Genetic polymorphisms of HLA-DP and isolated anti-HBc are important subsets of occult hepatitis B infection in Indonesian blood donors: a case-control study

Occult hepatitis B virus (HBV) infection (OBI) is defined as the presence of HBV DNA in the serum and/or liver of HBsAg-negative individuals. Since its discovery in 1963 and its subsequent association with HBV infection, hepatitis B surface antigen (HBsAg) remains the main serological marker in routine laboratory tests for the detection of infection. It greatly reduced HBV transmission due to blood transfusions, as transfusion of HBsAg positive blood is prohibited [1, 2]; however, numerous scientific papers have highlighted the presence of HBV infection in individuals who test negative for HBsAg and have detectable HBV DNA in the liver or blood [1‐3]. The prevalence of OBI, which has been reported worldwide, varies greatly across the globe, with higher rates reported in Asia; however, cases have also been reported in low HBV endemic areas [4]. Indonesia has the third-highest prevalence of HBV infection worldwide, with a moderate-to-high hepatitis B endemicity that affects 242 million people [5, 6].

Although it has been recognized since the 70s, OBI became a topic of interest in hepatology research in 1999, when a study published in The New England Journal of Medicine identified a large series of HBsAg-negative patients with chronic liver disease (CLD) who were positive for HBV genomes by testing liver biopsy specimens [7]. That study highlighted the clinical importance of OBI, which can promote or accelerate the progression of hepatitis C virus (HCV)-related chronic hepatitis to cirrhosis. In addition, it provided a new perspective on virological issues by showing that OBI viruses have no genetic mutations capable of preventing viral replication or HBsAg synthesis [8].

Significant advances in our understanding of the molecular mechanism underlying OBI have been made in the past decade [4]. Sequence variations in HBV genomes, including S gene variants (S-escape mutants), can result in conformational changes of HBsAg that render the protein undetectable by commercially available detection kits [4, 8]. In addition, in a small number of cases, OBI is linked to HBV mutants with defective replication activity or synthesis of S proteins, which cause the lack of detectable HBsAg despite the presence of HBV genomes [9, 10]. However, in most cases, OBI genomes are replication-competent viruses with genetic heterogeneity comparable to that of HBV isolates from individuals with “overt” (HBsAg-positive) infection. OBI status is therefore thought to result from a strong suppression of HBV replication and gene expression involving different mechanisms [8]; however, knowledge of the factors involved in the suppression of viral activity leading to the induction of OBI remains limited. Understanding these factors is important as it may provide new insights into HBV virology and reduce the probability of transmission.

HBV has indirect cytopathic properties, indicating that the host immune response is involved in regulating viral activity, including viral replication and disease progression [6]. One important finding of genome-wide association studies is that polymorphisms in the Human Leucocyte Antigen (HLA) DPA1/DPB1 genes, which are located on the short arm of chromosome 6, influence HBV infection [11, 12]. HLA-DP molecules belonging to HLA class II play an important role in adaptive host-immune responses, particularly in antigen presentation to CD4+ T helper cells [13, 14]. A recent study in Indonesia confirmed that HLA-DPA1 and HLA-DPB1 variants are associated with outcomes of HBV infection such as susceptibility, persistent infection, and disease progression [6]; however, to date, the effect of HLA-DP variants on OBI detection has not been investigated in an Indonesian population. It would be interesting to investigate the possible association of variations in HLA-DP loci with the suppression of HBV replication resulting in OBI, which is immune-mediated. The aim of the present study was to investigate the profiles and genetic influence of HLA-DPA1/DPB1 single nucleotide polymorphisms (SNPs) on the detection of OBI in the Indonesian population. This study focused on one SNP in HLA-DPA1 (rs3077) and three SNPs in HLA-DPB1 (rs3135021, rs9277535, and rs2281388). In addition, this study also investigated the prevalence of OBI and association between HBV antibodies and OBI findings in Indonesian blood donors.

The introduction of universal vaccination has strongly reduced vertical transmission in many countries; however, horizontal transmission is also an important route of HBV infection. In particular, blood transfusion from healthy donors with OBI is associated with the risk of horizontal transmission [22]. OBI is not only difficult to diagnose, but is also associated with a variety of clinical conditions. Several reports have indicated that OBI has similar infectivity and pathogenicity in the development of fulminant hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) with non-occult infection, and that it may affect the safety of blood transfusions and orthotopic liver transplantation (OLT) [3]. The role of OBI in chronic hepatitis C virus (HCV) infection is perhaps the most extensively studied, as OBI can promote or accelerate the progression of HCV-related CLD, as minimal lesions produced by the immune system in response to OBI may contribute to disease progression [23, 24]. Evidence validated by meta-analyses highlights the pro-oncogenic role of OBI and the risk of viral reactivation in immunocompromised OBI patients, which may lead to the (re)development of HBV-related liver disease [4, 8].

This study showed that the isolated anti-HBc serological status defined an important subset of OBI (Table 1). This result is consistent with that of previous studies in which isolated anti-HBc was identified as a significant serological marker of OBI [25, 26]. Isolated anti-HBc positivity refers to the detection of anti-HBc antibody without the usual accompanying markers (i.e., HBsAg if the infection is chronic and anti-HBs if the infection is resolved) [26]. The core antigen is a potent immunogen that elicits a strong and specific antibody response that is present during acute infection and usually persists for life; therefore, the presence of anti-HBc in the absence of any other marker is compatible with resolved or past hepatitis B infection [27, 28]. Several studies have highlighted the strong correlation between OBI and anti-HBc status [26, 29]. One investigation reported that of 16 patients with detectable serum HBV DNA at enrolment, 10 (63%) were positive for anti-HBc, whereas only 17 (42%) of 40 patients without serum HBV DNA tested positive for anti-HBc [30]. Similarly, studies show that the rate of HBV DNA is significantly high in anti-HBc–positive but anti-HBs–negative individuals, with a frequency of up to 60% in populations highly exposed to the virus [31]. The fact that OBI is more frequent in subjects positive for anti-HBc but negative for anti-HBs is presumably because these subjects lack the neutralizing effect of anti-HBs [32]; consequently, in the absence of hepatitis B DNA testing, isolated anti-HBc is an indicator of an important subset of OBI [25, 26]. Our study highlighted the superiority of anti-HBc over other HBV markers in predicting latent HBV infection in apparently healthy individuals and indicated that implementation of anti-HBc screening would improve the safety of blood donation.

In the present study, most OBI genomes were genotype B3. This is in accordance with previous studies showing that in Indonesia the most common HBV genotype is HBV B3 (HBV/B3), followed by HBV/C1, especially in Java island [21, 33].

The mechanisms underlying the low HBV DNA levels in the absence of detectable HBsAg in OBI remain unclear; however, both host and viral factors are important for viral replication suppression and for maintaining the control of HBV infection [34]. Several studies have suggested that host factors play a major role in the induction and maintenance of the occult status of HBV infection [24], as implied by an in vitro study showing that once the viruses are removed from the host’s liver microenvironment, the survival abilities of OBI isolates, in terms of replication, transcription, and protein synthesis, can be fully restored [35]. Further evidence was provided by a large-scale study that demonstrated the presence of potent and multi-specific HBV-specific T cell responses in OBI subjects. In that study, HBV-specific Th1 responses were quantitatively stronger in OBI than in inactive carriers and were similar or even higher against HBV antigens than those in patients with previous HBV resolution [34]. Therefore, the OBI phenomenon not only indicates the lack of complete clearance of HBV, but also reflects the ability of the host immune system to control leftover viruses in the liver after clinical resolution of disease through an efficient T-cell mediated response [34, 36].

The present study hypothesized that high levels of HLA-DPA1 and HLA-DPB1 expression will favour OBI development. HLA-DP is a heterodimer of specialized glycoproteins that deliver foreign peptides to the surface of the cell, and it belongs to a major isotype of HLA class II. It consists of two chains, the α (DPA) and β (DPB) chains, and plays a vital role in adaptive immunity [6]. The genes encoding the α and β chains (designated A and B, respectively) are expressed on the surface of antigen presenting cells, whereas in the liver their expression is limited to a small population of Kupffer cells, the resident macrophages of the liver [12]. These molecules encode proteins that are crucial for presenting HBV peptides to CD4⁺ helper T-cells by promoting T-cell allorecognition and peptide binding, resulting in an enhanced immune response and HBV clearance [37, 38]. CD4⁺ T cells also stimulate and preserve CD8⁺ cytotoxic T cells, which directly clear HBV-infected cells. As a consequence, a reduction in HLA-DP expression might interfere with antigen presentation and impair adaptive and cellular immune responses to HBV infection [6].

The present results support a previous theory on the impact of efficient T cell mediated immune responses on OBI induction and maintenance. We have shown that the minor T allele of rs3077 was the prominent allele detected in the OBI seropositive group in all the genetic models analyzed (Table 3). Furthermore, the T/A variants of rs3077 and rs9277535 in the haplotype model were also associated with a higher probability of OBI detection (Table 4). These findings are supported by previous studies that reported the involvement of HLA-DP polymorphisms in regulating antigen presentation, both at the cellular function and gene expression levels [39].

A previous study suggested that HLA-DP variants could influence the level of mRNA expression of HLA-DPA1 and HLA-DPB1 molecules in the liver, whereas other studies showed that a variant in the 3′ untranslated region (UTR) may affect mRNA stability through binding of regulatory factors or regulation by microRNAs [12, 37, 40]. The most convincing and well-investigated SNPs related to HBV infection and clearance, especially in the Asian population, are rs3077 and rs9277535, which are separated by approximately 22 kb and located within the 3′-UTR of HLA-DPA1 and HLA-DPB1, respectively [11, 12, 41‐43]. Because these SNPs are not located in the HLA-DP coding region, their biological effects are investigated by indirectly affecting the antigen-binding site through alterations in microRNA-binding sites leading to changes in HLA-DP gene expression [12]. This change could affect the stability and translation of mRNA by altering transcription factor binding. The minor “T” allele of rs3077 and minor “A” allele of rs9277535 are associated with increased mRNA expression of their respective genes [12, 38]. Therefore, high HLA-DPA1 and HLA-DPB1 expression might be more effective for presenting viral antigens to CD4⁺ helper T-cells, which may increase the capacity of the immune response to suppress viral replication [38]. The present study confirmed that rs3077-T and rs9277535-A, which were associated with increased mRNA expression of the HLA-DP gene, may favour a stronger immune response to suppress HBV replication, which leads to OBI.

Wasityastuti et al. confirmed that in the Indonesian population, HLA-DP variants exert a protective effect against HBV infection by reducing the susceptibility to HBV and increasing the spontaneous resolution of HBV infection [6]. This conclusion strengthens the evidence that the outcome of HBV infection could be influenced by the physical binding of HBV-derived peptides and their subsequent recognition by CD4⁺ helper T-cells, which is dependent on HLA-DP polymorphisms [14]; however, to the best of our knowledge, the present study is the first to examine the roles of HLA-DP variants in OBI.

This study reported a weak LD between rs9277535 and rs3077 (D’ = 0.59; Fig. 2), suggesting that the effects of these SNPs are likely to be independent [12]; however, the difference in LD values between the Indonesian and Japanese populations (Fig. 2) may be caused by the number of ethnic groups in Indonesia, which has a more heterogeneous population than Japan [6]. Japan is dominated by Mainland Japanese (98.5%), whereas Indonesia consists of three dominant ethnic groups (Javanese, 40.06%; Sundanese, 15.51%; and Malay, 3.7%) and more than 10 smaller ethnic groups [6, 44]. This multi-ethnic population provides a higher opportunity for mixture and gene flow that contribute to LD reduction [45]; however, this study found no association between HLA-DPA1 rs3135021 and OBI detection, although the haplotype association model indicated that the major allele “G” increased the probability of OBI. This result might be attributable to the sample size and the genetic diversity of the populations. A phylogenetic tree of 54.794 autosomal SNPs in 1928 individuals representing 75 populations shows that the Indonesian clade clustered separately from the Japanese, Korean, Chinese, Taiwanese, and Thai clades with a high bootstrap value. Indonesia, which is located near the equator, has a higher haplotype diversity than other populations of Northern latitudes [6, 46]. Accordingly, HLA-DPB1 rs2281388 deviated from HWE, which could be related to laboratory or genotyping factors, sample stratification, or random evolutionary changes that might lead to unreliable results [6, 47, 48].

In conclusion, HLA-DP variants were associated with OBI detection in seropositive Indonesian blood donors. The minor allele of rs3077 (T) in the HLA-DPA1 gene was related to an increased risk of OBI. A combination of haplotype markers (TGA for rs3077–rs3135021–rs9277535) was also associated with OBI detection. The HBV serological marker (isolated anti-HBc) may play an important role in predicting latent HBV infection. The results suggest that the combination of SNP genotyping in the HLA-DP gene and HBV serological marker testing may be a valuable screening method in the blood donation setting to prevent OBI transmission through blood donors.