Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4⁺ T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P < 0.001) response kinetics and superior ADCC (P < 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.

IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

需要采取策略来提高HIV-1包膜（Env）抗原（Ag）的免疫原性，以延长HIV-1疫苗诱导的更长寿命的B细胞应答的时间。HIV-1 Env gp140（天然或未切割的分子）或gp120单体蛋白引起相对较差的B细胞反应，这种反应是短暂的。我们假设CD4受体在T辅助细胞上的Env结合导致对T细胞募集的无反应作用，因此缺乏强，健壮和持久的B记忆反应。为了验证这一假设，我们通过与3-kDa CD4小蛋白模拟物进行稳定的交联来封闭gp140的CD4结合位点（CD4bs），从而阻止gp140在CD4 ⁺上的连接T细胞同时保留可被抗体依赖性细胞毒性（ADCC）效应子反应靶向的CD4诱导型（CDi）中和表位。重要的是，与接受gp140免疫的动物相比，恒河猴的免疫始终如一地产生出色的B细胞（P <0.001）反应动力学和卓越的ADCC（P <0.014）。在所检查的细胞因子中，CD4bs阻塞组的Ag特异性白介素4（IL-4）T辅助酶联免疫吸附点（ELISpot）检测增加得更早（P= 0.025）。重要的是，CD4bs阻塞的gp140抗原诱导了优异的B细胞和ADCC反应，并且升高的B细胞反应被证明具有显着的持久性，在免疫后持续超过60周。

重要性迄今为止，尚未尝试开发能够诱导有效且持久的B细胞反应的HIV疫苗。抗原特异性B细胞的发育和亲和力成熟通过B细胞与T滤泡辅助细胞之间的关键相互作用而发生在淋巴滤泡的生发中心。HIV包膜以可溶性脱落抗原或抗原-抗体复合物的形式结合T细胞上的CD4受体，从而导致这些专门的CD4阳性T细胞的活化受到损害。我们提出，CD4结合损伤是造成B细胞对基于HIV包膜的疫苗相对较差的应答的部分原因。为了验证这一假设，我们封闭了包膜抗原的CD4结合位点，并将其与当前使用的未封闭的包膜蛋白进行了比较。