Study On The Neutralization Characteristics Of HIV-1 Envelope Proteins And Their Modified Virus Strains From An HIV-1 Infected Individual With Broadly Neutralization Activity

In our previous study,DRVIA7(A7),a VRC01-like broadly monoclonal neutralizing antibody targeting CD4bs,was isolated from an HIV-1 infected individual(DRVI01)with broadly neutralization activity.A systematic analysis of the development of A7 over five years showed that the heavy chain of the antibody matured rapidly in two years,but the development of light chain of the antibody was blocked by the glycan of loop D and V5 region of gp120 protein,which resulted in the limited neutralization breadth of the A7 lineage antibodies.If we can identify the envelope glycoprotein(Env)which binds to the early antibody of A7 lineage,or develop the immunogen which is gradually becoming more neutralization-resistant through Env modification,we may be able to induce the A7-like antibody and even help the antibody to cross the barrier of glycan.Based on the previous studies on A7 antibody,the neutralization characteristics of HIV-1 Envolope glyproteins and their modified virus strains at 6 time points in five years were systematically analyzed in the present study.In first part of the study,we obtained 156 full-length envelope protein genes by single genome amplification(SGA)technique,identified 68 functional genes and prepared pseudoviruses.Sequence analysis and pseudovirus neutralization test showed that the increasing diversity of envelope protein sequences in DRVI01 was consistent with the appearance and maturation of A7 lineage antibodies.The broadly and potent neutralization activity and the ability of the viruses escape from concurrent autologous plasma neutralization are consistent with higher amino acid variation at antibody recognition sites of the envelope protein,suggesting a continuous coevolution between virus and antibody.Sequence analysis also found several characteristics of envelope protein which may be beneficial to the CD4bs antibody recognition,such as the short V1V2 region of envelope protein at early four time points,the fewer glycosylation sites and the lower NXT:NXS ratio of glycosylation motif.The role of these characteristics in neutralizing antibody development needs further study.Sequence alignment analysis revealed the hot spot region of amino acid mutation during the evolution of virus at five time points,and analyzing the influencing factors behind the mutation hotspots,could provide clues for further isolating new neutralizing antibodies.In the second part of the study,in the study of the resistance mechanism to VRCO1 and A7 reconstructed antibodies of envelope proteins derived from DRVI01,we screened and identified the key amino acid sites affecting the sensitivity of VRC01.Previous studies reported that N464 amino acids in V5 region did not affect VRC01 neutralization sensitivity.However,we found in this study that the single point mutation of N464T can reverse the mutant from VRC01 sensitivity to resistance.And the single point mutation of T464N reversed the mutant from VRC01 resistance to sensitivity.The N460,E279D,R282K point mutation changed the neutralization sensitivity of DRVI01 pseudovirus to VRC01 from resistance before mutation to sensitivity to VRC01.Based on the neutralization results of the mutant viruses and A7 antibody,we designed four immunogens which could bind to the A7 lineage antibodies with different neutralization breadth,hoping to simulate the development and maturation of A7 lineage antibodies in vivo.In the third part of the study,the potential neutralizing antibody analysis based on the evolutionary characteristics of envelope proteins,by analyzing the amino acid sequence of 156 full-length envelope proteins of DRVI01,the variation of key amino acids in bNAbs epitope was screened.In reference to the neutralization sensitivity of the virus strain before and after the mutation against concurrent autologous plasma,it is inferred that in addition to CD4bs neutralization specificity in DRVI01,there may also be neutralization specificity against gp41 fusion peptides and gp120-gp41 interface,which provides reference information for future antibody isolation experiment.