Pure and Authentic HIV-1 Env Immunogens

描述（由申请人提供）：广泛中和抗体（bnAb）预计将成为疫苗引发的针对 HIV-1 的保护性免疫的重要组成部分。然而，迄今为止所有引发此类反应的尝试都失败了。该领域缺乏进展可能与候选免疫原的真实性不足有关。免疫原性研究的一致发现是，虽然免疫血清能够有效地与指标免疫原结合，但很少有抗体能够识别病毒颗粒上发现的包膜糖蛋白 (Env) 的天然形式。由于 nAb 具有选择性结合这些 Env 三聚体的能力，因此我们假设 Env 三聚体可能是唯一能够在疫苗环境中选择性且可靠地诱导 nAb 的抗原。由于分离纯天然环境三聚体存在问题，迄今为止检验这一假设是不可能的。例如，颗粒疫苗的表面带有许多非功能形式的 Env 以及天然三聚体。这些 Env 的替代形式是“混杂的”，因为它们被非中和抗体（非 nAb）识别。因此，它们会干扰 nAb 的发育。在这里，我们描述了完全消除非功能性 Env 的新方法，这将使我们能够分离和测试纯正的 Env 三聚体作为疫苗。我们的具体目标是： 具体目标 1：生产带有真正的 Env 三聚体作为 Env 唯一形式的病毒样颗粒。我们正在尝试从颗粒中消除非功能性的 Env，只留下完整的三聚体。新的战略使我们更接近实现这一目标。 具体目标 2：生成纯正 Env 三聚体的可溶形式。我们正在研究纯化真正的可溶性环境三聚体的方法。将表征纯可溶性三聚体的抗原拓扑和稳定性。 具体目标 3：评估兔子中纯正的三聚体免疫原。连续几组动物的免疫结果将指导 nAb 反应的一致性、幅度和广度的改进。 nAb 反应的动力学、持续时间和特异性将得到表征。 具体目标 4：使用纯三聚体 VLP 作为“诱饵”来拯救广泛中和的单克隆抗体。 BmAb 是疫苗设计的有用工具。纯正的 Env 三聚体可能是选择记忆 B 细胞的理想“诱饵”，从而可以拯救新的 bnmAb。我们将生产荧光染料标记的诱饵，并与一个小组合作，该小组最近展示了一种流式细胞术方法，可以从感染 HIV-1 的捐赠者身上拯救新的 bnmAb。任何新鉴定的 bnmAb 的中和效力、广度和特异性都将被确定。我们还将检查 IgG 序列以及与种系的分歧程度。

DESCRIPTION (provided by applicant): Broadly neutralizing antibodies (bnAbs) are expected to be a crucial component of vaccine-elicited protective immunity against HIV-1. However, all attempts to elicit such responses to date have failed. The lack of progress in this area could relate to the insufficient authenticity of candidate immunogens. A consistent finding of immunogenicity studies has been that while immune sera efficiently bind to the index immunogen, few Abs recognize the native form of Envelope glycoprotein (Env) found on virus particles. Because nAbs have the select ability to bind to these Env trimers, we hypothesize that Env trimers may in turn be the only antigen capable of selectively and reliably inducing nAbs in a vaccine setting. Testing this hypothesis has until now been impossible, due to problems in isolating pure native Env trimers. For example, particulate vaccines bear many non-functional forms of Env on their surfaces, as well as native trimers. These alternative forms of Env are "promiscuous", in that they are recognized by non-neutralizing Abs (non-nAbs). As a result, they interfere with the development of nAbs. Here we describe new methods to completely eliminate non-functional Env, which will allow us to isolate and test pure authentic Env trimers as vaccines. Our Specific Aims are: Specific Aim 1: To produce virus-like particles bearing authentic Env trimers as the sole form of Env. We are attempting to eliminate non-functional Env from particles, leaving only intact trimers. New strategies have brought us close to achieving this goal. Specific Aim 2: To generate soluble forms of pure authentic Env trimers. We are investigating methods to purify authentic soluble Env trimers. The antigenic topology and stability of pure soluble trimers will be characterized. Specific Aim 3: To evaluate pure authentic trimer immunogens in rabbits. The results from immunizations of successive groups of animals will guide improvements in the consistency, magnitude and breadth of nAb responses. The kinetics, duration and specificity of nAb responses will be characterized. Specific Aim 4: To rescue broadly neutralizing monoclonal antibodies using pure trimer VLPs as "bait". BnmAbs are useful tools for vaccine design. Pure authentic Env trimers may be ideal "baits" for selecting memory B cells, allowing new bnmAbs to be rescued. We will generate fluorochrome-labeled baits and work in collaboration with a group who has recently demonstrated a flow cytometry method to rescue new bnmAbs from HIV-1-infected donors. The neutralization potency, breadth and specificity of any newly identified bnmAbs will be determined. We will also examine IgG sequences and the extent of divergence from the germline.