Synthetic HIV Vaccine Targeting Glycopeptide Neutralizing Epitopes

描述（由申请人提供）：能够诱导广泛中和抗体的有效艾滋病毒疫苗仍然是防治艾滋病毒/艾滋病流行的一个重要目标。最近发现了一类新的甘聚糖反应性广泛中和抗体（bNAbs），以PG9、PG16、PGT121和PGT128为代表，为新型表位疫苗的设计提供了线索。这些bNAbs以显著的广度和效力中和原代HIV-1毒株，并且似乎靶向位于HIV-1 gp120可变结构域（V1/V2或V3）的糖肽表位。突变、生化和结构研究表明，PG9和PG16的表位是由两个n -聚糖（位于N160和N156/N173糖基化位点）和一个位于V1/V2结构域的肽块组成的糖肽，而PGT128的表位似乎涉及N332和N301位点的n -聚糖。然而，由于gp120糖基化的异质性，进一步表征多糖特异性变得复杂。为了进一步确定聚糖的特异性，我们采用合成方法构建了携带已定义聚糖的均匀的V1V2和V3糖肽，从而鉴定出在N160位点携带Man5GlcNAc2聚糖的环状糖肽表位和在N156/N173位点携带唾液化n -聚糖的环状糖肽表位作为PG9/PG16的最小表位。我们还设计并合成了环状V3糖肽，证实了位于N332位点的Man9/Man8GlcNAc2聚糖的本质，用于PGT128的识别。通过合成这些糖肽表位的能力，我们建议设计新的免疫原，并假设在噬菌体Qb（一种具有良好特征的病毒样颗粒）上以高度有序的重复模式显示糖肽表位将导致免疫原能够引发糖肽特异性中和抗体。我们将通过追求三个具体目标来检验这一假设。目的1：设计合成携带多价V1V2和V3糖肽表位的qb基免疫原。免疫原的构建包括化学酶合成炔糖肽，并通过铜催化叠氮化物-炔环加成（CuAAC）键合反应与叠氮化物标记的Qb偶联；目的二是通过抗体结合研究评价合成免疫原的抗原性；目的3评价合成疫苗在家兔体内的免疫原性。采用elisa和病毒中和试验对免疫兔抗血清进行分析。这些实验将评估糖肽表位免疫原免疫中和抗体反应的幅度、广度和持续时间。这项研究很可能在设计真正有效的HIV-1疫苗方面产生重要的新见解。

DESCRIPTION (provided by applicant): An effective HIV vaccine capable of inducing broadly neutralizing antibodies remains a significant goal in combating the HIV/AIDS epidemic. Recent discovery of a new class of glycan-reactive broadly neutralizing antibodies (bNAbs), represented by PG9, PG16, PGT121, and PGT128, has shed light on design of novel epitope-based vaccine. These bNAbs neutralize primary HIV-1 strains with remarkable breadth and potency and appear to target glycopeptide epitopes located in the variable domains (V1/V2 or V3) of HIV-1 gp120. Mutational, biochemical and structural studies suggest that the epitopes of PG9 and PG16 are glycopeptides consist of two N-glycans (at the N160 and N156/N173 glycosylation sites) and a patch of peptide in the V1/V2 domain, while the epitopes of PGT128 appear to involve the N-glycans at the N332 and N301 sites. However, further characterization of the glycan specificity was complicated by the heterogeneity in glycosylation of gp120. We used a synthetic approach to constructing homogeneous V1V2 and V3 glycopeptides carrying defined glycans for further defining the glycan specificity, which led to the identification of a cyclic glycopeptide epitope carrying a Man5GlcNAc2 glycan at N160 and a sialylated N-glycan at N156/N173 site as the minimal epitopes for PG9/PG16. We also designed and synthesized cyclic V3 glycopeptides that confirm the essence of a Man9/Man8GlcNAc2 glycan at the N332 site for PGT128 recognition. With the ability to reconstitute these glycopeptide epitopes via synthesis, we propose to design novel immunogens and hypothesize that displaying the glycopeptide epitopes on bacteriophage Qb (a well-characterized virus-like particle) in a highly ordered repetitive pattern will lead to immunogens capable of eliciting glycopeptide-specific neutralizing antibodies. We will test this hypothesis through pursuing three specific aims. Aim 1 is to design and synthesize Qb-based immunogens carrying polyvalent V1V2 and V3 glycopeptide epitopes. Construction of the immunogens includes chemoenzymatic synthesis of alkyne-glycopeptides and their subsequent conjugation with azide-tagged Qb via the copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction; Aim 2 is to evaluate the antigenicity of the synthetic immunogens by antibody binding studies; and Aim 3 is to evaluate the immunogenicity of the synthetic vaccines in rabbits. The anti-sera from immunized rabbits are analyzed by ELISAs and viral neutralization assays. These experiments will assess the magnitude, breadth and duration of neutralizing antibody responses from immunizations with the glycopeptide epitope-based immunogens. This study is likely to yield important new insights in the design of a truly effective HIV-1 vaccine.