rapid engineering of fmd vaccine and challenge viruses

Rapid Engineering of FMD Vaccine and Challenge Viruses

Lee, S-Y 1,2, Lee, Y-J1., Kim, R-H1, Park, J-N1, Park, M-E1,2, Ko, M-K1, Choi, J-H2, Chu, J-Q3, Lee K-N1, Kim, S-M1, Tark, D1, Lee, H-S1, Ko, Y-J1, Seo, M-G1, Park, J-

W 1, Lee, M-H 1, Lee , J-S2, Kim, B1

2017 GFRA, Incheon, KOREA, OCTOBER 25-27, 2017

1 Animal and Plant Quarantine Agency, Republic of Korea 2 College of Veterinary Medicine, Chungnam National University, Republic of Korea, 3 Clinical Research Center of the Affiliated Hospital of Guangdong Medical College,China, 4 Korea Zoonosis Research Institute, Chonbuk National University, Republic of Korea

PARK, Jong-Hyeon 1 Ph.D., D.V.M.

[email protected]

Objectives and Methods

• We studied effective strategies for customized vaccine and challenge tool for protection and evaluation of specific serotypes or subtypes of FMDV.1. Producing the recovered virus by Infectious clones for

7 serotypes2. Pathogenesis test of recovered virus in mice and pigs3. Pig immunization with the antigen and challenge test

using the recovered virus

cDNA infectious clones for FMDV serotypes

(b)

(e)

P3 3’UTRA18O Manisa-FG

O (Om-O-PanAsia2)

Asia1 (Om-AsMOG)

A (Om-A22)

C (Om-C-Ob)

SAT1 (Om-SAT1-SA)

SAT2 (Om-SAT2-SAU)

SAT3 (Om-SAT3-SA)

C21

S-fragT7

IRES L P1 P2

O/PAK/44/2008 (Synthetic)

Asia1/MOG/2005

A22 Iraq/24/64

C1 Oberbayern (Synthetic)

SAT1/5sa/61 (Synthetic)

SAT2/SAU/6/00 (Synthetic)

SAT3/2sa57/59 (Synthetic)

VP4 VP2 VP3 VP1

P1

The capsid-coding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes .The seven serotype viruses were rescued successfully.

7 Serotypes

Synthesis of P1 region (5)

Amplified (2)

1. Virus recovery and characterization by infectious clones

Result (1)

2. Pathogenesis of the viruses in animalsfor possibility as a challenge virus

3. Immunization of experimental vaccine in pigs (and others) and challenge

O Manisa-FG

Asia1 (Om-AsMOG)A (Om-A22) C (Om-C-Ob)

SAT1 (Om-SAT1-SA) SAT2 (Om-SAT2-SAU) SAT3 (Om-SAT3-SA)

O (Om-O-PanAsia2)Smallest plaque

Virus plaques

Electron microscopy of the viruses

24 ~ 29 nm

O Manisa-FG O (Om-O-PanAsia2) Asia1 (Om-AsMOG)A (Om-A22)

C (Om-C-Ob) SAT1 (Om-SAT1-SA) SAT2 (Om-SAT2-SAU) SAT3 (Om-SAT3-SA)

Using strain-specific primers O-Manisa

O-PanAsia2

A

Asia1

C

SAT1

SAT2

SAT3

Common

Virus typing by strain-specific PCR

Omamisa

-FG

Om-O-P

anAsia

2

Om-A22

Om-AsM

OG

Om-C-O

bOm-S

AT1-S

AOm-S

AT2-S

AUOm-S

AT3-S

AOman

isa-W

T

104

105

106

107

108BHK-21BGK

IBRS-2ZZR

LFBK

Viruses

Copy

num

bers

of F

MDV

RNA

/0.1

ml

0 2 4 6 8 10 12 14 16 18 20 22 24100

101

102

103

104

105

106

O manisa-FGOm-O-PanAsia-2Om-A22Om-AsMOG

Om-C-ObOm-SAT1-SAOm-SAT2-SAU

O manisa-WT

Copy

num

bers

of F

MDV

RNA

/0.1m

l

Om-SAT3-SA

Hours after Infection

Virus growth in various cells

In ZZR cell

36 hpi

** *

O1 Manisa

O –PA2 Asia1 C SAT1

C

Lateral flow assay 2 for Ag(PBM kit) designed for 4 serotypes

T

Confirmation of different antigens using rapid test kit

C

Lateral flow assay1 for Ag(Svanova Kit) designed for 7 serotypes

TPosi

Posi

+ + -

Net

O. D

O manisa-FG

O-PanAsia2

A22

Asia1/MOG

C Obe

SAT1 SAU

SAT2 SASAT3 SA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

O A Asia1 C SAT1 SAT2 SAT3 Pos.

Each chimeric FMDV showed its serotype-specific antigenicity

Antigen-binding reaction by Ag-ELISA

Serological relationships among FMDV recombinants by cross-virus neutralization (VNT) using reference

anti-sera

Reference anti-sera against

following strains

Viruses used in cross VNT and the reciprocal arithmetic titers

O Manisa-

FG

Om-O-PanAsia2

Om-A22

Om-AsMO

GOm-C- Ob

Om-SAT1-SA

Om-SAT2-SAU

Om-SAT3-SA

O1Manisa 724 362 - - - - - -A22 16 - 90 - - - - -Asia-Shamir - - - 90 - - - -C-Resende 16 - - - 181 - - 16SAT1-BOT 1/68 64 22 - 22 16 362 - -SAT2-ZIM 5/81 - - - - - - 1448 -SAT3-ZIM 9/80 - - - - - - - 362

- : reciprocal titer of <16 (negative cutoff of VNT).


Result (2)



0 1 2 3 4 5 6 7 80

20

40

60

80

100 O manisa-FGOm-Wild typeOm-O-PanAsia-2Om-A22Om-Asia-MogOm-C-ObOm-SAT1-SAOm-SAT2-SAUOm-SAT3-SANon-treatment

Suckling mouse (n=10)

Days Post Infection

Surv

ival

rat

e (%

)Survival rate in suckling mice (7 days-old)

C As iaSAT3SAT1

SAT2

O Manisa

O PA-2

• High virulence ; C, O PA-2, Asia1, SAT1, SAT3• Low virulence : SAT2, O1 Manisa

O Man

isaOm-O

-Pan

Asia2

Om-A22

Om-Asia

MOGOm-C

-Ob

Om-SAT1-S

AOm-S

AT2-SAU

Om-SAT3-S

ANon-Trea

tmen

t

100

101

102

103

104 Adult mouse (n=3)

Recombinant viruses

Cop

y nu

mbe

rs o

f FM

DV

RN

A /

0.1m

l ser

a

****** ** *** ***

0 1 2 3 4 5 6 7 8 9 10 11 1280

85

90

95

100

105

110

115

Om-O-PanAsia2Om-A22Om-AsMOGOm-C-ObOm-SAT1-SAOm-SAT2-SAUOm-SAT3-SA

O manisa-FGAdult mouse (n=3)

Non-treatment

Days Post Infection

Bod

y W

eigh

t (%

)

Pathogenesis in adult mice (C57 BL/6)

Body weight Viremia

C > O PA-2 > A22 > SAT1, O Manisa, Asia1, SAT3, SAT2

C

A22

O PA-2

A. Clinical Score

0 2 4 6 8 1002468

10121416


O manias-FGSwine (n=2)

Days post infectionC

linic

al s

core

B. Nasal Discharge

0 2 4 6 8 101

10

100

1000

10000


O manias-FG

Days post infection

Nas

al d

isch

arge

of v

irus

by

rRT-

PCR

/0.1

ml

C. Viremia

0 2 4 6 8 101

10

100

1000

10000


O manias-FG

Days post infection

Vire

mia

by

rRT-

PCR

/0.1

ml

Pathogenesis in Pigs (1)

SAT2 and O manisa ; Low virulence

SAT2

O Manisa

O man

isa-FG

Om-O-P

anAsia

2

Om-A22

Om-AsM

OGOm-C

-Ob

Om-SAT1-S

AOm-S

AT2-SAU

Om-SAT3-S

A

10 0

10 1

10 2

10 3

10 4

10 5

0

10

20

30

40

50

60

70

Clinical score (sum)

Virus in nasal swab (sum)Swine (n=2) Virus in sera (sum)

Viruses

Viru

s de

tect

ion

by r

RT-

PCR

/0.1

ml

Clinical score

Comparison of clinical indexes (clinical score/ virus detection in swab and sera)

Pathogenesis in Pigs (2)

C, A22, O PanAsia2, SAT1, SAT3> Asia1 > O manisa, SAT2

O Manisa-FG

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80O Manisa-WTOm-PanAsia2NSP Antibody

Days Post Infection

VN ti

ters

(log

)

Percent inhibition % (N

SP)

Om-PanAsia2

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-PanAsia2O Manisa-WTNSP antibody

Days Post Infection

VN ti

ters

(log

)


SP)

Om-A22

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-A22A Malaysia97NSP antibody

Days Post Infection

VN ti

ters

(log

)


SP)

Om-AsMOG

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-AsMOGAsia1 ShamirNSP antibody

Days Post Infection

VN ti

ters

(log

)


SP)VN titers and NSP antibody

Serology in the infected pigs

NSP AntibodyHomo. SP Antibody Hetero. SP Antibody

6 dpi 5 dpi

5 dpi 5 dpi

1 day delayed detection

Om-SAT3 SA

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-SAT3 SASAT3 ZIM4NSP antibody

Days Post Infection

VN ti

ters

(log

)


SP)

Om-C Ob

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-C ObC3 ResendeNSP Antibody

Days Post Infection

VN ti

ters

(log

)


SP)

Om-SAT1 SA

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-SAT1 SASAT1 BOTNSP Antibody

Days Post Infection

VN ti

ters

(log

)


SP)

Om-SAT2 SAU

0 2 4 6 8 10 12 14

1.5

2.0

2.5

3.0

3.5

0

20

40

60

80Om-SAT2 SAUSAT2 ZIMNSP antibody

Days Post Infection

VN ti

ters

(log

)


SP)

Serology in the infected pigs

NSP AntibodyHomo. SP Antibody Hetero. SP Antibody

5 dpi 5 dpi

7dpi 5 dpi

Cross-virus neutralization of type-specific antibodies from the virus infected pigs

Chimeric FMDV-infected groups

(pig #, n=2)

Viruses used in cross VNT1 and the titers

O Manisa

Om-O-PanAsi

a2

Om-A22

Om-AsMO

G

Om-C-Ob

Om-SAT1-

SA

Om-SAT2-SAU

Om-SAT3-SA

O Manisa (#223, 233) 256/512 45/181 -2, - -, - -, - -, - -, - -, -

Om-PanAsia2 (#215,235) 128/256 724/512 -, - -/16 -, - -, - -, - -, -

Om-A22 (#224, 236) -/90 -/128 256/90 -/- -, - -, - -, - -, -

Om-AsMOG (#200, 222) 16/16 181/- -, - 181/90 -, - -, - -, - -, -

Om-C-Ob (#212, 218) -, - -, - -, - -, - 512/512 -, - -, - -, -

Om-SAT1-SA (#219, 220) -, - -, - -, - -, - -, - 181/512 -, - 16/-

Om-SAT2-SAU (#291, 234) -, - -, - -, - -, - -, - -, - 362/1024 -, -

Om-SAT3-SA (#189, 217) -, - -, - -, - -, - -, - -, - -, - 362/181

1 VNT: Virus neutralization test in sera from the pigs 12 days post-infection of each chimeric FMDV2 - : reciprocal titer of <16 (negative cutoff of VNT).

3 4 50.0

0.2

0.4

0.6

0.8

1.0 Pig-CS/Ad. mice-BWLPig-CS/Suc.mice-FRAd. mice-BWL/Suc.mice-FR

Swine/Adult mice/Suckling mice

Days Post Infection

Cor

rela

tion

(r) o

f clin

ical

inde

xes

betw

een

anim

al s

pece

sCorrelation of clinical indexes among animals

(Pig / Suckling mice/ Adult mice)

• adult mice ; body weight loss• suckling mice ; fetal rate• pigs ; clinical score

3day 5dayPig /Suc.miceAd mice/ Suc mice

0.830.92


Result (3)



0 1 2 3 4 5

1.0

1.5

2.0

2.5

3.0

3.5 Goats (n=4)

O Manisa-wtOm-PanAsia2Om-A22Om-AsMOG

Weeks post vaccination

VN ti

ters

(log

)

O(O PA-2)+ A+ Asia1

0 1 2 3 4 5

1.0

1.5

2.0

2.5

3.0

3.5Goats (n=4)

Om-C-ObOm-SAT1-SAOm-SAT2-SAUOm-SAT3-SA

Weeks post vaccinationVN

tite

rs (l

og)

C+ SAT1+SAT2+ SAT3

Immune responses by experimental vaccine in goats

O manisa- Hetero

Asia

A22O PA2

SAT2CSAT3

SAT1

5 µg /dose/ serotypes

0 8 142228 1 2 3 4 5 6 7 8 9 10

1.5

2.0

2.5

3.0

3.5 Control groupVaccination group

Challenge

O1m-O-PA2

DPV/DPI

VN ti

ters

(log

10)

0 8 142228 1 2 3 4 5 6 7 8 9 10

1.5

2.0

2.5

3.0

3.5Control groupVaccination group

Challenge

O1m-A22

DPV/DPI

VN ti

ters

(log

10)

0 8 142228 1 2 3 4 5 6 7 8 9 10

1.5

2.0

2.5

3.0

3.5Control groupVaccination group

Challenge

O1m-Asia1 MOG

DPV/DPI

VN ti

ters

(log

10)

( VN titers )

VN titers in immunized experimental trivalent vaccine and challenged pigs

5 dpi/vac6 dpi/con

5 dpi/vac5 dpi/con

5 dpi/vac5 dpi/con

• Immunized with 7.5 µg /dose/ serotypes and • challenged the recovered viruses.

1 2 3 4 5 6 7 8 9 10100

101

102

103

104

105

0

5

10

15

Clinical score

#134

Clinical ScoreSeraNasal Swab

Days post challenge

Cop

y nu

mbe

rs o

f vira

l RN

A /0

.1m

l

1 2 3 4 5 6 7 8 9 10100

101

102

103

104

105

0

5

10

15C

linical score#135

Days post challenge

Cop

y nu

mbe

rs o

f vira

l RN

A /0

.1m

l

1 2 3 4 5 6 7 8 9 10100

101

102

103

104

105

0

5

10

15

Clinical score

#136

Days post challenge

Cop

y nu

mbe

rs o

f vira

l RN

A /0

.1m

l

1 2 3 4 5 6 7 8 9 10100

101

102

103

104

105

0

5

10

15

Clinical score

#137

Days post challenge

Cop

y nu

mbe

rs o

f vira

l RN

A /0

.1m

l

1 2 3 4 5 6 7 8 9 10100

101

102

103

104

105

0

5

10

15

Clinical score

#138

Days post challenge

Cop

y nu

mbe

rs o

f vira

l RN

A /0

.1m

l

1 2 3 4 5 6 7 8 9 10100

101

102

103

104

105

0

5

10

15

Clinical score

#139

Days post challenge

Cop

y nu

mbe

rs o

f vira

l RN

A /0

.1m

l

Trivalent –vaccine injection groups (n=4) : protected

Pigs vaccinated with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge

Clinical characterization in immunized and challenged pigs

Control groups (n=4), ( Challenged with 3 rec viruses) : not protected

0 1 2 3 4 5 6 7 8 9 10343536373839404142 Control group (n=2)

Vaccination group (n=4)

DPI

Bod

y te

mpe

ratu

re (o C

)

Body temperature in immunized and challenged pigs

Vaccinated

control

Summary• This strategy will be a useful tool for rapidly

generating customized FMD vaccines or challenge viruses. • Using synthetic or amplified genes for 7 serotype

viruses against epidemic strains. • The virus has an antigenic similarity between

chimeric FMDV and wild types• We can get new challenge viruses against various

serotypes depend on P1 surface protein

• Especially it will provide a useful source for vaccine study in the countries which have prohibited introduction of FMDVs.

rapid engineering of fmd vaccine and challenge viruses

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