Novel DNA Manufacturing Process: Cell-Free Production and Testing of a New Multiv

描述(申请人提供)：在家禽中出现高致病性禽流感病毒，并将禽流感病毒直接传播给人类的病例数量不断增加，这些病毒有可能在大流行中传播，因此对公众健康构成重大威胁。目前，针对高致病性禽流感H5N1感染的预防和治疗选择有限。许多努力都集中在疫苗的开发上。DNA的免疫原性是在1990年首次被描述的，DNA疫苗代表了疫苗接种的一个非常有前途的未来。传统上，DNA疫苗是通过成熟的发酵过程生产的。CytoGenix开发了一种替代技术，即无细胞生产技术(SynDNAM)，用于以最小的工作量和快速的周转时间合成大量(克)DNA，用于小批量生产疫苗。在不需要细菌扩大生产的情况下，这项技术可以应用于最少的非治疗性DNA序列。因此，这一过程本质上比质粒发酵“更清洁”，最终产品需要最低限度的提纯。这一第一阶段提案的总体目标是验证我们的SynDNAM工艺用于DNA疫苗合成的使用。作为利用我们的无细胞大规模生产方法生产的核酸预防性药物的第一个目标，我们选择了人类甲型流感病毒，更具体地说，是潜在的大流行毒株流感A/越南/1203/04，H5N1。几份报告--包括我们自己的实验--表明，表达流感病毒蛋白质的DNA疫苗可以单独在接种疫苗的动物中引发强大的免疫反应，基本上防止它们在感染活病毒时生病。我们打算在体外合成并测试一种DNA疫苗，当注射到雪貂(选择的人类流感动物模型)中时，能够表达其中两种蛋白质，目的是诱导免疫反应，以预防这种潜在的大流行流感病毒株。我们的目标是1)用我们的SynDNAM过程生产一种DNA疫苗，并通过2)分析触发的免疫反应和3)优化免疫方案来测试其在活体病毒攻击的雪貂中的有效性。如果成功，我们的结果将为实施无细胞合成和测试使用我们的大规模生产过程制造的DNA疫苗的标准实验方案铺平道路。这将使我们能够将针对新流感病毒株和其他病毒病原体的新DNA疫苗的合成和测试常规化，并利用我们的生产过程简化DNA疫苗的制备。此外，它还将为以低成本快速生产有效疫苗奠定基础，这种技术可以很容易地转移到流行病威胁最大的发展中国家。从拟议的实验中收集的数据还将为遵循FDA开发/测试生物制品的“两个动物模型规则”的第二阶段实验奠定基础，在这种情况下，人类功效试验是不可行的或不符合道德的。与公共卫生相关：目前，没有针对大流行性流感的强效疫苗可供普通公众使用。如果成功，这项提议中描述的实验将为合成和生产新的流感DNA疫苗铺平道路，这些疫苗可以迅速适应新的病毒株，以及其他传统方法尚未生产出有效疫苗的病毒病原体，如艾滋病毒、疟疾和结核病。

DESCRIPTION (provided by applicant): The emergence of highly pathogenic avian influenza viruses in domestic poultry and the increasing number of cases of direct transmission of avian influenza viruses to humans are a significant threat to public health because of the potential for pandemic spread of these viruses. Currently, preventive and therapeutic options for highly pathogenic avian influenza H5N1 infections are limited. Many efforts have focused on the development of vaccines. The immunogenic potential of DNA was first described in 1990 and DNA vaccines represent a very promising future for vaccination. Traditionally, DNA vaccines are manufactured through a well established fermentation process. CytoGenix has developed an alternative, i.e. a cell-free production technology (synDNATM) for the synthesis of large quantities (grams) of DNA for vaccine production in small volumes with minimal effort and rapid turn around time. Without the need for bacteria to ramp-up production, this technology can be applied with a minimum of non-therapeutic DNA sequences. This process is therefore inherently "cleaner" than plasmid fermentation and the final product requires minimal purification. The overall goal of this Phase I proposal is to validate the use of our synDNATM process for the synthesis of DNA vaccines. As a first target for the development of nucleic acid-based prophylactic made using our cell-free large scale production method, we have chosen the human influenza A virus, more specifically the potential pandemic strain influenza A/Vietnam/1203/04, H5N1. Several reports - including our own experiments - have shown that DNA vaccines expressing proteins from the flu virus can individually trigger a robust immune response in vaccinated animals; essentially preventing them from falling ill upon infection with the live virus. We intend to synthesize in vitro and test a DNA vaccine capable of expressing two of these proteins when injected into ferrets (the human influenza animal model of choice) for the purpose of eliciting an immune response that will protect against this potential pandemic flu strain. Our objectives are to 1) generate a DNA vaccine with our synDNATM process and test its efficacy in ferrets with live virus challenges by 2) analyzing the immune response triggered and 3) optimizing the immunization regimen. If successful, our results will pave the way for the implementation of standard experimental protocols for the cell-free synthesis and testing of DNA vaccines made using our large scale production process. This will enable us to routinize the synthesis and testing of new DNA vaccines against new influenza viral strains and other viral pathogens as well as simplify the preparation of DNA vaccines using our production process. Moreover, it will set the stage for the rapid production of efficacious vaccines at low cost, with a technology that can be easily transferred to developing countries where the threat of pandemics is at its highest. The data gathered from the proposed experiments will also set the stage for phase II experiments following the FDA "two animal model rule" for the development/testing of biologicals where human efficacy trials are not feasible or ethical. PUBLIC HEALTH RELEVANCE: Currently, no robust vaccines against pandemic flu are available to the general public. If successful, the experiments described in this proposal will pave the way for the synthesis and production of new DNA vaccines against influenza that can be rapidly adapted to new viral strains as well as other viral pathogens to which effective vaccines prepared by conventional methods have yet to be produced i.e. HIV, malaria and tuberculosis.