Immunization of mice with DNA- and recombinant adenovirus-based SARS-CoV vaccines and expression of SARS-CoV envelope protein via E. coli system

The severe acute respiratory syndrome (SARS) is a newly emerged disease with high infectivity and morbidity, which caused massive outbreak worldwide from 2002 to 2003. The causative agent for SARS has been identified as a novel coronavirus named SARS-associated coronavirus (SARS-CoV). Clinical trials on SARS patients demonstrated that these people developed neutralizing antibodies against virus structural proteins. To date there is no efficient treatment for SARS, so the development of preventive vaccines is urgently necessary.
Recombinant adenovirus (ADV) is a widely used viral vector system and is known for effective delivery of foreign genes and high-level protein expression. In this study, we immunized Balb/c mice with DNA and recombinant adenovirus-based SARS vaccines. We used the recombinant ADV，Adv/SME，as vaccine which contains three of SARS-CoV structure protein genes, spike (S), membrane (M) and envelope (E). At the first part of this study，we prepared DNA and recombinant adenovirus vaccines, and evaluated the gene expression of DNA vaccine in 293A cells. First of all, the plasmid DNA vaccine，pShuttle/SME，was purified by massive preparation. Reverse transcription-polymerase chain reaction and western blotting were carried out to detect the expression of S, M and E genes in 293A cells transfected with pShuttle/SME. The mRNA expression of three genes was detectable; however, respective protein expression was non-detectable via western blot assay. On the other hand, high-titer Adv/SME was amplified in 293A cells. Two immunization strategies were performed in eight-weeks Balb/c mice to compare their efficiency in inducing antibody responses. The single shot immunization of recombinant adenovirus vaccine is so-called one-shot vaccination, while three DNA vaccines priming then one recombinant adenovirus boosting is so-called prime-boost vaccination. Collection of mouse sera was accompanied with the vaccination regimen.
For preparing antigen sources to detect antibody responses of immunized mice，the GST-E fusion protein was expressed in E. coli and was confirmed by western blotting. The GST-E fusion protein could be detected by rabbit anti-E antisera and mouse anti-GST monoclonal antibody. Then we measured the antibody responses of immunized mice via western blotting using GST-E fusion protein. It was shown that no specific anti-E antibody was detected after one-shot vaccination. Similarly, there was no significant differences between experimental and control sets after prime-boost vaccination, either.