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

尽管目前领先的HIV-1疫苗通常会引发有效的NAb，但存在两个问题：1）NAb通常靶向毒株- 特异性“聚糖孔”，限制宽度，2）NAb在疫苗接种者中不一致。关于后一点， 我们发现，消除聚糖头基冲突后，所有接种疫苗的人体内的2级NAb一致， 动物，这表明NAb通常无法导航聚糖。与此形成鲜明对比的是，来自HIV-1的广泛NAb（bNAb） 1+供体经常接触聚糖，而不是避免它们。因此，我们假设，通过促进 NAb-聚糖接触，我们可能会提高疫苗的广度和一致性。在这里，我们建议按顺序 异源初免-加强（SHPB）免疫，以尝试引发针对2个保守蛋白/聚糖表位的bNAb (V2&融合肽; FP）。这两个位点都适应多种NAb结合模式，促进表位集中 接近。在目标1中，我们将鉴定一组V25多样的、多V2 bNAb敏感的、良好表达的三聚体。 虽然高三聚体表达是必需的，但这样的Env是不常见的。获得有用的三聚体的两种方法是： 1)以测试各种V2敏感的“特殊”菌株; 2）KI V2敏感性转化为高表达菌株。选定环境 将被修改为：1）插入聚糖孔，2）KI常见FP变体1（FP 8var 1）序列/KO N611 聚糖（调节FP暴露）。在目标2中，我们将尝试使用病毒样颗粒（VLP）改进疫苗NAb 表达来自Aim 1的三聚体。CH 01 HC KI BL 6小鼠表达CH 01 NAb重链（HC）的UCA， 可以与不同小鼠LC联合收割机组合的小鼠HC库。这些小鼠可以潜在地产生NAb， 像FP这样的目标。由于早期事件形成NAb反应，在初始KLH-FP 8v 1引发后， 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，以尽量减少冲突 或免疫原性外源聚糖以促进早期NAb-聚糖接触。以后的VLP镜头将保持一致 扩大之前枪击案的NAB范围5只小鼠将在中间时间点处死，其余小鼠在之后处死。 这是最后一枪，让我们研究NAb个体发育最佳预充方案将主要由以下因素确定： 中和动力学、效力、广度和一致性，以及来自分子遗传学标准。这种疗法将是 在CH 01前体通过过继转移降低至生理频率的小鼠中重新测试， HC x Balb/c F1小鼠也表现出降低的CH 01 HC频率，这是一个更强大的遗传背景， 促进NAb发育。在确定了有效的预激注射后，将对组装的SHPB方案进行测试 在相同的F1小鼠中尝试提高宽度。变量将包括重叠或不重叠的顺序 注射，增加菌株多样性，增加表位严格性。最后，我们将在Trianni测试领先的方案， 表达多克隆人IgG的小鼠。将监测疫苗和非疫苗菌株的血清中和。 将使用VLP探针拯救MAb并分析SHM、V基因使用和H/L配对。NAb滴度，宽度， 将研究聚糖变化、特异性、聚糖阵列活性和个体发育的影响。总的来说，我们希望 通过确定诱导聚糖依赖性、V2和FP靶向NAb宽度的方案来推进HIV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.