Study On The Characteristics Of Protective T Cell Responses In HLA-B~*13 Positive CRF01_AE Subtype HIV-1 Infected MSM In China

Objective:AIDS is a severe infectious disease that endangers human health caused by human immunodeficiency virus-1.Although highly effective antiretroviral therapy can significantly reduce the morbidity and mortality of HIV-infected patients,there is still no curable strategy or effective vaccine for AIDS due to the existence of a HIV latency.The human leukocyte Antigen-I?HLA-I?-restricted cytotoxic T lymphocyte?CTL?response plays a critical role in the control of human immunodeficiency virus type1?HIV-1?replication.Many current vaccination strategies are focused on the identification of immunogens that can induce T cell responses against HIV.However,the polymorphism of the epidemic circulating HIV-1 in different populations and the discrepancy in host genetic background,the development of vaccines is more difficult than that of other pathogenic vaccines.However,the high genetic variability of HIV-1 is one of the major challenges associated with the development of an effective T cell-based vaccine.Therefore,characterization of the polymorphisms of epitopes in different subtypes and exploring possible protective immune responses are of great significance for the design of T cell vaccine based on epitope.The interaction between virus and host is a dynamic process in HIV-infected people.In one aspect,HLA-restricted T cell response can inhibit virus replication.Virus-specific T-cell responses could be detected in acute HIV infection.Studies have shown that the emergence of CTL responses in acute HIV infection is accompanied by a significant decrease in viral load,specific T cell response in chronic stage is associated with the length of asymptomatic survival.In addition,due to the high degree of genetic variability,and continuously mutation under the immune selection pressure,some escape mutations are frequently transmitted,and they might be accumulating in some populations,resulting in the gradually loss of the protective T-cell response,which may be related to the rapid disease progress.Therefore,it was helpful to improve the effectiveness of vaccine or immunotherapy by fully understand the characteristics of epitope variation of the epidemic circulating strain and the dynamic changes of protective T cell response in this population.In recent years,the HIV-1 epidemic among men who have sex with men?MSM?in China has increased rapidly.CRF01_AE has become the predominant subtype in most regions,followed by the CRF07_BC and B subtypes as well as other recombinants.It has been reported that disease progression in HIV-1-infected MSM is relatively fast.Considering the role of HLA-restricted T cell response in virus control,it is still unclear whether the rapid disease progression in HIV-1-infected MSM in China is associated with the adaptation of HIV-1,whether the protective immune responses still exist in this population remains unclear.Therefore,we will further analyze the variation of the well-reported protective HLA,namely,B^*57,B^*5801,B^*27,B^*51 and B^*13 in different subtype among MSM in China,and the role of epitope variation in rapid disease progression was evaluated.We discovered that the high distribution frequency of B^*13and low mutation rates in B^*13 restricted epitopes,hence,the subjects were further targeted to B^*13 positive CRF01_AE early infected HIV-1 individual among MSM.The occurrence and evolution of HLA-B^*13 restrictive T cell response within one year of infection were explored individually.Next-generation sequencing technology was preformed to analyze the association between HIV evolution and T-cell responses,reveal the occurrence and accumulation of escape mutation,and exploring the potential protective immune response in MSM,and might help with the design of an epitope-based vaccine and with immunotherapy.Methods1 SubjectsPart 1:Subjects were recruited since 2008 from a large-scale prospective HIV-negative MSM high-risk population cohort at our hospital in Liaoning Province.HIV-1 infection was regularly screened with ELISA and affirmed with a Western blot assay.Antibody-negative samples were tested for HIV-1 RNA with 24 minipool nucleic acid amplification testing?NAAT?.Once HIV nucleic acid test was positive,patients were recruited for screening and affirmative test again.The time of infection was estimated as follows:For subjects whose screening and validated tests were positive,estimated time of infection was the intermediate time of the period between the last negative screening test and the first antibody-positive test;For subjects whose screening test was positive,affirmative test results are uncertain and high-risk behavior uncertainties,estimated time of infection was the 30 days before;For subjects whose screening test was negative but nucleic acid test was positive.estimated time of infection was 14 days before the time of nucleic acid positive;For subjects with a reliable history of high-risk sexual behavior estimated time of infection was predicted according to the history of high-risk sexual behavior.The subjects were classified based on the results of the HIV-1-specific RNA testing,including antigen and antibody tests in plasma,according to the system described by Fiebig et al.By 2012,54 subjects of early HIV infection were recruited.The inclusion criteria were 1)18-65 years old;2)Estimated time of HIV infection within 6 months;3)CD4⁺T cell counts>350 cells/ml at the first time after seroconversion,and being naive to antiretroviral therapy.Part2 and Part3:Individuals who were HIV-1 negative but had MSM high risk behaviors were continually followed up until 2014,12 HLA-B^*13 MSM infected CRF01_AE subtype were enrolled.Inclusion criteria:1).18-65 years old;2).HLA genotype is HLA-B^*13;3.HIV-1 subtype were CRF01_AE;4.Estimated time of HIV infection within 6 months;5)Being antiretroviral therapy naive from the time of enrollment until 1 year.Exclusion criteria:1)Other drug tests within 60 days before enrollment;2)Co-infection and active infection.Whole blood,plasma,PBMC samples were obtained at the first enrollment,3 month and 12 months after infection,All subjects provided informed consent for this study.This study was approved by the Medical Research Ethics Committee of the First Affiliated Hospital of China Medical University.Experimental methods1.CD4⁺T cell countThe CD4⁺T cell counts a determined using a FACS Calibur?flow cytometer?Becton-Dickinson,USA?.2.Vrological measurementsPlasma HIV-1 VLs were measured with the COBAS AmpliPrep/COBAS TaqMan HIV-1test?Roche,Germany?.3.DNA extractionGenomic DNA was extracted from anticoagulated whole blood using the QIAamp Blood Kit?Qiagen,USA?.4.HLA class I genotypingHLA class I genotyping was performed with Micro SSP^TM Generic HLA class I DNA typing trays?One Lambda,USA?with two-digit allele specificities according to the manufacturer's instructions Due to the opposite effects that HLA-B^*5801 and HLA-B^*5802 have on HIV disease progression,HLA-B^*58 was further distinguished at four-digit specificities by amplification and sequencing of exon 2-5?including introns2-4?5.RNA extractionHIV-1 RNA was extracted from the plasma samples collected at enrollment?earliest available sample after infection?using the QIAamp^?Viral RNA Mini Kit?Qiagen,Germany?.6.Nearly full-length HIV-1 genome sequencing and phylogenetic analysesReverse transcription of the RNA to single-stranded cDNA was performed using SuperScript III?Invitrogen,USA?according to the manufacturer's instructions.Nearly full-length genome fragments?637-9613 nt relative to HXB2?were amplified by nested PCR.The cDNA generated served as a template for PCR amplification of 5-kb fragments corresponding to the 3'or 5'half of the viral genome Single-genome amplification?SGA?and sequencing of the HIV-1 DNA were performed to acquire a single virus sequence from quasispecies.HIV-1 reference strains were downloaded from the Los Alamos HIV Sequence Database to validate the HIV subtypes using phylogenetic analyses based on pol sequences using the neighbor-joining algorithm in MEGA software.7.Synthetic HLA-B^*13 restricted peptideBased on about 54 Nearly full-length sequences of Liaoning acute CRF01-AE subtype infected subjects,we used the BioEdit to create the consensus sequence,and screened 6 HLA-B^*13 restricted epitope namely Gag-HQSLSPRTL?HL9?,Gag-VQNAQGQMV?VV9?,Gag-GQMREPRGSDI?GI11?,Gag-RQANFLGRL?R L9?,Pol-GQDQWTYQI?GI9?,Pol-RQYDQILIEI?RI9?,which were synthesized by the Sigma-Aldrich,US.peptides presenting amino acid variants which are different from Pol-GI9,Gag-GI11,Gag-VV9 sequence were also synthesized by GL Biochem,China8.IFN-?ELISPOT assayELISPOT assays were performed according to the manuscript?BD ELISPOT,USA?to detect the HIV-1-specific IFN-?-secreting cells among the PBMCs.PBMCs were plated at 10⁶/ml per well with peptides at a final concentration of 5?g/ml.Phytohemagglutinin at a concentration of 5?g/ml was used as a positive control,and medium alone was used as a negative control.Spots were quantified using the ImmunoSpot@Analyzer?Cellular Technology Ltd,USA?.The number of specific IFN-Y-secreting T cells was expressed as spot-forming cells?SFCs?per 106 PBMC inputs.9.Amplication of targeting geneTargeting genes was amplified by nested PCR method.The first round amplification primers of gag gene were:5'-ATCTCTAGCAGTGGCGCCCGAACAG-3'and5'-TAATGCTTYTATTTTYTCTTYTGTYAATGGC-3'.The first round amplification primers of pol gene were:5'-TGGAAATGTGGRAARGARGGAC-3'and 5'-CCTGTHTGCAGHCCCCAATATGTT-3'.The second round primers of Pol-GI9were:5'-AGCTGGACTGTCAATGATATAC-3'and5'-TTTCCCCATATTACTATGCT-3';ThesecondroundprimersofGag-GI11were:5'-CAAGGTTTCWGTCATCCARTTTTT-3'and P:5'-CAGAGCCAACAGCCCCACCA-3'and The second round primers of Gag-VV9 were:5'-WGAYACCAARGAAGCYTTAGA-3'amd 5'-GTTCCTGCTATRTCACTWCCCCTYGGTTC-3'.10.PCR products validationPCR products were visualized by 1%agarose gel electrophoresis11.PCR products purification and Library QuantificationThe PCR products were purified according to the instructions of Beckman purification kit developed by Beckman Coulter Company.QUBIT fluorescence quantitative analyzer was used to quantify the PCR products accurately.KAPA Library Quantification Kits were performed for accurate qPCR-based library quantification.12.NGSNGS were performed according to the manuscript of Truseq Nano DNA HT Library Prep protocol.In brief,the main step was repairing Ends and Size Selection,then adenylate 3'Ends,Ligate Indexed Paired-End adapters,PCR Amplification,Validate Library,Normalize and pool Libraries,finally sequencing on Miseq platform.13.Bioinformatics AnalysisQIIME2 software was used to obtain representative sequence14.statistical analysisnonparametric Mann-Whitney test statistical analysis was used in comparisons between groups,Spearman rank testing was used to assess all correlations.Survival Kaplan-Meyer analysis was performed using a log-rank test.c2 tests were used to compare the proportions of different groups.The proportions of escape mutations between the different groups was analyzed using Fisher's exact tests.Hierarchical cluster analysis was performed to analyze the correlations between the mutation features of the epitopes in each subject and the HIV subtypes using Euclidean distance in Cluster 3.0software.All the statistical analyses and graphical presentations were carried out in SPSS18.0 software?Chicago,IL?and GraphPad Prism 5.0?La Jolla,CA?.Results:1 CRF01_AE HIV-1 strains that lack wild-type protective T cell epitopes restricted by classic protective HLA alleles may be associated with rapid disease progression in HIV-infected MSM in Chinaa)A total of 54 early-HIV-infected MSM were recruited in this study.Three HIV subtypes were identified among the 54 subjects:CRF01_AE?88.89%?,CRF07_BC?5.56%?and B?5.56%?.We then grouped patients according to the presence of the well-known protective HLAs,namely,B^*13,B^*51,B^*27,B^*57 and B^*5801.The results showed that The median age,Fiebig stage,VL,CD4⁺T cell counts,the CD4⁺T cell percentage and CD4/CD8 ratio of the subjects at the baseline as well as the HIV subtypes were all comparable between the protective HLA group and the nonprotective HLA group?P>0.05?.Kaplan-Meier survival analysis further confirmed that the time taken for the CD4⁺T cell counts to decrease to less than 350 cells/?l and the VL to increase to greater than 10⁵ copies/ml were not significantly different between the two groups?P=0.971 and P=0.081?.These results suggested that the“protective”HLAs lost their viral control function among the early-HIV-1-infected Chinese MSM.b)Thirty-two well-studied“protective”HLA-restricted epitopes were analyzed.The analysis showed that 96.88%?31/32?of the epitopes had polymorphisms,ranging from1.85%to 100%with an average variation rate of 70.02%.We found that the variations in the epitopes were rather common in both groups.In 96.88%?31/32?of the epitopes,the variation rates in the HLA-matched group and the HLA-unmatched group were not significantly different?p>0.05?c)To further explore the associations of the variation features in 32 epitopes and HIV subtypes,a hierarchical cluster analysis was performed according to the variation statuses of the 32 epitopes in each subject.We identified 4 clusters,in 96.30%?52/54?of the subjects,the variation clusters were consistent with those of the other subjects infected with the same HIV subtypes.This result indicated that the variation features were subtype specificd)We found that the variation rates of the epitopes in both the CRF01_AE-1 and the CRF01_AE-2 were significantly higher than those in the non-CRF01_AE group?76.82%vs.48.96%,P=0.004,and 71.27%vs.48.96%,P=0.010,respectively?.We found that subjects infected with the CRF01_AE-1 and the CRF01_AE-2 exhibited significantly faster disease progression than those infected with the non-CRF01_AE subtype?P=0.035?.Hence,the CRF01_AE HIV-1 strains that lacked wild-type protective T cell epitopes restricted by the classic protective HLA alleles may be associated with rapid disease progression.2 Exploring the association of T Cell Response of CRF01_AE Subtype infected HLA-B^*13 MSM with Disease Progressa)By analyzing the longitudinal T-cell responses of 6 B^*13 individuals at 3 month and12 individuals at 1 year.We found that at 3 month the frequency of recognition of Pol-GI9,Gag-GI11 and Gag-VV9 epitopes were relatively high,were 50.00%,50.00%and 33.33%respectively;Mean magnitude were 583.33±792.43?425.00±727.92 and±958.95?SFU/10⁶ PBMC?.Besides,we found that at 1 yea the frequency of recognition of Pol-GI9,Gag-GI11 and Gag-VV9 epitopes were relatively high,were 75.00%,58.33%and 50.00%respectively;Mean magnitude were 26.67±759.36?390.83±666.28 and 610.00±908.91?SFU/10⁶ PBMC?.Hence,Pol-GI9,Gag-GI11 and Gag-VV9could be considered immunodominant epitopes for B^*13 individuals.b)In this study,we investigated the dynamic of the magnitude and breadth of B^*13restricted T cell response to immunodominant epitopes in 6 early infected patients.The total magnitude and frequency of recognition was not significantly different from that at1 year?1423.33±1378.31 vs 1916.67±878.97 SFU/10⁶ PBMC,P=0.397;44.44%vs72.22%,P=0.093?,and magnitude of every single peptide was not significantly different from that at 1 year?P>0.05?.The results showed that Pol-GI9,Gag-GI11 and Gag-VV9immunodominant epitope-specific T cell responses emerged at 3 months,and could lasting for at least 1year.c)Pol-GI9 and Gag-VV9 T-cell responses might be related to faster disease progression;Gag-GI11 T-cell responses may be related to slower disease progression,which might still have protective effect.In B^*13 positive CRF01_AE subtype HIV early infected MSM,there was a trend that the magnitude of Pol-GI9 specific T cell response was positively correlated with VL at 3months?r=0.786,P=0.064?.At 3 months,the CD4⁺T cell count of the responding group with Gag-VV9 epitope was significantly lower than that of the non-responding group?246.50±83.5 vs.555.75±48.54,P=0.026?,which might be related to the rapid disease progression.There was a trend that the relative magnitude of Gag-GI11 specific T cell response was positively correlated with CD4⁺T cell counts at 1 year?r²=0.288,P=0.072?,which might be related to the slow disease progression.3.Analysis of mutation driven by B^*13 restricted T cell response in CRF01_AE infected MSMa)The mutation rate of Pol-GI9 epitope was low in population level,D490G mutation was only detected in one patient?321145?.It was found that the Pol-GI9 specific T cell response did not show functional cross-recognition efficiency of D490G varied peptide,suggesting that D490G mutation might be a complete escape mutation.b)The mutation rate of amino acids in Gag-GI11 epitope was relatively low,only 3patients had M228I mutation.Which accounting for 75.68%,29.71%and 3.10%respectively.It was found that the Gag-GI11 specific T cell response showed functional cross-recognition efficiency of M228I varied peptide in patient 320019 and 325029.c)High mutation rates of amino acids was found in Gag-VV9 epitope?58.33%?,4types of amino acid mutations,including?M142W,V143T?,V143T,A139T and V143A were found.The results showed that?M142W,V143T?,V143T,A139T and V143A mutations could partly escape T cell response.Considering that Gag-VV9 specific T cell response was associated with accelerating disease progression and the possibility of escaping,Gag-VV9 might not be suitable for T-cell vaccineConclusion:1.In conclusion,we found that the well-known protective HLA-restricted epitopes in early-HIV-infected MSM subjects in China had a large number of variations,mainly concentrated in the CRF01_AE subtype,demonstrating the loss of protective T cell responses at the population level.This result may be one of reasons why disease progression is rapid in HIV-1-infected MSM.2.Pol-GI9,Gag-GI11 and Gag-VV9 could be considered immunodominant epitopes for B^*13 individuals.Pol-GI9 and Gag-VV9 T-cell responses might be related to faster disease progression;Gag-GI11 T-cell responses may be related to slower disease progression,which might still have protective effect.3.The mutation rate of amino acids in Gag-GI11 epitope was relatively low,Priority should be given to this epitope for T-cell vaccine as a result of the functional cross-recognition efficiency of M228I varied peptide.Pol-GI9 and Gag-VV9 might not be suitable for T cell vaccine or immunotherapy.