fo

r th

e de

term

inat

ion

of

IgG

inhi

bitio

n of

al

um

adso

rbed

birc

h po

llen

alle

rgoi

ds. T

his

assa

y ha

s be

en s

tudi

ed

for

use

as a

sta

bilit

y te

st a

nd w

heth

er t

he a

ssay

has

bee

n su

ffici

ent

sens

itive

to d

etec

t a

2 tim

es r

educ

tion.

Fin

ally,

the

assa

y wa

s va

lidat

ed a

ccor

ding

the

curre

nt g

uide

lines

.

Met

hods

:Th

e ne

wly

deve

lope

d Ig

Gin

hibi

tion

assa

y us

es Ig

Gan

tibod

ies

spec

ific

for

birc

h po

llen

alle

rgoi

dsth

at

were

ob

tain

ed

by

imm

uniza

tion

of r

abbi

ts w

ith g

luta

rald

ehyd

em

odifi

ed e

xtra

cts

(alle

rgoi

d) o

f birc

h po

llen.

The

ass

ay s

tarts

with

a p

re-in

cuba

tion

of a

mixt

ure

of a

nti-a

llerg

oid

seru

m a

nd d

iffer

ent c

once

ntra

tions

of

alu

m-a

dsor

bed

birc

h po

llen

alle

rgoi

d(A

LU-B

). Af

ter

pre-

incu

batio

n th

e m

ixtur

e is

add

ed t

o an

birc

h al

lerg

oid

coat

ed

mic

rotit

erpl

ate

to b

ind

free

alle

rgoi

d-sp

ecifi

c Ig

G. S

ubse

quen

tly,

boun

d Ig

Gis

quan

titat

ivel

y m

easu

red

by a

n HR

P co

njug

ate

and

stai

ned

with

TM

B. T

he e

xten

t to

whic

h th

e Ig

Gan

tibod

ies

bind

to

the

pla

te i

n pr

esen

ce o

f AL

U-B

is

co

mpa

red

with

the

m

axim

um a

mou

nt o

f Ig

Gan

tibod

ies

that

bin

ds in

abs

ence

of

ALU

-B (

E-m

ax).

Resu

lts a

re f

inal

ly ex

pres

sed

as p

erce

ntag

e in

hibi

tion

rela

tive

to t

he u

ninh

ibite

d va

lue.

The

50%

inhi

bitio

nva

lue

is us

ed a

s re

ad o

ut. A

n in

hou

se b

irch

alle

rgoi

dre

fere

nce

sam

ple

is u

sed

for d

ay-to

-day

cor

rect

ion

and

as a

ssay

con

trol.

Suita

bilit

y of

the

ass

ay w

as d

eter

min

ed w

ith d

iffer

ent

PUR-

B ba

tche

s an

d ba

tche

s st

ored

at e

leva

ted

tem

pera

ture

s.

Res

ults

:Th

e bi

rch

polle

n al

lerg

oid

prep

arat

ion

indu

ced

high

spe

cific

IgG

titer

s in

ra

bbits

, wh

ich

was

dem

onst

rate

d by

EL

ISA

and

imm

unob

lotti

ng(F

ig.

1).

Imm

unob

lotti

ngsh

ows

that

the

rab

bit

anti

seru

m re

cogn

ises

birc

h ex

tract

, birc

h al

lerg

oid

prep

arat

ions

an

d th

e re

leva

nt b

irch

alle

rgen

Bet

v1

(Fig

. 1B)

. Usi

ng d

iffer

ent

conc

entra

tions

of A

LU-B

mixe

d wi

th th

e ra

bbit

seru

m re

sulte

d in

do

se-re

spon

se c

urve

s on

birc

h al

lerg

oid

polle

n co

ated

micr

otite

rpl

ates

. By

test

ing

supe

rnat

ant o

f PUR

-B b

atch

es it

was

pro

ven

that

the

dos

e-re

spon

secu

rves

wer

e m

ainl

y ca

used

by

ALU

-B

and

not b

y un

boun

d m

ater

ial s

ince

sup

erna

tant

con

tain

s a

mor

e th

an 1

00 ti

mes

lowe

r sig

nal (

Fig

2). T

able

1 s

hows

a s

igni

fican

t in

crea

se in

the

50%

IgG

inhi

bitio

n va

lue

when

two

times

dilu

ted

batc

hes

are

anal

ysed

, ind

icat

ing

that

the

sens

itivity

of t

he a

ssay

is

suf

ficie

nt to

det

ect a

50%

redu

ctio

n in

act

ivity

. Sto

rage

of t

he

ALU

-B v

acci

ne a

t va

rious

ele

vate

d te

mpe

ratu

res

resu

lted

in

incr

ease

d 50

% I

gGin

hibi

tion

valu

es i

ndica

ting

a re

duct

ion

of

IgG

bind

ing

to t

he e

pito

pes

(Fig

3, T

able

2).

Valid

atio

n of

the

assa

y sh

owed

that

the

assa

y is

spe

cific

for b

irch,

has

a n

orm

al

dist

ribut

ion

of p

oint

s, s

hows

a g

ood

prec

isio

n an

d is

rob

ust

(Tab

le 3

). Th

e as

say

is s

till

unde

r ev

alua

tion

to e

stab

lish

whet

her

the

IgG

inhi

bitio

n EL

ISA

is s

uita

ble

for

long

itudi

nal

mon

itorin

g of

sta

bilit

y of

the

adso

rbed

pro

duct

Conc

lusi

on:

The

newl

y de

velo

ped

and

valid

ated

IgG

inhi

bitio

n as

say

is su

itabl

e fo

r m

easu

ring

IgG

bind

ing

to P

UR-B

bat

ches

and

is

able

to a

t lea

st d

etec

t a tw

o-fo

ld re

duct

ion

in p

oten

cy.

The

assa

y sh

owed

re

duce

d Ig

Gbi

ndin

g to

te

mpe

ratu

re

stre

ssed

ALU

-B v

acci

ne, s

ugge

stin

g th

at th

e as

say

is s

tabi

lity

indi

catin

g.

Figu

re 2

. IgG

inhi

bitio

n cu

rves

of 2

bat

ches

AL

U-B

(b

lue

curv

es)

and

IgG

inhi

bitio

n cu

rves

mea

sure

d in

the

ir su

pern

atan

t (re

d an

d or

ange

cur

ves)

.

Figu

re 3

. The

gra

ph re

pres

ents

IgG

inhi

bitio

n cu

rves

of a

PU

R-B

bat

ch s

tore

d at

5°C

, 25°

C,

37 °C

for 2

mon

ths

and

at 9

0°C

for 1

and

4

hrs.

The

dot

ted

line

show

s th

e 50

% in

hibi

tion

of th

e di

ffere

nt c

urve

s.

Res

ults

Valid

atio

npa

ram

eter

s

CV

=16

%In

ter-a

ssay

prec

isio

nTe

mpe

ratu

reco

nditio

nsar

e cr

ucia

lR

obus

tnes

s

CV

= 15

%In

tra-a

ssay

prec

isio

n

Nor

mal

dist

ribut

ion

of p

oint

s; a

ncho

r poi

nts

appr

oach

uppe

r an

d lo

wera

sym

ptot

eM

odel

Spec

ificfo

rALU

-B a

nd n

otto

oth

erPU

RET

HAL

pre

para

tions

Spec

ificity

Tabl

e 3.

Val

idat

ion

resu

lts o

f the

50%

IgG

inhi

bitio

n as

say

for A

LU-B

bat

ches

.

90T0

137

140

148

94 112

50%

IgG

inhi

bitio

nva

lue

in A

Ueq

/ml

% re

cove

rySt

orag

eco

nditi

ons

66 64 61

37° °°°C

, 2 m

onth

s

90° °°°C

, 1 h

our

90° °°°C

, 4 h

ours

96 80

5° °°°C

, 2 m

onth

s

25° °°°C

, 2 m

onth

s

Lane

1: M

arke

r; La

ne 2

: Bi

rch

polle

n ex

tract

; La

ne 3

-5: 3

diff

eren

t Bi

rch

polle

n al

lerg

oid

batc

hes.

The

box

in

dica

tes

the

Bet

v1

band

Figu

re 1

A. D

irect

ELI

SA o

f th

e ra

bbit

anti-

seru

m te

sted

on

Birc

h po

llen

extra

ct a

nd o

n al

lerg

oid.

The

ser

um s

hows

dos

e-re

spon

ses

to b

oth

prep

arat

ions

with

hig

her

affin

ity f

or

the

alle

rgoi

dpr

epar

atio

n.

Figu

re 1

B.I

mm

unob

loto

f rab

bit s

erum

im

mun

ized

with

birc

h po

llen

alle

rgoi

d. T

he

anti-

seru

m is

test

ed o

n bi

rch

polle

n ex

tract

an

d on

thre

e di

ffere

nt b

irch

polle

n al

lerg

oid

prep

arat

ions

..

Nor

mal

dose

½N

orm

aldo

se

Batc

h B

128

AU

eq/m

l

286

AU

eq/m

l

50%

IgG

inhi

bitio

nva

lue

CV

7.4%

5.5%

Nor

mal

dose

½N

orm

aldo

se

Batc

h A

128

AU

eq/m

l

294

AU

eq/m

l

50%

IgG

inhi

bitio

nva

lue

CV

1.5%

2.0%

Tabl

e 1.

50%

IgG

inhi

bitio

n va

lues

of

2 ba

tche

s AL

U-B

, al

so

test

ed

when

1:

1 di

lute

d in

form

ulat

ion

buffe

r.

12

34

51

23

45

0,0

1,0

2,0

3,0

4,0

5,0

1010

010

0010

000

1000

0010

0000

0

seru

m d

ilutio

nOD value (450 nm)

Birc

h po

llen

extra

ctBi

rch

polle

n al

lerg

oid

Tabl

e 2.

The

50%

IgG

inhi

bitio

n va

lues

of e

ach

curv

e ar

e pr

esen

ted

in th

e ta

ble.

The

dec

reas

e of

50%

IgG

inhi

bitio

n wa

s ca

lcula

ted

in re

latio

n to

the

ALU

-B s

ampl

e an

alys

ed a

t t0.

CV

of a

ll m

easu

rem

ents

is b

elow

10%

-100102030405060708090100

110

100

1000

1000

010

0000

ALU-

B in

AUe

q/m

l

IgG inhibition (%)

5°C

25°C

37°C

90°C

, 1 h

r 9

0°C

, 4 h

rs

020406080100

110

100

1000

1000

010

0000

ALU-

B in

AUe

q/m

l

IgG inhibition (%)

ALU-

B ba

tch

ASU

P AL

U-B

batc

h A

ALU-

B ba

tch

BSU

P AL

U-B

batc

h B

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

y

572

-An

antib

ody-

base

d Te

chni

que

for S

tabi

lity

Stud

ies

on

Alum

Ads

orbe

d B

irch

polle

n al

lerg

oid

prep

arat

ions

E. K

erkv

liet,

C. F

rans

o, A

. Seg

aar a

nd R

. van

den

Hou

tH

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds

Back

grou

nd a

nd a

im:

IgE

base

d po

tenc

y as

says

for

alle

rgen

s ar

e es

sent

ial t

ests

for

mon

itorin

g st

abilit

y in

ext

ract

s. H

owev

er,

thes

e Ig

Epo

tenc

y as

says

can

not

be

used

for p

oten

cy d

eter

min

atio

n of

alle

rgoi

d-ba

sed

prod

ucts

, si

nce

they

es

sent

ially

la

ck

IgE

reac

tivity

. Fu

rther

mor

e, a

lum

iniu

m c

an d

eter

iora

te b

indi

ng o

f ant

ibod

ies

to

prot

eins

atta

ched

to a

sol

id p

hase

. We

prev

ious

ly d

evel

oped

an

IgG

base

d in

hibi

tion

assa

y fo

r m

onito

ring

stab

ility

of a

lum

ad

sorb

ed m

ite p

repa

ratio

ns.

A sim

ilar

assa

y ha

s no

w be

en

deve

lope

d fo

r th

e de

term

inat

ion

of

IgG

inhi

bitio

n of

al

um

adso

rbed

birc

h po

llen

alle

rgoi

ds. T

his

assa

y ha

s be

en s

tudi

ed

for

use

as a

sta

bilit

y te

st a

nd w

heth

er t

he a

ssay

has

bee

n su

ffici

ent

sens

itive

to d

etec

t a

2 tim

es r

educ

tion.

Fin

ally,

the

assa

y wa

s va

lidat

ed a

ccor

ding

the

curre

nt g

uide

lines

.

Met

hods

:Th

e ne

wly

deve

lope

d Ig

Gin

hibi

tion

assa

y us

es Ig

Gan

tibod

ies

spec

ific

for

birc

h po

llen

alle

rgoi

dsth

at

were

ob

tain

ed

by

imm

uniza

tion

of r

abbi

ts w

ith g

luta

rald

ehyd

em

odifi

ed e

xtra

cts

(alle

rgoi

d) o

f birc

h po

llen.

The

ass

ay s

tarts

with

a p

re-in

cuba

tion

of a

mixt

ure

of a

nti-a

llerg

oid

seru

m a

nd d

iffer

ent c

once

ntra

tions

of

alu

m-a

dsor

bed

birc

h po

llen

alle

rgoi

d(A

LU-B

). Af

ter

pre-

incu

batio

n th

e m

ixtur

e is

add

ed t

o an

birc

h al

lerg

oid

coat

ed

mic

rotit

erpl

ate

to b

ind

free

alle

rgoi

d-sp

ecifi

c Ig

G. S

ubse

quen

tly,

boun

d Ig

Gis

quan

titat

ivel

y m

easu

red

by a

n HR

P co

njug

ate

and

stai

ned

with

TM

B. T

he e

xten

t to

whic

h th

e Ig

Gan

tibod

ies

bind

to

the

pla

te i

n pr

esen

ce o

f AL

U-B

is

co

mpa

red

with

the

m

axim

um a

mou

nt o

f Ig

Gan

tibod

ies

that

bin

ds in

abs

ence

of

ALU

-B (

E-m

ax).

Resu

lts a

re f

inal

ly ex

pres

sed

as p

erce

ntag

e in

hibi

tion

rela

tive

to t

he u

ninh

ibite

d va

lue.

The

50%

inhi

bitio

nva

lue

is us

ed a

s re

ad o

ut. A

n in

hou

se b

irch

alle

rgoi

dre

fere

nce

sam

ple

is u

sed

for d

ay-to

-day

cor

rect

ion

and

as a

ssay

con

trol.

Suita

bilit

y of

the

ass

ay w

as d

eter

min

ed w

ith d

iffer

ent

PUR-

B ba

tche

s an

d ba

tche

s st

ored

at e

leva

ted

tem

pera

ture

s.

Res

ults

:Th

e bi

rch

polle

n al

lerg

oid

prep

arat

ion

indu

ced

high

spe

cific

IgG

titer

s in

ra

bbits

, wh

ich

was

dem

onst

rate

d by

EL

ISA

and

imm

unob

lotti

ng(F

ig.

1).

Imm

unob

lotti

ngsh

ows

that

the

rab

bit

anti

seru

m re

cogn

ises

birc

h ex

tract

, birc

h al

lerg

oid

prep

arat

ions

an

d th

e re

leva

nt b

irch

alle

rgen

Bet

v1

(Fig

. 1B)

. Usi

ng d

iffer

ent

conc

entra

tions

of A

LU-B

mixe

d wi

th th

e ra

bbit

seru

m re

sulte

d in

do

se-re

spon

se c

urve

s on

birc

h al

lerg

oid

polle

n co

ated

micr

otite

rpl

ates

. By

test

ing

supe

rnat

ant o

f PUR

-B b

atch

es it

was

pro

ven

that

the

dos

e-re

spon

secu

rves

wer

e m

ainl

y ca

used

by

ALU

-B

and

not b

y un

boun

d m

ater

ial s

ince

sup

erna

tant

con

tain

s a

mor

e th

an 1

00 ti

mes

lowe

r sig

nal (

Fig

2). T

able

1 s

hows

a s

igni

fican

t in

crea

se in

the

50%

IgG

inhi

bitio

n va

lue

when

two

times

dilu

ted

batc

hes

are

anal

ysed

, ind

icat

ing

that

the

sens

itivity

of t

he a

ssay

is

suf

ficie

nt to

det

ect a

50%

redu

ctio

n in

act

ivity

. Sto

rage

of t

he

ALU

-B v

acci

ne a

t va

rious

ele

vate

d te

mpe

ratu

res

resu

lted

in

incr

ease

d 50

% I

gGin

hibi

tion

valu

es i

ndica

ting

a re

duct

ion

of

IgG

bind

ing

to t

he e

pito

pes

(Fig

3, T

able

2).

Valid

atio

n of

the

assa

y sh

owed

that

the

assa

y is

spe

cific

for b

irch,

has

a n

orm

al

dist

ribut

ion

of p

oint

s, s

hows

a g

ood

prec

isio

n an

d is

rob

ust

(Tab

le 3

). Th

e as

say

is s

till

unde

r ev

alua

tion

to e

stab

lish

whet

her

the

IgG

inhi

bitio

n EL

ISA

is s

uita

ble

for

long

itudi

nal

mon

itorin

g of

sta

bilit

y of

the

adso

rbed

pro

duct

Conc

lusi

on:

The

newl

y de

velo

ped

and

valid

ated

IgG

inhi

bitio

n as

say

is su

itabl

e fo

r m

easu

ring

IgG

bind

ing

to P

UR-B

bat

ches

and

is

able

to a

t lea

st d

etec

t a tw

o-fo

ld re

duct

ion

in p

oten

cy.

The

assa

y sh

owed

re

duce

d Ig

Gbi

ndin

g to

te

mpe

ratu

re

stre

ssed

ALU

-B v

acci

ne, s

ugge

stin

g th

at th

e as

say

is s

tabi

lity

indi

catin

g.

Figu

re 2

. IgG

inhi

bitio

n cu

rves

of 2

bat

ches

AL

U-B

(b

lue

curv

es)

and

IgG

inhi

bitio

n cu

rves

mea

sure

d in

the

ir su

pern

atan

t (re

d an

d or

ange

cur

ves)

.

Figu

re 3

. The

gra

ph re

pres

ents

IgG

inhi

bitio

n cu

rves

of a

PU

R-B

bat

ch s

tore

d at

5°C

, 25°

C,

37 °C

for 2

mon

ths

and

at 9

0°C

for 1

and

4

hrs.

The

dot

ted

line

show

s th

e 50

% in

hibi

tion

of th

e di

ffere

nt c

urve

s.

Res

ults

Valid

atio

npa

ram

eter

s

CV

=16

%In

ter-a

ssay

prec

isio

nTe

mpe

ratu

reco

nditio

nsar

e cr

ucia

lR

obus

tnes

s

CV

= 15

%In

tra-a

ssay

prec

isio

n

Nor

mal

dist

ribut

ion

of p

oint

s; a

ncho

r poi

nts

appr

oach

uppe

r an

d lo

wera

sym

ptot

eM

odel

Spec

ificfo

rALU

-B a

nd n

otto

oth

erPU

RET

HAL

pre

para

tions

Spec

ificity

Tabl

e 3.

Val

idat

ion

resu

lts o

f the

50%

IgG

inhi

bitio

n as

say

for A

LU-B

bat

ches

.

90T0

137

140

148

94 112

50%

IgG

inhi

bitio

nva

lue

in A

Ueq

/ml

% re

cove

rySt

orag

eco

nditi

ons

66 64 61

37° °°°C

, 2 m

onth

s

90° °°°C

, 1 h

our

90° °°°C

, 4 h

ours

96 80

5° °°°C

, 2 m

onth

s

25° °°°C

, 2 m

onth

s

Lane

1: M

arke

r; La

ne 2

: Bi

rch

polle

n ex

tract

; La

ne 3

-5: 3

diff

eren

t Bi

rch

polle

n al

lerg

oid

batc

hes.

The

box

in

dica

tes

the

Bet

v1

band

Figu

re 1

A. D

irect

ELI

SA o

f th

e ra

bbit

anti-

seru

m te

sted

on

Birc

h po

llen

extra

ct a

nd o

n al

lerg

oid.

The

ser

um s

hows

dos

e-re

spon

ses

to b

oth

prep

arat

ions

with

hig

her

affin

ity f

or

the

alle

rgoi

dpr

epar

atio

n.

Figu

re 1

B.I

mm

unob

loto

f rab

bit s

erum

im

mun

ized

with

birc

h po

llen

alle

rgoi

d. T

he

anti-

seru

m is

test

ed o

n bi

rch

polle

n ex

tract

an

d on

thre

e di

ffere

nt b

irch

polle

n al

lerg

oid

prep

arat

ions

..

Nor

mal

dose

½N

orm

aldo

se

Batc

h B

128

AU

eq/m

l

286

AU

eq/m

l

50%

IgG

inhi

bitio

nva

lue

CV

7.4%

5.5%

Nor

mal

dose

½N

orm

aldo

se

Batc

h A

128

AU

eq/m

l

294

AU

eq/m

l

50%

IgG

inhi

bitio

nva

lue

CV

1.5%

2.0%

Tabl

e 1.

50%

IgG

inhi

bitio

n va

lues

of

2 ba

tche

s AL

U-B

, al

so

test

ed

when

1:

1 di

lute

d in

form

ulat

ion

buffe

r.

12

34

51

23

45

0,0

1,0

2,0

3,0

4,0

5,0

1010

010

0010

000

1000

0010

0000

0

seru

m d

ilutio

n

OD value (450 nm)

Birc

h po

llen

extra

ctBi

rch

polle

n al

lerg

oid

Tabl

e 2.

The

50%

IgG

inhi

bitio

n va

lues

of e

ach

curv

e ar

e pr

esen

ted

in th

e ta

ble.

The

dec

reas

e of

50%

IgG

inhi

bitio

n wa

s ca

lcula

ted

in re

latio

n to

the

ALU

-B s

ampl

e an

alys

ed a

t t0.

CV

of a

ll m

easu

rem

ents

is b

elow

10%

-100102030405060708090100

110

100

1000

1000

010

0000

ALU-

B in

AUe

q/m

l

IgG inhibition (%)

5°C

25°C

37°C

90°C

, 1 h

r 9

0°C

, 4 h

rs

020406080100

110

100

1000

1000

010

0000

ALU-

B in

AUe

q/m

l

IgG inhibition (%)

ALU-

B ba

tch

ASU

P AL

U-B

batc

h A

ALU-

B ba

tch

BSU

P AL

U-B

batc

h B

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

y

572

-An

antib

ody-

base

d Te

chni

que

for S

tabi

lity

Stud

ies

on

Alum

Ads

orbe

d B

irch

polle

n al

lerg

oid

prep

arat

ions

E. K

erkv

liet,

C. F

rans

o, A

. Seg

aar a

nd R

. van

den

Hou

tH

AL A

llerg

y BV

, Lei

den,

The

Net

herla

nds

Back

grou

nd a

nd a

im:

IgE

base

d po

tenc

y as

says

for

alle

rgen

s ar

e es

sent

ial t

ests

for

mon

itorin

g st

abilit

y in

ext

ract

s. H

owev

er,

thes

e Ig

Epo

tenc

y as

says

can

not

be

used

for p

oten

cy d

eter

min

atio

n of

alle

rgoi

d-ba

sed

prod

ucts

, si

nce

they

es

sent

ially

la

ck

IgE

reac

tivity

. Fu

rther

mor

e, a

lum

iniu

m c

an d

eter

iora

te b

indi

ng o

f ant

ibod

ies

to

prot

eins

atta

ched

to a

sol

id p

hase

. We

prev

ious

ly d

evel

oped

an

IgG

base

d in

hibi

tion

assa

y fo

r m

onito

ring

stab

ility

of a

lum

ad

sorb

ed m

ite p

repa

ratio

ns.

A sim

ilar

assa

y ha

s no

w be

en

deve

lope

d fo

r th

e de

term

inat

ion

of

IgG

inhi

bitio

n of

al

um

adso

rbed

birc

h po

llen

alle

rgoi

ds. T

his

assa

y ha

s be

en s

tudi

ed

for

use

as a

sta

bilit

y te

st a

nd w

heth

er t

he a

ssay

has

bee

n su

ffici

ent

sens

itive

to d

etec

t a

2 tim

es r

educ

tion.

Fin

ally,

the

assa

y wa

s va

lidat

ed a

ccor

ding

the

curre

nt g

uide

lines

.

Met

hods

:Th

e ne

wly

deve

lope

d Ig

Gin

hibi

tion

assa

y us

es Ig

Gan

tibod

ies

spec

ific

for

birc

h po

llen

alle

rgoi

dsth

at

were

ob

tain

ed

by

imm

uniza

tion

of r

abbi

ts w

ith g

luta

rald

ehyd

em

odifi

ed e

xtra

cts

(alle

rgoi

d) o

f birc

h po

llen.

The

ass

ay s

tarts

with

a p

re-in

cuba

tion

of a

mixt

ure

of a

nti-a

llerg

oid

seru

m a

nd d

iffer

ent c

once

ntra

tions

of

alu

m-a

dsor

bed

birc

h po

llen

alle

rgoi

d(A

LU-B

). Af

ter

pre-

incu

batio

n th

e m

ixtur

e is

add

ed t

o an

birc

h al

lerg

oid

coat

ed

mic

rotit

erpl

ate

to b

ind

free

alle

rgoi

d-sp

ecifi

c Ig

G. S

ubse

quen

tly,

boun

d Ig

Gis

quan

titat

ivel

y m

easu

red

by a

n HR

P co

njug

ate

and

stai

ned

with

TM

B. T

he e

xten

t to

whic

h th

e Ig

Gan

tibod

ies

bind

to

the

pla

te i

n pr

esen

ce o

f AL

U-B

is

co

mpa

red

with

the

m

axim

um a

mou

nt o

f Ig

Gan

tibod

ies

that

bin

ds in

abs

ence

of

ALU

-B (

E-m

ax).

Resu

lts a

re f

inal

ly ex

pres

sed

as p

erce

ntag

e in

hibi

tion

rela

tive

to t

he u

ninh

ibite

d va

lue.

The

50%

inhi

bitio

nva

lue

is us

ed a

s re

ad o

ut. A

n in

hou

se b

irch

alle

rgoi

dre

fere

nce

sam

ple

is u

sed

for d

ay-to

-day

cor

rect

ion

and

as a

ssay

con

trol.

Suita

bilit

y of

the

ass

ay w

as d

eter

min

ed w

ith d

iffer

ent

PUR-

B ba

tche

s an

d ba

tche

s st

ored

at e

leva

ted

tem

pera

ture

s.

Res

ults

:Th

e bi

rch

polle

n al

lerg

oid

prep

arat

ion

indu

ced

high

spe

cific

IgG

titer

s in

ra

bbits

, wh

ich

was

dem

onst

rate

d by

EL

ISA

and

imm

unob

lotti

ng(F

ig.

1).

Imm

unob

lotti

ngsh

ows

that

the

rab

bit

anti

seru

m re

cogn

ises

birc

h ex

tract

, birc

h al

lerg

oid

prep

arat

ions

an

d th

e re

leva

nt b

irch

alle

rgen

Bet

v1

(Fig

. 1B)

. Usi

ng d

iffer

ent

conc

entra

tions

of A

LU-B

mixe

d wi

th th

e ra

bbit

seru

m re

sulte

d in

do

se-re

spon

se c

urve

s on

birc

h al

lerg

oid

polle

n co

ated

micr

otite

rpl

ates

. By

test

ing

supe

rnat

ant o

f PUR

-B b

atch

es it

was

pro

ven

that

the

dos

e-re

spon

secu

rves

wer

e m

ainl

y ca

used

by

ALU

-B

and

not b

y un

boun

d m

ater

ial s

ince

sup

erna

tant

con

tain

s a

mor

e th

an 1

00 ti

mes

lowe

r sig

nal (

Fig

2). T

able

1 s

hows

a s

igni

fican

t in

crea

se in

the

50%

IgG

inhi

bitio

n va

lue

when

two

times

dilu

ted

batc

hes

are

anal

ysed

, ind

icat

ing

that

the

sens

itivity

of t

he a

ssay

is

suf

ficie

nt to

det

ect a

50%

redu

ctio

n in

act

ivity

. Sto

rage

of t

he

ALU

-B v

acci

ne a

t va

rious

ele

vate

d te

mpe

ratu

res

resu

lted

in

incr

ease

d 50

% I

gGin

hibi

tion

valu

es i

ndica

ting

a re

duct

ion

of

IgG

bind

ing

to t

he e

pito

pes

(Fig

3, T

able

2).

Valid

atio

n of

the

assa

y sh

owed

that

the

assa

y is

spe

cific

for b

irch,

has

a n

orm

al

dist

ribut

ion

of p

oint

s, s

hows

a g

ood

prec

isio

n an

d is

rob

ust

(Tab

le 3

). Th

e as

say

is s

till

unde

r ev

alua

tion

to e

stab

lish

whet

her

the

IgG

inhi

bitio

n EL

ISA

is s

uita

ble

for

long

itudi

nal

mon

itorin

g of

sta

bilit

y of

the

adso

rbed

pro

duct

Conc

lusi

on:

The

newl

y de

velo

ped

and

valid

ated

IgG

inhi

bitio

n as

say

is su

itabl

e fo

r m

easu

ring

IgG

bind

ing

to P

UR-B

bat

ches

and

is

able

to a

t lea

st d

etec

t a tw

o-fo

ld re

duct

ion

in p

oten

cy.

The

assa

y sh

owed

re

duce

d Ig

Gbi

ndin

g to

te

mpe

ratu

re

stre

ssed

ALU

-B v

acci

ne, s

ugge

stin

g th

at th

e as

say

is s

tabi

lity

indi

catin

g.

Figu

re 2

. IgG

inhi

bitio

n cu

rves

of 2

bat

ches

AL

U-B

(b

lue

curv

es)

and

IgG

inhi

bitio

n cu

rves

mea

sure

d in

the

ir su

pern

atan

t (re

d an

d or

ange

cur

ves)

.

Figu

re 3

. The

gra

ph re

pres

ents

IgG

inhi

bitio

n cu

rves

of a

PU

R-B

bat

ch s

tore

d at

5°C

, 25°

C,

37 °C

for 2

mon

ths

and

at 9

0°C

for 1

and

4

hrs.

The

dot

ted

line

show

s th

e 50

% in

hibi

tion

of th

e di

ffere

nt c

urve

s.

Res

ults

Valid

atio

npa

ram

eter

s

CV

=16

%In

ter-a

ssay

prec

isio

nTe

mpe

ratu

reco

nditio

nsar

e cr

ucia

lR

obus

tnes

s

CV

= 15

%In

tra-a

ssay

prec

isio

n

Nor

mal

dist

ribut

ion

of p

oint

s; a

ncho

r poi

nts

appr

oach

uppe

r an

d lo

wera

sym

ptot

eM

odel

Spec

ificfo

rALU

-B a

nd n

otto

oth

erPU

RET

HAL

pre

para

tions

Spec

ificity

Tabl

e 3.

Val

idat

ion

resu

lts o

f the

50%

IgG

inhi

bitio

n as

say

for A

LU-B

bat

ches

.

90T0

137

140

148

94 112

50%

IgG

inhi

bitio

nva

lue

in A

Ueq

/ml

% re

cove

rySt

orag

eco

nditi

ons

66 64 61

37° °°°C

, 2 m

onth

s

90° °°°C

, 1 h

our

90° °°°C

, 4 h

ours

96 80

5° °°°C

, 2 m

onth

s

25° °°°C

, 2 m

onth

s

Lane

1: M

arke

r; La

ne 2

: Bi

rch

polle

n ex

tract

; La

ne 3

-5: 3

diff

eren

t Bi

rch

polle

n al

lerg

oid

batc

hes.

The

box

in

dica

tes

the

Bet

v1

band

Figu

re 1

A. D

irect

ELI

SA o

f th

e ra

bbit

anti-

seru

m te

sted

on

Birc

h po

llen

extra

ct a

nd o

n al

lerg

oid.

The

ser

um s

hows

dos

e-re

spon

ses

to b

oth

prep

arat

ions

with

hig

her

affin

ity f

or

the

alle

rgoi

dpr

epar

atio

n.

Figu

re 1

B.I

mm

unob

loto

f rab

bit s

erum

im

mun

ized

with

birc

h po

llen

alle

rgoi

d. T

he

anti-

seru

m is

test

ed o

n bi

rch

polle

n ex

tract

an

d on

thre

e di

ffere

nt b

irch

polle

n al

lerg

oid

prep

arat

ions

..

Nor

mal

dose

½N

orm

aldo

se

Batc

h B

128

AU

eq/m

l

286

AU

eq/m

l

50%

IgG

inhi

bitio

nva

lue

CV

7.4%

5.5%

Nor

mal

dose

½N

orm

aldo

se

Batc

h A

128

AU

eq/m

l

294

AU

eq/m

l

50%

IgG

inhi

bitio

nva

lue

CV

1.5%

2.0%

Tabl

e 1.

50%

IgG

inhi

bitio

n va

lues

of

2 ba

tche

s AL

U-B

, al

so

test

ed

when

1:

1 di

lute

d in

form

ulat

ion

buffe

r.

12

34

51

23

45

0,0

1,0

2,0

3,0

4,0

5,0

1010

010

0010

000

1000

0010

0000

0

seru

m d

ilutio

n

OD value (450 nm)

Birc

h po

llen

extra

ctBi

rch

polle

n al

lerg

oid

Tabl

e 2.

The

50%

IgG

inhi

bitio

n va

lues

of e

ach

curv

e ar

e pr

esen

ted

in th

e ta

ble.

The

dec

reas

e of

50%

IgG

inhi

bitio

n wa

s ca

lcula

ted

in re

latio

n to

the

ALU

-B s

ampl

e an

alys

ed a

t t0.

CV

of a

ll m

easu

rem

ents

is b

elow

10%

-100102030405060708090100

110

100

1000

1000

010

0000

ALU-

B in

AUe

q/m

l

IgG inhibition (%)

5°C

25°C

37°C

90°C

, 1 h

r 9

0°C

, 4 h

rs

020406080100

110

100

1000

1000

010

0000

ALU-

B in

AUe

q/m

l

IgG inhibition (%)

ALU-

B ba

tch

ASU

P AL

U-B

batc

h A

ALU-

B ba

tch

BSU

P AL

U-B

batc

h B

In re

latio

nto

this

pres

enta

tion,

I de

clare

the

follo

win

g, re

alor

perc

eive

dco

nflic

tsof

inte

rest

: th

e pr

esen

ter i

s an

empl

oyee

of H

AL A

llerg

yIn

rela

tion

to th

is p

rese

ntat

ion,

I de

clar

e th

e fo

llow

ing,

real

or p

erce

ived

con

flict

s of

inte

rest

: the

pre

sent

er is

an

empl

oyee

of H

AL A

llerg

y.EA

ACI C

ongr

ess

2012

B i o c h e mic a l c h ara c t e ri z a t i o n o f c h e mic a l l y m o d i f i e d Ara h 2 is o f o r ms; s tru c tu r e - f u n c t i o n r e l a t i o n s h i p s

34

D. Luykx (1) , D. Apostolovic (1), H. Warmenhoven (1), D. Verbart (1), G.A.H. de Jong (2), S.J. Koppelman (1).

(1) HAL Allergy BV, Leiden, The Netherlands, (2) TNO, Zeist, The Netherlands.

BackgroundModified forms of major peanut allergens Ara h2 and Ara h6 were shown to be less allergenic than their native counterparts, making them potentially suitable for peanut allergy immunotherapy. Two isoforms of Ara h2 are known: heavy isoform of appr. 20 kDa and a light isoform of appr. 17 kDa. Furthermore, Ara h6 is an allergen of appr. 15 kDa, homologous to Ara h2. This study aims to describe the biochemical characteristics and IgE-binding potency of the modified allergens Ara h2-heavy isoform, Ara h2-light isoform, and Ara h6 in comparison with their native counterparts.

MethodsAra h2-heavy isoform, Ara h2-light isoform, and Ara h6 were purified to homogeneity to obtain the native allergens. To obtain the modified allergens, the native allergens were modified by reduction of the disulfide bonds and subsequent alkylation of the free Cys residues. The modified allergens as well as their native counter parts were analysed for change in molecular weight, secondary structure, and allergenicity, using SDS-PAGE, far-UV CD spectroscopy, and an IgE-binding assay respectively.

ResultsThe modification of the allergens was optimized by investigating the kinetics, resulting in a consistent procedure for modification. All three allergens showed an increase in apparent molecular weight and unfolding of protein structure upon reduction and alkylation. Consequently, the IgE-binding potency was decreased about a hundred-fold for each of the tested allergens.

ConclusionsUnderstanding the biochemical nature of the modification of the major peanut allergens Ara h2 and Ara h6 allows to optimize the process to reduce their allergenicity.

EAACI, 16-20 June 2012, GenevaAbstract number: 1448, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 71 - Clinical and experimental applications of molecular allergology

35

Poster Session 71 - Clinical and experimental applications of molecular allergology

B i o c h e mic a l c h ara c t e ri z a t i o n o f c h e mic a l l y m o d i f i e d Ara h 2 is o f o r ms; s tru c tu r e - f u n c t i o n r e l a t i o n s h i p s

Figu

re 2

: Far

UV

CD

spe

ctra

of n

ativ

e (b

lack

) and

mod

ified

(blu

e) A

rah2

-hea

vy is

ofor

m(A

), Ar

ah2

-ligh

t iso

form

(B) a

nd A

rah6

(C).

1448

-Bio

chem

ical

cha

ract

eriz

atio

n of

che

mic

al m

odifi

catio

n of

Ara

h2

isof

orm

s: s

truc

ture

-func

tion

rela

tions

hips

H. W

arm

enho

ven1

& D

. Apo

stol

ovic

1 , D

. Luy

kx1 ,

D. V

erba

rt1, A

. de

Jong

2 , T.

Cirk

ovic

Vel

icko

vic3

, S. K

oppe

lman

1

1 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s, 2 TN

O Q

ualit

y of

Life

, Zei

st, T

he N

ethe

rland

s, 3 U

nive

rsity

of B

elgr

ade,

Bel

grad

e, S

erbi

a

Figu

re 1

: SD

S-P

AGE

patte

rns

of A

rah2

-hea

vy is

ofor

m(A

), Ar

ah2

-ligh

t iso

form

(B),

and

Ara

h6 (C

) afte

r red

ucin

g th

e al

lerg

ens

with

0.5

or 5

.0 m

MD

TT fo

r diff

eren

t per

iods

of

time

(0-6

0 m

in).

Mol

ecul

ar w

eigh

t mar

ker (

M).

Figu

re 3

: Int

rinsi

c flu

ores

cenc

e sp

ectr

a of

nat

ive

(bla

ck) a

nd m

odifi

ed (r

ed) A

rah2

-he

avy

isof

orm

(A),

Ara

h2-li

ght i

sofo

rm(B

) and

Ara

h6 (C

).

C.

M

0

0

.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0 6

0 m

in

0.5

mM

DTT

5 m

MD

TT

M

0

0.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0

60 m

in

M

0

0

.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0 6

0 m

in

B.

A.

B

.

C.

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

A.

B.

C

.

010203040506070

290

310

330

350

370

390

Wav

eleng

th (nm

)

Fluorescence intensity (105 LU)

010203040

290

310

330

350

370

390

Wav

eleng

th (nm

)

Fluorescence intensity (105 LU)

Bac

kgro

und

& A

im:

Mod

ified

form

s of

maj

or p

eanu

t alle

rgen

s Ar

ah2

and

Ara

h6 w

ere

show

n to

be

less

alle

rgen

ic th

an th

eir

nativ

e co

unte

rpar

ts. I

n th

is w

ay th

ey a

re

pote

ntia

lly s

uita

ble

for

pean

ut a

llerg

y im

mun

othe

rapy

. Tw

o is

ofor

ms

of

Ara

h2 a

re k

now

n: a

hea

vy is

ofor

mof

~20

kD

aan

d a

light

isof

orm

of ~

17

kDa.

Ara

h6 w

ith a

mol

ecul

ar w

eigh

t of ~

15 k

Da

is h

omol

ogou

s w

ith A

rah2

. Th

is s

tudy

aim

s to

inv

estig

ate

the

bioc

hem

ical

cha

ract

eris

tics

of

redu

ced

and

alky

late

dAr

ah2

isof

orm

sin

com

paris

on w

ith t

heir

nativ

e co

unte

rpar

ts a

nd to

stu

dy th

e ki

netic

s of

the

mod

ifica

tion.

The

refo

re, t

he

thre

e m

odifi

ed a

llerg

ens

wer

e an

alyz

ed f

or a

cha

nge

in m

olec

ular

wei

ght,

prot

ein

stru

ctur

e an

d al

lerg

enic

ityby

usi

ng S

DS-

PAG

E, m

ass

spec

trom

etry

(M

S),

CD

and

flu

ores

cenc

e sp

ectro

scop

y, a

nd a

n Ig

E-bi

ndin

g as

say.

Met

hods

:M

odifi

catio

n: A

rah2

-hea

vy i

sofo

rm,

Ara

h2-li

ght

isof

orm

and

Ara

h6

wer

e m

odifi

ed b

y re

duct

ion

of t

he d

isul

phid

e bo

nds

with

0.5

or

5m

MD

TT

and

subs

eque

nt

alky

latio

n of

th

e fre

e C

ysre

sidu

es

with

io

doac

etam

ide

(50

mM

).

SDS-

PAG

E: N

on-re

duce

d, p

artia

lly r

educ

ed a

nd f

ully

red

uced

sam

ples

w

ere

appl

ied

onto

14%

pol

yacr

ylam

ide

gels

and

sta

ined

with

Coo

mas

sie

Blue

.

Mas

s sp

ectro

met

ry:

Tryp

ticpe

ptid

es o

btai

ned

from

the

sam

ples

wer

e se

para

ted

via

liqui

d ch

rom

atog

raph

y be

fore

ele

ctro

spra

yio

nisa

tion

and

ente

ring

the

LTQ

Orb

itrap

XL h

ybrid

mas

s sp

ectro

met

er.

The

ioni

sed

pept

ides

wer

e fra

gmen

ted

to re

veal

the

(mod

ified

) am

ino

acid

seq

uenc

e fo

r ide

ntity

. MS

was

als

o ap

plie

d fo

r int

act n

ativ

e an

d m

odifi

edal

lerg

ens.

CD

: Far

-UV

CD

spe

ctra

wer

e re

cord

ed fr

om 1

90 to

260

nm

.

Fluo

resc

ence

spe

ctro

scop

y: F

luor

esce

nce

spec

tra w

ere

reco

rded

fro

m

290

to 3

90 n

m w

ith e

xcita

tion

at 2

80 n

m.

IgE

inhi

bitio

n as

say:

Th

e m

odifi

ed

alle

rgen

s co

mpe

te

with

th

eir

corre

spon

ding

nat

ive

form

coa

ted

to E

LISA

pla

tes

for

bind

ing

to s

erum

Ig

E. Res

ults

:Th

e ki

netic

s of

the

pean

ut a

llerg

en m

odifi

catio

n w

as s

tudi

ed w

ith S

DS-

PAG

E as

an

incr

ease

of m

olec

ular

mas

s w

as e

xpec

ted

durin

g re

duct

ion

and

alky

latio

n (F

ig. 1

). By

usi

ng 0

.5 m

MD

TT th

e th

ree

alle

rgen

s w

ere

tota

lly m

odifi

ed a

fter 3

0 m

in w

here

as 5

min

was

nee

ded

for m

odifi

catio

n w

hen

usin

g 5

mM

DTT

. Ac

cord

ing

to t

he S

DS-

PAG

E pa

ttern

s th

e in

crea

se o

f the

mol

ecul

ar m

ass

for t

he m

odifi

ed a

llerg

ens

appe

ars

to b

e ~4

kD

a. A

ccor

ding

to M

S, a

mas

s in

crea

se o

f 465

.6 D

aw

as d

eter

min

ed

for

Ara

h2.

Con

side

ring

the

pres

ence

of

8 C

ysre

sidu

es in

Ara

h2 a

nd

the

alky

latio

n (w

hich

add

s 57

Da

per

Cys

resi

due)

, th

is l

atte

r m

ass

shou

ld b

e co

rrect

. Th

e ov

eres

timat

ed m

ass

incr

ease

for

the

mod

ified

al

lerg

ens

obta

ined

with

SD

S-PA

GE

can

be e

xpla

ined

by

thei

r pr

otei

n st

ruct

ures

. Far

-UV

CD

spe

ctra

indi

cate

typi

cal

-hel

ix s

truct

ures

for

the

nativ

e al

lerg

ens

(i.e.

, el

liptic

ityat

208

and

222

nm

)w

here

as t

ypic

al

defo

lded

prot

ein

stru

ctur

es w

ere

obse

rved

for

the

mod

ified

alle

rgen

s (F

ig.

2).

Def

olde

dan

d th

eref

ore

stre

tche

d pr

otei

ns m

igra

te r

elat

ivel

y sl

ow t

hrou

gh t

he S

DS-

PAG

E ge

l in

com

paris

on t

o fo

lded

pro

tein

s re

sulti

ng in

ban

ds th

at c

orre

spon

d to

rela

tivel

y hi

gher

mol

ecul

ar m

asse

s th

an e

xpec

ted.

The

def

olde

dpr

otei

n st

ruct

ures

of t

he m

odifi

ed a

llerg

ens

are

supp

orte

d by

fluo

resc

ence

spe

ctro

scop

y (F

ig. 3

). Af

ter f

ull r

educ

tion

of th

e th

ree

alle

rgen

s, th

e in

trins

ic fl

uore

scen

ce in

tens

ity d

ecre

ases

and

a

smal

l shi

ft of

the

fluor

esce

nce

emis

sion

max

imum

(Em

ax) i

s ob

serv

ed

for

the

Ara

h2-li

ght i

sofo

rm a

nd A

ra h

6 (i.

e., 3

and

8 n

m, r

espe

ctiv

ely)

. Th

ese

findi

ngs

indi

cate

that

the

Trp

and

Tyr r

esid

ues

in A

rah2

, and

Tyr

re

sidu

es i

n Ar

ah6

(Ar

ah6

doe

s no

t co

ntai

n Tr

p) a

re g

ettin

g m

ore

expo

sed

to t

he b

uffe

r. Th

is o

ccur

s du

ring

defo

ldin

gof

pro

tein

s. T

he

defo

lded

prot

ein

stru

ctur

es

of

the

pean

ut

alle

rgen

s re

sult

in

a co

nsid

erab

le d

ecre

ase

of a

llerg

enic

ityas

the

rela

tive

rest

pot

ency

(Ig

E)

of m

odifi

ed A

rah2

-hea

vy is

ofor

m, A

rah2

-ligh

t iso

form

and

Ara

h6 w

as

dete

rmin

ed to

0.0

1, 0

.02

and

0, re

spec

tivel

y.

Con

clus

ion:

Red

uctio

n an

d al

kyla

tion

of A

rah2

and

Ara

h6 r

esul

ts i

n co

mpl

ete

mod

ifica

tion

of a

ll is

ofor

ms

resu

lting

in u

nfol

ded

prot

ein

stru

ctur

es.

A.

0102030

290

310

330

350

370

390

Wave

length

(nm)

Fluorescence intensity (105 LU)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy

EAAC

I Con

gres

s 20

12

Figu

re 2

: Far

UV

CD

spe

ctra

of n

ativ

e (b

lack

) and

mod

ified

(blu

e) A

rah2

-hea

vy is

ofor

m(A

), Ar

ah2

-ligh

t iso

form

(B) a

nd A

rah6

(C).

1448

-Bio

chem

ical

cha

ract

eriz

atio

n of

che

mic

al m

odifi

catio

n of

Ara

h2

isof

orm

s: s

truc

ture

-func

tion

rela

tions

hips

H. W

arm

enho

ven1

& D

. Apo

stol

ovic

1 , D

. Luy

kx1 ,

D. V

erba

rt1, A

. de

Jong

2 , T.

Cirk

ovic

Vel

icko

vic3

, S. K

oppe

lman

1

1 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s, 2 TN

O Q

ualit

y of

Life

, Zei

st, T

he N

ethe

rland

s, 3 U

nive

rsity

of B

elgr

ade,

Bel

grad

e, S

erbi

a

Figu

re 1

: SD

S-P

AGE

patte

rns

of A

rah2

-hea

vy is

ofor

m(A

), Ar

ah2

-ligh

t iso

form

(B),

and

Ara

h6 (C

) afte

r red

ucin

g th

e al

lerg

ens

with

0.5

or 5

.0 m

MD

TT fo

r diff

eren

t per

iods

of

time

(0-6

0 m

in).

Mol

ecul

ar w

eigh

t mar

ker (

M).

Figu

re 3

: Int

rinsi

c flu

ores

cenc

e sp

ectr

a of

nat

ive

(bla

ck) a

nd m

odifi

ed (r

ed) A

rah2

-he

avy

isof

orm

(A),

Ara

h2-li

ght i

sofo

rm(B

) and

Ara

h6 (C

).

C.

M

0

0

.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0 6

0 m

in

0.5

mM

DTT

5 m

MD

TT

M

0

0.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0

60 m

in

M

0

0

.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0 6

0 m

in

B.

A.

B

.

C.

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

A.

B.

C

.

010203040506070

290

310

330

350

370

390

Wav

eleng

th (nm

)

Fluorescence intensity (105 LU)

010203040

290

310

330

350

370

390

Wav

eleng

th (nm

)

Fluorescence intensity (105 LU)

Bac

kgro

und

& A

im:

Mod

ified

form

s of

maj

or p

eanu

t alle

rgen

s Ar

ah2

and

Ara

h6 w

ere

show

n to

be

less

alle

rgen

ic th

an th

eir

nativ

e co

unte

rpar

ts. I

n th

is w

ay th

ey a

re

pote

ntia

lly s

uita

ble

for

pean

ut a

llerg

y im

mun

othe

rapy

. Tw

o is

ofor

ms

of

Ara

h2 a

re k

now

n: a

hea

vy is

ofor

mof

~20

kD

aan

d a

light

isof

orm

of ~

17

kDa.

Ara

h6 w

ith a

mol

ecul

ar w

eigh

t of ~

15 k

Da

is h

omol

ogou

s w

ith A

rah2

. Th

is s

tudy

aim

s to

inv

estig

ate

the

bioc

hem

ical

cha

ract

eris

tics

of

redu

ced

and

alky

late

dAr

ah2

isof

orm

sin

com

paris

on w

ith t

heir

nativ

e co

unte

rpar

ts a

nd to

stu

dy th

e ki

netic

s of

the

mod

ifica

tion.

The

refo

re, t

he

thre

e m

odifi

ed a

llerg

ens

wer

e an

alyz

ed f

or a

cha

nge

in m

olec

ular

wei

ght,

prot

ein

stru

ctur

e an

d al

lerg

enic

ityby

usi

ng S

DS-

PAG

E, m

ass

spec

trom

etry

(M

S),

CD

and

flu

ores

cenc

e sp

ectro

scop

y, a

nd a

n Ig

E-bi

ndin

g as

say.

Met

hods

:M

odifi

catio

n: A

rah2

-hea

vy i

sofo

rm,

Ara

h2-li

ght

isof

orm

and

Ara

h6

wer

e m

odifi

ed b

y re

duct

ion

of t

he d

isul

phid

e bo

nds

with

0.5

or

5m

MD

TT

and

subs

eque

nt

alky

latio

n of

th

e fre

e C

ysre

sidu

es

with

io

doac

etam

ide

(50

mM

).

SDS-

PAG

E: N

on-re

duce

d, p

artia

lly r

educ

ed a

nd f

ully

red

uced

sam

ples

w

ere

appl

ied

onto

14%

pol

yacr

ylam

ide

gels

and

sta

ined

with

Coo

mas

sie

Blue

.

Mas

s sp

ectro

met

ry:

Tryp

ticpe

ptid

es o

btai

ned

from

the

sam

ples

wer

e se

para

ted

via

liqui

d ch

rom

atog

raph

y be

fore

ele

ctro

spra

yio

nisa

tion

and

ente

ring

the

LTQ

Orb

itrap

XL h

ybrid

mas

s sp

ectro

met

er.

The

ioni

sed

pept

ides

wer

e fra

gmen

ted

to re

veal

the

(mod

ified

) am

ino

acid

seq

uenc

e fo

r ide

ntity

. MS

was

als

o ap

plie

d fo

r int

act n

ativ

e an

d m

odifi

edal

lerg

ens.

CD

: Far

-UV

CD

spe

ctra

wer

e re

cord

ed fr

om 1

90 to

260

nm

.

Fluo

resc

ence

spe

ctro

scop

y: F

luor

esce

nce

spec

tra w

ere

reco

rded

fro

m

290

to 3

90 n

m w

ith e

xcita

tion

at 2

80 n

m.

IgE

inhi

bitio

n as

say:

Th

e m

odifi

ed

alle

rgen

s co

mpe

te

with

th

eir

corre

spon

ding

nat

ive

form

coa

ted

to E

LISA

pla

tes

for

bind

ing

to s

erum

Ig

E. Res

ults

:Th

e ki

netic

s of

the

pean

ut a

llerg

en m

odifi

catio

n w

as s

tudi

ed w

ith S

DS-

PAG

E as

an

incr

ease

of m

olec

ular

mas

s w

as e

xpec

ted

durin

g re

duct

ion

and

alky

latio

n (F

ig. 1

). By

usi

ng 0

.5 m

MD

TT th

e th

ree

alle

rgen

s w

ere

tota

lly m

odifi

ed a

fter 3

0 m

in w

here

as 5

min

was

nee

ded

for m

odifi

catio

n w

hen

usin

g 5

mM

DTT

. Ac

cord

ing

to t

he S

DS-

PAG

E pa

ttern

s th

e in

crea

se o

f the

mol

ecul

ar m

ass

for t

he m

odifi

ed a

llerg

ens

appe

ars

to b

e ~4

kD

a. A

ccor

ding

to M

S, a

mas

s in

crea

se o

f 465

.6 D

aw

as d

eter

min

ed

for

Ara

h2.

Con

side

ring

the

pres

ence

of

8 C

ysre

sidu

es in

Ara

h2 a

nd

the

alky

latio

n (w

hich

add

s 57

Da

per

Cys

resi

due)

, th

is l

atte

r m

ass

shou

ld b

e co

rrect

. Th

e ov

eres

timat

ed m

ass

incr

ease

for

the

mod

ified

al

lerg

ens

obta

ined

with

SD

S-PA

GE

can

be e

xpla

ined

by

thei

r pr

otei

n st

ruct

ures

. Far

-UV

CD

spe

ctra

indi

cate

typi

cal

-hel

ix s

truct

ures

for

the

nativ

e al

lerg

ens

(i.e.

, el

liptic

ityat

208

and

222

nm

)w

here

as t

ypic

al

defo

lded

prot

ein

stru

ctur

es w

ere

obse

rved

for

the

mod

ified

alle

rgen

s (F

ig.

2).

Def

olde

dan

d th

eref

ore

stre

tche

d pr

otei

ns m

igra

te r

elat

ivel

y sl

ow t

hrou

gh t

he S

DS-

PAG

E ge

l in

com

paris

on t

o fo

lded

pro

tein

s re

sulti

ng in

ban

ds th

at c

orre

spon

d to

rela

tivel

y hi

gher

mol

ecul

ar m

asse

s th

an e

xpec

ted.

The

def

olde

dpr

otei

n st

ruct

ures

of t

he m

odifi

ed a

llerg

ens

are

supp

orte

d by

fluo

resc

ence

spe

ctro

scop

y (F

ig. 3

). Af

ter f

ull r

educ

tion

of th

e th

ree

alle

rgen

s, th

e in

trins

ic fl

uore

scen

ce in

tens

ity d

ecre

ases

and

a

smal

l shi

ft of

the

fluor

esce

nce

emis

sion

max

imum

(Em

ax) i

s ob

serv

ed

for

the

Ara

h2-li

ght i

sofo

rm a

nd A

ra h

6 (i.

e., 3

and

8 n

m, r

espe

ctiv

ely)

. Th

ese

findi

ngs

indi

cate

that

the

Trp

and

Tyr r

esid

ues

in A

rah2

, and

Tyr

re

sidu

es i

n Ar

ah6

(Ar

ah6

doe

s no

t co

ntai

n Tr

p) a

re g

ettin

g m

ore

expo

sed

to t

he b

uffe

r. Th

is o

ccur

s du

ring

defo

ldin

gof

pro

tein

s. T

he

defo

lded

prot

ein

stru

ctur

es

of

the

pean

ut

alle

rgen

s re

sult

in

a co

nsid

erab

le d

ecre

ase

of a

llerg

enic

ityas

the

rela

tive

rest

pot

ency

(Ig

E)

of m

odifi

ed A

rah2

-hea

vy is

ofor

m, A

rah2

-ligh

t iso

form

and

Ara

h6 w

as

dete

rmin

ed to

0.0

1, 0

.02

and

0, re

spec

tivel

y.

Con

clus

ion:

Red

uctio

n an

d al

kyla

tion

of A

rah2

and

Ara

h6 r

esul

ts i

n co

mpl

ete

mod

ifica

tion

of a

ll is

ofor

ms

resu

lting

in u

nfol

ded

prot

ein

stru

ctur

es.

A.

0102030

290

310

330

350

370

390

Wave

length

(nm)

Fluorescence intensity (105 LU)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy

EAAC

I Con

gres

s 20

12

Figu

re 2

: Far

UV

CD

spe

ctra

of n

ativ

e (b

lack

) and

mod

ified

(blu

e) A

rah2

-hea

vy is

ofor

m(A

), Ar

ah2

-ligh

t iso

form

(B) a

nd A

rah6

(C).

1448

-Bio

chem

ical

cha

ract

eriz

atio

n of

che

mic

al m

odifi

catio

n of

Ara

h2

isof

orm

s: s

truc

ture

-func

tion

rela

tions

hips

H. W

arm

enho

ven1

& D

. Apo

stol

ovic

1 , D

. Luy

kx1 ,

D. V

erba

rt1, A

. de

Jong

2 , T.

Cirk

ovic

Vel

icko

vic3

, S. K

oppe

lman

1

1 HAL

Alle

rgy

BV, L

eide

n, T

he N

ethe

rland

s, 2 TN

O Q

ualit

y of

Life

, Zei

st, T

he N

ethe

rland

s, 3 U

nive

rsity

of B

elgr

ade,

Bel

grad

e, S

erbi

a

Figu

re 1

: SD

S-P

AGE

patte

rns

of A

rah2

-hea

vy is

ofor

m(A

), Ar

ah2

-ligh

t iso

form

(B),

and

Ara

h6 (C

) afte

r red

ucin

g th

e al

lerg

ens

with

0.5

or 5

.0 m

MD

TT fo

r diff

eren

t per

iods

of

time

(0-6

0 m

in).

Mol

ecul

ar w

eigh

t mar

ker (

M).

Figu

re 3

: Int

rinsi

c flu

ores

cenc

e sp

ectr

a of

nat

ive

(bla

ck) a

nd m

odifi

ed (r

ed) A

rah2

-he

avy

isof

orm

(A),

Ara

h2-li

ght i

sofo

rm(B

) and

Ara

h6 (C

).

C.

M

0

0

.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0 6

0 m

in

0.5

mM

DTT

5 m

MD

TT

M

0

0.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0

60 m

in

M

0

0

.5

1

2

5

10 3

0 6

0 0

.5

1

2

5 1

0 3

0 6

0 m

in

B.

A.

B

.

C.

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

-40-200204060

260

250

240

230

220

210

200

190

Wave

lengt

h (nm

)

Ellipticity (mDeg)

A.

B.

C

.

010203040506070

290

310

330

350

370

390

Wav

eleng

th (nm

)

Fluorescence intensity (105 LU)

010203040

290

310

330

350

370

390

Wav

eleng

th (nm

)

Fluorescence intensity (105 LU)

Bac

kgro

und

& A

im:

Mod

ified

form

s of

maj

or p

eanu

t alle

rgen

s Ar

ah2

and

Ara

h6 w

ere

show

n to

be

less

alle

rgen

ic th

an th

eir

nativ

e co

unte

rpar

ts. I

n th

is w

ay th

ey a

re

pote

ntia

lly s

uita

ble

for

pean

ut a

llerg

y im

mun

othe

rapy

. Tw

o is

ofor

ms

of

Ara

h2 a

re k

now

n: a

hea

vy is

ofor

mof

~20

kD

aan

d a

light

isof

orm

of ~

17

kDa.

Ara

h6 w

ith a

mol

ecul

ar w

eigh

t of ~

15 k

Da

is h

omol

ogou

s w

ith A

rah2

. Th

is s

tudy

aim

s to

inv

estig

ate

the

bioc

hem

ical

cha

ract

eris

tics

of

redu

ced

and

alky

late

dAr

ah2

isof

orm

sin

com

paris

on w

ith t

heir

nativ

e co

unte

rpar

ts a

nd to

stu

dy th

e ki

netic

s of

the

mod

ifica

tion.

The

refo

re, t

he

thre

e m

odifi

ed a

llerg

ens

wer

e an

alyz

ed f

or a

cha

nge

in m

olec

ular

wei

ght,

prot

ein

stru

ctur

e an

d al

lerg

enic

ityby

usi

ng S

DS-

PAG

E, m

ass

spec

trom

etry

(M

S),

CD

and

flu

ores

cenc

e sp

ectro

scop

y, a

nd a

n Ig

E-bi

ndin

g as

say.

Met

hods

:M

odifi

catio

n: A

rah2

-hea

vy i

sofo

rm,

Ara

h2-li

ght

isof

orm

and

Ara

h6

wer

e m

odifi

ed b

y re

duct

ion

of t

he d

isul

phid

e bo

nds

with

0.5

or

5m

MD

TT

and

subs

eque

nt

alky

latio

n of

th

e fre

e C

ysre

sidu

es

with

io

doac

etam

ide

(50

mM

).

SDS-

PAG

E: N

on-re

duce

d, p

artia

lly r

educ

ed a

nd f

ully

red

uced

sam

ples

w

ere

appl

ied

onto

14%

pol

yacr

ylam

ide

gels

and

sta

ined

with

Coo

mas

sie

Blue

.

Mas

s sp

ectro

met

ry:

Tryp

ticpe

ptid

es o

btai

ned

from

the

sam

ples

wer

e se

para

ted

via

liqui

d ch

rom

atog

raph

y be

fore

ele

ctro

spra

yio

nisa

tion

and

ente

ring

the

LTQ

Orb

itrap

XL h

ybrid

mas

s sp

ectro

met

er.

The

ioni

sed

pept

ides

wer

e fra

gmen

ted

to re

veal

the

(mod

ified

) am

ino

acid

seq

uenc

e fo

r ide

ntity

. MS

was

als

o ap

plie

d fo

r int

act n

ativ

e an

d m

odifi

edal

lerg

ens.

CD

: Far

-UV

CD

spe

ctra

wer

e re

cord

ed fr

om 1

90 to

260

nm

.

Fluo

resc

ence

spe

ctro

scop

y: F

luor

esce

nce

spec

tra w

ere

reco

rded

fro

m

290

to 3

90 n

m w

ith e

xcita

tion

at 2

80 n

m.

IgE

inhi

bitio

n as

say:

Th

e m

odifi

ed

alle

rgen

s co

mpe

te

with

th

eir

corre

spon

ding

nat

ive

form

coa

ted

to E

LISA

pla

tes

for

bind

ing

to s

erum

Ig

E. Res

ults

:Th

e ki

netic

s of

the

pean

ut a

llerg

en m

odifi

catio

n w

as s

tudi

ed w

ith S

DS-

PAG

E as

an

incr

ease

of m

olec

ular

mas

s w

as e

xpec

ted

durin

g re

duct

ion

and

alky

latio

n (F

ig. 1

). By

usi

ng 0

.5 m

MD

TT th

e th

ree

alle

rgen

s w

ere

tota

lly m

odifi

ed a

fter 3

0 m

in w

here

as 5

min

was

nee

ded

for m

odifi

catio

n w

hen

usin

g 5

mM

DTT

. Ac

cord

ing

to t

he S

DS-

PAG

E pa

ttern

s th

e in

crea

se o

f the

mol

ecul

ar m

ass

for t

he m

odifi

ed a

llerg

ens

appe

ars

to b

e ~4

kD

a. A

ccor

ding

to M

S, a

mas

s in

crea

se o

f 465

.6 D

aw

as d

eter

min

ed

for

Ara

h2.

Con

side

ring

the

pres

ence

of

8 C

ysre

sidu

es in

Ara

h2 a

nd

the

alky

latio

n (w

hich

add

s 57

Da

per

Cys

resi

due)

, th

is l

atte

r m

ass

shou

ld b

e co

rrect

. Th

e ov

eres

timat

ed m

ass

incr

ease

for

the

mod

ified

al

lerg

ens

obta

ined

with

SD

S-PA

GE

can

be e

xpla

ined

by

thei

r pr

otei

n st

ruct

ures

. Far

-UV

CD

spe

ctra

indi

cate

typi

cal

-hel

ix s

truct

ures

for

the

nativ

e al

lerg

ens

(i.e.

, el

liptic

ityat

208

and

222

nm

)w

here

as t

ypic

al

defo

lded

prot

ein

stru

ctur

es w

ere

obse

rved

for

the

mod

ified

alle

rgen

s (F

ig.

2).

Def

olde

dan

d th

eref

ore

stre

tche

d pr

otei

ns m

igra

te r

elat

ivel

y sl

ow t

hrou

gh t

he S

DS-

PAG

E ge

l in

com

paris

on t

o fo

lded

pro

tein

s re

sulti

ng in

ban

ds th

at c

orre

spon

d to

rela

tivel

y hi

gher

mol

ecul

ar m

asse

s th

an e

xpec

ted.

The

def

olde

dpr

otei

n st

ruct

ures

of t

he m

odifi

ed a

llerg

ens

are

supp

orte

d by

fluo

resc

ence

spe

ctro

scop

y (F

ig. 3

). Af

ter f

ull r

educ

tion

of th

e th

ree

alle

rgen

s, th

e in

trins

ic fl

uore

scen

ce in

tens

ity d

ecre

ases

and

a

smal

l shi

ft of

the

fluor

esce

nce

emis

sion

max

imum

(Em

ax) i

s ob

serv

ed

for

the

Ara

h2-li

ght i

sofo

rm a

nd A

ra h

6 (i.

e., 3

and

8 n

m, r

espe

ctiv

ely)

. Th

ese

findi

ngs

indi

cate

that

the

Trp

and

Tyr r

esid

ues

in A

rah2

, and

Tyr

re

sidu

es i

n Ar

ah6

(Ar

ah6

doe

s no

t co

ntai

n Tr

p) a

re g

ettin

g m

ore

expo

sed

to t

he b

uffe

r. Th

is o

ccur

s du

ring

defo

ldin

gof

pro

tein

s. T

he

defo

lded

prot

ein

stru

ctur

es

of

the

pean

ut

alle

rgen

s re

sult

in

a co

nsid

erab

le d

ecre

ase

of a

llerg

enic

ityas

the

rela

tive

rest

pot

ency

(Ig

E)

of m

odifi

ed A

rah2

-hea

vy is

ofor

m, A

rah2

-ligh

t iso

form

and

Ara

h6 w

as

dete

rmin

ed to

0.0

1, 0

.02

and

0, re

spec

tivel

y.

Con

clus

ion:

Red

uctio

n an

d al

kyla

tion

of A

rah2

and

Ara

h6 r

esul

ts i

n co

mpl

ete

mod

ifica

tion

of a

ll is

ofor

ms

resu

lting

in u

nfol

ded

prot

ein

stru

ctur

es.

A.

0102030

290

310

330

350

370

390

Wave

length

(nm)

Fluorescence intensity (105 LU)

In re

latio

nto

this

pres

enta

tion,

I de

clar

eth

e fo

llow

ing,

real

orpe

rcei

ved

conf

licts

of in

tere

st:

the

pres

ente

r is

anem

ploy

ee o

f HAL

Alle

rgy

EAAC

I Con

gres

s 20

12

In re

latio

n to

this

pre

sent

atio

n, I

decl

are

the

follo

win

g, re

al o

r per

ceiv

ed c

onfli

cts

of in

tere

st: t

he p

rese

nter

is a

n em

ploy

ee o

f HAL

Alle

rgy.

EAAC

I Con

gres

s 20

12

Su m m ar y

Pro duct information .

SUBLIVAC ®

VenomenHAL ®

Composition: SUBLIVAC ®, sublingual drops, contains allergen extracts that are specifically prescribed for the patient on the basis of a diagnostic examination. Indication: Treatment of IgE-mediated allergy in patients with symptoms of allergic rhinitis, allergic conjunctivitis and/or allergic asthma, caused by allergens. Dosage and administration: The treatment is started with a daily dose of one drop. This dose is then increased every day with one drop until the highest daily dose of five drops is reached. The treatment is continued with five drops. The drops are to be kept under the tongue for at least 1 minute (preferably 2 - 3 minutes) before they are swallowed. The treatment should be continued for 3 to 5 years in order to remain as symptom-free as possible after the completion of the course of treatment. Contraindications: Acute infections of the eye, airways or organs involved in the immune system, secondary changes in the lungs(e.g. lung emphysema or bronchi-ectasie), severe immunopathological diseases or malignancies (e.g. autoimmune diseases of the kidneys, thyroid glands or the nervous system, rheumatism, tuberculosis and HIV), immunodeficiencies (including as a consequence of immunosuppressants), severe uncontrolled asthma with FEV

1 under 70%, or hypersensitivity

to any of the excipients. Warnings and precautions: If the treatment with pollen extracts is started during the pollen season, there is an increased risk of side effects. Take special care in case of pregnancy, inflammations in the mouth and after removal teeth or molars. Side effects: Local reactions in the mouth and throat, stomach upset and diarrhoea. Worsening of allergic reactions such as rhinitis, conjunctivitis, coughing and atopic eczema. Intensified systemic reactions (such as shortness of breath, generalised urticaria or Quincke’s oedema) can develop in rare cases. Intensified allergic reactions can particularly develop in very hypersensitive patients. These symptoms generally arise within 30 minutes after intake of the drops. Conditions for storage: Store in a refrigerator (2°C - 8°C).When the product is in use, it can be stored for a maximum of 6 months below 25 °C. Package: A SUBLIVAC ® Initial and maintenance treatment set consists of one bottle with dropper containing 24 ml fluid. The complete product information is available upon request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: April 2011

36

Composition: VenomenHAL® contains 120 micrograms/vial freeze-dried bee or wasp venom; powder and solvent for solution for injection.Indications: Diagnostic use (prick-testing and intracutaneous testing) and for causal treatment of patients with IgE-mediated insect venom allergy who have suffered a systemic reaction after a bee or wasp sting. Dosage and administration: The 120 µg freeze-dried insect venom should be dissolved with 1.2 ml solvent to 100 µg/ml. The dilution series are prepared by diluting 0.5 ml dissolved venom with 4.5 ml solution. Skin testing: The skin tests are performed by means of end point titration. It always begins with the skin-prick test to determine the patient’s level of sensibility. The intracutaneous test determines the individual starting concentration for treatment. Immunotherapy: Usually the initial treatment is carried out in hospital by administering several subcutaneous injections with increasing doses daily, starting with 0.1 ml of 0.0001 µg/ml until after day 5 the maintenance dose of 1 ml of 100 µg/ml is reached. If in-patient treatment is not possible, immunotherapy may be performed in outpatients, with one injection every 7 days at slowly increasing doses and concentrations. Treatment with the maintenance dose is continued as an outpatient procedure, the injection interval being extended gradually from 7 to 14, 21 and finally 28 days. It is advised to carry out the treatment over a period of 3 - 5 successive years. Contraindications: Skin testing: Secondary infections of the skin, hypersensitivity to any of the excipients. Relative: Acute severe allergic reactions, treatment with ß-blockers, pregnancy, cardiovascular diseases with increased risk if using adrenalin. Immunotherapy: Acute inflammatory diseases/feverish infection at the target organ; active tuberculosis; secondary alterations of the target organ (emphysema, bronchi-ectasis and others); autoimmune disorders e.g. of the kidneys, thyroid gland, nervous system and rheumatic diseases); immune deficiencies (e.g. that caused by immunosuppresants); severe uncontrolled asthma, particularly with an FEV

1 persistently below 70%; cardiovascular diseases

with increased risk if using adrenalin; use of ß-blockers or ACE inhibitors, clinical active malignant tumor, hypersensitivity to any of the excipients. Special warnings and special precautions for use: Treatment with immunotherapy injections should only be performed by physicians qualified in allergology. Appropriate emergency treatment for shock must be immediately available during and after every injection. The patient must remain under medical supervision after the injection for 30 minutes. Side effects: Especially in the case of patients with a high degree of sensitization, intensified allergic reactions may occur. These symptoms generally arise within 30 minutes of receiving the injection. Intensified local reactions at the injection site. Reappearance of patient specific allergic symptoms as mild systemic reactions (itching of eyes, sneezing, coughing, atopic eczema), intensified systemic reactions (shortness of breath, generalized urticaria, Quincke’s Oedema), in extreme rare cases also anaphylactic shock. Occasionally delayed reactions occur (type III and IV). Package: A VenomenHAL® set consists of 6 vials with each 120 µg freezed-dried bee or wasp venom and 6 vials with each with 1.2 ml solvent. A DILUENT set contains 10 vials with each 4.5 ml diluent. The complete product information is available upon request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: May 2011

PUReTHAL ®

Composition: PUReTHAL ® contains 20,000 AUM/ml modified pollen allergen extracts, 20,000 AUeq/ml of modified mites allergen extract respectively adsorbed onto aluminium hydroxide; suspension for subcutaneous injection. Indications: Treatment of immediate type allergic disorders (IgE-mediated), such as allergic rhinitis, allergic conjunctivitis and allergic bronchial asthma, which are triggered by allergenic substances. Dosage and administration: The therapy is started with a subcutaneous injection of 0.05 ml. After the first injection the dosage is increased stepwise to a maximum dose of 0.5 ml that is finally administered in monthly intervals. Always check by aspiration that the injection needle has not entered a blood vessel. Do not exceed the maximum dose of 0.5 ml. It is advised to carry out the treatment over a period of 3 - 5 successive years. Contraindications: Acute inflammatory diseases/feverish infection at the target organ, secondary changes of the target organ (emphysema, bronchi-ectasia and others), autoimmune disorders (e.g. of the kidneys, thyroid gland, nervous system and rheumatic diseases), immune deficiencies (e.g. that caused by immuno-suppressants), severe uncontrolled asthma particularly with an FEV

1 persistency below 70%, cardiovascular failure

with increased risk if using adrenalin, clinical active malignant tumor, hypersensitivity to any of the excipients. Special warnings and special precautions for use: Treatment with immunotherapy injections should only be performed by physicians qualified in allergology. Appropriate emergency treatment for shock must be immediately available during and after every injection. The patient must remain under medical supervision after the injection for 30 minutes. Special care should be taken in case of treatment with β-blockers, pregnancy and lactation, use of the product in children below the age of 5. Prophylactic immunization should be carried out no sooner than 7 days after the last injection. Side effects: Especially in the case of patients with a high degree of sensitization, intensified allergic reactions may occur. These symptoms generally arise within 30 minutes of receiving the injection. Intensified local reactions at the injection site. Reappearance of patient specific allergic symptoms as mild systemic reactions (itching of eyes, sneezing, coughing, atopic eczema), intensified systemic reactions (shortness of breath, generalized urticaria, Quincke’s oedema), in extreme rare cases also anaphylactic shock. After use subcutaneous knots and swellings (granulomas) at the injection site may be observed. Package: PUReTHAL ® is delivered in a 6 ml multidose vial with stopper and sealed with an aluminium cap. The complete product information is available on request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: May 2011

SUBLIVAC ®

VenomenHAL ®

Basisinformationen SUBLIVAC ® FIX / SUBLIVAC ®

Zusammensetzung: Lösungen zur sublingualen Immuntherapie. SUBLIVAC ® enthält Allergenextrakte nach individueller ärztlicher Rezeptur. Sonstige Bestandteile: Glycerol, Wasser, 6-Aminohexansäure (ε-Amino-Capronsäure/EACA), Dinatrium-hydrogenphosphat, Natrium dihydrogenphosphat, Pfefferminzöl. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). Gegenanzeigen, absolute: Akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); schwere Autoimmunerkrankungen; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 70% Sollwert; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; Infektionen des Mund-/Rachenraumes; nach zahnärztlicher Behandlung (z.B. Zahnentfernung). Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Exazerbation der patientenspezifischen allergischen Symptomatik. Allergische Lokal- und/oder Allgemeinreaktionen; Bauchschmerzen und Durchfall; Exazerbation eines atopischen Ekzems. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012

Basisinformationen VenomenHAL® Biene / VenomenHAL® WespeZusammensetzung: VenomenHAL® Biene, Wirkstoff: Bienengift. VenomenHAL® Wespe, Wirkstoff: Wespengift. 6 Durchstechflaschen enthalten jeweils 120 µg reines, gefriergetrocknetes Bienen- bzw. Wespengift. Sonstige Bestandteile: HSA (humanes Serum Albumin) und Mannitol. Lösungsmittel: 6 Durchstechflaschen enthalten jeweils 1,2 ml einer Lösung von Natriumchlorid, Phenol, HSA und Wasser zur Injektion. Anwendungsgebiete: Hauttestung und Therapie von Patienten mit IgE - vermittelter Insektengiftallergie, bei denen systemische Reaktionen nach Bienen- oder Wespenstich aufgetreten sind. Gegenanzeigen: Akute Entzündungsprozesse / Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); Autoimmunerkrankungen; aktive Tuberkulose; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 70 % Sollwert; Herz- und Kreislauferkrankungen mit erhöhtem Risiko bei der Anwendung von Adrenalin; Behandlung mit ß-Blockern und mit ACE-Hemmern; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Sensibilisierung gegenüber einem der sonstigen Bestandteile des Arzneimittels. Während der Schwangerschaft ist von der Einleitung einer Behandlung mit VenomenHAL® Biene oder VenomenHAL® Wespe abzusehen. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Gebrauchs- und Fachinformation. Ferner gelten die bekannten Gegenanzeigen für die Durchführung von Hauttestungen (siehe Gebrauchs- und Fachinformation). Die genannten Kontraindikationen sollten gegen die Gefährdung des Patienten durch einen eventuellen Insektenstich abgewogen werden. Nebenwirkungen: Lokal- und/oder Allgemeinreaktionen bis hin zum anaphylaktischen Schock. Patienten nach der Injektion mindestens 30 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Gelegentlich treten auch Reaktionen vom verzögerten Typ (Typ III und IV nach Coombs und Gell) auf. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Gebrauchs- und Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Warnhinweise: Hyposensibilisierungsimpfstoffe zur Injektion dürfen nur durch allergologisch weitergebildete bzw. allergologisch erfahrene Ärzte verschrieben und angewendet werden. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012

PUReTHAL ®

Basisinformationen PUReTHAL ®

Zusammensetzung: Suspensionen zur subkutanen Injektion, enthalten an Aluminiumhydroxid adsorbierte, mit Glutaraldehyd chemisch modifizierte allergene Substanzen aus Pollen (20.000 AUM/ml) oder Milben (20.000 AUeq/ml). Sonstige Bestandteile: NaCl, Phenol, Aluminiumhydroxid, Wasser zur Injektion. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). Gegenanzeigen, absolute: Akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); Autoimmunerkrankungen; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 70% Sollwert; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Erkrankungen mit Kontraindikationen gegen die Anwendung von Adrenalin; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; nicht für Kinder unter 5 Jahren; Behandlung mit ß-Blockern. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Exazerbation der patientenspezifischen allergischen Symptomatik. Lokal- und/oder Allgemeinreaktionen bis hin zum anaphylaktischen Schock. In Einzelfällen Granulombildung am Injektionsort, Schwäche, Schwindel, Konzentrationsstörungen, Kopfschmerzen, Magen-Darm-Beschwerden, Gelenkschmerzen, Fieber. Patienten nach der Injektion mindestens 30 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Nebenreaktionen können auch noch zu einem späteren Zeitpunkt auftreten. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Warnhinweise: Hyposensibilisierungsimpfstoffe zur Injektion dürfen nur durch allergologisch weitergebildete bzw. allergologisch erfahrene Ärzte verschrieben und angewendet werden. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012

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N o t e s

N o t e s

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Ad d r e ss e s

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Romania

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