Sequence Characteristics Of HIV-1B’ Envelope Proteins And Its Potential Correlation With Broadiy Neutralizing Activities

Background It is evidenced that passive transfer of broadly neutralizing antibodies completely blocked infection by a chimerical simian-human immunodeficiency virus in nonhuman primate studies and passive transfer of broadly neutralizing antibodies delayed HIV-1rebound after cessation of antiretroviral therapy in clinical study. These previous studies in animal models and humans reaffirm that broadly humoral immunity responses may be required to provide immune protection against HIV-1infection. However, exiting HIV-1envelope protein vaccines cannot effectively induce broadly neutralizing antibodies, this represents a major obstacle to thedevelopment of HIV vaccine. HIV-1viruses protect themselves from the effect of neutralizing antibodies via a variety of evolutionary mechanisms. For example, by changing the sequence length of the envelope sub-region to alter or affect the exposure of epitopes; forming gp120-gp41trimmer to cover a great quantity of monomeric proteins epitopes; and the Surface of membrane proteins highly glycosylated, so it is less recognized by the immune system. In spite of the mechanism of immune escape, different clades of HIV-1strains can be neutralized by neutralizing antibody from some chronic HIV-1infection patients, implyingthat there may be common neutralization epitopes in different HIV clades. Therefore, it is important to get in-depth knowledge of the sequence characteristics of epitopes targeted by broadly neutralization antibodies.ObjectivesThis study intends to identify samples with broadly neutralizing activities from HIV-1B’infected formor plasma donors, to amplify the gp160genes by single genome amplification and analyze the sequence characteristics and itspotential correlation with broadly neutralizing activities. MethodsThe neutralizing activities weremeasured using a panel of25Env-pseudoviruses including HIV-1clade B, C, A, CRF07_BC and CRF01_AE strains against plasma samples from a former plasma donor cohort.Viral RNAs wereextractedfrom plasma samples and their cDNAsynthesized.The subtype and genetic diversitywere determined based on the sequence. The sequence length of variable loops, the glycosylation sites (PNGS) number of variable loop sequences, net charge of V3loop sequences, the usage of top tetrapeptides and the usage of coreceptorwere analyzed.The sequence mutation of epitopes of monoclonal antibodies (2F5,4E10,10E8) targeting the gp41region, epitopes of mAbs (PG9, PG16) recognizing V1V2sub-region, epitopes of mAbs (2G12, PGT127/128)identified polysaccharides and epitopes of mAbs (b12, VRC01) targetingCD4binding region were prelimarily investigated.Characteristic siteswere screened by software at gp120and gp41regions.Results1. We chose6samples with broadly neutralizing activity (BCN sample),2samples with none broadly neutralizing (NBCN sample), and1sample with moderate broadly neutralizing activity (MBCN sample)for further analysis. The6BCN samples were able to neutralize more than80%test strains (25strains), in which the neutralizing breadthfor clade B strains is the biggest.the neutralizing capacity for AE viruses is theweakest. The neutralizing’ breadth of the2NBCN samples was below50%for thetested viruses.And theneutralizing breadth of the1MBCN sample was69.6%for the tested viruses.2. All these9samples were infected by HLV-1B’based on phylogenetic analysis.The genetic diversity of the gp160sequences from these9samples mainly lied inV1V2, V4, V5sub-regions, withthe highest APD in V1/V2. 3. Comparing with the control group, BCNstrains have a longer sequence length and more glycosylation sites at V1V2, V4regions,less net charge at V3loop.4. The preliminary analysis of sequence mutations of monoclonal antibody epitopes is as follows.2F5epitope E/KLDKWA (662-667HXB2) was seen in6BCN samples.34.4%sequences in CBJC495sample have K665N which determine the resistance of antibody2F5. Except for one sequence, the4E10epitope is WFxl (T/S) xxLW (672-680HXB2) in the remaining147sequences from the6BCNsamples and2NBCN samples. And this is consistent with the reportedepitope WFx (I/L)(T/S) XX (L/l) W (672-680HXB2) of4E10. N671, Y681,1682, R683are important for10E8. All sequences in sample CBJC438, CBJC437, CBJC515, CBJC495and CBJC473are R683K.8of17SGA sequences in CBJC501have N671S and R683K mutation while10E8epitope lack in one sequence. As Reported, there is directly correlation between glycosylation deletion of N295and2G12resistance. All the sequences in CBJC501and some sequences in CBJC438hadN295D, N2951, N295E.Glycosylation predictions indicate that few sequences in CBJC515and CBJC433lack of glycosylation at site295. N301and N332are key sites of PGT127and PGT128epitope. All of the SGA sequences in CBJC433have N332T.20%of sequences of CBJC438have N332D mutation,80%of CBJC438sequences have N332T mutation.23.8%of sequences in CBJC515are unglycosylated at N332. PG9mainly recognizes N156.N160and the V1V2region polysaccharides’Strand C’.ln CBJC501sample there are N160D mutations CBJC438and CBJC437each exists a sequence with N160D mutation. D185, S364, P369were identified by B12. Site185showed a great diversity in148squences. All of CBJC495sequences have D185S mutation, all of CBJC473sequences have D185N mutation, all of CBJC515 sequences have D185mutation, and other sequences have D185N, D185E mutation.D368gp120and G54VRC01are critical for forming hydrogen bonds for VRC01and gp120glycoprotein interaction, so the368is an important locus of VRC01epitope. In this study all148sequences are highlyconservative for the D at site368. BCN samples have escape mutations in key sites of10E8,2G12, PGT127/128and PG6/PG9epitope, and the proportion is77.8%,30.6%,27.8%and13.9%respectively. NBCN samples have mutations in key sites2F5and10E8epitopes, and the proportion is28.9%and100%respectively.5. By comparing the specific mutationsbetween BCN sequences and NBCN sequences,38sites and8sites were identifedin the gp120region and gp41region respectively, which may be associated with broadly neutralization activity, and8covariate patterns were identifed at gp120region.Conclutions1. HIV-1viruses may escape neutralization via increasing sequence lengthand the glycosylation of V1V2and V4loopand reducing V3ring static charge.2. Sequence analysis indicates that there may be broadly neutralizing antibodies targeting different antigen epitopes in the studying samples.3.46sites and8co-variation patterns in env genes may be associated with broadly neutralization activities.