Tepjanta Patcharin, Saethang Thammakorn, Fujiyama Kazuhito, Misaki Ryo, Kimkong Ingorn
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand.
Department of Computer Science, Faculty of Science, Kasetsart University, Bangkok, Thailand.
PLoS One. 2025 Jan 6;20(1):e0316328. doi: 10.1371/journal.pone.0316328. eCollection 2025.
The "a" determinant, a highly conformational region within the hepatitis B virus large surface protein (LHBs), is crucial for antibody neutralization and diagnostic assays. Mutations in this area can lead to conformational changes, resulting in vaccination failure, diagnostic evasion, and disease progression. The "a" determinant of LHBs contains a conserved N-linked glycosylation site at N320, but the mechanisms of glycosylation in LHBs remain unclear. This study aimed to investigate the impact of amino acid substitutions at N320 on antigenicity, three-dimensional (3D) structures of LHBs, immunogenic epitopes, and HBV DNA levels. LHBs were mutated by substituting asparagine 320 with proline, cysteine, lysine, and glutamine. The reactivity of the mutants with antibodies was evaluated by western blotting and immunofluorescence staining. Results showed increased binding affinity in N320C, N320Q, and particularly N320P mutants compared to the wild type, likely attributed to conformational changes predicted by the I-TASSER server and further refined by the GalaxyRefine server. Analysis conducted using the IEDB server indicated that the N320P mutation increased the antigenic index, whereas the N320C mutation significantly decreased it. Conversely, the N320K and N320Q mutations exhibited minor effects on antigenicity. Our observations also identified N320P as a potential B-cell epitope and a binding epitope for MHC-I (T-cell epitope). Furthermore, mutating the conserved N-linked glycosylation site at position N320 to proline significantly increased the secretion of HBV DNA in virions. This study enhances our understanding of the impact of a single amino acid mutation at N320 on antibody interaction, LHBs conformation, immunogenicity, and HBV DNA replication. These insights hold promise for advancements in HBsAg detection and the development of vaccines against hepatitis B virus.
“a”决定簇是乙肝病毒大表面蛋白（LHBs）内的一个高度构象区域，对抗体中和及诊断检测至关重要。该区域的突变可导致构象变化，从而引发疫苗接种失败、诊断逃避及疾病进展。LHBs的“a”决定簇在N320处含有一个保守的N - 连接糖基化位点，但LHBs中糖基化的机制仍不清楚。本研究旨在探讨N320处氨基酸替代对抗原性、LHBs的三维（3D）结构、免疫原性表位及乙肝病毒DNA水平的影响。通过将天冬酰胺320分别替换为脯氨酸、半胱氨酸、赖氨酸和谷氨酰胺来突变LHBs。通过蛋白质免疫印迹法和免疫荧光染色评估突变体与抗体的反应性。结果显示，与野生型相比，N320C、N320Q尤其是N320P突变体的结合亲和力增加，这可能归因于I - TASSER服务器预测并经GalaxyRefine服务器进一步优化的构象变化。使用IEDB服务器进行的分析表明，N320P突变增加了抗原指数，而N320C突变则使其显著降低。相反，N320K和N320Q突变对抗原性的影响较小。我们的观察结果还确定N320P是一个潜在的B细胞表位和MHC - I（T细胞表位）的结合表位。此外，将N320位置保守的N - 连接糖基化位点突变为脯氨酸可显著增加病毒粒子中乙肝病毒DNA的分泌。本研究增进了我们对N320处单个氨基酸突变对抗体相互作用、LHBs构象、免疫原性及乙肝病毒DNA复制影响的理解。这些见解有望推动乙肝表面抗原检测的进展及乙肝病毒疫苗的研发。
