Immunogenicity of the Membrane-Proximal Region of HIV-1 gp41

描述(申请人提供)：3个广中和(NT)单抗2F5、4E10和Z13的表位位于HIV-1被膜蛋白gp41(MPR)的膜近端区域。此外，这些单抗中的两个比其他三个广谱NT单抗中和更多的HIV-1初级分离株。这使MPR成为中和的主要场所，并因此成为开发艾滋病保护性疫苗的主要目标。然而，也有证据表明，自然感染过程中产生的MPR特异性抗体并没有中和，尽管这还没有经过严格的测试。可能只有某种形式的MPR会引起NT抗体。大部分或全部MPR似乎与质膜相互作用，核磁共振研究表明，该区域在胶束中形成螺旋结构，但不在水溶液中。面对针对2F5线性表位的强烈抗体反应，反复尝试用含有2F5线性表位的工程多肽或融合蛋白免疫均未引起NT活性。最近的结构研究表明，2F5和4E10单抗的表位可能包括蛋白质和脂肪。因此，我们和其他人假设，如果MPR以脂质或质膜的形式呈现给免疫系统，它可能会诱导NT抗体。此外，其他特征，如gp41的其他区域，或细胞膜的特定区域，可能有助于中和敏感包膜尖峰上的MPR的结构。因此，我们设计了一组14个不同的gp41片段，并分析了它们的表达。我们已经开始证明，这些蛋白质中的一种如预期的那样在细胞表面表达。在这项建议中，我们描述了三个特定的目标：(I)鉴定位于细胞表面的gp41片段，并确定它们是否形成三聚体及其与单抗2F5和4E10的相对亲和力；(Ii)使用编码这些gp41结构中最有希望的DNA来免疫兔子；(Iii)测试产生的免疫血清中和HIV-1初级分离株的能力，并确定这些NT反应是否是MPR所特有的。我们的目标是确定在细胞表面的背景下，MPR是否可以引发NT抗体。如果成功，我们将利用我们的结果开发一种针对MPR的疫苗。因此，该R03提案解决了NIAID的一个核心公共卫生目标，即在细胞表面的背景下确定作为疫苗开发目标的MPR的有效性。

DESCRIPTION (provided by applicant): The epitopes for 3 broadly-neutralizing (Nt) MAbs, 2F5, 4E10 and Z13, are located in the membrane proximal region of the HIV-1 envelope protein, gp41 (MPR). Moreover, two of these MAbs neutralize a larger spectrum of HIV-1 primary isolates than any of the other three broadly-Nt MAbs. This makes the MPR a prime site for neutralization, and as such, a prime target for the development of a protective vaccine against AIDS. However, there is also evidence that the MPR-specific Abs produced during natural infection are not neutralizing, though this has not been rigorously tested. It may be that only a certain form of the MPR will elicit Nt Abs. Most or all of the MPR appears to interact with the plasma membrane and NMR studies show that this region forms a helical structure in micelles, but not in aqueous solution Repeated attempts to immunize with engineered peptides or fusion proteins bearing the 2F5 linear epitope have all failed to elicit Nt activity, in face of strong Ab responses against the epitope. Recent structural studies indicate that the epitopes for 2F5 and 4E10 MAbs may comprise both protein and lipid. Thus, we and others have hypothesized that the MPR may elicit Nt Abs if it is presented to the immune system in the context of lipid or the plasma membrane. Moreover, other features, such as other regions of gp41, or specific areas of the cell membrane, may contribute to the structure of the MPR on neutralization-sensitive envelope spikes. Thus, we have designed a panel of 14 different gp41 fragments, and have analyzed their expression. We have begun to show that one of these proteins is expressed on the cell surface, as intended. In this proposal we describe 3 specific aims: (i) to identify gp41 fragments that are located on the cell surface, and to determine whether they form trimers and their relative affinity for MAbs 2F5 and 4E10; (ii) to use the DNA encoding the most promising of these gp41 constructs to immunize rabbits; and (iii) to test the resulting immune sera for their ability to neutralize HIV-1 primary isolates, and to determine if these Nt reactivities are specific for the MPR. Our goal is to determine if Nt Abs can be elicited by the MPR in the context of the cell surface. If successful, we will then use our results to develop a MPR-targeting vaccine. Thus, this RO3 proposal addresses a central public health goal of the NIAID, in determining the validity of the MPR, in the context of the cell surface, as a target for vaccine development.