Expression And Immunity Of Human Respiratory Syncytial Virus F Protein

Human respiratory syncytial virus(hRSV)is the primary cause of severe respiratory tract disease in infants and the elderly.The annual global economic burden of hRSV infection is estimated at more than $80 billion.HRSV vaccines have always faced significant challenges of inadequate safety or immunogenicity.Since the discovery of hRSV in the 1950s,domestic and overseas scientists have conducted a large number of experiments on hRSV vaccine.Seven hRSV vaccines that have entered clinical trials have been declared failure,including inactivated vaccines,subunit vaccines and vector vaccines.There is still no approved vaccine,so there is an urgent need to develop a safe and effective hRSV vaccine to prevent viral infection.HRSV fusion protein(F)is highly conserved,and the induced antibodies can simultaneously inhibit the infection of hRSV A/B subtype.In 2013,researchers made significant progress in the protein structure and epitopes of F protein.They resolved the conformation of F protein and found the Pre-F specific neutralizing antibody epitope 0.The epitope(?)elicited a higher titer of neutralizing antibodies in mice than other epitopes.An important aim of current vaccine research is to induce neutralizing antibody targeting pre-F.Pre-F protein has been expressed in insect cells and mammalian cell expression systems,but there has been no reported expression in yeast.In this study,RGF protein was expressed in Pichia pastoris GS115 and RBF protein was expressed in Escherichia coli BL21.The immunogenicity of RGF protein and RBF protein was compared in mice through intramuscular injection,and a murine monoclonal antibody was prepared.Meanwhile,the protective effect of fusion protein CTA1-DD-RBF was explored in mice after nasal immunization.The results are as follows:Escherichia coli has clear genetic background,fast reproduction and low production cost.Using Escherichia coli BL21,we expressed the prefusion F protein(pre-F)and postfusion F protein(Post-F),called RBF and Post-RBF,respectively.The RBF protein contains sequences encoding F residues 26-105 and 146-513(replacing two furin cleavage sites and P27 with a variable linker,GSGSG)with the C-terminal T4 fibritin trimerization motif(Foldon).To increase stability of the trimeric prefusion conformation and retain prefusion-specific neutralizing epitopes(?),a S190F-V207L pair mutation and two disulfide bonds(S155C and S290C,A149C and Y458C)were incorporated into the sequence.The plasmid pET32a can achieve the fusion expression of thioredoxin(Trx)and RBF protein,and the RBF protein exists in the form of trimer after the removal of Trx and His tags by thrombin cleavage.The results showed that the antibody subclasses were IgG2b and IgM and the light chain is ?.F protein has a complex structure and a large number of glycosylation sites.So far,there has been no report on the expression of F protein in yeast.To our knowledge,this is the first report demonstrating that hRSV F(RGF)can be successfully expressed in Pichia.pastoris GS115 by using codon optimization.Under laboratory conditions,the RGF yield in Pichia.pastor is was only approximately 1.5 mg/L.The RGF and RBF proteins were highly homogenous trimer structure under negative stain-electron microscopy.The KD(M)for RBF and the 5C4 antibody was 1.26×10-9,which is lower than that for Post-RBF and the 5C4 antibody(1.27×10-7)but higher than that for RGF and 5C4(1.86×10-10).Intramuscular injection of BALB/c mice with aluminum adjuvant and F protein(RGF or RBF),which could induce mice to produce high-titer neutralizing antibody and significantly reduce pulmonary virus titer and pathological damage in the lungs(P<0.001).In addition,the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice.Compared to mice immunized with formalin-inactivated RSV(FI-RSV),mice immunized with RGF or RBF exhibited superior protective immunity and induced a balanced Thl-biased immune response.hRSV infects via the respiratory mucosal surfaces,so a good mucosal adjuvant helps produce a better protective effect.Mucosal adjuvant CTA1-DD comprise the CTA1 subunit of Cholera toxin(CT)and two immunoglobulin(Ig)binding domains(DD)of staphylococcal protein A with low toxicity.CTA1-DD induces cellular and humoral immune responses.In this study,we have constructed and purified the recombinant protein CTA1-DD-RBF composed of CTA1-DD mucosal adjuvant and the prefusion F protein(RBF)using Escherichia coli BL21.The nontoxic mucosal adjuvant CTA1-DD is effective against many pathogens.Intranasal immunization with CTA1-DD-RBF could stimulate hRSV F-specific IgG1,IgG2a5 sIgA,neutralizing antibodies and T cells immunity.Compared to formalin-inactivated hRSV(FI-RSV),intranasal immunization with CTA1-DD-RBF induced Th1 immunity response with little lung immunopathology.Moreover,the protective immunity of CTA1-DD-RBF was superior to RBF protein,which was confirmed by serum-neutralizing activity and viral clearance after challenge.Based on the above results,this study compared the immunogenicity of RGF and RBF proteins for the first time and clarified the advantages and disadvantages of the two proteins,laying a foundation for the development of hRSV vaccine based on pre-F protein.The protective effect of CTA1-DD-RBF protein after nasal immunization was explored,and it was confirmed that CTA1-DD-RBF protein is a promising mucosal vaccine,providing a new direction and idea for the research and development of hRSV vaccine.