Immunodominance Of Epitopes And Protection Efficacy Of RBD Antigen Are Differentially Altered By Different Adjuvants And Immune Pathways

The Severe Acute Respiratory Syndrome Coronavirus 2（SARS-Co V-2）,also known as the novel coronavirus,is a highly infectious virus that can cause Coronavirus disease2019（COVID-19）.As of December 30th,2022,the virus has caused over 600 million infections and more than 6.6 million deaths worldwide.Currently,the main antigenic component of vaccines against COVID-19,apart from inactivated vaccines,is the Spike（S）protein on the surface of SARS-Co V-2.The S protein,through its Receptor Binding Domain（RBD）,recognizes the host cell surface receptor Angiotensin Converting Enzyme2（ACE2）and mediates the entry of the virus into host cells.Mutations in the sequence amino acid variations in the RBD structure can lead to changes in the species specificity and infectivity of the virus.RBD contains the most immunodominant neutralizing epitope of the entire SARS-Co V-2 virus,and more than 90%of neutralizing antibodies in COVID-19 patients are induced by RBD antigen.RBD can elicit an effective immune response in the body,thereby protecting the body from virus invasion.However,the immune-dominant epitopes of RBD and their mechanisms of action under different adjuvants and immunization routes are not yet clear.In order to improve the protective effect of COVID-19 vaccines with RBD as the main antigen,this paper studies the immunodominant response spectrum of RBD under different adjuvants and immunization routes,providing a theoretical basis and practical basis for the optimization and design of COVID-19 vaccines.The purpose of the study:The purpose of this paper is to explore the immunodominant B cell response induced by RBD under different adjuvants and immunization routes,and to clarify the protective immunodominant peptide under different adjuvants and immunization routes,providing a theoretical basis and practical basis for the optimization and design of COVID-19 vaccines.Research methods:（1）Expression and purification of RBD protein.The encoding sequence（319-529）of RBD region obtained from Pub Med（ID number MN908947）was cloned into the Bam H1and Xho1 sites of the pc DNA3.1 vector and further transfected into HEK293F cells for recombinant protein expression.The recombinant RBD was purified by Ni²⁺affinity chromatography,and the expression of the recombinant RBD was validated by SDS-PAGE electrophoresis and its purity was identified.Determination of the concentration of recombinant RBD protein.The concentration of recombinant RBD protein was determined by the BCA method.（2）Analysis of the immune protective effect of recombinant RBD under different adjuvants and immunization routes in animals.An animal model was established in 6-8week old female BALB/c mice immunized with recombinant RBD at 0 d,14 d,and 21 d.The RBD-specific Ig G neutralizing antibody titer and its subtype were measured by ELISA under different adjuvants and immunization routes.（3）The neutralizing antibodies produced by RBD in the serum of mice immunized through different immunization routes with different adjuvants can block the binding of ACE2 to cells,which is crucial for preventing infection.At the same time,this antibody has cross-protective effects against SARS-Co V-2 strains.（4）Design of 18-amino acid overlapping peptides covering the full length of the protective antigen RBD of SARS-Co V-2 using the stepwise overlapping peptide method,and synthesis of peptide segments.The purity of the synthesized peptides is greater than95%,and they are dissolved in dimethyl sulfoxide（DMSO）to 1 mg/m L,stored at-80℃for standby.Experimental results show that RBD_61-78and RBD_97-114demonstrate significant epitope dominance in mice immunized intranasally with RBD+Adda Vax,while _RBD145-162demonstrates significant immunological advantages in mice immunized intramuscularly with RBD+Adda Vax.RBD_49-66and RBD_145-162demonstrate significant immunological advantages in mice immunized intramuscularly with RBD+Al（OH）₃,while RBD_1-18,RBD_139-156,and RBD_145-162demonstrate significant immunological advantages in mice immunized intramuscularly with RBD+AS03.（5）The crystal structure of the RBD protein was obtained from the Protein Database（PDB）,and the positions of the six epitopes identified in this experiment were located on the surface of the protein crystal structure.This is more conducive to the antigen and specific antibody approaching to trigger an immune response.（6）The amino acid sequences of RBD in randomly selected SARS-Co V-2 wild-type strains and mutant strains including Alpha,Beta,Gamma,Delta,and Omicron were compared in the Gen Bank database.The sequences of the six immunodominant epitopes identified in this study are highly conserved,with amino acid sequence identities up to99.5%.Conclusion:This study shows that adjuvants and immunization routes can greatly influence the immunodominance of epitopes and the protective effect of RBD.The two new adjuvants,Adda Vax and AS03,and the six identified immunodominant epitopes are important for the development and optimization of COVID-19 vaccines.