IMMUNOGENICITY & STRUCTURES OF RHINOVIRUS-HIV CHIMERAS

提出的工作的目标是在原子细节序列中刻画 以及定义HIV-1免疫原的结构元素。使用这项技术 对于随机系统突变，我们能够生成 显示HIV-1序列的嵌合型人鼻病毒(HRV) 长度、序列和构象。诱变计划已经被 旨在解释HIV-1序列的大量自然分布。 我们正在使用免疫选择从“分子”中分离抗原嵌合体。 该系统识别显示HIV-1表位在 既有抗原性又有免疫原性的限制性构象。其中之一 HRV14：HIV-1嵌合体包含不匹配的HIV-1序列 这是任何已知的HIV-1分离物(指定为DN-6)。这种嵌合体引发了 一种抗HIV-1中和反应，是已报道的最强反应之一 用于任何艾滋病疫苗系统，并有效地中和了三个中的两个 测试了HIV-1毒株。 将建设三个特定的HRV14：HIV-1文库：两个侧重于 来自gpl20的HIV-1V3环，其中一个专注于保守的中和 来自gp41的表位。我们描述了一个“跨分支”的V3循环库 通过编码氨基酸残基含有V3环序列的复合体 存在于所有支系中。另一个V3循环库将重点放在分支B上 序列。第三个文库将包括插入保守的gp41 “ELDKWA”免疫原，以产生免疫显性显示 心率变异性表面的序列。与HIV-1相关的嵌合病毒 抗原性将从这些文库中免疫选择使用 中和抗HIV-1抗体。免疫选择的嵌合体是 被具有不同特异性的抗HIV-1抗体中和得好的话 用于免疫豚鼠，在一些情况下，还用于免疫黑猩猩。 HRV14：HIV-1嵌合体诱导的抗血清将被检测 中和实验室适应的艾滋病毒和初级分离株的能力- 1. 我们将确定MOST的子集的三维结构 使用X射线结晶学的免疫原性嵌合体。两个HRV14：HIV-1嵌合体 (包括DN-6)已经结晶并使X射线至少衍射到 3.0埃分辨率。这些嵌合体的结构将是 第一个揭示HIV-1 V3环序列的人，毫无疑问 免疫原性构象。一些嵌合体将被结晶成 一种或多种抗HIV单抗的Fab片段能够 中和多种HIV-1毒株。这些结构将会产生 对免疫原性的结构决定因素的洞察，并有望 有助于我们理解明显相似的序列在 免疫反应的强度和特异性。这些信息 从这些研究中学到的东西将直接适用于 针对艾滋病的有用疫苗。嵌合HRV的一个强项：HIV系统 它几乎可以用来呈现任何已识别的表位(或 其模拟表位)，鉴定具有免疫学特征的病毒 它模仿HIV的结构，并能够确定其结构 原子水平上的免疫原性HIV序列。

The goal of the proposed work is to characterize in atomic detail sequence and structural elements that define HIV-1 immunogens. Using the technique of random systematic mutagenesis, we are able to generate libraries of chimeric human rhinoviruses (HRVs) that display HIV-1 sequences with many lengths, sequences, and conformations. The mutagenesis schemes have been designed to account for the large natural distribution of HIV-1 sequences. We are using immunoselection to isolate antigenic chimeras from "molecular haystacks". This system identifies chimeras that display HIV-1 epitopes in constrained conformations that are both antigenic and immunogenic. One of the HRV14:HIV-1 chimeras contains an HIV-1 sequence that does not match that of any known HIV-1 isolate (designated DN-6). This chimera elicited an anti-HIV-1 neutralizing response that is among the strongest reported for any AIDS vaccine system and was effective in neutralizing two of three HIV-1 strains tested. Three specific HRV14:HIV-1 libraries will be constructed: two focus on the HIV-1 V3 loop from gpl20 and one focuses on a conserved neutralizing epitope from gp41. A "cross-clade" V3 loop library is described that will contain composites of V3 loop sequences by encoding amino acid residues that exist in all clades. Another V3 loop library will focus on clade B sequences. A third library will involve insertion of the conserved gp41 "ELDKWA" immunogen in order to generate an immunodominant display of this sequence on the surface of HRV. Chimeric viruses with relevant HIV-1 antigenicity will be immunoselected from these libraries using neutralizing anti-HIV-1 antibodies. Immunoselected chimeras that are neutralized well by anti-HIV-1 antibodies with diverse specificities will be used to immunize guinea pigs and, in a number of cases, chimpanzees. Antisera elicited by HRV14:HIV-1 chimeras will be assayed for their ability to neutralize both laboratory-adapted and primary isolates of HIV- 1. We will determine the three-dimensional structures of a subset of the most immunogenic chimeras using X-ray crystallography. Two HRV14:HIV-1 chimeras (including DN-6) have been crystallized and diffract X-rays to at least 3.0 Angstroms resolution. The structures of these chimeras will be the first to reveal HIV-1 V3 loop sequences that are unquestionably in immunogenic conformations. A number of chimeras will be crystallized with Fab fragments of one or more anti-HIV monoclonal antibodies capable of neutralizing multiple strains of HIV-1. These structures will yield insights into structural determinants of immunogenicity and will hopefully aid in our understanding of how apparently similar sequences differ in the strength and specificity of the immune responses elicited. The information learned from these studies will be directly applicable to the design of useful vaccines against AIDS. A strength of the chimeric HRV:HIV system is that it can be used to present virtually any identified epitope (or mimotope thereof), identify viruses with immunological characteristics that mimic those of HIV, and enable the determination of the structures of the immunogenic HIV sequences at the atomic level.