Exploring antibody recognition of the V3 region on HIV-1 to guide vaccine design

描述（由申请方提供）：能够中和多种HIV-1分离株感染性的单克隆抗体（mAb）已能够发现病毒包膜刺突上的相对保守位点，这些位点构成潜在的疫苗设计靶点。这些mAb也代表了指导所述疫苗设计的工具。MAb B4 e8结合包膜刺突gp 120亚基上V3环尖端的中心，并中和中等范围的病毒。B4 e8表位在HIV-1亚型B中出现的频率较低（约35%），这解释了其中和活性较低的原因。然而，B4 e8表位的变异，所有这些都涉及单个Arg Gln改变，在除亚型B之外的HIV-1亚型中以显著更高的频率（>70%）发生。这种改变大大降低了B4 e8的结合亲和力，因此，具有Gln的非亚型B病毒被B4 e8中和得很差。相反，具有Arg的非亚型B病毒被很好地中和。基于上述观察结果，我们假设B4 e8片段，即相当于B4 e8表位的V3尖端片段，也可能在B4 e8识别较差的病毒上可接近。 本资助申请的目的是确定B4 e8片段作为疫苗靶点对V3的潜在重要性。在本提案的R21探索阶段，我们将追求3个目标来解决这一点。在目标1中，将构建B4 e8突变体的组合文库，并利用酵母表面展示技术来鉴定互补位内与抗原功能性相互作用所需的残基。在目的2中，将通过体外抗体进化产生对非B亚型V3序列具有更高亲和力的B4 e8变体，并评价其对非B病毒的中和活性。在目标3中，将应用计算蛋白质设计来探索工程化抗原的可行性，所述工程化抗原有利地呈递由mAb B4 e8识别的V3区段，并评估选择抗原组合促进在动物中引发靶向B4 e8表位的抗体应答的能力。 R21阶段目标的实现将为不同HIV亚型的B4 e8片段的可及性提供关键的见解，因此，对疫苗靶点发现具有影响。因此，该提案的R33开发阶段侧重于免疫原设计和优化。在该阶段的目标1中，将进行抗原设计改进，从而应用从抗体应答中获得的对R21期免疫原的认识，并且将利用来自R21期的B4 e8变体作为模板来设计进一步的抗原，以靶向亲本抗体不识别的高频非亚型B V3序列。在目的2中，将用这些抗原免疫动物，并分析血清抗体应答，以改善交叉中和活性和与抗体模板匹配的特异性。公共卫生相关性：需要一种有效的疫苗来阻止HIV-1的全球传播。本申请的第一阶段试图确定病毒表面上蛋白质的相对保守区段是否可被抗体接近，以及此类抗体是否可以中和大量病毒分离株的感染性。如果是这样的话，所确定的片段将构成艾滋病毒疫苗研究的一个有希望的目标，因此，在应用的第二阶段，将大力追求优化实验性候选疫苗的设计和测试，以引发针对该目标片段的中和抗体。

DESCRIPTION (provided by applicant): Monoclonal antibodies (mAbs) capable of neutralizing the infectivity of a broad range of HIV-1 isolates have enabled the discovery of relatively conserved sites on the viral envelope spike that constitute potential vaccine design targets. These mAbs also represent tools to guide said vaccine design. MAb B4e8 binds central to the tip of the V3 loop on the gp120 subunit of the envelope spike and neutralizes a modest range of viruses. The B4e8 epitope occurs somewhat infrequently (~35%) in HIV-1 subtype B, explaining its somewhat modest neutralizing activity. However, variations of the B4e8 epitope, all of which involve a single Arg Gln alteration, occur at substantially higher frequency (>70%) in HIV-1 subtypes other than subtype B. This alteration reduces B4e8 binding affinity considerably and, accordingly, non-subtype B viruses with the Gln are neutralized poorly by B4e8. In contrast, non-subtype B viruses with an Arg are neutralized well. Based on the observations outlined above we hypothesize that the B4e8 segment, i.e. the V3 tip segment equivalent to the B4e8 epitope, may also be accessible on viruses that are recognized poorly by B4e8. The purpose of this grant application is to determine the potential importance of the B4e8 segment on V3 as a vaccine target. In the R21 exploratory phase of this proposal we will pursue 3 aims to address this point. In Aim 1, combinatorial libraries of B4e8 mutants will be constructed and yeast surface display technology will be utilized to identify residues within the paratope that are required for the functional interaction with antigen. In Aim 2, B4e8 variants with higher affinity for non-subtype B V3 sequences will be generated by in vitro antibody evolution and evaluated for neutralizing activity against non-B viruses. In Aim 3, computational protein design will be applied to explore the feasibility of engineering antigens that present the segment of V3 recognized by mAb B4e8 favorably and evaluate the ability of select antigen combinations to promote the elicitation in animals of antibody responses targeted to the B4e8 epitope. Achievement of the objectives in the R21 phase will provide critical insight into accessibility of the B4e8 segment on different HIV subtypes and, thus, have implications for vaccine target discovery. The R33 developmental phase of this proposal focuses therefore on immunogen design and optimization. In Aim 1 of this phase, antigen design improvements will be made, thereby applying insight gained from antibody responses to R21-phase immunogens, and B4e8 variants from the R21 phase will be utilized as templates to design further antigens to target high-frequency non-subtype B V3 sequences not recognized by the parental antibody. In Aim 2, animals will be immunized with these antigens and serum antibody responses will be analyzed for improved cross-neutralizing activities and specificities matching the antibody templates. PUBLIC HEALTH RELEVANCE: An effective vaccine is required to stop the global spread of HIV-1. The first phase of this application seeks to determine whether a relatively conserved segment of a protein on the surface of the virus is accessible to antibodies and whether such antibodies can neutralize the infectivity of a significant range of viral isolates. If so, the identified segment would constitute a promising target for HIV vaccine research and, therefore, in the second phase of application, optimization of the design and testing of experimental vaccine candidates to elicit neutralizing antibodies to the target segment will be pursued vigorously.