The introduction of positively-charged residues at the five-fold axis of foot-and-mouth virus SAT-type capsids enhances infection of cultured cells

Transcription

1 The introduction of positivelycharged residues at the fivefold axis of footandmouth virus SATtype capsids enhances infection of cultured cells Melanie Chitray Agricultural Research Council Onderstepoort Veterinary Institute South Africa

2 Introduction A biological characteristic of SAT viruses that complicates protective efficacy of FMD vaccines is the intra and interserotype variability. One of the challenges in the control of FMD is to timely and costeffectively 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 BHK21 cell culture. However, not all field strains can be adapted to suspension BHK21 cell cultures and do not necessarily produce desirable amounts of stable antigen. The production of customengineered 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 cellculture receptor binding sites and antigenstabilising mutations. This will facilitate fast and effective cellculture adaptation of engineered viruses: Speedy amplification within a few passages in BHK21 cells to create master virus seed stocks. Circumvents need to isolate on primary cell lines for further adaptation.

3 CHO677 CHO745 CHOlec2 (13) Int +, HS x x x x x x x x x x x x x x 10 4 Int, HS + CS + SA + HS HS CS SA

4 Adaptation of SAT viruses to cultured cells SAT1 A B VP SAT2 Courtesy of Abhay Kotecha University of Oxford

5 Adaptation of field virus (SAT2/Sau/6/00) 1. Plaque morphology: mediummilky to large clear plaques = indicative of adaptation 2. Genetic characterisation: BHKadapted SAT2/SAU/6/00 vs parental strain P1 region: 5 amino acid substitutions accumulated in encoded proteins CT1: BHK21 VP1 F. F. Maree Capsid protein Amino acid change Observed structural effect (predicted model) 58x BHK21 VP3 D193N Residues overshadowed by surface VP2 T99A loops; Side chains face inwards; not exposed. Minor role in adaptation. CT1 BHK58 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 CT = Calf thyroid cells BHK = Baby hamster kidney cells VP2 V50L D55N T99A Modeled structure of SAT2/SAU/6/00 crystallographic protomer VP3 D193N

6 AIM To investigate the effect of amino acids identified to enhance cell culture adaptation on intraserotype (vsat2 ΔSAT2/Sau ) and interserotype (vsat2 ΔSAT1/NAM ) FMDV chimeric viruses in cell entry Effect of the residue changes on cell entry? BHK21, CHOK1, CHO677, CHO745 cells, CHOlec2

7 LOG TITRE vsat2 Results Introduction of interserotype vsat2 ΔSAT1/NAM/307/98 mutations VP4 VP2 VP3 VP1 E135K E135K,E175K Plaque assays of the psat2/nam/307/98 Mutants BHK21 cells CHOK1 cells CHO677 cells CHO745 cells CHOlec2 cells CELL LINES NAM307/98WT vsat2nam1c135 vsat2nam1c135,175 VP1 VP BHK21: Integrin +, HS + CHOK1: HS +, CS +, SA + CHO677: HS, CS +, SA + CHO745: HS, CS, SA + CHOlec2: HS +, CS +, SA VP2

8 LOG TITRES Results Introduction of intraserotype (vsat2 ΔSAT2/Sau ) mutations vsat2 VP4 VP2 VP3 VP1 V50L D55N Plaque assays of vsat2/sau1d50, BHK21 cells CHOK1 cells CHO677 cells CHO745 cells CHOlec2 cells CELL LINES BHK21: Integrin +, HS + CHOK1: HS +, CS +, SA + CHO677: HS, CS +, SA + CHO745: HS, CS, SA + CHOlec2: HS +, CS +, SA vsat2sau_wt vsat2sau1d50,55

9 LOG TITRE Introduction of intraserotype (vsat2 ΔSAT2/Sau ) mutations vsat2 VP4 VP2 VP3 VP1 Plaque assays of vsat2/sau1d158 T158K BHK21: Integrin +, HS CHOK1: HS +, CS +, SA + 3 CHO677: HS, CS +, SA + 2 CHO745: HS, CS, SA + 1 CHOlec2: HS +, CS +, SA BHK21 cells CHOK1 cells CHO677 cells CHO745 cells CHOlec2 cells CELL LINES vsat2sau_wt vsat2sau1d158

10 LOG TITRE Introduction of intraserotype (vsat2 ΔSAT2/Sau ) mutations vsat2 VP4 VP2 VP3 VP1 E83K Plaque assays of vsat2/sau1d BHK21 cells CHOK1 cells CHO677 cells CHO745 cells CHOlec2 cells CELL LINES BHK21: Integrin +, HS + CHOK1: HS +, CS +, SA + CHO677: HS, CS +, SA + CHO745: HS, CS, SA + CHOlec2: HS +, CS +, SA vsat2sau_wt vsat2sau1d83

11 LOG TITRE Introduction of intraserotype (vsat2 ΔSAT2/Sau ) mutations vsat2 VP4 VP2 VP3 VP1 KGG110112KRR Plaque assays of vsat2/sau1d BHK21: Integrin +, HS CHOK1: HS +, CS +, SA CHO677: HS, CS +, SA + 2 CHO745: HS, CS, SA BHK21 cells CHOK1 cells CHO677 cells CHO745 cells CHOlec2 cells CELL LINES vsat2sau_wt vsat2sau1d CHOlec2: HS +, CS +, SA

12 Energetically favourable binding site GRID Courtesy of Abhay Kotecha University of Oxford VP Heparan Sulphate molecule

13 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, KGG110112KRR 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.

14 Acknowledgements Dr Francois Maree Prof Jacques Theron Dr Sonja Maree Dr Peninnah Nsamba