Preliminary Study On Antibody Activity And B Cell Characteristics Induced By SARS-CoV-2 β Mutant S Protein

Objective(s): In this study,the S protein trimers of SARS-CoV-2 B.1.351,B.1.618.3 and 2003 SARS-CoV(hereinafter referred to as 03 SARS)were used as immunogens to immunize mice with different immune combinations,and the antibody activity was studied by serum antibody titer and cross-reaction.Subsequently,B cells producing monoclonal antibodies against S protein were screened,and B cell characteristics were studied by analyzing the germline genes of the antibody variable region.This study provides a theoretical basis for the study of broad-spectrum antibodies and universal vaccines.Methods:(1)The S protein gene sequence was ligated to the expression vector by homologous recombination.The protein was expressed in CHO-S cells and purified by nickel ion affinity chromatography.The purified protein was subjected to SDSPAGE and enzyme-linked immunosorbent assay(ELISA)to identify its molecular size and function.(2)The identified protein was used to immunize mice according to different combinations,and Freund ’s adjuvant was selected as the immune adjuvant.According to the experimental design,divided into the following groups : the first group of mixed immunization B.1.351 and 03 SARS three times,each interval of 14 days;in the second group,the first two immunizations were 03 SARS,and the third immunizations were B.1.351 and B.1.618.3,with an interval of 14 days.The third group was immunized with B.1.351 and B.1.618.3 three times with an interval of 14 days.Serum antibody activity and cross reaction were detected by ELISA.(3)Mice with higher antibody titers were selected for hybridoma test to screen hybridoma cells that produced specific antibodies.The culture supernatant was collected for antibody purification.Ig M antibody was pre-packed with Ig M Focurose 6HP pre-packed column and purification instrument.Ig G used r Protein A Beads filler and gravity column.The molecular weight,purity and specificity of the purified antibody were identified by SDS-PAGE.Antibody binding activity and cross-reaction were detected by ELISA.(4)Total RNA was extracted from hybridoma cells and reverse transcribed into c DNA.The variable region sequences of antibody light chain and heavy chain were amplified by PCR using non-specific primers.The sequences were ligated into PMDTM19-T vector for sequencing.Then the gene sequences were aligned on the website to analyze the germline genes,CDR3 region length and mutation rate.Results:(1)The S protein trimers of B.1.351 and B.1.618.3 mutants were successfully expressed,with molecular sizes of 180 k Da(monomer)and 540 k Da(trimer).The purity and function meet the requirements.In the ACE2 binding experiment,B.1.351 showed stronger binding force than B.1.618.3.(2)After immunization,the results of the first group of mixed immunization of B.1.351 and 03 SARS showed that the serum antibodies had high binding to the three proteins,and there was no significant difference(P > 0.05).The second group immunized twice with 03 SARS,and the third immunization with B.1.351 and B.1.618.3 showed that the binding to 03 SARS was higher than the latter two,but there was no significant difference(P > 0.05).The results of the third group of mixed immunization B.1.351 and B.1.618.3 showed that the binding of these two proteins was significantly higher than that of 03 SARS,and there was a significant difference(P < 0.05).(3)Five hybridoma cells producing specific monoclonal antibodies were screened by hybridoma technique.P1C1,P3G3,P2B4 and P2B7 were Ig M type,and P3H1 was Ig G type.The affinity of Ig M antibody to various mutant proteins was low,but the crossreactivity was good.Ig G antibody had good affinity for γ-RBD,RBD(E484K),α-RBD,β-RBD and Omicron-S,and the EC50 were 4.057 ng/ml,6.738 ng/ml,29 ng/ml,9.415 ng/ml and 25.85 ng/ml,respectively.(4)By comparing the germline and mutation rate of the light chain and heavy chain variable regions of the five antibodies,there was no mutation in the heavy chain of P2B4,and the rest had a small amount of mutation;only P3H1 had mutations in the light chain,and the rest had no mutations.Conclusion(s): The affinity of B.1.351 to ACE2 was higher than that of B.1.618.3.The immunized mouse serum and the screened monoclonal antibody have certain cross activity to various mutants.Light chain mutation rate may affect antibody activity.This topic provides more strategies for the study of vaccines and antibodies.