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方面进行了大量而漫长的努力，但 实现强有力和持续的保护仍是未知数。发现和描述了许多 广泛中和抗体（bnAb），中和基因多样性病毒，以及观察到， bnAb可以被动地保护非人灵长类动物免受感染，这产生了乐观情绪， 选择的包膜（Env）免疫原将诱导广泛保护性的中和抗体（nAb）。 最近的研究表明，Env免疫原（i）保留天然三聚体的特征和（ii）基于 对来自个体的变体的研究是值得的。我们建议使用患者知情 通过DNA/修饰的安卡拉牛痘（MVA）免疫随后蛋白加强递送Env免疫原 以确定Envs的自然历史和呈现如何塑造遗传和功能抗体 景观在恒河猴（RM）。Env免疫原集来源于两个HIV-1感染者， 个体，由于其早期抗体反应的不同性质而选择，其可以通过 疫苗接种，以及随后的“精英”或“穷人”nAb宽度的发展。单克隆抗体来源于 感染后7个月，这两个个体在生殖系使用、克隆性、结合 亲和性和自体中和。因此，详细了解抗体反应是如何 受免疫原选择和呈现形式的影响和改变将确保疫苗的努力可以 驱动bnAb，同时避免产生功能有限的抗体的途径。 DNA/MVA，一种在RM中产生稳健和持久的Env特异性抗体应答的疫苗平台， 人类，并提供了对SIV攻击的保护，将用于提供连续的患者源性 Env免疫原。这些免疫接种之后将进行由gp 120或“天然”gp 120组成的蛋白质加强。 柔性连接的（NFL）gp 140稳定的三聚体。我们随后的多参数分析将包括抗体 种系使用，体细胞超突变，克隆谱系的发育和寿命，结合亲和力（KD）， 和中和病毒或促进Fc介导的信号传导的能力，以及免疫后激活 生殖中心（GC）B细胞和T滤泡辅助细胞（Tfh）。达到前所未有的水平， 分辨率，我们的分析将发生在单B细胞和单克隆抗体水平，检查 浆母细胞、记忆B细胞和存在于骨髓中的长寿浆细胞。根据结果 在这些分析中，我们将从免疫猴中选择单个B细胞用于RNA-Seq转录组 分析以将B细胞生物学与抗体功能能力联系起来。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应答， 蛋白质将进一步增加这些。相比之下，来自“穷人”中和剂的免疫原将揭示机制 对所需抗体反应的障碍。

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.