BBVU Virus Reference Department Centre for Infections Epitope profiling from HBsAg plasma BBVU Virus Reference Department Centre for Infections 25 April 2017
Drug associated changes 131 123 121 s-s- 124 126 158 Drug associated changes 131 133 99 134 161 137 --s--s- 149 164 107 -s-s- 138 D 139 s-s- 147 141 145 195 142 144 143 196 210 198
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 Mab 3 P2D3 A F P P P T D G T A K T N T C C C C T T C C R 147 124 138 C 122
Epitope profiling from HBsAg plasma Wild type, rtV173L/sE164D , rtM204V/sI195M relative epitope density 27% 30% 43%
Epitope profiling from HBsAg plasma Mab 3 Mab 2 Mab 1 63% 37% 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
Epitope profiling from HBsAg plasma
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
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
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
Acknowledgements Samreen Ijaz Richard Sloan Mathew Beale Renata Szypulska Siew Lin Ngui Samir Dervisevic National Blood Service