Hepatitis B virus immune escape mutants

描述（由申请人提供）：全世界有近4亿人慢性感染B型肝炎病毒（HBV），并有肝硬化或肝癌的风险，需要进行肝移植。用HBV包膜蛋白免疫可预防HBV感染，而B型肝炎免疫球蛋白（HBIG）单独或与核苷类似物如拉米夫定一起可降低移植肝脏的再感染率。然而，由于中和逃逸突变体的出现，突破性感染可能发生在疫苗接受者和肝移植接受者中，通常伴随少量野生型病毒。这些突变体包含病毒包膜蛋白α决定簇中的氨基酸变化，病毒包膜蛋白是中和抗体的主要靶标。我们的初步研究表明，α决定簇周围的结构变化可能会损害病毒体分泌，但不能排除共感染野生型病毒或第二位点突变恢复病毒体分泌的可能性。在本研究中，我们拟分析21个α决定簇突变体和7个与耐药性相关的突变体的病毒体分泌效率。对于病毒体分泌缺陷或几乎缺陷的突变体，尽管在患者血液中可检测到，我们将确定是否可以通过少量野生型包膜蛋白或通过引入M133 T突变来克服缺陷，M133 T突变产生额外的N-连接糖基化位点。M133 T突变可以与经典的G145 R免疫逃逸突变的病毒体分泌缺陷共同进化并拯救该缺陷。此外，通过与Lok博士的合作，我们将直接验证在患者血液中发现的免疫逃逸突变体是包膜病毒颗粒，还是主要是无包膜核心颗粒。病毒体分泌是病毒生命周期的一部分，对于病毒感染性和向新宿主的传播至关重要。因此，确定免疫逃逸突变体可能存在的分泌缺陷，以及该缺陷是否可以通过共感染野生型病毒或二次突变来克服，可能有助于预测免疫逃逸突变体带来的未来威胁，并设计更好的遏制策略。如果通过共感染野生型病毒或补偿性第二位点突变恢复病毒体分泌，则可以在不久的将来在可感染HBV的细胞系中测试它们在中和抗体存在下的感染性。本研究将为HBV感染及其相关肝癌的防治提供重要信息。 公共卫生相关性：预防B型肝炎病毒感染的疫苗有时会因为逃逸突变体的出现而失败。我们希望确定这些突变体从感染细胞释放病毒的效率以及共感染野生型病毒或其他突变的影响。所获得的知识将有助于调整疫苗策略，以更好地控制这些突变体。

DESCRIPTION (provided by applicant): Nearly 400 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at risk for cirrhosis or liver cancer, which necessitates liver transplantation. Immunization with HBV envelope proteins prevents HBV infection, and hepatitis B immune globulin (HBIG), alone or together with nucleoside analogs such as lamivudine, reduces the re- infection rate of the grafted liver. However, breakthrough infection can occur in both vaccine recipients and liver graft recipients due to the emergence of neutralization escape mutants, often together with small amounts of wild-type virus. Such mutants contain amino acid changes in the alpha determinant of viral envelope proteins, the primary target of neutralizing antibodies. Our preliminary studies suggest that structural changes around the alpha determinant may impair virion secretion, but the possibility of the co-infecting wild-type virus or second-site mutations restoring virion secretion cannot be excluded. In the present study, we propose to analyze the virion secretion efficiencies of 21 alpha determinant mutants and 7 mutants associated with drug resistance. For mutants with defective or nearly defective virion secretion, which are nevertheless detectable in patient blood, we will determine whether the defect can be overcome by a small amount of wild-type envelope proteins or by introduction of an M133T mutation, which generates an extra N-linked glycosylation site. The M133T mutation can coevolve with and rescue the virion secretion defect of the classic G145R immune escape mutation. Furthermore, through a collaboration with Dr. Lok, we will directly verify whether the immune escape mutants found in patient blood are enveloped virus particles, or primarily as nonenveloped core particles. Virion secretion is part of viral life cycle critical for viral infectivity and transmission to new hosts. Therefore, establishing possible secretion defect of the immune escape mutants and whether the defect can be overcome by co-infecting wild-type virus or secondary mutations may help predict the future threat posed by immune escape mutants and design better containment strategies. If virion secretion is restored by co-infecting wild-type virus or a compensatory second-site mutation, their infectivity in the presence of neutralizing antibodies can be tested in the near future in a cell line infectible with HBV. This study will provide important information for the prevention and treatment of HBV infection and associated liver cancer. PUBLIC HEALTH RELEVANCE: The vaccines to prevent hepatitis B virus infection sometimes fail because escape mutants arise. We would like to determine the efficiency of such mutants to release virus from infected cells and the impact of co-infecting wild-type virus or additional mutations. The knowledge obtained will help adjust the vaccine strategy for better control of these mutants.