| ObjectiveTo preliminarily study the immune response to RBD protein in people vaccinated with inactivated COVID-19 vaccine,and further study the humoral and cellular immune responses induced by RBD protein as well as the immune mechanism of interaction and to provide a deeper insight into the potential immunobiological responses induced by SARS-CoV-2 RBD vaccine and provide a theoretical basis for the design of future vaccination strategies.Methods1.Bioinformatics analysis of SARS-CoV-2 RBD protein(1)NCBI(https://www.ncbi.nlm.nih.gov/protein/)to retrieve the SARS-CoV-2,Delta mutant strains,Omicron mutant strains,SARS-CoV-1,HoC43 Spike and RBD protein amino acid sequence.(2)Online tools EXPASY(https://www.expasy.org/)to predict protein primary structure,Protparam(http://web.expasy.org/protparam/)analysis of the physicochemical property SARS-CoV-2 RBD protein.(3)SWISS-MODLE(https://swissmodel.expasy.org/interactive)online software download SARS-CoV-2,Delta,Omicron mutant strains of RBD-ACE2 complex structure PDB file:7a97,7w98,7wk4 and Pymol software were used for tertiary structure simulation,ACE2 binding site and RBD mutation site labeling.(4)Protein blast(https://blast.ncbi.nlm.nih.gov/blast.cgi),amino acid homology,Clustalw for amino acid sequence alignment.(5)RBD protein was purified by affinity chromatography.2.Immune response to RBD protein in human vaccinated with Corona Vac COVID-19 vaccine(1)Object of studySixty-four participants(35 unvaccinated donors and 29 vaccinated donors)were enrolled in this study.Human was vaccinated with CoronaVac vaccine,and prime vaccination included two doses of vaccine,the interval was 1 month.The interval between primary and booster vaccination was 6 months.We collected samples before vaccination(unvaccinated)and 40 days after basic immunization(40d dose2),6 months after baseline immunization(180d dose2),and 60 days after booster immunization(60d dose3).This study was approved by the Ethics Committee of Ningxia Medical University(2022-Z052).(2)Detection of RBD protein-specific humoral immunity induced by COVID-19 inactivated vaccine①SARS-CoV-2 RBD protein specific antibodies and tilter in plasma were determined by ELISA.② ELISA was used to detect the cross-reaction of vaccine induced antibody with Delta mutant and SARS-CoV-1 RBD protein.③ Elispot detected the vaccine induced SARS-CoV-2 RBD-specific memory B cells after RBD protein and R848+IL-2 stimulated PBMC in vitro.(3)Monitoring RBD protein-specific cellular immunity induced by COVID-19 inactivated vaccine① Flow cytometry detected the changes of SARS-CoV-2 RBD-specific CD4+T and CD8+T cells induced by vaccine after RBD protein stimulated PBMC in vitro.②We used FACS to analyze CD4+T and CD8+T cells difference,and used ELISA to analyzed the expression of the secreted cytokines after RBD protein stimulated PBMC in vitro.(4)The correlation between vaccine-induced RBD specific CD4+T cells and humoral immunity was analyzed by matrix correlation.(5)We used CBA to detect the plasma cytokine profile induced by vaccine.3.Mechanism of action of SARS-CoV-2 RBD protein induced by mouse Tfh cells in humoral immune responses(1)Construction of animal model:We injected 100 μL volume containing 20 μg RBD protein into C57BL/6 mice,the group included control group and RBD protein immune group with 6 mice in each group.Alum was used as adjuvant.Basal immunization was conducted with recombinant RBD protein for 3 times,while the control group was immunized with PBS.The interval was 2 weeks.We detected at day 7 and 14 after immunization.Other mice rested for 4 weeks and were immunized with recombinant RBD protein twice,PBS was injected into the control group,the interval was 2 weeks.The detected time was similar prime vaccination.This study was approved by the Ethics Committee of Ningxia Medical University(2023-2597).(2)The importance of CD4+T cells in humoral immunity of recombinant RBD protein was reverse-verified in mice with CD4 gene deficiency① FACS was used to analyzed the phenotypes of lymphocytes.② We performed ELISA to analyse antibodies and antibody titers before and after adoptive transfer of CD4+T cells following vaccination with recombinant RBD protein.(3)Monitoring humoral immunity induced by recombinant RBD protein① We monitored the dynamics of RBD-specific antibodies and and the changes of antibody titers by ELISA.② Elispot detected the changes of antigen-specific memory B cells after RBD protein and R848+IL-2 were stimulated in vitro.(4)Monitoring cellular immune response induced by recombinant RBD protein① After stimulation of RBD protein in vitro,ELISPOT detected the changes of antigene-specific T cells expressing IFN-γ and IL-4.② We performed FACS to detected the dynamic kinites of Tfh cells from spleen and lymph node.③ We performed flow cytometry to detected the dynamic kinites of Tfh cell subsets from spleen and lymph node.④FACS was performed to detected the subtypes of Tfh cell in secondary lymphoid organ.⑤ We monitored the production of functional cytokines IFN-y,IL-2,IL-10,IL-21 and PD-1 through flow cytometry,and the expression of transcription factor Bcl-6 in Tfh cells was detected by qRT-PCR after RBD protein stimulated lymphocytes in vitro.(5)Importance of Tfh cells induced by SARS-CoV-2 RBD protein in vaccine-induced humoral immunity① We analyze the correlation between Tfh cells and different B cell subsets secondary lymphoid organ,and the correlation between Tfh cell subsets and antibodies was analyzed by matrix correlation.② The percentage of Tfh cells,plasma cells and plasma blasts were detected by flow cytometry with or without αIL-21R blocker,and ELISA was performed to detected the levels of antigen-specific antibodies from culture supernatant after RBD protein stimulated lymphocytes in spleen and lymph nodes in vitro.③ To investigate whether Tfh cells were added with memory B cell differentiation,Tfh cells and non-Tfh cells were co-cultured with memory B cells sorted by magnetic beads,respectively.We used FCAS to detected MBC differentiated into antibody-secreting cells.The expression of IgG and IL-21 in culture supernatant was detected by ELISA.Results1.Bioinformatics analysis of SARS-CoV-2 RBD protein(1)SARS-CoV-2 RBD protein is a hydrophilic protein,accounting for 10%a helix,28%β folding and 62%random curling.(2)Amino acid homology analysis showed that no matter Spike or RBD protein,the Delta mutant had higher homology with SARS-CoV-2(99.14%,99.06%)than Omicron mutant(96.37%,93.02%),followed by SARS-CoV-1(76.12%,74.41%)and HoC43 had the lowest homology(37.79%,25.49%).(3)Analysis of RBD and ACE2 binding sites showed that 17 RBD residues of SARS-CoV-2 RBD protein contacted 19 ACE2 residues,and the Delta variant had multiple mutations in S protein,including two replacement L452R and T478K replacements in RBD.There were 15 mutations in Omicron RBD,among which K417N,G446S,S477N,T478K,E484A,Q493R,G496S,Q498R,N501Y and Y505H were located in RBM.(4)Phylogenetic tree results showed that amino acid sequence of S protein of SARS-CoV-2 strain had little difference among different countries and regions.2.Immune response to RBD protein in people vaccinated with inactivated COVID-19 vaccine(1)Detection of RBD protein-specific humoral immunity induced by COVID-19 inactivated vaccine①Anti-RBD IgG and IgM antibodies were detected in 100%(29/29)and 83%(24/29)of subjects at day 40 of the two doses,and remarkable decrease at 6 months after the two doses.IgG and IgM antibodies were detected in 28%(8/29)and 38%(11/29)of the individuals,but antibody levels were still higher than in the unvaccinated population.After the third booster dose,the response rates of IgG and IgM increased to 100%(29/29)and 93%(27/29),respectively.At day 40 after the second dose of vaccine,83%(24/29)of subjects had detected IgA antibodies.It is nearly undetectable six months after the second dose.After booster vaccination,the IgA antibody response rate increased to 48%(14/29),and the vaccineinduced IgG antibody was mainly IgG1.②Anti-delta and SARA-CoV-1 IgG was detected in 58%(17/29)and 48%(14/29)of subjects at day 40 of two doses and only in 13%(4/29)of individuals at 6 months after two doses.No anti-SAES-CoV-1 IgG was detected.After the third dose of booster inoculation,the anti-Delta and SARA-CoV-1 IgG antibody response rates were 75%(22/29)and 37%(11/29),and there was no cross-reaction in IgM and IgA.③ Relative increases in the frequency of RBD-specific memory B cells 40 days after two doses of basal immunization were still detected 6 months after two doses,and the third dose of enhanced immunization induced a stronger memory B cell response compared to the unvaccinated cohort.(2)RBD protein-specific cellular immune response induced by COVID-19 inactivated vaccine① AIM+(CD 137+OX4O+)CD4+T cell response increased significantly on day 40 and day 60 after basic two-dose inoculation,and the reaction of AIM+CD4+T cells at 6 months after prime vaccination was remarkable lower than that at day 40.AIM+(CD69+CD137+)CD8+T cells also showed the same trend.The frequency of AIM+CD4+T cells was significantly higher than that of AIM+CD8+T cells at each study time point.AIM+CD4+T and CD8+T cells were detected in 6%and 10%of unvaccinated individuals,respectively.② On day 40 after two doses,significant Thl cell polarization and weak Th17 cell polarization were detected.The same phenomenon was observed on day 60 after booster inoculation,but no polarization of CD8+T cells was observed.ELISA results showed that the levels of IFN-γ,IL-2 and TNF-α induced by the vaccine increased significantly on day 40 of two-dose immunization and day 60 of three-dose booster immunization,and weak expression of IL-17A was detected.We did not find change of IL-4 after vaccination.We found a small number of T cells expressing TNF-α,IL-2,and IL-17A in unvaccinated people.③Correlation results showed that AIM+CD4 was positively correlated with AIM+CD8 cells,RBD specific memory B cells,IgG and IgA after inactivated vaccine.④ Cytokine profile showed that TNF-α and IL-17A expressed higher cytokines in 40d dose2 group and 60d dose3 group,which was consistent with the previous ICS results.The key cytokine to initiate the Th1 response,IL-12p70,increased significantly after booster vaccination,and there was a strong association between primary and early induction cytokines of booster vaccination,but the correlation and complexity between cytokines decreased gradually over time.3.Mechanism of action of SARS-COV-2 RBD protein induced by mouse Tfh cells in humoral immune responses(1)Humoral immune response induced by recombinant RBD protein① We did not notice that IgM and IgG increase after the vaccination.The levels of IgM continued to decrease from 2w to 7w after booster immunization but were still higher than those in the control group.The IgG level remained stable until 7 w after booster immunization and did not show a downward trend.Almost no IgA was detected in the first immunization,which was significantly increased at the first week of enhanced immunization and continuously decreased at the seventh week of enhanced immunization.② The levels of IgG1,IgG2a and IgG2b increased after prime immunization,and the antibody levels remained stable until 7 w after booster immunization.After the initial immunization with IgG2c antibody,the antibody level continued to increase at 1 and 2 w,but no significant increase was observed at 1 w of enhanced immunization,and the antibody level increased at 2 w,lasting until 7 w.IgG3 was not increased at 1 w,increased at 2 w after prime immunization,and increased significantly at 1 w after booster immunization,and decline at 7 w but was still higher than prime immunization.③ The dynamic changes of B-cell subpopulation induced by RBD protein were not consistent in lymph nodes and spleen.The frequencies of GC B and MBC in lymph nodes and PB and GC B in spleen were increased after primary vaccination,and the frequencies of PC,GC B and MBC in lymph nodes and PC,PB,GC B and MBC in spleen were significantly increased after booster vaccination.④ The frequency of RBD-specific MBC was relative increased after prime vaccination,and significantly increased after booster vaccination.(2)The importance of CD4+T cells in humoral immune response induced by SARS-CoV-2 RBD protein was verified in mice with CD4 gene deficiency.ELISA results showed that the absence of CD4+T cells resulted in a significant decrease in vaccine-induced IgG,IgM,and IgA antibody levels,while the adoptive transfer of CD4+T cells resulted in a significant increase in antibody levels.(3)Cellular immune response induced by recombinant RBD protein① The number of SARS-CoV-2 RBD specific T cells producing IFN-γ increased significantly after the primary immunization,and the reaction did not improve after booster vaccination.Expressed T cells expressing IL-4 was not detected after primary or booster immunization.② The dynamic changes of Tfh cells induced by SARS-CoV-2 RBD protein in lymph nodes and spleen were similar.The number of Tfh cells was increased after prime vaccination,and significantly increased after booster vaccination.The RBD protein mainly induced CXCR5+ICOS+Tfh cells activation.The trend in lymph nodes was similar to spleen.The CD154 expression level of Tfh cells increased at day 7 following vaccination with RBD protein,however the level was decreased at day 14.The expression level of CD 154 in CXCR5+ICOS+Tfh cells from lymph nodes was always higher than that in spleen.We did not notice significant change of Tfh subgroups in lymph nodes after prime vaccination.While CXCR3+(Tfh1 and Tfh 1-17)and CXCR3-(Tfh2 and Tfh17)Tfh subgroups in spleen were increased.The Tfh subgroups of CXCR3+(Tfh1 and Tfh 1-17)and CXCR3-(Tfh2 and Tfh17)in lymph nodes and spleen were significantly increased.The response of CXCR3+(Tfh1 and Tfh1-17)subset was stronger than CXCR3-(Tfh2 and Tfh17)subset.③ Cytokine detection results showed that Tfh cells expressed increased IFN-γ at the early stage of the initial immunization,and IFN-γ,IL-2 and IL-10 increased at the early stage of the enhanced immunization.PD-1 increased both after the initial immunization and the enhanced immunization,and the increase was more significant at the early stage of the immunization.④ The expression of IL-21 was increased at 1w after the prime immunization and after booster immunization.Bcl-6 was relatively increased after the initial immunization and significantly increased after enhanced immunization.(3)Importance of Tfh cells induced by SARS-CoV-2 RBD protein in vaccine-induced humoral immunity① We observed a positive correlation between Tfh cells,PB and GC B cells in spleen,and between Tfh cells and GC B cells in lymph node after prime vaccination.In addition,we observed a positive correlation between Tfh cells and MBC in lymph node and between Tfh cells and PC in spleen after booster vaccination.② Inhibition of IL-21/IL-21R signaling pathway significantly decreased Tfh cells frequency in lymph nodes and spleen,the levels of RBD-specific antibodies in culture supernatant were remarkable inhibited,and the number of plasma cells and plasma blasts in secondary lymphatic organs were significantly decreased.③ Tfh cells but not non-Tfh cells could promote the differentiation of MBC into antibody-secreting cells,and the levels of IgG antibody and IL-21 were significantly increased in the group of Tfh cells co-culture with memory B cells.Conclusion1.The construction of SARS-CoV-2 RBD protein were analyzed,and it was found that RBD protein was an ideal vaccine antigen choice,which laid a foundation for further research on the specific immune response of RBD protein.2.COVID-19 inactivated vaccine induced adaptive responses specific to SARS-CoV-2 RBD,including persistent memory B cells,multifunctional CD4+T cells dominated by Thl,and extensive and complex cytokine patterns,and antigen-specific CD4+T cells were correlated with humoral immunity.These observations showed that RBD protein had strong immunogenicity as a vaccine target antigen3.SARS-CoV-2 RBD protein induced a durable humoral immune response and triggered a GC response;By regulating molecules important for the initiation,follicular homing and auxiliary function of Tfh cells to mediate functional Tfh cell responses,Tfh cells regulate vaccine-induced humoral immune responses through the IL-21/IL-21R signaling pathway,and assist the differentiation of memory B cells into antibody secretory cells,emphasizing the important role of Tfh cells in the protective humoral immune immunity induced by SARS-CoV-2 recombinant RBD protein. |
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