Humoral And Cellular Immune Response In COVID-19 Patients

COVID-19 caused by SARS-COV-2 is a typically heterogeneous disease with clinical manifestations ranging from asymptomatic infection,mild influenza-like symptoms,pneumonia,severe acute respiratory distress syndrome,and even death.Most patients present with fever,dry cough and fatigue.Mild patients are characterized by low fever and fatigue,while severe patients usually develop dyspnea and/or hypoxemia 1 week after symptom onset,while critical patients rapidly develop into acute respiratory distress syndrome,sepsis,metabolic acidosis,bleeding dysfunction and multiple organ failure.COVID-19 rapidly spred around the world at the end of 2019,and on 11 March 2020,the World Health Organization(WHO)considered the current COVID-19 outbreak to be a global pandemic.As of February 2022,there were 406 million confirmed cases of COVID-19 and nearly 5.8 million deaths,putting enormous strain on human health,healthcare systems,social stability and the global economy.The host’s immune system plays a key role in defending against SARS-CoV-2 infection.The natural immune system quickly recognizes the infection and triggers adaptive immunity.Humoral immunity eliminates viruses in the circulatory system,and cellular immunity kills viruses in cells.The two work together to defend against viral infection,and protect the body from damage.Are both humoral and cellular immunity involved in the immune response to SARS-COV-2 infection?How do their immune responses interact?Does the immune response persist after recovery,how long can it last,and is it effective in protecting the human from reinfection?Obviously,there are still many mysteries to be solved about the characteristics of immune response in COVID-19 patients and their immune function.Therefore,it is urgent to understand the immune protection mechanism of COVID-19patients,so as to lay a foundation for the prevention,control and treatment of COVID-19and the development and evaluation of candidate vaccines.In this study,we evaluated the characteristics of humoral and cellular immune responses in severe patients in acute stage,mild patients in early recovery stage,and mild and severe patients followed up after recovery.First,Ig M and Ig G antibody reactions in serum of patients were measured by ELISA and analysis of Ig G subtype was conducted.Then,neutralising antibody titers in serum of patients were detected using pseudovirus-based neutralization assay,and neutralising potency of serum was further evaluated by live SARS-CoV-2 neutralization assay.Secondly,a SARS-CoV-2 proteome peptide microarray was used to screen and compare the antibody epitopes in serum of patients.Then,flow cytometry was used to analyze and compare the PBMCs of patients with different immune cell types,macrophage types,memory cell types and virus-specific cytokine expression.Finally,ELISpot was used to evaluate the virus-specific immune responses of patients.Part Ⅰ Characteristics of humoral and cellular immune responses of severe COVID-19patients in acute stage.First,to confirm the humoral immune response of severe COVID-19 in acute stage,SARS-CoV-2 S-RBD-and NP-specific Ig M and Ig G antibody responses were detected.The results showed that S-RBD-and NP-specific Ig M antibodies and S-RBD-and NP-specific Ig G antibodies were detected in 70%(7/10)of patients,and the level of NP-specific Ig G antibody was more significant than that of S-RBD.Then,in order to determine the subtype of Ig G antibody,we analyzed two subtypes of Ig G antibody,Ig G1 and Ig G3.The results showed that the NP-and S-RBD-specific Ig G antibodies were mainly Ig G1 subtype.Subsequently,in order to evaluate the neutralising antibody response of the patients,NAT50of the serum samples was measured,and the results showed that 70%(7/10)of the patients were positive for neutralizing antibody,and NAT50 varied greatly between individuals.We then analyzed the different immune cells in PBMCs and assessed T cell function in order to determine whether severe COVID-19 in acute stage developed SARS-COV-2specific cellular immune responses.The results showed that the percentage of CD4~+T cells in patients was significantly increased,while the percentage of CD8~+T cells was significantly decreased,leading to a significant increase in CD4:CD8 ratio in severe patients.There was no significant difference in the percentage of NKT cells,but a significant decrease in the percentage of NK cells.Although the percentage of T cells(CD3~+CD56~-)in PBMCs increased,the absolute number was significantly lower than that in healthy controls.In addition,the expression of IFNγ~+CD4~+T cells was significantly decreased,while the expression of IL-17A and TNFαwas similar.The expression of IFNγand TNFαin CD3~-T cells was significantly decreased.Finally,IFNγELISpot analysis was performed on PBMCs and no SARS-COV-2 specific T cells were detected in any severe COVID-19 patients.Part Ⅱ Characteristics of humoral and cellular immune responses of mild COVID-19patients in early recovery.First,to confirm whether humoral immune response maintained in mild COVID-19 patients in early recovery,we evaluated SARS-CoV-2 S-RBD-and NP-specific Ig M and Ig G antibody responses.The results showed that S-RBD-and NP-specific Ig M and Ig G antibodies were detected in 100%(14/14)of the patients,and the Ig G antibody levels were higher than Ig M in the follow-up patients 2 weeks after discharge.Then,in order to determine the subtypes of Ig G antibody,we analyzed three subtypes of Ig G antibody,Ig G1,Ig G2 and Ig G3.The results showed that the main NP-and S-RBD-specific Ig G antibodies were Ig G1 subtypes,and no NP-or S-RBD-specific Ig G2 antibodies were detected in all patients.Subsequently,in order to evaluate the neutralising antibody response,NAT50 was measured,and the results showed that 100%(8/8)of the patients who had just been discharged were positive,and 83%(5/6)of the patients who had been followed up 2 weeks after discharge were positive,and NAT50 varied greatly among different individuals.Then,to confirm the presence of SARS-CoV-2-specific cellular immune responses in the mild COVID-19 patients in early recovery,we analyzed different immune cells in PBMCs and evaluated the function of T cells.The results showed that the percentage of different immune cells in PBMCs was similar.Then,the results of IFNγELISpot showed that S-RBD-specific T cells were the most widely distributed and the number of N-protein specific T cells was the most abundant,and the number of N-protein specific T cells was positively correlated with the level of neutralising antibodies.Part Ⅲ Dynamic characteristics of humoral and cellular immune responses in recovery COVID-19 patients.First,to evaluate the dynamic responses of humoral immunity over time in recovery COVID-19 patients,SARS-CoV-2 S-RBD-specific Ig M,Ig G,and neutralising antibody were detected.The results showed that the positive percentage of Ig M antibody decreased gradually from 100%during 5～7 weeks to 50%during 34～42 weeks after symptom onset,while the positive percentage of Ig G and neutralising antibody maintained 100%from 5～7weeks to 34～42 weeks after symptom onset,and Ig G and neutralising antibody titers both maintained high until 34～42 weeks after symptom onset.The titer of Ig G antibody did not decrease significantly over time in both mild and severe patients.In addition,the correlation between Ig G and neutralizing antibodies increased from 5～7 weeks to 11～13 weeks,and then maintained till weeks 34～42.We then compared antibody levels between mild and severe patients.The results showed that Ig G antibody titers of severe patients were significantly higher than that of mild patients from 5～7 weeks to 11～13 weeks after symptom onset,and significantly higher than that of mild patients during 14～16 weeks and 34～42weeks.In addition,in order to screen antibody epitopes in serum of recovery COVID-19patients and to compare the differences between mild and severe patients,a SARS-CoV-2proteome peptide array assay was conducted.The results showed that a total of 184 Ig G epitopes were identified,which were mainly distributed in S protein and ORF1ab.Of which,S-82,N-21,N-17 and N-40 antibody epitopes on structural proteins existed in the serum of most patients.Compared to the mild COVID-19,the expression of 35 antibody epitopes was significantly different in the severe,of which 30 antibody epitopes were up-regulated and only 5 antibody epitopes were down-regulated.Among the up-regulated antibody epitopes,peptide S-58 and N-39 were the most significantly up-regulated,and the following peptides ORF3A-22,S-56,ORF1AB-389,N-17,S-126,N-3,N-13 and ORF1AB-511,were also significantly up-regulation.Additionally,the expression level of up-regulated antibody epitopes was more significant than that of the down-regulated ones.Then,we analyzed the different immune cells and SARS-CoV-2-specific cellular immunity.Compared with healthy controls,the percentage of activation of SARS-CoV-2 S/N-specific T cells in recovered patients was significantly higher.And the activation percentage of S/N-specific CD8~+T cells in recovered patients was significantly higher than that of CD4~+T cells.Notably,SARS-CoV-2 specific T-cell responses were strongly biased toward the expression of Th1 cytokines,included the cytokines IFNγ,TNFαand IL2.Moreover,the secreted IFNγand IL2 level in severe patients was higher than that in mild patients.Additionally,the number of IFNγ-secreting S-specific T cells in recovered patients were higher than that of N-specific T cells.Overall,the SARS-CoV-2 S/N-specific T-cell responses in recovered patients were strong,and virus-specific immunity was present until 14-16 weeks after symptom onset.In conclusion,we systematicly charactered the humoral and cellular immunity in symptomatic COVID-19 patients in different progression of the disease.Most patients can develop humoral immunity in acute stage,and high-efficiency and high-affinity antibodies can maintain at least 10 months.Cellular immunity impaired in severe patients during the acute stage,but was detectable in mild patients during the recovery stage,and maintained for at least 4 months in patients with significant Th1 biased.In addition,there was significant heterogeneity in humoral and cellular immune responses,and the immune response in severe patients was significantly stronger than that in mild patients.Our research supports the protective immunity of SARS-COV-2,deepens our understanding of the pathogenesis of COVID-19,and provides new insights and ideas for the future development and optimization of SARS-COV-2 antiviral drugs and vaccines.