The Regularity And Application Of Humoral Immune Response In COVID-19 Infections

【Objective】 The coronavirus disease 2019(COVID-19),caused by the SARS-Co V-2 in December 2019,has spread worldwide.As of March 19,2021,there has been 121,474,329 cases and 2,684,173 deaths worldwide.The COVID-19 pandemic has brought great damage to the physical and mental health of people around the world and the stable development of society.Therefore,scientific prevention and control strategies,accurate diagnostic methods,effective treatment strategy and vaccines are the keys to prevent the spread of the epidemic.This study aims to systematically explore the specific antibodies of asymptomatic infections and COVID-19 patients with different severity and clinical outcomes,in order to fully understand the humoral immune response in COVID-19 infections.Further,elucidating the level and kinetic of the specific antibody in COVID-19 infections is significant for understanding the immune response between SARS-Co V-2 and infections,and has great significance for COVID-19 diagnosis,treatment and vaccines.【Methods】 1.To establish an antibody response landscape for Ig M and Ig G,we detected 20 SARS-Co V-2 specific antibodies in 2,360 sera from 783 COVID-19 patients and 601 control sera using the SARS-Co V-2 protein microarray.Further,we analyzed the levels and dynamic changes of specific antibodies in patients with different severity and clinical outcomes(non-severe,severe-survivors and severe-nonsurvivors),to understand the role of SARS-Co V-2 specific antibodies in disease progression.2.123 COVID-19 patients with different severity and clinical outcome were randomly selected from 1056 inpatients in Tongji Hospital during the epidemic.The levels of neutralizing antibody in each serum were detected by the pseudotyped virus neutralization assay and further correlated with the onset time,severity,clinical treatment,laboratory biomarker and clinical outcome,so as to fully understand the kinetics of neutralizing antibody and their clinical significance in disease progression.3.The SARS-Co V-2 protein microarray was used to detect the levels of 20 SARS-Co V-2 specific antibodies in 2,360 sera of 1,034 COVID-19 patients on admission.Cox proportional hazards model and Kaplan-Meier survival analysis were used to explore the association between them.Further,we analyzed the levels and dynamic changes of specific antibodies in patients with different severity and clinical outcomes(non-severe,severe-survivors and severe-nonsurvivors).A computational cross-validation model was finally used to verify the prediction of mortality risk.4.We actively screened 11,766 epidemiological suspected individuals by nucleic acid testing(NAT)and serological testing.63 asymptomatic infections were enrolled in the study.At the same time,63 healthy people and 51 mild patients were included as controls.The SARS-Co V-2 protein microarray and pseudovirus neutralizing antibody assay were used to detect the levels of 20 SARS-Co V-2 protein specific antibodies and neutralizing antibodies.Further,we depicted the levels and dynamics changes of asymptomatic infections and mild patients with exposure or onset time.【Results】1.A comprehensive antibody landscape against SARS-Co V-2 proteome in COVID-19 patients has been constructed.(1)S and N proteins induced the strongest antibody response,and showed different dynamic changes in patients with different severity and clinical outcomes.S1 Ig G gradually increased after the onset in non-severe patients,and reached a stable level at about 25 days,and then maintained for 60 days.In severe-survivors,S1 Ig G rose rapidly after symptom onset,reaching a peak at about 25 days,and then began to decline slowly.In severe-nonsurvivors,S1 Ig G also rose rapidly,reaching a peak at about 25 days,but then declined rapidly.(2)In addition,non-structural proteins and accessory proteins(NSP1,NSP7,NSP8,Rd Rp,ORF3 b,and ORF9b)also induced Ig G response.However,these Ig G responses and dynamic changes of non-structural proteins and auxiliary proteins were different from S and N proteins.The Ig G responses of non-structural/accessory proteins were related to the severity of the disease and clinical outcomes.About 20 days after symptoms onset,the Ig G responses of these proteins dropped sharply in non-severe,severe-survivors and severe-nonsurvivors.2.The kinetics of neutralizing antibody in COVID-19 patients were established.(1)The levels and dynamic changes of neutralizing antibody in patients with different severity and clinical prognosis were different.During the antibody rising period,the rate of neutralizing antibody production in mild/moderate patients was significantly lower than that of patients in other groups.In the antibody stable phase,severe/ critical patients tended to have higher neutralizing antibody titers than mild/moderate patients.During the decline period,the decline rate of neutralizing antibodies in critical-deceased patients was the fastest,while the mild/normal patients was the lowest.Besides,the level of neutralizing antibodies in severe and critical-discharged patients declined slowly and fall to the same level as that of mild/normal patients,and these three groups may last for a longer time.(2)The relationship of the dynamic changes of neutralizing antibody during clinical progression with clinical biomarkers and treatment regimens were established.Mild/moderate patients did not need any kinds of oxygen therapy and recovered quickly with normal peripheral blood lymphocytes.Neither inflammatory factor storm nor inflammatory reaction were induced in these groups.Severe patients gradually recovered with the inhalation of nasal oxygen or mask oxygen with only abnormal lymphocytes during the acute phase.n Ab of these patients gradually declined but maintained at a medium or high level.Critical patients with low lymphocytes and high levels of inflammatory markers such as IL-6,CRP and D-dimer,usually suffered from severe breathing difficulties.With the assistant treatment non-invasive mechanical ventilation,these clinical biomarkers gradually restored to the normal levels and their neutralizing antibodies also remained at a medium or high level.Even if mechanical ventilation or even ECMO support treatment was used,critical-deceased patients could not prevent the progress to the death,because their lymphocytes progressively dropped to very low level,CRP maintained high levels,and the level of IL-6 progressively rose.As a result,the neutralizing antibody in the serum drops sharply,and the disease eventually worsens.Our results indicated that neutralizing antibodies might play a vital role in the disease recovery,and a stable level of neutralizing antibodies could prevent disease exacerbation or death.3.A method to predict clinical outcome based on early Ig G antibody response was established.(1)A comprehensive analysis of the relationship between SARS-Co V-2 specific Ig G antibodies and clinical outcomes.Cox proportional hazards model revealed that the Ig G antibody of non-structural proteins and accessory proteins(NSP1,NSP4,NSP7,NSP8,NSP9,NSP10,Rd Rp,NSP14,ORF3 b and ORF9b)were significantly positive correlated with the mortality risk of COVID-19,and Kaplan-Meier survival analysis and principal component analysis further confirmed this conclusion.(2)In addition,the levels of these antibodies in the serum of patients after admission were relative to the severity of the disease.The levels of antibodies in severe-nonsurvivors were higher than that of severe-survivors and non-severe patients.The dynamic changes showed that the antibody levels of severe-nonsurvivors were higher than that of severe-survivors and non-severe patients in early stage.(3)By calculating the cross-validation model,the predicted AUC of these 10 Ig G were from 0.62 to 0.71.The findings indicated that early high levels of non-structural/ accessory proteins(NSP1,NSP4,NSP7,NSP8,NSP9,NSP10,Rd Rp,NSP14,ORF3 b,and ORF9b)specific Ig G antibody levels increased the mortality risk of COVID-19 and could be used as potential indicators for COVID-19 death prediction.4.The diagnosis and antibody kinetics of COVID-19 asymptomatic infections(1)We actively screened by NAT from 11,766 epidemiologically suspected individuals.There were only 12 asymptomatic individuals with positive NAT but without any relevant clinical symptoms and radiological changes of the lung.We further conducted a serological survey with the sera collected from all participants,and another 51 asymptomatic infections were discovered with positive Ig G or Ig M but without any relevant clinical symptoms and radiological changes of the lung.Therefore,63 asymptomatic infections were included in this study.63 healthy individuals with negative results for both NAT and serological testing were selected as negative controls.51 mild patients without any preexisting conditions were also screened from 1056 patients during hospitalization in Tongji Hospital as positive controls.(2)63 asymptomatic infections were classified into four subgroups according to the results of nucleic acid testing and serological testing.Interestingly,81%(51/63)asymptomatic infections had negative NAT results,only 19% were NAT positive,NAT combined with Ig M antibody detection significantly improved the sensitivity of detection of asymptomatic infections from 19% to 55.5%.The combination of serological testing and NAT significantly raised the detection sensitivity.(3)Compared with healthy controls,asymptomatic infections and mild patients induced stronger Ig M/Ig G antibodies,especially against S1 and N proteins.Although asymptomatic infections and mild cases had similar Ig M profiles against 20 proteins of SARS-Co V-2 by clustering analysis,mild patients tended to induce stronger Ig M responses against S1 and N proteins than asymptomatic infections.Besides,mild patients tended to induce stronger Ig G responses against S1 and N proteins than asymptomatic infections.(4)S1-specific Ig M response was induced in asymptomatic infections on the 7 days after exposure,which peaked on days from 17 days to 25 days,and then disappeared after two months.Early to 1d after symptoms onset,Ig M antibody against the N protein rapidly evolved and persisted at a high level in mild patients,while S1 specific Ig M responses were increased persistently until 29 days after symptoms onset.In addition,S1 or N specific Ig M and Ig G responses in mild patients maintained for at least 65 days.(5)38.1%(24/63)asymptomatic individuals did not produce neutralizing antibody,while 61.9%(39/63)of asymptomatic infections only produced low titers of neutralizing antibody.Mild patients produced higher levels of neutralizing antibody than that of asymptomatic infections.In addition,as early as 7 days after exposure,neutralizing antibody rapidly evolved in asymptomatic individuals,peaked on days from 10 days to 25 days,then decayed rapidly within our observed period.As early as1 day post symptom onset,mild patients also produced low level of neutralizing antibody,and then,the titer rose persistently until 22 days and maintain high levels for at least 65 days.Our results indicated that asymptomatic infections also need vaccine immunity.【Conclusion】 Our research gives a profile of humoral immune response to SARS-Co V-2.It is of great significance for early diagnosis,prognosis monitoring,clinical treatment,convalescent serum treatments,and vaccine development.