Preparation And Protective Efficacy Of Recombinant Respiratory Syncytial Virus Vaccine In BALB/C Mice

Respiratory syncytial virus（RSV）is a leading cause of lower respiratory tract viral disease in infants,old and other immunodeficiency people.It is responsible for an estimated 30 million RSV infection and high rate of RSV reinfection per year.More than 90% of infants infect RSV and RSV infection accounts for 70% of the hospitalization rate such as bronchiolitis,pneumonia and infant asthma.In old people,RSV infection leads to obstructive pulmonary disease and heart and lung complication.There remains no licensed vaccine product,despite a long-term dedicated research.Therefore,WHO will be the development of RSV vaccine as an urgent solution problems.The development of RSV vaccine has been dampened.In the 1960 s,a formalin-inactivated RSV（FI-RSV）vaccine was found to be poorly protective and was associated with disease enhancement.Followed by the use of traditional methods developed a variety of RSV vaccine.Although achieved some success,but mostly stop in clinical trials.Subunit vaccines are considered to be the most likely to develop vaccines for the prevention of elderly infection with RSV,while attenuated live vaccines are the most likely candidate vaccine for the prevention of infant infection with RSV.Many international studies have also explored these two vaccine forms.Subsequently,the cold-adapted virus was isolated by Qing Yan et al.and a KM516-AS vaccine was developed but it is not yet been applied to the clinic research.rA2DM2-2 and rA2cp248/404/1030ΔSH vaccine by using reverse genetics approach induced immunogenicity and immunogenic protection with an unapproved safety issue during the first phase of clinical research.A subunit vaccineof MedImmune and Novatiby using full-length F protein（PFP）was shown to be safe and immunogenic in human clinical trials,but it fails to solve the Th2 bias immune response problem.Recently,a 112 amino acid（aa）（aa 412-524）derived from the F protein is used alone or is constructed into expression vector or is used as a new chimeric protein,the new recombinant protein can induce Th1/Th2 balance in mucosal immune response.In addition to the frequently used protective antigen（F protein）,the vaccine requires the co-administration of an adjuvant.Less amount of adjuvant by nasal delivery elicits optimal mucosal and systemic immune response.The development of a new RSV vaccine candidate is the focus of the study.First,we constructed the recombinant RSV F self-assembled nanoparticle（NP）vaccine to explore the assembly of RSV F protein during immune response and immune protection effect.In recent years,the application of nanotechnology to vaccine is a hot spot.This technology be estimated have a market value of more than 100 billion.Whether it is through the activation of nanoparticles to strengthen the immune system,or as a delivery system or immune stimulating adjuvant to enhance the immune response.I suggest that nanoparticles are expected to revolutionize that treatment of the disease and the delivery of the biologically.Second,in order to create recombinant live attenuated vaccine chimeric RSV neutralizing epitope was included with the use of influenza reverse genetics.Lastly,the immunogenicity and immunoprotective effect were evaluated.Preparation and protective efficacy of respiratory syncytial nanoparticles vaccine in BALB/c miceWe prepared NPs from multiple F protein oligomers by insect expression system.The immunogenicity and immunoprotection of BABL/c mice were examined via intranasal immunization.Objective:To generate recombinant RSVFNPsvaccine;To identify immunogenicity and immunoprotection of recombinant vaccine;To explore the practicality and feasibility of NP Vaccine.Methods:We designed the aa sequence of RSV（wild type）F protein by using bioinformatics techniques and the segment underwent codon optimization.The pFastBacⅠ-F plasmid was constructed and the recombinant bacillus Bacmid-F and Bacmid-F were transfected into sf9 cells to obtain a stable expressing baculovirus system.The expression protein was analysed by Western blot（WB）,the F protein was purified by affinity chromatography and the assembly of the NPs was confirmed by electron microscopy.The specific antibody titers and neutralizing antibody against RSV in immunized BALB/c mice were determined by indirect-ELISA and micro neutralization method,respectively.Apart from humoral immunity,cellular immunity,such as IFN-γ and IL-4 level in mouse spleen cell suspension,was performed using ELISPOT method.The protective effect of recombinant RSV-F NPs was determined by detecting the changes in body weight,pathology,the amount of cytokine production and viral load in mice after RSV（A2 strain）challenge.Results:The transfected MOI was 0.1 and the recombinant virus TCID50 was 108/100μl.WB identified a specific band at 65 KD for the expressed recombinant RSV F protein.Electron microscopic observation of NP structure was "rosette" shape,which was a successful assembly of nanoparticles.The results showed that RSV specific antibodies（IgG,IgG1,IgG2a）and cellular immune response were efficiently induced by immunization.The group of 30μg NPs IgG immune titer reached 103.682±0.05,significantly higher than 10μg NPs（P<0.001）.Less than 1 of IgG1/IgG2 a suggested that recombinant RSV F NPs vaccine can induce a Th1 cellular immune response in mice.Compared with the muscle immunization group,the mice immunized with intranasal route produced s IgA specific antibody,the group of 30μg NPs lung titerreached 102.3±0.7.In vitro and neutralization experiments showed that 30μg NPs can stimulate the 102±0.13 level ofneutralizing antibody whereas the amount of IFN-γ and IL-4 cytokines secreted by spleen cells were significantly increased.After the challenge,the nasal pneumonia virus load of mice immunized decreased,and there is no changes in the body weight and enhance respiratory disease（ERD）.RSV F NPs can reduce the nasal pneumonia virus load and have effective of body weight changes,there were no significant pathological changes in the lungs and not led to ERD.Conclusion: Recombinant RSV-F NPs vaccine was successfully prepared and it can effectively stimulate to produce mucosal immunity,cellular immunity and humoral immune response and immunogenic protection in BABL/c mice.Recombinant Influenza Virus Expressing a Fusion Protein Neutralizing Epitope of RSV Confers Protection In MiceIn this study,recombinant chimeric FLU/RSV/NA-3F attenuated live vaccine was prepared by using cold adapted influenza virus as vector.Immunogenicity and immunoprotection were evaluated in BABL/c mice.Objective: To prepare recombinant chimeric FLU/RSV/NA-3F attenuated live vaccine and evaluate the immunogenicity and immunoprotection.To develop safe and effective recombinant RSV Live attenuated vaccine.Methods: The NA gene of A/California/07/2009（H1N1）strain was modified by bioinformatics method.The RSV F protein neutralized epitope II was inserted into the NA gene sequence recombinant NA-3F gene.Recombinant plasmids were co-transfected to MDCK/COS1 cells with 6 plasmids of A/Ann Arbor / 6/60（H2N2）and HA of A/California/07/2009（H1N1）.After amplification on the chicken embryo,the recombinant chimeric FLU/RSV/NA-3F virus was identified by hemagglutination test（HI）,TCID50,western blot and electron microscopy.BABL/c mice were immunized with two doses of 104 TCID50 and 10⁶TCID₅₀.The specific antibody and the s IgA antibody titer in nasal lung lavage fluid were detected by ELISA.The neutralizing antibody against influenza was detected in the serum of mice by HI and neutralizing antibody against RSV virus was detected by neutralization experiments.The levels of IFN-γ and IL-4 cytokines were detected by ELISPOT.The immunoprotection of recombinant FLU/RSV/NA-3F vaccine strain was carried out by detecting the changes of body weight,lung virus load and lung pathology.Results: The recombinant FLU/RSV/NA-3F attenuated live vaccine strain was successfully prepared.Western blot showed a successful expressed chimeric protein ata specific band of 63 KD.the recombinant virus was consistent with the characteristics of influenza virus from electron microscopy observation.The titer of the purified virus was1: 29 and the TCID50 was 10⁸TCID₅₀/ml.The results showed that the feature of recombinant FLU/RSV/NA-3Fattenuated live vaccine strain is similar to that of A/California/07/2009（H1N1）live attenuated vaccine strain,such as the 33℃ of the optimum temperature IgG,IgG1 and IgG2 a antibodies for RSV can be produced in both group,but the group of 10⁶TCID₅₀ was associated with more higher IgG titers than the 10⁴TCID₅₀ group reached103.372 ± 0.126.The result（IgG1/IgG2 a <1）indicated that recombinant FLU/RSV/NA-3F attenuated live vaccine can induce cellular immune response to Th1 type.Immunized mice produced significant s Ig A specific antibody,the titer of the nasal irrigation fluid reached102.1±0.5,and the titer of the lung was102.9.Neutralization experiments showed that the recombinant vaccine could produce neutralizing antibody against RSV with titer of101.75±0.053.HI experiment showed that it could produce neutralizing antibody against influenza virus1:640.The levels of IFN-γ and IL-4 cytokines secreted by spleen cells were significantly increased.The viral load in the lung tissue of 10⁶TCID₅₀ group was 101.147±0.02 and the viral load in the lung tissue of mice was 102.181±0.02.There is no significant changes in body weight and pathological aspects in the lungs.In the passive immunization,the viral load in the lung tissue of the mice was103.1±0.015（P <0.01）.Conclusion:Recombinant chimeric FLU/RSV/NA-3F attenuated live vaccine was successfully prepared and it can effectively stimulate mucosal immunity,cellular immunity and humoral immune response in BALB/c mice.Passive immunization experiments show that immuned sera played a role in reduced replication and lung tissue lesions in RSV lungs.