Using DNA/MVA/protein immunization of rhesus macaques to investigate how the background of the HIV-1 envelope and nature of the protein boost shape the genetic and functional antibody landscape.

摘要(项目摘要/摘要) 尽管在开发艾滋病毒疫苗上进行了强有力而漫长的努力，但免疫相关性是必要的 实现强有力和持续的保护仍是未知数。众多生物的发现和表征 广谱中和抗体(BNAbs)中和遗传多样性的病毒，观察到 在非人类灵长类动物中，bNAbs可以被动地预防感染，这在战略上产生了乐观情绪 选定的包膜(Env)免疫原将诱导具有广泛保护性的中和抗体(NAB)。 最近的研究表明，(I)保留天然三聚体特征和(Ii)基于 关于发展出bNAbs的个体的变体是有价值的追求。我们建议利用患者知情的方式 通过DNA/改良安卡拉牛痘(MVA)免疫和蛋白质增强免疫传递环境免疫原 确定环境病毒的自然历史和表现形式如何塑造基因抗体和功能性抗体 恒河猴(Rm)的景观。环境免疫原集来自两个感染了HIV-1的人 个体，由于其早期抗体反应的不同性质而被选择，这可以被模仿 疫苗接种，以及随后的“精英”或“差”NAB广度的发展。来源于以下来源的单抗 这两个个体在感染7个月后在生殖系使用、克隆、结合 亲和力和自体中和能力。因此，对抗体反应的详细了解 受免疫原选择和呈现形式的影响和改变，将确保疫苗努力能够 被驱动到bNAbs，同时避免产生功能有限的抗体的途径。 DNA/MVA，一种疫苗平台，在RM和MVA中产生强大和持久的环境特异性抗体反应 人类，并提供了针对SIV挑战的保护，将被用于提供患者衍生的序贯 环境免疫原。在这些免疫接种之后，将增加由gp120或天然蛋白组成的蛋白质。 柔性连接(NFL)gp140稳定的三聚体。我们随后的多参数分析将包括抗体 种系利用，体细胞超突变，克隆谱系的发育和寿命，结合亲和力(KD)， 以及中和病毒或促进Fc介导的信号转导以及免疫后激活的能力 生发中心(GC)B细胞和T滤泡辅助细胞(TFH)。达到前所未有的水平 我们的分析将在单个B细胞和单抗水平上进行，检查 存在于骨髓中的浆母细胞、记忆B细胞和长寿的浆细胞。根据结果 在这些分析中，我们将从免疫的猴子中选择单个B细胞进行RNA-Seq转录组 分析B细胞生物学与抗体功能的关系。我们的总体假设是 来自“精英”中和剂的免疫原将引发功能性中和抗体反应，而三聚体 蛋白质将进一步增强这些功能。相比之下，来自“差的”中和剂的免疫原将揭示机理。 阻碍了理想的抗体反应。

Abstract (Project Summary/Abstract) Despite a strong and lengthy effort focused on developing an HIV vaccine, immune correlates necessary to achieve robust and sustained protection remain unknown. The discovery and characterization of numerous broadly neutralizing antibodies (bnAbs) that neutralize genetically diverse viruses, and the observation that bnAbs can passively protect against infection in nonhuman primates has generated optimism that strategically selected envelope (Env) immunogens will induce neutralizing antibodies (nAbs) that are broadly protective. Recent studies suggest that Env immunogens that (i) preserve features of the native trimer and (ii) are based on variants from individuals that developed bnAbs are worthy pursuits. We propose to utilize patient-informed Env immunogens delivered via DNA/modified vaccinia Ankara (MVA) immunization followed by protein boost to determine how the natural history and presentation of the Envs shape the genetic and functional antibody landscape in rhesus macaques (RM). The Env immunogen sets are derived from two HIV-1 infected individuals, chosen due to the disparate nature of their early antibody responses, which can be mimicked by vaccination, and subsequent development of ‘elite’ or ‘poor’ nAb breadth. Monoclonal antibodies derived from these two individuals 7 months after infection display striking differences in germline usage, clonality, binding affinity, and autologous neutralization. Thus, a detailed understanding of how antibody responses are influenced and altered by the immunogen choice and presentation form will ensure that vaccine efforts can be driven towards bnAbs, while simultaneously avoiding pathways that produce antibodies with limited function. DNA/MVA, a vaccine platform that produces robust and durable Env-specific antibody responses in RM and humans, and has provided protection against SIV challenge, will be used to deliver sequential patient-derived Env immunogens. These immunizations will be followed by a protein boost consisting of either gp120 or ‘native flexibly linked’ (NFL) gp140 stabilized trimers. Our ensuing multi-parametric analysis will include antibody germline usage, somatic hypermutation, development and longevity of clonal lineages, binding affinity (KD), and capacity to neutralize virus or facilitate Fc-mediated signaling, as well as post-immunization activation of germinal center (GC) B cells and T follicular helper cells (Tfh). To achieve an unprecedented level of resolution, our analysis will take place at the single B cell and monoclonal antibody level, examining plasmablasts, memory B cells, and long lived plasma cells residing in the bone marrow. Based on the results of these analyses, we will select individual B cells from immunized monkeys for RNA-Seq transcriptome analysis to connect B cell biology with antibody functional capacity. Our overall hypothesis is that the immunogens from the ‘elite’ neutralizer will elicit functional neutralizing antibody responses, and the trimer protein will further augment these. By contrast, immunogens from the ‘poor’ neutralizer will reveal mechanistic roadblocks to the desired antibody responses.