Modulating antigenic and immunogenic properties of HIV Env by altering signal sequence

项目摘要 RV 144试验的适度保护与靶向V1 V2和V3的非中和抗体相关 HIV Env区域和抗体依赖性细胞毒性（ADCC）活性。这些发现重新激发了 对HIV疫苗方法的兴趣，可以诱导保护性抗体，中和和非中和， FC功能能力。本研究将探讨Env信号序列（SS）在调节细胞凋亡中的作用。 HIV Env引发具有Fc功能的保护性抗Env Ab的能力。这项研究是基于我们的 初步发现，通过将HIV分离株AA 05的SS与另一个分离株AC 02的SS交换， 产生的gp120（AA05 - 02SS）诱导V1V2和V3抗体应答，其宽度增加， 滴度，最重要的是免疫小鼠中更大的Fc功能。gp120诱导的Abs AA05 - 02SS 与JRFL、MN、HxB2和A244的V2肽交叉反应，而WT gp120 AA05与V2弱结合 只有HXB2。值得注意的是，AA 05 - 02 SS诱导的Ab显示V1V2特异性ADCP活性，而WT gp120 AA 05没有。事实上，SS-交换改变了AA 05上高甘露糖和复杂聚糖的比例， 02SS与WT AA 05 gp120，如质谱法所示;这些变化发生在 v1v2，c2，v3和v4环。我们还发现，将AA 05和AC 02 SS交换到HIV REJO Env 使REJO病毒对V1V2特异性mAb的中和更具抗性。SS诱导的Env变化 与单克隆抗体和凝集素对Env识别改变相关的免疫原性和病毒中和表型 对高甘露糖和复合糖有特异性。在一项单独的研究中，将单个突变引入Env SS REJO或JRFL也影响病毒体相关Env上N-聚糖的寡糖组成， 反过来调节Env识别和病毒对V1V2和V3特异性mAb中和的敏感性。因此，我们认为， 我们提出了一个总体假设，即通过改变Env SS，我们可以调节HIV的糖基化 Env影响表位暴露/稳定性和免疫原性，并通过选择特定的SS或 SS残基，我们可以产生具有增强的引发功能性Ab的能力的Env免疫原。我们将 通过评估SS交换/突变Env免疫原的表位暴露变化来测试这一想法， 通过用Ab探测和通过高能C-陷阱解离质量在N-聚糖糖中强制改变来确定稳定性 光谱法（目标1）。我们将用选择的SS修饰的Env免疫原免疫小鼠，并比较它们的免疫效果。 诱导具有Fc功能的Env特异性Ab的能力。我们还将识别与以下内容相关的SS签名： 功能性Ab的诱导。将使用产生具有或不具有Fc功能的Ab的免疫方案 以分离mAb（目的2）。我们将评估疫苗诱导的抗体的保护效力，这些抗体由SS- 使用人源化小鼠模型在被动转移/HIV攻击实验中修饰的Env免疫原（目的 3）。这项研究的数据将提供有关HIV Env SS的重要信息，可用于设计更多 有效的HIV疫苗，能够引发针对HIV的保护性Ab应答。

Project Summary Modest protection of RV144 trial correlated with non-neutralizing antibodies targeting the V1V2 and V3 region of HIV Env and antibody-dependent cellular cytotoxicity (ADCC) activity. These findings reinvigorated the interest in HIV vaccine approaches that can induce protective Abs, neutralizing and non-neutralizing, with Fc functional competency. This proposal will investigate the role of Env signal sequence (SS) in modulating the capacity of HIV Env to elicit protective anti-Env Abs with Fc functions. The proposed study is based on our preliminary findings that, by swapping the SS of HIV isolate AA05 with SS from another isolate AC02, we produced gp120 (AA05-02SS) that induced V1V2 and V3 Ab response with increased breadth, higher titers, and most importantly greater Fc function in immunized mice. Abs induced by gp120 AA05-02SS cross-reacted with V2 peptides of JRFL, MN, HxB2 and A244, while the WT gp120 AA05 bound weakly to V2 of HxB2 only. Notably, Abs induced by AA05-02SS displayed V1V2-specific ADCP activity while the WT gp120 AA05 did not. Indeed, SS-swap altered the proportion of high-mannose and complex glycans on the AA05- 02SS vs WT AA05 gp120 as shown by mass spectrometry; these changes were at N-glycans that are in the V1V2, C2, V3 and the V4 loop of gp120. We also found that swapping AA05 and AC02 SS onto HIV REJO Env rendered REJO virus more resistant to neutralization by V1V2-specific mAbs. SS-induced changes of Env immunogenicity and virus neutralization phenotype correlated with altered Env recognition by mAbs and lectins specific for high-mannose and complex sugars. In a separate study, single mutations introduced to the Env SS of REJO or JRFL also affected oligosaccharide compositions of N-glycans on virion-associated Env, which in turn modulated Env recognition and virus sensitivity to neutralization by V1V2- and V3-specific mAbs. Hence, we propose an overall hypothesis that, by altering the Env SS, we can regulate the glycosylation of HIV Env to impact on epitope exposure/stability and immunogenicity, and by selecting a particular SS or SS residue/s we can generate Env immunogen with enhanced capacity to elicit functional Abs. We will test this idea by assessing the SS-swapped/mutant Env immunogens for changes in epitope exposure and stability by probing with Abs and for changes in N-glycan sugars by high energy C-trap dissociation mass spectrometry (Aim 1). We will immunize mice with selected SS-modified Env immunogens and compare their capacity to induce Env-specific Abs with Fc functions. We will also identify the SS signature associated with induction of functional Abs. Immunization regimen that produced Abs with or without Fc functions will be used to isolate mAbs (Aim 2). We will evaluate the protective efficacy of vaccine-induced Abs elicited by SS- modified Env immunogens in passive transfer/HIV challenge experiments using humanized mouse model (Aim 3). Data from this study will provide vital information about HIV Env SS that can be exploited to design more effective HIV vaccine capable of eliciting protective Ab response against HIV.