Development of nucleic acid-based vaccines against gonorrhea

摘要 淋病每年影响全球超过8700万人。2019年，超过60万例淋病病例 报告给CDC的数据显示，自2009年美国的历史低点以来增加了92%。然而，真正的发病率 据估计，美国每年约有150万例。抗生素耐药性的传播已经严重 有限的治疗选择-目前，头孢曲松是唯一获准的一线治疗药物。一种安全有效的 迫切需要淋病疫苗。脂寡糖（LOS）是最丰富的分子上的 淋球菌表面，并在细菌毒力中发挥多方面的作用。mAb 2C7识别的LOS表位 Ng突变体（因此称为2C7表位）在体内由> 95%的Ng表达;不表达2C7表位的Ng突变体在体内由> 95%的Ng表达。 表位在小鼠中被减弱。我们的小组已经开发了2C7表位的肽模拟物（模拟表位）， 当被配置为多抗原肽（MAP）时，其增强杀菌性Ab并减弱Ng定植， 小鼠此外，我们已经阐明了MAP作为补体介导的杀伤（杀伤）的作用机制。 通过膜攻击复合物），强烈提示血清杀菌活性作为机制相关性 保护。在与Evaxion Biotech的合作项目中，我们筛选了30种抗原， EDEN，Evaxion专有的计算机平台，用于小鼠免疫原性、杀菌活性和功效 阴道定植模型。这些研究确定了两种细胞分裂蛋白作为有希望的候选者， 体内功效当被配置为融合蛋白（嵌合体）时，细胞分裂蛋白表现出改善的活性。 与单个蛋白质相比，它在体内具有更高的免疫活性，目前是我们的主要蛋白质疫苗候选者。dna和mrna 疫苗已被证明在抗击新冠肺炎大流行方面非常成功。这些平台引发了高 保护水平，已被证明是安全的，是快速适应和经济的。在这里，我们将利用这两个 提供保护性淋球菌抗原的新疫苗平台。多价疫苗的优势在于 更广泛的菌株覆盖范围，提高疗效，也将提高耐药性发展的障碍。在 目的1，我们将评估含有2C7 LOS模拟表位（PEP 1）的DNA疫苗构建体，该模拟表位被配置为 八聚体多联体（PEP1_OCT）、细胞分裂蛋白嵌合体或两者的组合。血清将 评估抗体滴度和补体依赖性杀菌活性，以及应答的持久性。 将与目前正在开发的这些疫苗的蛋白质版本进行比较。粘膜 还将测量（阴道）Ab应答。将通过ELISpot测量T细胞应答。根据数据， 在体外，将评估前导DNA疫苗制剂在小鼠阴道定殖模型中的功效。 在目标2中，将使用mRNA疫苗递送平台表达目标1中描述的构建体。如在 目的1、检测血清和阴道液中的抗体滴度、血清杀菌活性、T细胞反应 以及前导mRNA疫苗制剂在淋病小鼠模型中的功效。成功完成 这项提案中的工作将确定一种先导淋球菌DNA和/或mRNA疫苗，用于进一步的临床前研究。 发展

ABSTRACT Gonorrhea affects over 87 million people annually globally. In 2019, over 600,000 cases of gonorrhea were reported to the CDC, a 92% increase since the historic low in the US in 2009. However, the true incidence of cases annually in the US is estimated to be ~1.5 million. The spread of antimicrobial resistance has severely limited treatment options – currently, ceftriaxone is the only approved first line of treatment. A safe and effective vaccine against gonorrhea is urgently needed. Lipooligosaccharide (LOS) is the most abundant molecule on the gonococcal surface and plays multifaceted roles in bacterial virulence. A LOS epitope recognized by mAb 2C7 (therefore called the 2C7 epitope) is expressed by >95% of Ng in vivo; Ng mutants that do not express the 2C7 epitope are attenuated in mice. Our group has developed a peptide mimic (mimitope) of the 2C7 epitope, which when configured as a multi-antigen peptide (MAP) elicits bactericidal Abs and attenuates Ng colonization in mice. Further, we have elucidated the mechanism of action of MAP as complement-mediated killing (killing through membrane attack complex), strongly suggesting serum bactericidal activity as a mechanistic correlate of protection. In a collaborative venture with Evaxion Biotech, we screened 30 antigens that were identified by EDEN, Evaxion’s proprietary in silico platform, for immunogenicity, bactericidal activity and efficacy in the mouse vaginal colonization model. These studies identified two cell division proteins as promising candidates based on efficacy in vivo. When configured as a fusion protein (chimera), the cell division proteins showed improved ac- tivity in vivo compared to individual proteins and currently is our lead protein vaccine candidate. DNA and mRNA vaccines have proven very successful in the fight against the Covid-19 pandemic. These platforms elicit high levels of protection, have proven safe, are rapidly adaptable and economical. Here, we will leverage these two newer vaccine platforms to deliver the protective gonococcal antigens. A multivalent vaccine has the advantage of broader strain coverage, improved efficacy and would also raise the barrier for development of resistance. In Aim 1, we will evaluate DNA vaccine constructs that contain the 2C7 LOS mimitope (PEP1) configured as an octameric concatemer (PEP1_OCT), the cell division protein chimera, or a combination of the two. Serum will be assessed for antibody titers and complement-dependent bactericidal activity, as well durability of responses. Comparisons will be made with the protein versions of these vaccines currently under development. Mucosal (vaginal) Ab responses will also be measured. T cell responses will be measured by ELISpot. Based on data in vitro, the lead DNA vaccine formulation(s) will be assessed for efficacy in the mouse vaginal colonization model. In Aim 2 the constructs described in Aim 1 will be expressed using an mRNA vaccine delivery platform. As in Aim 1, we will evaluate Ab titers in serum and vaginal fluid, measure serum bactericidal activity, T cell responses and efficacy of the lead mRNA vaccine formulation(s) in the mouse model of gonorrhea. Successful completion of the work in this proposal will identify a lead gonococcal DNA and/or mRNA vaccine for further pre-clinical development.