Evaluation Of The Different Algorithms For Detecting Syphilis In HIV-infected People And Study Of The Role Of Treponema Pallidum Membrane Protein Tp47 On HIV Transmission Through The Genital Tract

In recent years,the incidence of syphilis has increased significantly,especially among HIV-infected people.Currently,there are three algorithms for screening of syphilis:traditional algorithm,reverse algorithm and European Centre for Disease Prevention and Control(ECDC)algorithm.No studies have analyzed the different algorithms for detecting syphilis in HIV-positive people.Previous studies have shown that syphilis infection can increase the susceptibility to HIV,but the mechanism of how syphilis promote HIV infection is not clear.Therefore,we conducted the following research:Part 1.We conducted a cross-sectional study to assess the impact of different syphilis screening algorithms in a HIV-positive population.Sample size was estimated to be 677 using N=Z2?P(1-P)/?2,assuming 19.8%syphilis prevalence in HIV infected patients,with 3%precision and 95%level of confidence.Each sample was simultaneously tested for Toluidine red unheatedserum test(TRUST),Treponema pallidum particle agglutination(TPPA),and Treponema pallidum enzyme-linked immunosorbent assay(TP-ELISA).The results of seroprevalence of syphilis were interpreted following different algorithms respectively.Among the 865 HIV-infected patients collected,the seroprevalence of syphilis was 24.9%in the reverse screening process,and the seroprevalence of syphilis was 14.2%in the traditional screening procedure.The difference between them was statistically significant.(p<0.0001).The overall compliance rate between the reverse screening process and the ECDC screening process was 99.8%.Part 2.In the serological test of syphilis,there are problems such as serum fixation and false negatives due to the absence of antibodies at the initial stage of infection.In order to establish a method for detecting antigen of Treponema pallidum to assist in the diagnosis of syphilis,the monoclonal antibody against Treponema pallidum membrane protein Tp47 was prepared in this study.Firstly,Tp47 protein was expressed and purified by gene recombination technology to immunize BALA/c mice,and 5 monoclonal antibodies against Tp47 protein were obtained by hybridoma fusion technology.Isotype of the 1E2B4 clone was of IgG2a,and the other were of IgG1.Among them,the 1E2B4 clone had the highest antibody titer and was up to 1:4x105 measured by ELISA.According to Western Blot and immunoprecipitation experiments,these mAbs can specifically recognize the target antigen,The ELISA and colloidal gold strip were established to detect recombinant Tp47 protein by using 1B3F2 clone and 1E2B4 clone.The sensitivity of the ELISA was 6 ng/ml,which had good specificity and potential clinical application value.Part 3.In order to explore the effects of Treponema pallidum infection on HIV sexual infection and related mechanisms,we stimulated Endl/E6E7 cells by using Tp47-the major membrane protein of Treponema pallidum to mimic the effect of Treponema pallidum on HIV transmission in the genital tract.After stimulation with Tp47 protein,Endl/E6E7 cells showed increased expression of IL-6 and IL-8 at the protein and mRNA levels(p<0.05),and increased intracellular viral antigen levels after HIV infection(p<0.05).The mRNA seq was employed to screen DEGs between the tp47 pretreatment group and the simple virus-infected group.The biological process involved in the DEGs were mainly enriched in "regulating cytokine synthesis","regulating immune-related cytokines","cell movement",and "protein import".Their molecular function were mainly related to " cytokine receptor binding".The results of mRNA seq were highly reliable,which were verified by qPCR,In summary:(1)We advocate the reverse algorithm(or the ECDC)for the screening of syphilis in HIV-infected populations.The tradition algorithm underestimates the seroprevalence of syphilis in HIV-infected individuals.(2)We obtained 5 anti-Tp47 monoclonal antibodies,which can recognize Tp47 protein sensitively and specifically.Using the autonomous developed anti-Tp47 mAb,the ELISA and colloidal gold test strip were established to detect recombinant Tp47 protein,which have good application prospects in the diagnosis of syphilis(3)Treponema pallidum membrane protein Tp47 may enhance the transmission of HIV through the reproductive tract by promoting the expression of inflammatory factors.