Targeting Immunogenicity to the MPER Hinge and C-helix for BNAb Elicitation

项目概要-总体 虽然明显需要疫苗，该疫苗引发针对人免疫缺陷病毒的广泛中和抗体（BNAb）。 一种高度变异的逆转录病毒HIV-1能够阻止人与人之间和全球范围内的传播，是实现这一目标的手段 仍然难以捉摸天然免疫过程中针对三聚体gp 160脆弱位点产生的抗体 感染促使逆转录病毒进一步突变，使个体准种多样化。一个明显的例外是 膜近端外部区域（MPER）部位是隐形的，主要浸没在脂质中， 在半融合/融合期间显示。因此，免疫压力不会混淆或促进病毒感染。 逃跑然而，天然产生的抗体的缺乏被假定是以下原因的结果： 考虑到脂质反应性、多特异性甚至自身反应性， 某些抗MPER mAb。相反，我们最近的小鼠免疫原性研究已经产生了germinal 使用脂质体阵列MPER的中心衍生的和T细胞依赖的、亲和力成熟的抗MPER抗体 无临床后遗症或显著抗脂质反应性的节段。这些发现与重新- 评估了HIV-1患者中自然产生的人抗MPER抗体的更高患病率。这个PO 1 专注于产生具有有效BNAb特异性的抗MPER反应，创造免疫原， 捕获在三聚体解旋的过渡期间产生的天然MPER构象状态，从 紧凑的环状gp 160碱基与平面病毒膜上的构象异构体的结合，具有最大MPER暴露。这 在项目1中，应使用Reinherz小组（DFCI）的分子免疫学方法实现目标， 结合项目2中Irvine组（MIT）的生物材料专业知识， 组分A-D、NMR（瓦格纳，哈佛）、电子显微镜（EM）（Walz，Rockefeller）、EPR（Song， NHMFL）和微刻（Love，MIT）。免疫原设计的优化将通过以下方式实现： 通过使用合成肽核酸在纳米疫苗中的脂质膜上定位MPER (PNA)通过NMR、EPR、EM和BIAcore测量来检查茎、酰基链和TM片段接头。在 此外，最佳的纳米级MPER段间距和组织将使未承诺的共同 BNAb的祖先（UCA）。抗体附着的载体空间将由生物材料配方控制 聚合物聚（乙二醇）（PEG）的“空间云”和利用纳米盘嵌入的gp 160在 异源免疫策略。通过单细胞显微雕刻拯救的引发的mAb的接近角 将进行评估，并与自然产生的BNAb进行比较。将化学佐剂共递送至 激活STING和ICOS通路将增加生发中心的CD 4 TFH， 超突变通过分析原液血清评估疫苗诱导的长期浆细胞发育 使用常规小鼠的IgG和单个骨髓（BM）浆细胞微雕刻以及完全IG- 基因座人源化小鼠（KyMouse）将允许在相关的临床前模型系统中进行免疫原调整。

Project Summary - Overall While there is an obvious need for vaccines eliciting broadly neutralizing antibodies (BNAbs) against the highly mutable retrovirus HIV-1 to stem person-to-person and global spread, the means to achieve this goal have remained elusive. Antibodies produced against trimeric gp160 sites of vulnerability during natural infection drive retroviral mutation further, diversifying quasispecies in individuals. One apparent exception is the membrane proximal external region (MPER) site, which is stealth, largely immersed in lipid and only revealed during hemifusion/fusion. Hence, immune pressures are not confounding or contributing to viral escape. However, a paucity of naturally arising antibodies has been postulated to be a consequence of deletion at the earliest B cell checkpoints in view of lipid reactivity, polyspecificity and even autoreactivity of certain anti-MPER mAbs. To the contrary, our recent mouse immunogenicity studies have generated germinal center-derived and T cell-dependent, affinity-matured anti-MPER antibodies using liposome-arrayed MPER segments without clinical sequelae or significant anti-lipid reactivity. These findings are consistent with the re- evaluated greater prevalence of naturally arising human anti-MPER antibodies in HIV-1 patients. This PO1 focuses on engendering anti-MPER responses with potent BNAb specificities, creating immunogens that capture native MPER conformational states arising during transitions of trimer unwinding, going from the compact torus-like gp160 base to conformers on the flat viral membrane with maximum MPER exposure. This goal shall be achieved using molecular immunology approaches of the Reinherz group (DFCI) in Project 1 in conjunction with the biomaterials expertise of the Irvine group (MIT) in Project 2 and utilizing technology components A-D, NMR (Wagner, Harvard), electron microscopy (EM) (Walz, Rockefeller), EPR (Song, NHMFL) and microengraving (Love, MIT), respectively. Optimization of immunogen design will be achieved by orientation of the MPER on lipid membranes in nanovaccines through the use of synthetic peptide nucleic acid (PNA) stalks, acyl chain and TM segment linkers and vetted by NMR, EPR, EM and BIAcore measurements. In addition, optimal nanoscale MPER segment spacing and organization will engage uncommitted common ancestors (UCAs) of BNAbs. Vector space of antibody attachment will be controlled by biomaterial formulation of polymer poly(ethylene glycol) (PEG) "steric clouds" and utilization of nanodisc-embedded gp160 in heterologous immunization strategies. Approach angles of elicited mAbs rescued by single-cell microengraving will be assessed and compared with those of naturally arising BNAbs. Co-delivery of chemical adjuvants to activate the STING and ICOS pathways shall augment CD4 TFH in germinal centers to drive somatic hypermutation. Assessment of vaccine-induced long-term plasma cell development by analysis of bulk serum IgG and single bone marrow (BM) plasma cell microengraving using conventional mice as well as complete Ig- locus humanized mice (KyMouse) will allow for immunogen tuning in a relevant pre-clinical model system.