O'Rourke Sara M, Sutthent Ruengpung, Phung Pham, Mesa Kathryn A, Frigon Normand L, To Briana, Horthongkham Navin, Limoli Kay, Wrin Terri, Berman Phillip W
Department of Biomolecular Engineering, Baskin School of Engineering, University of California Santa Cruz, Santa Cruz, California, United States of America.
National HIV Repository and Bioinformatics Center (Thailand), Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
PLoS One. 2015 Mar 20;10(3):e0119608. doi: 10.1371/journal.pone.0119608. eCollection 2015.
Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain β-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.
了解对中和抗体敏感性和抗性的分子决定因素对于开发旨在预防HIV感染的疫苗至关重要。在本研究中，我们采用遗传学方法来表征HIV包膜蛋白中赋予中和敏感性或抗性的自然发生的多态性。从感染CRF01_AE病毒的个体构建了源自病毒准种的密切相关包膜基因文库。用含有广泛中和抗体的血浆筛选这些文库，并选择中和敏感和抗性变体进行序列分析。体外诱变使我们能够鉴定出三名个体中导致对这些抗体中和产生抗性的单个氨基酸变化。所有这三个突变在四链V1/V2结构域β-折叠结构中A链C末端与B链N末端之间的高变连接肽附近产生了N-连接糖基化位点（两个在N136，一个在N149）。尽管N136先前已被认为与广泛中和单克隆抗体的结合有关，但该糖基化位点似乎抑制了HIV-1感染受试者血浆中中和抗体的结合。先前的研究报道，与从慢性感染中分离出的病毒相比，传播奠基者病毒中V1/V2结构域的长度较短且糖基化位点较少。我们的结果表明，基于CRF01_AE分支病毒重组包膜蛋白的疫苗免疫原可能通过包含缺乏这些糖基化位点的包膜蛋白而得到改善。这一策略可能会提高在部分成功的RV144 HIV疫苗试验中使用的疫苗的效力，在该试验中，两种CRF01_AE免疫原（源自A244和TH023分离株）在N136和N149处均具有糖基化位点。
