the introduction of positively-charged residues at the ... · • a biological characteristic of...
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The introduction of positively-charged residues at the
five-fold axis of foot-and-mouth virus SAT-type capsids
enhances infection of cultured cells
Melanie Chitray
Agricultural Research Council
Onderstepoort Veterinary Institute
South Africa
Introduction
• A biological characteristic of SAT viruses that complicates protective efficacy of FMD vaccines is the
intra- and inter-serotype variability.
• One of the challenges in the control of FMD is to timely and cost-effectively produce vaccines
tailored for specific geographical regions that will afford adequate protection against circulating
antigenic subtypes of FMDV field strains.
• In addition, the production of new vaccines is critically dependant upon adaptation of viruses from
the field for growth in BHK-21 cell culture.
• However, not all field strains can be adapted to suspension BHK-21 cell cultures and do not
necessarily produce desirable amounts of stable antigen.
• The production of custom-engineered FMD vaccines can be facilitated using infectious cDNA
technology, which not only makes it possible to engineer chimeric viruses containing the antigenic
region of a field strain, but also allows for the introduction of cell-culture receptor binding sites and
antigen-stabilising mutations.
• This will facilitate fast and effective cell-culture adaptation of engineered viruses: Speedy
amplification within a few passages in BHK-21 cells to create master virus seed stocks.
• Circumvents need to isolate on primary cell lines for further adaptation.
3.1 x 106
5.5 x 104
3.2 x 103
1.24 x 103
9.4 x 103
3.5 x 105
2.7 x 105
1.7 x 105
7.5 x 105
1.37 x 105
1.05 x 105
(13)
1.58 x 105
CHO-677 CHO-745
1.02 x 104
6.4 x 103
CHO-lec2
Int+, HS+
Int-, HS+
CS+
SA+ HS-
HS-
CS- SA-
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Adaptation of SAT viruses to cultured cells
SAT2
A B
SAT1
VP3
135
175
Courtesy of Abhay Kotecha
University of Oxford
2. Genetic characterisation: BHK-adapted SAT2/SAU/6/00 vs parental strain
• P1 region: 5 amino acid substitutions accumulated in encoded proteins
Capsid
protein
Amino acid
change
Observed structural effect (predicted
model)
VP3 D193N Residues overshadowed by surface
loops; Side chains face inwards; not
exposed. Minor role in adaptation. VP2 T99A
VP1 V50L Surface exposed; Conservative
change.
VP1 D55N Surface exposed; Change in local
surface charge.
VP1
T158K Surface exposed; Change in local
surface charge to strong (+); C-
terminal base of GH loop; within
known epitope.
T158K
V50L
D55N
T99A D193N
VP1
VP2
VP3
F. F. Maree
Adaptation of field virus (SAT2/Sau/6/00)
Modeled structure of SAT2/SAU/6/00
crystallographic protomer
BHK-21
CT1:
CT1 BHK58
CT = Calf thyroid cells
BHK = Baby hamster kidney cells
58x BHK-21
1. Plaque morphology: medium-milky to large clear plaques = indicative of adaptation
AIM
To investigate the effect of amino acids identified to
enhance cell culture adaptation on intra-serotype
(vSAT2ΔSAT2/Sau) and inter-serotype (vSAT2ΔSAT1/NAM) FMDV
chimeric viruses in cell entry
- Effect of the residue changes on cell entry? BHK-21, CHO-K1, CHO-677, CHO-745 cells, CHO-lec2
1110
1112
2074
Results Introduction of inter-serotype vSAT2ΔSAT1/NAM/307/98 mutations
VP4 VP2 VP3 VP1
E135K E135K,E175K
vSAT2
VP1
VP2
VP3
BHK-21: Integrin+, HS+
CHO-K1: HS+, CS+, SA+
CHO-677: HS-, CS+, SA+
CHO-745: HS-, CS-, SA+
CHO-lec2: HS+, CS+, SA-
6.1
0 0 0 0
8.4
0 0 0 0
6.9
0 0 0 0 0
1
2
3
4
5
6
7
8
9
BHK-21 cells CHO-K1 cells CHO-677 cells CHO-745 cells CHO-lec2 cells
LO
G T
ITR
E
CELL LINES
Plaque assays of the pSAT2/Nam/307/98 Mutants
NAM307/98WT vSAT2NAM1C135 vSAT2NAM1C135,175
Results Introduction of intra-serotype (vSAT2ΔSAT2/Sau) mutations
VP4 VP2 VP3 VP1 vSAT2
5.2
0 0 0 0
6.4
0 0 0 0 0
1
2
3
4
5
6
7
BHK-21 cells CHO-K1 cells CHO-677 cells CHO-745 cells CHO-lec2 cells
LO
G T
ITR
ES
CELL LINES
Plaque assays of vSAT2/Sau1D50,55
vSAT2Sau_WT vSAT2Sau1D50,55
V50L-
D55N
BHK-21: Integrin+, HS+
CHO-K1: HS+, CS+, SA+
CHO-677: HS-, CS+, SA+
CHO-745: HS-, CS-, SA+
CHO-lec2: HS+, CS+, SA-
VP4 VP2 VP3 VP1 vSAT2
T158K
5.2
0 0 0 0
6.8
4.5
0 0
4.1
0
1
2
3
4
5
6
7
8
BHK-21 cells CHO-K1 cells CHO-677 cells CHO-745 cells CHO-lec2 cells
LO
G T
ITR
E
CELL LINES
Plaque assays of vSAT2/Sau1D158
vSAT2Sau_WT vSAT2Sau1D158
Introduction of intra-serotype (vSAT2ΔSAT2/Sau) mutations
BHK-21: Integrin+, HS+
CHO-K1: HS+, CS+, SA+
CHO-677: HS-, CS+, SA+
CHO-745: HS-, CS-, SA+
CHO-lec2: HS+, CS+, SA-
5.2
0 0 0 0
6.4 6.6
0 0
6.2
0
1
2
3
4
5
6
7
BHK-21 cells CHO-K1 cells CHO-677 cells CHO-745 cells CHO-lec2 cells
LO
G T
ITR
E
CELL LINES
Plaque assays of vSAT2/Sau1D83
vSAT2Sau_WT vSAT2Sau1D83
VP4 VP2 VP3 VP1 vSAT2
E83K
Introduction of intra-serotype (vSAT2ΔSAT2/Sau) mutations
BHK-21: Integrin+, HS+
CHO-K1: HS+, CS+, SA+
CHO-677: HS-, CS+, SA+
CHO-745: HS-, CS-, SA+
CHO-lec2: HS+, CS+, SA-
Introduction of intra-serotype (vSAT2ΔSAT2/Sau) mutations
5.2
0 0 0 0
8.6 8.7
0 0
5.7
0
1
2
3
4
5
6
7
8
9
10
BHK-21 cells CHO-K1 cells CHO-677 cells CHO-745 cells CHO-lec2 cells
LO
G T
ITR
E
CELL LINES
Plaque assays of vSAT2/Sau1D110-112
vSAT2Sau_WT vSAT2Sau1D110-112
KGG110-112KRR
VP4 VP2 VP3 VP1 vSAT2
BHK-21: Integrin+, HS+
CHO-K1: HS+, CS+, SA+
CHO-677: HS-, CS+, SA+
CHO-745: HS-, CS-, SA+
CHO-lec2: HS+, CS+, SA-
Energetically favourable binding site - GRID
VP1 110-112
Heparan Sulphate
molecule
Courtesy of Abhay Kotecha
University of Oxford
Summary
• The virus strains adapted via frequent cell culture passage resulted in some strains possibly
utilising chondroitin sulphate for cell entry and two of these viruses could be using sialic acid
as a receptor. The α5β1 and αvβ5 receptor usage is also unknown. Further investigation is
required.
• The introduction of the positively charged mutations for each of the chimeras, increased the
affinity of the mutants to utilise the integrin receptors for cell entry.
• The change in plaque morphology of the WT vs the chimera viruses observed is indicative of
adaptation.
• Amino acid changes in VP1 positions: T158K, E83K, KGG110-112KRR allows for heparan
sulphate usage without the need for consistent cell culture passaging.
This study has shown to be beneficial for SAT type vaccine production where viruses that
were previously impossible to adapt to cell culture can be designed with improved growth
properties.
Acknowledgements
Prof Jacques Theron
Dr Francois Maree
Dr Sonja Maree
Dr Peninnah Nsamba
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