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

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 bec ause 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 p roduction 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 pandemi c 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 preventin g 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 elici ting 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 proce ss. 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 sta ge 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 sta ge 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 availa ble 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.