Structural Approaches to HIV-1 Immunogen Design and BNAb Analysis

摘要 在HIV-1包膜蛋白上只有少数几个部位代表了 广谱中和抗体(BNAbs)的诱导。四个与gp120亚基相关，而一个与gp120亚基相关 这些位点位于gp41的膜近端外区(MPER)。我们之前已经证明了 MPER是一种保守的结构单元，由两个螺旋组成，由带有串联关节的铰链隔开 促进病毒的半融合和融合。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bNAbs显示出异常的HIV-1毒株。 和分支中和广度，如单抗10E8所示。直到最近，人们还不知道如何引出 抗MPER特异性抗体。作为完整的gp160三聚体的一部分，mper的免疫原性也很差。 或者在分离的gp41亚单位免疫原的背景下。因此，抗MPER抗体不是最多的 在自然感染期间经常出现特异性。这种劣势并不令人惊讶，因为 MPER在很大程度上是脂质浸泡的。然而，最近，隐形脂质体排列在孤立的 CD4T细胞表位和TLR配体的结合能有效地促进MPER的产生。 小鼠骨髓(BM)浆细胞的发育，包括产生体细胞抗体的细胞 显微雕刻和单细胞聚合酶链式反应分析显示CDRH3基因高度突变，长度较长。免疫原性 这一部分由残基可及性主导，并受立体化学调节。其后果是 后者是在制造插入MPER片段的过程中可能产生不想要的特异性 进入类脂膜。这里，通过添加具有相关膜倾斜角的跨膜(TM)片段 由计算预测和结构/生物物理验证以及移除暴露的 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或变异的TM序列来插入MPER 变成纳米盘和脂质体。MPER残基的取向和深度将通过核磁共振和EPR进行评估 方法并结合分子电子显微镜和X射线进行迭代调整和精炼 用抗MPER抗体进行的结晶学研究。在AIM II中，使用常规小鼠和小鼠 完成小鼠免疫球蛋白基因座的人源化，我们将建立免疫原和疫苗时间表，以 产生bNab诱导，并与自然感染HIV-1的诱导进行比较。现有脂质体 配方、新型纳米盘和不断发展的生物材料阵列将用于免疫，然后是MPER 诱导抗体的残基特异性、提取活性、天然刺激物结合和HIV-1中和活性 将通过检测原装免疫球蛋白和单个骨髓浆细胞抗体进行评估。几种MPER的优化 将执行用于诱导针对M亚群分支的高效bNAb的序列，以准备 未来的纳米疫苗临床试验。

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.