| BackgroundSevere acute respiratory syndrome coronavirus 2(SARS-CoV-2)belongs to the genus beta coronavirus,and its infection causes a novel coronavirus pneumonia(COVID-19)that poses a serious threat to all human health.Mass basal and booster immunization with first-generation COVID-19 vaccine can contain the pandemics of this virus.However,the emerging Variants of Concern(VOCs)of COVID-19 evade the immune protection of the vaccine and increase the risk of reinfection.The neutralization effect of existing monoclonal antibodies against VOCs decreased obviously.There is an urgent need to develop next-generation vaccines and more efficient and stable monoclonal antibodies against VOCs.Objective and significanceRe-evaluate the immunogenicity characteristics of COVID-19 convalescent patients infected with original strain naturally,and provide new ideas and insights for vaccine development;And combined with Illumina Miseq high-throughput sequencing and bioinformatics technology to screen SARS-CoV-2 monoclonal antibody.Method1.Sample collection:We collected 384 blood samples of COVID-19 cases from 21 hospitals in Guangdong Province for antibody level determination and correlation study;And 12 blood samples of COVID-19 convalescent cases from Guangdong Second People’s Hospital and Foshan Fourth People’s Hospital for the construction of immune repertoire and screening of monoclonal antibodies.2.Evaluation of immunogenicity characteristics of COVID-19 convalescent patients:the cross-reaction levels between SARS-CoV-2 and six other coronaviruses were measured by ELISA.The correlation between neutralizing antibody and IgA,IgM and IgG antibodies was analyzed by neutralization test and Spearman rank correlation,and the decline of neutralizing antibody of VOCs were evaluated at the same time.In addition,the factors influencing the decline of neutralizing antibodies were analyzed by univariate analysis and multiple linear regression models.3.Construction of immunome repertoire and screening of monoclonal antibodies:Peripheral blood mononuclear cells were collected from convalescent COVID-19 patients and total RNA was extracted.The iR kit of arm-PCR technology was used to construct the immunome repertoire and sequenced by Illumina MiSeq platform.Combined with bioinformatics methods,protein docking,structure simulation and function prediction were performed to screen monoclonal antibodies targeting RBD,then expression and purification.Finally,the performance of monoclonal antibodies was evaluated by Biolayer Interferometry and neutralization test.In addition,the data of BCR-IGH were analyzed.Main results1.SARS-CoV-2 showed strong cross-reactivity with SARS-CoV(S-IgM 51.32%,S-IgG 78.25%,N-IgM 89.52%,N-IgG 100.00%),but not with MERS-CoV(S-IgM 25.59%,S-IgG 14.89%,N-IgM 28.23%,N-IgG 26.18%),with different crossreactivity with the four seasonal coronaviruses.2.There is a strong correlation between neutralizing antibody and IgG(r=0.667,p<0.001),and IgG may be used as an alternative marker for neutralizing antibody production.3.The neutralizing ability of serum of COVID-19 convalescent patients to VOCs decreased significantly,and age,fever and hormone therapy were independent risk factors for the decline of neutralizing antibody.4.The SARS-CoV-2 immunome repertoire was successfully constructed by using PBMC of COVID-19 convalescent patients,and 23 monoclonal antibodies were screened and successfully expressed.5.Individuals with mild illness may have better diversity of immunome repertoire.Conclusions1.The humoral immune antibody induced by SARS-CoV-2 original strain has weak neutralization effect on VOCs,which hinders the protective effect of the first generation vaccine.2.This study combined Illumina MiSeq high-throughput sequencing and bioinformatics methods to rapidly screen and successfully express 23 monoclonal antibodies from PBMC of COVID-19 convalescent patients,demonstrating the feasibility of this approach and providing a powerful tool for SARS-CoV-2 research. |
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