Studies Of Interaction Between KIR/HLA-Bw4 Alleles And The Progression Of HIV-1 Infection In Chinese Population

ObjectiveKIR alleles and HLA genes,known as important host genetic factors,have an impact on the progression of HIV-1 infection.KIRs,a diverse family of recognition receptors,expressing on NK ceils regulate NK activity via the recognition of specific HLA class I molecules on the surface of target cells.They mainly act in self or nonself-recognition of NK ceils and the regulation of NK cell-mediated cytolysis. The KIR and HLA gene complexes are both highly polymorphic.Several studies abroad showed that homozygosity for HLA-Bw4 allele was significantly associated with the ability to delay the disease progression and to maintain a normal CD4 T cell count,as well as the activating KIR allele KIR3DS1 in combination with HLA-Bw4-80Ile was associated with delayed progression to AIDS.Certain result,additionally,revealed an association between the co-expression of KIR3DL1 and B57~*S(includes HLA-Bw4-80Ile)and delayed progression to AIDS.Whether or not KIR genes are associated with disease progression to AIDS in Chinese population,it has not been reported.In this study,we investigated the gene frequency of KIR and HLA-Bw4 alleles in Chinese individuals infected by HIV-1(B' subtype)through blood transmission,to define the difference of the gene frequency between typical progressors and slow progressors and the relation between the disease,progression and KIR/HLA-Bw4.Our results might provide scientific basis for slowing the disease progression and developing new therapy.Materials and Methods1.Study objectsEighty-one HIV-1-seropositive subjects were recruited in our study.All of them were Chinese Han population and from Henan,Jilin and Liaoning province.They were all infected by HIV-1 B' subtype and transmitted through paid blood donor route.Blood samples were collected and serologic status was determined by ELISA with confirmation by Western blot.43 SP remain asymptomatic and clinically healthy for at least ten years and their counts of CD4~+ T cells remain above 500 cells/μl for three times(at interval of more than 6 months).38 TP have clinical symptoms or counts of CD4~+ T cells decrease to less than 500 cells/μl after 5 years since infection.The study was approved by the Research and Ethics Committee of China Medical University. Informed consent was obtained from each participant at recruitment.2.Extraction of Genomic DNA samplesGenomic DNA samples were obtained from 1ml EDTA-anticoagulated peripheral whole blood by using QIAamp DNA Mini Kits according to the manufacturer's instructions.3.CD4~+ absolute countThe 20μl TriTEST reagents CD4/CD8/CD3 were added to the CD4 absolute vials and 50μl whole blood was added then.After 15-min incubation at dark at room temperature,the erythrocytes were lysed by using FACS lysing solution.After 15-min incubation at room temperature again,the vials were taken to perform FACS analysis by using Multiset software.The absolute count and the percentage of CD4~+,CD8~+ and CD3~+ T cells were calculated automatically.4.Genotyping assay of KIR allelesGenomic DNA was typed by PCR-SSP(polymerase chain reaction sequence-specified primer)using KIR Genotyping SSP Kit.The presence or absence of KIR alleles were determined by referring to the work sheet according to the product fragment.5.Genotyping assay of HLA-B geneHLA-B genotyping was performed by the PCR-SSP method.The genomic DNAs were used as the template.The amplification primers Bx1 and BINT3 allowed amplification of a HLA-B-specific product of about 1000bp.The whole amplification product was purified using QIAquick Gel Extraction Kit.BEX2F acted as the sequencing primer.The purified amplification product was used as a template for direct cycle-sequencing reactions. 6.Analysis of nucleotides and amino acids of HLA-B sequenceWe analyzed the HLA-B sequences and the reference sequences by use of the BioEdit software.We translated the nucleotides into amino acids and judge the HLA-B serological epitopes according four variable amino acids spanning positions 80-83 of the second exon.7.Statistical analysisSPSS11.5 software was used to assay the data.The gene frequencies were calculated by direct counting and were assessed by the x~2 test with Yates's correction or Fisher's exact test.The odds ratio(OR)was calculated by the cross-product ratio.Exact confidence intervals(CI)of 95%were obtained.P values＜0.05 was considered significant.Results1.The relationship between KIR allelic and disease progressionKIR2DS3 allele frequency was significantly higher in TP group(26.3%)than in SP group(7.0%)(P=0.018,OR=4.762,95%CI=1.201-18.883).2.HLA-B allelic associations with disease progressionHLA-Bw4 homozygote frequency was significantly lower in TP group(7.9%)than in SP group(30.2%)(P=0.012,OR=0.198,95%CI=0.051-0.761).3.Association between disease progression and co-expression of KIR3DS1/KIR3DL1 and HLA-Bw4-80I geneThere was no statistically significance between the 2 groups with the co-expression of KIR3DS1/KIR3DL1 and HLA-Bw4-80I Gene.Conclusion1.KIR2DS3 might potentially be associated with accelerated HIV disease progression in Chinese population.2.Homozygosity for HLA-Bw4 allele might be associated with delayed disease progression in Chinese population.