HIV Vaccines Based on GP120-CD4 Mimetic Complexes

描述（由申请人提供）：阻止艾滋病毒流行的最终手段是一种预防性疫苗，它可以阻止病毒在普通人群中的传播。现在人们普遍认为，这种疫苗必须引起抗艾滋病毒抗体反应以及细胞免疫才能提供保护。为了实现这一目标，有必要确定一种免疫原，这种免疫原将引发能够预防（中和）世界范围内发现的原发性HIV毒株感染的广泛中和抗体。我们的方法侧重于CD4结合诱导gp120的“受限”过渡状态结构。在之前的研究中，我们发现交联的gp120可溶性CD4复合物在猕猴体内引发了抗体，这些抗体可以中和各种初级分离株，而不考虑Clade。我们还表明，这些“广泛中和”的抗体是通过亲和层析从免疫血清中分离出来的，这种亲和层析含有与CD4模拟微小蛋白（CD4M9）连接的gp120的约束单链复合物（称为SCBaL/M9）。因此，单链gp120-CD4模拟复合物值得探索作为疫苗亚基免疫原，在人体内引发广泛中和抗体。然而，在初步实验中，SCBaL/M9诱导广泛中和抗体的效率低于单链gp120-CD4复合物（FLSC）。这可能意味着SCBaL/M9的很大一部分在稳态条件下不能维持链内相互作用，因此不能在gp120上呈现引发广泛中和抗体所需的关键约束决定因子。这种不稳定性与CD4M9已知的特性是一致的，CD4M9对gp120的结合亲和力仅为CD4的1%左右。与此一致的是，我们的初步研究表明SCBaL/M9的分子内相互作用比FLSC更不稳定。因此，我们将在本项目中评估的中心假设是，我们将通过产生高度稳定的分子内复合物的序列修饰来提高SCBaL/M9的免疫原性。为了探索这一假设，本项目的目的1将是设计和评估SCBaL/M9的修饰版本，以提高链内结合稳定性。目的2将比较修饰复合物与SCBaL/M9在兔体内的免疫原性。将进行结合和功能测定，以验证没有任何修饰的免疫原引起与人或兔CD4发生交叉反应的抗体。我们期望这些努力产生新的候选免疫原，可以在各种疫苗环境中可行地用于引发广泛中和抗体。

DESCRIPTION (provided by applicant): The ultimate means for stopping the HIV epidemic is a prophylactic vaccine that blocks virus transmission in the general population. It is now accepted that such a vaccine will have to elicit anti-HIV antibody responses as well as cellular immunity to provide protection. To meet this goal, it is necessary to identify an immunogen that will elicit broadly neutralizing antibodies capable of preventing (neutralizing) infection by primary HIV strains found worldwide. Our approach focuses on the "constrained" transition state structure of gp120 induced by CD4 binding. In previous studies, we showed that crosslinked gp120-soluble CD4 complexes elicited antibodies in macaques that neutralized a wide variety of primary isolates regardless of Clade. We also showed that these "broadly neutralizing" antibodies were isolated from immune sera by affinity chromatography with a constrained single chain complex (called SCBaL/M9) containing gp120 linked to a CD4 mimetic miniprotein (CD4M9). Thus, single chain gp120-CD4 mimetic complexes warrant exploration as vaccine subunit immunogens to elicit broadly neutralizing antibodies in humans. However, in preliminary experiments SCBaL/M9 elicited broadly neutralizing antibodies less efficiently than a single chain gp120-CD4 complex (FLSC). This could mean that a significant portion of SCBaL/M9 fails to maintain an intrachain interaction under steady state conditions and consequently does not present the key constrained determinants on gp120 that are needed to elicit broadly neutralizing antibodies. Such instability is consistent with the known properties of CD4M9, which has a binding affinity for gp120 that is only about 1% that of CD4. In agreement, our preliminary studies show that the intramolecular interactions in SCBaL/M9 are less stable than in FLSC. Accordingly, our central hypothesis, which we will evaluate in this project, is that we will improve the immunogenicity of SCBaL/M9 by sequence modifications that produce highly stabilized intramolecular complexes. In order to explore this hypothesis, Aim 1 of this project will be to design and evaluate modified versions of SCBaL/M9 for improved intrachain binding stability. Aim 2 will be to compare the immunogenicity of modified complexes versus SCBaL/M9 in rabbits. Binding and functional assays will be performed to verify that none of the modified immunogens elicits antibodies that crossreactive with human or rabbit CD4. We expect these efforts to yield new candidate immunogens that can be feasibly used to elicit broadly neutralizing antibodies in a variety of vaccine contexts.