Natural history of HBV infection (adapted from Torbenson Lancet 2003)

1. Natural history of HBV infection(adapted from Torbenson Lancet 2003) Time Chronic infection 10E9 No Serol. marker Healthy carriage Viral load Occult chronic infection 10E3 Anti-HBc Anti-HBe Recovery occult DNA Anti-HBs Low Immune response High

2. Differential diagnosis of occult HBV Marker Recovered Chronic Anti-HBc Positive Positive Anti-HBe + ++ Anti-HBs Positive Negative Viral load (copies/ml) <10E3 <10E4 PC stop codon No Yes BCP mutations Few Frequent Secondary response Yes No to HBsAg

3. Distribution of HBV DNA load in 126 HBsAg + and 12 HBsAg - samples from Ghana, West Africa 45 11% of DNA+ samples are HBsAg - 40 35 30 25 Number of samples 20 15 10 5 0 <10 <1E2 <1E3 <1E4 <1E5 <1E6 <1E7 <1E8 <1E9 HBV DNA load (IU/ml)

4. Investigation of 7 occult HBV infections from Ghanaian blood donors Rapid test HBsAg negative : 5107 EIA neg HBV DNA pos = 12 2d sample negative 4 Studied 7 insufficient volume 1 Presence of virion by Ab capture No signal 1 no capture 3 Capture + 3 Sequencing

5. Characterisation of 7 occult HBV ID DNA load Anti-HBc Anti-HBe Anti-HBs IU/ml 1237 13 Neg 979 16 Pos Neg Neg 1240 26 Pos Pos Neg 1252 6 Neg 245 117 Pos Neg Neg 1504 17 Neg 3461 29 Neg

6. Virus in plasma incubated with mouse mAb against ‘a’ determinant overnight + Add magnetic beads with sheep anti-mouse IgG covalently bound Capture on magnet Wash Lyse captured particle and extract nucleic acid Capture of HBV particles from plasma using magnetic beads Q-PCR

7. Preliminary data on occult HBV infection ID Viral anti-HBsAg S seq BCP/PC Complete load capture sequence genome 1237 13 0 0 0 979 16 0 0 0 1240 26 0 0 0 1252 6 0 + 0 245 117 + + + + 1504 17 + 0 + 3461 29 + + + 0

8. F F F F H H H F 100 100 G G E G E E G G E 245-12 245-21 100 100 E A D A A 100 D 100 A 100 D 100 A D D B C C B C C B C B B HBV whole genome Neighbour-Joining tree Sample 245 VL 117 IU/ml Complete sequence of 2 clones 0.01 substitutions/site

9. Main sequence features in 4 OBI ID BCP Pre-core Pre-S1/Pre-S2 ‘a’ 1762-64 1896 SC 245-12 WT + WT S140L 245-21 WT + C 2930 del. S140L 1504 WT WT - - 1252 - - - T131A 3461 WT WT - WT

10. 110 120 130 140 K01252 C P L I P G S S T T S T G P C R T C T T L A Q G A S M F P S C C C S K P S D G N C K03461 C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C S K P S D G N C K00245-12 C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C L K P S D G N C K00245-21 C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C L K P S D G N C X75657E C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C S K P S D G N C X75664E C P L I P G S S T T S T G P C R T C M T L A Q G T S M F P S C C C S K P S D G N C AB106564E C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C S K P A D G N C AB091255E C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C S K P S D G N C AB091256E C P L I P G S S T T I T G P C R T C T T L A Q G T S M F P S C C C S K P S D G N C C P L I P G S S T T S T G P C R T C T T L A Q G T S M F P S C C C S K P S D G N C Loop 1 Loop 2 Loop 3 Loop 4 Substitutions in the ‘a’ loops of HBV genotype E

11. Anti-HBs response to HBV vaccine of 2 asymptomatic donors with anti-HBc only Primary injection 2d injection 5 4 3 2 1 0 Anti-HBs (S/CO) 0 10 20 30 40 50 Days after primary vaccination

12. Conclusions • Molecular studies of occult HBV are difficult • Most include enveloped virions • Viral load is consistently <500 IU/ml • Detection requires single donation screening • Serologically most seem to be anti-HBc only • BCP and PC wild type suggest recovered infection despite absence of detectable anti-HBs • No evidence of ‘a’ determinant escape was found

13. 0.005 substitutions/site Partial S region (490 bp) Neighbour-Joining Tree B B B C B B B D C C D C C D 100 D D C D 100 98 G G G A 100 G 99 G G 100 A A 3461 245-21 A 100 E A A 245-12 E 1252 E E E 95 100 E F F F H F F H H F H