Study On The Characteristics Of HIV-1 Envelope Proteins And The Mechanism Of Neutralization Resistance To PGT135 In Chronically Infected Individuals With Highly Broad Neutralizing Activity

An effective HIV-1 vaccine needs to induce broadly neutralizing antibody(bNAb)response.Though it is still difficult to induce the production of bNAbs through vaccine immunization,a proportion of infected individuals can generate broadly neutralizing activity during the natural HIV-1 infection,indicating that the human immune system can recognize the conserved epitopes on the viral envelope protein and produce bNAbs.This brings new hope for the induction of similar antibody responses through vaccination to effectively control HIV-1 infection.To analyze the characteristics of Env protein and their correlation with broadly neutralizing activity in the infected individuals who produce broadly neutralizing antibodies from natural infections is still the main way to decipher the mechanism of bNAbs production.Studying the escape mechanism of viruses to bNAbs and analyzing the epitope specificities of HIV-1 bNAbs in the infected individuals will help to understand the co-evolution of the virus and antibodies,to isolate novel bNAbs and provide clues for the design of HIV-1 vaccines.In the first part of the study,seven HIV-1 chronically infected patients were divided into highly broad cross-reactive neutralizing activity(hBCN+)and non-highly broad cross-reactive neutralizing activity(hBCN-)groups based on whether their neutralization breadth was greater than 90%.The full-length envelope protein(Env)gene of the infected person was amplified by single-copy genome amplification(SGA)method.Analysis of the Env protein sequence characteristics of the hBCN+group,hBCN-group,B subtype chronic infection group(B-SP)and China B subtype group(B-database)strains showed that compared with hBCN-group,the V1 and V4 regions of hBCN+strains are significantly longer and have a higher degree of glycosylation.V1 length and N-glycosylation sites of the hBCN+group strain is not only higher than that of the hBCN-group,but also higher than that of the Chinese subtype B(B-Database)sequence and the chronically infected donor's sequence(B-SP),which has a longer length and more glycosylation sites.In addition,it also contains a high proportion of extra cysteine.The disulfide bond composed of extra cysteine in the VI region of the hBCN+group may play an important role in the stability of the V1 region and the adjacent V2 region.Understanding the characteristics of the envelope protein(Env)of the rare infected individuals with neutralization breadth of more than 90%among individuals chronically infected with HIV-1 B' strains can provide valuable information for the development of vaccines against the epidemic strains.In the second part of the study,we explored the mechanism of neutralization escape of the PGT135 antibody of the viral strains of CBJC515 in the hBCN+group with longitudinal time-point samples and the previous research basis.Previous studies found that all the strains of CBJC515 are resistance to PGT135 neutralization.In this study,site-directed mutagenesis,fragment chimerization and neutralization experiments proved that the 2005 strain escaped PGT135 neutralization by losing the N332 glycan site,while the 2006 and 2008 strains may escape PGT135 neutralization througlh the V1,V4,C2 or V1-V3 region changes.By comparing the consensus sequence and point mutation verification with another chronically infected individual CBJC437 whose viral strains all can be neutralized by PGT135,it was found that a small number of strains in 2006 and 2008 can also escape through N398/N611 glycans in a strain-specific manner.We used SWISS-MODEL and pymol software to simulate the spatial structure of these strains bound to the PGT135 antibody,and we found that these changes may have changed the conformation of the key contact area through distal changes,thereby affecting antibody neutralization.It is speculated that these abnormal escape pathways may help prolong the exposure of bNAb epitopes and promote the development of bNAbs.In addition,the chimeric experiment also allows us to explore the co-evolution and functional maintenance between different regions of Env.Studies have confirmed that the V1V2 region has an important impact in the function of the envelope protein,and the V3 region can promote the recovery of protein function and buffer harmful polymorphisms in other regions.The third part of the study further analyzed the potential specificity of neutralizing antibodies against V1V2 glycans,V3 glycans and MPER regions in CBJC515.The specificity of neutralizing antibodies against V1V2-glycan epitopes and V3-glycan epitopes in CBJC515 was analyzed using mutant strains.The results of neutralization experiments and sequence analysis suggested that CBJC515 may not have neutralizing antibodies targeted V1V2-glycan epitopes.There was a neutralizing antibody activity against V3 glycans in plasma samples at the time of 20090519,but the existence of this type of neutralizing antibody could not be clearly determined at the time of 20081118,implying that during the period from 20081118 to 20090519,the maturation process of neutralizing antibodies against V3-glycan epitopes occurred in CBJC515.Through SGA sequence analysis and evolutionary analysis,we found similar phenomena to the previously reported V3-glycan bNAb development,such as the rapid switch of N334-N332,the slow change of N332-N334,and the shortened V1 region,the long V1 region to retain N332 glycosylation site and the phenomenon of incomplete neutralization,suggesting that using assistant evolutionary lineage and Env variants with gradually growth of the V1 loop may have important significance for the induction of this type of bNAb.By using plasma and MPER synthetic peptides ELISA binding experiments,competitive neutralization experiments,and analysis of neutralizing antibody specificity through mutant strains constructed at key sites 677 and 693,the specificity of neutralizing antibodies against the MPER epitope could not be clearly identified in the plasma of CBJC515.The main conclusions of this study are as follows:1.The hBCN+group had unique V1 region.V1 length and N-glycosylation sites of the hBCN+group strain was not only higher than the hBCN-group,but also higher than the Chinese subtype B(B-Database)sequence and the chronically infected donor's sequence(B-SP),which has a longer length and more glycosylation sites.In addition,it also contains a high proportion of extra cysteine.2.Through site-directed mutagenesis and fragment chimerization,we found extreme variation may be needed by HIV-1 to escape from PGT135 without changing the epitope itself,for example,through longer V1 region,changing V4/C2/V1-V3,and 611/398 glycosylation site.The chimeric experiment emphasized the extensive interference of V1V2 region on function,and the key role of the V3 region to maintain the diversity of Env by buffering harmful polymorphisms.3.We identified that this patient developed neutralizing specificity for V3-glycan epitopes from 20081118 to 20090519.