A Novel Approach for Mycobacterium Tuberculosis Vaccine Development

描述(申请人提供)：结核分枝杆菌(Mtb)是结核病(TB)的病原体，是一种细胞内细菌病原体，每年导致约170万人死亡，感染世界三分之一的人口。目前使用最广泛的疫苗是卡介苗，它是牛分枝杆菌的一种活的无毒变种，牛分枝杆菌是引起反刍动物结核病的近亲物种。给免疫抑制的人注射卡介苗会传播并导致严重的危及生命的感染。这种固有的安全性RIS仍然存在于第二代卡介苗中，这种疫苗经过改良后可以表达结核分枝杆菌抗原和/或使细菌在宿主细胞内存活能力增强的因子。因此，需要一种安全和更有效的结核病疫苗。在这项提议中，我们寻求开发一种基于呼吸道病毒副流感病毒5(PIV5)的新型结核病疫苗。副流感病毒5(PIV5)是一种副粘病毒，被认为是引起狗儿咳嗽的一个促成因素，目前还不知道它会在人类身上引起任何疾病。PIV5的一些特性使其成为一种有吸引力的疫苗候选载体。首先，含有活PIV5的狗舍咳嗽疫苗已经在狗身上使用了30多年。人类可能由于与狗的密切接触而接触到这种病毒。然而，还没有记录的人类疾病与该病毒有关。其次，它可以在许多细胞中以高滴度产生，包括已被批准用于疫苗生产的Vero细胞。第三，PIV5可以感染人类细胞系和原代人类细胞。第四，在我们最近的研究中，我们发现预先存在的对PIV5的免疫力并不会对基于PIV5的疫苗产生的免疫力产生负面影响。第五，表达流感病毒内部蛋白NP的PIV5对小鼠的致死性流感病毒攻击具有保护作用。由于NP介导的保护性免疫依赖于细胞免疫，因此基于PIV5的疫苗能够诱导保护性细胞免疫反应。第六，在我们的初步研究中，在表达HIV Gag蛋白的PIV5(PIV5-Gag)和表达Gag的痘苗病毒(MVA-Gag)的并列比较中，PIV5-Gag在小鼠中诱导了比MVA-Gag更好的细胞免疫应答。最后，鼻腔注射PIV5是一个非常好的特征，可以激发强大的粘膜免疫反应，因此是接种呼吸道病原体疫苗的理想选择。该载体以前没有被评估为结核分枝杆菌疫苗载体。我们推测PIV5作为活载体将诱导免疫以预防结核分枝杆菌感染。在这项提案中，我们计划演示使用PIV5作为结核分枝杆菌疫苗开发载体的原则证明。我们将致力于以下具体目标：制备和分析表达Mtb抗原85A(PIV5-85A)和85B(PIV5-85B)的重组PIV5，并在体内评价重组病毒的免疫原性和免疫效果。在这项研究结束时，我们希望确定表达结核分枝杆菌抗原的PIV5是否为良好的结核分枝杆菌疫苗候选。如果我们在这项研究中确定了有效的基于PIV5的候选疫苗，我们将进一步 在豚鼠和非人类灵长类动物等更相关的动物模型上测试候选动物。我们还将继续修改病毒载体，使候选疫苗发挥最大效果。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is an intracellular bacterial pathogen that kills approximately 1.7 million people each year and infects one-third of the world's population. The most-widely used vaccine in use today is BCG, which is a live avirulent variant of M. bovis, a closely related species that causes tuberculosis in ruminants. BCG administration to an immunosuppressed individual can disseminate and lead to serious life-threatening infection. This inherent safety ris remains in second- generation BCG vaccines modified to express Mtb antigens and/or factors that imbue the bacteria with enhanced survival inside host cells. Thus, there is a need for a safe and more effective TB vaccine. In this proposal, we seek to develop a novel TB vaccine based on parainfluenza virus 5 (PIV5), a respiratory virus. Parainfluenza virus 5 (PIV5), a paramyxovirus, is thought to be a contributing factor for causing kennel cough and is not known to cause any illness in humans. Several characteristics of PIV5 make it an attractive vaccine candidate vector. First, kennel cough vaccines containing live PIV5 have been used in dogs over 30 years. Humans are likely exposed to this virus due to close contact to dogs. Yet, no recorded illness in humans has been associated with the virus. Second, it can be produced in high titers in many cells including Vero cells that have been approved for vaccine production. Third, PIV5 can infect human cell lines and primary human cells. Fourth, in our recent study, we have found that pre-existing immunity against PIV5 does not negatively affect immunity generated by a PIV5-based vaccine. Fifth, PIV5 expressing NP, an internal protein of influenza virus, protected against lethal influenza virus challenge in mice. Since NP-mediated protective immunity relies on cell-mediated immunity, the results suggest that PIV5-based vaccine is capable of inducing protective cellular immune responses. Sixth, in a side-by-side comparison of PIV5 expressing HIV Gag protein (PIV5-gag) and a vaccinia virus (MVA) expressing Gag (MVA-Gag), PIV5-Gag induced better cellular immune responses than MVA-Gag in mice in our preliminary studies. Finally, the intranasal administration of PIV5 is a very good feature for eliciting robust mucosal immune responses, and thus ideal for vaccinating against respiratory pathogens. This vector has not previously been evaluated as an Mtb vaccine vector. We hypothesize that PIV5 as a live vector will induce immunity to prevent Mtb infection. In this proposal, we plan to demonstrate the proof-of principal for using PIV5 as a vector for Mtb vaccine development. We will focus our efforts on the following specific aims: generate and analyze recombinant PIV5 expressing Mtb antigens 85A (PIV5-85A) and 85B (PIV5-85B) and evaluate immunogenicity and efficacy of recombinant viruses in vivo. At the end of this study, we expect to have determined whether PIV5 expressing Mtb antigens are good Mtb vaccine candidates. If we identify efficacious PIV5-based vaccine candidates in this study, we will further test the candidates in more relevant animal models such as guinea pig and non-human primate. We also will continue to modify the viral vector to make the vaccine candidate maximally effective.