| Background:Severe acute respiratory syndrome coronavirus type 2（SARS-CoV-2）is the pathogen of Corona Virus Disease 2019（COVID-19）.Since it was discovered at the end of 2019,it has spread rapidly around the world.By June 2022,it had infected more than500 million people and caused more than 6 million deaths.SARS-CoV-2 caused serious public health problems.With the rapid spread of SARS-CoV-2 variants,the epidemiological situation has become increasingly serious.COVID-19 patients showed different disease states after infection.Most COVID-19patients showed mild or moderate symptoms such as fever and cough,while a small number of patients developed acute respiratory distress syndrome,acute heart injury,multiple organ failure,and secondary infection,resulting in serious irreversible injury.Strengthening the research on the mechanism of severe illness of COVID-19 is very important to effectively reduce the occurrence of severe illness,and it is also the key to the success of epidemic prevention and control.Studies have shown that severe cytokine storms will occur in severe COVID-19 patients,indicating that the occurrence of severe illness is closely related to the immune status of patients.In-depth analysis of the immune status of COVID-19 patients in vivo,especially of severe COVID-19 patients,is important for elucidating the mechanism of serious illness in COVID-19 patients,preventing the occurrence of serious illness,and reducing the SARS-CoV-2 epidemic.Objective:In this study,moderate and severe COVID-19 patients were enrolled to compare the distribution of antibodies and cytokines,determine the difference of antibody typing in different COVID-19 patients,clarify the neutralization of antibodies to different SARS-CoV-2 variants in different COVID-19 patients,and analyze the internal mechanism of the changes of antibodies and cytokines in COVID-19 patients.The monoclonal binding antibody against SARS-CoV-2 N protein was prepared by B cell immortalization technique to provide support for the diagnosis,treatment,prevention,and control of COVID-19.Methods:（1）Sample collection.According to the Chinese Government Diagnosis and Treatment Guideline(8th edition),different clinical types of COVID-19 patients,healthy controls and vaccine vaccinators were recruited.After signing the informed consent forms,professional interviewers investigated the epidemiology information and collected peripheral blood samples.（2）Antibody titer detection and typing.Using wild type SARS-CoV-2 live virus（Beta CoV/Beijing/AMMS01/2020）and 9 pseudoviruses,the neutralization titer of plasma antibody was obtained by limited dilution method to observe cytopathic effect or detect fluorescence value.The level of binding antibody in peripheral blood of different subjects was detected by anti-N protein and anti-S protein IgG antibody ELISA quantitative detection kit.The second antibodies against IgA,IgM,IgG1～4,IgD,and IgE were used to detect the typing of anti-N protein and anti-S protein antibodies in patients with COVID-19 and vaccine recipients.（3）Detection of bacterial and fungal infection.The contents of BDG,LPS,i FABP,GP73 in COVID-19 patients and healthy people were detected by ELISA,to systematically analyze the immune characteristics of COVID-19 patients.（4）Detection of cytokine.The contents of IL-1β,IL-2,IL-4,IL-6,IL-8,IL-10,IL-18,IL-33,CCL2,CCL3,CCL4,CXCL1,CXCL2,CXCL6,CXCL9,CXCL10,IFN-γand TNF-αin COVID-19 patients and healthy people were detected by Ella-Simple ELISA system and Luminex system.（5）Correlation between HERV-K and IFN in COVID-19 patients.The transcriptional levels of HERV-K gag,env,pol genes and IFN related genes IFNB1,ISG15,IFIT1 in Vero E6 cells infected with SARS-CoV-2 were detected by q PCR method and verified in peripheral blood cells of COVID-19 patients.The transcriptional level of HERV-K gene after SARS-CoV-2 infection was confirmed by next-generation sequencing and GO analysis.（6）Antibody preparation of B cell immortalization technique.PBMCs were isolated from peripheral blood of COVID-19 patients,and B cells were immortalized by EBV infection.The specific binding antibodies to SARS-CoV-2 N protein and S protein were identified by multiple rounds of cloning and antibody binding specificity analysis.The RNA of the cell line was extracted,and reverse transcribed to amplify the light and heavy chain variable region sequence of the antibody.The antibody expression vector was constructed and transfected into 293F cells.The supernatant was collected and the antibody was purified by protein A/G column,and the specificity and affinity of the antibody were analyzed.Results:（1）A total of 82 COVID-19 patients,29 vaccinated populations,and 32 healthy controls were recruited.According to the clinical symptoms,the COVID-19 patients included 1 mild COVID-19 patient,49 moderate COVID-19 patients,30 severe COVID-19 patients,and 2 critical COVID-19 patients.There was no statistical difference in gender composition,average age,and disease history between moderate COVID-19patients and severe COVID-19 patients.（2）The results of neutralization antibody detected by wild type SARS-CoV-2showed that the titer of neutralization antibody and the concentration of plasma IgG-S and IgG-N antibodies in severe patients were significantly higher than those in moderate patients（P<0.05）.The content of IgG-N in moderate COVID-19 patients was 13.30times higher than that of IgG-S antibody,and the content of IgG-N antibody in severe COVID-19 patients was 16.72 times higher than that of IgG-S antibody（P<0.05）.The level of plasma IgG-S antibody was positively correlated with the titer of neutralizing antibody in both moderate and severe COVID-19 patients（P<0.05）.（3）The results of antibody typing showed that the positive rate of anti-N protein antibody in moderate and severe COVID-19 patients was IgA>IgM>IgG,and that of anti-S protein antibody was IgG>IgA>IgM.IgA-S,IgA-N,and IgM-N antibodies were all positive in COVID-19 patients,but IgM-S was negative in 2 severe patients.No specific IgE positive antibodies were detected in plasma of all COVID-19 patients.The positive rate of IgG3-S antibody in COVID-19 patients was higher than that of IgG3-N,and the positive rate of IgG4-N antibody was higher than that of IgG4-S antibody（P<0.05）.The positive rate of IgG4-N antibody in COVID-19 patients was 67.01%,while the IgG4-N antibodies of SARS-CoV-2 vaccinated patients were all negative.（4）Mann-Whitney test showed that the i FABP of moderate and severe COVID-19patients was lower than that of healthy subjects,while the levels of BDG,LSP,and GP73were higher than those of healthy controls（P<0.05）.So COVID-19 patients had bacterial and fungal infections without intestinal flora ectopic.（5）Mann-Whitney test showed that the levels of IL-1β,IL-2,CXCL1,and CXCL6in moderate and severe COVID-19 patients were lower than those in healthy controls,while the contents of IL-4,IL-6,CCL2,CCL4,IFN-γand TNF-αwere higher than those in healthy controls（P<0.05）.The levels of IL-1β,IL-6,and CXCL9 in severe COVID-19 patients were significantly higher than those in moderate COVID-19 patients（P<0.05）.（6）The plasma neutralizing antibody titers of COVID-19 patients were detected by SARS-CoV-2 live virus and Wuhan pseudovirus,respectively,and the results showed that there was a high correlation between them（r=0.9716,P<0.05）.The plasma neutralization ability of Wuhan SARS-CoV-2 infected patients to Beta,Gamma,Kappa,Delta,Lambda,and Omicron pseudoviruses was lower than that of Wuhan strains,which respectively decreased by 5.30,3.38,3.11,2.31,4.02,32.32 times（P<0.05）.（7）By Mann-Whitney test,the expression of HERV-K gag,env,pol genes and IFN related genes IFNB1,ISG15,IFIT1 increased in COVID-19 patients and Vero E6 cells infected with SARS-CoV-2（P<0.05）.GO analysis showed that HERV-K was related to the secretion of IFN.（8）12 immortalized cell lines from B cells of COVID-19 patients were obtained.The light and heavy chain variable region sequence of 2 antibodies against SARS-CoV-2N protein was obtained by amplification and sequencing.Anti-SARS-CoV-2 N protein antibody was efficiently expressed and purified in vitro.The kinetic constants of the 2pairs of antibody KD respectively were 1.42×10-8 M and 1.31×10-8 M.Conclusion:（1）The levels of antibodies and cytokines in plasma of COVID-19 patients changed significantly after infection,and the imbalance of immune factors was more common in severe patients,indicating that the imbalance of immune response may be the internal mechanism of severe patients.In-depth analysis of the immune characteristics of COVID-19 patients is important for preventing severe diseases and reducing SARS-CoV-2epidemic.（2）There was a significant difference in antibody typing between vaccinated patients and COVID-19 patients,suggesting that there is a difference in the mechanism of immune induction between vaccine immunization and innate infection.SARS-CoV-2 specific antibody typing may be used to identify newly infected and vaccinated people.（3）Plasma from COVID-19 patients infected with the Wuhan strain showed reduced neutralization of 8 globally prevalent SARS-CoV-2 variants,and the newly emerged variants had stronger immune escape ability,suggesting that anti-SARS-CoV-2 vaccine design and development should further increase targeting.（4）The high transcriptional level of the HERV-K gene in COVID-19 patients may be involved in the activation of IFN by the c GAS-STING pathway,which provides a starting point for further study of the mechanism of innate immunity in the process of SARS-CoV-2 virus infection.（5）The specific humanized monoclonal antibody against SARS-CoV-2 N protein was successfully prepared,indicating that the antibody can be rapidly prepared by immortalized B cells in patients.The application of this technique provides technical support for the prevention and control of possible emerging pathogens in the future. |
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