IMMUNOGENICITY & STRUCTURES OF RHINOVIRUS--HIV CHIMERAS

所提出的工作的目标是在原子细节序列的特征 和定义HIV-1免疫原的结构元件。使用该技术 随机系统诱变，我们能够产生文库， 嵌合人鼻病毒（HRV），其展示HIV-1序列， 长度序列和构象诱变方案已经被 设计用于解释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， 干草堆”。该系统鉴定了在细胞中展示HIV-1表位的嵌合体， 限制性构象，其既是抗原性的又是免疫原性的。之一 HRV 14：HIV-1嵌合体含有与HIV-1序列不匹配的HIV-1序列， 任何已知HIV-1分离物（命名为DN-6）。这种嵌合体引发了 一种抗HIV-1中和反应， 对于任何艾滋病疫苗系统， 检测的HIV-1菌株。 将建立三个特定的HRV 14：HIV-1文库：两个侧重于 HIV-1 V3环来自gp 120，一个集中于保守的中和环， 来自GP 41的表位。描述了“交叉进化枝”V3环文库，其将 含有编码氨基酸残基的V3环序列的复合物 存在于所有进化枝中另一个V3循环库将集中于进化枝B 序列的第三个文库将涉及保守的gp 41 “ELDKWA”免疫原，以产生这种免疫显性展示。 HRV表面的序列。具有相关HIV-1的嵌合病毒 抗原性将从这些文库中免疫选择， 中和抗HIV-1抗体。 免疫选择嵌合体， 被具有不同特异性的抗HIV-1抗体很好地中和， 可以用来免疫豚鼠，在一些情况下，也可以免疫黑猩猩。 将测定由HRV 14：HIV-1嵌合体引发的抗血清的 能够中和实验室适应性和原发性HIV分离株， 1. 我们将确定一个子集的三维结构的最 免疫原性嵌合体。两个HRV 14：HIV-1嵌合体 （包括DN-6）已经结晶，并将X射线吸收到至少 3.0令人不安的决议。这些嵌合体的结构将是 第一次揭示HIV-1 V3环序列，毫无疑问， 免疫原性构象。许多嵌合体将被结晶化， 一种或多种抗HIV单克隆抗体的Fab片段，其能够 中和多种HIV-1病毒这些结构将产生 深入了解免疫原性的结构决定因素， 帮助我们理解在不同的基因组中， 所引发的免疫应答的强度和特异性。的信息 从这些研究中学到的知识将直接适用于设计 预防艾滋病的有效疫苗 嵌合HRV：HIV系统的优势 它可用于呈递几乎任何鉴定的表位（或 其模拟表位），鉴定具有免疫学特征的病毒 模拟艾滋病毒的结构，并能够确定 在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.