HIV-1 P24 Research Articles

Outcomes of acutely HIV-1-infected individuals following rapid antiretroviral therapy initiation.

Few data exist on the benefits and acceptability of rapid initiation of antiretroviral treatment in acute HIV infection (AHI). We analysed a large cohort of acutely infected HIV patients starting antiretroviral therapy (ART) to determine uptake, linkage into care and time to achieve viral suppression. Case notes of all individuals diagnosed with AHI between May 2014 and October 2015 at 56 Dean Street, a sexual health clinic in London, UK were reviewed. AHI was defined through documentation of plasma HIV RNA positivity only, plasma HIV RNA and p24 antigen positivity with a negative HIV enzyme immunoassay (EIA) test or HIV EIA test switching from negative to positive within 6 weeks. Between-group comparisons of time to viral suppression according to ART chosen were performed using the log-rank test. We identified 113 individuals with AHI. Linkage to care was 95%. 77% of patients started ART at first medical appointment: all men who have sex with men, median age 35 years, median viral load (VL) log10 6.45, median CD4+ T-cell count 483 cells/mm3. Median time from diagnosis to ART initiation was 20 days. At 24 weeks, no patients had discontinued ART; 99% of patients achieved viral suppression by 24 weeks, with a median time to documented VL suppression of 74 days. Viral suppression was more rapid with integrase inhibitors compared with other regimens (median 41 versus 88.5 days, P<0.05). In acute HIV infection, individuals demonstrated high ART uptake and rapid VL suppression suggesting that early treatment with antiretrovirals is acceptable and efficacious.

T cells with low CD2 levels express reduced restriction factors and are preferentially infected in therapy naïve chronic HIV-1 patients.

Introduction: Restriction factors (RFs) suppress HIV-1 in cell lines and primary cell models. Hence, RFs might be attractive targets for novel antiviral strategies, but their importance for virus control in vivo is controversial. Methods: We profiled the expression of RFs in primary blood-derived mononuclear cells (PBMC) from therapy-naïve HIV-1 patients and quantified infection. Results: Overall, there was no correlation between individual RF expression and HIV-1 status in total PBMC. However, we identified a T cell population with low levels of intracellular CD2 and reduced expression of SAMHD1, p21 and SerinC5. CD2low T cells with reduced RF expression were markedly positive for HIV-1 p24. In contrast, CD2+ T cells were less infected and expressed higher levels of RFs. CD2low T cell infection correlated with viral loads and was associated with HIV-1 disease progression. Conclusions: In untreated therapy naïve chronic HIV-1 patients, RF expression in T cells is associated with CD2 expression and seems to influence viral loads. Our study suggests that RFs help to control HIV-1 infection in certain T cells in vivo and supports the potential for RFs as promising targets for therapeutic intervention.

Effects of heme degradation products on reactivation of latent HIV-1.

Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.

Peptidic β-sheets induce Congo red-derived fluorescence to improve the sensitivity of HIV-1 p24 detection

We employ a peptide which contains a 2-phenylalanine core component to efficiently form β-sheets, which can combine with Congo red molecules and produce strong fluorescence with high sensitivity to ensure an earlier diagnosis of HIV infection.

Fluorescent silver nanoparticle based highly sensitive immunoassay for early detection of HIV infection

Fluorescent silver nanoparticles have been employed in an immunoassay to detect HIV p24 antigen with high sensitivity in the linear range of 10–1000 pg mL−1.

Screening ssDNA aptamers against HIV P24 antigen using agarose beads as carriers

In this study, carboxylated agarose beads were used as screening carriers combined with the subtractive systematic evolution of ligands by exponential enrichment (SELEX) technology to obtain an ssDNA secondary library, which could specifically bind to HIV P24 antigen after nine screening rounds from a random ssDNA library. An electrophoretic mobility shift assay (EMSA) and quantitative real-time PCR assay (RT-qPCR) was performed to monitor in real-time the binding specificity of the ssDNA secondary library to the HIV P24 antigen. The obtained ssDNA secondary library was converted to the corresponding dsDNA library, ligated to a PMD18-T vector, and transformed into E. coli DH5α and sequenced. The results demonstrated that after nine rounds of screening, two aptamer sequences specifically bound to the HIV P24 antigen were obtained.

Centrifugal Microfluidic Platform for Rapid, Multiplexed Detection of TB and HIV Biomarkers in Whole Blood Samples

Infection with Mycobacterium Tuberculosis represents a significant threat to people with immune disorders, such as HIV-positive individuals, and can result in significant health complications or death if not diagnosed and treated early. We present a centrifugal microfluidic platform for multiplexed detection of tuberculosis and HIV biomarkers in human whole blood with minimal sample preparation and a sample-to-answer time of 30 minutes. This multiplexed assay was developed for the detection of two M.tuberculosis secreted proteins, whose secretion represents an active and ongoing infection, as well as detection of HIV p24 protein and human anti-p24 antibodies. The limit of detection for this multiplex assay is in the pg/mL range for both HIV and M.tuberculosis proteins, making this assay potentially useful in the clinical diagnosis of both HIV and Tuberculosis proteins indicative of active infection. Antigen detection for the HIV assay sensitivity was 89%, the specificity 85%. Serological detection had 100% sensitivity and specificity for the limited sample pool. The centrifugal microfluidic platform presented here offers the potential for a portable, fast and inexpensive multiplexed diagnostic device that can be used in resource-limited settings for diagnosis of TB and HIV.

Introducing a cleavable signal peptide enhances the packaging efficiency of lentiviral vectors pseudotyped with Japanese encephalitis virus envelope proteins

Research into the properties of Japanese encephalitis virus (JEV) has been facilitated by use of pseudotyped viruses. The signal peptide is a key determinant for membrane targeting and membrane insertion, which could affect packaging of pseudotyped viruses. In this study, we generated three lentiviral vectors pseudotyped with JEV envelope proteins that co-express either a strong signal peptide from vesicular stomatitis virus (VSV)-G (VSVMEpv) or a weak signal peptide of JEV (SPMEpv), or a virus without a signal peptide in front of the JEV prM/E (MEpv). Western blot demonstrated that JEV E protein and HIV p24 were present in the same particles of the three pseudotyped JEV-E based lentiviral vectors. Electron microscopy revealed that the three pseudotyped JEV-E based lentiviral vectors were 120–180nm in diameter. Real-time quantitative reverse transcriptase polymerase chain reaction showed that the titer of VSVMEpv was 17-fold higher than that of MEpv, while the titer of SPMEpv was six-fold higher than that of MEpv. Inclusion of a signal peptide enhanced packaging efficiency of pseudotyped JEV-E based lentiviral vectors. With a strong signal peptide helping they generate a higher number of viral particles. Green fluorescent protein and luciferase expression showed that the transduction titer or relative fluorescence units of VSVMEpv, SPMEpv and MEpv were not significantly different. We suggest that the signal peptide does not influence the infectivity of pseudotyped JEV-E based lentiviral vectors. In addition, our findings indicated that pseudotyped JEVs show preferential tropism for BHK-21 cells, supporting the mimic function displayed by parental JEV. Therefore, our study provided a cost-effective method to generate pseudotyped JEV-E based lentiviral vectors, which may represent a valid model to investigate some of the infectious properties of JEV.

Array-in-well binding assay for multiparameter screening of phage displayed antibodies.

Phage display is a well-established and powerful tool for the development of recombinant antibodies. In a standard phage display selection process using a high quality antibody phage library, a large number of unique antibody clones can be generated in short time. However, the pace of the antibody discovery project eventually depends on the methodologies used in the next screening phase to identify the clones with the most promising binding characteristics e.g., in terms of specificity, affinity and epitope. Here, we report an array-in-well binding assay, a miniaturized and multiplexed immunoassay that integrates the epitope mapping to the evaluation of the binding activity of phage displayed antibody fragments in a single well. The array-in-well assay design used here incorporates a set of partially overlapping 15-mer peptides covering the complete primary sequence of the target antigen, the intact antigen itself and appropriate controls printed as an array with 10×10 layout at the bottom of a well of a 96-well microtiter plate. The streptavidin-coated surface of the well facilitates the immobilization of the biotinylated analytes as well-confined spots. Phage displayed antibody fragments bound to the analyte spots are traced using anti-phage antibody labelled with horseradish peroxidase for tyramide signal amplification based highly sensitive detection. In this study, we generated scFv antibodies against HIV-1 p24 protein using a synthetic antibody phage library, evaluated the binders with array-in-well binding assay and further classified them into epitopic families based on their capacity to recognize linear epitopes. The array-in-well assay enables the integration of epitope mapping to the screening assay for early classification of antibodies with simplicity and speed of a standard ELISA procedure to advance the antibody development projects.

HIV persists in CCR6+CD4+ T cells from colon and blood during antiretroviral therapy.

Objectives:The objective of this article is to investigate the contribution of colon and blood CD4+ T-cell subsets expressing the chemokine receptor CCR6 to HIV persistence during antiretroviral therapy.Design:Matched sigmoid biopsies and blood samples (n = 13) as well as leukapheresis (n = 20) were collected from chronically HIV-infected individuals receiving antiretroviral therapy. Subsets of CD4+ T cells with distinct differentiation/polarization profiles were identified using surface markers as follows: memory (TM, CD45RA−), central memory (TCM; CD45RA−CCR7+), effector (TEM/TM; CD45RA−CCR7−), Th17 (CCR6+CCR4+), Th1Th17 (CCR6+CXCR3+), Th1 (CCR6−CXCR3+), and Th2 (CCR6−CCR4+).Methods:We used polychromatic flow cytometry for cell sorting, nested real-time PCR for HIV DNA quantification, ELISA and flow cytometry for HIV p24 quantification. HIV reactivation was induced by TCR triggering in the presence/absence of all-trans retinoic acid.Results:Compared with blood, the frequency of CCR6+ TM was higher in the colon. In both colon and blood compartments, CCR6+ TM were significantly enriched in HIV DNA when compared with their CCR6− counterparts (n = 13). In blood, integrated HIV DNA levels were significantly enriched in CCR6+ versus CCR6− TCM of four of five individuals and CCR6+ versus CCR6− TEM of three of five individuals. Among blood TCM, Th17 and Th1Th17 contributed the most to the pool of cells harboring integrated HIV DNA despite their reduced frequency compared with Th2, which were infected the least. HIV reactivation was induced by TCR triggering and/or retinoic acid exposure at higher levels in CCR6+ versus CCR6− TM, TCM, and TEM.Conclusion:CCR6 is a marker for colon and blood CD4+ T cells enriched for replication-competent HIV DNA. Novel eradication strategies should target HIV persistence in CCR6+CD4+ T cells from various anatomic sites.

Humoral Antibody Responses to HIV Viral Proteins and to CD4 Among HIV Controllers, Rapid and Typical Progressors in an HIV-Positive Patient Cohort.

The purpose of this study was to assess humoral antibody responses as a function of disease progression (DP) in a well-defined HIV+ cohort. We quantified antibodies to HIV-1 gp120, Gag, and CD4 receptor by enzyme-linked immunosorbent assay in sera from a cohort of 97 HIV+ subjects at defined stages of DP. We also measured antibody-dependent cellular cytotoxicity (ADCC) as a function of the clinical status of the patients. We purified antibodies to CD4 and gp120 and assessed them for specificity, ability to block gp120 binding to target cells, ability to block virus infection, and ability to facilitate ADCC. All of the HIV+ patient samples were positive for antibodies to HIV gp120 and p24 and 80% showed evidence of hypergammaglobulinemia. Approximately 10% of cohort members were positive for antibodies to CD4, but we noted no significant correlation relevant to DP. There were statistically significant differences between the groups concerning the level of humoral response to gp120 and Gag. However, we observed no distinction in ability of anti-gp120 antibodies purified from each group to neutralize infection. In addition, there was a statistically significant difference in ADCC, with elite controllers exhibiting significantly lower levels of ADCC than the other five groups. We detected IgA anti-gp120 antibodies, but did not correlate their presence with either DP or ADCC levels. The results are consistent with the interpretation that the humoral antibody response to the antigens assessed here represents a signature of the level of viremia but does not correlate with clinical status of HIV infection.

Development of carbon dot based microplate and microfluidic chip immunoassay for rapid and sensitive detection of HIV-1 p24 antigen

A highly sensitive, precisely specific, environmentally friendly, high-throughput, microwell-plate and microchip-based sandwich assay was developed to detect HIV-1 p24 antigen, a protein biomarker using fluorescent carbon dots. High quantum yield carbon dots were synthesized using citric acid and ethylenediamine as carbon and nitrogen sources by a single-step hydrothermal reaction. The desired amine groups confirmed by FTIR on the carbon dots were coupled to streptavidin by amine–amine coupling reaction using glutaraldehyde. The detection range of the carbon dot based immunoassay (CDIA) was found to be between 20 and 1000 pg/mL in a linear dose-dependent manner. CDIA tested for HIV negative plasma samples showed no false positive results in the detection of HIV-1 p24 antigen. The CDIA was extended to develop a microfluidic carbon dot immunoassay (μCDIA) which exhibited analytical sensitivity in the range of 30–1000 pg/mL. The CDIA and μCDIA can easily be adapted to a lab-on-a-chip platform for use in resource limited settings and can also be multiplexed for the detection of other pathogens like TB and Hepatitis.

Effect of Glu12-His89 Interaction on Dynamic Structures in HIV-1 p17 Matrix Protein Elucidated by NMR.

To test the existence of the salt bridge and stability of the HIV-1 p17 matrix protein, an E12A (mutated at helix 1) was established to abolish possible electrostatic interactions. The chemical shift perturbation from the comparison between wild type and E12A suggested the existence of an electrostatic interaction in wild type between E12 and H89 (located in helix 4). Unexpectedly, the studies using urea denaturation indicated that the E12A substitution slightly stabilized the protein. The dynamic structure of E12A was examined under physiological conditions by both amide proton exchange and relaxation studies. The quick exchange method of amide protons revealed that the residues with faster exchange were located at the mutated region, around A12, compared to those of the wild-type protein. In addition, some residues at the region of helix 4, including H89, exhibited faster exchange in the mutant. In contrast, the average values of the kinetic rate constants for amide proton exchange for residues located in all loop regions were slightly lower in E12A than in wild type. Furthermore, the analyses of the order parameter revealed that less flexible structures existed at each loop region in E12A. Interestingly, the structures of the regions including the alpha1-2 loop and helix 5 of E12A exhibited more significant conformational exchanges with the NMR time-scale than those of wild type. Under lower pH conditions, for further destabilization, the helix 1 and alpha2-3 loop in E12A became more fluctuating than at physiological pH. Because the E12A mutant lacks the activities for trimer formation on the basis of the analytical ultra-centrifuge studies on the sedimentation distribution of p17 (Fledderman et al. Biochemistry 49, 9551–9562, 2010), it is possible that the changes in the dynamic structures induced by the absence of the E12-H89 interaction in the p17 matrix protein contributes to a loss of virus assembly.

Control of early HIV-1 infection associates with plasmacytoid dendritic cell-reactive opsonophagocytic IgG antibodies to HIV-1 p24.

We have previously demonstrated that HIV-1 p24-specific plasmacytoid dendritic cell-reactive opsonophagocytic antibody (PROAb) responses associate with control of chronic HIV infection. Here, we examined whether HIV-1 p24-specific PROAbs associate with control of early HIV infection and their relationship with HIV-1 p24-specific IgG subclasses. Plasma collected at 8 and 52 weeks following primary HIV-1 infection was obtained from antiretroviral therapy-naïve patients who were classified as 'good' (plasma HIV-1 RNA < 5000 copies/ml; n = 17) or 'poor' (HIV-1 RNA > 50 000 copies/ml; n = 15) controllers at week 52. HIV-1 p24-specific PROAb responses were assayed using a plasmacytoid dendritic cell line (Gen2.2), and HIV-1 p24-specific IgG3, IgG1 and IgG2 levels were assayed by ELISA. HIV-1 p24-specific PROAb responses increased in 'good controllers' (P = 0.01) but remained unchanged in 'poor controllers' between weeks 8 and 52. Of the HIV-1 p24-specific IgG subclasses measured, only IgG1 increased over this time period in 'good controllers' alone (P = 0.003), which correlated with the increase in HIV-1 p24-specific PROAb responses (r = 0.83, P < 0.0001). Depletion of IgG1 from IgG preparations of 'good controllers' resulted in the inhibition of HIV-1 p24-specific PROAb responses. In the total patient cohort, plasma HIV-1 RNA levels at week 52 correlated inversely with changes in HIV-1 p24-specific PROAb responses (r = -0.37, P = 0.04) and IgG1 (r = -0.51, P = 0.003) levels between weeks 8 and 52. Control of early HIV-1 infection was associated with an increase in HIV-1 p24-specific PROAb responses, which was mediated by HIV-1 p24-specific IgG1 antibodies. These findings provide further evidence that antibodies to HIV core proteins may contribute to control of HIV-1 infection.

Immune Activation at Sites of HIV/TB Co-Infection Contributes to the Pathogenesis of HIV-1 Disease.

Systemic immune activation is critical to the pathogenesis of HIV-1 disease, and is accentuated in HIV/TB co-infected patients. The contribution of immune activation at sites of HIV/TB co-infection to viral activity, CD4 T cell count, and productive HIV-1 infection remain unclear. In this study, we measured markers of immune activation both in pleural fluid and plasma, and in T cells in pleural fluid mononuclear cell (PFMC) and peripheral blood mononuclear cell (PBMC) in HIV/TB co-infected subjects. The relationship between soluble and T cell activation markers with viral load in pleural fluid and blood CD4 T cell count were assessed. The T cell phenotype and activation status of HIV-1 p24 + T cells in PFMC and PBMC from HIV/TB patients were determined. We found that T cell and macrophage-specific and non-specific soluble markers of immune activation, sCD27, sCD163, IL1Ra, and sCD14, were higher in pleural fluid as compared to plasma from HIV/TB co-infected subjects, and higher as compared to pleural fluid from TB mono-infected subjects. Intestinal fatty acid-binding protein, a marker of intestinal tract damage, in plasma from HIV/TB co-infected patients was not different than that in HIV+ subjects. Expression of HLADR and CD38 double positive (HLADR/CD38) on CD4 T cells, and CD69+ on CD8 T cells correlated with pleural fluid viral load, and inversely with blood CD4 T cell count. Higher expression of HLADR/CD38 and CCR5 on CD4 T cells, and HLADR/CD38 and CD69 on CD8 T cells in PFMC were limited to effector memory populations. HIV-1 p24+ CD8 negative (includes CD4 + and double negative T cells) effector memory T cells in PFMC had higher expression of HLADR/CD38, Ki67, and CCR5 compared to HIV-1 p24- CD8 negative PFMC. Cumulatively, these data indicate that sites of HIV/TB co-infection are the source of intense immune activation.

Acetone and methanol fruit extracts of Terminalia paniculata inhibit HIV-1 infection in vitro

In this study, we report the in vitro anti-HIV1 activity of acetone and methanol extracts of fruit of Terminalia paniculata. Cytotoxicity tests were conducted on TZM-bl cells and peripheral blood mononuclear cells (PBMC), the CC50 values of both the extracts were ≥260 μg/mL. Using TZM-bl cells, the extracts were tested for their ability to inhibit replication of two primary isolates HIV-1 (X4, Subtype D) and HIV-1 (R5, Subtype C). The activity against HIV-1 primary isolate (R5, Subtype C) was confirmed using activated PBMC and by quantification of HIV-1 p24 antigen. Both the extracts showed anti-HIV1 activity in a dose-dependent manner. The EC50 values of the acetone and methanol extracts of T. paniculata were ≤10.3 μg/mL. The enzymatic assays were performed to determine the mechanism of action which indicated that the anti-HIV1 activity might be due to inhibition of reverse transcriptase (≥77.7% inhibition) and protease (≥69.9% inhibition) enzymes.

A Spirulina maxima-derived peptide inhibits HIV-1 infection in a human T cell line MT4

Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS). Anti-HIV agents targeting various steps in HIV life cycle have been developed; however, so far, no effective drugs have been found. We show here that a peptide isolated from Spirulina maxima (SM-peptide) inhibits HIV-1 infection in a human T cell line MT4. SM-peptide inhibited HIV-1IIIB-induced cell lysis with a half-maximal inhibitory concentration (IC50) of 0.691 mM, while its 50 % cytotoxic concentration (CC50) was greater than 1.457 mM. Furthermore, the SM-peptide inhibited the HIV-1 reverse transcriptase activity and p24 antigen production. This suggests that SM-peptide is a novel candidate peptide, which may be developed as a therapeutic agent for acquired immunodeficiency syndrome patients.

Denovo HIV infection diagnoses in a Department of Dermatology and Venereology in Lisbon, Portugal.

Background Portugal has the highest prevalence rate of HIV infection in Western Europe. The proportion of patients with a late diagnosis, carried out in full-blown AIDS stage, remains high. Skin and mucous membrane manifestations are not rare in these patients. Objective A demographic, clinical, and laboratorial characterization of patients with de novo HIV infection diagnosis made in the Department of Dermatology and Venereology of a central hospital in Lisbon, Portugal. Methods Retrospective review of medical records of adult patients newly diagnosed with HIV infection (reactive immunoassay for antibodies to HIV-1/HIV-2 or HIV p24 antigen) in the Dermatology and Venereology Department of a Portuguese central hospital in the period between January 2005 and December 2013. Results During the study period, 97 new cases were diagnosed, 70 men and 27 women. The median age at diagnosis was 36 years. Of the total, 50 cases were diagnosed with a concomitant sexually transmitted infection (STI), more frequently syphilis and ano-genital HPV infection. The remaining 47 patients were diagnosed with other dermatological conditions such as prurigo nodularis, psoriasis, and Kaposi's sarcoma. The duration of complaints that lead to medical attention ranged from <1 week to 8 years, being significantly lower in patients diagnosed with a concomitant STI ( p < 0.01). Basal viral load was also lower in this group of patients ( p < 0.05). Of all the new diagnosed cases, 80% of patients are currently retained in care. Conclusion This study grants a descriptive overview of de novo HIV infection diagnoses performed by dermatovenereologists in a central hospital in Lisbon, Portugal. As in the past, the importance of Dermato-Venereology for HIV infection diagnosis remains present in daily clinical practice.

Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China.

The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560–616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.

Expansion and Productive HIV-1 Infection of Foxp3 Positive CD4 T Cells at Pleural Sites of Hiv/Tb Co-Infection

CD4 T-cells expressing Foxp3 are expanded systemically during active tuberculosis (TB) regardless of HIV-1 co-infection. Foxp3+ CD4 T cells are targets of HIV-1 infection. However, expansion of HIV-1 infected Foxp3+ CD4 T cells at sites of HIV/TB co-infection, and whether they contribute to promotion of HIV-1 viral activity is not known. Pleural fluid mononuclear cells (PFMC) from HIV/TB co-infected patients with pleural TB were characterized by immune-staining and FACS analysis for surface markers CD4, CD127, CCR5, CXCR4, HLA-DR and intracellular expression of Foxp3, HIVp24, IFN-γ and Bcl-2. Whole PFMC and bead separated CD4+CD25+CD127- T cells were assessed for HIV-1 LTR strong stop (SS) DNA by real-time PCR, which represents viral DNA post cell entry and initiation of reverse transcription. High numbers of HIV-1 p24 positive Foxp3+ and Foxp3+CD127- CD4 T cells were identified in PFMC from HIV/TB co-infected subjects. CD4+Foxp3+CD127- T cells displayed high expression of the cellular activation marker, HLA-DR. Further, expression of the HIV-1 co-receptors, CCR5 and CXCR4, were higher on CD4+Foxp3+T cells compared to CD4+Foxp3- T cells. Purified CD4+CD25+CD127- T cells isolated from PFMC of HIV/TB co-infected patients, were over 90% CD4+Foxp3+T cells, and exhibited higher HIV-1 SS DNA as compared to whole PFMC, and as compared to CD4+CD25+CD127- T cells from an HIV-infected subject with pleural mesothelioma. HIV-1 p24+ Foxp3+ CD4+T cells from HIV/TB patients higher in Bcl-2 expression as compared to both HIV-1 p24+ Foxp3- CD4 T cells, and Foxp3+ CD4+T cells without HIV-p24 expression. Foxp3+ CD4 T cells in PFMC from HIV/TB co-infected subjects are predisposed to productive HIV-1 infection and have survival advantage as compared to Foxp3 negative CD4 T cells.