The introduction of positively-charged residues at the ... · • A biological characteristic of SAT viruses that complicates protective efficacy of FMD vaccines is the intra- and

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


LO

G T

ITR

ES

CELL LINES

Plaque assays of vSAT2/Sau1D50,55

vSAT2Sau_WT vSAT2Sau1D50,55

V50L-

D55N







T158K

5.2

0 0 0 0

6.8

4.5

0 0

4.1

0

1

2

3

4

5

6

7

8


LO

G T

ITR

E

CELL LINES

Plaque assays of vSAT2/Sau1D158

vSAT2Sau_WT vSAT2Sau1D158

Introduction of intra-serotype (vSAT2ΔSAT2/Sau) mutations






5.2

0 0 0 0

6.4 6.6

0 0

6.2

0

1

2

3

4

5

6

7


LO

G T

ITR

E

CELL LINES

Plaque assays of vSAT2/Sau1D83

vSAT2Sau_WT vSAT2Sau1D83


E83K








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


LO

G T

ITR

E

CELL LINES

Plaque assays of vSAT2/Sau1D110-112

vSAT2Sau_WT vSAT2Sau1D110-112

KGG110-112KRR







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

Documents

The introduction of positively-charged residues at the ... · • A biological characteristic of SAT viruses that complicates protective efficacy of FMD vaccines is the intra- and