Study On Novel Nucleic Acid Vaccines For SARS

Severe Acute Respiratory Syndrome (SARS), also called infectious atypical pneumonia in China, is a new emerging respiratory infectious disease infected by SARS-CoV (SARS associated coronavirus). During 2002-2003, SARS spread to over 30 countries, causing 9096 cases. Effective vaccines have not developed for SARS.Nucleic acid vaccine (DNA vaccine) is the emphasis in research of SARS vaccine. Unlike conventional vaccines employing either killed virus or purified antigens, nucleic acid vaccine can efficiently elicit humoral and cellular immune responses, and have preponderances in simple preparation and low cost. Currently, there are several research about SARS DNA vaccines with S and N gene. Mice immunized with these DNA vaccines produce humoral and cellular immune responses. Results also showed DNA vaccines with SARS-CoV S or N genes have low immunity.In view of above considerations, this study constructed recombinant plasmids containing S, N and codon-optimized S2 and N gene fragments of SARS-CoV and compared their immnogenicities in BALB/c mice, and observed the effect of codon-optimization and DNA priming-protein boosting on immunogenicity of SARS-CoV N gene in BABL/c mice.Firstly, S1 gene (2307bp), S2 gene (1632bp) and N gene (1269bp) were amplified by RT-PCR from SARS-CoV BJ01 strain, and cloned into the pVAX1 vector, and recombinant plasmids pVS1, pVS2, pVN were constructed. The codon usage frequency of SARS-CoV was analysised and results showed that the codon usage is different between human genome and S/N gene of SARS-CoV. To increase the expression level of the S2 and N protein fragments in mammalian cells, we replaced all codons by those more frequently used in human genome without modifying the protein sequence. The codon-optimized S2 (opt-S2) and N gene(opt-N) have the higher G+C% than wild genes. Opt-S2 and opt-N gene fragments weresynthesized and cloned into pVAX1 vector, and recombinant plasmid DNAs pVeS2 and pVeN were constructed. The recombinant plasmids were transiently transfected into BHK21 cells. Specific proteins of SARS were detected in BHK21 cells and specific reactivities to the SARS-CoV antisera were confirmed.To observed the effect of codon-optimization on immunogenicity of SARS-CoV S gene in BALB/c mice, five groups of five-week-old female mice were immunized with plasmids pVAXl, pVSl, pVS2, pVSl+pVS2 and pVeS2 by intramuscular injection in the hind leg muscles. Specific antibodies against SARS-CoV could be detected by IFA in vaccinated mice at day 30 post-priming, and there is few differents on IFA antibody titers in sera from mice immunized with plasmid pVeS2 and pVS2. Neutralizing antibody titer of sera from mice immunized with pVeS2 on day 90 post-priming is higher than group immunized with pVS2. To observe cellular immune responses of vaccinated mice, spleen cells were isolated from mice and stimulated with inactivated SARS-CoV in vitro. Four days later, the proliferation powers were determined. The results showed that simulation indexes (11.85±1.54) from mice immunized with pVeS2 were higher than pVS2 group and other groups. In addition, the proportions of CD4\ CD8+ in splenocytes of mice immunized with pVeS2 were also changed. Our data suggested opt-S2 gene of SARS could elicit stronger humoral and cellular immune responses and codon-optimization enhanced the immunogenicity of S2 fragment.To observed the effect of codon-optimization and DNA priming-protein boosting on immunogenicity of SARS-CoV N gene in BABL/c mice. Five-week-old female mice were immunized twice with recombinant plasmids pVN or pVeN by intramuscular injection in the hind leg muscles, and boosted with recombinant N protein. The antibody titers were detected by ELISA. Specific anti-N IgG antibody titer of mice immunized with pVeN is higher than mice immunized with pVN. The antibody titer in pVN group or pVeN group could rise remarkably after boosting with N protein. Data suggested that both codon-optimization and DNA priming-protein boosting could enhance humoral immune response. To observe cellular immune...