Study On Gp41 Neutralizing Epitope Mutations And Neutralizing Antibody Levels Of HIV-1-infected Individuals In China

ObjectiveBy 1981,with the first HIV-1-infected cases reported,AIDS has dramatically increased on the global.For HIV extremely high genetic variability,scientists have not found cure for AIDS and effecitive vaccine yet.Ideal vaccine should induce specificity neutralizing antibodies and CTL responces.Neutralizing antibody generated from B cell and can neutralize viral resulted in lost infectivity.The major immunogen of HIV-1 is gp160,which is made up of gp120 and gp41.Transmembrane subunit gp41 is conserved relatively and involved in the virus-mediated membrane fusion,which results in viral entry into target cell.For it is capable of inducing neutralizing antibodies and it is an attractive therapeutic target.So far,several potent neutralizing antibodies have been raised,conserved region MPER(membrane proximal-external region) in gp41 recognized by 2F5 and 4E10,were shown to be effective in vitro and also in animal models.Recently,passive transfer of cocktail of neutralizing mAbs(2G12,2F5 and 4E10) was proved to effectively delay HIV-1 rebound in acutely infected individuals after cessation of antiretroviral therapy(ART).The success of passive immunization with neutralizing mAbs greatly encourages the development of the mAb-response-based AIDS vaccines.Unfortunately,with the disease progression,the viral inhibitory activity of neutralizing antibody was replaced by the appearance of resistant virus.Moreover,high genetic variability of HIV-1 is probably a great obstacle for antibody therapy and vaccine development.Some HIV-1 strains are of general resistance to antibody neutralization,and loss of the relevant epitope on envelope subunits may be an important reason for this resistance.It have been observed that antibody titers to this epitope in sera from AIDS patients were significantly lower than those in sera from CRF0 1_AE asymptomatic subjects which were collected in the same year.While whether the 2F5 neutralizing antibody is related to disease progression need to be fiLrther study.For vaccine-mediated protection,high levels of neutralizing antibodies appear to contribute to protection against HIV-1.Some studies have reported better neutralization of primary HIV-1 isolates by SP sera than by sera from HIV-1 progressors.Neutralizing antibodies in SP are really involved in delaying disease progression in HIV-1-infected individuals,and the knowledge of their epitopes might help to design immunogens able to induce such antibodies upon immunization. Currently,phage display is a rapidly progressing biotechnology that allows presentation and reproduction of diverse panels of engineered peptides and proteins.Diversity between people of different races on immunity and heredity exist in HIV-1-infected individuals.HIV-1 epidemic pattern in China differ from any other countries,so we cannot copy the results of abroad studies.We are in great need to make clear the mutation of HIV-1 neutralizing epitopes and levels of neutralizing antibodies. Neutralization and amino acids mutation within epitopes of gp41 in Chinese has been no such investigation reported.We studied neutralizing antibody levels and amino acids mutation of conserved neutralization epitopes of HIV-1 in common HIV-infected individuals,in China.The aim of this study also was to identify the epitopes for these neutralizing antibodies from SP,as these should represent immunogens potentially able to elicit neutralizing antibodies upon vaccination.Materials and Methods1.Study populationNinety-Two HIV-1-infected individuals from Henan,Yunnan and Liaoning were enrolled in this study.They acquired HIV-1 infection through blood charged transmission,drug injection transmission and sexual contact transmission.HIV-1 infection was screened by anti-HIV ELISA(Vironostika,Organon Tenika,The Netherlands) and confirmed by Western blot(Genelab Diagnostics,Singapore).These patients were stratified according to US CDC recommendation(1993).Among these persons,24 were slow progressors(SP)(with asymptomatic and stable CD4 T-cell counts＞500 cells/μl beyond ten years),34 were asymptomatic(AS)(CD4 T-cell counts＜500 cells/μl but more than 200 cells/μl and the subject had no AIDS defined symptoms) and 34 were AIDS patients(CD4 T-cell counts＜200 cells/μl or CD4 T-cell counts gave indications of AIDS).Only ARV treatment-na(i|¨)ve patients were included. HIV-1 subtype was determined by phylogenetic analysis based on part of env,pol and gag gene regions.Include 54 HIV-1 B′-clade infected individuals,24 HIV-1 CRF B′C-clade infected individuals and 10 HIV-1 CRF01 AE-clade infected individuals.HIV-1 Written informed consent was obtained before the enrollments from the studied participants.This study was approved by the institutional review board at the China Medical University.2.CD4⁺T cells countsCD4⁺T cell counts were measured using TriTEST CD4FITC/CD8PE/CD3PerCP reagent with 20μl anticoagulated whole blood.After incubation for 15 minutes in the dark at room temperature,FACS lying solution was added and then incubated.CD4⁺ T cell counts and corresponding ratios were obtained by flow cytometer analysis with FACS MMLTISET software.3.Viral load assayPlasma HIV RNA was quantified with Amplicor HIV-1 Monitor test,version 1.5 (Roche Diagnostics,Rotkreuz Switzerland) by an ultra-sensitive modification.4.DNA and RNA extraction,cDNA synthesisBoth HIV-1 DNA from peripheral blood and RNA from plasma were extracted with QIAamp Viral DNA and RNA Mini Kit(Qiagen,German) according to the manufacture's recommendations.RNA was reverse transcribed into cDNA with a reverse transcriptase kit(TAKARA,Japan).Two separate plasma from patients collected in June/2005 and June/2006 were used for analysis of gp41 genes variation.5.Nested PCR amplification and sequencingHIV-1 gp41 gene(461bp) was amplified with outer primers gp41-1(5′-TCT TRG GAG CAG CAG GAA GCA CTA TGG G- 3′HIV-1_HXB2 7789-7816nt) and gp41-2 (5′-AAC GAY AAJ GGT GAR TAT CCC TGC CTAA- 3′HIV-1_HXB2 8374-8347nt),and followed by nested PCR with inner primers gp41-3(5′-ACA ATT ATT GTC TGG TAT AGT GCA RCA GCA - 3′HIV-1_HXB2 7850-7879nt) and gp41-4(5′-TTA AAC CTA YCAAGC CTC CTA CTA TCA TTA - 3′HIV-1_HXB2 8310-8281nt).6.Products PurifiedThe PCR products were confirmed throμgh 1.5%agarose gel electrophoresis in present of 0.5μg/ml ethidium bromide and photographed under ultraviolet illumination. Products were purified with QIAquick Gel Extraction Kit(Qiagen,Germany). 7.Construction of gp41 clonesThe amplified gp41 fragments(461bp) from plasma RNA were cloned into pCR4TOPO^(?) TA vectors(Invitrogen,USA),and cloning sequences of the quasispecis viruses were confirmed by DNA sequencing by the dideoxy-chain termination method.8.Sequencing reactions50 fmoles of purified PCR products were sequenced directly with primers gp41-s1 (5′-CAA TTA TTG TCT GGT ATA GTG C-3′,HIV-1_HXB2 7851-7872nt ) and gp41-s2 (5′-CAA GCC TCC TAC TAT CAT TA-3′HIV-1_HXB2,8300-8281 nt),and by dideoxy chain termination method with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and AmpliTaq DNA polymerase(Perkin-Elmer,France) on an automated sequencer 377(Applied Biosystems,USA) according to the manufacturer's recommendations.9.Phylogenetic analysisSequences were edited and aligned by BIOedit software.Phylogenetic analysis was conducted by MEGA3.1 software.Amino acid sequences of gp41 gene were obtained from the translation of nucleotide acid sequences and compared with the reference sequences(HXB2)[GenBank:K03455]with neutralization epitopes.The sequences determined in the present study have been deposited in the Genbank nucleotide database(accession numbers:EU542544-542575,EU569776-569829).10.Neutralization assayNeutralization assays were performed in 96-well culture plates by incubating virus stock with diluted plasma specimen,heat-inactivated at 56℃for 30 min. Approximately 500TCID₅₀(determined by Spearman- Karber equation assay) of HIV-1 was added to each well.After incubation for 30 min at 37℃,PBMC(1.0×10⁵ cells) was added to each well.PBMC were maintained in IL-2 culture medium containing 1μM indinavir,and the cells were fed in IL-2 culture medium on day 1^st.On day 7^th, supernatant were harvested,p24-Ag was examined.11.ELISAThe peptides 4(ELDKWA)-C(5μg/ml) and gp41(0.1μg/ml) were coated overnight on a microtiter plate at 4℃.Nonspecific binding was blocked for more that 2 hours by incubation with PBS(3%BSA).After washing with PBS-Tween 20(0.05%Tween 20), normal serum and sample with different dilution were added and incubated 1 hour at 37℃.After washing,peroxidase-conjugated goat anti-human immunogloulins was added.TMB solutiong was added and stopped by H₂SO₄(2M),and the absorbance at 450 nm was read.12.Plasma IgG purificationResuspend Dynabeads M-280 Tosylactivated well by pipetting or vortexing for approximately.Dissolve the goat anti human IgG antibody in 0.1M H₃BO₃ pH9.5.The Dynabeads M-280 Tosylactivated was coated by goat anti human IgG.Add plasma sample,IgG of plasma would bind and ready for use,store the coated Dynabeads at 4℃. Experience according to the manufacture's protocols.13.Panning ProcedureDilute 1.5×10¹¹ phage(10μl of original library) with 1ml of TBST and add to beads coated with plasma IgG Panning procedure based on instruction manual of rapid screening of peptide ligands with a phage display peptide library,NEB.The first and second plasma was P2 and P9.The third sample was plasma of HIV-1 negtive.Tween concentrations were increased stepwise(0.1,0.3,0.5%) in 3 rounds of panning.Pick plaques from plates having no more than 100 plaques,incubate and sequence.14.Plaque amplification and purify elutePick plaques from plates having no more than～100 plaques.Incubate tubes at 37℃with shaking for 4.5～5 hours.Transfer the culture and spin 10 minutes at 10,000 rpm,and then re-spin.Piped the upper 80%of the supernatant and add 1/6 volume of PEG/NaCl,4℃for over night.Spin PEG precipitation 15 minutes at 10,000 rpm and re-spin.Suspend the pellet in 1 ml TBS,re-precipitate with 1/6 volume of PEG/NaCl, and re-spin.Suspend the pellet in 5091 TBS.Store at 4℃.15.Assaying selected clones by ELISA96 mexisorp plate coated with M13 antibody,4℃for overnight.Fill each well completely with Blocking Buffer 5mg/ml BSA for at least 1 hour,add phage incubated at 37℃for 1 hour,additionally,per phage clone should be used for HIV-1 positive and negative plasma pools.Add HRP conjugated goat anti-human IgG substrate to each well,TMB solution was added and stopped by H₂SO₄(2M),read the absorbance at 450nm.Assays were run in triplicates. 16.Rapid purification of sequencing template and sequencAdd 200μl PEG/NaCl,centrifuge and discard supernatant.Suspend pellet thoroughly in lodide buffer and add ethanol.Wash pellet in 70%ethanol,dry briefly under vacuum.Suspend pellet in 30μl TE buffer.Objective DNA gene(170bp) was amplified with the primer phd1-(5′-TTC GCA ATT CCT TTA GTG-3′) and phd2-(5′-TTT GTC GTC TTT CCA GAC GT-3′).The phd2 primer is recommended for dideoxy sequencing.Determine the amino acid sequence from this strand using the genetic code shown in manual.Amino acid sequences of phage compared with the reference sequences(B.TH.90.BK132 ACC AY173951) with neutralization epitopes.17.Phage ELISAPlates were coated overnight at 4℃with 100μl/well gp41 epitope 0.1μg/ml.The next day,plates were blocked at 4℃with 200μl/well blocking solution.And then washed three times with PBST(I%Tween-20).100μl/well plasma of HIV-1 negative control(1:100) and 100μl/well selected phages mix 1:1 was added in plate.Plates were washed,incubated with 100μl/well HRP-goat anti human IgG conjugate and read as described above.Assays were run in triplicate.18.Statistical analysisComparisons and analysis between DNA sequences and RNA sequences were completed by 2-tailed Fisher's exact test.The results were obtained comparably by test.Neutralization activity was calculated as percent inhibition.The significance of the differences in IC50 values was calculated by student's t-text.The nonparametric Mann-Whitney U test was used to assess differences in neutralizing activity between pool sera groups.Results1.Mutations of ELDKWA and NWFDIT epitopes in gp41 of Chinese HIV-1-infected individuals.All of 92 amino acid sequences,especially in the mAb 2F5 and 4El0 epitopes on the gp41 region from proviral DNA were analyzed.For 2F5 epitopes,mutation levels of ELDKWA was 20/92,while the frequency of 4E10 epitope mutation was 34/92, respectively frequencies were 21.73%and 36.95%.The most common mutation were E662A/K/S(14.1%/3.3%/3.3%),K665S/E/Q(17.4%/2.2%/1.1%),A667K/N(16.3% /1.1%)in 2F5 epitope.Mutations N671S/D(13.0%/1.1%),D674S/G/N(3.3%/2.2% /1.1%),T676S(16.3%) were found in 4E10 peptide.2.Compared mutations of ELDKWA and NWFDIT epitopes in gp41 among clade B′,CRF_B′C and CRF01_AE.The frequencies of ELDKWA motif were 3/54(5.55%),16/24(66.66%) and 0/10 (0%) came from respectively clade B′,CRF_B′C and CRF01_AE(P＜0.01).And the frequencies of NWFDIT epitope were clade B′16/54(29.62%),CRF_B′C 14/24 (58.33%) and CRF01 AE 3/10(30%)(P<0.05).3.Comparison of neutralization epitopes among SP,AS and AIDS patients.The differences in the frequency of neutralizing epitope mutation on NWFDIT motif between SP,AS,and AIDS patients were of statistical significant.A higher frequency of the neutralizing epitope mutation on 4E10 epitope was observed in AIDS patients than that in AS and in SP(P＜0.01).In contrast,the mutation difference in epitope ELDKWA among these three groups was not statistically significant(P＞0.05).4.Sequence evolution in HIV-1-infected persons.In view of the fact that the neutralizing resistance-associated mutations in proviral DNA and plasma HIV-1 RNA were inconsistent,we compared sequences each obtained from both DNA and RNA contemporary(the 1^st time point,June 2005).RNA sequence showed a little discrepancy with proviral DNA sequence,in which neutralizing epitope variances,S676T,N671S and E662A,existed in RNA sequences in three patients. Meanwhile,we observed RNA sequences evolution of 16 persons from the cohort of 54 treatment-naive HIV-1-infected individuals in one year.The major mutations,E662A (1/16) and D674E(1/16),on ELDKWA epitope and the mutations N671S(1/16) and T676S(4/16) on NWFDIT epitope,were discovered(the 2^nd time point,June 2006), which are associated with the escape from the neutralizing antibodies.We observed thirty-two cloning sequences from three patients' viral RNA samples collected at two time points.Phylogenetic tree revealed highly specific clustering of cloning sequences from all patients from two time points except sample HA114 that showed more diversity of sequence than others.No mutations on 2F5 and 4El0 epitopes were observed in RNA sequences obtained from the time point 1 in all three persons. However,one year later(the 2^nd time point),different levels of diversity on these epitopes occurred in these patients,among whom one did not show any diversity (HAS48) and the other two had low and high diversities,respectively.Mutation E662A (4 of 5 clones) occurred in patient HAS31,while mutations E662K(4 of 8 clones), K665E(5 of 8 clones),D674S(1 of 8 clones) and D674N(3 of 8 clones) occurred in patient HAS114.5.Comparison of neutralizing antibody levels among HIV-infected individuals,AIDS patients and SP.2FS-like antibody levels of SP were noticeable higher than that of AIDS patients, and that of HIV-infected individuals also higher than AIDS patients,but showed no significant difference(P＞0.05) between HIV-infected individuals and AIDS patients; The gp41 antibody titers showed no difference among three groups;The IC50 mean titers for SP,HIV-infected individuals and AIDS patients were respectively 160.33, 104.29 and 60.67.The neutralization titers of SP were significantly higher than that for HIV-infected individuals(P＜0.05).The neutralization titers showed no significant difference(P＞0.05) between HIV-infected individuals and AIDS patients;There was significant correlation with 2F5-like antibody titers and gp41 antibody.2F5-like antibody titers was no association with the neutralization titers(P＞0.05).6.Selection of HIV-1 minotopes.To identify vaccine-relevant HIV-1-specific mimotopes,phage libraries exposing peptides of different length and conformation were screened with immobilized IgG from plasma of two SP with broadly neutralizing antibodies.For each screening,48 single-phage clones were tested for specificity by ELISA.Peptide sequences were then compared to linear HIV-1 protein sequence and HIV-1 Protein structure files.The sequence of Clone 14 is identical to Clone 15 and Clone 31 which located in gp120 C1 region.Clone 10 is located in gp120 V1 loop and Clone 22 occurs on gp120 C2 region, Clone 23 is located in gp120 V4 loop.Another six peptide epitope occur on the envelope gp41 glycoprotein.Notably,Clone 40 sequence occurs gp41 C-terminal heptad repeats,it has higher inhibition ratio titer of antibody to HIV-1 gp41.The described approach could be applicable for immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy. Conclusion1.92 HIV-1-infected individuals from China were demonstrated diversify mutations in 2F5 and 4El0 neutralizing epitopes in HIV-1 env gp41;The mutation degrees of amino acids in conserved neutralizing epitopes were different in different subtypes.2.The neutralizing epitopes mutations of 4E10 have correlation with the disease progression.Mutation of neutralizing epitope of plasma viral RNA naturally may be contributed to partly or full resistant to neutralizing antibody.3.There was significant correlation with the levels of 2F5 neutralizing antibody and gp41 antibodies.There was no significant correlation with the levels of 2F5 antibody and neutralizing antibody.The levels of 2F5-like antibodies may influence on disease progression of HIV-infected individuals.There should be other neutralizing antibodies that may arrest disease from progressing.4.By the use of serum IgG antibodies of SP to screen the phage random polypeptide library,we can obtain multiple phage mimetic peptides of HIV related antigen epitope.The Clone located in gp41 conserved C-terminal heptad repeats could inhibit antibody binding to HIV-1 gp41.This epitope should provide the basis for detecting HIV-1 specific antibodies programs.