Structural Approaches to HIV-1 Immunogen Design and BNAb Analysis

摘要 HIV-1包膜蛋白上只有少数几个位点代表了HIV-1感染的易感区域。 广泛中和抗体（BNAb）的激发。其中四个与gp 120亚基相关， 这些位点位于gp 41的膜近端外部区域（MPER）。我们以前表明， MPER是一种保守的结构元件，由两个螺旋组成，由具有串联接头的铰链分开， 促进病毒半融合和融合。MPR特异性BNAb通过干扰gp 41发挥其抗病毒作用 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菌株 和进化枝中和宽度，如mAb 10 E8所示。直到最近，人们还不知道如何引出 抗MPER特异性Abs. MPER作为完整gp 160三聚体的组分， 刺突或在分离的gp 41亚单位免疫原的情况下。因此，抗MPER抗体不是最 在自然感染期间引起的频繁特异性。这种亚优势并不奇怪，因为 MPER在很大程度上是脂质浸入的。然而，最近，隐形脂质体阵列分离的 发现MPER和运载结合的CD 4 T细胞表位和TLR配体有效地促进MPER-1表达。 小鼠中的特异性骨髓（BM）浆细胞发育，包括产生具有体细胞的Ab的那些 如通过微刻和单细胞PCR分析所显示的，CDRH 3的超突变和冗长。免疫原 该片段的结构由残基可及性主导，并由立体化学调节。的后果 后者是在制造性插入MPER片段期间可能产生不需要的特异性 形成脂质膜这里，通过附加具有相关膜倾斜角的跨膜（TM）片段， 通过计算预测和结构/生物物理验证以及去除暴露的 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或变体TM序列插入MPER， 变成纳米盘和脂质体。将通过NMR和EPR评估MPER残基的方向和深度 方法和反复调整和完善结合分子电子显微镜和X射线 使用抗MPER Abs的晶体学研究。在Aim II中，使用常规小鼠以及KyMouse， 完成小鼠IG基因座的人源化，我们将建立免疫原和疫苗时间表， 产生BNAb激发，与自然HIV-1感染产生的BNAb激发进行比较。现有脂质体 制剂、新型纳米盘和不断发展的生物材料阵列将用于免疫，然后用于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 将通过检查原液IgG和单个BM浆细胞Ab进行评估。几种MPER的优化 将进行用于诱导针对M亚群进化枝的高效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.