BBVU Virus Reference Department Centre for Infections

1. Epitope profiling from HBsAg plasma BBVU Virus Reference Department Centre for Infections

3. 123 121 124 s-s- 126 158 Drug associated changes 131 133 99 134 161 --s--s- 149 137 164 -s-s- D 107 s-s- 138 139 147 141 145 195 142 144 143 196 210 198

4. Epitope profiling from HBsAg plasma • Map HBsAg epitopes using 3 monoclonal antibodies which bind to discrete epitopes • Coat solid phases individually and detect captured HBsAg Mab 2 H3F5 Mab 1 D2H5 T S G Q M F A Mab 3 P2D3 P P D T P G T K A N T C T C C T C C T C R 147 138 124 C 122

5. 27% 43% 30% Epitope profiling from HBsAg plasma Wild type, rtV173L/sE164D , rtM204V/sI195M relative epitope density

6. Mab 3 Mab 2 37% 63% Mab 1 Epitope profiling from HBsAg plasma rtV173L/sE164D + rtM204V/sI195M Total loss of one epitope induced by two mutations neither of which alone has a detectable effect DRUG INDUCED MUTANTS BEHAVE LIKE VACCINE ESCAPE MUTANTS

7. Epitope profiling from HBsAg plasma

9. Epitope profiling from HBsAg plasma • Natural HBsAg mutants still cause diagnostic difficulties— • HBsAg neg/low reactivity samples BUT HBeAg and HBV DNA pos P120Q, N131P, K160N, E164G F20S, Q30R, F134S, G145R, Y200F T116N, M133T, T134S, Y200F Q129P, F134L, G145R, S154P Q101R, I126T and G145R • These samples have been identified since the beginning of 2009

10. Epitope profiling from HBsAg plasma • Virus genotype was important • Mutation phenotype differs between genotypes • G145R in genotype B backbone – D2H5 epitope ablated • G145R in genotype C backbone – D2H5 epitope is maintained • Influence of backbone • HBsAg mutants need to be studied in the context of their own backbone • SDM of a lab backbone will lead to inappropriate conclusions

11. Epitope profiling from HBsAg plasma • C-terminal changes associated with drug resistance modulate epitope profile • I195M reduced D2H5 reactivity • I195M + G145R restored D2H5 reactivity • ? HBsAg structure • Downstream C’ mutations do impact on HBsAg phenotype CONCLUSIONS: • Sequence analysis not sufficient to give predictions for HBsAg loss • Phenotyping through epitope profiling more appropriate • Polydisperse particulate solid phase offers primary phenotypic screening

12. Acknowledgements • Samreen Ijaz • Richard Sloan • Mathew Beale • Renata Szypulska • Siew Lin Ngui • Samir Dervisevic • National Blood Service