Relationship Between Mutations In S Protein And Coexistence Of HBsAg And HBsAb In Patients With Chronic Hepatitis B Virus Infection

Hepatitis B virus (HBV) is a serious and prevalent global public health problem. It is estimated that more than 2 billion people have been infected with HBV worldwide, and more than 350 million are chronic carriers of HBV. About 686 thousand patients with chronic hepatitis B (CHB) died from HBV-related liver disease annually, such as hepatic failure, liver cirrhosis, and hepatocelluar carcinoma. According to the World Health Organization (WHO), China is among areas with a high incidence of HBV infection. According to published estimates, there are 93 million patients with chronic HBV infection in our country, including about 20 million CHB patients.The HBV genome is a partially double-stranded circular DNA, which consists of a full-length negative strand and a shorter positive strand. The negative strand contains four open reading frames (ORF), which are S region, C region, P region, and X region. The S gene encoding the major hepatitis B surface (HBsAg) locates in the S region. The full-length S protein consists of 226 amino acid residues, and has been divided into subregions corresponding to structural domains:the N-terminal region (aa 100 to 99), the major hydrophilic region (MHR)(aa 100 to 169), and the C-terminal region (aa 170 to 226). The "a" determinant (aa 124 to 147), which is responsible for the stability, the conformational structure, and the immunogenicity of HBsAg, locates in the MHR. It comprises the most important site of recognition of HBsAg by antibodies and immune response cells.Clinically, the emergence of circulating HBsAg indicates the HBV infection, and the appearance of HBsAb usually represents resolution of infection and is regarded as the symbol of immunity to HBV infection. Traditionally, HBsAb is neutralizing antibody, which can neutralize and clears HBsAg. Therefore, it should be impossible to detect HBsAg and HBsAb simultaneously in the same serum. However, the coexistence of HBsAg and HBsAb in patients with chronic HBV infection has been studied and reported from time to time. Previous studies indicated that patients with HBsAg+/ HBsAb+ have a more aggressive risk of liver cirrhosis and hepatocellular carcinoma than patients with HBsAg+/ HBsAb-. Until now the mechanism underlying the simultaneous detection of HBsAg and HBsAb is not clear. Therefore, the aims of this study were to evaluate the relationship between the contradictory serological pattern of the coexistence of HBsAg and HBsAb and amino acid substitutions in S protein.Part I The clinical features of patients with simultaneous HBsAg and HBsAbThis part was to explore the clinical features of patients with simultaneous HBsAg and HBsAb. Fifteen patients were served as the experimental group as carrying both HBsAg and anti-HBs, meanwhile,22 patients with solely positive for HBsAg made up the control group. Serum specimens from all patients were collected and stored at -70?. The enzyme-linked immunosorbent assay and the chemiluminescent microparticle immunoassay were employed to qualitatively and quantitatively measure the HBV serological markers. Serum alanine aminotransferase (ALT) was detected by velocity method and the serum HBV DNA level was quantitatively detected by real-time PCR. Data were analyzed using SPSS 17.0 statistical software. There were no significant differences between the two groups with regard to age, gender, serum ALT level, HBsAg title, HBeAg positive rate, genotype, serum HBV DNA level.Part II Amino acid mutation analysis of S protein in patients with concurrent HBsAg and HBsAbThis section was to analyze the amino acid sequence of S protein for the two groups, so as to explore the relationship between the coexistence of HBsAg and HBsAb and mutations in S protein. Serum HBV DNA was extracted and the primers sequences were designed and synthesized. PCR was carried out and the PCR products were identified, purified and cloned before sequencing. The obtained sequences were analyzed by Chromas software and Omiga genetic analysis software. Following this, the obtained amino acid sequences were compared with the standard reference sequences from the NCBI website. Finally, the differences of amino acid variability between the two groups were analyzed using SPSS 17.0 software. Results showed the most frequent mutation in the two groups was located at position sl26. Only patients from the experimental group have the substitutions T/I126A/S, P127T, G130E/N, M133S, F134I, T140I, and G145R, which were confirmed to be related to the alteration of immunogenicity of HBsAg. When considering the N-terminal (p=0.029) and the MHR (p=0.000) of S protein, the amino acid variability of experiment group was markedly increased compared to that of control group. However, no statistically significant difference was found in the C-terminal. Importantly, mutations were mainly found in the "a" determinant and the amino acid variability of experiment group was remarkably higher than that of control group (p=0.008). There are two loops in the "a" determinant. Mutations gathered in loop1 and experiment group has the higher amino acid variability (p=0.006). However, there was no statistically significant difference in loop2 between the two groups. This research revealed that high amino acid variability in and/or around the "a" determinant contributes to the emergence of coexisting HBsAg and HBsAb. These results not only provide the theory evidence for exploring the mechanism of mutations causing the alteration of immunogenicity of HBsAg, but also are beneficial to clinical follow-up, prognosis judgement, and prevention of disease progression to advanced liver diseases.In summary, there was no statistically significant difference in the clinical features between the experimental group and the control group. Besides, high amino acid variability in and/or around the "a" determinant contributes to the emergence of coexisting HBsAg and HBsAb.