Synthetic HIV Vaccine Targeting Glycopeptide Neutralization Epitopes

描述（由申请人提供）：能够诱导广泛中和抗体的有效HIV疫苗仍然是抗击HIV/AIDS流行病的重要目标。近年来发现了一类新的聚糖反应性广泛中和抗体（bNAb），以PG 9、PG 16、PGT 121和PGT 128为代表，为基于表位的新型疫苗的设计提供了新的思路。这些bNAb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 gp 120的可变结构域（V1/V2或V3）中的糖肽表位。突变、生物化学和结构研究表明，PG 9和PG 16的表位是由两个N-聚糖（在N160和N156/N173糖基化位点）和V1/V2结构域中的肽片组成的糖肽，而PGT 128的表位似乎涉及N332和N301位点的N-聚糖。然而，聚糖特异性的进一步表征由于gp 120糖基化的异质性而变得复杂。我们使用合成方法构建携带确定聚糖的同质V1 V2和V3糖肽，以进一步确定聚糖特异性，这导致鉴定在N160处携带Man 5GlcNAc 2聚糖和在N156/N173位点处携带唾液酸化N-聚糖的环状糖肽表位作为PG 9/PG 16的最小表位。我们还设计并合成了环状V3糖肽，其证实了在N332位点的Man 9/Man 8 GlcNAc 2聚糖对于PGT 128识别的本质。与通过合成重建这些糖肽表位的能力，我们建议设计新的免疫原，并假设展示噬菌体Qb（一个良好的特征病毒样颗粒）上的糖肽表位在一个高度有序的重复模式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：设计并合成Qb多价V1、V2和V3糖肽表位免疫原。免疫原的构建包括炔糖肽的化学酶促合成及其随后通过铜催化的叠氮化物-炔环加成（CuAAC）点击反应与叠氮化物标记的Qb缀合;目的2是通过抗体结合研究评估合成免疫原的抗原性;目的3是评估合成疫苗在兔中的免疫原性。用ELISA和病毒中和试验分析免疫兔的抗血清。这些实验将评估来自用基于糖肽表位的免疫原免疫的中和抗体应答的幅度、宽度和持续时间。这项研究可能会在设计真正有效的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.