Dynamic Immunological Characteristicsin COVID-19 Recovered Patientsand Inactivated Vaccine Recipients

Background and AimsThe study aimed to assess the persistence of humoral and cellular immune responses in COVID-19 recovered patients,and evaluate the safety and immunogenicity of inactivated vaccines in these individuals.Besides,the characteristics and durability of antibody response against the VOCs were studied in healthy individuals who received the third homologous booster dose of inactivated vaccine.MethodsPart Ⅰ:During one-year post symptom onset,we longitudinally collected 162 samples from76 patients and quantified IgM and IgG antibodies recognizing the nucleocapsid(N)protein or the spike protein(S)by chemiluminescent immunoassay(CLIA).The neutralization activity against wild type(WT)and B.1.351 VOC was detected by live virus neutralization assay.The proportion,phenotype,and function of immune cells in recovered patients were measured by flow cytometry.The S,membrane(M),and N peptide pools of SARS-CoV-2 were used to stimulate PBMCs in vitro,and to detect the virus-specific T cell response.Part Ⅱ:A total of 149 samples were collected from SARS-CoV-2 recovered individuals(n=51)and healthy controls(n=63)who were pre-vaccinated or had completed the inactivated vaccination.The safety of vaccination was assessed using a standardized questionnaire.Characterizing the IgG antibody against the receptor-binding domain(RBD)of spike protein(Anti-S-RBD IgG),as well as the neutralizing antibodies(NAbs)against the SARS-CoV-2 WT and VOCs by CLIA.SARS-CoV-2 RBD-specific memory B cell response was detected by ELISpot assay.Antigen-specific T cell response against SARS-CoV-2 overlapping peptides was also detected.Part Ⅲ:Healthy subjects(n=38)who completed three doses of inactivated vaccine were recruited and a total of 63 samples were collected.The samples were divided into four groups based on the time after the third dose: 1-3 months(n=17),4-6months(n=9),7-9months(n=11),and 10-12months(n=26)post-vaccination.CLIA was used to detect Anti-S-RBD IgG antibodies and NAbs against WT and VOCs.ResultsPart Ⅰ:1.There was no statistical difference in the proportion,phenotype,and function of immune cells between recovered patients and healthy individuals except for the proportion of Treg cells was still higher in recovered patients.2.Anti-N IgG(88.2%)and Anti-S IgG(90.8%)were still detectable in most patients one year after recovery,and the antibody titers were relatively stable.However,only 43% and22.6% of patients had neutralizing activity against WT and B.1.351,and the neutralization titers against B.1.351 had decreased significantly.3.The magnitude and breadth of T cell response decreased gradually over time,and about half of the patients could not detect a positive T cell response at one year of recovery.Part Ⅱ:1.The overall incidence of adverse reactions was 42.9% in SARS-CoV-2 recovered individuals.Injection-site pain and fever were the most common local and systemic adverse reactions,with an incidence of 31.4% and 11.4% respectively.There were no serious adverse reactions and the symptoms can be self-resolved.2.A single dose of inactivated vaccine significantly improved the neutralization ability against WT and VOCs(Alpha,Beta,Delta,and Omicron)in recovered patients,whereas the second dose did not increase the antibody response additionally.This robust antibody response lasts for at least three months.There was no significant difference in NAbs titers among recovered patients with different ages,gender,and disease severity.3.The number of memory B cells secreting RBD-specific IgG antibody in recovered patients was significantly higher than that in healthy subjects who received two vaccine doses.4.After vaccination,S-,M-and N-specific CD4+ T cell responses could be detected in100%,100%,and 91.7% of recovered patients respectively,and CD8+ T cell responses could be detected in 66.7% of recovered patients.Both CD4+ and CD8~+T cells increased the breadth of response to the peptide pools and the frequency of secreting cytokines.Part Ⅲ:1.Compared with two doses of inactivated vaccine,the third dose significantly increased the seropositivity and NAbs titer against WT and VOCs in healthy subjects.2.After the third dose,antibody titers waned gradually over time.Correlative analyses displayed an average of 19.80%,20.70%,18.60%,10.50%,and 17.40% decline in antibody titers per 30 days for the Anti-S-RBD IgG,WT,Alpha,Beta,and Delta variants,respectively.3.Most individuals still have relatively stable NAbs titers against WT,Alpha,Beta,and Delta variants 10-12 months after booster vaccination,but can hardly neutralize Omicron and are prone to breakthrough infection.Conclusion1.In patients infected with SARS-CoV-2,the dysregulation of proportion,phenotype,and function in immune cells had recovered one-year post symptom onset;However,the humoral and cellular immune responses significantly decreased,so vaccination is necessary to improve the protection.2.Inactivated vaccine has favorable safety in COVID-19 recovered patients.A single dose can reactivate robust humoral immune response,memory B cell response,and specific T cell response.3.The third dose of inactivated vaccine significantly increased the NAbs against VOCs in healthy individuals,and the neutralizing capacity against most VOCs except Omicron was stable and long-lasting.