| Background:Human respiratory syncytial virus(HRSV)is a major cause of bronchiolitis and pneumonia in Children under 5 years old and in the elderly,and can lead to hospitalization and death in severe cases.Currently,there is only one approved HRSV vaccine,while multiple HRSV vaccines are still in clinical trials,including subunit,vehicle,particle and live attenuated vaccines.Therefore,it is essential to further develop safe and effective HRSV vaccines.The fusion glycoprotein(F)and attachment glycoprotein(G)of HRSV are the main target antigens in HRSV vaccine research,both of which have been shown to induce neutralizing antibody production.Due to the relative conservation of the F protein sequence and the presence of multiple antigenic epitopes,it occupies a dominant position in current clinical HRSV subunit vaccine and protective monoclonal antibody research.However,the G protein has slow research progress due to its highly variable sequence at the central conserved region,high glycosylation,and reported disease-enhancing effects(Enhanced respiratory disease,ERD)in animal models.To address the issue of glycosylation in the G protein,studies have shown that protein expressed in prokaryotic systems can avoid glycosylation,thus enhancing the immunogenicity of the G protein.At the same time,choosing appropriate adjuvants can also improve the immune effect of the G protein.In addition,using key epitope peptides of the G protein is also a feasible solution.Peptide vaccines have a simple composition and can contain different specific antigenic epitopes,which can effectively avoid protein regions that cause hypersensitivity reactions or induce ERD.This suggests that a peptide vaccine may be another vaccine strategy to address the highly glycosylated G protein and ERD.In addition,the cost of peptide vaccines is lower,and the production process is relatively simple,so they can be rapidly prepared to respond to the large-scale spread of HRSV.Objective:The aim of this study is to avoid glycosylation of the G protein through expression in a prokaryotic system,and to explore the effects of different new adjuvants on the immune effect of the G protein.In addition,by screening for effective antigenic epitope peptides in the G protein through extensive literature review and the use of bioinformatics software,the immunogenicity and safety of these peptides will be evaluated,providing scientific basis for the selection of adjuvants for HRSV subunit vaccines based on the G protein and the design of peptide vaccines.Methods:Firstly,this study expressed the membrane-proximal region of the HRSVA-Long strain G protein consisting of amino acids 67-298(REG)in Escherichia coli and co-immunized BALB/c mice with REG in combination with four different adjuvant formulations(BFA03 adjuvant,B-type Cp G OND+AL adjuvant combination,C-type Cp G OND+AL adjuvant combination,and CD4+ T cell epitope peptide+C-type Cp G OND+AL adjuvant combination)by injecting 100 μL of immunogen into the thigh muscles of each mouse.Boost immunization was performed on day 28 after the primary immunization to explore the immunological effects of the four different adjuvant combinations with REG.Secondly,this study used bioinformatics websites(such as IEDB)and literature review to screen for conserved regions in the G protein sequence and selected three epitope peptides,namely Peptide1(P1,aa173-186),Peptide2(P2,aa44-159),and Peptide3(P3,aa184-198).BFA03 adjuvant,which was identified in the previous study to enhance immunogenicity,was used to co-immunize BALB/c mice with P1,P2,and P3 epitope peptides alone,as well as an equal mixture of P1,P2,and P3 epitope peptides,to explore the immunological effects of individual and mixed epitope peptides.Finally,due to the poor immunogenicity of P1,P2,and P3 epitope peptides,this study explored the reason and selected a long epitope peptide,Peptide4(P4,aa169-207),which contains P1 and P3,for immunogenicity and protective effect studies in BALB/c mice using the adjuvant combination of CPG+AL,which has good adsorption capacity,to investigate the immunological effects of multiple epitope long peptides.The immunological evaluation of BALB/c mice in this study mainly used experimental methods such as enzyme-linked immunosorbent assay(ELISA),enzyme-linked immunospot assay(ELISpot),HRSV challenge protection assay,real-time quantitative PCR assay,plaque reduction neutralization test,and lung tissue pathological sectioning.Results:1.Immunizing BALB/c mice with REG protein combined with a new BFA03 adjuvant can induce the strongest binding antibodies,but there was no significant difference in the level of neutralizing antibodies produced by the two groups compared to the REG protein combined with B-type Cp G OND+AL adjuvant group.Only these two adjuvant combinations could reduce the lung virus load after mouse attack,and the difference was statistically significant(P<0.05).According to the analysis of the lung pathological evaluation results after mouse attack,all adjuvant combinations with REG protein in this study did not show effective protective effects in reducing HRSV attack-induced lung pathology compared to the PBS group.Therefore,the results suggest that when selecting G protein to develop HRSV vaccines and screening for highly potent neutralizing antibodies,further optimization of adjuvant selection is needed to reduce lung pathology.2.Whether immunizing mice with P1,P2,P3 peptides alone or in combination,the immunogenicity of the three epitope peptides was weak,with binding antibody titers less than1:50 and neutralizing antibody levels below 1:8.In addition,there was no statistically significant difference in the effect of combining or not combining the BFA03 adjuvant.Therefore,it is speculated that the poor immunogenicity is related to the small number of amino acids in the three epitope peptides themselves,and it may also be related to the poor effect of the BFA03 adjuvant in enhancing peptide immunogenicity.3.In mice immunized with P4 epitope peptide,the average EC50 value of binding antibodies in the 14 th day after the first immunization was 1517,while after the second immunization,the average EC50 value of binding antibodies in mouse serum was as high as 46697.2 in the 14 th day,and the geometric mean titer of neutralizing antibodies was 126.Although P4 did not show the ability to reduce lung virus load in mice four days after the attack,the lung pathological results showed that compared with the CPG+AL group used alone,the P4 epitope peptide could significantly reduce lung pathology,suggesting that the P4 epitope peptide can effectively reduce lung damage.Conclusion:This study investigated the immunogenicity of different adjuvant combinations on a prokaryotic-expressed h RSV G protein vaccine.While some novel adjuvants were able to induce high levels of neutralizing antibodies,they did not effectively reduce pulmonary pathological damage.This suggests that the choice of adjuvants is crucial for vaccine development and future efforts should focus on optimizing adjuvants to improve vaccine efficacy against h RSV G protein.In this study,we conducted immunological research on chemically synthesized B-cell epitope peptides and found that linear epitope short peptides have weak immunogenicity.The length,amino acid sequence,structure,and surface properties of the peptides are critical factors that affect their immunogenicity.We identified the long peptide P4 as having strong immunogenicity because longer peptide sequences may contain multiple epitopes,making them more likely to be recognized and activated by B-cells.This experiment provides a foundation for evaluating the immunogenicity of HRSV G protein B-cell epitope peptides,which can be used for future research on HRSV Gprotein peptide vaccines.Overall,this study provides scientific evidence for the development of HRSV G protein subunit vaccines.It is recommended that adjuvant selection should be given priority in the development of HRSV G protein vaccines,while in peptide vaccine research,the focus should be on selecting longer peptides with multiple binding epitopes.figure [13] table [4] reference [116]... |
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