Construction And Immunogenicity Of Recombinant Adenovirus-SARS Coronavirus (SARS CoV) Spike Glycoprotein

The Severe Acute Respiratory Syndrome (SARS), also called Infectious atypical pneumonia (IAP), was originated from province of China in November 2002, and then spread over the world rapidly. This disease was an urgent, complicated, severe, rapid pneumonia with infected virus systemic, and caused other organs damaged, function prostrated, extremely strong infectivity, people have easy catch, high mortality. It is the most influential new infection that impairs people's health seriously. People had not enough recognition and defense, as the SARS-CoV has never been reported in the history of people's diseases. So the SARS could spread rapidly, and people have easy catch, high mortality, few hundreds of people have died from the disease. Until now, there is no specific treatment and no effective vaccines or therapies currently available. Inactivated SARS-CoV by irradiating is the most facile and convenient vaccine, but inactivated vaccine presumably possess a potential risk which results in the infection of the vaccinated. There is something need to be studied with instancy, such as the mechanism of SARS, variation of immunity function of patients, most important is to develop a new efficacious vaccine to prevent SARS recurring.In order to best define an effective SARS vaccine approach, it is important to consider the correlates of protection from coronaviruses in animal models and any evidence for immunity that may be emerging from clinical experience. We must also examine the host-pathogen interaction and ask whether specific antigens (proteins derived from the pathogen) or host responses, such as antibodies and T cell response, provide any insight into the type of vaccine that should be developed.The spike(S) protein of SARS CoV is the type-I transmembrane glycoprotein thatis responsible for recognizing and binding its receptor of host cells, and directing the fusion between viral and cell membranes. Two functional domains at the amino (SI) and carboxy (S2) termini of S protein are conserved among the coronaviruses, especially with the more conserved S2 domain. The S protein is also the major antigenic determinant for coronaviruses. Therefore, we targeted the S2 fragment and the full length of S protein for construction of effective vaccines of SARS.We focused on using adenovirus (Ad)-based gene transfer vectors that can induce both humoral and cellular immune responses to develop an effective vaccine. Adenoviral vectors have particular advantages for use as in vivo gene transfer vehicles, including a broad host range, the ability to infect both dividing and non-dividing cells, and ease of high-titer purification. Ad5 vector system is a useful vehicle for both efficient delivery and long-term stable transduction of therapeutic genes to both dividing and non-dividing cells. Their ability to generate CTL responses and mucosal immunity may be particularly significant for protection against SARS coronavirus (SARS CoV) infection.Here, we described the construction, identification, production, and the immune characterization of two SARS recombinant adenoviruses expressing the spike glycoprotein gene(S and S2 protein) of SARS-CoV strain CUHK-SulO. The researches include: (1) Synthesis of the full length of S and its truncated fragments of SARS. (2) Expressions of two fragments (SI and S2) in E.coli. (3) Construction of the two recombinant adenoviruses of S glycoproteins. (4) Identification and detection of the recombinant adenoviruses by IFA and western blotting. (5) The antibody against SARS -CoV spike-protein was determined by ELISA.We tested the hypothesis that expression of the SARS-CoV spike glycoprotein (S), or its component part S2, in Ad5 vector can elicit cellular and humoral immune responses against S in immunized mice. A synthetic spike gene was made by an overlapping PCR and ligation with endonucleases strategy with codon optimization for mammalian cells. An Ad vector expressing S or S2 driven by a CMV promoter was constructed and protein expression was confirmed by Western blotting and IFA analysis. To evaluate the immune responses stimulated by these two vaccines, twodoses of the vectors were administered by intraperitoneal routes to BALB/c mice. The serum antibody titers were measured by ELISA .The results showed that immunization with Ad vaccines expressing the SARS-CoV S or S2 elicit high titers of SARS-CoV antibodies in mice.In this study, the adenovirus was employed as vector to transfer S and S2 proteins of SARS into293 cells and BALB/C mice, to investigate the biological implications and immune effects of the two recombinant adenoviruses in vitro and in vivo. We intended to determine whether the two adenoviruses had influence on the humoral immune response in mice.1. Construction of recombinant adenoviruses of S, SI and S2 of SARSBy overlapping and ligation with endonucleases technique, fl, f2, f3 and f4 fragments were ligated together to obtain the full length of S gene. The fl plus f2, is SI, while O plus f4, is S2. The three fragments were cloned into the shuttle vector pshuttle-CMV;the resultant constructs were electrotransformed into E.coli strain BJ5183, and the recombinant adenoviral plasmids were cleaved with Pad to expose their inverted terminal repeats and transfected into 293 packaging cell to generate viruses. Three strains of recombinant adenoviruses carrying S, SI and S2 were obtained.2. Biological characteristics of recombinant adenovirusesThe resultant recombinant adenoviruses showed typical adenovirus morphology with a diameter about 70 nm under electron microscope. The integration of SARS S, SI and S2 into the adenovirus genome was identified by PCR. The viral genomes were stable when tested by PCR during the passage in 293 cells.3. Expression of SARS S gene in 293 cellsBy IFA and Western blot analysis, the expressions of SARS antigens were detected in cell lysates or culture medium of 293 cells infected by each of the three strains of recombinant adenoviruses, respectively. The antigenicity of S, SI and S2 protein was analyzed by western blot using the horse against SARS. When 293 cells were infected with RAd-S, RAd-Sl and RAd-S2 at various multiplicity of infection (MOI), the expressions of S protein and its truncated fragments increased with MOI. Additionally,we infected 293 cells with the three strains of recombinant adenoviruses at same MOI. The results showed that the expression of S protein in 293 cells infected with RAd-S and RAd-S2 was strong. However, the expression of RAd-Sl was so weak that it can be seldom detected4. The immune response of BALB/C mice induced by recombinant adenoviruses Four of seven groups of BALB/C mice were intraperitoneally inoculated with RAd-S, RAd-Sl and RAd-S2, and the other three groups with wild Ad5 and PBS as control, respectively. Samples of mice serum were taken and tested for antibodies to SARS antigens by ELISA. The mice in experimental groups (rAd-S and rAd-S2 group) produced anti-SARS efficiently, indicating that these two recombinant adenoviruses may have influence on the humoral immune response to SARS S protein. There is no antibody production of rAd-S 1 because of no expression of SI in 293 cells. All samples of mice serum were tested negative for anti-SARS. IgG specific for SARS were positive in serum samples.In this study, we have artificially synthesized the full length of S protein gene and its fragments,Sl and S2 as the targets to construct SARS recombinant adenovirus vaccines and the SARS S protein and its truncated fragment (S2) were transferred and expressed successfully in 293 cells and BALB/C mice. The results demonstrated the immune effects in mice immunized with the two recombinant adenoviruses. Both the two recombinant adenoviruses, rAd-S and rAd-S2, have the ability to induce humoral in BALB/c mice. These two recombinant adenoviruses would probably be good choices of candidate vaccines to prevent from SARS infection.