Alphavirus replicon vectors have been used in successful immunization and challenge experiments for a wide range of pathogens in rodent, avian, equine and primate species. Especially in acute infection models, these vectors, expressing immunizing genes from pathogenic viruses, have demonstrated the capacity for strong and protective immunization. A Phase I clinical trial with a Venezuelan equine encephalitis virus (VEE) replicon vaccine, expressing the gag gene from clade C human immunodeficiency virus (HIV), was initiated in 2003. We propose an analogous alphavirus replicon vaccine approach using immunizing genes of the SARS coronavirus (SARS-CoV) to develop a safe and effective vaccine against this virus. Specifically, we will 1) Clone the E, M, N and S genes of the SARS coronavirus, as well as X ORFs 1-5, into VEE and South African Arbovirus 86 (AR86) replicon vectors and evaluate these immunogens in mice, ferrets and nonhuman primate models, 2) Produce and test alternative forms of SARS immunogens, including SARS virus-like particles (SARS-VLP), for potential use in second generation vaccines, and 3) Clone and express native and mutated forms of the SARS coronavirus ORFs X1 and X3, interferon antagonist genes, from alphavirus replicon vectors for reagent production and as potential vaccine components. In Preliminary Studies we have expressed all the SARS-CoV structural and X ORF genes in the context of the VEE vectors, have demonstrated the induction of neutralizing antibodies in mice with the S construct, and have early evidence of SARS-VLP production using the baculovirus expression system in Sf9 cells. This overall development program is designed to rapidly produce a safe and effective vaccine candidate based on the alphavirus vectors as well as provide refinements for use in second generation vaccines for SARS-CoV.
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