Globally Appropriate Genome Reduced Killed Whole Bacterial HIV Vaccines

关于HIV疫苗开发的广泛共识要求设计抗原以诱导生产 抗体，如已知的广谱中和(BN)单抗(MAb)。两个环境BN单抗 结合部位，膜近端外区(MPER)和融合肽(FP)是有吸引力的靶点 因为它们是线型多肽。我们开发了一种新的、低成本、全球适用的疫苗平台：KILL 全细胞(KWC)基因组减少型大肠杆菌(GREC)，疫苗抗原在细菌表面表达 使用革兰氏自动转运器。使用合成生物学，可以在大约3周内制造出可测试的候选疫苗 ~每个50美元。使用该平台生产的疫苗每剂成本约为1美元，可以在全球现有工厂生产， 并且有宽大的冷链要求。我们使用KWC grec平台制造了冠状病毒FP疫苗 并在动物模型上显示了临床疗效。我们建议使KWC grec HIV疫苗具有靶向 MPER和Fp。我们的初步数据表明，我们可以用这种方法表达HIV MPER和FP AGS 用MPER来源的抗原诱导小鼠的HIV中和血清。我们假设KWC GREC平台， 针对MPER和FP，将产生安全、有效、低成本、全球适用的艾滋病毒疫苗。 在第一阶段，目标1，我们将利用生物工程合成编码MPER和FP AGS的DNA 加强Ag暴露和抗原性的策略。我们将把这些DNA克隆到我们的表达载体中， 转化为GREC以制造KWC候选疫苗。使用高度中和和相对非中和 ，我们将测试候选疫苗，选择那些在结合中和方面表现出最佳差异的疫苗。 用于体内测试的中和性较低的单抗。在目标2中，我们将给小鼠接种疫苗以评估疫苗的免疫原性， 比较不同的候选疫苗，单独和组合，以确定最好的疫苗和 使用ELISA、ELISPOT和PhenoSense中和试验的剂量/路线/佐剂 具有代表性的病毒小组。 进入第二阶段的决策之门：生产单一或联合疫苗，以诱导血清感染 在对≥的表观中和试验中，免疫小鼠产生IC50和GT；100的小鼠免疫75%， 对于75%的≥-1env参考毒株，而来自对照免疫小鼠(免疫小鼠)的血清 不表达病毒抗原的细菌)在基线对照上没有显示中和。 在第二阶段中，我们将在目标3中进行非人灵长类(NHP)模型研究，以评估安全性， 免疫原性，以及通过疫苗诱导中和抗体反应的能力。我们希望我们的疫苗 将诱导特定的HIV抗原结合抗体、HIV抗原特异性T细胞反应和BN中和抗体 NHP中的反应，这意味着该疫苗将在人类身上安全有效。KWC疫苗有很长的时间 历史，制造成本低，可以在全球现有设施中生产，我们预计我们的工作 可以迅速转化为安全有效、廉价、全球适用的预防性艾滋病毒疫苗。

A broad consensus regarding HIV vaccine development calls for engineering antigens that elicit production of antibodies like the known Broadly Neutralizing (BN) Monoclonal Antibodies (MAbs). Two Env BN MAb binding sites, the Membrane Proximal External Region (MPER) and Fusion Peptide (FP), are attractive targets because they are linear peptides. We developed a new, low cost, globally appropriate vaccine platform: Killed Whole Cell (KWC) Genome-Reduced E. coli (grEc), with vaccine antigens expressed on bacterial surfaces using Gram- autotransporters. Using synthetic biology, testable vaccine candidates can be made in ~3 wks for ~$50 each. Vaccines made with the platform will cost ~$1/dose, can be produced in existing factories globally, and have forgiving cold chain requirements. We made coronavirus FP vaccines using the KWC grEc platform and showed clinical efficacy in an animal model. We propose to make KWC grEc HIV vaccines targeting MPER and FP. Our preliminary data show that we can express HIV MPER and FP Ags with this approach and elicit HIV neutralizing sera in mice using an MPER-derived Ag. We hypothesize that the KWC grEc platform, targeting MPER and FP, will yield safe, effective, low cost, globally appropriate HIV vaccines. In PHASE 1, Aim 1, we will synthesize DNAs encoding MPER and FP Ags, employing bioengineering strategies to enhance Ag exposure and antigenicity. We will clone these DNAs into our expression plasmid and transform into grEc to make KWC candidate vaccines. Using highly neutralizing and relatively non-neutralizing MAbs, we will test candidate vaccines, selecting those that show the best difference in binding neutralizing vs. less neutralizing MAbs for in vivo testing. In Aim 2, we will vaccinate mice to assess vaccine immunogenicity, comparing the different candidate vaccines, alone and in combination, to identify the best vaccines and dose/route/adjuvant using ELISAs, ELIspot assays, and PhenoSense neutralization assays against a representative panel of viruses. Decision Gate to progress to Phase 2: Production of a single or combination vaccine that elicits sera in vaccinated mice yielding an IC50 >100 in the PhenoSense neutralization assay for ≥75% of mice immunized, for ≥75% of HIV-1 Env reference strains, while sera from control immunized mice (mice immunized with bacteria not expressing viral antigen) show no neutralization above baseline control. In PHASE 2 we will, in Aim 3, conduct a non-human primate (NHP) model study to assess the safety, immunogenicity, and ability to elicit a neutralizing Ab response by the vaccines. We expect that our vaccines will induce specific HIV Ag-binding Abs, HIV antigen-specific T cell responses, and BN neutralizing Ab responses in NHP, implying that the vaccine will be safe and effective in humans. KWC vaccines have a long history, are inexpensive to make, and can be produced globally in existing facilities, we anticipate that our work can be quickly translated into safe and effective, inexpensive, globally appropriate, prophylactic HIV vaccines.