HIV Vaccines Based on GP120-CD4 Mimetic Complexes

描述(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，它阻止病毒在普通人群中的传播。现在公认的是，这样的疫苗必须引起抗艾滋病毒抗体反应和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，它将产生广泛的中和抗体，能够预防(中和)世界各地发现的主要艾滋病毒毒株的感染。我们的方法集中在由CD_4结合诱导的gp120的“受限”过渡态结构。在以前的研究中，我们表明，交联型gp120可溶性CD4复合体能在猕猴体内激发抗体，中和各种初级分离株，而不考虑Clade。我们还发现，用含有gp120的限制性单链复合体(称为SCBaL/M9)，通过亲和层析从免疫血清中分离到这些“广中和”抗体，gp120连接到一个CD4模拟小蛋白(CD4M9)。因此，单链gp120-CD4模拟复合体作为疫苗亚单位免疫原值得探索，以在人类中诱导广泛的中和抗体。然而，在初步实验中，SCBaL/M9诱导广谱中和抗体的效率低于单链gp120-CD4复合体(FLSC)。这可能意味着在稳态条件下，SCBaL/M9的很大一部分未能维持链内相互作用，因此不存在gp120上的关键限制性决定簇，而gp120是诱导广泛中和抗体所需的。这种不稳定性与CD4M9的已知性质是一致的，CD4M9与gp120的结合亲和力仅为CD4的1%左右。我们的初步研究表明，SCBaL/M9中的分子内相互作用不如FLSC中的稳定。因此，我们的中心假设，我们将在本项目中进行评估，即我们将通过序列修改来提高SCBaL/M9的免疫原性，从而产生高度稳定的分子内复合物。为了探索这一假设，本项目的目标1将设计和评估SCBaL/M9的修饰版本，以改善链内结合稳定性。目的2比较修饰后的复合物与SCBaL/M9在兔体内的免疫原性。将进行结合和功能分析，以验证修改后的免疫原都不会引起与人或兔的CD4交叉反应的抗体。我们预计这些努力将产生新的候选免疫原，可用于在各种疫苗环境中诱导广泛的中和抗体。

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.