Development of a mucosal vaccine to prevent Clostridium difficile infection using papilloma pseudovirus as a vector.

摘要 艰难梭菌是卫生保健环境中腹泻的主要原因，有50万感染病例， 每年一次。艰难梭菌感染和疾病(CDI)通常发生在抗生素治疗后， 消除了大部分肠道共生微生物区系，并为艰难梭菌提供了定植的机会 我的直觉。如果定植的艰难梭菌产生毒素，许多患者就会发生CDI，并出现症状 从腹泻到危及生命的伪膜性结肠炎。CDI的发病率在#年呈上升趋势 很大程度上是因为抗生素的使用增加了。此外，CDI在约20%的接受治疗的患者中复发，使得 一种严重且昂贵的疾病，没有治愈方法，也没有有效的经批准的疫苗。这样做的目的是 建议开发一种保护性疫苗来预防CDI及其在医疗保健中的传播 环境。虽然一些艰难梭菌疫苗处于第二阶段/第三阶段的临床试验，但它们针对的是毒素，但将 不能阻止艰难梭菌在肠道粘膜中的定植，也不能阻止其传播。我们认为最理想的 保护性疫苗将产生针对毒素的强烈的粘膜IgA抗体反应，而且 一种防止艰难梭菌定居的药物。乳头瘤病毒是一种小的DNA病毒，其中主要的 衣壳蛋白L1与次要衣壳蛋白L2组装成病毒样颗粒(VLP)。这些VLP 感染粘膜表面，不具传染性，但具有很强的免疫原性，使其成为理想的载体 粘膜疫苗。我们已经从衣壳蛋白L1和L2中培育出乳头状瘤假病毒(PSV)， 含有表达艰难梭菌毒素A和B的受体结合域(RBD)的质粒，我们有 初步数据显示，这些疫苗可以诱导中和粘膜IgA，并保护小鼠免受 艰难梭菌的挑战。在目标1中，我们将建立表达毒素A、B的RBD的PSV(来自不同的菌株) 和二元毒素。我们将在老鼠和仓鼠身上对它们进行免疫。我们将确定一种佐剂，它将增加 小鼠和金黄地鼠的粘膜抗体应答和产生长期免疫记忆 CDI动物模型标准。在目标2中，我们将产生表达表面分子Flid或High的PSV 分子量SLP，将影响定植的分子，并决定这些疫苗是否能够诱导 抑制艰难梭菌定植的抗体。在目标3中，我们将给小鼠和仓鼠接种一种 将所有PSV和ACCHANGE与不同菌株的产毒艰难梭菌孢子混合，以确定CDI 和艰难梭菌的定植被阻止。如果成功，粘膜疫苗将产生一种强大的 中和粘膜对毒素和表面抗原Flid和SLP的IgA反应。这样一种疫苗 将对改善卫生保健环境中数千名患者的卫生保健做出重大贡献， 并节省数十亿美元的医疗保健成本。

Abstract Clostridium difficile is a major cause of diarrhea in health care settings, with one-half million cases of infection, annually. Clostridium difficile infection and disease (CDI) usually occurs after antibiotic treatment, which eliminates much of the intestinal commensal microbiota and provides C. difficile the opportunity to colonize the gut. If the colonizing C. difficile produces toxins, many patients develop CDI and suffer from symptoms ranging from diarrhea to life-threatening pseudomembranous colitis. The incidence of CDI is on the rise, in large part because of increased use of antibiotics. Further, CDI recurs in ~20% of treated patients, making this a serious and expensive disease, for which there is no cure and no effective approved vaccine. The goal of this proposal is to develop a protective vaccine to prevent CDI and also, its transmission in the health care environment. While a few C. difficile vaccines are in Phase II/III clinical trials, they target the toxins, but will not prevent colonization of C. difficile in the intestinal mucosa, nor its transmission. We think that the optimal protective vaccine would be one that generates a strong mucosal IgA antibody response against toxins, and also one that prevents colonization of C. difficile. Papillomaviruses are small DNA viruses in which the major capsid protein, L1, assembles with the minor capsid protein, L2 into virus-like particles (VLP). These VLPs infect mucosal surfaces, are not infectious but are strongly immunogenic, making them ideal vectors for mucosal vaccines. We have developed papilloma pseudoviruses (PsV) from capsid proteins L1 and L2, that contain plasmids expressing the receptor binding domain (RBD) of the C. difficile toxins A and B, and we have preliminary data showing that these vaccines can induce neutralizing mucosal IgA and protect mice from challenge by C. difficile. In Aim 1, we will develop PsV expressing RBD of toxins A, B (from different strains) and binary toxin. We will immunize them in mice and hamsters. We will identify an adjuvant that will increase the mucosal Ab response and generate long-term immunologic memory in mice and hamsters, the gold standard for CDI animal models. In Aim 2, we will generate PsV expressing surface molecules FliD or high molecular weight SLP, molecules that will affect colonization, and determine if these vaccines can induce antibodies that inhibit colonization of C. difficile. In Aim 3, we will vaccinate mice and hamsters with a mixture of all the PsV and challenge with toxogenic C. difficile spores from different strains to determine if CDI and colonization of C. difficile are prevented. If successful, the mucosal vaccine will generate a robust neutralizing mucosal IgA response against both toxins and the surface antigens FliD and SLP. Such a vaccine would make a major contribution to improving the health care of thousands of patients in health care settings, and save billions of dollars in health care costs.