| Background:Human respiratory syncytial virus(hRSV)is one of the major pathogens that cause acute lower respiratory tract illness(ALRI)in infants,elderly individuals,and immunocompromised individuals.The fusion protein(F)on the surface of the virus is a highly conserved and antigenic type Ⅰ transmembrane glycoprotein,which can exist in two conformations,pre-fusion(Pre-F)and post-fusion(Post-F).Pre-F protein contains six antigenic sites(φ,Ⅰ,Ⅱ,Ⅲ,Ⅳ,and Ⅴ),while Post-F has only four antigenic sites(Ⅰ,Ⅱ,Ⅲ,and Ⅳ).Antigenic site φ(aa62-69,aa196-209)is a conformational epitope that exists only in Pre-F protein and can induce antibodies with the highest neutralizing activity.Therefore,many hRSV vaccines currently in clinical trials are designed based on Pre-F protein,including recombinant protein vaccines,recombinant vector vaccines,particle vaccines,and attenuated live vaccines.As of March 2023,the Food and Drug Administration(FDA)in the United States has received two applications for the approval of hRSV recombinant protein vaccines;On May 3 of the same year,the US FDA approved the world’s first RSV vaccine,"Arexvy" developed by GlaxoSmithKline(GSK),for the prevention of lower respiratory diseases caused by RSV infection in the elderly population.Currently,research on hRSV peptide vaccines is relatively limited.Compared with other types of vaccines,peptide vaccines have several advantages,including simple composition,low cost,and ease of production,which enable rapid preparation to address hRSV epidemics.Each peptide is an immunogenic peptide molecule composed of tens to dozens of amino acids,containing multiple antigenic B cell and/or T cell-specific epitopes,which can target host production of humoral and cellular immunity,and effectively avoid protein regions that cause host hypersensitivity reactions or enhanced respiratory disease,thereby having good safety.Additionally,during respiratory virus infections,the nasal mucosa is one of the important sites that directly contacts the external environment.Immunity through intranasal administration can directly act on the respiratory mucosa,stimulating the production of local immune responses,promoting immune cell aggregation,and playing a barrier-protective role,thereby quickly and directly blocking pathogen invasion.Although further research on peptide vaccines is needed,they have broad application prospects in the development of hRSV vaccines.Objective and Methods:The aim of this study was to screen for immunogenic peptide epitopes of the hRSV F protein and develop corresponding peptides.Immunization of mice using different adjuvants and immunization routes(including intramuscular injection and nasal drop immunization)was performed to evaluate their immunogenicity and provide a scientific basis for the development of hRSV peptide vaccines.In this study,linear and conformational peptide epitopes of the hRSVF protein were identified by extensive literature analysis and sequence alignment using bioinformatics software such as IEDB and Alphago.Peptides were synthesized using chemical synthesis or expressed in HEK293 cells.Various assays were used,including enzyme-linked immunosorbent assay(ELISA),enzyme-linked immunospot assay(ELISpot),virus challenge protection assay,real-time quantitative PCR assay,plaque reduction neutralization assay,and lung tissue histopathology assay to evaluate the immune efficacy of peptides in mice.Results1 Screening of Linear Epitope Peptides and Evaluation of their ImmunogenicityIn this study,we designed and screened two linear epitope peptides,PA(F protein,aa216-244)and PB(F protein,aa110-136),based on the hRSV F protein.We evaluated the immunogenicity of PA,PB,a mixture of the two peptides(PM),and a concatenation of the two peptides(PL)in mice.The peptides were mixed with Al+CpG adjuvant and administered via intramuscular injection to induce immune responses.All four peptide formulations induced high-titer binding antibodies and reduced lung virus loads.After challenge with live hRSV virus,the PB-immunized mice had a shorter time to recover their body weight and lower lung virus loads compared to the PM and PL groups and the control group(P<0.05).When PM and PL were administered via both intranasal and intramuscular routes,the intranasal group showed better weight recovery and less lung pathology than the intramuscular group.In summary,our study demonstrates that intramuscular immunization with PB peptide provides better protection in mice than PM and PL,while intranasal immunization is superior to intramuscular immunization.2 Synthesis and expression of conformational epitope φ and immunogenicity evaluationIn this study,a conformational epitope peptide Pφ was designed based on the Pre-F protein antigenic site φ.The Fc fragment of an antibody was connected to Pφ and expressed in HEK293 cells to obtain the antigen Fc-φ.Pφ was immunized in mice with different adj uvants(AL+CpG,BFA03,CpG)via intramuscular injection and nasal drops.The results showed that all experimental groups could produce high binding antibodies,and compared with the control group,nasal immunization with Pφ could reduce lung pathology in mice(P<0.05).After virus challenge,the lung virus load was lowest in the nasal immunization(Pφ+CpG)group,significantly lower than other immunization groups,once again demonstrating that nasal immunization produces better protective effects than intramuscular injection.3 Evaluation of the immunogenicity of mixed peptides through intranasal immunizationCompared with intramuscular injection,intranasal immunization has several advantages,such as generating an immune response in the nasal mucosa to form a protective barrier,being non-invasive,low cost,reducing the risk of blood-borne disease transmission,and being more readily accepted by humans.However,the safety of intranasal immunization remains a challenge.Therefore,this study further verified the safety of the screened peptides PB and Pφ for intranasal immunization and explored the effect of Pre-F protein intranasal immunization.The experimental results showed that after intranasal immunization with CpG adjuvant,all experimental groups(Pφ,PB,Pφ+PB,PB+Pre-F,Pre-F)showed no significant lung pathological damage after challenge.Although the recovery of mouse body weight was better after immunization with peptide PB,the degree of reduction in lung viral load was not significant.After immunization with peptide Pφ and mixed peptide(PB+φ),there was no statistically significant difference in lung viral load between groups,but both were better than peptide PB alone.The combination of mixed peptides and protein(Pφ+PB,PB+Pre-F)immunized with CpG adjuvant produced higher binding and neutralizing antibodies than single peptides or proteins(Pφ,PB,and Pre-F).ConclusionsIn summary,this study identified two immunogenic peptides,the linear epitope peptide PB and the conformational epitope peptide Pφ,which were found to be safe and effective in combination with adjuvants when administered through both intranasal and intramuscular routes in BALB/c mice.Intranasal immunization was found to be superior to intramuscular injection because it mimics the natural viral infection process,induces respiratory mucosal protection,and establishes the first line of defense against viral infection.In combination with mucosal adjuvants,it can induce long-lasting protective effects.Therefore,this study provides basic data for further research on hRSV peptide vaccines,especially for intranasal immunization.Figure[22]Table[3]Reference[111]... |
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