Objective To compare the performance of fourth generation HIV antigen/antibody combined detection reagents for HIV early infection samples,international HIV seroconversion panel samples and routine clinical screening samples.Methods Thirty seven early HIV infected samples from the followup gays in Shen Yang between 2009 and 2011,66 seroconversion panel samples from BBI company (U.S.A),NABI company(U.S.A) and NIBSC company(U.K) and 703 routine HIV screening samples in the first hospital of China medical university in October 2010 were collected.All kinds of samples were tested by three diagnostic reagents based on chemiluminescence assay (CLIA),electrochemiluminescence assay (ECLIA) and enzyme-linked immunosorbent assay (ELISA) respectively.The detection sensitivity and specificity of these assays were analyzed.Results For 59 early infected and seroconversion samples,the sensitivities of both ECLIA and CLIA reagent were 96.61％ (95％ CI 91.5％-100.0％),higher than that of the ELISA kit (95％ CI 75.0％-92.9％) (x2 =5.341,P ＜ 0.05),which is 83.93％ ; Comparison among the three reagents for different subtypes of the antibody seroconversion samples showed that ECLIA had the highest sensitivity while CLIA was the lowest ; Detection sensitivity of the three reagents for the P24 antigen is CLIA ＞ ECLIA ＞ ELISA; With detection of 703 clinical routine screening samples,the specificities of three reagents were 100％ (CLIA),99.86％ (ECLIA) and 99.71％ (ELISA) respectively.Conclusions For the sensitivity of the fourth HIV diagnostic reagents CLIA and ECLIA are better than ELISA.The former two reagents are more suitable for identifying earlier HIV infection in clinic.

Objective To evaluate the detectability of HIV antigen-antibody in the window period of acute infection by three HIV antigen-antibody assays.Methods Twenty-two samples of HIV seroconversion serum panels and thirty-seven HIV acute infected plasm samples from our laboratory collected from cohort study of men who have sex with men between 2009 and 2011,were assayed by ECLIA,CLIA and ELISA methods.All assays were evaluated for the ability to detect HIV in the window period,and the sensitivity of each assay for acute samples was analyzed.Chi square test was used for statistical analysis.Results The ability of detecting HIV in the window period of each assay was different.For HIV seroconversion serum panels,the results of ECLIA and CLIA assays were consistent,and the window period was shortened at least 1 to 5 days compared with ELISA assay.For HIV acute samples,all were HIV positive by ECLIA or CLIA assay,but for ELISA assay,94.6％ was positive.For samples before seroconversion,ECLIA and CLIA assay had the same sensitivity (93.5％),which is superior to ELISA assay (71.0％) (x2 =5.14,P ＜0.05).Conclusion The ability of detecting HIV in the window period was different for each assay.The results of ECLIA and CLIA assay are consistent,superior to ELISA assay.

Objective To investigate the possible association of HLA-DQ alleles with pelvic inflammatory disease (PID) caused by Chlamydia trachomatis in Han nationality in Northern China. Methods Thirty five Han Chinese patients with PID caused by Chlamydia trachomatis and 98 unrelated healthy individuals from the same area were genotyped for HLA-DQ gene alleles by the PCR-sequence specific oligonucleotide (SSO) method. Results Compared with the healthy controls, the frequencies of HLA-DQA1*0501 and HLA-DQB1*0301 allele were significantly increased and also associated with the antibody response to Chlamydia trachomatis heat-shock protein 60 (CHSP60). Conclusions These results may provide clues to reveal the susceptibility gene of chlamydial pelvic inflammatory disease in China and the immunogenetic mechanisms of the disease.

Objective To explore the polymorphism of nef gene and conservation level of functionally important domains of nef as well as their influences on HIV-1 disease progression of HIV-1 B'infected individuals in northern China.Methods 30 long term nonprogressors(LTNPs)and 42 typical progressors (TPs)were selected.Provirus DNA was extracted from whole blood sample.The full nef gene was amplified by nested-PCR.PCR product was sequenced directly after purification.Phylogenetic analysis and amino acid sequence mutation was applied on nef sequences to explore the differences between LTNPs and TPs.Results At position 15,the S15R/K/N substitution was detected.The frequency of TPs(64.29%)wsa higher than LTNPs(33.33%,P<0.01,0R=3.60);R21K/E/H/I.Q,TPs and LTNPs mutation frequency was 59.52%and 93.33%(P<0.005,OR=0.11);At position 39,K39R/E/N was only detected in TPs(23.81%,P<0.005).Conclusion No significant deletion or defect associated with disease progression wsa detected in nef gene of HIV-1 B'.But it suggested that K/E/H/I/Q mutation at 21 st amino acid of nef associated the disease nonprogression.R/K/N at 15 th amino acid of nef and R/E/N mutation at 39th amino acid of nef associated the disease progression in.HIV-1 B'.All domaias of nef amino acids sequences were comparatively conservative.

Objective To investigate the level of B7-H1 and its counter-receptor PD-1 expression in mDC and different subsets of T lymphocytes in HIV infected individuals in China and to analyze the correlation between the level of B7-H1/PD-1 and disease progression, and to demonstrate that PD-1/PD-L1-dependent inhibition is operating in HIV infected patients.Methods Percentage of B7-H1 and PD-1 expression in mDC, CD+4 T cells and CD+8 T cells from thirty-six untreated HIV infected patients and 20 health controls were selected and detected by flow-cytometry, its correlations with CD+4 T cell absolute counts and plasma viral loads were analyzed.Results The percentage of B7-H1 expression in mDC, CD+4 T cells and CD+8 T cells (mean 15.21, mean 20.63, mean 13.5)were higher than that of health controls (all P<0.05).The percentage of PD-1 expression in CD+4 T cells and CD+8 T cells (mean 17.91, mean 19.21)were higher than that of health controls (P<0.05, P<0.05). The level of B7-H1 and PD-1 expression were inversely correlated with CD+4 T-cell counts(mDC+B7-H1+:r=-0.647, P<0.01;CD+4B7-H1+:r=-0.489, P=0.002;CD+8B7-H1+:r=-0.372, P=0.026;CD+4PD-1+:r=-0.374, P=0.025;CD+8PD-1+:r=-0.455, P=0.005) and positively correlated with HIV viral load(mDC+B7-H1+:r=0.662, P<0.01;CD+4B7-H1+: r=0.426, P=0.01;CD+8B7-H1+:r=0.531, P=0.001;CD+4PD-1+:r=0.362, P=0.03;CD+8PD-1+:r=0.380, P=0.022).Conclusion The level of B7-H1 and PD-1 expression was associated with HIV disease progression, which provides a useful marker to define disease progression of HIV infection.

ObjectiveTo screen mimetic HIV-1 neutralizing epitopes from plasma with high level neutralizing antibody,and to provide useful information for further study of the interaction between antigen and antibody.MethodsIn order to gain neutralizing antibody recognized mimotopes, we detected neutralizing antibodieslevelsof 11HIV-1infectedslowprogressorsbyPBMC-basedneutralization assays.High-titer HIV-neutralizing antibodies from plasma of SPs was used as the ligand for biopanning by phage-displayed random peptide library.Positive phage clones was evaluated by ELISA,sequenced,and analyzed for homology to HIV-1 env by local BLAST to deduce the neutralizing peptide.ResultsTwenty-two clones were obtained consistent with requirement through three rounds biopanning.After comparison analysis,twelve clones include C8 were obtained as mimotopes of neutralizing antibody,C40 located in gp41Ⅱ cluster with the highest titer by inhibition ratio may be as neutralizing epitope.Conclusion By the use of IgG antibodies from SPs to screen the phage random polypeptide library,one can acquire multiple phage mimetic peptides of HIV related antigen epitope.(Chin J Lab Med,2012,35:838-842 )

Objective To evaluate the performance of the third generation ELISA and the fourth generation ELISA for HIV-1 diagnosis assays on acute and early HIV-1 infected samples.Methods Sixtyseven acute/early HIV-1 infected samples were collected from the follow-up gays with seroconversion in Shen Yang city and from clinical patients in the First Affiliated Hospital of China Medical University with incomplete HIV-1 specific bands in western blot between 2008 and 2010.Third generation ELISA,fourth generation ELISA,western blot and HIV-1 viral load detecting were used for detecting these samples.The sensitivity,consistency were compared between third generation ELISA and fourth generation ELISA to detect the seroconversion samples and the window periods were abserved.Chi square test was used for statistical analysis.Results In the 67 acute/early HIV-1 infected samples,56 were HIV positive and 11 were HIV negative by the third generation ELISA.The sensitivity of the third generation ELISA was 83.6％ (95％ CI:72.5％ -91.5％); 63 were HIV positive,1 was at gray zone and 3 were HIV negative by the fourth generation ELISA.The sensitivity of the fourth generation ELISA was 94.0％ (95％ CI:85.4％ -98.3％),higher than the third generation ELISA(x2 =16.1,P ＜0.01).The consistency of the third generation ELISA and the fourth generation ELISA was 86.6％ ( 95％ CI:76.0％ - 93.7％).The earliest third generation ELISA positive sample was the sample collected 16 days after HIV infection and the earliest fourth generation ELISA positive sample was the sample collected 9 days after HIV infection.There was significantly different on the window periods between the third generation ELISA and the fourth generation ELLSA Conclusion The fourth generation ELISA had a higher sensitivity and shorter window period on acute/early HIV infected samples than the third generation ELISA,which is more suitable for the HIV early infection screening on high risk populations.

Objective To examine the expression of TLR7/8 in monocytes purified from HIV-1 infected individuals and to study its association with disease progression. Methods Sixty-three HIV-1 infected individuals and 18 normal controls were enrolled. Monocytes were purified by MACS system and RNA of them was extracted by RNA mini kit of QIAGEN company. TLR7/8 expression was tested by real-time RT-PCR with ABI7500. Results It was found that the expression of TLR7 was strongly correlated with absolute CD4 count (r =0.614, P＜0.01) , so was TLR8 (r =0.419, P＜0.01). The expression of TLR7 in slow progressor (SP) group was higher than that in HIV-1 infected patients group, AIDS patients group and normal group (P ＜ 0. 05 ) . HIV group and normal group were strongly higher than AIDS group (P ＜ 0. 05). It was no significant differentiation of expression of TLR7 between HIV infection group and normal control group. The expression of TLR8 in SP group and normal group were significantly higher than that in AIDS group (P ＜ 0. 05). The expression of TLR8 was no singnificantly difference between SP group and HIV group or normal control group, so was it between HIV group or normal control group and AIDS group. Conclusion The expression of TLR7/8 in monocytes from HIV-1 infected patients significantly correlated with disease progression.

Objective To study the association of CD4+CD8+Foxp3+ regulatory T cells with the HIV long term non-progressors(LTNP) in China. Methods Seventy-four HIV-1 infected patients (LTNP group, HIV group and AIDS group)and 16 normal controls were enrolled and the frequency of CD4+CD25+Foxp3+ regulatory T cells were detected by flow cytometry. To study the correlation between CD4+CD25+Foxp3+ regulatory T cells and disease progression, the absolute CD4+ T cells, viral load, apoptosis and activation of T cells were also examined. Results The frequency of CD4+CD25+Foxp3+ regulatory T cells in LTNP group was significantly lower than that in HIV and AIDS group (P<0.05). The frequency of CD4+CD25+Foxp3+ regulatory T cells was inversely related to CD4+ T cells and closely related to viral load and CD38, CD95 expression on CD4, CD8+ T cells (P<0.05). Conclusion The frequency of CD4+CD25+Foxp3+ regulatory T cells of HIV infected LTNP is significantly lower than typical progressors, which indicates that alternation of regulatory T cells may play a protective role in LTNP.

Objective To detect the alternation of the level of CTLA-4 expression in T cells and regulatory T cells, and to study the relationship between CTLA-4 expression in T cells and regulatory T cells and disease progression of HIV infected Chinese. Methods Fifty-eight untreated HIV-1 infected patients were enrolled and the level of CTLA-4 expression in T cells and regulatory T cells were detected by flowcytometry. CD4+T cell numbers, viral load, level of CD95, HLA-DR, CD38 expression on T cells were measured to study the relationship between the level of CTLA-4 expression and disease progression of HIV infected patients. Results We found that the level of CTLA-4 expression in CD4+T cells continuously increased in long term nonprogressors (LTNP), asymptomatic HIV infected patients (HIV) and AIDS patients (P<0.05). The level of CTLA-4 expression in CD4+T cells was significantly correlated with CD4+T cell counts, the frequency of CD8+ CD38+T cells, CD4+CD95+T cells and CD8+CD95+T cells (P<0.05). There had no difference in the level of CTLA-4 expression in CD8+T cells among all groups and neither did we find the relationship between the level of CTLA-4 expression on CD8+ T cells and the CD4 counts, viral load, activation or apoptosis of T cells. The level of CTLA-4 expression in regulatory T cells of LTNP group was significantly lower than that of HIV and AIDS group (P<0.05). The level of CTLA-4 expression in regulatory T cells was significantly correlated with CD4 counts, the frequency of CD4+HLA-DR+T cells and CD8+HLA-DR+T cells (P<0.05). Conclusion The level of CTLA-4 expression in CD4+T cells and regulatory T cells is correlated with disease progression and the level of the activation of immune system of HIV infected Chinese and may play a role in the immune balance in HIV infection.