Eliciting neutralizing antibodies and B cell responses using novel HIV Env immunogens in non-human primates

中和抗体可能是有效的HIV-1疫苗所必需的。然而，很少有候选疫苗 疫苗接种后有效地引发广泛中和抗体（bNAb）。最近，VRC工作组 在豚鼠和非人灵长类动物（NHP）中引发融合肽导向的bNAb。同样，我们最近 异源NFL（未裂解）三聚体-脂质体初免后在兔中引发bNAb：在Scripps加强 我们在引发免疫中用靶向N-聚糖缺失激活了B细胞应答 （Dubrovskaya等人，Immunity 2019）。我们分离了两种概括血清活性的兔bNAb。这些 bNAb针对Env脆弱性的两个不同位点。中和反应的激发是 通过靶向N-聚糖缺失、高密度脂质体阵列和异源三聚体恢复剂增强 增强环境在豚鼠的独立实验中，我们在多种动物中也使用 N-聚糖缺失，异源Env NFL初免：加强方法。最近的这些成果令人鼓舞 成功地解决了一个长达30年的问题。近天然三聚体穗的新时代 模拟物，加上微粒阵列，结构知情设计和Env特异性B的高分辨率分析 细胞和淋巴结应答，为更有效地引发bNAb提供了新的机会。我们提出了一个 综合的，多方面的方法，融合了世界领先的艾滋病毒Env三聚体设计，分析， 接种疫苗后的B细胞应答和抗体，NHP免疫组织分析和基于EM的分析正在进行 免疫应答和高分辨率Ab：三聚体相互作用。我们将使用有序的三聚体素数： 在兔子和豚鼠中引发bNAb，在NHP中引发这种反应，在小范围内转化为成功。 使用项目1（Wyatt）中的新型免疫原设计和呈递以及NFL的免疫接种， 通过核心B（Silvestri）将三聚体转化为NHP。我们将使用通过EM评估的“实时”血清Fab与三聚体的结合， 与基于NGS的快速mAb互补的核心C（Ward）中的多克隆IgG表位作图（EMPEM） 在项目2（Karlsson Hedestam）中进行克隆、测序和功能表达。与VRC合作 （Mascola）我们将定义非中和性mAb以掩盖不需要的非中和性表位或更好地 展示交叉中和mAb的表位。我们将比较NFL三聚体-脂质体与细胞表面NFL三聚体 阵列表达的激动人心的mRNA脂质包封技术。我们将评估是否在 与成年猕猴相比，幼年NHP B细胞库将更好地产生bNAb，如在哺乳动物中观察到的。 人类感染为了遵循我们在三聚体-脂质体疫苗接种后发现的第2层CD 4 b-定向的bNAb， 称为E70，我们将通过NHP中的定向NFL三聚体去糖基化来靶向CD 4 b。根据我们最近的 在发现非常广泛中和的疫苗诱导的mAb 1C 2之后，我们还将关注gp 41：120三聚体 接口.利用这些初步线索，我们将采取多方面、跨部门的综合办法 以指导在用接近天然的、未裂解的NFL Env三聚体接种后NHP中bNAb的引发。

Neutralizing antibodies are likely to be required for an effective HIV-1 vaccine. However, few candidate vaccines efficiently elicit broadly neutralizing antibodies (bNAbs) following vaccination. Recently, the VRC working group has elicited fusion peptide-directed bNAbs in guinea pigs and non-human primates (NHPs). Similarly, we recently elicited bNAbs in rabbits following heterologous NFL (uncleaved) trimer-liposome prime:boosting at Scripps where we activated B cell responses with targeted N-glycan deletions in the priming immunizations (Dubrovskaya et al, Immunity 2019). We isolated two rabbit bNAbs that recapitulate the serum activity. These bNAbs are directed against two distinct sites of Env vulnerability. The elicitation of neutralizing responses was enhanced by targeted N-glycan deletion, high-density liposomal array and heterologous trimer restorative boosting Env. In independent experiments in guinea pigs, we have elicited bNAbs in multiple animals also using a N-glycan deletion, heterologous Env NFL prime:boost approach. These recent outcomes are encouraging inroads toward the successful solution of a 3-decade-long problem. The new era of near-native trimeric spike mimics, coupled with particulate array, structure-informed design and high-resolution analysis of Env-specific B cell and lymph node responses, afford new opportunities to more efficiently elicit bNAbs. We propose an integrated, multi-faceted approach blending the expertise of world leaders in HIV Env trimer design, analysis of B cell responses and Abs following vaccination, NHP immune tissue analysis and EM-based analysis of ongoing immune responses and high-resolution Ab:trimer interactions. We will use well-ordered trimer prime:boosting that elicited bNAbs in rabbits and guinea pigs to elicit such responses in NHPs, translating success in small animals to NHPs using novel immunogen design and presentation in Project 1 (Wyatt) and immunization of NFL trimers into NHPs via Core B (Silvestri). We will use “real-time” serum Fab-to-trimer binding evaluated by EM polyclonal IgG epitope mapping (EMPEM) in Core C (Ward) in complement with rapid mAb NGS-based mAb cloning, sequencing and functional expression in Project 2 (Karlsson Hedestam). In collaboration with the VRC (Mascola) we will define either non-neutralizing mAbs to mask unwanted non-neutralizing epitopes or to better display the epitopes of cross-neutralizing mAbs. We will compare NFL trimer-liposomes to cell surface NFL trimer array expressed from the exciting mRNA lipid encapsulation technology. We will assess if immunization in the juvenile NHP B cell repertoire compared to adult macaques will better generate bNAbs as is observed during human infection. To follow our discovery of a tier 2 CD4bs-directed bNAb following trimer-liposome vaccination, termed E70, we will target the CD4bs by directed NFL trimer deglycosylation in the NHPs. Based on our recent discovery of the very broadly neutralizing vaccine-induced mAb, 1C2, we will also focus on the gp41:120 trimer interface. Leveraging these initial leads, we will undertake a multifaceted, cross-component integrated approach to guide the elicitation of bNAbs in NHPs following vaccination with near-native, uncleaved NFL Env trimers.