Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the “a” determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications.

Hepatitis B virus (HBV) infection is a major public health problem in most countries, with approximately 2 billion people worldwide showing exposure to the virus, nearly 300 million carrying HBV chronic infection and over 1 million deaths per year due to HBV-related end-stage liver disease, liver cirrhosis and liver cancer[1-5].

HBV is an enveloped Hepadnavirus with an incomplete double-stranded DNA genome of 3.2 Kb[6]. Eight genotypes, with a distinct geographical distribution, have been identified to date. Genotype A prevails in north-western Europe and in the United States, genotypes B and C in Asia, genotype D in the Mediterranean basin, the Middle East, and India, genotype E in Western Africa, genotype F in South and Central America, genotype G in the United States and France, genotype H in Northern Latin America[7], genotype I in Laos, Vietnam, Eastern India[8,9] and North-Western China[10] and genotype J in Japan[11,12].

The worldwide prevalence of chronic HBV infection in the general population borders 5%, but it differs widely from one geographical area to another, from 0.1%-2.0% in the United States and Western Europe, from 2.0%-8.0% in Eastern Mediterranean countries and Japan, and from 5.0%-20.0% in South-Eastern Asia and sub-Saharan Africa[1,13].

Risk factors for HBV infection include transfusion of unscreened blood, renal dialysis, sexual promiscuity, sharing or re-using syringes among injection drug users, tattooing, piercing, working or residing in a health-care setting, living in a correctional facility and long-term household or intimate non-sexual contact with an HBsAg-positive individual. In highly endemic areas the majority of hepatitis B surface antigen (HBsAg) chronic carriers acquire HBV infection at birth or in the first decade of life, whereas in countries with a low endemicity HBV transmission occurs mostly in adulthood due to unprotected sexual contact, syringe sharing or parenteral exposure to contaminated medical equipment[7,14-17].

A vaccine against HBV became available in 1982, and ten years later the World Health Organization recommended universal vaccination of newborn babies with the HBsAg produced by yeast cells into which the genetic code for HBsAg had been inserted. The complete vaccination schedule induces protective antibody levels in more than 95% of infants, children and young adults.

The emergence of single or multiple amino acid substitutions in the HBsAg region has been found in infants born to HBsAg-positive mothers who underwent passive/active immunoprophylaxis at the birth, in HBsAg-positive liver transplant recipients treated with hyperimmune anti-HBs immune globulin and in patients who experienced loss of HBsAg after anti-HBV therapy.

This review article focuses on the impact of HBsAg mutants on vaccine escape, failure of diagnostic tests to detect HBsAg, and on the development of hepatocellular carcinoma (HCC).

Human HBV is the prototype member of the Hepadnaviridae family, which includes a variety of avian and mammalian viruses sharing similar genomic organization, organ tropisms and a unique strategy of genome replication[14].

HBV is one of the smallest enveloped animal viruses with a diameter of 42 nmol/L consisting of an outer lipid envelope and an icosahedral nucleocapsid core composed of proteins. The nucleocapsid encloses the viral DNA and a DNA polymerase acting also as a reverse transcriptase[17]. The outer envelope contains the embedded proteins HBsAg, pre-S1 and pre-S2 involved in the viral binding of, and entry into susceptible cells. HBV is also called “Dane particle” after the name of the researcher who first observed it on electron microscopy together with filaments and 22 nmol/L spherical bodies in the serum of infected individuals[18]. HBV infects the hepatocytes, whereas the filaments and spherical bodies do not contain the viral DNA and do not infect the liver cells. These filaments and spherical bodies show the same HBsAg reactivity as the surface of HBV and are considered to be produced by HBsAg in excess during the life cycle of the virus[19].

The HBV genome consists of 3200 base pairs of partially double-stranded circular DNA containing four (P, C, S and X) overlapping open reading frames (ORF) with a nucleotide diversity of ≥ 8% in different genotypes[15,16]. The P gene codes for the viral polymerase/reverse transcriptase. It has four domains: A terminal domain, which serves as a protein primer for reverse transcription of pre-genomic viral RNA, a spacer region without no apparent function, the polymerase domain, which has reverse transcription activity, and the RNase H domain, responsible for the degradation of the RNA template during reverse transcription.

The core (C) gene codes for HBcAg, the major structural protein of the nucleocapsid. The preC/C ORF is transcribed into a precore/core fusion protein. During entry into the endoplasmic reticulum, 19 amino acids are cleaved from the N-terminal end of the precore protein by a signal peptidase. When transported into the Golgi compartment, additional amino acids are removed from the C-terminal end by intra-Golgi proteases to form the HBe antigen. This antigen, which is secreted into the serum, is used as a marker of active HBV replication in clinical practice. The possibility that the circulating HBe antigen may suppress the immune response and favor HBV replication has been hypothesized[14] but never proven, and the biological function of this protein, if any, remains unknown. The preS/S ORF encodes the envelope proteins HBsAg, pre-S1 and pre-S2. The X gene codes for potent transactivating factors of viral and cellular genes (HBxAg), some of which possibly correlated to the development of HCC[20,21].

The preS/S ORF encodes three different structurally related envelope proteins, termed the large (L), middle-sized (M) and small (S) protein, that is synthesized from alternative initiation codons. The three proteins share the same carboxy-terminus but have different amino terminal extensions. In particular, the S protein corresponding to the HBsAg consists of 226 amino acids (aa), the M protein contains an extra N-terminal extension of 55 aa, and the L protein has a further N-terminal sequence of 108-119 aa compared with the M protein[22]. The enhancer and basic core promoter regions of S region overlap with the X gene.

HBsAg is an envelope glycoprotein that is currently the primary element for diagnosis and target of immunoprophylaxis of HBV infection. The dominant epitopes of HBsAg, which are the targets of neutralizing B-cell responses, reside in the ‘‘a’’ determinant (aa 124-147) within the major hydrophilic region (MHR).

Several mutations in the S region have been described and those most frequently reported in the literature are listed in Table 1. In most cases, they were aa substitutions due to a single mutation, but nucleotide deletions have also been reported. The majority of mutations were located in the S region, but some mutations were also identified in the pre-S1 or pre-S2 regions. Mutations in the S region have been found in various HBV genotypes, while those in pre-S1 or pre-S2 have been frequently observed in patients with HBV genotype C (Table 1)[23-60].

Some HBsAg mutants have been associated with major biological or clinical events such as immune escape, failure to detect HBsAg and the development of HCC.

The association of single or multiple aa substitutions in the HBsAg region with failed protection in infants who received passive/active prophylaxis and in HBsAg-positive liver transplant patients undergoing continuous passive immunoprophylaxis should alert clinicians to the possible onset of acute hepatitis B or a reactivation of a previous HBV infection, respectively, in these cases.

Similarly, the possibility that some subjects resulting HBsAg-negative may harbor HBV infection because an aa substitution has made the presence of HBsAg undetectable with the commercially available assays should be taken into account by clinicians and healthcare personnel working in laboratories and blood banks.

Although several studies reported an association between HBsAg mutations and HCC, the data on this point are not conclusive because most of the studies were performed in south-eastern Asia, some of them were very small, most of them were cross-sectional and a few reported data contrasting with those from the majority of studies. A large worldwide study, planned on the basis of the data available, would almost certainly improve our knowledge on this topic.