Preparation And Protective Efficacy Of Recombinant Respiratory Syncytial Virus Vaccine In Balb/c Mice

Respiratory syncytial virus (RSV) is the most major pathogen causing lowerrespiratory disease in infants and the elderly. A worldwide respiratory tract infectioncaused by RSV is more than thirty million each year. RSV infection in infants lessthan2years is more than90%. Meanwhile, RSV is also one of the primary pathogenscausing pneumonia and death in elderly and the immunodeficiency.RSV is mainly spread by droplets or direct contact with patients. Upperrespiratory tract infection symptoms first appear, usually with runny nose, cough, lossof appetite, fever and other symptoms. After1-3days there performs significantlylower respiratory tract involvement, such as severe bronchiolitis caused by airwayobstruction and respiratory distress. It has caused worldwide attention, and hadbecome a global public health problem.As we all know, vaccine is an important means of prevention and control ofinfectious diseases. RSV vaccine development still needs a big breakthrough, andWHO has also comfirmed that RSV vaccine being included in the world’s mostpriority vaccines. After half a century research, there still no licensed RSV vaccine. Inthe1960s there first appeared formalin inactivated vaccine (FI-RSV).But it causedsevere immune-enhancing in vaccinated populations, and ultimately ended in failure;Soon afterwards there developed live attenuated vaccine and vector live vaccine, butbecause of its safety concern, it still need more clinical experiment; Subunit vaccine isprepared according to RSV surface glycoprotein such as F and G protein, but it alsohas some problems to be solved such as the presence of immunogenicity, immuneeffects instability and so on; In recent years VLP vaccine has a very rapid developed,but because of the way it vaccined it can not stimulate the body produce mucosalimmune response, so it also need to seek innovation and breakthroughs. Althoughmany types of vaccines are being developed, but there is still no certified RSVvaccine, so the development of a safe and effective RSV vaccine is very important. Meanwhile, the major route of traditional vaccination is intramuscular injection,which motivate very limited cellular immune response and it can not produce mucosalimmune response. Therefore, find a new way of immunization has become animportant breakthrough in RSV vaccine development.This study is based on the construction of new RSV vaccine strains, committed toaddressing the problem such as the imbalance of Th1/Th2, the limition of cellularimmunity, lack of mucosal immune response and so on. This study includes twoaspects: First, according to the status that currently RSV VLP vaccine can not producemucosal immune response, we carry out a new immunization way of intranasalhoping for producing long-lasting cellular and mucosal immunity responses; Second,RSV and influenza usually has mixed infections in clinical, we conduct a chimericRSV and influenza vaccine formulations as "dual vaccine" to provide a new direction.Preparation and protective efficacy of respiratory syncytial virus-like particles(RSV VLPs) vaccine in Balb/c miceVirus-like particles (VLPs) has become a new direction of vaccine research.Influenza matrix protein M1and the RSV fusion protein (F) or attachmentglycoproteins (G) are combined to form virus-like particles (RSV-VLPs).We use Bac-to-Bac system to expresse RSV-VLPs candidate vaccine strainswhich contants influenza M1protein skeleton, and RSV F, G protein. And intranasallyimmunized6-8week female BALB/c mice to evaluate the immunogenicity andprotective effacy of candidate vaccine.Vector NTI AdvanceII system were used to analysis the sequence. We usecomplete recombinant Bacmid-M1-F/Bacmid-M1-G to transfected insect cells, andexpresse the recombinant RSV-VLPs vaccine. Sucrose density gradientcentrifugation were used to purify the recombinant RSV-VLPs vaccine. Afterintranasal immunization we use ELISA assay to detect the level of serum IgG, IgG1,IgG2a and nasal lavage lung sIgA antibody titers, ELISPOT assay were used in thedetection of IFN-γ and IL-4level in mouse spleen cell suspension, etc. Two weeksafter boost, we choose RSV A2strains to attack the vaccine mice，and evaluate theimmune protective vaccine candidates by detecting changes in body weight, lungviral load and lung biopsy.Western blot result showed that recombinant RSV-VLPs vaccines target proteinswere stably expressed. And electron microscopy observation showed recombinantRSV-VLPs vaccine has particle characteristics. The results of animal experiments confirmed that the recombinant RSV-VLPs vaccine in mice can produce high titersof RSV-specific antibody, and the levels of cytokines IFN-γ and IL-4rise. Thecandidate vaccine strain RSV-VLPs can be considered as good immunogenicity. Thechallenge experiment results show that the candidate vaccine RSV-VLPs can reducenasal and lung viral load, reducing lung injuries and corrected weight changes. Itshowed good immune protection in RSV A2strain challenge. The recombinantRSV-VLPs vaccine is one of the most worthy-study candidate vaccines.Construction and protective efficacy of recombinant clod-adapt influenzavaccine strain as a vector for FLU-RSV vaccineIn recent years, the reverse genetics technology has a very rapid development, aswell as the influenza virus as a vaccine delivery vector system had attracted more andmore attention. It has been successfully applied in such as HIV, GFP, IL-2etc.MedImmune developed palivizumab mainly based on RSV A type F proteinepitope263-275amino acids, it is the world’s first RSV-specific humanizedmonoclonal antibodies, palivizumab play an important role in RSV prevention andearly intervention process.In this sdudy we use attenuated cold-adapted influenza vaccine strains as aplatform to express fitted RSV F protein and antibody epitopes. And intranasallyimmunized6-8week female BALB/c mice to evaluate the immunogenicity andprotective effacy of the candidate vaccine.We use reverse genetics technology to transform the attenuated cold-adaptedinfluenza vaccine strain NA gene by insert the RSV F epitope II. The recombinantNA-F fragment were used with other seven fragments PB1, PB2, PA, HA, NA, NPand M rearranged to rescue the recombinant FLU-RSV vaccine strains. We usesucrose density gradient centrifugation to purify the recombinant FLU-RSV vaccine.After intranasal immunization we use ELISA assay to detect the level of serum IgG,IgG1, IgG2a and nasal lavage lung sIgA antibody titers, ELISPOT assay were usedin the detection of IFN-γ and IL-4level in mouse spleen cell suspension, etc. Twoweeks after boost, we choose RSV A2strains to attack the vaccine mice，andevaluate the immune protective vaccine candidates by detecting changes in bodyweight, lung viral load and lung biopsy.The results confirmed the identification of recombinant FLU-RSV vaccine strainhas stable expression of RSV F protein, electron microscopy confirm that therecombinant FLU-RSV vaccine strain has particle characteristics. We named the recombinant FLU-RSV vaccine rFLU/RSV/NA-F. Vivo and in vitro replication assayshowed rFLU/RSV/NA-F vaccine strain has shown no statistical difference withattenuated cold-adapted H1N1vaccine strain. Animal experiments confirmed rFLU/RSV/NA-F vaccine strain can induce RSV-specific Th1-type immune response,while effectively produce mucosal immune response, confirmed thatrFLU/RSV/NA-F vaccine strain has good immunogenicity. The virus challengeexperiment results show that the candidate vaccine rFLU/RSV/NA-F can reducenasal and lung viral load and lung injuries. It showed good immune protection inRSV A2strain challenge. It confirmed that rFLU/RSV/NA-F vaccine strain has asignificant protective effect, which can effectively resist RSV infection. We willfurther test the recombinant rFLU/RSV/NA-F vaccine strain in cotton and monkey.