| Background:The diversity and latent infection of HIV-1 are the main reasons for the failure of traditional antiviral therapy to completely eradicate the virus in infected people.At the same time,long-term antiviral therapy will bring heavy economic burden,toxic side effects and virus resistance.Therefore,finding new antiviral drugs,such as developing antibody drugs,has become one of the research focus.Broadly neutralizing antibodies,especially VRC01 class bNAbs directed to CD4-binding site,are capable of neutralizing a variety of HIV-1 epidemic strains.Passive administration of bNAbs can protect animal from infection,and it can also effectively control the rebound of the virus after infected person stop taking antiretroviral drugs.In vaccine design,the isolation of bNAbs is helpful for the design of immunogens that can induce bNAbs.In recent years,it has also been found that some nNAbs can mediate the ADCC activity which kills HIV-1 infected cells and effectively clears the virus reservoir.Objective:To isolate mAbs against CD4bs and HIV-1 membrane protein from PBMCs of chronically HIV-1 infected individuals in China,and identify the function of mAbs and its evolutionary mechanism,which may provide important information for reverse immunogen design and provide candidates for the development of therapeutic mAbs.Methods:ELISA was used to screen samples that might contain antibodies against CD4bs from chronically HIV-1 infected patients with different neutralizing activities.The antigen-specific B cells were screened from PBMCs by flow cytometry,and the heavy and light variable region genes of mAbs were obtained by reverse transcription and nested PCR.IMGT website was used to analyze antibody germline allele and SHM.MAbs were obtained by molecular cloning and protein expression.ELISA and BLI were used to identify the epitope and kinetic characterization of antigen-antibody binding.The neutralization ability of mAbs were detected by neutralization assay.Gp 160 genes of plasma HIV-1 viruses were amplified by SGA and CD4bs were analyzed.I-TASSER website and PyMol software were used to simulate the protein structure of antibody and antigen.Unbiased B cell repertoires were amplified by 5' RACE PCR and were deeply sequenced by Illumina Miseq PE300 method.Mafft software,IQ Tree software,Highlighter Tool,Consensus Maker Tool and R language were used to analyze the evolution mechanism of antibodies.The affinity between mAbs and target cell was detected by flow cytometry,and ADCC activity was detected by non-radioactive cytotoxic assay.Results:1.Screening plasma samples containing antibodies directed to CD4bsThree samples,CBJC261,CBJC263 and CBJC451,which may contain antibodies against CD4bs,were screened out from 220 samples of HIV-1 chronically infected patients whose plasma with different neutralizing activities.The plasma of CBJC263 with moderate broadly neutralizing activity,and CBJC261 and CBJC451 with broadly neutralizing activity.2.Isolation and Identification of a VRC01 class antibody and study on the evolution mechanism of its light chainNine B cells were selected from PBMCs of CBJC263,and four mAbs were expressed,among them 263A9 was a HIV-1 specific antibody.The heavy and light chain of 263A9 originated from the IGHV1-02*04 and IGKV1-33*01 germlines,with SHM of 18.75%and 14.70%respectively.The CDR3 of 263A9 light chain contains 5 amino acids.All this meet the genetic characteristics of VRC01 class bNAbs.And the epitope of 263A9 was identified as CD4bs,so 263A9 is a VRC01 class mAb with limited neutralizing breadth 8.33%.The neutralization breadth of 263A9H+VRC01L was 81.8%,while 263A9L+VRC01H did not have neutralization ability,indicated that the light chain of 263A9 suffered function deficit.The 263A9 light chain was mutated at important sites to VRC01 class bNAbs according to the reports,but the mutant antibodies did not have neutralization ability.The distance between the N-terminal D1(the first amino acid at the N-terminal)of 263A9 light chain and the N461 of V5 ring is shorter than the distance between the N-terminal E1 of VRC01 light chain and N461,which may form an unfavorable structure.LCDR1 of 263A9 light chain is far away from GP120 relative to VRC01.These may be the main reasons for the dysfunction of 263A9 light chain.The main structural differences between 263A9L and VRC01L are the N terminal of LFR1,LCDR1 and LCDR3.Seven kinds of chimeric antibodies were constructed by using LFR1,LCDR1 and LCDR3 of VRC01 to replace the regions of 263A9,respectively or jointly.It was found that the neutralization breadth of antibodies only increased to 33.33%.Structural analysis showed that although LCDR1 and LCDR3 in 3#were the same as VRC01,3#LCDR1 formed a different conformation with VRC01's LCDR1,which was still relatively far from gp120.The sequence of LFR1 in 7#(263A9L-VRC01 LFR1 LCDR1 LCDR3)was the same as that of VRC01's LFR1,while the N-terminal of 7#formed a similar structure to that of 263A9.This explained why 3#and 7#did not save the 263A9 function well,and indicated the special coordination between the different regions of the 263A9 light chain.Unbiased B cell repertoires showed that the proportions of IGVH1-2 and IGVK1-33 in heavy and Kappa chains were 1.52%and 2.18%,respectively.The evolutionary tree of IGVK1-33 gene showed that the light chain of 263A9 and bNAbs from the same germline had significantly different evolutionary pathways,suggesting that 263A9 light chain was an off-track light chain.K11R mutation in LFR2 and S7T in LFR3 may be the key mutations causing the off-track evolution of 263A9 light chain.3.Isolation and functional identification of monoclonal antibodies that mediate ADCCSeven and eight B cells were selected from PBMCs of CBJC261 and CBJC451 respectively,and mAbs were expressed,among them 451-B4 was HIV-1 specific antibody.The variable region gene of 451-B4 heavy and light chain were derived from VH1-24*01 and VL1-40*01,respectively,and with SHM both of 6.60%.451-B4 was a CD4bs-directing mAb identified by ELISA,linear peptide library assay and BLI.The neutralizing activity of 451-B4 was limited to 8.33%.451-B4 specifically binds to 8E5 cells and able to mediate ADCC activity,which is weaker than that of 10E8 and comparable to that of VRC01.4.Isolation and functional identification of HIV-1 envelope specific monoclonal antibodiesSeven HIV-1 membrane protein specific mAbs were screened from a chronically HIV-1 infected person whose plasma with broadly neutralizing activity.The heavy chain genes of the mAbs were derived from IGHV1-69*09,IGHV1-08*01,IGHV1-46*01,IGHV4-34*01,IGHV4-61*01 and IGHV3-43*02 families,with SHM of 10.76%to 21.05%.The light chain genes of the mAbs were derived from IGKV3-20*01,IGKV1-39*01,IGKV1-NL1*01,IGKV3-15*01 and IGKV1-9*01 families,with SHM 6.03%to 18.64%.MAbs 508-B1,508-C1,508-C5,508-D4 and 508-E5 mainly recognize the gp41 region.508-D6 and 508-F1 combine with gp120.508-D4 and 508-F1 could neutralize pseudoviruses CNE8 and X2278 in Global Panel respectively,with IC50 38.1?g/mL and 41.7 ?g/mL.Conclusions:1.A VRC01 class antibody 263A9 was successfully isolated from a sample whose plasma with moderate neutralizing activity.The adverse structure of the D1 at the N end of 263A9 light chain and the N461 of V5 loop,and the farther of LCDR1 from GP120 relative to VRC01 may be the main reasons for the dysfunction of 263A9 light chain.Neither site-directed mutagenesis nor fragment exchange could well save light chain function of 263A9,indicated that the maturation of light chain require the synergistic action in each segment.Evolution analysis of IGKV1-33 sequences in CBJC263 antibody repertoire suggested that 263A9 light chain represent an off-track evolution.The analysis of characteristic loci in the evolution of 263A9 light chain showed that K11R in LFR2 and S7T in LFR3 might be the key mutations that caused the off-track evolution of 263A9 light chain.In the design of reverse vaccinology,the K11R in LFR2 and the S7T in LFR3 region should be avoided,which may effectively avoid the off-track evolution.This provide reference information for vaccine design.2.An antibody 451-B4,which recognizes CD4bs and could mediate ADCC activity,was successfully isolated,providing an alternative antibody drug for the treatment of HIV-1 infection.3.Seven HIV-1 membrane protein specific mAbs were obtained,of which 5 mAbs recognizes gp41 and 2 mAbs combine with gp120.Two antibodies have limited neutralizing activity.This group of antibodies can provide a technical reserve for HIV-1 detection reagents and antibody drug development. |
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