Study On The Built-in CpG Adjuvant And The Prefusion Conformation F To Enhance The Immunogenicity Of RSV Vaccine

Human respiratory syncytial virus(RSV)is an enveloped,non-segmented,negative-sense,single-stranded RNA virus in the Pneumoviridae family.It causes respiratory disease throughout life with greatest disease burden in infants and the elderly.RSV is estimated to cause 30 million lower respiratory tract infections and at least 60,000 deaths worldwide each year in children<5 years of age.Despite RSV being discovered over 60 years ago,an effective vaccine is still unavailable.The first vaccine,a formalin-inactivated,alum adjuvanted RSV(FI-RSV),was evaluated in infants and young children in the 1960s.Unfortunately,this vaccine caused enhanced respiratory disease(ERD)resulting in a high rate of hospitalization and two deaths associated with peribronchiolar mononuclear cell infiltration with an excess of eosinophils.Two features of the FI-RSV vaccine that may have contributed to ERD were the induction of antibodies with poor neutralizing activity and the Th2 polarized memory response.The poor neutralizing activity of FI-RSV-induced antibodies may have resulted from lack of prefusion F(Pre-F)epitopes on the viral surface.A Th2 polarized immune response with enhanced pulmonary inflammation including eosinophilia has been a feature of RSV-challenged animals following FI-RSV vaccination.This experience with the FI-RSV vaccine candidate and subsequent studies suggest that a vaccine should induce antibodies with good neutralizing activity and a Thl rather than a Th2-biased memory response.The innovative results and main research contents of this study are summarized as follows:Firstly,we describe a strategy to improve RSV DNA vaccines by inserting the CpG-C motif into the plasmid DNA.Following immunization of BALB/c mice,the stronger Thl polarized and antigen-specific humoral and cellular immune responses are induced with better protection against RSV infection.By inserting 5 or 20 copies of the CpG-C motif into the plasmid encoding RSV F,designated pVAX1-F-CpG5 or pVAX1-F-CpG20,we evaluated their ability to enhance the antibody response and direct a Thl predominant immune response.Our results indicated that the build-in CpG-C motif resulted in higher titer of serum and neutralizing antibodies and a more Thl-biased response in BALB/c mice.Twenty copies of CpG-C were most effective at decreasing pulmonary pathology,virus replication and weight loss in RSV challenged mice following immunization with the DNA vaccine.Secondly,P27 is retained at F2 C-terminus in the mutant M1-F to stabilize the Pre-F state.The expression level of M1-F protein is similar to wild type F,but it is found that more Pre-F epitopes exist in the transfected 293T cells by plasmid encoding M1-F protein rather than F protein.In this study,furin cleavage sites in F protein were mutated to analyze their influence on F protein conformation.The results indicated that the downstream site II is important for the expression of F.A sharply decreased expression of F were observed when either the single site ? or both sites were mutanted.However,the expression level of M1-F bearing site I mutant was similar to wide type F.And,on 293T cells transfected with plasmids encoding M1-F,there were more Pre-F epitopes found compared with plasmid encoding wild type F.Thirdly,we design series of mutants based on M1-F protein to keep Pre-F construction more stable than M1-F itself.The resultant mutant with thermostable Pre-F conformation and high expression has been found and has the potential to enhance the efficacy of RSV vaccine in BALB/c mice.To optimize the thermostability of M1-F mutant,an additional disulfide bond was introduced.Among the modified mutants,two mutants of M1-F-1,M1-F-5 displayed relative high expression level and were further analyzed the stability in different temperatures.Both of the mutants showed a higher stability in site(?)after incubation at 55? for 10 minutes than F and the parent Ml-F.M1-F-5 also kept the site V more stable than F,M1-F and M1-F-1 at this temperature.Taking into account the expression level,M1-F-1 had more epitope 0 left after incubation of 2 days at 37? and 4 days at 4? or-20?.Additionally,despite F could induce higher level of serum antibody,Ml-F-1 mutant exhibted better efficacy at increasing neutralizing antibody titer and decreasing pulmonary pathology and virus replication following immunization in BALB/c mice.In summary,the built-in CpG adjuvant is an effective method to induce the enhanced immunogenicity and Thl-biased immune response of RSV DNA vaccine.And,the mutant M1-F-1 with Pre-F conformation,characteristic of high expression and fine thermostability,is also obtained by mutating the furin cleavage site I and introducing disulfide bond.Following immunization of BALB/c mice by the plasmid encoding M1-F-1,the elevated neutralizing antibody response and potent protective immunity were induced.These investigations are significant for the development of RSV vaccine candidate with independent intellectual property right.