The hepatitis B surface antigen (HBsAg) is a multifunctional glycoprotein composed of large (LHB), middle (MHB), and small (SHB) subunits. HBsAg isoforms have numerous biological functions during HBV infection—from initial and specific viral attachment to the hepatocytes to initiating chronic infection with their immunomodulatory properties. The genetic variability of HBsAg isoforms may play a role in several HBV-related liver phases and clinical manifestations, from occult hepatitis and viral reactivation upon immunosuppression to fulminant hepatitis and hepatocellular carcinoma (HCC). Their immunogenic properties make them a major target for developing HBV vaccines, and in recent years they have been recognised as valuable targets for new therapeutic approaches. Initial research has already shown promising results in utilising HBsAg isoforms instead of quantitative HBsAg for correctly evaluating chronic infection phases and predicting functional cures. The ratio between surface components was shown to indicate specific outcomes of HBV and HDV infections. Thus, besides traditional HBsAg detection and quantitation, HBsAg isoform quantitation can become a useful non-invasive biomarker for assessing chronically infected patients. This review summarises the current knowledge of HBsAg isoforms, their potential usefulness and aspects deserving further research.
Infection with the hepatitis B virus (HBV) remains a global health problem despite successful vaccination programs. Almost two billion people worldwide have been infected with HBV at some point in their lives, and around 296 million live with chronic infection. Complications related to chronic infection are still a source of significant morbidity and mortality of approximately 820,000 annually, primarily due to liver cirrhosis and hepatocellular carcinoma (HCC).
The natural history of chronic HBV infection is exceptionally complex and progresses nonlinearly through five distinct phases: (1) hepatitis B e antigen (HBeAg)-positive chronic HBV infection, (2) HBeAg-positive chronic hepatitis B (CHB), (3) HBeAg-negative chronic HBV infection, (4) HBeAg-negative active CHB and (5) the HBsAg-negative phase. Patients in the last phase can experience viral reactivation in cases of severe immunosuppression. The current management of chronic HBV infection includes two therapeutic regimes: pegylated interferon α (PEG-IFNα) and nucleos(t)ide analogues (NAs). The treatment goal determined by all guidelines is the improvement of the long-term outcomes by the persistent inhibition of HBV replication. Unfortunately, a complete, sterilising cure is not achievable by current therapy approaches because the suppression of viral replication does not affect the persistence of the HBV mini-chromosome—the covalently closed circular DNA (cccDNA) in the nucleus of the hepatocyte or the viral DNA integrated into the host genome. Thus, an alternative endpoint of therapy followed today is a “functional cure”, defined by a persistently undetectable serum HBV DNA and hepatitis B surface antigens (HBsAg) with or without seroconversion to corresponding antibodies.
The human HBV is the prototype member of the family Hepadnaviridae, which includes a variety of similar avian and mammalian viruses. The mature infectious HBV particle, originally called a “Dane particle”, consists of a nucleocapsid core enclosed in a glycolipid envelope. The nucleocapsid comprises partially double-stranded circular DNA attached to endogenous polymerase and an icosahedral capsid, while the envelope is made of a lipid bilayer bearing three different surface proteins.
The three viral surface proteins large (LHB), middle (MHB), and small (SHB) constitute the HBV surface antigen (HBsAg). It is a multifunctional glycoprotein and the major antigen of the viral envelope, responsible for eliciting humoral and cellular immune responses during infection. HBsAg isoforms are involved in many biological functions during HBV infection—from initial and specific viral attachment to the hepatocytes to establishing chronic infection with their immunomodulatory properties. The genetic variability of domains encoding HBsAg isoforms is responsible for their altered synthesis and presentation. Based on accumulated evidence, this may play a role in the pathogenesis of specific liver conditions. In diagnosis, the quantitation of HBsAg isoforms has been perceived as a potentially valuable tool for evaluating the chronic infection phase and monitoring to the treatment response. The immunogenic properties of surface proteins were long ago recognised as crucial for the development of vaccines, and the presence of different HBsAg isoforms has a role in the immune response they elicit. Some of the new therapeutic approaches that have been developed in recent years target the production, assembly and secretion of HBsAg isoforms. This review summarises the current knowledge of HBsAg isoforms, their potential usefulness and their aspects deserving further research.
The organisation of the HBV genome includes four partially overlapping open reading frames (ORFs): S, P, C and X. The S ORF is composed of pre-S1, pre-S2 and S genes and is responsible for the synthesis of three surface proteins included in the HBsAg. The longest P ORF encodes the viral enzyme essential for the viral life cycle—HBV polymerase. The whole C ORF, including the pre-C and C regions, is translated into the precursor protein, which yields the hepatitis B e protein (HBeAg), a soluble antigen. The C region encodes the hepatitis B core antigen (HBcAg) or C protein, representing the viral capsid’s primary structural protein.
Soon after its discovery in the 1960s, it became evident that the newly recognised hepatitis virus was highly variable. Ten serotypes (also known as HBsAg subtypes) were identified based on the amino acid variability of the HBsAg. In the 1980s, HBV genotypes were recognised based on a sequence divergence of more than 8% over the entire genome. Thus far, 10 HBV genotypes (A–J) have been identified. For isolates of genotypes A, B, C, D and E, based on a genome sequence divergence of 4% to 8%, an additional classification of sub-genotypes was introduced, and so far, more than 40 have been recognised. The genotypes, sub-genotypes and serotypes have distinct geographical distributions. HBV genotypes and serotypes result from the evolutionary drift of the viral genome as a consequence of a long-term adaptation of the virus to genetic determinants of different host populations.
On the other hand, HBV is a virus prone to variability that arises spontaneously due to its unique life cycle. The reasons for this spontaneous variability lie in an error-prone viral reverse transcriptase and a very high replication rate. The estimated mutation frequency of HBV is approximately 10-fold higher than for other DNA viruses (1.4 to 3.2 × 10−5 substitutions/site/year). Accordingly, HBV exists as a quasi-species population whose composition is determined by the host immune response and antiviral therapy or vaccination.
HBV also plays a role in natural infection with the hepatitis D virus (HDV). HDV is a satellite virus that does not code envelope glycoproteins but depends on HBsAg to form complete virions and, hence, can infect humans only as a co-infection or a superinfection with HBV. Its genome, a single-stranded RNA, is associated with two isoforms of the hepatitis delta antigen—small (S-HDAg) and large (L-HDAg)—to form ribonucleoprotein (RNP). HDV RNA has >70% internal base pairing, enabling folding into a partially double-stranded rod-like structure. HDV virion assembly depends on the interaction of RNP with HBsAg isoforms.
Surface proteins are all encoded by a single open reading frame—S-ORF divided by three start codons into the pre-S1, pre-S2 and S domain. Two sub-genomic mRNAs are responsible for the translation of the surface proteins: 2.4 kb sub-genomic mRNA, transcribed from pre-S1, pre-S2 and S domains, for LHB and 2.1 kb sub-genomic mRNA, transcribed from pre-S2 and S domains, for MHB and SHB. Surface proteins’ mRNAs are mainly transcribed from cccDNA but can also be transcribed from the parts of the integrated viral genome.
The three surface proteins share the same C-terminus but have different N-terminal extensions. The SHB consists of 226 amino acids (aa), the MHB protein contains an extra 55 aa at the N-terminus, while the LHB protein has an additional 108 or 119 aa (depending on the genotype) at the N-terminus, relative to MHB. The ratio between LHB, MHB and SHB in the envelope of mature virions is 1:1:4. The synthesis of envelope proteins is diverse from genome replication and occurs in the endoplasmic reticulum (ER), where they integrate into the ER membrane. Four transmembrane domains are within the S-domain of all three envelope proteins, connected by internal and external loops. In the ER, the three envelope proteins undergo co- and post-translation modification in the form of N-glycosylation. All three proteins are N-glycosylated at asparagine 146 in the S-domain, while MHB is N-glycosylated at asparagine 4 of the pre-S2 domain. The LHB shows no N-glycosylation at pre-S2, but its pre-S1 region is myristoylated at the G2 residue. The N-glycosylation sites have significant roles in the viral life cycle. In addition, in HBV genotypes C and D, there is an O-glycosylation site at T37 within pre-S2.
The transmembrane topology of surface proteins is essential for their antigenicity and function in viral morphogenesis and release. Unlike MHB and SHB, the LHB protein exhibits two transmembrane topologies. LHB’s pre-S1 and pre-S2 domains can be transported from the luminal to the cytoplasmic side of the ER by using myristoylated residue at position 2 in pre-S1 as an anchor. Approximately half of LHB proteins have their pre-S-domain on the cytoplasmic side of the ER, while the other half have their pre-S on the ER luminal side. During the step of envelope formation, the pre-S1 on the cytoplasmic side is essential for the interaction between the LHB and the newly formed capsid. A short sequence on the junction between pre-S1 and pre-S2 in LHB, called the matrix domain (MD), is crucial for binding with the capsid and forming complete virions.
The pre-S1 exposed on the luminal side of the ER will end up on the surface of the Dane particle, where aa 2–48 serves as a viral anti-receptor for the bile receptor, sodium taurocholate co-transporting polypeptide (NTCP), recognised as the major HBV attachment molecule on the surface of the hepatocytes. The specificity of binding to NTCP is improved by the myristoylation of the N-terminus of the pre-S1. The HBV virion initi
