Analysis Of BCR Characteristics Of Antigen-specific Single Memory B Cells In Patients With Long-term HIV-1 Infection

Objective:Human immunodeficiency virus type 1,HIV-1 antigen-specific B cell receptor(BCR)of single memory B cell was analyzed objective to understand the BCR characteristics and functional correlation of memory B cells,so as to provide theoretical basis for exploring the humoral immune mechanism of broad-spectrum neutralizing antibodies and antibody affinity maturation in patients with long-term non progression of HIV-1 infection,and further provide new ideas for the prevention and treatment of HIV-1 infection.Methods:The antigen-specific single memory B cells were sorted by flow cytometry The variable region genes of BCR heavy chain and light were amplified by reverse transcription,nested polymerase chain reaction(nested PCR)and touch up nested PCR,Then the PCR products were recovered and purified by agarose gel electrophoresis,they were digested,linked to expression vector,transformed into E.coli competent cells,and clones were picked up and sequenced,Paired heavy chain and light chain plasmids were expressed in 293T cells and 293F cells,respectively Recombinant protein A was used to purify the antibody secreted by 293F cells,and the purified antibodies were identified by SDS polyacrylamide gel electrophoresis(SDS-PAGE)and Coomassie brilliant blue staining,The binding and neutralizing activities of the antibodies were detected by enzyme linked immunosorbent assay(ELISA)and TZM-bl cell neutralization assay.Results:(1)407 heavy chain variable regions and 258 light chain variable regions of PT15 antigen-specific single memory B cells were obtained by gene amplification;(2)Touch-up nested PCR can effectively improve the positive rate of light chain variable region gene amplification;(3)different antigen-specific single memory B cells have different sources of BCR heavy light chain genes,AE,YU2-gp140 specific single memory B cells have rare types of BCR light chain,and the types of germline genes are more abundant;(4)different sorting antigens can get single memory B cells BCR heavy chain variable region SHM;(5)somatic hypermutation(SHM)of heavy chainand light variable region gene from different embryo lines mostly occurred in the complementary determining region,The light chain variable region gene SHM mostly occurs in CDR1、CDR2,the heavy light chain variable region gene with high level of SHM had high mutation in CDR1 region;(6)with the increase of time,the BCR gene of memory B cells in pt sample appeared the phenomenon of old germline gene disappearance and new germline gene production,and the length of SHM and CDR3 increased;(7)eight antibodies were successfμlly expressed in T cells;(8)293F cells expressed and expressed successfμlly Purified three monoclonal antibodies P1A2-2、P1B4-1、P1A9-1;(9)monoclonal antibodies P1A2-2、P1B4-1、P1A9-1 have high binding activity to different subtypes of HIV-1 pseudovirus,low binding activity to gp120,and weak neutralization activity to global panel HIV-1 pseudovirus.Conclusions:The neutralization antibody needs to mature for a long time and evolve under the stimulation of various antigens.The long-term progress of HIV-1 infection provides an appropriate immune environment for the production and maturity of broad-spectrum neutralization antibody.The BCR characteristics of single memory B cells with different sorting antigens and different time points sμggest that the memory B cells of the long-term non progressors of HIV-1 infection gradually evolve towards the trend of increasing broad-spectrum reactivity and affinity under the condition of long-term exposure to different antigens.