Inducing HIV-1 NAb Breadth by Native Trimer Prime-Boost Vaccination

尽管主要的HIV-1疫苗现在通常会引发有效的nabs，但有两个问题：nabs通常针对毒株-- 特定的“糖链空洞”，有限的宽度；2)接种疫苗的人之间的nabs不一致。关于后一点， 我们发现，在所有接种的疫苗中，消除葡聚糖头基团冲突显示出一致的第二层nabs 动物，这表明nabs经常不能导航糖链。与之形成鲜明对比的是，来自HIV的广泛NAb(BNAbs)- 1+捐赠者经常接触多糖，而不是避开它们。因此，我们假设，通过促进 NAB-葡聚糖接触，我们可能会提高疫苗的广度和一致性。在这里，我们提出了顺序 异源Prime-Boost(SHPB)免疫诱导针对2个保守蛋白/糖链表位的bNAbs (V2&融合肽；Fp)。这两个位点都适应多种NAB结合模式，促进了表位的集中 接近了。在目标1中，我们将确定一组5不同的、多V2bNAb敏感的、表达良好的三聚体。 虽然高三聚体表达是必不可少的，但这样的环境很少见。获得有用的三聚体的两种方法是： 1)检测各种V2敏感的“特殊”菌株；2)将KI V2敏感的菌株转化为高表达菌株。选定的环境 将被修改为：1)堵塞糖链空洞，2)KI共同的FP变体1(FP8var1)序列/KO N611 多糖(调节FP暴露)。在目标2中，我们将尝试使用病毒样颗粒(VLP)来改进疫苗NAB CH01 HC Ki BL6小鼠表达CH01 NAB重链(HC)的UCA 一个可以与不同的小鼠LC相结合的小鼠HC曲目。这些小鼠可以潜在地产生nabs来 像FP这样的额外目标。因为早期事件形成NAB反应，在最初的KLH-FP8v1引发各种 的概念将在随后的3个VLP镜头中进行测试，包括糖苷酶消化的VLP以最大限度地减少冲突 或免疫原性的外来多糖，以促进NAB-多糖的早期接触。以后的VLP拍摄将保持一致 以扩大从早期投篮中产生的NAB。5只小鼠将在中间时间点被处死，其余的在 最后一枪，让我们研究NAB个体发育。最好的引爆方案主要由以下因素决定 中和动力学，效力，广度和一致性，来自分子遗传学标准。这个养生法将是 在CH01前体通过过继转移降低到生理频率的小鼠中重新测试 Hc x Balb/c F1小鼠也表现出CH01 Hc频率降低&更稳健的遗传背景 促进NAB的发展。在确定了有效的引爆注射后，将测试组装的SHPB方案 在相同的F1小鼠中，试图提高广度。变量将包括重叠或非重叠序列 疫苗，增加菌株多样性，并增加表位的严格性。最后，我们将在特里亚尼测试领先的养生法 表达人多克隆抗体的小鼠。将监测疫苗和非疫苗株的血清中和情况。 将使用VLP探针拯救单抗，并分析SHM、V基因使用和H/L配对。NAB效价，广度， 将研究糖链变化、特异性、糖链排列活性和个体发育的影响。总的来说，我们希望 通过确定诱导糖依赖、V2和FP靶向NAB广度的方案来推进艾滋病毒疫苗。

Although leading HIV-1 vaccines now routinely elicit potent NAbs, 2 problems are 1) NAbs usually target strain- specific "glycan holes", limiting breadth, 2) NAbs are inconsistent among vaccinees. Regarding the latter point, we have found that eliminating glycan head group clashes reveals consistent tier 2 NAbs in all vaccinated animals, suggesting that NAbs often fail to navigate glycans. In stark contrast, broad NAbs (bNAbs) from HIV- 1+ donors frequently contact glycans rather than avoid them. Therefore, we hypothesize that, by promoting NAb-glycan contacts, we might improve vaccine breadth & consistency. Here, we propose sequential heterologous prime-boost (SHPB) immunizations to try to elicit bNAbs to 2 conserved protein/glycan epitopes (V2 & fusion peptide; FP). Both sites accommodate multiple NAb binding modes, facilitating epitope focused approaches. In Aim 1, we will identify a panel of 5 diverse, multi-V2 bNAb-sensitive, well-expressed trimers. Although high trimer expression is essential, such Envs are uncommon. 2 ways to obtain useful trimers will be: 1) to test various V2-sensitive "special" strains & 2) KI V2-sensitivity into high-expressing strains. Selected Envs will be modified to: 1) Plug glycan holes, 2) KI the common FP variant 1 (FP8var1) sequence/KO the N611 glycan (regulates FP exposure). In Aim 2, we will try to improve vaccine NAbs, using virus-like particles (VLPs) expressing trimers from Aim 1. CH01 HC KI BL6 mice express the UCA of the CH01 NAb heavy chain (HC) amid a mouse HC repertoire that can combine with diverse mouse LCs. These mice can potentially generate NAbs to additional targets like FP. Since early events shape NAb responses, following initial KLH-FP8v1 priming a variety of concepts will be tested in 3 subsequent VLP shots, including glycosidase-digested VLPs to minimize clashes or immunogenic foreign glycans to promote early NAb-glycan contacts. Later VLP shots will be kept consistent to expand NAbs arising from earlier shots. 5 mice will be sacrificed at an intermediate timepoint & the rest after the final shot, allowing us to study NAb ontogeny. The best priming regimen will be determined primarily from neutralization kinetics, potency, breadth & consistency, & from molecular genetics criteria. This regimen will be re-tested in mice where CH01 precursors are reduced to physiologic frequency by adoptive transfer & in CH01 HC x Balb/c F1 mice that also exhibit reduced CH01 HC frequency & a more robust genetic background to potentiate NAb development. Having identified effective priming shots, assembled SHPB regimens will be tested in the same F1 mice to try to improve breadth. Variables will include overlapping or non-overlapping sequential shots, increasing strain diversity, & increasing epitope stringency. Finally, we will test leading regimens in Trianni mice expressing polyclonal human IgG. Serum neutralization of vaccine & non-vaccine strains will be monitored. MAbs will be rescued using VLP probes & analyzed for SHM, V gene use, & H/L pairings. NAb titers, breadth, effects of glycan changes, specificity, glycan array activity & ontogeny will be investigated. Overall, we hope to advance HIV vaccines by identifying regimens that induce glycan-dependent, V2 & FP-targeted NAb breadth.