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

项目摘要 靶向V1V2和V3的非中和抗体与RV144试验的适度保护相关 HIV Env区和抗体依赖的细胞毒性(ADCC)活性。这些发现重新激发了人们的活力 对艾滋病毒疫苗方法的兴趣，这种方法可以诱导保护性抗体，中和和非中和，与 FC职能能力。这项建议将研究环境信号序列(SS)在调制中的作用 HIV Env诱导具有Fc功能的保护性抗Env抗体的能力。建议的研究是基于我们的 初步发现，通过将HIV分离株AA05的SS与另一分离株AC02的SS交换，我们 产生gp120(AA05-02SS)，诱导V1V2和V3抗体应答，幅度更大，更高 效价，最重要的是在免疫的小鼠中有更强的Fc功能。Gp120 AA05-02SS诱导的ABS 与JRFL、MN、HxB2和A244的V2多肽发生交叉反应，而WT gp120 AA05与V2结合较弱 仅限于HxB2。值得注意的是，AA05-02SS诱导的抗体显示出V1V2特异性的ADCP活性，而WT gp120 AA05没有。的确，SS-SWAP改变了AA05上的高甘露糖和复杂多糖的比例- 02SS与WT AA05 gp120的质谱图；这些变化发生在N-糖链上 Gp120的V1V2、C2、V3和V4环。我们还发现，将AA05和AC02 SS交换到HIV Rejo Env 使Rejo病毒对V1V2特异性单抗具有更强的中和能力。生长抑素对环境的影响 免疫原性和病毒中和表型与单抗和凝集素改变的环境识别相关 专为高甘露糖和复合糖。在另一项研究中，将单一突变引入环境SS Rejo或JRFL的作用也影响病毒粒子相关环境蛋白上N-糖链的寡糖组成，在 转而调节环境识别和病毒对V1V2和V3特异性单抗的中和敏感性。因此， 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 环境影响表位暴露/稳定性和免疫原性，并通过选择特定的SS或 SS残留物/S，我们可以产生具有增强能力的环境免疫原，以诱导功能性抗体。我们会 通过评估SS交换/突变的Env免疫原的表位暴露变化来测试这一想法 Abs探针的稳定性和高能C-陷阱解离质量法研究N-糖链的变化 光谱分析(目标1)。我们将用精选的SS修饰的Env免疫原免疫小鼠，并比较它们的 诱导具有Fc功能的Env特异性抗体的能力。我们还将标识与以下内容关联的SS签名 功能性抗体的诱导。将使用产生具有或不具有Fc功能的抗体的免疫方案 分离单抗(目的2)。我们将评估SS-1诱导的疫苗诱导的抗体的保护效果。 利用人源化小鼠模型(AIM)进行被动转移/HIV攻击实验中的改良Env免疫原 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 有效的艾滋病毒疫苗能够诱导针对艾滋病毒的保护性抗体反应。

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.