| BackgroundIn view of the important role of neutralizing antibodies in the prevention of HIV-1 infection,it is an important goal of HIV vaccine research to design immunogens that can induce efficient broadly neutralizing antibody responses.Membrane protein is the sole target of HIV-1 neutralizing antibodies,so the study of the characteristics of membrane protein gene(env)in samples with broadly neutralizing activity will be critical in the design of HIV vaccine immunogens.In this study,we investigated the evolutionary characteristics of membrane protein sequences and the neutralization phenotype of pseudoviruses from an HIV-1 infected patient with broadly cross-reactive neutralization,which will provide helpful information for the study of evolutionary escape of broadly neutralization and the design of membrane protein immunogen to induce broadly neutralizing antibody responses.ObjectiveTo analyze the evolutionary characteristics of HIV-1 membrane protein genes from an individual with broadly cross-reactive neutralization activity,and to explore the neutralization sensitivity of Env-pseudoviruses from different time points against autologous plasma samples and representative broadly monoclonal neutralizing antibodies(bmNAbs).MethodsViral RNA extraction,reverse transcription,single genome amplification(SGA)of full length env genes and sequencing were performed on plasma samples from an individual(CBJC515)with broadly cross-reactive neutralization activity at 5 consecutive time points of 20050816,20060418,20070424,20081118 and 20090519.Sequencer,MEGA,BioEdit and other bioinformatic softwares and Geno2pheno(coreceptor),Weblogo and other online tools were used to analyze the sequence characteristics of env genes.The representative env genes selected from 3 time points of 20050816,20060418 and 20081118,were cloned into pcDNATM3.1 Directional TOPO expression vector and identified.The env expression plasmid was co-transfected into 293T/17 cells with HIV-1 backbone plasmid(pSG3?env)to prepare pseudoviruses.And the neutralization phenotype of the pseudoviruses were identified through neutralization experiments with autologous plasma samples and representative bmNAbs.Results1.Totally,120 SGA sequences of env genes were obtained from plasma samples of 5 consecutive time points of CBJC515,and 11,4 and 13 functional env clones were constructed at 3 time points of 20050816,20060418 and 20081118,respectively.Phylogenetic analysis of env sequences confirmed that all 120 env genes were clustered to B.CN.RL42 and evolved into two separate lineages:the dominant cluster and the inferior cluster.The sequences at same time point were clustered with each other,and some sequences at different time points were crossed.2.The genetic distance of each variable loop of SGA sequence of different years continued to evolve and increased overtime.The number of V3 loop amino acids and the number of N-glycosylation sites were highly conserved,and the V1V2 variable loop length and N-glycosylation sites increased with time.The proportion of X4 virus increases with time.The proportion of X4 viruses in the dominant cluster was generally higher than that in the inferior cluster.The crown tetrapeptide was dominated by GPGR,and their proportion increased with disease progression.The crown tetrapeptide of the dominant viruses were GPGR,and GLGR and GQGR were in the inferior viruses.3.The neutralization sensitivity of pseudoviruses constructed in this study to autologous plasma at 8 consecutive time points was similar to that of neutralization testing using different subtype pseudoviruses,neutralization sensitivity showed a fluctuation of first increase(0-15 month),subsequent decline(15th to 32th month),and then rise(32-45 month).The pseudovirus is less sensitive against the concurrent and earlier plasma samples,and the sensitivity increased against later plasma samples.4.All Env-pseudotyped viruses constructed at three time points were sensitive to 10E8 and VRC01 but were highly resistant to PGT135.Most of the viruses were sensitive to 12A21,PGT121 and 2G12.X4 viruses were more sensitive to 2G12 and 12A21 than R5 viruses but more resistant to VRC01.The dominant clusters of viruses were more resistant to VRC01 and 10E8 than the inferior cluster,with the inferior cluster were more sensitive to 2G12,12A21 and PGT121,respectively.5.The sensitivity of Env-pseudotyped viruses to VRC01 was negatively correlated with the number of amino acids in V1V2 and PNGS on gp120;sensitivity to 2G12 and 12A21 was positively correlated with the number of amino acids in V1V2;and sensitivity to 2G12 and PGT121 was positively correlated with the number of PNGS.Conclusion1.Two separate lineages of viruses:the dominant cluster and the inferior cluster were found in the study subject by phylogenetic analysis of env sequences.The virus escapes the neutralization selection pressure by increasing the V1V2 variable loop length and the number of N-glycosylation sites,tropism transformation,and evolves in different directions.2.The neutralization sensitivity of the virus to autologous plasma at consecutive time points showed a flunctuation of first increase(0-15 month),subsequent decline(15th to 32th month),and then rise(32-45 month).3.Among the six bmNAbs tested in this study,the Env-pseudotyped viruses were sensitive to 10E8,PGT121 and VRC01,but were all highly resistant to PGT135.10E8 has stronger neutralization ability than 12A21,2G12 and VRC01 against these pseudoviruses.And PGT121 has stronger neutralization ability than 12A21 and 2G12 to these pseudoviruses.4.The inferior lineages of viruses were more sensitive to VRC01 and 10E8 than the dominant lineages of viruses;conversely,the dominant viruses were more sensitive to PGT121,12A21 and 2G12 than the inferior viruses;pseudoviruses,and even pseudoviruses from the same time point,differed significantly in sensitivity to specific bmNAbs,suggesting the complexity of the neutralization phenotype between the virus quasi-species. |
PDF Full Text Request