Structural Approaches to HIV-1 Immunogen Design and BNAb Analysis

HIV-1 免疫原设计和 BNAb 分析的结构方法

• 批准号: 9982761
• 负责人: ELLIS L REINHERZ
• 金额: $ 128.98万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-05 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982761
• 关键词: 3-Dimensional; Affinity; Antibodies; Antibody Formation; Antigenic Variation; Antigens; Antiviral Agents; B-Lymphocytes; Binding; Biocompatible Materials; Biophysics; Bone Marrow; C-terminal; CD4 Positive T Lymphocytes; Cell Maturation; Cell-Matrix Junction; Cells; Characteristics; Chemicals; Chronic; Clinical; Conserved Sequence; Cryoelectron Microscopy; Development; Electron Microscopy; Elements; Epitopes; Excision; Formulation; Fostering; Frequencies; Future; HIV Envelope Protein gp120; HIV-1; Human; Immunization; Immunization Schedule; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Individual; Infection; Joints; Knowledge; Ligands; Lipid Binding; Lipids; Liposomes; Masks; Membrane; Membrane Lipids; Memory B-Lymphocyte; Methods; Molecular; Molecular Conformation; Monoclonal Antibodies; Mus; Mutation; Plasma Cells; Polysaccharides; Preparation; Production; Recombinants; Roentgen Rays; Schedule; Side; Site; Specificity; Structure; Subgroup; T-Lymphocyte Epitopes; Time; Transmembrane Domain; Vaccination; Vaccine Design; Vaccines; Variant; Viral; adduct; alpha helix; design; env Gene Products; gp160; immunogenic; immunogenicity; improved; nanodisk; nanovaccine; neutralizing antibody; novel; particle; plasma cell development; programs; stereochemistry

ABSTRACT There are only a handful of sites on the HIV-1 envelope protein that represent regions of vulnerability for elicitation of broadly neutralizing antibody (BNAbs). Four are associated with the gp120 subunit while one of the sites resides in the membrane proximal external region (MPER) of gp41. We previously showed that the MPER is a conserved structural element consisting of two helices separated by a hinge with tandem joints that facilitates viral hemifusion and fusion. MPER-specific BNAbs exert their anti-viral effect by disturbing gp41 conformational change required for viral entry into host cells. These BNAbs manifest extraordinary HIV-1 strain and clade neutralization breadth as exemplified by the mAb 10E8. Until recently, it was not known how to elicit anti-MPER specific Abs. The MPER is poorly immunogenic either as a component of the intact gp160 trimeric spike or in the context of an isolated gp41 subunit immunogen. Hence, anti-MPER Abs are not the most frequent specificity elicited during natural infections. This subdominance is not surprising given the fact that the MPER is lipid immersed to a significant degree. However, very recently, stealth liposomes arraying the isolated MPER and cargoing conjoint CD4 T cell epitope and TLR ligands were found to effectively foster MPER- specific bone marrow (BM) plasma cell development in the mouse, including those producing Abs with somatic hypermutation and lengthy CDRH3 as shown by microengraving and single-cell PCR analysis. Immunogenicity of this segment is dominated by residue accessibility and modulated by stereochemistry. The consequence of the latter is that unwanted specificities can be engendered during fabricated insertion of the MPER segment into a lipid membrane. Here, by appending transmembrane (TM) segments with relevant membrane tilt angles guided by computational predictions and structural/biophysical verification as well as removal of exposed misguiding chemical adducts, we shall program the desired nanovaccine immunogenicity for B cell memory and plasma cell development. In Aim I, we shall use native HIV-1 or variant TM sequences to insert the MPER into nanodiscs and liposomes. Orientation and depth of MPER residues will be assessed by NMR and EPR methods and iteratively adjusted and refined in conjunction with molecular electron microscopy and X-ray crystallographic studies using anti-MPER Abs. In Aim II, using conventional mice as well as the KyMouse with complete humanization of the mouse Ig loci, we shall establish both immunogens and vaccine schedules to engender BNAb elicitation, comparing them with those arising from natural HIV-1 infection. Existing liposome formulations, novel nanodiscs and evolving biomaterial arrays will be used for immunization and then MPER residue specificity, extraction activity, native spike binding and HIV-1 neutralizing activity of elicited antibodies will be assessed by examining bulk IgG and single BM plasma cell Abs. Optimization of several MPER sequences for induction of highly potent BNAbs against M subgroup clades will be performed in preparation for future clinical nanovaccine trials.

抽象的 HIV-1 包膜蛋白上只有少数位点代表易受感染的区域 广泛中和抗体（BNAb）的引发。其中四个与 gp120 亚基相关，而其中之一 该位点位于 gp41 的膜近端外部区域 (MPER)。我们之前表明 MPER 是一种保守的结构元件，由两个螺旋组成，两个螺旋由具有串联接头的铰链隔开， 促进病毒半融合和融合。 MPER 特异性 BNAb 通过干扰 gp41 发挥抗病毒作用 病毒进入宿主细胞所需的构象变化。这些 BNAb 表现出非凡的 HIV-1 毒株 和进化枝中和宽度，如 mAb 10E8 所示。直到最近，人们还不知道如何引出 抗 MPER 特异性抗体。 MPER 作为完整 gp160 三聚体的组成部分，免疫原性较差 尖峰或在分离的 gp41 亚基免疫原的背景下。因此，抗 MPER 抗体并不是最重要的。 自然感染期间经常引起特异性。考虑到这一事实，这种次要地位并不令人惊讶 MPER 在很大程度上是脂浸的。然而，最近，隐形脂质体排列分离的 发现 MPER 和货物联合 CD4 T 细胞表位和 TLR 配体可有效促进 MPER- 小鼠中特定骨髓 (BM) 浆细胞的发育，包括那些产生体细胞抗体的浆细胞 微雕刻和单细胞 PCR 分析显示超突变和长 CDRH3。免疫原性 该片段的结构由残基可及性主导并受立体化学调节。的后果 后者是在 MPER 片段的制造插入过程中可能会产生不需要的特异性 进入脂质膜。在这里，通过附加具有相关膜倾斜角度的跨膜（TM）片段 以计算预测和结构/生物物理验证以及暴露的去除为指导 误导化学加合物，我们将为 B 细胞记忆编程所需的纳米疫苗免疫原性 和浆细胞发育。在目标 I 中，我们将使用天然 HIV-1 或变体 TM 序列来插入 MPER 成纳米盘和脂质体。 MPER 残基的方向和深度将通过 NMR 和 EPR 评估 方法并结合分子电子显微镜和X射线进行迭代调整和完善 使用抗 MPER 抗体进行晶体学研究。在 Aim II 中，使用传统鼠标以及 KyMouse 为了完成小鼠 Ig 位点的人源化，我们将建立免疫原和疫苗方案 产生 BNAb 诱导，并将它们与自然 HIV-1 感染引起的 BNAb 进行比较。现有脂质体 配方、新型纳米圆盘和不断发展的生物材料阵列将用于免疫接种，然后进行 MPER 引发抗体的残基特异性、提取活性、天然刺突结合和 HIV-1 中和活性 将通过检查大量 IgG 和单个 BM 浆细胞抗体进行评估。几个MPER的优化 将进行用于诱导针对 M 亚组进化枝的高效 BNAb 的序列，以准备 未来的纳米疫苗临床试验。