Globally Appropriate Genome Reduced Killed Whole Bacterial HIV Vaccines

关于艾滋病毒疫苗开发的广泛共识要求工程抗原， 抗体，如已知的广泛中和（BN）单克隆抗体（MAb）。双Env BN MAb 结合位点，膜近端外部区域（MPER）和融合肽（FP），是有吸引力的靶点 因为它们是线性肽。我们开发了一种新的、低成本的、全球适用的疫苗平台： 全细胞（KWC）基因组减少E.大肠杆菌（grEc），疫苗抗原表达在细菌表面 使用革兰氏自动转运体。使用合成生物学，可测试的候选疫苗可以在3周内制成， 每人50美元。使用该平台生产的疫苗成本约为1美元/剂，可在全球现有工厂生产， 并有宽容的冷链要求。我们使用KWC grEc平台制备了冠状病毒FP疫苗 并在动物模型中显示出临床疗效。我们建议使KWC grEc艾滋病毒疫苗靶向 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 Ag， 使用MPER衍生的Ag在小鼠中引发HIV中和血清。我们假设KWC grEc平台， 以MPER和FP为靶点，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，我们将合成DNA编码MPER和FP抗原，采用生物工程 增强Ag暴露和抗原性的策略。我们将这些DNA克隆到我们的表达质粒中， 转化成grEc来制造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和免疫应答方面表现出最佳差异的疫苗。 用于体内测试的中和MAb较少。在目标2中，我们将接种小鼠以评估疫苗免疫原性， 比较单独和组合的不同候选疫苗，以确定最佳疫苗， 剂量/途径/佐剂，使用ELISA、ELIspot测定和PhenoSense中和测定， 代表性的病毒组。 进入第2阶段的决定关口：生产一种单一或联合疫苗， 在PhenoSense中和试验中，≥75%的免疫小鼠产生IC 50>100的免疫小鼠， 对于≥75%的HIV-1 Env参考菌株，而来自对照免疫小鼠的血清（用 不表达病毒抗原的细菌）没有显示出高于基线对照的中和作用。 在第2阶段，我们将在目标3中进行非人灵长类动物（NHP）模型研究，以评估安全性， 免疫原性和通过疫苗引发中和Ab应答的能力。我们希望我们的疫苗 将诱导特异性HIV Ag结合Ab、HIV抗原特异性T细胞应答和BN中和Ab 在NHP中的反应，这意味着疫苗将是安全和有效的人类。KWC疫苗有很长的 历史，制作成本低廉，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.