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