Approaches to inducing broadly neutralizing antibodies with immunogens mimicking steric occlusion of the MPER as configured on the HIV-1virion surface

摘要 鉴于HIV-1结构的复杂性，开发预防性艾滋病疫苗是一项艰巨的任务。 包膜（Env）蛋白以及由免疫驱动的病毒准种之间的广泛抗原变异 逃生机制此外，针对Env三聚体上的非中和表位的免疫显性， 病毒粒子表面的唯一病毒抗原，通过以下方式引发广泛中和抗体（bnAb）： 接种疫苗特别困难。尽管如此，分离的bnAb的免疫学和结构表征仍然是不确定的。 从HIV-1感染的供体中获得的新的抗原决定簇已经引导了额外的中和靶表位，沿着新的方法， 疫苗战略。gp 41亚基的膜近端外部区（MPER）是一种有吸引力的bnAb 如4 E10、10 E8和DH 511和2F 5所示， 单克隆抗体然而，包括肽、蛋白质支架或MPER/脂质体的MPER免疫原疫苗具有 所有这些都不能引起中和活性，表明病毒粒子上四级结构的不完全模拟 面此外，MPER的可及性是有限的，被gp 160三聚体胞外域屏蔽， 病毒膜从上到下，导致MPER的免疫原性差， 三聚体免疫原。gp 140三聚体免疫原的封闭而非开放构型已被证明是 这对于引发针对gp 120中表位的bnAb是重要的。同样，我们最近的MPER/脂质体结果 提示，用当前疫苗制剂提供给B细胞受体的不受限制的接近角度是 这是有问题的，导致大多数Ab的诱导没有中和活性或gp 160三聚体反应性。 因此，MPER免疫原必须模拟空间闭塞，并强制限制抗体对MPER的可及性。 以类似于病毒粒子上的GP 160四级结构构型所施加的方式 面鉴于MPER N-末端区域的序列变异，我们将制定策略， 增强针对Aim 1中MPER C-末端区域的亚显性Ab应答。基因敲入（KI）小鼠 产生具有长CDRH 3环的抗体的模型，外在因素如环状二GMP佐剂，ICOSL 和持续的抗原供应将单独或组合测试其对增强免疫功能的影响。 MPER C-末端区域特异性Ab。在目标2中，我们将利用纳米磁盘技术作为平台， 用于将gp 160组装到天然膜样环境中以引发或加强MPR特异性Ab 以期望的接近角响应，并消除偏离目标的矢量响应。结合 在目标1中优化的疫苗方案中，我们将寻求互补的异源免疫策略， 小鼠和兔模型以促进4 E10/10 E8样bnAb的诱导。DNA C粒子和 MPERTM/脂质体免疫原将进一步不利于gp 120 -41定向的显性不期望的扩增。 由gp 160/纳米盘引发的Ab应答，同时促进诱导足够的bnAb血清滴度 有必要的接近角度将确定MPR特异性bnAb的遗传和生物物理特征。

ABSTRACT Development of a preventive AIDS vaccine is a daunting task given the structural complexity of the HIV-1 envelope (Env) protein as well as extensive antigenic variation among viral quasispecies driven by immune escape mechanisms. Moreover, immunodominance toward non-neutralizing epitopes on the Env trimer, the sole viral antigen on the virion surface, makes the elicitation of broadly neutralizing antibody (bnAb) by vaccination particularly difficult. Nonetheless, immunological and structural characterizations of isolated bnAbs from HIV-1 infected donors have guided additional neutralizing target epitopes along with new approaches in vaccine strategies. The membrane proximal external region (MPER) of gp41 subunit is an attractive bnAb target given its linear and conserved epitope sequences as exemplified by 4E10, 10E8 and DH511 and 2F5 mAbs. However, MPER immunogen vaccines including peptides, protein scaffolds or MPER/liposomes have all failed to elicit neutralizing activities, suggesting incomplete mimicry of the quaternary structure on the virion surface. Furthermore, the accessibility of the MPER is limited, being shielded by gp160 trimer ectodomain from above and the viral membrane from below, contributing to the poor immunogenicity of the MPER elicited by trimer immunogens. A closed rather than open configuration of gp140 trimer immunogens has proven to be important for elicitation of bnAbs directed to epitopes in gp120. Likewise, our recent MPER/liposome results suggest that the unrestricted approach angle afforded to the B cell receptor with current vaccine formulation is problematic, resulting in induction of a majority of Abs without neutralizing activity or gp160 trimer reactivity. Therefore, MPER immunogens must mimic spatial occlusion and enforce limited Ab accessibility to the MPER in a manner analogous to that imposed by the quaternary structural configuration of gp160 on the virion surface. Given sequence variations in the N-terminal region of the MPER, we will develop strategies to augment subdominant Ab responses directed to the MPER C-terminal region in Aim 1. A knock-in (KI) mouse model generating antibodies with long CDRH3 loops, extrinsic factors such as cyclic di-GMP adjuvant, ICOSL and persistent antigen supply will be tested independently or in combination for their impact on augmenting MPER C-terminal region-specific Abs. In Aim 2, we shall exploit nanodisc technology that serves as a platform for the assembly of gp160 into a native membrane-like environment to prime or boost MPER-specific Ab responses with a desirable approach angle and to eliminate off-target vector responses. In conjunction with optimized vaccine regimen in Aim 1, we shall pursue complementary heterologous immunization strategies in mouse and rabbit models to foster the induction of 4E10/10E8-like bnAbs. DNA C-particle and MPERTM/liposome immunogens will further disfavor expansion of gp120-41 directed dominant undesirable Ab responses elicited by the gp160/nanodisc, while facilitating the induction of sufficient serum titers of bnAbs with requisite approach angles. Genetic and biophysical features of MPER-specific bnAbs will be determined.