Studies Of Potential Linkage Between HLA Class Ⅰ-B Alleles And Disease Progression Of HIV Infection In China Population

PrefaceThe interaction between host and HIV-1 determine the termination of HIV-1 infected individuals. In general, most HFV-1 infected individuals would progress to AIDS in 8-10 years, but about 5% of infected individuals remain asymptomatic and clinically healthy for a long periord. These status was called long-term nonprogressors(LTNP). Some investigators proposed that the mechanism of LTNP might be genetically controlled. Several studies in abroad showed that specific human leukocyte antigen (HLA)-B alleles have been found to be associated with the rate of disease progression. For example, HLA-B*57 and HLA-B*27 are associated with slower disease progression and with low virus load, whereas HLA-B*35 is associated with a faster rate of disease progression in Caucasion population. Whether or not this conclusion will fit the patients of china, which is no reported.In this study, our polymerase chain reaction sequence specified primer (PCR-SSP) based on HLA-B genotyping of 291 typical progressors (TPs) and 67 LTNPs revealed some evidences that HLA-B locus polymorphism can influence the rate of disease progression in Chinese HIV-1-infected individuals.Meterials and Methoads1. Study objectsSubjects from two different cohorts were included in our study: a cohort of LTNP, and a cohort of TP. 358 untreated HIV-1 infected patients were from Liaoning, Jilin, Henan, Xinjiang and Yunnan Province in China population. Blood samples were collected and serologic status was determined by ELISA(Vironostika,Organon Tednika,The Netherlands) with confirmation by Western blot(Genelab Diagnostics, Singapore). Sixty-seven LTNPs remains asymptomatic and clinically healthy for at least ten years and their counts of CD4+ T cells remain above 500 cells/μl. 291 TPs have clinical symptoms or counts of CD4+ T cells decrease to less than 500 cells/μl after 5-10 years since infection. All of them were informed of the comprehensive process and purpose of our research before informed consent was obtained.2. Reagent(l)Whole blood cell DNA extraction kit: QIAamp DNA Mini kit (QIAgen,Germany)(2)PCR kit: TaKaRa Taq^TMDR001 AM (Biotech Co.Lltd,Japan)(3)DNA molecular weight standard: 100bp DNA ladder (GIBCOBRL, American)(4) HLA Class I DNA Typing Tray: Micro SSP^TM Generic HLA Class I DNATyping Tray (One Lamda, American)3. Equipments(1) Gel image analytical system: Ultra-Violet Product (UVP. British)(2) PCR apparatus: GeneAmp PCR System 9600(PERKIN ELMER, American)(3) Electrophoresis apparatus: POWER PAC 300 (BIORAD, American)(4) Ultraviolet spectrophotometer: U2001 (HITACHI, Japan)4. CD4+ absolute countThe 20ul TriTEST regents CD4/CD8/CD3 was added to the CD4 absolute vial and 50μl whole blood was added then. After a 15-min incubation at room temperature, the erythrocytes were lysed by using FACS lysing solution. After another 15-min incubation at room temperature, 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.5. Extraction of Genomic DNA samplesGenomic DNA samples were obtained from 1ml peripheral whole blood by using QIAamp DNA Mini Kits (Qiagen,Valencia, CA) according to standard techniques.6. Genotyping assay of Human Leukocyte Antigen-BHLA-B genotyping was performed by the polymerase chain reaction sequence-specified primer (PCR-SSP) method using commercial PCR-SSP kit(Micro SSP; One Lambda, CA) according to the manufacturer's instructions.7. Statistical analysisLTNPs and TPs were compared for baseline characteristics using the nonparametnc Mann-Whitney U test for continuous variables (CD4+ cell counts and viral loads). Allele frequencies were calculated as (h + 2H)/2N, (H is the number of homozygous genotypes, h is the number of heterozygous genotypes, and N is the total number of the sample population.) HLA-B alleles were compared between the 2 groups using the x² or Fisher exact test. The odds ratio (OR) as an estimate of risk and the Fisher's exact test were used to determine the strength of the allele-specific associations in the LTNP vs TP groups. The OR is used to estimate risk in case-control studies. An OR <1 indicates protection, whereas an OR >1 indicates increased risk. P values <0.05 was considered significant.Results1. Immunological and virologyical information on the 2 Study Groups: The CD4+ T cell counts of LTNPs and TPs was 660±19 cells/μl and 294±7 cells/μl; the plasma viral load between these 2 groups was significantly different (P<0.01): 3.39±0.13 lg copies/ml in SPs versus 4.29±0.05 lg copies/ml in TPs (P<0.01).2. Human Leukocyte Antigen Allelic Associations With Disease Progression: HLA-B* 15 allele frequency was significantly higher in TP group (17.2%)than in LTNP group (8.2%) (B*15:P=0.0096,OR=2.32,95%CI= 1.17—4.72). And then HLA-B*67 allele frequency was significantly higher in LTNP group (4.5%)than in TP group (1.2%) (B*67::P = 0.021,OR = 0.26,95%CI = 0.08⁰.89). None of other alleles was found to have a different distribution between the 2 study groups.Conclusion1. HLA-B* 15 alleles is potentially associated with faster HIV disease progression in China population.2. HLA-B*67 may be associated with slower disease progression in China population.