A Study On Formaldehyde Inactivated Vaccine Of Respiratory Syncytial Virus With Presentation Of Pre-fusion F Protein

Respiratory syncytial virus(RSV)is the most common cause of bronchiolitis and pneumonia in infants and young children.So far,there is no a safe and effective licensed RSV vaccine.The F protein in the FI-RSV vaccine has been shown to mainly present a post-F conformation rather than a pre-F conformation,which may be one of the reasons why vaccinees cannot produce high-level antibodies.Therefore,the purpose of this research is to stabilize the pre-F of virus particles by exploring suitable inactivation and fixation methods,so that the vaccine can stimulate the body to produce high neutralizing antibodies,and with suitable adjuvants to help stimulate a balanced Th cell response and promote antibody affinity maturation.In the end,it is hoped that a new generation of inactivated vaccines can be developed.This thesis first explored the effects of a series of inactivated fixatives on pre-F based on infected cells,and found that the fixatives such as formaldehyde and paraformaldehyde can stabilize the pre-F on the surface of the infected cells with suitable treatment parameters.After preliminary verification on the mouse model,this study found that the immunogen with stabilized pre-F could stimulate higher serum neutralization and could provide more powerful protection for mice during challenge,and cause no serious pathological damage to the lungs.Furthermore,this paper established a dot immunoblotting method to detect pre-F on viral particles,and successfully transferred the optimal fixative treatment parameters obtained from the infection cell platform to the virus,and found that these optimal fixative parameters could also stabilize pre-F on virions.This paper also found that high salinity conducive to maintaining pre-F can be used as storage conditions for vaccines.Subsequently,this thesis used the F protein antibody library obtained by screening to establish a double antibody sandwich ELISA that can be used for the quantification of viral particles pre-F.Through testing,it was found that the content and proportion of virus pre-F obtained from the production of existing parameters was very low,which means a still huge room for improvement in the proportion of pre-F virus particles.In this thesis,through detailed exploration of parameters including virus strains,culture environment,multiplicity of infection and culture time,production methods,etc.,we found the best combination of production parameters that can particularly increase the proportion of pre-F on virus particles.The proportion of F increased to more than 5 times of the original,and the vaccine pre-F-high with a high proportion of pre-F was prepared.This paper subsequently compared the immunogenicity of pre-F-high and pre-F-low vaccines on cotton rats and found that compared to pre-F-low vaccines,preF-high vaccines could stimulate significantly higher levels of serum neutralization,and elicit a higher and longer-lasting antibody response than DS-CAV1(recombinantly expressed pre-F stable modification).After this study,the advantages of pre-F-high vaccine and pre-F-low vaccine in a series of dose gradient experiments were compared under the combination of aluminum adjuvant,and it was found that pre-F-high vaccine can stimulate higher levels of Serum neutralization and higher pre-F specific antibodies.The serum antibodies elicited by pre-F-high vaccine have a higher affinity with pre-F;when the vaccinated cotton rats are challenged with RSV,the pre-F-high vaccine can more completely inhibit the replication of pulmonary viruses,even in an extremely low dose.The pre-F-high vaccine at the same dose can better avoid severe lung pathology.In general,the pre-F-high vaccine provided significantly safer and more effective protection against RSV challenge for the vaccinated cotton rats,but a certain dose of pre-F-high combined with aluminum adjuvant still cannot completely avoid the occurrence of lung pathology.Therefore,this paper also compared the safety to mice inoculated with pre-F-high adjuvanted with different adjuvants when facing RSV challenge,and found that compared with the Alhydrogel(aluminum adjuvant)group,the CpG/MPLA adjuvant group stimulated a higher level of pre-F binding antibodies and the affinity of which were greatly improved.This adjuvant group stimulated a higher level of serum neutralization and stimulated a Th1-biased T cell response.Pulmonaey pathology of mice in the CpG/MPLA group and MPLA group were the lightest when facing RSV infection.Finally,this paper also discussed the effect of pre-F-specific high neutralizing antibody on the transmission of RSV virus among cells.By simulating the transmission of cell-free virions and pseudopodia,this study found that a high concentration of 5C4 antibody can completely neutralize the spread of cell-free virions.Through ultraresolution technology,it was found that the high neutralizing antibody could block the virions transmitted by pseudopodia on the cell membrane and prevent them from establishing a successful infection.This study also analyzed the virus spread among cells in the presence of high concentrations of high neutralizing antibodies,and found that high concentrations of 5C4 antibody also hard to completely inhibit the infection from infected cells to uninfected cells.It implyed an unknown way of spreading between cells of the RSV virus that was less sensitive to neutralizing antibodies.Then this study found that tunneling nanotube(TNT)-like plasmodesmata exist among the infected cells and neighboring cells,and RSV N protein was in the tunnels.In addition,movability of infected cells increased.The study inferred that it may be a new mode of spreading virus from cell to cell by transmitting viral material through tunnelling nanotube-like plasmodesmata.After,the time sequence of the occurrence of these three transmission modes of RSV was analyzed,and it was found that TNT-like plasmodesmata transmission could begin in the early stage of infection with no need for the formation of progeny virions,while pseudopodia transmitting virions and cellfree virions need to wait until the progeny virions were formed about 11 hours post the infection.This study finally analyzed the composition of TNT-like plasmodesmata and found that its composition included a large amount of F-actin and a small amount of βtubulin;we also analyzed the composition of viral materials that may be transmitted through plasmodesmata,and found that N Protein,P protein,L protein,M2-1 protein,mRNA,and genome RNA were all located in the tunnels,which suggested that the virus replication factory(inclusion body)might be transmitted through the plasmodesmata.This suggests that once the virus build infection in the primary cells,it could quickly initia intercellular diffusion through TNTs,which may happen earlier than the assembly of the virus particles.However,once TNTs are formed,the inhibitory effect of neutralizing antibody on viral diffusion will be significantly inhibited.In summary,this paper has obtained the optimal fixative treatment parameters that could be used to stabilize the pre-F protein on the virions,and obtained the production method of the virus with a high pre-F proportion.Based on these,an attempt was made to develop a new-generation inactivated RSV vaccine with a high pre-F proportion,and combining with several adjuvants,the study completed the preliminary evaluation of the protection and safety of the new vaccine in cotton rat and mouse models.Compared to the low pre-F proportion vaccine,the high pre-F proportion vaccine showed excellent protection and safety,and avoided the occurrence of serious lung pathology while suppressing viral infection.This paper also discovered a new RSV transmission mode that was not sensitive to high neutralizing antibody,which may be one of the reasons why the low-dose vaccine cannot completely suppress the pathology despite the production of high efficiency neutralizing antibody.