Heydarchi Behnaz, Center Rob J, Bebbington Jonathan, Cuthbertson Jack, Gonelli Christopher, Khoury Georges, Mackenzie Charlene, Lichtfuss Marit, Rawlin Grant, Muller Brian, Purcell Damian
a Department of Microbiology and Immunology , The University of Melbourne at The Peter Doherty Institute for Infection & Immunity , Melbourne , VIC , Australia.
MAbs. 2017 Apr;9(3):550-566. doi: 10.1080/19420862.2016.1270491. Epub 2016 Dec 20.
We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.
我们从一头牛体内分离出了HIV-1包膜（Env）特异性记忆B细胞，这头牛在长期接种HIV-1 Env gp140三聚体疫苗后，产生了具有广泛中和活性的高滴度多克隆免疫球蛋白G（IgG）。我们从这些记忆B细胞中克隆出了与牛IgG匹配的重链（H）和轻链（L）可变（V）基因，并构建了具有人恒定（C）区/牛V区嵌合或全牛C区和V区的IgG单克隆抗体（mAb）。在42个筛选出的Ig⁺记忆B细胞中，两种单克隆抗体（6A和8C）显示出与gp140 Env的高亲和力结合。对这两种单克隆抗体的全牛和人嵌合异构体的表征表明，它们具有高度的型特异性，仅能结合可溶性未切割的AD8 gp140三聚体和共价稳定的AD8 SOSIP gp140切割三聚体，而不能结合单体gp120。V基因的基因组序列分析表明，6A单克隆抗体的第三个重链互补决定区（CDRH3）长度为21个氨基酸，而8C CDRH3长度为14个氨基酸。6A和8C的整个重链可变区（VH）与现有最佳匹配种系V基因的差异分别为27%和25%，6A和8C的CDRH3区体细胞突变率分别为47.62%和78.57%，表明与其他物种的CDRH3相比，体细胞超突变水平较高。对6A和8C的VH基因进行丙氨酸诱变表明，CDRH3中的半胱氨酸和色氨酸氨基酸对抗原结合至关重要。因此，这些牛疫苗诱导的抗HIV抗体具有一些精英人类广泛中和抗体的显著结构特征，如CDRH3大小和亲和力成熟过程中的体细胞突变。然而，虽然6A和8C单克隆抗体抑制了可溶性CD4与gp140 Env的结合，但它们没有重现多克隆抗体对HIV感染的中和活性。
