Preparation Of Recombinant SARS-CoV-2 S Protein Multi-epitope Antigen And Study Of Its Immune Efficacy After Formulation With Different Adjuvants

To date,the severe acute respiratory syndrome-coronavirus 2(SARS-CoV-2)pandemic has been a major global public health event,infecting more than 750 million people and causing over 6.8 million deaths as of 16February 2023(https://covid19.who.int/).Currently,SARS-CoV-2 has evolved into new variants of BQ and XBB subtypes that escape most of the neutralising antibodies elicited by the vaccine and are highly immune evasive.SARS-CoV-2 enters host cells by binding its spike(S)protein to the angiotensin-converting enzyme 2(ACE2)receptor on the host cell membrane and fusing with it through transmembrane serine protease 2(TMPRSS2)-mediated cleavage and activation of the S protein.The S protein is a key molecule for SARS-CoV-2 infection and a major target for vaccine or monoclonal antibody development.However,as SARS-CoV-2continues to spread globally,its genome remains relatively stable but still undergoes frequent mutations,especially in the receptor binding domain(RBD)of the S protein.Moreover,the S protein contains important neutralising antigenic epitopes that are essential for designing multi-epitope anti-SARS-CoV-2 vaccines.In addition to the antigen,however,the adjuvant component plays a crucial role in modulating vaccine-induced immune responses.Vaccine adjuvants can enhance antigen-specific immunity by rapidly inducing innate immune responses,increasing antigen uptake and processing by antigen-presenting cells and promoting adaptive immune responses.Therefore,selecting an appropriate adjuvant to formulate with the antigen is vital for vaccine development.In this study,we screened and analyzed B cell epitopes of SARS-CoV-2 S proteins by bioinformatics methods,produced a recombinant SARS-CoV-2 S protein multi-epitope antigen vaccine by recombinant DNA technology and formulated it with different vaccine adjuvants to evaluate its immunogenicity in mice and compare the effects and mechanisms of different adjuvants.The details of this research are as follows.1.Prediction of B cell epitopes of SARS-CoV-2 S proteins by bioinformatics analysisWe first selected an appropriate reference strain of SARS-CoV-2 from NCBI website.Then we screened B cell epitope prediction results from two different algorithms and combined them with secondary and tertiary structures of SARS-CoV-2 S protein itself.We also considered highly conserved regions in S protein structure and excluded epitopes with an amino acid mutation frequency higher than 0.4‰.Finally,we identified four B cell epitopes(343–358,369–386,406–423 and 472–489)with high conservation and low mutability on the surface of S protein.2.Design,construction,expression and purification of recombinant multi-epitope protein GSE3 of SARS-CoV-2 S proteinTo optimize the design of a recombinant multi-epitope protein that can elicit specific immunity against SARS-CoV-2,we incorporated laboratory-predicted CD4~+T cell epitopes and combined T cell and B cell epitopes into a recombinant multi-epitope SARS-CoV-2 spike protein(GSE3)that conforms to our structural model.We then constructed the initial GSE1 sequence by overlap PCR,generated the recombinant expression vector p ET28a-GSE3 by two rounds of plasmid digestion and ligation,and successfully produced the recombinant multi-epitope protein GSE3 after induction expression identification.3.Production of recombinant multi-epitope protein GSE3 vaccine and study of its immunological activityWe used GSE3 and a commercialized recombinant spike protein(Sp)as vaccine antigens and formulated them with six oil-in-water emulsion adjuvants(AS08,T3,T4,T7,T8 and P111)that were developed in our laboratory to produce vaccines and immunize mice.We then measured the level of antigen-specific antibody production in serum from each group by ELISA.During this period,we identified the LPS component of the GSE3protein and investigated the sequential immunization in the group of mice that were immunized with the GSE3 vaccine.Finally,we performed cross reactivity using GSE3 immune serum and Sp protein.The results showed that all six adjuvants induced effective specific immunity in both batches of mice,but with different immune effects:AS08 and P111 groups had the best immune effects while T4 and T8 groups had the worst.Sequential immunization was superior to conventional immunization in terms of antibody production levels(p<0.05).GSE3 vaccine immune serum could bind to commercially available recombinant S proteins,demonstrating the rationality of the GSE3 design and the feasibility of developing a recombinant multi-epitope GSE3 vaccine.4.To study the relationship between vaccine-induced antibody production and Neutrophils/B cells ratio at the local immune site.To explore how vaccine-induced antibody production relates to local immune neutrophil/B cell ratio,we used peritoneal immunization instead of intramuscular immunization as a background for previous laboratory studies and detected changes in cell ratio in peritoneal lavage cells(PLCs)1 hour after peritoneal immunization by flow cytometry.The results revealed that both neutrophils and B cells increased to varying degrees in PLCs of immunocompetent mice,accounting for about 80%of total cell count;moreover,B cell number increase correlated positively with antibody production at later stage of immunization(p<0.001).This phenomenon also applied to na?ve mice.We measured cytokine expression in their PLCs by real-time PCR.The results suggest that neutrophils recruited locally by immunization induce a potent inflammatory response that inhibits B cell activation factor(BAFF)activation of B cells for antibody production;they also provide experimental data to validate our adjuvant primary screening platform.In summary,our recombinant multi-epitope SARS-CoV-2 S protein(GSE3)has good immunogenicity and immunologic specificity.it lays groundwork for further development of a broad-spectrum recombinant multi-epitope SARS-CoV-2 vaccine and also offers a novel idea for establishing an adjuvant primary screening platform.