Improving Production of Attenuated Malaria Sporozoite Vaccines through Genetic Mo

描述（由申请人提供）：疟疾每年造成估计5亿例临床病例和高达270万例死亡，使非洲的GDP减少>1%，并且对包括军事人员在内的旅行者来说是一个严重的风险。Sanaria的目标是为两个主要市场开发和商业化>90%保护性减毒恶性疟原虫（Pf）子孢子（SPZ）疫苗，潜在年收入> 10亿美元，即发达国家的旅行者和发展中国家的幼儿。 用辐射减毒（RA）PfSPZ免疫可以保护人类志愿者长达10个月免受Pf攻击。 没有其他实验性疟疾疫苗能提供类似的保护。 迄今为止，RA PfSPZ疫苗的开发被认为是不切实际的，因为通过临床上可接受的肠胃外途径免疫人类，产生足够量的PfSPZ，以及产生和表征满足疫苗的监管和商业标准所需的无菌、纯化、稳定、冷冻保存的RA PfSPZ被认为是不可行的。 Sanaria已经克服了所有这些问题，就新药研究申请（IND）计划与FDA会面，并将在2008年评估其RA PfSPZ疫苗的人体安全性，免疫原性和保护效力。 常规生产大量PfSPZ的一个主要考虑因素是蚊子中Pf感染的强度;如果感染率增加，则产量成正比增加，而无需额外的努力。 最近在蚊子分子遗传学领域的一些进展为Sanaria提供了显著增加PfSPZ产量的独特机会。昆虫科学家在了解昆虫对外来微生物感染的反应方面取得了相当大的进展。在蚊子中，三个基因（TEP 1，LRIM 1，SRPN 6）最近被证明在限制某些疟原虫感染的强度方面发挥重要作用。 也就是说，这些基因在蚊子抵抗感染的努力中发挥作用。 消除这些基因的表达会导致昆虫更容易受到感染。 我们建议评估的具体作用，这些基因在感染安。stephensi通过Pf，然后创建An的行。stephensi，其中这些基因中的一些或全部的表达被消除。 由这种修饰产生的蚊子将对Pf感染极其敏感，在唾液腺中产生持续更高数量的子孢子。 将在蚊子中使用已建立的瞬时基因敲低技术（向成年雌性注射双链RNA）评估三种靶基因。 根据这些数据，将使用已建立的蚊子基因整合技术创造永久性改造的蚊子。 将评估经修饰的蚊子的Pf感染表型。 在这个项目中产生的蚊子将是原型，证明这种方法的可行性，以提高子孢子的生产。该项目将导致一个第二阶段项目，其中将构建新的改良蚊子，优化转基因的时间和空间表达模式，纳入自动防故障转基因稳定特征，从基因载体中消除外来转基因，并纳入生物安全转基因，导致蚊子只能在实验室中生长。这些蚊子将在大规模子孢子生产工作的背景下进行评估。 公共卫生相关性-项目叙述：疟疾每年造成5亿例临床病例和1-3百万例死亡，每年造成非洲GDP损失>1%，是旅行者和军事人员的严重关切。Sanaria的目标是为两个主要市场开发和商业化一种保护性>90%的疟疾疫苗，年收入可能超过10亿美元：1）来自发达国家的旅行者; 2）发展中国家的婴儿，幼儿和少女。 这一项目的成功将大大降低疫苗的开发和生产成本，并缩短这种疟疾疫苗的上市时间。

DESCRIPTION (provided by applicant): Malaria causes an estimated 500 million clinical cases and up to 2.7 million deaths per annum, reduces the GDP of Africa by >1% and is a serious risk for travelers including military personnel. Sanaria's goal is to develop and commercialize a >90% protective attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine for two primary markets with potential annual revenues >$1 billion, travelers from the developed world and young children in the developing world. Immunization with radiation-attenuated (RA) PfSPZ can protect human volunteers for up to 10 months against Pf challenge. No other experimental malaria vaccine has given comparable protection. The development of a RA PfSPZ vaccine has to date been viewed as impractical because it was not considered feasible to immunize humans by a clinically acceptable parenteral route, produce adequate quantities of PfSPZ, and produce and characterize the aseptic, purified, stable, cryopreserved RA PfSPZ needed to meet regulatory and commercial standards for a vaccine. Sanaria has overcome all these problems, met with the FDA regarding plans for an Investigational New Drug application (IND) and will assess in humans the safety, immunogenicity and protective efficacy of its RA PfSPZ vaccine in 2008. A major consideration for routine production of large quantities of PfSPZ is the intensity of Pf infection in the mosquito; if the infection rates are increased, production is increased in direct proportion for no additional effort. Recent advances in a number of areas of mosquito molecular genetics provide Sanaria with unique opportunities to significantly increase PfSPZ production. Insect scientists have made considerable progress toward understanding how insects respond to infections by foreign microbes. In mosquitoes, three genes (TEP1, LRIM1, SRPN6) have recently been shown to play a major role in limiting the intensity of some Plasmodium infections. That is, these genes play roles in the mosquito's efforts to resist infection. Eliminating the expression of these genes results in insects that are more susceptible to infection. We propose to assess the specific role these genes play in the infection of An. stephensi by Pf and then create lines of An. stephensi in which the expression of some or all of these genes is eliminated. The mosquitoes resulting from such modifications will be extremely susceptible to Pf infection, yielding consistently higher numbers of sporozoites in the salivary glands. The three target genes will be assessed using established transient gene knockdown technologies in mosquitoes (double-stranded RNA injections into adult females). Based on those data, permanently modified mosquitoes will be created using established mosquito gene integration technologies. Modified mosquitoes will be assessed with respect to their Pf infection phenotype. The mosquitoes produced in this project will be prototypes that demonstrate the viability of this approach to enhancing sporozoite production. This project will lead to a phase II project in which new modified mosquitoes will be constructed that optimize temporal and spatial expression patterns of transgenes, incorporate fail-safe transgene stabilization features, eliminate foreign transgenes from the gene vector and incorporate a biosafety transgene that results in mosquitoes that can only be grown in the laboratory. These mosquitoes will be evaluated within the context of large-scale sporozoite production efforts. PUBLIC HEALTH RELEVANCE - PROJECT NARRATIVE: Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel. Sanaria's goal is to develop and commercialize a >90% protective malaria vaccine for two primary markets with a potential for >$1 billion annual revenues: 1) Travelers from the developed world; 2) Infants, young children, and adolescent girls in the developing world. Success in this project will significantly decrease the cost of development and production of the vaccine, and reduce the time to market for this malaria vaccine.