Protective Effect And Mechanism Of Conservative RBD9.1 Antigen Peptide Vaccine On SARS-CoV-2 Mutant Strains&Study On The Mechanism Of Immune Tolerance Induced By RBD Vaccine Boostr

Part 1: PROTECTIVE EFFECT AND MECHANISM OF CONSERVATIVE RBD9.1 ANTIGEN PEPTIDE VACCINE ON SARS-Co V-2 MUTANT STRAINSBackground: The emergence of the highly pathogenic coronavirus severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)and its rapid international spread have created a severe global public health emergency.Vaccines are considered one of the most effective and feasible strategies for liberating socially enforced isolation,economic disruption recovery and herd immunity.With the acceleration of clinical trials,in March 2021,countries around the world have implemented the SARS-Co V-2 vaccine broad population vaccination plan.Unfortunately,in the past year,SARS-Co V-2 mutant strains have appeared in different countries successively,such as: B.1.1.7,P.1,B.1.351,B.1.617.1 and B.1.617.2.Compared with the wild-type sequence of SARS-Co V-2,the RBD protein sequences of these SARS-Co V-2 mutants contain multiple amino acid point mutations,such as K417 N,E484K,N501 Y.Studies have shown that mutation sites in these receptor binding domain(RBD)regions have antagonized most of the neutralizing antibodies currently on the market,greatly weakening the protective effect of currently available vaccines,especially mutations at sites 484 and 501.The results showed that the neutralizing ability of vaccine serum against mutants was significantly reduced compared to that of SARS-Co V-2 wild-type.It is of great significance to study vaccines based on more conserved B cell epitopes and T cell epitopes on the RBD-ACE2 interaction surface for the prevention of SARS-Co V-2 mutant infection.Contents of this study:(1)Definition of the conserved SARS-Co V-2 RBD region T/B cell surface receptor(BCR/TCR)epitope antigen(RBD9.1).(2)Evaluation of the ability of RBD9.1 vaccine to induce neutralizing antibodies in mice.(3)Identification of key amino acids of RBD9.1 immunogenicity.(4)Mechanism exploration of RBD9.1 vaccine-induced humoral immune response.(5)Evaluation of RBD9.1 vaccine-induced cellular immune response.(6)Assessment of RBD9.1-induced long-acting memory B cells and T cell immune response.(7)Assessment of ADE effect induced by RBD9.1 vaccine serum.The purpose of the above studies is to screen out the conserved antigenic peptide RBD9.1 and further evaluate the protective effect of the induced humoral and cellular immune responses on mutant strains in vivo.Methods:(1)Identification of the conserved SARS-Co V-2 RBD region BCR/TCR epitope antigen(RBD9.1): Collecting the peripheral blood of 31 patients who recovered from COVID-19,the plasma and peripheral blood mononuclear cell(PBMC)were separated.RBD9.1-binding ability were detected by peptide ELISA.The neutralization of serum were respectively detected by the RBD-ACE2 competition binding assay and pseudovirus neutralization assay.RBD9.1region conservation was performed by comparing the amino acid sequences of the mutant and wild-type SARS-Co V-2.(2)Evaluation of the ability of RBD9.1 vaccine to induce neutralizing antibodies in mice:RBD9.1 and the carrier were conjugated to immunize mice,and the serum Enzyme linked immunosorbent assay(ELISA),RBD-ACE2 competition binding experiment and pseudovirus neutralization experiment were used to detect antigen-specific antibody titers and neutralization in serum respectively.(3)Identification of key amino acids of RBD9.1 immunogenicity: Each amino acid of RBD9.1 was mutated to A and biotinylated,and the key amino acids bound by RBD9.1 and serum antibodies were detected by ELISA.(4)Exploration the mechanism of RBD9.1 vaccine-induced humoral immune response: flow cytometry,immunofluorescence,Enzyme Linked Immunospot Assay(ELISPOT),ELISA and other experiments were performed to detect the proportion and number of RBD-specific plasma cells,germinal center B cells and Tfh cells.(5)Evaluation of cellular immune response induced by RBD9.1vaccine: After RBD9.1 stimulation,the ratio of reactive CD69+CD8+ T cells and CD137+CD8+ T cells induced by splenocytes and the secretion of interleukin-2(IL-2)and Interferon-γ(IFN-γ)were detected by Flow cytometry and ELISA respectively.(6)Exploration of RBD9.1-induced long-term memory B cell and T cell immune responses: After the last immunization,the disappearance of antigen-specific antibodies were detected by ELISA in mouse serum;The serum neutralization were detected by competition ELISA;The proportion and frequency of antigen-specific memory B cells on different days after the last immunization were detected by flow cytometry,ELISPOT and ELISA respectively;Splenocytes stimulated by RBD9.1 peptide,the reactive percentage of CD69+CD8+ T cells or CD137+CD8+ T cells were detected by flow cytometry and the secretion of IL-2 and IFN-γ in the supernatant were detected by ELISA.(7)Evaluation of ADE effect induced by RBD9.1 vaccine serum: mix vaccine serum and SARS-Co V-2pseudovirus,and co-incubate with daudi cells expressing FC receptor.The copy of pseudovirus in daudi cells were detected by bioluminescence.Results:(1)After collecting sera from 31 patients who recovered from COVID-19,the RBD9.1 binding ability of serum and the neutralization ability of serum were detected respectively.The results showed that the RBD9.1 binding ability of serum and the neutralization ability of serum is positively correlated;By sequence comparison,the amino acid sequence of RBD9.1 is conserved in wild-type and mutant strains.(2)RBD9.1 and keyhole limpet hemocyanin(KLH)were coupled to immunize mice,the serum could detect RBD-specific antibody and neutralizing capable,and showed sustained neutralizing effect in wild-type and mutant strains.(3)By coating the biotinylated RBD9.1mutant peptide,the S451 and S454 sites are the key amino acids for the binding of RBD9.1 to serum antibodies.(4)The ratio and function of RBD-specific plasma cells,germinal center B cells and Tfh cells were detected by flow cytometry,immunofluorescence,ELISPOT and ELISA.The results showed that RBD9.1 could secrete antigen-specific antibodies by inducing the germinal center reaction to generate antigen-specific plasma cells.(5)Splenocyte induction after immunization was stimulated by RBD9.1,the percentage of reactive CD69+CD8+ T cells and CD137+CD8+ T cells,and the secretion of IL-2 and IFN-γ were detected by flow cytometry and ELISA respectively.(6)After the last immunization of RBD9.1,antigen-specific antibodies in the mice serum can last up to half a year in vivo.Antigen-specific memory B cells of different days after the last immunization were detected by flow cytometry,ELISPOT and ELISA.It was found that the antigen-specific memory B cells induced by RBD9.1 could persist for a long time in vivo.Spleen cells stimulated by RBD9.1 on the 94 th day after the last immunization,the percentage of reactive CD69+CD8+ T cells and CD137+CD8+ T cells,and the secretion of IL-2 and IFN-γ were detected by flow cytometry and ELISA respectively.(7)Mix with serum and SARS-Co V-2 pseudovirus,and combined with daudi cells expressing FC receptors.By chemiluminescence,it was found that RBD9.1vaccine serum did not observed ADE effect in wild-type,B.1.1.7,P.1and B.1.617 mutants.Conclusions: Through this study(1),it was found that the 20 amino acid RBD9.1 peptide contains both B cell and T cell epitopes,and induces both B cell and T cell immune responses in Balb/c mice.(2)The sequence of RBD9.1 is conserved in mutants and can induce sustained neutralization,which provides a theoretical basis for the preparation of mutant strain-specific vaccines.(3)RBD9.1 can form long-term antigen-specific immune memory and protection after immunization,which is conducive to the long-term protection of RBD9.1 antigen peptide vaccine.(4)RBD9.1 binding ability with the convalescent serum of COVID-19 is proportional to the neutralization ability of the serum,which provides reliable and practical information for SARS-Co V-2 mutant vaccine development.Part 2: STUDY ON THE MECHANISM OF IMMUNE TOLERANCE INDUCED BY RBD VACCINE BOOSTRBackground: The COVID-19 epidemic has ravaged the world,and the continuous evolution of SARS-Co V-2 mutants has brought enormous challenges to control the global epidemic.Although the COVID-19 vaccine can effectively reduce the infection rate and severe rate of SARS-Co V-2 mutant strains.Booster vaccines or new vaccinations are necessary due to viral diversification and vaccine-induced immunity decline leading to large-scale breakthrough infections with Delta or Omicron mutants after vaccination.In order to control the outbreak as early as possible,the Food and Drug Administration(FDA)has authorized the use of booster vaccinations for adults after the completion of the basic vaccination.The use of a booster shot appeared to be effective,as preliminary studies showed that three doses of the Pfizer Bio Ntech m RNA vaccine neutralized the Omicron variant with approximately 40 folds reduction in neutralizing activity compared to wild-type,while two doses showed little protection.However,the applicable conditions of the booster needle,the frequency of inoculation,possible side effects,and whether it can be used as an important means to defend against the infection of mutant strains need to be further explored.Immune tolerance is usually the antigen-specific humoral and cellular immune responses that the body produces due to repeated stimulation of self-antigens or foreign antigens that cannot or inefficiently induce the body.After long-term infection of the liver by HBV,the body will induce humoral immune tolerance to the HBV virus,which is manifested as being unable to induce the body to produce specific antibodies to HBV surface antigen.In addition,long-term continuous stimulation of hepatitis B virus will induce the up-regulation of the expression of exhausted molecules such as PD-1 and CTLA-4 on the surface of CD8+ T cells,so that CD8+ T cells cannot clear the hepatitis B virus in hepatocytes,thereby inducing cellular immune tolerance.Repeated stimulation of antigens can induce immune tolerance in the body.It is not clear,then,whether vaccination with a COVID-19 booster vaccine after the basic vaccination program will induce immune tolerance of the adaptive immune system.Therefore,an in-depth understanding of the humoral and cellular immune responses induced by SARS-Co V-2 vaccine booster injections is helpful for a more comprehensive understanding of the protective immune response mechanism induced by SARS-Co V-2 vaccine booster injections,and it is helpful for the global use of homologous booster vaccines providing an important rationale for countries defending against mutant strains.Contents of this study:(1)Evaluation of the ability of RBD vaccine booster to produce RBD-specific antibodies;(2)Evaluation of serum neutralization ability of RBD vaccine booster;(3)Evaluation of RBD vaccine booster to induce humoral immune response;(4)Evaluation of CD4+ T cell immune response induced by RBD vaccine booster;(5)Evaluation of CD8+ T immune response induced by RBD vaccine booster;(6)Evaluation of immune tolerance induced by RBD vaccine booster.The above research aims to explore the humoral and cellular immune responses induced by RBD booster vaccine in vivo and its mechanism of action.Methods:(1)Evaluation of the ability of RBD vaccine booster to generate RBD-specific antibodies: According to the conventional RBD immunization strategy,the RBD immunization group was divided into a normal immunization group and a booster immunization group.Antigen-specific Ig G,Ig G1 and Ig G2 a antibody titers and neutralization in serum were detected by serum ELISA,respectively.(2)Evaluation of serum neutralization ability of RBD vaccine booster immunization: The antigen-specific antibody titer and neutralization capability of serum were detected by RBD-ACE2 competition binding assay and pseudovirus neutralization experiment respectively.(3)Evaluation of the ability of RBD vaccine booster to induce humoral immune response: The proportion and number of plasma cells,antigen-specific memory B cells,germinal center B cells and Tfh cells were detected by flow cytometry,immunofluorescence,ELISPOT and ELISA were used to detect.(4)Evaluation of CD4+ T cell immune response induced by RBD vaccine booster: Flow cytometry was used to detect the proportion of Tn,Tcm,Tem and Te subsets of CD4+ T cells in the RBD extended immunization group,as well as the proportions of PD-1 and LAG-3.(5)Evaluation of CD8+ T cell immune response induced by RBD vaccine booster: The proportion of Tn,Tcm,Tem and Te subsets of CD8+ T cells in the RBD extended immunization group was used to detect by flow cytometry,as well as the proportions of PD-1 and LAG-3 in Te sub-population.(6)Identification of immune tolerance induced by RBD vaccine booster: The proportion of Treg in splenocytes and the secretion of IL-10 in mice serum were respectively detected by flow cytometry and ELISA.Results:(1)According to the conventional RBD immunization strategy,the RBD immunization group were divided into the conventional group and the extended immunization group.By serum ELISA,the titers of antigen-specific Ig G,Ig G1 and Ig G2 a antibodies in the serum of the extension group were significantly lower than those of the normal immune group.(2)Evaluation of serum neutralization ability of RBD vaccine booster immunization: The antigen-specific antibody titer were detected by ELISA.The results of RBD-ACE2 competition binding experiment and pseudovirus neutralization experiment showed that the neutralization capability of the extended group was significantly lower than that of the normal immune group.(3)The results of flow cytometry,immunofluorescence,ELISPOT and ELISA showed that the RBD vaccine booster can induce the reduction of germinal center response and the secretion of antigen-specific antibodies.(4)Flow cytometry results showed that for CD4+ T cells,the Tcm and Tem subsets of extended group were lower than that of the conventional group,and along with the high expression of PD-1 in the Te subset.(5)Evaluation of CD8+ T cell immune response induced by RBD vaccine booster: flow cytometry was used to detect the proportion of Tn,Tcm,Tem and Te subsets of CD8+ T cells in the RBD extended immunization group,as well as the express of PD-1 and LAG-3.The results showed that in the extension group,the Tem subgroup of CD8+ T cells decreased,accompanied by the high expression of PD-1 in the Te subgroup.(6)Identification of immune tolerance induced by booster injection of RBD vaccine: Flow cytometry and ELISA were used to detect the percentage of Treg in the spleen cells and the expression of IL-10 in immunized mice.The results showed that the proportion of Treg and the secretion of IL-10 were both increased in the extended group.Conclusions: This study found that(1)prolonged RBD vaccine booster inoculation can inhibit the formation of germinal centers in vivo and further fail to produce antigen-specific humoral immune responses,thereby failing to produce RBD-specific antibodies and inducing humoral immune tolerance in the body.(2)Extended RBD vaccine booster inoculation can inhibit the activation of antigen-specific T cells and induce the up-regulation of inhibitory receptors PD-1 and LAG-3,leading to the formation of cellular immune tolerance in the body.(3)Prolonged RBD vaccine booster inoculation promotes the proliferation of Treg and the secretion of IL-10 in vivo to generate an immunosuppressive microenvironment,thereby promoting the formation of adaptive immune tolerance.