A Lyme disease vaccine using newly identified in-vivo expressed antigens

描述（由申请人提供）：莱姆病是美国最常见的节肢动物传播疾病，2009年报告的确诊和疑似病例超过38,000例。莱姆病的病原体是伯氏疏螺旋体，由肩胛骨蜱传播。莱姆病病例的数量在过去15年中增加了两倍。这种疾病有时在早期阶段难以诊断，导致治疗延误。不及时治疗的患者可能会出现严重、持久的症状，并且对抗生素治疗没有反应。预防蜱虫接触的控制措施不足以预防该病。我们建议开发一种疫苗来控制人群中的莱姆病。在被注射到哺乳动物宿主的皮肤后，伯氏疏螺旋体细菌在传播到其他器官之前在局部繁殖。我们现在第一次能够从感染小鼠的皮肤中分离出足够数量的细菌，以鉴定在皮肤中高水平表达的蛋白质。我们认为，针对这些在莱姆病早期急性期表达的蛋白质进行免疫接种，将非常有效地预防细菌的增殖和传播。在感染小鼠皮肤中鉴定的15种膜蛋白将使用标准细菌表达系统以重组形式生产。这些蛋白质在伯氏疏螺旋体菌株中是保守的，这应确保我们的疫苗对所有流行的伯氏疏螺旋体菌株有效。然后在佐剂存在的情况下，将每种抗原给予C57Bl/6小鼠。我们将证明这种免疫方案导致抗伯氏疏螺旋体抗体的出现。然后将用104个活的疏螺旋体挑战免疫的动物。挑战21天后，我们将收集组织样本，以确定它们是否含有活的伯氏疏螺旋体。我们还将定量检测这些样品中伯氏疏螺旋体DNA的存在。一种蛋白抗原被认为具有保护作用(1)如果攻毒后免疫小鼠的组织中不能培养出螺旋体，或者(2)如果细菌负荷（根据检测到的DNA量确定）至少减少了50%。在这些实验结束时，我们将选择多达四种抗原进行进一步研究。第二阶段的实验将确定最佳配方和给药方法。我们还将研究抗原是否可以组合以提供对伯氏疏螺旋体的全面保护。

DESCRIPTION (provided by applicant): Lyme disease is the most common arthropod-borne disease in the United States, with over 38,000 confirmed and probable cases reported in 2009. The causative agent of Lyme disease is the spirochete Borrelia burgdorferi, which is transmitted by the tick of the Ixodes scapularis tick. The number of Lyme disease cases has tripled over the past fifteen years. The disease is sometimes difficult to diagnose in its early stages, leading to delayed treatment. Patients who are not treated in a timely fashion may suffer from severe, long lasting symptoms, and fail to respond to antibiotic therapy. Control measures designed to prevent contact with ticks are not sufficient to prevent the disease. We propose to develop a vaccine to control Lyme disease in human populations. After being injected into the skin of a mammalian host, Borrelia bacteria multiply locally before disseminating to other organs. We have now for the first time been able to isolate sufficient amounts of bacteria from the skin of infected mice in order to identify proteins expressed at high levels in the skin. We believe that immunization against these proteins, which are expressed in the early acute phase of Lyme disease, will be highly effective in preventing bacterial proliferation and dissemination. Fifteen membrane proteins identified in the skin of infected mice will be produced in recombinant form using standard bacterial expression systems. These proteins are conserved among strains of Borrelia, which should ensure that our vaccine is effective against all strains of circulating Borrelia. Each antigen will then be administered to C57Bl/6 mice in the presence of an adjuvant. We will demonstrate that this immunization protocol results in the appearance of anti-Borrelia antibodies. Immunized animals will then be challenged with 104 live Borrelia spirochetes. Twenty-one days after challenge we will collect tissue samples to determine whether they contain live Borrelia. We will also quantitatively detect the presence of Borrelia DNA in these samples. A protein antigen will be considered protective (1) if spirochetes cannot be cultured from the tissues of immunized mice after challenge or (2) if the bacterial load, determined from the amount of DNA detected, is reduced by at least 50%. At the conclusion of these experiments we will select up to four antigens for further study. Phase II experiments will determine the optimal formulation and method of delivery. We will also examine whether antigens can be combined to provide full protection against Borrelia.