Immunogenecity And Immunoprotection Of The Chimeric Attenuated Influenza Vaccines Containing RSV Epitopes

Aim Identify three chimeric attenuated influenza vaccines containing RSVepitopes, study their safety, immunogenicity and immune protection primitively, andexpect to find an efficient and simple immune pathway.Methods The experiments of HA and HI were used to detect if the viruses weretransfected successfully. Totally, viruses were tested by RT-PCR, SDS-PAGE, indirectimmunofluorescence, electron microscopy, then expanded in chick embryo. After that,they were purified by sucrose density gradient centrifugation and the titer wasdetermined through TCID50. Animal experiments were done to study their safety,immunogenicity and immune protection preliminarily.Results The results of HA and HI experiments indicating that the viruses weretransfected successfully. Moreover, the gene sequences of recombinant viruses wereconsistent with plasmids, which were identified by RT-PCR. The SDS-PAGEelectrophoresis showed the main components of the influenza viruses were existed.Then, Indirect Immunofluorescence Assay （IFA） testing pointed out that the skeleton ofthe recombinant viruses were A/PR/8/34, and the results of electron microscopyindicated that the morphology of the recombinant viruses were similar to the wild typeinfluenza viruses. The recombinant viruses were purified by sucrose density gradientcentrifugation and the TCID50of RSV-FLU/NS1-F was10^-7.82, RSV-FLU/NS1-G was10^-7.5, RSV-FLU/NS1-F+G was10^-8. In animal experiments, BALB/c mice wereinoculated with10^-5TCID₅₀through nasal cavity. On days1,2,3,5,7and10afterinoculation, mice lungs, nasal turbinates and brains load with viruses were titrated todetermine the safety of recombinant viruses. The results also showed that there were noviruses were found in brain, and in nose, RSV-FLU/NS1-F+G was not found on the2ndday and RSV-FLU/NS1-F, RSV-FLU/NS1-G disappeared on the3rd day. And noviruses were detected in lungs after10days. There were no mice death. All these resultsshowed that the recombinant viruses were safe. High HI titer as well as some level ofRSV-specific IgG and sIgA could be detected in all of BALB/c mice which wereinoculated with the recombinant viruses. IgG1, IgG2a results and the ELISpot tests ofIL-4and IFN-γ showed that the recombinant viruses could induce humoral immune andcellular immune at the same time. The result of cytokines in serum showed that the IL-4and IFN-γ secreted from experimental group were higher than the control group. The evaluation of viruses immune protection indicated RSV-FLU/NS1-F andRSV-FLU/NS1-G could protect mice from lung injury, but the protective effect ofRSV-FLU/NS1-F+G was not effective.Conclusion Three chimeric attenuated influenza vaccines containing RSVepitopes were transfected successfully and their genome were identified correctly. Andthe three attenuated vaccines showed good safety and immunogenicity, of Which,RSV-FLU/NS1-F and RSV-FLU/NS1-G could protect mice from lung injury effectively,but RSV-FLU/NS1-F+G could not.