Protective Characteristics Of Plasma Antibodies In SARS-CoV-2 Infected Individuals

BackgroundNew coronavirus pneumonia caused by Severe Acute Respiratory Syndrome Coronavirus type 2(SARS-CoV-2)has been spreading rapidly worldwide since late2019.The virus belongs to the β-coronavirus family and is an enveloped positivestranded RNA virus with a size of about 30 kb.It encodes a set of structural proteins,including spike protein(S protein),nucleocapsid proteina(N protein),membrane protein(M protein),and envelope protein(M protein),Envelope protein(E protein);and non-structural proteins(most of which comprise the viral replication and transcriptional complexes)and accessory proteins.Structural proteins,together with the lipid bilayer from the host,form an enveloped virus(or viral particle)that delivers viral genomic RNA into the cell.These structural proteins are critical to the assembly and pathogenicity of the virion.Among them,the S protein is the critical first step in the attachment of the virus to the host cell receptor and constitutes the spines on the surface of the viral particle.The antibody is a "Y" shaped structure consisting of two light and two heavy chains,and papain hydrolyzes two identical fragments of antigen binding(Fab)and a fragment crystallizable(Fc).The specific antibodies induced by SARS-CoV-2infection can exert antiviral effects through a variety of functions,including Fabmediated neutralization,Fc-mediated effector functions,Fc-mediated effector functions including complement dependent cytotoxicity(CDC),and Fc-mediated effector functions.cytotoxicity(CDC),antibody-dependent cell-mediated cytotoxicity(ADCC),and antibody-dependent cell-mediated phagocytosis(ADCP).phagocytosis(ADCP),etc.Currently,the protective humoral immune response is thought to be dominated by neutralizing antibodies(Nabs),whose main role is to prevent infection by blocking viral attachment and cell entry.However,recently,Fc-mediated ADCC function has been reported for protection against various infectious agents and pathogenesis.At the same time,plasma therapy in recovery period is also an important method to deal with new and sudden infectious diseases,especially severe acute respiratory syndrome(SARS)and other diseases causing severe and high mortality.The use of plasma in the treatment of severe SARS-CoV-2 cases has been reported,and the passive input of protective antibodies to clear the virus is one of the important mechanisms.However,the selection of time points for plasma collection during SARS-CoV-2 recovery has been rarely reported.In this study,we propose to establish a simple ADCC assay to detect plasma antibody ADCC activity in SARS-CoV-2 infected patients and to determine the specific antibody production in plasma samples from the same SARS-CoV-2 infected patients as well as the neutralizing activity of plasma antibodies.The correlation of ADCC activity with antibody titer and neutralizing activity was analyzed to investigate the protective characteristics of plasma antibodies in SARS-CoV-2infected patients.Also,it is a guideline for determining the timing of plasma sample collection in recovering patients for clinical treatment.Part Ⅰ.SARS-CoV-2 infection induces specific antibody production and determination of plasma antibody neutralizing activity ObjectiveTo analyze the specific antibody production induced by SARS-CoV-2 infection using a capture ELISA method,and to determine the plasma antibody neutralization activity using a flow-based pseudo-viral neutralization assay to analyze the plasma protective antibody profile of SARS-CoV-2 infected patients.Methods1.Plasma-specific antibody assayA capture ELISA method,anti-tag antibody was used to capture the C-terminal tagged recombinant SARS-CoV-2 S,S1 and RBD proteins,was used to analyze a total of 38 plasma samples from 28 infected patients with SARS-CoV-2 infection to induce the production of specific antibodies.2.Plasma antibody neutralization activity assayFlow-based pseudo-viral neutralization assay was used to analyze the ability of plasma antibodies to neutralize the virus in a total of 38 SARS-CoV-2-infected plasma samples from 28 infected patients.Results1.Plasma-specific antibody productionThe positive conversion rate of S-,S1-and RBD-specific antibody assays was97.4%(37/38)and 100%(26/26)for samples tested at more than 2 weeks time points.2.Plasma antibody neutralizing activity was measuredSamples with antibody titers >1:320 accounted for 26.3%(10/38)and samples with antibody titers <1:320 accounted for 73.7%(28/38).Among them,samples with antibody titers <1:320 were divided into two groups according to whether neutralizing activity could be detected in plasma samples;55.3%(21/38)of plasma samples could detect neutralizing activity,and 18.4%(7/38)of plasma samples did not detect neutralizing activity.Comparing the two groups of plasma samples with neutralizing activity,there was a significant difference in neutralizing activity(IC50)(p<0.01),which did not seem to be related to the time of sample collection(p>0.01).There were eight infected patients with plasma samples from two or more time points whose plasma antibody neutralizing activity decreased significantly with the time of recovery.Conclusion1.SARS-CoV-2 infection induces specific antibody production,including antibodies targeting the S1 and RBD regions.2.The trend of antibody titers with recovery time was dynamically observed,and it was found that antibody titers peaked at 3-4 weeks.3.The neutralizing activity of plasma samples with antibody titers >1:320 [ IC50:749.6(396.5 to 3772)] was higher than that of plasma samples with detectable neutralizing activity with antibody titers <1:320 [ IC50 of 81.4(11.56 to 228.4)],with a statistically significant difference,p<0.01..Part Ⅱ Establishment of plasma antibody ADCC activity assay and activity determination in SARS-CoV-2 infected patients ObjectiveIn this experiment,we first established a simple method for the ADCC activity of SARS-CoV-2 specific antibodies,and then used the successfully established method to detect the ADCC activity of plasma antibodies in 38 plasma samples from the same28 SARS-CoV-2 infected patients,and also analyzed the correlation between plasma antibody ADCC activity and antibody titer and neutralization activity in SARS-CoV-2infected patients.To further explore the characteristics of plasma protective antibodies in SARS-CoV-2 infected patients and to provide guidance on the timing of plasma collection for therapeutic use in recovery patients.Methods1.Construction and identification of target cellsthe WT SARS-CoV-2 S protein expression plasmid was transfected into HEK293 T cells,and the cells were collected after 48 h.The expression of WT SARSCoV-2 S protein was identified using Western Blot method.2.Establishment and optimization of antibody ADCC activity assayHEK293T cells successfully expressing WT SARS-CoV-2 S protein were used as target cells,and Jurkat cells stably expressing Fc γ RIIIa-V158 were used as effector cells to establish a simple method for antibody ADCC activity assay.And the effect on plasma antibody ADCC activity of SARS-CoV-2 infected patients was observed using different effector cell to target cell ratios.3.Antibody ADCC activity assayA total of 38 plasma antibody ADCC activities of SARS-CoV-2 infected patients from 28 infected cases were assayed by the above method.4.Correlation analysis: correlationswere calculated using the SPSS software nonparametric Spearman correlation test,and P<0.01 was considered a statistically significant difference.Results1.Construction and identification of target cellsThe target cells were successfully constructed and identified by Western Blot method to express WT SARS-CoV-2 S protein.2.Establishment and optimization of the method for detecting ADCC activity of antibodiesWe successfully established a method to detect the ADCC activity of antibody,and optimized the optimal experimental condition of 4:1 ratio of effector cells to target cells.3.Detection of ADCC activity in plasma samplesADCC activity could be detected in 86.8% of plasma samples,and the detection rate of ADCC in plasma samples within 2 weeks was 50%(5/10)and more than 2weeks was 100%(28/28).4.Correlation between plasma antibody ADCC activity and S-specific antibody titersPlasma antibody ADCC activity was positively correlated with S-specific antibody titers with a correlation coefficient of r=0.686(p<0.01),and plasma antibody ADCC activity was positively correlated with S1 and RBD-specific antibody titers with correlation coefficients of r=0.535 and r=0.471(p<0.01),respectively.5.Correlation between plasma antibody ADCC activity and neutralizing activityPlasma antibody ADCC activity was positively correlated with neutralizing activity with a correlation coefficient of r=0.573,(p<0.01).Conclusion:1.Successfully prepared and identified by Western Blot the target cells used in the establishment of the antibody ADCC assay.2.Successfully established and optimized the antibody ADCC assay with the optimal experimental conditions of 4:1 ratio of effector cells to target cells.3.86.8%(33/38)of plasma samples from SARS-CoV-2 infected patients showed detectable ADCC activity,and the trend with recovery time was consistent with the specific antibody titer,peaking at 3-4 weeks.It provides a reference for the timing of plasma collection from patients recovering for clinical treatment.4.Plasma antibody ADCC activity in SARS-CoV-2 infected patients can be derived from RBD,S1 or S2 specific antibodies.5.Some plasma antibodies of SARS-CoV-2 infected patients have both neutralizing activity and ADCC effect.6.Antibody ADCC activity assay can be added when evaluating plasma for rescue treatment.