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BBVU Virus Reference Department Centre for Infections

This study examines the epitope profiling of HBsAg in plasma, utilizing three monoclonal antibodies to map specific epitopes. It highlights the impact of drug-associated mutations, including the loss of epitopes induced by combinations of mutations that do not affect epitopes individually. Natural HBsAg mutants complicate diagnostics. The findings assert the importance of genotype in mutation phenotypes and suggest that sequence analysis alone is inadequate for predicting HBsAg losses. Epitope profiling through phenotyping is recommended for accurate understanding.

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BBVU Virus Reference Department Centre for Infections

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

  2. 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

  3. 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

  4. 27% 43% 30% Epitope profiling from HBsAg plasma Wild type, rtV173L/sE164D , rtM204V/sI195M relative epitope density

  5. 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

  6. Epitope profiling from HBsAg plasma

  7. 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

  8. 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

  9. 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

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

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