| Adenoviral vectors have been widely used in gene therapy and recombinant vaccine development. Adenovirus is a double stranded DNA virus without envelope. There are more than 50 types of human andenovirus (HAdV), which are clasified into seven distinct species (A-G). HAdV can target different human organs or tissues, and cause various diseases. The commonly used adenovirus vectors are constructed based on HAdV5, a HAdV-C virus which induces infection on upper respiratory tract. HAdV5 targets to the respiratory epithelial cells, by binding to the coxsackie-adenovims receptor (CAR) on the cellular surface. A novel HAdV41 vector system has been established by our group. HAdV41, a member of HAdV-F (enteric adenovirus), has natural gastrointestinal tropism. The target of HAdV41 maybe intestinal epithelial cell, and the cellular reporter of HAdV41 has not been fully elucidated. The recombinant vaccines based on HAdV5 and HAdV41 vectors may trigger different immune responses due to the differences of the two viruses in biological characteristics.Middle East respiratory syndrome coronavirus (MERS-CoV), first reported in 2012, is an emerging coronavirus causing a severe acute respiratory syndrome (SARS)-like disease with high fatality. MERS-CoV has been becoming a significant public health threat, while no vaccine or effective treatment options exist to prevent a potential MERS-CoV pandemic. The spike (S) protein of MERS-CoV, a component of the virion membrane, which plays an important role in viral attachment and entry into target cells, is the main neutralizing antigen of coronaviruses.In this study, we constructed recombinant HAdV5 and HAdV41 carrying MERS-CoV S gene, and administered them to mice intramuscularly or intragastrically as MERS-CoV vaccines. The systemic and mucosal antigen-specific immune responses induced were evaluated. To elucidate the biodistribution of recombinant HAdV5 and HAdV41 in mice, we constructed the vectors carrying both GFP and firefly luciferase reporter genes. After infecting mice intramuscularly (i.m.) or intragastrically (i.g.), biodistribution of the vectors was studied through detecting the expression of reporter gene by using in vivo imaging and in vitro luciferase assay.1. The immune response in mice elicited by the recombinant adenovirus vaccinesObjective Observe the immune response of recombinant MERS-CoV vaccines in mice and evaluate the feasibility of using HAdV41 and HAdV5 as oral vaccine vectors. Methods Human codon optimized S protein gene of MERS-CoV was inserted into the multiple cloning sites (MCS) of the shuttle plasmids, and the adenviral plasmids were generated by homologous recombination of the shuttle and backbone plasmids in E. coli BJ5183 strain. Recombinant adenoviruses bearing MERS-CoV S gene (HAdV41-MERS-S and HAdV5-MERS-S) were rescued and amplified in 293 TE32 cells and 293 cells, respectively. Control viruses carrying GFP gene (HAdV41-GFP and HAdV5-GFP) were constructed similarly. Genomic DNAs of the recombinant adenoviruses were extracted by the Hirt method and identified by restriction analysis and PCR amplification. The expression of MERS-CoV S protein in HAdV41-MERS-S-or HAdV5-MERS-S-infected cells was detected by Western blotting and indirect immunofluorescence assay. The viruses were purified with two rounds of CsCl ultracentrifugation, the viral particle (vp) titers were calculated by measuring the genomic DNA content, and the infectious titers (IU/ml) were determined by limiting dilution assay on 293 cells. Balb/C mice (N= 10 per group) were immunized with 5×109 vp of HAdV41-MERS-S or 1×109 vp of HAdV5-MERS-S intramuscularly (i.m.) or intragastrically (i.g.). Half number of the mice in each group was sacrificed 4 weeks post-immunization, and the rest mice were sacrificed 16 weeks post-immunization. Anti-MERS-S IgG and neutralizing antibody were examined in sera, and anti-MERS-S IgA were examined in sera, lung lavage fluid, small intestine and large intestine by ELISA. Functional antigen-specific T cell responses were examined in the spleen and pulmonary lymphocytes of the mice. Results The recombinant plasmids were successfully constructed and the recombinant viruses were rescued in packaging cells. The results of restriction analysis and the PCR amplification were consistent with the predicted fragments, demonstrating the viral genomic DNAs were correct. The expression of MERS-CoV S protein was confirmed by Western blot and indirect immunofluorescence. HAdV41-MERS-S was purified in 2.3 ml with the viral particle (vp) titer of 5.71 ×1011 vp/ml and Infectious titer of 2.7×109 IU/ml; HAdV5-MERS-S was purified in 2.3 ml with the viral particle titer of 3.57x1012 vp/ml and infectious titer of 1.3x1011 IU/ml. And the control adenoviruses were constructed and purified similarly. Four weeks after a single dose administration of HAdV41-MERS-S or HAdV5-MERS-S via i.m. route, MERS-S specific IgG antibodies were induced in all mice (n=5), while neutrlizing antibodies were elicited in 20% HAdV41-MERS-S immunized mice and 100% HAdV5-MERS-S immunized mice. No antigen-specific IgA antibody was detected. In the spleen and pulmonary lymphocytes isolated from immunized mice, functional antigen-specific T cell responses could be stimulated. Antigen-specific IgG, neutrlizing antibodies and T cell responses could be detected 16 weeks post vaccination. For the mice immunized via i.g. route, administration by either HAdV41-MERS-S or HAdV5-MERS-S induced antigen-specific IgG in 60% immunized mice (n= 5), and neutrlizing antibodies were elicited in 40% HAdV41-MERS-S immunized mice and 60% HAdV5-MERS-S immunized mice. Antigen-specific IgA could be detected in serum and on a variety of mucosal surfaces (including lung lavage fluid, the small intestine, and the large intestine); antigen-specific T cell responses were not detected. Antigen-specific IgG, IgA and neutrlizing antibodies could still be detected 16 weeks post vaccination. Conclusion After a single dose administration of HAdV41-MERS-S or HAdV5-MERS-S via i.m. route, functional antigen-specific T cell responses were stimulated and could persist for several months in spleen and lung, but no antigen-specific IgA antibody was detected. In contrast, for mice immunized via i.g. route, administration by either HAdV41-MERS-S or HAdV5-MERS-S induced antigen-specific IgA in serum and on mucosal surfaces (including lung, the small intestine and the large intestine), and antigen-specific T cell responses were not detected. Both rHAdV-based vaccines induced systemic humoral immune responses (IgG and neutralising antibodies) regardless of the route of administration.2. The biodistribution of adenovirus vectors in miceObjective Observe the biodistribution of adenovirus HAdV41 and HAdV5 in mice. Methods Using pLEGFP-Cl and pGL4.17 as template, GFP and firefly luciferase genes were cloned with PCR amplification and inserted into the MCS of shuttle plasmids to construct psh41GFP-T2A-Luc and psh5GFP-T2A-Luc (The coding sequences of the two genes was linked with T2A sequence). Plasmid psh41GFP-T2A-Luc and psh5GFP-T2A-Luc were confirmed by restriction analysis, PCR amplification and sequencing. Adenovirus plasmid pAdV41GFP-T2A-Luc and pAdV5GFP-T2A-Luc were generated by homologous recombination of shuttle and backbone plasmids in E. coli BJ5183 strain. The recombinant viruses, HAdV41GFP-T2A-Luc and HAdV5GFP-T2A-Luc, were rescued and amplified in 293 TE32 cells and 293 cells, respectively. Genomic DNAs of the recombinant adenoviruses were extracted by the Hirt method and identified by restriction analysis and PCR amplification. The expression of GFP and firefly luciferase was detected by fluorescence microscopy observation and luciferase assay in HAdV41GFP-T2A-Luc or HAdV5GFP-T2A-Luc infected A549 cells. Viruses were purified with CsCl ultracentrifugation, the viral particle titer was calculated by measuring the genomic DNA content, and the infectious titers (IU/ml) were determined by limiting dilution assay on 293 cells. Balb/C mice were infected with 5×109 vp of HAdV41GFP-T2A-Luc or 1×109vp of HAdV5GFP-T2A-Luc via intramuscular (i.m.) or intragastric (i.g.) route. At 4 h,18 h,48 h and 7 d post infection, the fire luciferase activity in mice were determined using in vivo imaging; after that,2 mice of each group were sacrificed,17 tissues were collected, homogenized and lysed to release active firefly luciferase, and the luciferase activity in different tissues were detected by using luciferase assay. Results pAdV41GFP-T2A-Luc and pAdV5GFP-T2A-Luc were successfully constructed, and HAdV41GFP-T2A-Luc and HAdV5GFP-T2A-Luc viruses were successfully rescued. The results of restriction analysis and the PCR amplification were consistent with the predicted fragments, demonstrating the viral genomic DNAs were correct. In HAdV41GFP-T2A-Luc and HAdV5GFP-T2A-Luc infected A549 cells, green fluorescence was observed and active luciferase was detected, suggesting the expression of GFP and luciferase. HAdV41GFP-T2A-Luc was purified in 1.65 ml with the viral particle titer of 7.6×1010 vp/ml and infectious titer of 2.9×108 IU/ml; HAdV5GFP-T2A-Luc was purified in 2.11 ml with the viral particle titer of 4.2×1011 vp/ml and infectious titer of 3.1×1010 IU/ml. Strong fluorescence and high luciferase activity could be detected in the injection site by in vivo imaging and luciferase assay after the infection of mouse with HAdV41GFP-T2A-Luc or HAdV5GFP-T2A-Luc via i.m. For mice infected with HAdV5GFP-T2A-Luc, the luciferase activity could be detected modestly in the liver and spleen. After infection by HAdV41GFP-T2A-Luc via i.g., weak fluorescent in the snout of the mice could be seen by in vivo imaging at 18 h post infection, and the luciferase activity could be modestly detected in peyer’s patch, large intestine and lower small intestine at 4h and 18h post infection. For mice infected with HAdV5GFP-T2A-Luc via i.g., weak fluorescence in the snout of the mice could be observed at 18 h and 48 h post infection; the luciferase activity could be modestly detected in peyer’s patch, large intestine and lower small intestine as well as in lung and archea. Conclusion In mice infected via i.m.route, HAdV41 and HAdV5 distributes mainly in injection site; and in mice infected with HAdV41 and HAdV5 via i.g. route, the expression of transgene is weak and mainly located in intestinal tract.In conclusion, we constructed rHAdV41 and rHAdV5 carrying MERS-CoV S gene, which could elicit systemic and mucosal antigen-specific immune responses in mice after a single dose administration. We observed the biodistribution of rHAdV41 and rHAdV5 in mice by tracing the expression of reporter gene, partially explaining the weaker immune responses triggered by rHAdV via i.g. route. Our study will benefit the development of MERS-CoV vaccine. This is the first report of rHAdV41 biodistribution in mice, which improved our understanding of HAdV41 virology and and laid a foundation for developing HAdV41 as a useful vaccine vector. The results also suggest that mouse is not the ideal animal model for evaluation of rHAdV41 as an oral vaccine vector. |
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