Presentation of Structural Determinants of the 2F5 Neutralization Epitope

描述(由申请人提供)： 开发能够产生针对不同基因亚型的HIV-1初级分离株的中和抗体的包膜蛋白免疫原仍然是HIV-1疫苗领域最困难的挑战。Gp41胞外区保守的膜-近端外区含有两个广泛中和的人类单抗2F5和4E10的表位，因此是HIV-1疫苗设计的重要靶点。然而，到目前为止，所有通过使用非结构多肽免疫原来诱导抗gp41抗体的尝试都失败了，这些抗体与感染人类产生的抗体相当。成功可能需要更深入地了解gp120/gp41复合体的结构基序，该结构基序能稳定病毒粒子表面的成熟包膜三聚体。目前的研究假设，受体介导的gp41激活涉及一个受调控的结构转换序列，其中有一个或多个可能揭示隐蔽中和表位的途径上的中间体(S)。我们最近发现了一个包含2F5表位的四链螺旋线圈结构(C43)，这表明2F5和4E10表位的结构方面可能是瞬时暴露的，并需要刺激中和抗体反应。这项研究计划的广泛和长期目标是使用蛋白质化学和结构生物学方法来设计和生产稳定形式的C43螺旋螺旋基序，以诱导广泛反应的中和抗体。这项研究的前提是，确定2F5中和表位最佳呈递的结构要求，可以鉴定出稳定的免疫原产物，从而产生有效的抗HIV-1免疫。这项研究的具体目的是：(1)设计和开发稳定版本的C43螺旋线圈结构域，该结构域含有2F5表位，用于免疫原性研究。我们将产生一个四链螺旋线圈的单链类似物，在其中四个螺旋螺旋发生突变，以确定有利于影响四聚体中螺旋间堆积相互作用的特定残基替换，以便稳定2F5表位的有序螺旋肽结构，从而保护表面暴露的侧链。(2)评价稳定的C43螺旋线圈变异体在小动物体内的免疫应答。我们将在兔和豚鼠身上进行免疫原性研究，以确定稳定的C43多肽蛋白是否涉及对动物的免疫。我们还将评估作为颗粒免疫原捕获到纳米微珠上的稳定盘绕分子的免疫原性。研究界和公共卫生界一致认为，预防性疫苗是控制全球艾滋病毒-1流行的明显的长期解决方案(1-8)。不幸的是，事实证明，这一目标难以实现，也没有这样的疫苗可用。克服这一重要的生物医学问题将需要新的和富有想象力的设计战略，使我们更接近成功的艾滋病疫苗的目标。

DESCRIPTION (provided by applicant): Development of envelope protein immunogens capable of eliciting neutralizing antibodies against primary HIV-1 isolates from different genetic subtypes remains the most difficult challenge in the field of HIV-1 vaccinology. The conserved membrane-proximal external region of the gp41 ectodomain bears the epitopes of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important target of HIV-1 vaccine design. However, all attempts to elicit anti-gp41 antibodies comparable to these produced in infected humans by using unstructured peptide immunogens have thus far failed. Success will likely require a deeper understanding of the structural motifs of the gp120/gp41 complex that stabilize the mature envelope trimer on the virion surface. Current thinking postulates that the receptor-mediated activation of gp41 involves a regulated sequence of structural transitions with one or more on-pathway intermediate(s) that may reveal cryptic neutralization epitopes. Our recent identification of a four-stranded coiled-coil structure (C43) encompassing the 2F5 epitope suggests that structural aspects of the 2F5 and 4E10 epitopes may be transiently exposed and required for stimulating neutralizing antibody responses. The broad, long-term objective of this research plan is to use protein chemistry and structural biology approaches to engineer and produce stable forms of the C43 coiled-coil motif for the induction of broadly reactive neutralizing antibodies. The premise of the proposed research is that definition of the structural requirements for optimal presentation of the 2F5 neutralization epitope can lead to the identification of stable immunogen products to generate effective anti-HIV-1 immunity. The Specific Aims of the proposed research are: (1) To design and develop stabilized versions of the C43 coiled-coil domain bearing the 2F5 epitope for immunogenicity studies. We will generate a single-chain analog of the tetraplex coiled coil in which the four mutagenesis to identify specific residue substitutions that favorably influence inter-helical packing interactions in the tetramer in order to stabilize an ordered helical peptide structure of the 2F5 epitope that preserves surface-exposed side chains. (2) To evaluate the immunological responses elicited by stabilized C43 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized involve immunization of the animals using the stabilized C43 peptide proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-8). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine.