Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.

HIV包膜糖蛋白（Env）表面被N-聚糖广泛屏蔽，限制了B细胞对保守的中和决定簇的识别。在选定的HIV感染个体中引发广泛中和抗体（bNAb）揭示，B细胞库可产生能够穿透聚糖盾的Abs。因此，我们试图确定靶向的N-聚糖缺失是否可能改变对Env的抗体应答。我们专注于保守的CD4结合位点（CD4bs），因为这是一个已知的中和决定簇，它没有糖基化作用以允许CD4受体参与，但被周围的N-聚糖环化。我们有选择地删除了排列良好的进化枝C 16055天然柔性连接（NFL）三聚体上CD4bs附近的潜在N聚糖位点（PNGS），以潜在地提高朴素B细胞的识别能力体内。我们产生了聚糖缺失的三聚体变体，该变体保持了天然样的构象和稳定性。使用一组由CD4bs定向的bNAb，我们证明了在N-聚糖缺失的三聚体变体上CD4bs的可及性得到改善。我们表明，缺少这些Env PNGS的假病毒对CD4bs特异性bNAb的中和作用更为敏感，但仍对未中和的mAb具有抵抗力。我们使用两种方法比较了缺失的聚糖和完全糖基化的NFL三聚体，进行了兔免疫原性实验。首先是删除4个PNGS位点，然后用完全糖基化的Env进行增强。第二个是删除4个位点，并在随后的增强中逐渐重新引入这些N-聚糖。我们证明，缺失16055 PNGS的三聚体会更快地引起血清抗体，以统计学上显着的方式更有效地中和CD4bs-近端PNGS缺失的病毒，并强烈趋向于完全糖基化的自体病毒的中和增加。此方法引发了血清IgG，该抗体能够交叉中和残基N276（天然或工程）上缺少N-聚糖的选定2级病毒，这表明有序三聚体的PNGS缺失是引发B细胞对此保守的中和决定簇反应的有前途的策略。