Study On The Screening And Function Of Broadly Neutralizing Antibodies Against SARS-CoV-2

BackgroundThe coronavirus disease 2019(COVID-19)is an acute respiratory infectious disease caused by the infection of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).SARS-CoV-2 has spread widely in more than 220 countries and regions around the world since the late 2019.It has caused 500 million infections and over 6 million deaths around the worldwide.The rapid global spread and transmission of SARS-CoV-2 are hypothesized to provide the virus with substantial opportunities for the natural selection of favorable mutations.As the most important means of prevention and protection,the COVID-19 vaccines effectively reduces the mortality and infection rate.But the emergence of SARS-CoV-2 varients,especially after the emergence of the Omicron varient,had escaped the neutralizing antibody induced by vaccines.Neutralizing monoclonal antibodies can block the infection of SARS-CoV-2and can be used for the prevention and treatment of COVID-19,but a series of SARS-CoV-2 variants has showed resistant to the neutralizing antibodies.Therefore,development of antibodies with broad-spectrum activities is a guideline for the prevention and treatment of COVID-19.ObjectiveBroadly neutralizing antibodies against SARS-CoV-2 receptor binding domain(RBD)were screened from the COVID-19 patients,which can be the candidate therapeutic antibodies for COVID-19 and provid guidance for research on antibody function and vaccine design.MethodPBMC and serum samples were collected from the patients infected by WT virus and recovered from COVID-19,and we evaluated the serum titers of recovered patients by pseudovirus neutralization test and surrogate virus neutralization test.We used the magnetic beads to sort memory B cells from PBMC and use the single-cell culture platform established in our laboratory for single-cell culture.We detected the binding activity of B cell-secreted antibody supernatant to RBD protein of SARS-CoV-2 wild type(WT)and beta variant.Then we lysed the antigen protein-specific cells binding to SARS-CoV-2 RBD to obtain the m RNA.And we obtained antibody heavy and light chain variable region genes by reverse transcription and nested PCR technology and clone the genes into expression vectors for expression.The function of the antibody was verified and studied by the vitro experiments such as enzyme-linked immunosorbent assay,virus neutralization experiment.ResultsThe patients recovered from the COVID-19 still had neutralizing antibodies against SARS-CoV-2 after six months.But there are differences between individuals.We successfully obtained eight strains of SARS-CoV-2 RBD specific neutralizing antibodies from patients who recovered from the COVID-19.And 3 antibodies could neutralize both SARS-CoV and SARS-CoV-2,and 5 antibodies could neutralize SARS-CoV-2 WT,Beta Delta and Omicron(except D1H8).Antibody D1F6 shows excellent neutralizing activity against known SARS-CoV-2 VOC strains(Variants of concern)and is a potential antibody for the treatment of COVID-19.Compared with other IGHV1-2-encoded antibodies,the aspartic acid at position 59 of the D1F6 heavy chain is critical for resistance to the E484 mutation in the RBD region of SARS-CoV-2.ConclusionsThe patient who has recovered from COVID-19 still retain neutralizing antibodies against SARS-CoV-2,and some of the broadly neutralizing antibodies with cross-neutralizing against SARS-CoV.The antibody D1F6 screened in this study is a specially broad-spectrum neutralizing antibody encoded by IGHV1-2 gene,which can provide some references for antibody research.It exhibits excellent neutralizing activity against known SARS-CoV-2 VOC strains and is expected to become a COVID-19 therapeutic antibody.