h7n9 influenza and enterovirus 71 (ev71) virus vaccine ... against c4 viruse challenge pbs 2+...

H7N9 influenza and Enterovirus 71 (EV71) virus vaccine development in Taiwan

The Head of R&DJuine-Ruey Chen Ph.D

國光生物科技(股)公司ADIMMUNE CORPORATION

Major Products (1) JEV vaccine, domestic market share : 100%

(2) Tuberculin PPD, domestic market share : 100%

(3) Tetanus toxoid, domestic market share : 50%

(4) Influenza vaccine, domestic market share : 30%

Founded in 1965 45 years of expertise in Vaccinology

Company location Taichung

Business Focus Vaccines, Diagnostics, & Biological Products

Primary Business Developer / Manufacturer / Distributor

Technology Partner Crucell , Kitasato Institute

The only cGMP manufacturer and company of human vaccine in Taiwan

Overview of the Adimmune

H7N9 raise pandemic concernsand the vaccine preparation

• By 29 May 2013, approximately 2 months after the initial report, the numberof laboratory-confirmed H7N9 infections reached 132, with 37 deaths.

• The case fatality proportion reached approximately 25%, which is a provisionalvalue because many patients remain hospitalized as of 8 May 2013 and thenumber of mild cases remains unknown.

• However, most patients had a history of recent exposure to poultry, generallyat live bird markets, however there is no evidence of sustained onwards virustransmission to other people.

• The concern for a pandemic caused by a newly emerged avian influenza A virushas led to clinical trials with H7N9 candidate vaccines .

• The Minister of health, Taiwan supported two grants for the Taiwan vaccineindustry to develop the H7N9 vaccines.



Conclusion: This inactivated subunit influenza A (H7N7) vaccine was safe but

poorly immunogenic in humans

The challenge of development of H7 influenza vaccine

(Sanofi Pasteur Inc)



Seed virus：E4 or E5 10 7~8 EID50

10-3 dilution 10-4 dilution 10-5 dilution 10-6 dilution

0.2mL/embryo egg 0.2mL /embryo egg 0.2mL /embryo egg 0.2mL /embryo egg

34±0.5℃

30hr48hr

52hr

5 eggs/group

Allantoic fluid collection

初離心移除卵蛋白8,000g 4℃ 30min

超高速離心收集病毒(Pellet down)

130,000g 4℃ 60min

30hr48hr

52hr

30hr48hr

52hr

30hr48hr

52hr

To estimate the HAU titers

Investigation of the best incubation condition for the H7N9 virus production

Improvement of the H7N9 vaccine efficacy in Adimmune Corp.

Al(OH)3

Squalene

w/o adjuvant

(1) Human 15µg/ dose test in mice= 0.5µg (2) Human 45ug/ dose 1.5 µg(3) Human 90µg/ dose 3.0 µg

Inactivated split and whole H7N9 virus vaccine





The electron micrograph of A/Mallard/Netherlands/12/2000-IBCDC-1(H7N7)

A/Shanghai/2/2013 IDCDC-RG32A (H7N9) inactivated split or whole viruses

How to prepare an effective influenza vaccine

with glycoprotein HA

Chen JR, Ma C, Wong CH. Trends Bio technol. 2011 Sep;29(9):426-34.

Purification of HA protein of H7N9 with Ni-NTA column

30 60 90 150 300 500 IM(mM)

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Adimmune

Digestion of H7N9 extracellular domain of HA with N-glycosidase

100

70

55

40

130

33

25

15

Endo H cleave only high mannose structures (n = 2-150, x =

(Man)1-2, y = H)

M 2 5 10 20 30 60 min

Endo H

100

70

55

40

130

170

PNGase F hydrolyzes nearly all types of N-glycan chains from

glycopeptides/proteins. [x = H or sugar(s)]

M 0 1 3 5 7 9 20 30 40 60 min

PNGase F

Purification and characterization of the H7N9 extracellular domain of HA

ADIM-rHA

ADIM-Whole

Quantification of the H7N9 vaccine bulk for clinical trial

Split H7N9 HA Standard curve

y = 9.7017x + 0.2575

R2 = 0.9911

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.9000

1.0000

0.0000 0.0200 0.0400 0.0600 0.0800

HA Conc (ug/ml)

OD45

0 Standard(RD SplitH7N9)

線性 (Standard(RDSplit H7N9))

336.06

Sample nameSample HAConc(μg/ml)

Sample totalprotein(μg/ml)

FAS13001 Split H7N9 312.84 246.8FAS13002 Split H7N9 291.2FAS13003 Split H7N9 318.55 284.4

ELISA SRIDMethods

322.5±12.1 274.1±23.9

Immunogenicity of H7N9 vaccine in mice

A

B

C

14 28 35 50



Inactivated H7N7 split and whole virus antigens combined with different adjuvants as a vaccine in mice (1)

(Split

) 0.5

ug w/o 3

(Split

) 0.5

ug +Al(O

H)

(Split

) 0.5

ug +Addavax 3

(whole

) 0.5

ug +Al(O

H)

(whole

) 0.5

ug +Addavax

(Split

) 1.5

ug w/o

(Split

) 1.5

ug +Al(O

H)3

(Split

) 1.5

ug +Addavax

(whole

) 1.5

ug +Al(O

H)3

(whole

) 1.5

ug +Addavax

(Split

) 3ug w

/o

(Split

) 3ug +

Al(OH)3

(Split

) 3ug +

Addavax

(whole

) 3ug +

Al(OH)3

(whole

) 3ug +

Addavax0

200

400

600

800

1000

1200

Ser

um

HI t

iters

(G

MT

)

0.5ug 1.5ug 3ug





(Split) 0.5ug

w/o 3

(Split) 0.5ug

+Al(O

H)

(Split) 0.5ug

+Add

avax

(who

le) 0

.5ug

w/o 3

(who

le) 0

.5ug

+Al(O

H)

(who

le) 0

.5ug

+Add

avax

Non

- immun

ized

con

trol

0

500

1000

1500

2000

2500

3000

Ne

utr

ali

za

tio

n t

ite

rs

(G

MT

)

(Split) 0.5ug

w/o 3

(Split) 0.5ug

Al(O

H)

(Split) 0.5ug

Add

aVax

(Who

le) 0

.5ug

w/o

(Who

le) 0

.5ug

Al(O

H)3

(Who

le) 0

.5ug

Add

aVax

Non

-immun

ized

con

trol

0

500

1000

1500

2000

2500

(Split) 0.5ug

w/o 3

(Split) 0.5ug

+Al(O

H)

(Split) 0.5ug

+Add

avax

(who

le) 0

.5ug

w/o 3

(who

le) 0

.5ug

+Al(O

H)

(who

le) 0

.5ug

+Add

avax

Non

- immun

ized

con

trol

0

500

1000

1500

Vaccine: H7N9 Shanghai-2Virus: H7N9 Shanghai-2

Vaccine: H7N9 Shanghai-2Virus: H7N9 Anhui-1

Vaccine: H7N9 Shanghai-2Virus: H7N7

H7N9 (Shanghai-2) vaccine not only elicited neutralizing antibodies against same strain of virus but also conferred cross-protection against

H7N9 (Anhui-1) and H7N7 viruses

HAI≧40



0 1 2 3 4 5 6 7 8 9 10111213140

102030405060708090

100110

AddaVax

0.5 g H7N9-S

0.5 g H7N9-S-Al(OH)3

0.5 g H7N9-S-AddaVax


Su

rvia

l R

ate

(%

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 140

10

20

30

40

50

60

70

80

90

100

110

days after infection

Bo

dy w

eig

ht

(%)

H7N9 Virus Challenge studies in vaccine immunized mice

0 1 2 3 4 5 6 7 8 9 10111213140

102030405060708090

100110

AddaVax

0.5 g H7N9-S

0.5 g H7N9-S-Al(OH)3



Su

rvia

l R

ate

(%

)

Positive Results Presented from H7N9 vaccine with adjuvants in Clinical Trial

Summary



1. The H7N9 inactivated virus vaccine is sufficient to confer cross-neutralizing immunity against H7N9 and H7N7 viruses

2. The Squalene-based H7N9 vaccine may be the best formulation for the further vaccine preparation

3. Adimmune alum-based H7N9 vaccine results showed that the seroprotection rate were met with the criteria for Phase II clinical and will conduct the phase III study in 2015.

The Development of EV71 Inactivated Virus Vaccine with

Single Use Bioreactor


Juine-Ruey Chen Ph.D陳俊叡

(A) Host：Vero cells(B) Virus：Validated virus seed from NHRI.(C) Adimmune has successfully established the EV71 vaccine production with 10L and 50L bioreactors.(D) Adimmune was supported by Taiwan government to speed up the progress of EV71 vaccine

development, 200L and 1000L production scale will be established in 2014.

The scale up the production scale for preparation of EV71 vaccine in phase II clinical trials

Production of EV71 vaccines with 200L single use bioreactor

R&D Department ManagerJuine-Ruey Chen Ph.D

陳俊叡


Adimmune

Production of EV71 vaccine with 200L Bioreactor has

been successfully established by Adimmune R&D in NHRI

Adimmune

Production of EV71 vaccine with 200L Bioreactor has been

successfully established by Adimmune R&D in NHRI

Virus challenge to test if the EV71 vaccine provide sufficient protection in mice


ELISpot

Cross-protection of Adimmune EV71(B4; E59) vaccine

EV71(C2) challengeEV71(B4) vaccination

PBS group

Vaccine group:

-- 2 g EV71+Al(OH)3

-- 6 g EV71+Al(OH)3

-- 2 g EV71+ AddaVAX

Current status (D35):

All die All survival

C2 (Asia strain)

Adimmune-B4

(Bioreactor)

Mice immunized vaccine Virus challenge

B4 (Taiwan)

C4 (China strain)

Adimmune EV71 (B4) vaccine conferred cross-protective

immunity against C2 and C4 viruses challenge

(A)

(B)

0 2 4 6 8 10 12 14 160

102030405060708090

100110

2 g B4+Al(OH)3

6 g B4+Al(OH)3

PBS

Vaccine: B4

Virus: C2

Su

rv

ial R

ate

(%

)

0 2 4 6 8 10 12 14 16-200

-100

0

100

200

2ug+Al(OH)3

6ug+Al(OH)3

PBS

days after infection

Bo

dy

We

igh

t (%

)Adimmune EV71 (B4) vaccine conferred cross-protective

immunity against C2 virus challengeAdimmune EV71 (B4) vaccine conferred cross-protective

immunity against C2 viruse challenge

0 1 2 3 4 5 6 7 8 9 1011121314150

102030405060708090

100110

PBS2ug+Al(OH)36ug+Al(OH)3

Vaccine: B4

Virus: C4

days post-infection

Su

rviv

al ra

te (

%)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15-50

0

50

100

150

PBS

2ug+Al(OH)36ug+Al(OH)3

days post-infection

Bo

dy

we

igh

t (%

)

6μg vaccine

2μg vaccine

PBS


immunity against C4 virus challenge

Day 6 post infection

(A)

(B)


immunity against C4 viruse challenge

PBS 2+ AddaVAX 2+AlPO4 6+AlPO4

Mock

C2

C4

The features of pathological changes in muscles of EV71 infected

hSCARB2-Tg mice.

In situ EV71 distribution in the brainstem of hSCARB2-Tg mice

The GMP pilot production plan of EV71 vaccine

http://www.flickr.com/photos/shoken/3489376734/

Thank you for your attention

http://www.flickr.com/photos/shoken/3489376734/

Welcome to Taiwan

h7n9 influenza and enterovirus 71 (ev71) virus vaccine ... against c4 viruse challenge pbs 2+...

Documents