Efficacy of Hepatitis B Vaccine Against Antiviral Drug-Resistant Hepatitis B Virus Mutants in the Chimpanzee Model

1. Efficacy of Hepatitis B Vaccine Against Antiviral Drug-Resistant Hepatitis B Virus Mutants in the Chimpanzee Model

2. Abstract Hepatitis B virus (HBV) mutants resistant to treatment with nucleoside or nucleotide analogs and those with the ability to escape from HBV-neutralizing antibody have the potential to infect HBV-vaccinated individuals

3. Abstract To address this potential serious public health challenge, we tested the efficacy of immunity induced by a commercial hepatitis B vaccine against a tissue culture-derived, clonal HBV polymerase mutant in HBV seronegative chimpanzees

4. Abstract The polymerase gene mutant contained a combination of three mutations (rtV173L, rtL180M, rtM204V), two of which resulted in changes to the overlapping viral envelope of the hepatitis B surface antigen (sE164D, sI195M)

5. Abstract Prior to the HBV mutant challenge of vaccinated chimpanzees, we established virologic, serologic, and pathologic characteristics of infections resulting from intravenous inoculation of the HBV polymerase gene mutant and the sG145R vaccine-escape surface gene mutant

6. Abstract Cloning and sequencing experiments determined that the three mutations in the polymerase gene mutant remained stable and that the single mutation in the surface gene mutant reverted to the wild-type sequence

7. Abstract Immunological evidence of HBV replication was observed in the vaccinated chimpanzees after challenge with the polymerase gene mutant as well as after rechallenge with serum-derived wild-type HBV (5,000 chimpanzee infectious doses administered intravenously), despite robust humoral and cellular anti-HBV immune responses after hepatitis B vaccination

8. Abstract ——Conclusion Our data showing successful experimental infection by HBV mutants despite the presence of high anti-HBs levels considered protective in the vaccinated host are consistent with clinical reports on breakthrough infection in anti-HBs-positive patients infected with HBV mutants.

9. Abstract——Conclusion In the absence of a protective humoral immunity, adaptive cellular immune responses elicited by infection may limit HBV replication and persistence

10. Background • Hepatitis B is a global health problem that related to chronic active hepatitis, liver cirrhosis, and primary liver cancer • Vaccination against HBV prevents new infections and efforts toward the control of chronic disease have involved the therapeutic inhibition of viral replication using analogs of nucleotides or nucleosides • Lamivudine was the first drug in this class to be licensed for the treatment of chronic hepatitis B and remains in widespread use

11. Mechanism of analogs of nucleotides or nucleosides Lamivudine Adefovir Entecavir Infective HBV particles Infective HBV particles Partial Double-stranded DNA HBsAg capsid DNA polymerase RT (-)-DNA wrapped Pre-genomemRNA A(n) cccDNA mRNA

12. Background Pol gene (HBV Polymerase gene) Its reverse transcriptase district contains seven functional sequence (A-G)，The main target area of nucleoside or nucleotide analogs widely used in clinical is located in the reverse transcriptase (RT) domain B and C, therefore resistant variantion is located in the domain B and C

13. Background Antiviral drug-resistant mutations selected during treatment with lamivudine in the HBV polymerase gene (Pol) clustered within its B domain (rtV173L, rtL180M) and in the C domain in the conserved YMDD motif (rtM204V)

14. It has been reported that these mutant viruses are transmissible and may have the potential to cause breakthrough infections among recipients of hepatitis B vaccine • The development of neutralizing antibody escape in those individuals may result from the changed amino acid composition of the HBV envelope protein, as the envelope and polymerase open reading frames overlap in the circular HBV genome

15. ORF S: coding for HBV envelope protein [neutralization of the “a” determinant of the hepatitis B surface antigen (HBsAg) protein] P: coding for DNA polymerase Hepatitis B virus genome

16. alterations in the neutralization of the “a” determinant of the hepatitis B surface antigen (HBsAg) protein have been reported in patients with mutations in the Pol-gene receiving therapy with lamivudine • A markedly reduced binding of anti-HBs antibodies to HBsAg with mutations corresponding to Pol-protein changes (rtV173L, rtL180M, rtM204V) has been demonstrated in vitro

17. These findings raise the possibility of the selection of lamivudine-resistant HBV mutants with antigenically modified HBsAg proteins that could act as vaccine escape mutants and infect vaccinated individuals in whom anti-HBs exerts a further positive selection pressure

18. The most common mutation in the S-gene product is glycine to arginine at position 145 (sG145R) • It has been suggested that the immunity induced by existing vaccines may not be protective against various HBV mutants with altered surface proteins because of the conformational nature of the “a” determinant • Furthermore, the existence of such HBV isolates and the potential of these mutants to infect hepatitis B-vaccinated individuals may lead to occult HBV infection and, consequently, have serious public health implications

19. Materials and Methods

20. Tissue Culture-Derived Mutant and Wild-TypeHBV Inocula • Point mutations were introduced by site-directed mutagenesis into a 1.5-times genome-length wt-HBV belonging to genotype A, subtype adw 2 • The sG145R mutation was produced in the S-gene with overlapping rtW153Q mutation in the Pol-gene（HBsAg Classical immune evasion strain ） • A combination of three HBV Pol-gene mutations, rtV173L, rtL180M, and rtM204V, was produced that correspond to sE164D and sI195M mutations in the S-gene (rtL180M does not result in an amino acid change in the S-gene)

21. Chimpanzee Infectivity Experiments Eleven colony-born chimpanzees (six females and five males, ranging in age from3 to 11 years and weighing between 11 to 37 kg), determined to be seronegative for markers of HBV infection, were used for infectivity, vaccination, and challenge studies All chimpanzees inoculated with the wild-type and mutant HBV inocula were followed up for 180 to 200 days after inoculation

23. Chimpanzee HBV Immunization and Challenge Experiments • Two chimpanzees (CH10364 and CH10369) were given a pediatric dose (10 μg/0.5mL) of a licensed recombinant hepatitis B vaccine at 0, 28, and 56 days • One control chimpanzee(CH10301) was vaccinated with hepatitis A vaccine • All three vaccinated chimpanzees were challenged with the clonal HBV Pol-gene mutant containing 9.8* 109 copies of HBV DNA 42 days after the third vaccine dose

24. Chimpanzee HBV Immunization and Challenge Experiments • the control chimpanzee was challenged 35 days after the hepatitis A vaccination • The two hepatitis B-vaccinated chimpanzees were further cross-challenged with 5,000 chimpanzee infectious doses (CID) of human serum-derived wt-HBV inoculum 216 days after the Pol-gene mutant challenge • A naive chimpanzee, CH10270, served as a control for the wt-HBV challenge and was also inoculated with 5,000 CIDs of the wt-HBV inoculum

25. Chimpanzee HBV Immunization and Challenge Experiments • Serial serum specimens were collected from all the chimpanzees twice weekly and whole blood samples for peripheral blood mononuclear cells (PBMCs) isolation were collected every 2 weeks • Serum alanine aminotransferase (ALT) levels were measured in fresh serum specimens. Individual cutoff values for ALT levels were established for each animal using at least 10 preinoculation measurements

26. Serological Markers of HBV Infection • All serum samples were tested for : HBsAg antibody to HBsAg (anti-HBs) total antibody to hepatitis B core antigen (anti-HBc) IgM anti-HBc hepatitis B e antigen (HBeAg) total anti-HBe • The levels of anti-HBs in samples collected during vaccination and challenge periods were also quantitatively measured

27. T-Cell Responses • PBMCs from the chimpanzees were enriched and tested in an interferon-ΥELISpot assay for reactivity to HBV surface and polymerase antigens • Millipore were precoated with antibodies to human interferon-Υ and 2*105 chimpanzee PBMCs in medium added to each well • Duplicate wells received pools of overlapping synthetic peptides that spanned either amino acids 67-237 of the HBsAg or amino acids 1-344 of the reverse transcriptase domain of the polymerase protein • Plates were incubated and developed for spot formation using a second antibody to interferon-Υ conjugated to enzyme followed by substrate • Spot-forming cells (SFCs) in the microtiter wells were quantified using a CTL analyzer • Responses were considered positive when an average of 10 SF Cover background was detected in the duplicate wells

28. Results

30. Wild-Type HBV

31. Sequencing studies showed that all clones from the inoculum and samples collected from the two infected chimpanzees at the beginning, during, and at the end of viremia maintained the wild-type sequence

33. HBV S-Gene Mutant (sG145R)

34. Sequencing studies of 24 clones from the first HBV DNA-positive serum sample postinoculation (day 98) showed the preservation of the inoculum sequence in only 33% of the clones, whereas 67% had the wild-type sequence

36. HBV Pol-Gene Mutant (rtV173L, rtL180M,rtM204V)

37. Sequencing studies showed that all clones from the two inocula and samples collected at the beginning, during, and at the end of viremia in the three infected chimpanzees showed the preservation of the rt173L, rt180M, and rt204V mutations

38. Protective Efficacy of HBV Vaccine The ability of a licensed hepatitis B vaccine to provide protection against challenge with the HBV Pol-gene mutant was evaluated in two chimpanzees (CH10364 and CH10369) that received the hepatitis B vaccine and a control chimpanzee (CH10301) that received the hepatitis A vaccine

39. Both hepatitis B vaccine recipients seroconverted to anti-HBs 1 week after the first dose of the vaccine, and anti-HBs levels were boosted to steady-state levels of >75 mIU/mL after the administration of the second and third vaccine doses

41. Neither humoral nor cellular immunity to HBV antigens was detected in the control chimpanzee CH10301 vaccinated with HAV vaccine

42. All three chimpanzees were challenged with the triple mutant to determine if coding changes in the corresponding S-gene (sE164D and sI195M) compromised protective immunity elicited by hepatitis B vaccination

45. Anti-HBc, which is a marker of exposure or infection, was detected on day 7 in CH10364 and days 7, 10, and 17 in CH10369

47. The hepatitis A-immunized control chimpanzee CH10301 was infected because HBV DNA was detected in serum from days 42 to 91, not exceeding 1 log IU/mL • The chimpanzee seroconverted to total anti-HBc from day 87 and anti-HBs from day 84 post infection. • Despite replication of the Pol-gene mutant virus in this chimpanzee, peripheral blood T-cell responses against the surface or polymerase proteins were not detected • No ALT elevation was observed during the follow-up period

48. The presence of a humoral immune response to the HBV core antigen in both ENGERIX-B-vaccinated chimpanzees and of circulating T-cells against the HBV polymerase antigen in one chimpanzee (CH10364) indicated that sterilizing immunity against the HBV Pol-gene mutant was lacking • Therefore, chimpanzees CH10364 and CH10369 were challenged with human-derived wt-HBV to determine if they were protected against challenge with a virus encoding intact (that is, unmutated) HBsAg

50. Challenge with the wt-HBV inoculum (HLD1, 5,000 CIDs) was carried out when anti-HBs levels were 63,610 mIU/mL and 18,868 mIU/mL in CH10364 and CH10369, respectively, and after loss of HBV mutant-stimulated T-cell responses in both chimpanzees