Development of nucleic acid-based vaccines against gonorrhea

摘要 淋病每年影响全球8700万人。2019年，超过60万淋病病例被 向美国疾病控制与预防中心报告，自2009年美国的历史低点以来增长了92%。然而，真正发生的 据估计，美国每年的病例约为150万例。抗菌素耐药性的传播已经严重 治疗选择有限--目前，头孢曲松是唯一被批准的一线治疗药物。一种安全有效的 目前迫切需要预防淋病的疫苗。脂低聚糖(LOS)是生物体中含量最丰富的分子 淋球菌表面，并在细菌毒力中发挥多方面的作用。单抗2C7识别的LOS表位 (因此称为2C7表位)由体内95%的Ng表达；不表达2C7的Ng突变体 表位在小鼠体内减弱。我们小组开发了一种2C7表位的多肽模拟(模拟表位)，它 当被配置为多抗原肽(MAP)时，可诱导产生杀菌抗体并减弱Ng在小鼠体内的定植 老鼠。进一步，我们阐明了MAP的作用机制是补体介导的杀伤 通过膜攻击复合体)，强烈表明血清杀菌活性作为一种机制相关 是一种保护。在与Evaxion Biotech的一项合作项目中，我们筛选了30种抗原，这些抗原被 Eden，Evaxion在硅胶平台上的专利，用于免疫原性、杀菌活性和对小鼠的疗效 阴道定植模型。这些研究确定了两个有希望的候选细胞分裂蛋白，基于 体内药效。当被配置为融合蛋白(嵌合体)时，细胞分裂蛋白显示出更好的活性。 与单个蛋白质相比，它具有体内活性，目前是我们的主要蛋白质疫苗候选者。DNA和mRNA 事实证明，疫苗在抗击新冠肺炎大流行方面非常成功。这些平台引发了高度的兴趣 保护级别已被证明是安全的，具有快速适应性和经济性。在这里，我们将利用这两个 提供保护性淋球菌抗原的较新疫苗平台。多价疫苗的优势在于 更广泛的菌株覆盖面，提高了药效，也将提高抗药性发展的障碍。在……里面 目的1，我们将评估包含2C7 LOS模拟表位(PEP1)的DNA疫苗构建物 八聚体串联蛋白(PEP1_OCT)，细胞分裂蛋白嵌合体，或两者的组合。血清意志 评估抗体效价和补体依赖的杀菌活性，以及反应的持久性。 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 (阴道)抗体反应也将被测量。T细胞反应将通过ELISpot进行测量。基于中的数据 在体外，铅DNA疫苗配方(S)将在小鼠阴道定植模型中进行疗效评估。 在目标2中，将使用信使核糖核酸疫苗递送平台表达目标1中描述的构建体。如中所示 目的1、检测血清和阴道液中的抗体效价，检测血清杀菌活性、T细胞反应 以及铅基因疫苗制剂(S)在淋病小鼠模型中的疗效。成功完成 这项建议中的工作将确定一种主要的淋球菌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.