HEPATITIS B: are escape mutants of concern?

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1 VACCINATION: AN EVOLUTIONARY ENGINE FOR SPECIES? Fondation Mérieux Conference Centre Veyrier-du-Lac, France November 25-27, 2013 HEPATITIS B: are escape mutants of concern? Alessandro ZANETTI Department of Biomedical Sciences for Health University of Milan, Italy

2 Hepatitis B: the burden 2 billion people have been globally infected with HBV Over 350 million chronically infected (6% of the global population; 14 million living in Europe) An estimated 700,000 people (36,000 in Europe) die each year 4.5 million new HB cases/year (of whom a quarter progress to CLD) WHO

3 Types of Hepatitis B vaccines I generation plasma derived (1981) II generation yeast-derived (1986) rec-dna III generation - Combined vaccines (HA + HB; hexavalent) - pres1-s2 vaccines - novel adjuvanted vaccines

4 Vaccination against hepatitis B Strategies for vaccination were initially targeted to groups at increased risk. Failure of such policies led WHO to recommend that all countries should introduce hepatitis B vaccination into their national immunization programs.

5 Countries having introduced HepB vaccine and infant HepB coverage, countries (including parts of India and the Sudan) have introduced HepB vaccine in their national infant immunization schedule <50% (5 countries, 3%) 50-79% (25 countries, 13%) 80-89% (35 countries, 18%) 90% (114 countries, 59%) HepB not in schedule (14 countries, 7%) WHO/UNICEF

6 Hepatitis B vaccination: immunogenicity Several hundred million vaccinations have been administered worldwide with an outstanding record of safety and efficacy. Vaccination has proved highly successful in reducing the disease burden, the development of the carrier state and the hepatitis B related morbidity and mortality

7 HEPATITIS B VIRUS

8 Hepatitis B genotypes and serotypes 8 genotypes (A-H) 4 major serotypes (adw, adr, ayw, ayr) 9 minor serotypes

9 The hepatitis B virus replication cycle From: Zoulim F, Locarnini S, Gastroenterology 2009.

10 Emergence of HBV mutants Following this strategy of replication involving a RT lacking proof-reading capacity, HBV shows greater mutability than other DNA viruses. Error-prone replication leads to 2x10 4 base substitutions per site per year. Mutations can occur in all 4 genes through: spontaneous errors of viral Pol as a consequence of pressure by the host immune system by exogenous factors including immunization or treatment with antiviral drugs.

11 Major hydrophilic region and 'a' determinant of surface gene on surface protein

12 S-gene mutants Neutralizing (i.e. protective) Abs induced by vaccination are targeted towards the conformational epitope of the a determinant. This provides protection against all HBV genotypes and subtypes conferring broad immunity. Alterations of residues within this region can lead to conformational changes that can allow replication of the mutated HBV in vaccinated people.

13 G145R features Point mutation from guanosine to adenosine at nt position 587resulting in aa substitution from G to R at position 145 of the a determinant. Since the G145R substitution alters the projecting second loop the a determinant, neutralizing Abs induced by vaccination are no longer able to recognize the mutated epitope. G145R was shown to be viable, infectious and pathogenic in chimpanzees.

14 Vaccine-escape HBV mutants S gene mutants may take advantage on the wild-type in escaping the immunity of vaccinated people. Mutants occur in presence of anti-hbs in: infants born to HBV+ carrier mothers treated with HBIG plus vaccine liver transplanted patients who received HBIG for prophylaxis HBsAg-negative asymptomatic carriers.

15 POL-gene mutants Availability of NAs has provided effective treatments to CHB patients, significantly reducing morbidity and mortality. The emergence of drug-resistant mutants can lead to treatment failure and progression to LD. The development of resistance begins with mutations in the Pol gene followed by a virological and then biochemical ( ALT) breakthrough and worsening of LD.

16 Primary antiviral drug resistance mutations From: Zoulim F, Locarnini S, Gastroenterology 2009.

17 Cumulative annual incidence (%) of resistance for Lamivudine, Telbivudine, Adefovir, Tenofovir and Entecavir Up to 20% 10 0 Years of therapy Lamivudine Entecavir (LAM res) Telbivudine HBeAg+ Telbivudine HBeAg- Adefovir HBeAg- Adefovir LAM res Tenfovir Entecavir (naive) Gastroenterology 2009.

18 Polymerase-HBsAg Overlap Genes encoding surface antigen and the polymerase overlap Polymerase Terminal Protein Spacer Reverse transcriptase RNAse H Surface PreS1 Pre S2 S a determinant Mutations occurring in the RT during antiviral therapy can result in changes in the surface gene Mutations selected in the S gene during immunoprophylaxis can result in changes in the RT

19 HBsAg Mutation Selected by LMV/ADV/ETV 99 D Y T T T/V S S/T Q G M L G S/T G P P P I/L L P V C K/R 121 -s-s- T Loop 1 of a determinant C 124 C T/M S M F/Y P S C C C S/T K P Loop 2 of a determinant T/I/A P/C A/V Q G N/T S/T I C T C P N G D I P W A S S F/L 172 Stop A/G K/R R F S V S F/Y W A W L W E 173 F L 161 L 194 F L 164 D A/V S/L LMV/L-dT ADV ETV I 176 V 196 L/S/Stop W M M/I 195 M

20 Public health significance of antiviral drug - associated vaccine escape mutants Such mutants have the potential to infect both naïve and immunized people affecting the efficacy of both the antiviral treatment and the vaccination programs. From: Zoulim F, Locarnini S, Gastroenterology 2009.

21 What is the meaning of this finding? Is it only a matter of academic research interest or is it a crucial problem with far-reaching implications in terms of public health?

22 Impact of hepatitis B mass vaccination in hyperendemic area: Taiwan July 1984: mass vaccination of newborns Taiwan HBsAg Anti-HBc 6-9 yrs yrs yrs 9,8% 20,6% 0,6% 2,9% Prevalence of HBsAg among individuals <20 yrs * 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 0,7x10 5 0,36x10 5 0,15 0,19 persons/yr Annual incidence (x 10 5 ) of HCC among children, 6-14 yrs* ; ** *Chang MH et al. N Engl J Med 1997;336:1855-9; **Chang MH et al. Clin Cancer Res 2005;11:

23 Cases/ Morbidity rate (x 10 5 inhabitants) of hepatitis B in Italy, according to age ( ) Age group: Total Vaccination Incidence x 10 5 in 2012: 0-14: 0, : 0,4 >24: 1,1 Total: 0,85

24 Prevalence of anti-hbc in individuals aged <30 years Impact of HBV vaccination in Italy Evidence shows that pratically no markers of HBV are currently reported in vaccinated people <30 years of age in Italy ,8% 5,8% Prevalence of anti-hbc in individuals aged <30 years <1%

25 Acute hepatitis B in vaccinated people in Italy (SEIEVA, ) ~15% Immigrants 5,671 cases 5,156 (91%) vaccination status known 5,000 (97%) subjects not vaccinated 156 (3%) subjects vaccinated mean age 35.9 yrs 67.7% males 325 (6.5%) escaped mandatory vaccination 37 (23.7%) properly vaccinated

26 Hepatitis B in vaccinated people in Italy (SEIEVA, ) 37 cases vaccinated properly Molecular characterization: - 14/21 wild type - 7/21 escape mutants 156 cases of hepatitis B in vaccinated people Correctly vaccinated 24%

27 Conclusions Vaccination has clearly proved to be very successful Cases of HB in fully vaccinated people are rare. Breakthrough infections caused by S-gene mutants are occasionally reported but at present they do not pose a serious threat to the established vaccination programs. The emergence of drug resistant mutants with alterations in the a determinant of the S protein is of concern since the pool of patients with CHB potentially in need of treatment with NA is huge. Global surveillance networks should be set up to monitor the epidemiological dynamics and pubic health impact of vaccine-escape/treatment-escape mutants.