DECEPTIVE IMPRINTING OF FMDV: MAPPING OF CAPSID HYPER-VARIABLE REGIONS BETWEEN TWO DISPARATE SAT2

STRAINS

Tovhowani RamulongoLia Rotherham, Pamela Opperman, Jacques Theron, Elizabeth Reider, Francois Maree

Introduction

~ SAT2 is the most prevalent and diverse of all SAT serotypes

~ Recent incursion into North Africa and the Middle East

~ FMDV uses deceptive imprinting to decoy host immune response from vital

conserved regions i.e. RGD

~ These decoy epitopes may convey some neutralizing activity or protection, but are

largely strain specific

~ Genetically modified viruses provide a valuable tool for the manipulation of the

biological properties of field and laboratory strains

~ This present a possible avenue towards design of safe and effective vaccines that

provide broader protection against the diverse SAT2 field strains

Introduction

~ Masking of immunodominant epitopes may allow the immune system to refocus and

recognize previously subdominant conserved epitopes that induce protection

~ Immune dampening strategy by replacing known hypervariable immunodominant epitope

regions between two disparate strains

~ SAT2/ZIM/7/83 (topotype II) previously used as vaccine strain and the devastating

SAT2/EGY/9/2012 (topotype VII)

~ This Egypt 2012 outbreak resulted in highest death rate of young animals

Aim

To dampen and refocus the immunity towards neutralizing

epitope regions of a SAT2 virus

Objectives

~To predict hypervariable immunodominant epitope regions

between two SAT2 viruses

~To replace predicted antigenic regions of SAT2/ZIM/7/83

with the corresponding regions of SAT2/EGY/9/2012

~To characterize the epitope replaced mutants

Prediction of antigenic sites

~ Systematic analysis of the capsid proteins of SAT2/EGY/9/2012 and

SAT2/ZIM/7/83

~ Emphasis on regions of high amino acid variability

~ Flexible exposed structural loops of the viral capsid

~ Previously identified antigenic sites on SAT2/ZIM/7/83 or types A and O

~ Predicted epitope region of SAT2/EGY/9/2012 were introduced into the pSAT2

plasmid; a SAT2/ZIM/7/83 infectious clone

Mutant viruses

GH-loop C-terminal

VP1VP3VP2

VAVDQMPS VVL

129IETDRLPK136 187AVF189

HTNKTSFV

VGDHTRVF

81LGEHERVW88

AKGG

108SHNN111

VYTKAA

135KYTQQS140

GH-loop

C-terminal

43 LTNRTAFA50

GH-loop

135 160

135

196 216

160 196 216

vEGYHNNSAT2

vEGYepiSAT2

vEGYTQQS SAT2

vEGYsite3 SAT2

vEGYEHERSAT2

vEGYVP2/VP3/VP1SAT2

vEGYGH-loopSAT2

vEGYGH-loopSAT2SAT2

vEGYC-termSAT2

vEGYGH & Ct SAT2

VP1VP3VP2 2ALab 3´UTR polyAIRESpolyCS-frag

SspI XmaI

ZIM/7/83

EGY/9/12

VP1 site3

Visual pentamer display

VP1 EHER

VP1 HNN

VP3 combo

VP1 TQQS

Cyan : VP1 Magenta : VP3Green : VP2

Plaques morphology

BHK-21 IB-RS-2 CHO-K1

no plaques

vEGYVP2/VP3/VP1SAT2

vEGYGH-loopSAT2SAT2

vEGYC-termSAT2

vEGYGH&Ct SAT2

no plaques

ZZ-R-127

vSAT2

Antigenicity of epitope-replaced viruses

Zim/7/83 sera pool SAR/3/04 sera pool0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

vSAT2

vEGYepiSAT2

vEGYsite3SAT2vEGYEHERSAT2vEGYHNNSAT2vEGYTQQSSAT2vEGYGH-loopSAT2vEGYC-termSAT2vEGYGH & CtSAT2vEGYVP2/VP3/VP1SAT2vEGYGH-loopSAT2SAT2

Ser

um

tit

res

Antigenic profiling with mAbs

0-45

45-80

80-100

ID5 GG1 GE11 GD12 DA10vEGYepiSAT2

vEGYsite3 SAT2

vEGYEHERSAT2

vEGYHNNSAT2

vEGYTQQSSAT2

vEGYGH-loopSAT2

vEGYC-termSAT2

vEGYGH&CtSAT2

vEGYVP2/VP3/VP1SAT2

vSAT2 used as reference with 100% binding

DA10 footprint

Opperman et al. 2014

Cyan : VP1 Magenta : VP3Green : VP2White : GH-loop

Conclusions~ CHO-K1 cells were unable to sustain replication of vEGYGH &CtSAT2 and vEGYGH-

loopSAT2SAT2 mutants

~ vEGYGH-loopSAT2SAT2

~ Reduction in cross reactivity against ZIM/7/83 and SAR/03/04 sera

~ vEGYVP3/VP3/VP1SAT2

~ Reduction in cross reactivity against ZIM/7/83 and SAR/03/04 sera

~ At least 60% reduction in reactivity to all mAbs

~ vEGYGH&CtSAT2

~ Increase in cross reactivity against SAR/03/04 sera

~ 30% reduction in binding to mAbs 1D5 and GD12

~ vEGYsite3SAT2

~ Reduction in cross reactivity against ZIM/7/83 and SAR/03/04 sera

~ 70% reduction in reactivity to mAb DA10

Collaborators

Oxford University – Drs Elizabeth Fry and Abhay Kotecha

Agricultural Research Council

University of Pretoria -Prof Jacques Theron

Pirbright Institute - Drs Bryan Charleston and Nick Juleaff

Plum Island (USDA) – Dr Elizabeth Reider

Thank you

• Supervision and support: Dr Francois Maree and Team

• Travel sponsors :DTRA

Whitehead Scientific

• GFRA for platform