Hepatitis B vaccination

Abstract

Hepatitis B virus is a worldwide leading cause of acute and chronic liver disease including cirrhosis and hepatocellular carcinoma. Effective vaccines have been available since the early ‘80s and vaccination has proved highly successful in reducing the disease burden, the development of the carrier state and the HB-related morbidity and mortality in the countries where vaccination has been implemented.

 

Neutralizing (protective) antibodies (anti-HBs) induced by vaccination are targeted largely towards the amino acid hydrophilic region, referred to as the common a determinant which is present on the outer protein coat or surface antigen (HBsAg), spanning amino acids 124–149. This provides protection against all HBV genotypes (from A to H) and is responsible for the broad immunity afforded by hepatitis B vaccination. Thus, alterations of residues within this region of the surface antigen may determine conformational changes that can allow replication of the mutated HBV in vaccinated people.

An important mutation in the surface antigen region was identified in Italy some 25 years ago in infants born to HBsAg carrier mothers who developed breakthrough infections despite having received HBIG and vaccine at birth. This virus had a point mutation from guanosine to adenosine at nucleotide position 587, resulting in aa substitution from glycine (G) to arginine (R) at position 145 in the a determinant. Since the G145R substitution alters the projecting loop (aa 139–147) of the a determinant, the neutralizing antibodies induced by vaccination are no longer able to recognize the mutated epitope. Beside G145R, other S-gene mutations potentially able to evade neutralizing anti-HBs and infect vaccinated people have been described worldwide.

In addition, the emergence of Pol mutants associated with resistance to treatment with nucleos(t)ide analogues can select viruses with crucial changes in the overlapping S-gene, potentially able to alter the S protein immunoreactivity. Thus such mutants have the potential to infect both naïve and immunized people, negatively affecting the efficacy of both the antiviral treatment and the vaccination programs.

Despite concern, at present the overall impact of vaccine escapes mutants seems to be low and they do not pose a public health threat or a need to modify the established hepatitis B vaccination programs. The development of novel NAs with a high barrier to resistance is warranted.

Keywords: :

• HBV
• HBV escape mutants
• Hepatitis B
• Hepatitis B vaccination
• drug resistant mutants

Introduction

Hepatitis B virus (HBV) is a leading cause of acute and chronic liver disease including cirrhosis and liver cancer, which ranks as the third cause of cancer deaths worldwide. WHO estimates that at least 2 billion people have been globally infected with HBV. Over 240 million (14 million living in Europe) are chronically infected. An estimate 500 000–700 000 (36 000 in Europe) people die each year for HBV-related diseases, and 4.5 million new cases of acute hepatitis B occur each year, and a quarter of these may progress to chronic liver disease.^1,2

Despite this impressive burden, hepatitis B is now considered a largely treatable and preventable disease, thanks to the availability of effective antiviral drugs and the adoption of several public health measures, including vaccination.

Safe and effective vaccines have been available since the early ’80 when the so called plasma-derived vaccines were first introduced and then replaced by DNA-recombinant vaccines around the mid-’80s.

WHO recommends to introduce hepatitis B vaccination into national childhood immunization programs and catch up programmes focused to people at increased risk of HBV exposure.³ At present, 181 countries have implemented this recommendation, and Italy was one of the first countries to do so, starting in 1991.

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 carrier state and the hepatitis B-related morbidity and mortality in the countries where vaccination has been implemented.⁴

Thus thanks to the use of antiviral drugs such as the last generation nucleos(t)ide analogues (NAs) for the treatment of chronically infected patients as well as the implementation of extensive programs of vaccination may lead in the end to the elimination of hepatitis B and hepatitis B-related diseases.

However, against this view, there are some concerns due to the peculiar biology of HBV, in particular its propensity to develop—through mutation—drug resistant and vaccine-escape mutant viruses which may potentially challenge the therapeutic and prevention programs currently in place.

Hepatitis B Virus (HBV)

Briefly, HBV is a 42 nm DNA virus (the so-called Dane particle), composed by an outer glycoprotein envelope containing HBsAg (hepatitis B surface antigen), an inner icosahedral core (HBcAg) surrounding a circular, partially double-stranded DNA molecule (composed by a complete minus strand and a partial plus strand) of approx 3.2 kb in length, and a large polymerase that functions as both a reverse transcriptase for synthesis of the negative DNA strand from pregenomic RNA and an endogenous DNA polymerase for synthesis of the positive DNA strand using the negative strand.^5-7

The super compact HBV genome contains four overlapping genes. The preS/S gene has 3 ORFs that encode 3 forms of HBsAg: the large (pre-S1), medium (pre-S2) and small (S) structural proteins of the viral envelope. The C gene has two ORFs (C and pre-C) encoding the HBcAg (hepatitis B core antigen) and the e protein, which is processed to produce soluble HBeAg (hepatitis e antigen). The X gene encodes a small protein with transactivator activity, while Pol (polymerase) gene encodes a large polymerase protein.⁸

On the basis of a minimal divergence of 8% of the complete genome sequences, eight HBV genotypes (from A to H) and a number of sub-genotypes—with a minimal genetic divergence of 4%—have been identified so far. In addition, there are 4 major subtypes: adw, adr, ayw, ayr, and 9 minor subtypes. HBV serotypes and genotypes have distinct geographical and ethnic distribution worldwide and may play a role in determining the severity of liver disease and treatment outcome of HBV infection.^9-15

The viral life cycle is complex since HBV adopts, at least in part, a retrovirus-like strategy involving reverse transcription of a pre-genome RNA to the negative strand DNA. After the DNA minus strand is formed the DNA polymerase starts to synthesize the positive strand. Virions are then formed via budding into the endoplasmic reticulum. Virus particles can either exit the cell or re-enter into the nucleus to initiate another round of replication which involves covalently closed circular DNA (cccDNA) formation followed by its transcription of mRNA, including pre-genome RNA.

Because of this strategy of replication, which involves a RT lacking proof-reading capacity, HBV shows greater mutability than other DNA viruses. Mutations can occur in all four genes through spontaneous errors of the viral polymerase or as a consequence of pressure by the host immune system or by exogenous factors including immunization or treatment with antivirals.

The HBsAg a Determinant

All HBV genotypes and serotypes share the common determinant a which spans aa 124–149 within the major hydrophilic region (MHR) and is in a form of two major and one minor loops with cysteine-disulphide bonds, protruding from the outer surface of the virus; the second hydrophilic loop (aa 139 to 147 or 149) is the major target for neutralizing anti-HBs produced following natural infection or vaccination.

Neutralizing (protective) antibodies induced by vaccination are targeted largely towards the conformational epitope of the a determinant. This provides protection against all HBV genotypes and subtypes and is responsible for the broad immunity afforded by HBV vaccination. Thus, alterations of residues within this region of the surface antigen can determine conformational changes that can allow replication of mutated viruses in vaccinated people (vaccine escape mutants or VEMs). In addition, such mutated viruses can be undetectable by the current diagnostic assays, posing a potential threat to the safety of blood supply.^16-19

S-Gene Mutants

An important mutation in the surface antigen region was first identified in Italy some 25 years ago in infants born to HBsAg carrier mothers who developed breakthrough infections despite the presence of protective levels of antibody produced in response to the administration of HBIG (hepatitis B iperimmune gamma globulins) plus vaccine received at birth.^20,21 This virus has a point mutation from guanosine to adenosine at nt position 587, resulting in aa substitution from glycine (G) to arginine (R) at position 145 (G145R) of the a determinant of the surface antigen. Since the G145R substitution alters the projecting second loop of the a determinant, the neutralizing antibodies induced by vaccination are no longer able to recognize the mutated epitope. G145R was shown to be viable infectious and pathogenic in chimpanzees.²²

Beside the prototype G145R, other S gene mutations (alone or in combination) across the entire a determinant potentially able to evade neutralizing anti-HBs have been identified worldwide rising concern that these mutant viruses may take advantage on the wild type in escaping the immunity of vaccinated people.^23-26 HBV infection with S-gene mutant viruses has been reported to occur in presence of protective levels of anti-HBs in infants born to HBV-infected mothers who received HBIG plus vaccine (suggesting that HBIG, when used, can be the major driving force for the selection of vaccine escape mutants), in liver transplanted patients who received HBIG for prophylaxis, and in HBsAg-negative chronic carriers of HBV. Globally speaking the emergence of G145R is an uncommon event more generally associated with the use of HBIG rather than vaccination.^27,28

In 2010 a survey conducted in Taiwan failed to detect an increased prevalence of vaccine escape mutants in a population of children and adolescents fully covered by universal infant immunization over a period of 20 years.²⁹

Of concern, a recent study carried out in China reported that HBV mutants capable of infecting people are emerging 13 years after the implementation of a successful universal vaccination program.^30,31 However, a more careful analysis of data from such study showed that following vaccination both the HBsAg carrier rate and prevalence of variants indeed decreased, even though the variant prevalence decreased at lower rate (71% versus 33%). In addition in this study a clear differentiation between proven- and not-proven VEMs among the HBsAg variants is lacking.^32,33

Nucleos(t)ide Analogues Resistant Mutants

The availability of nucleos(t)ide analogues able to suppress HBV replication by inhibiting the reverse transcriptase (rt) has provided effective treatments to patients with chronic hepatitis B, significantly reducing morbidity and mortality. However, the selection and emergence of drug-resistant HBV mutants can lead to treatment failure and progression to liver disease. The development of resistance due to mutations in the Pol gene is usually followed by a virological breakthrough (rise in HBV DNA levels of at least 1 log compared with the nadir value), a biochemical breakthrough (ALT elevation) and worsening of liver disease.^34,35

Mutations of the Pol gene located within the catalytic domain of the RT region are particularly common after treatment with Lamivudine (LMV). Evidence indicates that LMV resistance increases progressively over the time of treatment (up to 80% after 48 m of the treatment).³⁶

The rate of emergence of resistant HBV following treatment with other NAs, particularly Adefovir (ADV) and Telbivudine (LdT), is lower than that of LMV but still substantial in many cases.³⁶

Indeed, current chronic hepatitis B treatment guidelines recommend that Entecavir (ETV) or Tenofovir (TDF), drugs with high potency and the lowest rates of resistance, should be chosen as first-line treatment options to minimize the likelihood of resistance development and to increase the chances of achieving treatment goals.³⁴ Since the Pol-gene encoding the polymerase completely overlaps the S gene encoding the surface protein (Fig. 1), each mutation occurring in the Pol-gene implies possible changes in the envelope protein and vice versa. Thus, drug-resistant HBV mutants emerging under NAs therapy may also show mutations in the S protein (Table 1) causing alteration in the antigenicity of the protein.^35-37

Figure 1. Schematic representation of the overlap between the HBV polymerase and envelope open reading frames. The numbers indicate amino acid (aa) sites. Numbering is according to genotype D. The a determinant of HBsAg that is located between aa 124 and 149, and which includes the major antibody neutralization domain of HBV, is indicated.

Table 1. Major drug-resistant mutations in the HBV reverse-transcriptase (rt) resulting in structural changes in the HBsAg protein

Nucleos(t)ide	Pol-gene mutations	S gene mutations
S gene mutations	(Primary or compensatory)
LMV and LdT	rtM204V	sI195M
	rtM204I	sW196S/L/*
	rtV173L	sE164D
ADV	rtA181T^§#	sW172L/*
(^#LMV, ^§LdT)	rtA181V^§	sL173F
ETV	rtI169T	sF161H/L
	rtT184C	sL175F+sL176V
	rtT184L/S	sL175F
	rtT184G	sL176V
	rtT184M	sL176stop
	rtS202C/G	sS193F/L
	rtS202I	sV194F/S

^§,# Also induced by ^#LMV or ^§LdT; *stop codon.

HBV S escape variants are viable and pathogenic and may infect properly vaccinated people Thus, “NAs-resistant, possible vaccine escape mutants” have the potential to infect both naïve and immunized people, negatively affecting the efficacy of both the antiviral treatment and the vaccination programs.

What are the Public Health Implications of S-Gene and Pol-Gene Mutants?

The implementation of universal vaccination had a tremendous impact in terms of reduction in morbidity and mortality of hepatitis B and HB-related diseases.

In Taiwan, for example, the HBsAg prevalence in children under 15 of years of age decreased from 9.8% in 1984 (the year of introduction of mass vaccination) to less than 0.5% in 2009. The annual average incidence of HCC in children declined from 0.7 per 100 000 to less than 0.2 after 20 years of vaccination.^38,39

Similar successful results were reported in other hyper-endemic countries such as the Gambia, China, Singapore, and Alaska where the impact of vaccination in terms of reduction in the burden of hepatitis B, the rate of carrier state, and hepatitis B-related mortality has been impressive.

In Italy, where approx 20 million children have been vaccinated since implementation of vaccination in 1991, the overall morbidity rate per 100 000 inhabitants fell from 7 in 1990 to 1 in 2012. This decline was even more striking in 15–24-year-old individuals where incidence per 100 000 dropped from 17 to less than 0.4 in the same period of time.⁴⁰ Moreover several seroprevalence studies show a dramatic drop of HBV markers in vaccinated people, particularly those under 30 years of age.⁴¹ According to our national surveillance system (SEIEVA) breakthrough infections due to vaccine escape mutants are rarely reported.

On the whole, data collected from Italy and elsewhere show the success of vaccination as the most effective measure to control and prevent hepatitis B and its severe sequelae.

Conclusions

Vaccination has clearly proved to be very successful resulting in remarkable progress towards the prevention and control of hepatitis B and HBV-related diseases on global scale. Cases of hepatitis B in fully vaccinated people are generally 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 drugs resistant mutants with alteration in the a determinant of the S protein is of some concern. The development of novel NAs with a high barrier to resistance is warranted.

Global surveillance networks should be set up to monitor the epidemiological dynamics and public health impact of vaccine escape and treatment escape mutants.

Abbreviations:
aa	=	amino acid
ADV	=	Adefovir
ALT	=	alanine aminotransferase
anti-HBs	=	antibody to hepatitis B surface antigen
cccDNA	=	covalently closed circular DNA
ETV	=	Entecavir
HB	=	hepatitis B
HBcAg	=	hepatitis B core antigen
HBeAg	=	hepatitis B e antigen
HBIG	=	hepatitis B immunoglobulin
HBsAg	=	hepatitis B surface antigen
HBV	=	hepatitis B virus
HCC	=	hepatocellular carcinoma
LdT	=	Telbivudine
LMV	=	Lamivudine
MHR	=	major hydrophilic region
NA	=	Nuclos(t)ide analogue
ORF	=	open reading frame
RT	=	reverse transcriptase
TDF	=	Tenofovir
VEMs	=	vaccine escape mutants
WHO	=	World Health Organization

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.