05 december 2015 bbvu virus reference department centre for infections epitope profiling from hbsag...
TRANSCRIPT
18 April 2023
BBVU
Virus Reference Department
Centre for Infections
Epitope profiling from HBsAg plasma
99
123121 124
107
137
138 139 147
149
D164
145
144
158
s-s-
s-s--s-s-
--s--s-
195
196
198210
126
141
131
133134
142143
161
Drug associated changes
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
CCCCC
CT
TT
TT
T
T
P
R
PPA
AQ G
G
SM
F
DK
N
122
124 138147
C
Mab 3
P2D3
Mab 2
H3F5 Mab 1
D2H5
Epitope profiling from HBsAg plasma
27%
30%
43%
Wild type, rtV173L/sE164D , rtM204V/sI195M relative epitope density
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 mutationsneither of which alone has a detectable effect
DRUG INDUCED MUTANTS BEHAVE LIKEVACCINE ESCAPE MUTANTS
Epitope profiling from HBsAg plasma
Accession Number
118
119
120
123
130
131
133
134
139
140
141
142
143
144
145
164
166
172
173
175
180
189
190
191
194
195
196
197 P2D3 D2H5 H3F5
FN295497 M1 L M1 C 44.45 53.34 2.20FN295498 M2 A M2 D 32.29 24.43 43.28FN295499 M4 S M4 B 34.55 32.04 33.41FN295500 M5 T S M5 D 40.44 26.55 33.01FN295501 M6 S L M6 D 36.39 28.43 35.18FN295502 M7 S M7 B 34.27 35.69 30.05FN295503 M9 A M9 A 38.46 29.67 31.87FN295504 M10 A I M10 D -0.03 23.14 76.89FN295505 M11 N M11 C 40.41 22.08 37.51FN295506 M12 P M12 D 34.35 21.13 44.51FN295507 M13 L I M13 E 26.24 20.06 53.71FN295508 M15 I* M M15 A 51.67 18.58 29.75FN295509 M16 I L M16 E 16.55 18.21 65.24FN295510 M17 L V* F M17 E 39.42 9.67 50.91FN295511 M19 Y* M19 A 49.71 18.29 32.00FN295512 M20 L E R M20 D 48.38 0.38 51.24FN295513 M21 L R M21 D 48.64 -0.19 51.55FN295514 M22 L A M22 D 33.52 11.55 54.93FN295515 M23 L M23 D 37.82 9.73 52.45FN295516 M24 L M24 D 37.99 8.64 53.37FN295517 M25 L M25 D 34.94 13.10 51.96FN295518 M26 A G M26 A 65.60 1.41 32.99FN295519 M27 A M27 D 51.89 2.36 45.75FN295520 M28 E M M28 E 24.24 6.97 68.80FN295521 M31 R T M31 C 36.81 25.03 38.16FN295522 M32 R M32 B 75.60 0.30 24.10FN295523 M33 Stop* M33 D 36.03 21.64 42.32FN295524 M34 I H V M34 C 90.51 7.23 2.26FN295525 M35 I N S L K M35 D 94.56 -0.76 6.21FN295526 M36 A D M M36 D 25.49 23.10 51.40FN295527 M37 L* M37 C 36.59 21.35 42.06FN295528 M38 M M38 A 43.54 21.12 35.34FN295529 M39 T G M39 C 69.63 4.66 25.71
P2D3D2H5H3F5
Amino Acid Sequence Changes mAb Performance
ins
Sam
ple No
Sam
ple No
Genotype
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
• 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
Epitope profiling from HBsAg plasma
Acknowledgements
• Samreen Ijaz• Richard Sloan• Mathew Beale• Renata Szypulska• Siew Lin Ngui• Samir Dervisevic
National Blood Service