| BackgroundThe coronavirus disease 2019(COVID-19)caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spread rapidly around the world and caused a pandemic,which poses a significant threat to public health.Herd immunity by mass vaccination is critical to control outbreaks of COVID-19 successfully for long term,and the longevity of immunity after natural infection or vaccination is vital for vaccine design and development and for evaluation of future disease epidemics.However,the persistence of immunity following natural infection with SARS-CoV-2 remains unclear.The humoral and cellular immune response induced by virus infection is closely related to virus clearance,disease severity,vaccine response,and protection against future infection.In particular,the persistence of virus-specific neutralizing antibodies can guide the design and evaluation of vaccines.In addition,the adaptive immune response is closely related to the protection of viral re-infection.However,studies on the dynamic and persistence of SARS-CoV-2-specific antibodies,T cell,and memory cellular immune responses after natural infection are limited.Therefore,it is urgent to study the humoral and cellular immune responses to SARS-CoV-2 after natural infection,which can provide evidence for the development of vaccines and the formulation of disease prevention and control measures.ObjectivesThrough measurement of virus-specific antibody and memory T and B cells at different time points after natural infection of SARS-CoV-2,we aim to identify the kinetic of antibody responses after SARS-CoV-2 infection and the characteristics and persistence of virus memory T and B cell immune responses,which will provide important evidence for vaccine design and disease prevention and control.MethodsThe cross-sectional and cohort studies were employed in this study.Patients who were laboratory-confirmed and general healthy control population prior to the COVID-19 pandemic were enrolled from Shandong,Jiangsu and Zhejiang,China.All subjects were invited to give informed consent to participate in this study,blood samples were collected from the acute phase,and 3,6,9 and 14 months after SARS-CoV-2 infection.The serum and peripheral blood mononuclear cells(PBMCs)were isolated.The demographic,epidemiological and clinical information were collected from each subject.Serum immunoglobulin G(IgG)against SARS-CoV-2 spike and receptor-binding domain(RBD)and Immunoglobulin M(IgM)against SARS-CoV-2 RBD were measured by enzyme-linked immunosorbent assay(ELISA).Serum neutralizing antibodies(NAb)were measured by surrogate virus neutralization test,pseudovirus neutralization test,and live virus-based neutralization test.SARS-CoV-2-specific memory T and B cells were detected by Enzyme-linked immunospot assay(ELISpot)and flow cytometry(FC).Microsoft excel was used to establish the database.Graph Pad Prism 8.0 was used to analyze data and draw plots.Categorical variables were analyzed using theχ2 test or Fisher’s exact test.Mann-Whitney U test and Wilcoxon rank-sum test was used to compare the difference between the two independent samples and the two paired samples,respectively.Kruskal-Wallis test with false discovery rate method was used for multiple independent samples comparisons.Friedman test with false discovery rate method was used for multiple paired samples comparisons.Spearman’s rank correlation was used to determine correlation between analyzed parameters.All statistical tests were 2-sided with a significance level of 0.05.Results(1)Antibody and T cell response from the acute phase to 3 months after SARS-CoV-2 infectionWe conducted a prospective cohort of 25 SARS-CoV-2 infected patients at the early stage of the COVID-19 outbreak,and 112 serum samples were collected at different time points of 1～7 days,8～14 days,15～21 days,28 days and 3 months post-infection and 20 PBMCs samples at 3 months post-infection.The serum samples and PBMCs were tested for virus-specific antibodies and T-cell responses.The results showed that the positive rate of anti-RBD IgM antibodies was 33.3%one week after SARS-CoV-2 infection,and all patients were tested positive 2 weeks after SARS-CoV-2infection.The antibody level reached a peak 15 to 21 days after infection.The positive rate was 75%3 months after infection,and the level declined rapidly.Similar to the IgM antibody,anti-N IgG antibody were detected in 33.3%of patients one week after SARS-CoV-2 infection,peaked at 15 to 21 days after infection,all patients were detected positively from 2 weeks to 3 months after infection.16.7%patients were positive for anti-S IgG within one week,peaked at 28 days after infection with a GMT of 7014.6,anti-S IgG antibody remained detectable in 100%patients,with a GMT of 4345.1.The positive rate of anti-RBD IgG antibody was 8.3%one week after SARS-CoV-2infection,peaked at 28 days after infection with a GMT of 4036.5,and 95%of patients were tested positive for RBD IgG 3 months after infection with a GMT of 1513.6.Neutralizing antibody titers peak at 15 to 21 days after infection,with a GMT of 1280,and neutralizing antibody was detected in 85%patients 3 months after infection with a GMT of 697.9.Next,we investigated SARS-CoV-2-specific T cells on blood from 20recovered patients 3 months after SARS-CoV-2 infection.We observed spike-specific CD4~+and CD8~+T cells in 50%and 15%of patients and N-specific CD4~+and CD8~+T cells in 65%and 10%of patients 3 months after infection.We then characterized the phenotypic memory of SARS-CoV-2-specific CD4~+and CD8~+T cells,the IFN-γ~+CD4~+and CD8~+T cells were phenotypically effector memory and effector cells.(2)Immunological response to SARS-CoV-2 6 months after infectionWe conducted a cross-sectional study of 481 recovered patients 6 months after infection.We found that 37.0%,93.6 and 95.6%of patients maintained SARS-CoV-2-specific anti-RBD IgM,anti-N IgG and anti-S IgG,respectively.68.8%and 88.4%of patients maintained SARS-CoV-2-specific surrogate virus neutralizing antibody and pseudovirus neutralizing antibody,with mean inhibition rate of 42.03%and a GMT of235.5,respectively.Virus-specific T cell responses were measured in 105 randomly selected samples of PBMCs,and most recovered patients had SARS-CoV-2-specific CD4~+and CD8~+T cells at 6 months post-infection,with a more robust SARS-CoV-2-specific CD4~+T cells responses than CD8~+T cell response.Over 80%of antigen-specific CD4~+and CD8~+T cells in patients predominantly TNF-α-and IL-2-expressing.We found that SARS-CoV-2-specific memory CD4~+and CD8~+T cell responses were detected in most recovered patients,SARS-CoV-2-specific memory CD4~+cells were phenotypically effector memory T and central memory T cells,whereas late effector T and effector memory T cells for CD8~+T cells.Our study showed that virus-specific antibodies and memory T-cell immune responses remained detectable in the majority of recovered patients,and the antibody and T-cell responses remained at high levels 6months after SARS-CoV-2 infection.(3)Immunological response to SARS-CoV-2 9 months after infectionWe performed a cross-sectional study of 59 recovered patients 9 months after infection to evaluated antibody and T cell immune responses.We found that 98.3%of patients had detectable anti-N IgG antibodies approximately 1 year after infection,55.9%of patients had detectable anti-RBD IgG antibodies,with low binding anti-RBD antibody titer(GMT<500).In contrast,93.2%of patients showed a positive detection of anti-S IgG.66.1%of patients had antibody to inhibit RBD binding to ACE2,with mean inhibition rate of 46.4%and 89.8%of patients had detectable NAb,with a low GMT titer of 66.1.We found that 74.6%of patients had detectable RBD-specific IgG~+memory B cells,71.2%of patients had detectable IFN-γ-secreting T cells 9 months after SARS-CoV-2 infection.There was correlation between SARS-CoV-2-specific memory B cell response and anti-RBD IgG(r=0.350,P=0.007),anti-S IgG(r=0.330,P=0.01),and NAb-inhibition rate(r=0.456,P<0.001).Further analysis showed that the age,sex,and underlying medical conditions were not associated with antibody responses except for a higher anti-S IgG titer in female.Patients older than 60 years old had fewer memory B cells secreting RBD specific IgG than patients under 60 years old,age,sex,and underlying medical conditions were not associated with SARS-CoV-2-specific IFN-γ-secreting T cells.(4)Persistence of immunological response in recovered patients 14 months after SARS-CoV-2 infectionWe conducted a cross-sectional study of 545 recovered patients 14 months after SARS-CoV-2 infection and a cohort study of 253 recovered patients were followed between 6 and 14 months after SARS-CoV-2 infection.Cross-sectional analysis of SARS-CoV-2-specific antibodies revealed that 88.1%of patients were seropositive for anti-N IgG antibody positivity rate was,91.0%of patients were seropositive for anti-S IgG antibody 14 months after infection,and the GMT of anti-S IgG was 203.6;surrogate virus neutralizing antibodies and pseudovirus neutralizing antibodies were detectable in 61.8%and 70.6%of recovered patients,and the mean inhibition rate and GMT were 34.8%and 127.6,respectively.Longitudinal analysis of SARS-CoV-2-specific antibodies from 6 to 14 months after infection revealed a significant decrease in IgG,IgM,and neutralizing antibody levels 14 months after infection,with a 5.6-fold decrease in S-IgG antibody titers(P<0.0001)and a 4.2-fold decrease in pseudovirus neutralizing antibody(P<0.0001).There was no statistical difference between patient groups of different severity.In addition,we analyzed the serum neutralizing activity against Omicron variant in 48 paired sera of 6 months and 14 months after infection from 24 recovered patients.The results showed that 25%and 29%of recovered patients6 months after infection were positive for neutralizing antibodies against Omicron BA.1and BA.2,with a GMT of 19.4 and 19.2,GMT against the Omicron BA.1 and BA.2displayed a 25.9-fold(P<0.0001)and 26.1-fold(P<0.0001)decrease compared to GMT against the SARS-CoV-2 Wuhan-Hu-1,respectively.Only 8%and 21%of recovered patients 14 months after infection were detected positively for neutralizing antibodies against Omicron BA.1 and BA.2,with a GMT of 11.3 and 15.8,GMT against the Omicron BA.1 and BA.2 displayed a 10.9-fold(P<0.0001)and 7.8-fold(P<0.0001)decrease compared to GMT against the SARS-CoV-2 Wuhan-Hu-1,respectively.Virus-specific T cell responses were measured in 63 randomly selected samples of PBMCs,and most recovered patients had SARS-CoV-2-specific activated CD4~+and CD8~+T cells and SARS-CoV-2-S-specific IFN-γand TNF-αcytokines secreting CD4~+and CD8~+T cells at 14 months post-infection.ConclusionsMost recovered patients had detectable antibodies over one year after SARS-CoV-2 infection,but a low antibody titer was observed.Moreover,serum neutralizing activity to Omicron variants extensively decreased.Virus-specific B cell responses persists in over 70%of recovered patients 1 year after infection,Virus-specific T cell responses persists in over 50%of recovered patients 14 months after infection.These findings provide key evidence for development vaccination strategy in previously infected individuals and COVID-19 control and prevention in future. |
