1. BBVU Virus Reference Department Centre for Infections
Epitope profiling from HBsAg plasma
BBVU
Virus Reference Department
Centre for Infections
25 April 2017

3. 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

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
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

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

6. 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

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