Pure and Authentic HIV-1 Env Immunogens

描述（由申请方提供）：广泛中和抗体（bnAb）预计是疫苗诱导的抗HIV-1保护性免疫的关键组分。然而，迄今为止，所有争取这种答复的努力都失败了。这一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，虽然免疫血清有效地结合到索引免疫原，但很少有抗体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，我们假设Env三聚体反过来可能是唯一能够在疫苗环境中选择性和可靠地诱导nAb的抗原。由于分离纯天然Env三聚体的问题，到目前为止，测试这一假设是不可能的。例如，颗粒疫苗在其表面上携带许多非功能性形式的Env以及天然三聚体。Env的这些替代形式是“混杂的”，因为它们被非中和Ab（非nAb）识别。因此，它们干扰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的真实Env三聚体作为疫苗。我们的具体目标是： 具体目的1：生产携带真实Env三聚体作为Env唯一形式的病毒样颗粒。我们正试图从颗粒中消除非功能性Env，只留下完整的三聚体。新的战略使我们接近实现这一目标。 具体目的2：产生纯的真实Env三聚体的可溶形式。我们正在研究纯化真正的可溶性Env三聚体的方法。将表征纯可溶性三聚体的抗原拓扑结构和稳定性。 具体目标3：在兔中评价纯的真实三聚体免疫原。来自连续动物组的免疫接种的结果将指导nAb应答的一致性、幅度和广度的改进。将表征nAb应答的动力学、持续时间和特异性。 具体目标4：使用纯三聚体VLP作为“诱饵”拯救广泛中和的单克隆抗体。BnmAbs是疫苗设计的有用工具。纯的真正的Env三聚体可能是选择记忆B细胞的理想“诱饵”，允许拯救新的bnmAbs。我们将生产荧光染料标记的诱饵，并与一个最近展示了流式细胞术方法的小组合作，从HIV-1感染的供体中拯救新的bnmAbs。将确定任何新鉴定的bnmAb的中和效力、广度和特异性。我们还将检查IgG序列和与种系的分歧程度。

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.