Improving Production of Attenuated Malaria Sporozoite Vaccines through Genetic Mo

描述（由申请人提供）：疟疾每年造成约5亿临床病例和多达270万人死亡，使非洲国内生产总值减少100亿美元，对包括军事人员在内的旅行者构成严重风险。Sanaria的目标是为两个主要市场（发达国家的旅行者和发展中国家的幼儿）开发和商业化一种具有90%保护性的减毒恶性疟原虫（Pf）孢子子（SPZ）疫苗，其潜在年收入为10亿美元。用辐射减毒（RA） PfSPZ免疫可以保护人类志愿者免受Pf攻击长达10个月。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，因为通过临床可接受的肠外途径对人类进行免疫，生产足够数量的PfSPZ，并生产和鉴定无菌、纯化、稳定、低温保存的RA PfSPZ，以满足疫苗的监管和商业标准，这是不可行的。Sanaria已经克服了所有这些问题，与FDA就新药研究申请（IND）的计划进行了会面，并将于2008年对其RA PfSPZ疫苗的安全性、免疫原性和保护功效进行人体评估。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，产量就会成正比地增加，而不需要额外的努力。最近在蚊子分子遗传学的一些领域取得的进展为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（TEP1、LRIM1、SRPN6）最近被证明在限制某些疟原虫感染的强度方面发挥了主要作用。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。我们建议评估这些基因在An感染中发挥的具体作用。stephensi通过Pf，然后创建An的线。其中部分或全部这些基因的表达被消除。经过这种改造的蚊子将极易受到Pf感染，在唾液腺中产生持续较高数量的孢子虫。将利用已建立的蚊子瞬时基因敲除技术（向成年雌性蚊子注射双链RNA）对这三个目标基因进行评估。基于这些数据，将使用现有的蚊子基因整合技术创造出永久改造的蚊子。将对转基因蚊子的Pf感染表型进行评估。在这个项目中产生的蚊子将成为原型，证明这种方法在提高孢子体产量方面的可行性。该项目将进入第二阶段，届时将构建新的转基因蚊子，优化转基因的时空表达模式，结合故障安全转基因稳定特性，从基因载体中消除外来转基因，并结合生物安全转基因，使蚊子只能在实验室中生长。将在大规模孢子体生产工作的背景下对这些蚊子进行评价。

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.