| Respiratory syncytial virus(RSV)is one of the most important pathogens that can cause severe lower respiratory tract diseases.The susceptible population is infants,children,elderly and immunocompromised persons.Infants under 2 years of age have been infected with RSV at least once.Every year,2.5-4%of infants are hospitalized due to RSV infection.This has brought a heavy economic burden on the global health care business.In the United States alone,the annual RSV-related medical care costs up to $950 million.there is no vaccine or effective anti-viral drugs that can ameliorate RSV infection.Palivizumab(Synagis(?))is the only one approved for RSV prophylaxis that has been marketed by the FDA.However,due to its high price,the antibody is only used in the prevention of RSV infection in high-risk newborns such as immunodeficiency and congenital heart disease.For the study of RSV vaccines,in the United States in the 1960s,researchers used the formalin-inactivated whole virus vaccine(FI-RSV)against RSV to prevent the infection of RSV in some infants and young children.Subsequent re-exposure to RSV in these preimmune seronegative infants resulted in a significant increase in the frequency and severity of RSV-induced acute lower respiratory infections.In later studies,this enhanced RSV disease was referred to as enhanced RDS(ERD),which eventually leded to the death of two young children in the study.The failure of RSV inactivated vaccine had greatly hindered the development of RSV vaccine.In recent years,the emergence of various new vaccine design strategies have brought new opportunities and challenges to the field of RSV vaccines.Many subunit vaccines and live attenuated vaccines had entered clinical practice,they all ended in failure.The two major membrane proteins G protein and F protein on the surface of RSV virus are considered to be the most important immunogens for inducing neutralizing antibody response.Among them,G protein has a large difference between different types,and F protein is more conservative.Therefore,the F protein has always been the main target of vaccine research,and the successful application of the antibody against F protein Palivizumab also supports this view.With the in-depth analysis of the structure of the F protein,the researchers find that there are two conformations of the F protein of RSV,namely the pre-fusion conformation and the post-fusion conformation.The epitope recognized by Palivizumab is distributed on both conformations.Antibodies recognizing the pre-fusion conformation exhibit 10-100 folds higher in vitro neutralizing potency than Palizivumab,indicating that the pre-fusion conformation of the F protein is the more dominant target conformation for inducing a neutralizing antibody response.And the induced neutralizing antibody response after immunization with the genetically engineered,relatively stable pre-fusion F protein was also significantly better than that of post-fusion F protein by two-four folds.Further research also found that in the FI-RSV,the virus surface F protein was mainly in the post-fusion conformation,indicating that FI-RSV could not effectively induce highly efficient neutralizing antibodies against RSV,therefore,the reason for the failure of FI-RSV was likely to be due to the fact that weak neutralizing antibodies induced after immunization couldn't effectively inhibit the infection of RSV.At the same time,these weak neutralizing antibodies binded to antigen to produce a large number of immune complexes and accumulated in the respiratory tract and caused disease enhancement.Compared with other vaccine types,the antigen-inactivated epitope presented by the whole virus inactivated vaccine is the most comprehensive and closest to the natural state.Therefore,in this study we hope to try to find suitable inactivation conditions and vaccine preservation conditions to maximum retention of the pre-fusion F protein.The results show that the pre-fusion conformation of the F protein is extremely unstable on the surface of the natural RSV virus.Most of the pre-fusion conformation of the virus surface disappear after being left at 4? for 72 h.When inactivated with formaldehyde at a concentration of 0.2667%-0.0156%for 12 h,40%-60%of the pre-fusion F protein on the virus surface is retained,and the assay still maintain this ratio after 72 h of exposure.In the absence of formaldehyde inactivation of the virus surface,the pre-fusion F protein retain less than 20%under the same conditions.This shows that at the appropriate concentration of formaldehyde can simultaneously stabilize the pre-fusion F protein on the virus surface.We also found that the pre-fusion F protein could not be stabilized under FI-RSV treatment conditions(final formaldehyde concentration 0.01%),and the F protein on the virus surface was mainly post-fusion conformation after 72 hours of inactivation.In addition,we also found that salt solutions above 330 mM are also beneficial to the stability of pre-fusion F protein on the virus surface,but do not fixe the pre-fusion F protein,and the pre-fusion F protein will gradually decrease after removing the high salt solution.This shows that salt solutions above 330mM can be used as effective preservation conditions.Therefore,the inactivated virus finally obtained in this study combined with the preferred inactivation conditions and storage conditions can finally maintain 80%-90%of the surface pre-fusion F protein.Further mouse immunoassay results showed that from the post-immune serum neutralizing antibody levels,the neutralizing antibody levels of the preferred conditions at the same immunization dose were approximately four to five folds higher in the FI-RSV group and smaller at all immunization doses.Neutralization of mouse serum neutralizing antibodies can achieve effective serum neutralizing antibody levels that protect the infection.The subsequent challenge results also show that the positive group and the FI-RSV groups have significant weight loss on the 7th day after the challenge,which is about 20%lower,while the the preferred condition groupd have a slight weight loss on the second day after challenge of the low-dose immunization groups,the weight of the other groups are maintain at more than 95%.At the same time,the virus titer test results after the challenge showe that there are no obvious virus detections except for the control groups.Subsequently,we also perform pathological assessment of lung tissue in mice.From the pathological section of the lung,it is show that the inflammation of the preferred vaccine groups is weaker than that of the control group and the FI-RSV groups,indicating that the preferred vaccine has a good protective effect on mice.In summary,we finally hope that the optimized conditions and evaluation results obtained from the exploration can provide a solid theoretical and practical basis for the development of RSV vaccines. |
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