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rAAVs are one of the most promising viral vectors for gene therapy and genetic vaccinations. A vast number of studies in experimental animals (mice, dogs, pigs, nonhuman primates), and several clinical trials in humans, have provided convincing results for the efficacy of this genetic delivery system. The in vivo efficacy of AAV has not been evaluated in avian species yet. We have previously isolated and rescued an infectious clone of an avian AAV. We have also developed the vectors that will support high titer production of recombinant AAAV in a cell based system. Our initial in vitro studies, using rAAAV-GFP/bGal, have provided evidence for robust transduction of avian derived cells with this viral vector. We will now evaluate rAAAV as a transgene delivery system in chickens. In our experience with rodent models, the use of a recombinant virus that carries the human erythropoietin gene (rAAV-hEpo) is very effective in evaluating transduction efficiency, time course, and persistence of transgene expression, as well as host immune responses for the transgene. hEpo, will be secreted in the blood stream and can be easily measured with very sensitive and highly specific immuno-assays in serum samples. Highly purified recombinant hEpo is also readily available, and can be used in elisa assays to determine the antibody titers in serum samples of rAAAV-hEpo vaccinated chickens. hEpo is significantly diversified from the homologues chicken thrombopoetin and could potentially be immunogenic. It is worth noting however, that hEpo expressed in a eukaryotic host is highly glycosylated and not very immunogenic, thus, mimicking the nature of surfaced exposed viral antigens like the HA of avian influenza and the HN of NDV. Therefore, utilizing a rAAAV-hEpo could serve as useful model in the initial evaluation of rAAAV as a vaccine delivery system. In this MAES proposal, we will produce a rAAAV-hEPO and determine the effect of the delivery route on the kinetics of transduction, levels of transgene expression, and antibody response in young chickens. <P>
The following specific aims will be carried out: <OL> <LI> Production of a rAAAV-hEpo viral vector. We intend to produce ~1012 particles of recombinant virus by transfecting the necessary packaging plasmids in 293T cells. The viral particles will be purified, based on a previously validated protocol, using a successive series of discontinuous iodoxanol gradients, and continuous CsCl gradients. The number of recombinant virions will be determined with real time quantitative PCR and infectious titer will be determined in the DF1 chicken cell line. Purity of the viral preparation will be assessed with electron microscopy and coomassie staining of SDS-PAGE gels. <LI> Evaluate the in vivo kinetics of rAAAV-hEpo transduction and immune response. We will administer the viral vector in 7-day old pathogen free chickens at various doses (108-1010 particles/bird) either intramuscularly or orally. We will collect blood samples 10 days after the initial delivery and every 15 days thereafter for a period of 6 months. The blood samples will be used to measure the levels of hEpo, antibody responses against hEpo, and the AAAV itself.
NON-TECHNICAL SUMMARY: Traditional vaccines fall into two broad categories: a) live attenuated infectious material, and b) killed, inactivated, or subunit preparations. Live attenuated vaccines can produce a diverse and persistent immune response, and often are effective after only a single dose. However, they present safety concerns due to the risk of reverting to the virulent form. Inactivated or subunit vaccines do not pose such a threat, but often they are less immunogenic and require repeated boosting to induce an adequate immune response. In recent years, AAV vectors have received much attention in the field of gene therapy for their ability to establish long-lasting transgene expression in a variety of tissues. Some of the most impressive data have come from skeletal muscle and salivary glands in mice and nonhuman primates where transgene expression after a single injection persists for over one year. AAV vectors have a number of appealing qualities for vaccine development, including lack of pathogenicity and toxicity, absence of viral coding sequences from the vector genome, ability to infect dividing and non-dividing cells, highly efficient transduction of various cell types, and ability to elicit both T and B cell responses to the transgene. Moreover, AAV remains stable and infectious at a range of pH, proteases, and temperatures that make it suitable for oral administration. These features make AAV an ideal vaccine platform over other contemporary vector systems, as it enables the development of robust immune responses, due to long-term recombinant antigen expression. Recent advances in recombinant AAV production and purification systems have made the process cost effective. Current methods allow production of >1014 particles in a small laboratory setting. Therefore, the use of an AAAV vector as a genetic vaccine delivery system in poultry warrants further exploration. In our previously published research, we realized the significance that this vector may have as a vaccine delivery system in poultry, due to high transduction efficiency of avian derived cells in vitro. In our experience, the in vitro transduction profile of certain AAV serotypes does not always correlate with efficacy in whole animal experimentations. Our proposed research will be the first in vivo experiment in poultry using an AAV. It is designed to determine the minimal amount of viral vector that will result in sustained expression and induction of immune response. A low effective dose will increase the applicability of this viral vector in practice, making it more attractive from a cost perspective. Our proposed research will also determine the duration needed from the time of vaccine delivery till detection of humoral immune response and the preferred route of immunization. A relatively quick response (less than 4 weeks) is generally desired. We have little doubt that intramuscular administration will be effective. However, it would be preferable if oral administration could provide similar or even better results. Parenteral administration of vaccines is cumbersome and rarely applied in the field.
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APPROACH: Production of a recombinant AAAV vector requires 3 packaging plasmids. A bidirectional plasmid expressing 3 genes from the human adenovirus type 5, an expression plasmid that provides the AAAV rep and cap genes, and a shuttle plasmid that contains the AAAV ITRs. The transcriptional cassette of interest must be cloned in the shuttle vector. Primers containing a Pml1 site and an SpeI site will be used to PCR amplify a fragment of pAAV2-hEpo that contains the hEpo gene under the control of an RSV promoter and bGH poly(A) tail. The PCR product will be digested with Pml1 and SpeI restriction enzymes and will be directionally cloned in the same sites of the shuttle vector. The resulting plasmid (pAAAV-hEpo) will be cotransfected with helper and the packaging plasmids in HEK 293T cells grown in 150-cm dishes using CaPO4 co-precipitation at a ratio of 2:3:1. 48 hrs post-transfection, the recombinant virus will be purified from the cells using mild lysis, differential precipitation and continuous CsCl gradients. Purity of the prep will be evaluated by EM and SDS-PAGE followed by Coomassie staining to detect the characteristic VP1, VP2 and VP3 proteins of AAAV. Viral titers will be determined using real-time quantitative PCR in TaqMan assay using RSV primers and probe16 and infectivity will be assessed in Df1 cells. Once we prepare the rAAAV-hEpo viral vector and thoroughly characterize its purity and infectious titers, we will conduct our in vivo experiments to evaluate efficacy. Embryonated inbred eggs will be hatched in our laboratory and housed at the laboratory of animal and resources facility. 7 groups of chickens will be used. Each group will consist of five chickens. Three different doses of rAAAV-hEpo (108, 109, 1010 particles), will be administered in 7-day old birds, either intramuscularly or orally. One group will serve as control and will only receive 300 ul of PBS intramuscularly. For oral immunization, 500 ul of the viral solution will be administered to chickens by esophageal intubation with a blunt-tipped feeding needle. For intramuscular immunizations, 300 ul of the viral solution will be injected in the legs (150 ul/leg). The day before vaccination, blood samples will be collected from each bird. Ten days after vaccination, we will start collecting blood samples, twice/month for a total duration of six months. Bllod serum will be used to quantify the levels hEpo and chicken IgG responses to hEpo. Our proposed research will be the first in vivo experiment in poultry using an AAV. This class of viruses has shown enormous promise in humans and other species as a vaccine delivery system and/or gene therapy. Our pilot experiments will aid in determining whether rAAAV can induce a sustained transgene expression in vivo. Several vaccination programs of poultry flocks require repeated antigen administration in a year of laying cycle to maintain high antibody titer. The use of rAAAV may circumvent this problem. If our studies provide promising results, we intend to generate rAAAVs that will deliver potential useful vaccines, such as the HA of highly pathogenic avian influenza.