Production fo a safe EMCF-FMDV recombinant vaccine against FMD

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HAL Id: hal-02806663

https://hal.inrae.fr/hal-02806663

Submitted on 6 Jun 2020

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Production fo a safe EMCF-FMDV recombinant vaccine against FMD

Margot Carocci

To cite this version:

Margot Carocci. Production fo a safe EMCF-FMDV recombinant vaccine against FMD. Building Bridges Workshop, Jul 2011, Bejing, China. 28 diapos. �hal-02806663�

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Production of a safe EMCV-FMDV recombinant vaccine against FMD

UMR1161 (ANSES,ENVA,INRA) Maisons-Alfort, FRANCE

Margot Carocci Margot Carocci Margot Carocci Margot Carocci

Building Bridges, Beijing 2-4 July 2011

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Plan

Introduction

Why a recombinant vaccine EMCV-FMDV?

Results

I. production and characterization of EMCV∆2A

II. Virulence attenuation on human primary astrocytes III. Virulence attenuation on mice

IV. Production EMCV-FMDV Conclusion

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Why a recombinant vaccine EMCV-FMDV?

- A live attenuated vaccine: fast and good protection - Easy to produce less confinement, less expensive

- Distinguish easily vaccinated and infected animals

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FMDV / EMCV

• Picornaviridae :

- positive ssRNA, ~8kb

- Structural and genomic organization similar - Encode a single polyprotein,

- Similar viral cycle

They are the 2 Picornaviridae, of different genus (Aphtovirus / Cardiovirus), showing more similarity

Why a recombinant vaccine EMCV-FMDV?

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Genomic organization

Clivage sites

2A3C^pro L^pro

Why a recombinant vaccine EMCV-FMDV?

VPg VPgVPg

VPg polyCpolyCpolyCpolyC IRESIRESIRESIRES polyApolyApolyApolyA

L L L

L VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A 3B3B3B3B 3C3C3C3C 3D3D3D3D

P1 P2 P3

Structural proteins Non structurales proteins

???

?

Polyprotein PolyproteinPolyprotein Polyprotein

FMDVFMDVFMDV FMDV

L 1A1A1A1A L

LL

L VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A3B3B3B3B 3C3C3C3C 3D3D3D3D

??? EMCV ?

EMCVEMCV EMCV

VPg VPgVPg

VPg polyCpolyCpolyCpolyC IRESIRESIRESIRES polyApolyApolyApolyA

L L L

L VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A 3B3B3B3B 3C3C3C3C 3D3D3D3D L

L L

L VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A 3B3B3B3B 3C3C3C3C 3D3D3D3D

P1 P2 P3

Structural proteins Non structurales proteins

???

?

Polyprotein PolyproteinPolyprotein Polyprotein

FMDVFMDVFMDV FMDV

L 1A1A1A1A L

LL

L VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A3B3B3B3B 3C3C3C3C 3D3D3D3D

??? EMCV ?

EMCVEMCV EMCV

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Why a recombinant vaccine EMCV-FMDV?

Development of a chimera EMCV-FMDV virus : Production of an hybrid genome :

Non structural proteins EMCV

polyA

L 1A

L VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D

P1 - FMDV

FMDV O France2001 P2 P3

Capsid proteins

3 ’UTR 5 ’UTR

Non

EMCV

polyA

L 1A

L VP4 VP2 VP3 VP1

L VP4 VP2 VP3 VP1 2A2A 2B2B 2C2C 3A 3B 3C 3D

3A 3B 3C 3D

P1 - FMDV

FMDV O France2001 P2 P3

proteins 5 ’ 3 ’

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Towards a safe vaccine

Can infect many different animal species

Depending on the animal species and the viral strain, EMCV can cause Myocarditis

Reproduction disorders Diabetes

Encephalitis

=> Attenuation of EMCV virulence is needed

EMCV

: Encephalomyocarditis virus

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Plan

Introduction

Results

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B279 strain 210 passed on BHK-21 : non-virulent for pigs

(P.Denis and F. Koenen Arch Virol 2003)

C9 clone => a deletion in the 2A protein sequence.

Test virulence on mice C57Bl/6

I. EMCV ∆ 2A production and characterization

0 10 20 30 40 50 60 70 80 90 100

J1 J2 J3 j4 J7 J8 J9 J10 J14 J16 J18 J21 J22

Day post inoculation

Healthy mice (%)

279 210 210C2 210C3 210C4 C2 C3 C7 C9

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In vitro transcription

ΔΔ

ΔΔ2A2A2A2A EMCVEMCVEMCVEMCV

Incorporation of a deletion in 2A, using a reverse genetic previously developed in our lab (Bakkali

et al., 2002).

Transfection

EMCVΔ2A production

I. EMCV ∆ 2A production and characterization

EMCV∆2A

VP2 VP2 VP2

VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 VP4

VP4 VP4

VP4 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A3B3B3B3B 3C3C3C3C 3D3D3D3D LLLL VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A3B3B3B3B 3C3C3C3C 3D3D3D3D LLLL VP4VP4VP4VP4 VP2VP2VP2VP2 VP3VP3VP3VP3 VP1VP1VP1VP1 2A2A2A2A 2B2B2B2B 2C2C2C2C 3A3A3A3A3B3B3B3B 3C3C3C3C 3D3D3D3D LLLL

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I. EMCV ∆ 2A production and characterization

1,0E+02 1,0E+03 1,0E+04 1,0E+05 1,0E+06 1,0E+07

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

hpi hpi hpi hpi Virus titer (logVirus titer (logVirus titer (logVirus titer (log¹⁰¹⁰¹⁰¹⁰TCID50/ml)TCID50/ml)TCID50/ml)TCID50/ml)

EMCV ∆2A EMCV

One-step growth cycle

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EMCV Δ2A EMCV

Plaque assay on BHK-21

I. EMCV ∆ 2A production and characterization

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Plan

Introduction

Results

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EMCV

Able to infect a wide range of animal species.

Has been described to possibly infect and cause of head ache, vomiting, malaise, fever... In Human.

(Oberste et al, Emerg Inf Dis 2009)

Test infectivity of Human primary astrocytes by EMCV, and the attenuation of EMCV∆2A.

II. Virulence att. on human primary astrocytes

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Astrocytes 5hpi

EMCV can infect and replicate on human primary astrocytes

II. Virulence att. on human primary astrocytes

1,E+02 1,E+03 1,E+04 1,E+05 1,E+06 1,E+07

0 10

hpi

Virus titer (log10 TCID50/ml)

EMCV-VP1 Dapi

EMCV

EMCV∆2A EMCV-VP1

Dapi

EMCV∆2A is able to infect and replicate on human primary astrocytes but less efficiently.

1,E+02 1,E+03 1,E+04 1,E+05 1,E+06 1,E+07

0 10

hpi

Virus titer (log10 TCID50/ml)

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EMCV∆2A virus is less virulent than the wild type on Human primary Astrocytes

0 20 40 60 80

0,1 1 10 20

MOI

Cell viability 48hpi(%)

BHK infected with EMCV∆2A BHK infected with EMCV

Astrocytes infected with EMCV∆2A Astrocytes infected with EMCV

II. Virulence att. on human primary astrocytes

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Plan

Introduction

Results

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Clinical signs, death ?

Intraperitoneally inoculation

C57BL/6

II. Virulence attenuation on mice

4x10⁵pfu/ mice

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EMCV ∆2A does not induce any clinical sign in C57BL/6 mice

III. Virulence attenuation on mice

0 1 2 3 4 5 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Day post-inoculation

Healthy mice

EMCV EMCV∆2A

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III. Virulence attenuation on mice

1,0E-07 1,0E-06 1,0E-05 1,0E-04 1,0E-03 1,0E-02 1,0E-01 1,0E+00

1 2 3 4 7 8 9 22

Dpi

log10 (EMCV / 18S RNA)

EMCV EMCV∆2A

Detection of EMCV ∆2A in mice heart only at 3dpi 1 out of 5 mice heart & at 9dpi in 3 out of 5. No more at 22dpi.

in mice heart.

=>EMCV ∆2A is able to replicate in vivo.

1,0E-16 1,0E-14 1,0E-12 1,0E-10 1,0E-08 1,0E-06 1,0E-04 1,0E-02 1,0E+00

1 2 3 4 7 8 9

Dpi log10 (EMCV / 18S RNA) EMCV in lombar spinal cord

EMCV in thoracic spinal cord EMCV in brain

EMCV∆2A

in mice CNS.

EMCV ∆2A does not reach the mice CNS.

Number of viral RNA copy:

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Conclusion sur EMCV ∆ 2A

Virulence attenuation of EMCV∆2A : on BHK-21

on human primary astrocytes

(With first demonstration of human primary astrocytes sensitivity to EMCV.)

on mice

The EMCV∆2A virus is an attenuated virus,

Which should be a safe base for a recombinant EMCV-FMDV vaccine.

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Plan

Introduction

Results

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Shuttle plasmid with partial cDNA of EMCVdeleted in 2A protein.

Substitution of EMCV P1 by FMDV-P1 in the shuttle plasmid

Substitution du fragment genomique EMCV (P1-2A) par corresponding fragment from the shuttle vector (P1FA-2A∆)

ΔΔΔ

Δ2A2A2A2A EMCVEMCVEMCVEMCV

Δ ΔΔ Δ2A2A2A2A

EMCV EMCV EMCV EMCV 5’ncr+ L5’ncr+ L5’ncr+ L5’ncr+ L EMCVEMCVEMCV

EMCVP2 & P3 P2 & P3 P2 & P3 P2 & P3 + 3’ncr + 3’ncr+ 3’ncr + 3’ncr

FMDV FMDV FMDV FMDV P1P1 P1P1

Mutagenesis insertion restrictions sites PCRs from cDNA of EMCV.

Transfection in BSR-T7

FMDV FMDV FMDV FMDV P1P1P1 P1 EMCV

EMCV EMCV EMCV 2A+2B part 2C 2A+2B part 2C 2A+2B part 2C 2A+2B part 2C Δ Δ Δ Δ2A2A2A2A

EMCV EMCV EMCV EMCV part 5’ncr + L part 5’ncr + L part 5’ncr + L part 5’ncr + L

IV. Production of IV. Production of IV. Production of

IV. Production of

_EMCV_∆_2A-P1FMDV

Production of chimera virus?

?

pMC1

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IV. Production of EMCV IV. Production of EMCV IV. Production of EMCV

IV. Production of EMCV ∆ 2A ----P1FMDV P1FMDV P1FMDV P1FMDV

P1-FMDV

VP0-FMDV VP2-FMDV

M

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Mock

p1.26∆2A pMC1

pMC1

IV. Production of EMCV IV. Production of EMCV IV. Production of EMCV

IV. Production of EMCV ∆ 2A ----P1FMDV P1FMDV P1FMDV P1FMDV

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Virulence attenuation of EMCV∆2A : on BHK-21

on human primary astrocytes

(With first demonstration of human primary astrocytes sensitivity to EMCV.)

on mice

The EMCV∆2A virus safe base for a recombinant.

Transfection of pMC1 (recombinant cDNA):

Cytopathic effects and expression of P1-FMDV viral particle production ?

To be confirmed…

Conclusion

Conclusion Conclusion

Conclusion

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UMR 1161 (ANSES,ENVA,INRA) Maisons-Alfort, FRANCE

Stephan Zientara, Labib Bakkali Kassimi, Monique Guy.

Muriel Coulpier.

Acknowledgements

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Production fo a safe EMCF-FMDV recombinant vaccine against FMD

Plan

Why a recombinant vaccine EMCV-FMDV?

FMDV / EMCV

Why a recombinant vaccine EMCV-FMDV?

Why a recombinant vaccine EMCV-FMDV?

Why a recombinant vaccine EMCV-FMDV?

Towards a safe vaccine

EMCV

Plan

I. EMCV ∆ 2A production and characterization

I. EMCV ∆ 2A production and characterization

I. EMCV ∆ 2A production and characterization

I. EMCV ∆ 2A production and characterization

Plan

II. Virulence att. on human primary astrocytes

II. Virulence att. on human primary astrocytes

II. Virulence att. on human primary astrocytes

Plan

II. Virulence attenuation on mice

III. Virulence attenuation on mice

III. Virulence attenuation on mice

Conclusion sur EMCV ∆ 2A

Plan

IV. Production of IV. Production of IV. Production of

IV. Production of

IV. Production of EMCV IV. Production of EMCV IV. Production of EMCV

IV. Production of EMCV ∆ 2A ----P1FMDV P1FMDV P1FMDV P1FMDV

IV. Production of EMCV IV. Production of EMCV IV. Production of EMCV

IV. Production of EMCV ∆ 2A ----P1FMDV P1FMDV P1FMDV P1FMDV

Conclusion

Conclusion Conclusion

Conclusion

Acknowledgements

Thank you for your attention !