Severe acute respiratory syndrome (SARS) has been reported in Asia, North America, and Europe. In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of SARS. SARSCoV is a non-segmented, single-stranded, (+) sense RNA, which was 29.74 kb in one of the first sequenced isolates. There are no drugs or vaccine for treating or preventing SARS.
We aim to develop a vaccine to control infection with SARS-CoV by targeting a surface accessible protein. The correlate of protective immunity for virtually all effective vaccines against viruses is the titer of antibodies that neutralizes virus invasion into cells and/or development of the virus within the cells. For antibodies to neutralize virus they in general are directed against proteins or glycoproteins that are accessible on the surface of viruses when they are extracellular. The """"""""spike"""""""" (also called S or E2) glycoprotein of SARS-CoV is such a target. It is also attractive as a target, because it is thought to mediate invasion of the virus into host cells. The spike protein of one isolate is 1256 amino acids encoded by 3768 base pairs. Based on our experience with identifying biologically active regions of proteins that mediate invasion into cells, and are good targets for antibody mediated protective immunity, we selected a region of the SARS-CoV spike protein which we have designated Region I. It is represented by 2421 base pairs and codes for a protein of 807 amino acids. Our data indicate that N-gycosylation of such proteins reduces expression and immunogenicity of the proteins. Region I was selected in part because of paucity of N-glycosylation sites. However, there are 9 such sites in the native sequence. We therefore altered the the known sequence of Region 1 to eliminate these 9 potential N-glycosylation sites, and produced a synthetic gene based on this altered sequence. Our experience with expression of proteins in Pichia pastoris indicates that altering codon usage based on our proprietary information leads to enhanced expression of recombinant proteins and in vivo expression of proteins by DNA vaccines. We therefore will use the Region I Spike Protein SARS-CoV native no glycosylation (RI-native-NG) sequence as the basis for creating a second synthetic gene in which we will optimize the gene sequence to alter codon usage. This sequence is designated Region I Spike Protein SARS-CoV codon optimized no glycosylation (RI-optimized-NG). These synthetic DNA sequences, RI-native NG and RI-optimized-NG, will be used to construct DNA plasmids and to produce recombinant proteins in P. Pastoris that will be optimized for production and purification and used as immunogens. Mice and rhesus monkeys will be immunized with 3 doses of recombinant protein in adjuvant or a sequential (prime-boost regimen) of a DNA plasmid expressing the SARS-CoV spike protein followed by recombinant protein in adjuvant. Anti-sera will be assessed for antibodies to spike protein by ELISA and capacity to neutralize virus invasion of Vero cells in vitro. Successful completion of Phase I will be achieved when we have generated a robust method for producing and purifying high quality at high yield Region I rec. protein, and have demonstrated that anti-sera elicited by immunization neutralizes viral activity in vitro. Phase II will include a monkey immunization and challenge study and scale up and process development for cGMP production and clinical trials. Our long term goal of commercializing a SARS vaccine is enhanced by this collaboration among Protein Potential (recombinant protein and DNA plasmid development expertise), CDC (viral neutralization assays and knowledge of the disease), and NMRC (immunization of mice and monkeys, and clinical trials).
