Urine assay for tenofovir to monitor adherence in real time to tenofovir disoproxil fumarate/emtricitabine as pre-exposure prophylaxis.

OBJECTIVES: Tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) is approved for pre-exposure prophylaxis (PrEP) against HIV infection. Adherence is critical for the success of PrEP, but current adherence measurements are inadequate for real-time adherence monitoring. We developed and validated a urine assay to measure tenofovir (TFV) to objectively monitor adherence to PrEP. METHODS: We developed a urine assay using high-performance liquid chromatography coupled to tandem mass spectrometry with high sensitivity/specificity for TFV that allowed us to determine TFV concentrations in log10 categories between 0 and 10 000 ng/mL. We validated the assay in three cohorts: (1) HIV-positive subjects with undetectable viral loads on a TDF/FTC-based regimen, (2) healthy HIV-negative subjects who received a single dose of TDF/FTC, and (3) HIV-negative subjects receiving daily TDF/FTC as PrEP for 24 weeks. RESULTS: The urine assay detected TFV with greater sensitivity than plasma-based measures and with a window of measurements within 7 days of the last TDF/FTC dose. Based on the urine log-linear clearance after the last dose and its concordance with all detectable plasma levels, a urine TFV concentration > 1000 ng/mL was identified as highly predictive of the presence of TFV in plasma at > 10 ng/mL. The urine assay was able to distinguish high and low adherence patterns within the last 48 h (> 1000 ng/mL versus 10-1000 ng/mL), as well as nonadherence (< 10 ng/mL) extended over at least 1 week prior to measurement. CONCLUSIONS: We provide proof of concept that a semiquantitative urine assay measuring levels of TFV could be further developed into a point-of-care test and be a useful tool to monitor adherence to PrEP.

HCV viraemia associates with NK cell activation and dysfunction in antiretroviral therapy-treated HIV/HCV-co-infected subjects.

The impact of hepatitis C virus (HCV) RNA levels on immune status in chronically HCV mono-infected when compared to HIV/HCV co-infected on antiretroviral therapy (ART) remains poorly understood. A total of 78 African American subjects HCV viraemic/naïve to HCV treatment (33 HCV genotype 1 mono-infected, 45 ART-treated HIV/HCV genotype 1 co-infected) were studied. Clinical and liver enzyme measurements were performed. Whole blood was analysed for immune subset changes by flow cytometry. Peripheral blood mononuclear cells (PBMC) were used for same-day constitutive and in vitro Interferon (IFN)-α-induced signal transducer and activator of transcription (STAT) phosphorylation, K562 target cell lysis and K562 target cell recognition-mediated IFN-γ production. Statistical analysis was performed using R (2.5.1) or JMP Pro 11. While both groups did not differ in the level of liver enzymes, HIV/HCV had higher T-cell activation/exhaustion, and constitutive STAT-1 phosphorylation compared to HCV. In contrast, CD4+ FoxP3+ CD25+ frequency, IFN-αR expression on NK cells, as well as constitutive and IFN-α-induced direct cytotoxicity were lower in HIV/HCV. Linear regression models further supported these results. Finally, increase in HCV viral load and CD4+ T-cell count had an opposite effect between the two groups on NK cell activity and T-cell activation, respectively. HCV viral load in ART-treated HIV/HCV co-infection was associated with greater immune activation/exhaustion and NK dysfunction than HCV viral load alone in HCV mono-infection. The more pronounced immune modulation noted in ART-treated HIV-co-infected/untreated HCV viraemic subjects may impact HCV disease progression and/or response to immunotherapy.

Evidence for the innate immune response as a correlate of protection in human immunodeficiency virus (HIV)-1 highly exposed seronegative subjects (HESN).

The description of highly exposed individuals who remain seronegative (HESN) despite repeated exposure to human immunodeficiency virus (HIV)-1 has heightened interest in identifying potential mechanisms of HIV-1 resistance. HIV-specific humoral and T cell-mediated responses have been identified routinely in HESN subjects, although it remains unknown if these responses are a definitive cause of protection or merely a marker for exposure. Approximately half of HESN lack any detectible HIV-specific adaptive immune responses, suggesting that other mechanisms of protection from HIV-1 infection also probably exist. In support of the innate immune response as a mechanism of resistance, increased natural killer (NK) cell activity has been correlated with protection from infection in several high-risk cohorts of HESN subjects, including intravenous drug users, HIV-1 discordant couples and perinatally exposed infants. Inheritance of protective NK KIR3DL1(high) and KIR3DS1 receptor alleles have also been observed to be over-represented in a high-risk cohort of HESN intravenous drug users and HESN partners of HIV-1-infected subjects. Other intrinsic mechanisms of innate immune protection correlated with resistance in HESN subjects include heightened dendritic cell responses and increased secretion of anti-viral factors such as ß-chemokines, small anti-viral factors and defensins. This review will highlight the most current evidence in HESN subjects supporting the role of epithelial microenvironment and the innate immune system in sustaining resistance against HIV-1 infection. We will argue that as a front-line defence the innate immune response determines the threshold of infectivity that HIV-1 must overcome to establish a productive infection.

Interleukin 13 inhibits human immunodeficiency virus type 1 production in primary blood-derived human macrophages in vitro.

The mechanisms by which cellular immunity maintains the asymptomatic state after human immunodeficiency virus type 1 (HIV-1) infection are poorly understood. CD4+ T lymphocytes play a complex role in regulating anti-HIV effector pathways, including activation of macrophages, which are themselves implicated in clinical latency and pathogenesis of symptomatic acquired immune deficiency syndrome. We have found that a newly identified T helper type 2 lymphokine, interleukin 13 (IL-13), inhibits HIV-1ADA and Ba-L replication in primary tissue culture-derived macrophages but not in peripheral blood lymphocytes. Viral production in cells was measured by viral protein (p24) and reverse transcriptase levels, while entry was assessed by proviral DNA analysis at timed intervals after infection. Inhibition by IL-13 was dose and time dependent and not mediated through altered viral entry, reverse transcription, or viral release. IL-13 is therefore a candidate cytokine for the suppression of HIV infection within monocytes and macrophages in vivo.

HIV-1-negative female sex workers sustain high cervical IFNɛ, low immune activation, and low expression of HIV-1-required host genes.

Sex workers practicing in high HIV endemic areas have been extensively targeted to test anti-HIV prophylactic strategies. We hypothesize that in women with high levels of genital exposure to semen changes in cervico-vaginal mucosal and/or systemic immune activation will contribute to a decreased susceptibility to HIV-1 infection. To address this question, we assessed sexual activity and immune activation status (in peripheral blood), as well as cellular infiltrates and gene expression in ectocervical mucosa biopsies in female sex workers (FSWs; n=50), as compared with control women (CG; n=32). FSWs had low-to-absent HIV-1-specific immune responses with significantly lower CD38 expression on circulating CD4(+) or CD8(+) T-cells (both: P<0.001) together with lower cervical gene expression of genes associated with leukocyte homing and chemotaxis. FSWs also had increased levels of interferon-ɛ (IFNɛ) gene and protein expression in the cervical epithelium together with reduced expression of genes associated with HIV-1 integration and replication. A correlative relationship between semen exposure and elevated type-1 IFN expression in FSWs was also established. Overall, our data suggest that long-term condomless sex work can result in multiple changes within the cervico-vaginal compartment that would contribute to sustaining a lower susceptibility for HIV-1 infection in the absence of HIV-specific responses.

Proinflammatory response and IL-12 expression in HIV-1 infection.

HIV-1 infection elicits a broad range of host responses, many of which interfere with the regulatory pathways of gene expression of interleukin-12 (IL-12), a heterodimeric cytokine essential for cell-mediated immunity against microbial infection. The inhibition of IL-12 production by accessory cells after HIV-1 infection has been identified as a potential factor responsible for impaired innate and Th1 cell-mediated responses observed in AIDS patients. The mechanism by which HIV-1 infection suppresses IL-12 gene expression is largely uncharacterized. Here we review all pathways identified that could potentially mediate HIV-induced impairment of IL-12 gene expression, such as IL-10, transforming growth factor beta, interferon-alpha/beta, tumor necrosis factor alpha, Fc receptors, complement regulatory proteins, and receptors. Also discussed is the decreased CD40 ligand induction in CD4 T cells during HIV infection, which may have a strong impact on T cell-dependent IL-12 production that is critical for the establishment and maintenance of a Th1 response.

Chemokine immunobiology in HIV-1 pathogenesis.

The discovery of HIV-1-suppressive chemokines and the subsequent discovery of their cognate receptors as coreceptors for HIV-1 entry herald a paradigmatic shift in the study of HIV-1 pathogenesis. The presence of polymorphisms in chemokine receptor and chemokine genes associated with altered progression and susceptibility to the HIV-1 disease further underscores the potential importance that chemokines and their cognate receptors play in HIV-1 pathogenesis. It has become increasingly apparent that the immune system maintains a delicate balance between the positive and negative regulators that govern the chemokine and cytokine networks. Here we review the most recent developments in chemokine biology and relate how research into their structure, regulation, and the mechanism of their actions can shed light on the immnunopathogenesis of HIV-1 disease.

IL-13 acts on macrophages to block the completion of reverse transcription, inhibit virus production, and reduce virus infectivity.

An understanding of the immune suppression of HIV-1 replication in macrophages continues to be a major goal of AIDS research due to the central role this cell type has in AIDS pathogenesis. We have previously discussed the potential clinical benefits of the anti-inflammatory cytokine interleukin-13 (IL-13), which, unlike IL-4 or IL-10, had limited effects on T cell functions. In this report are extend our observations on the effects of IL-13 on HIV-1 replication in monocyte-derived macrophages (MDM) and show redundancy with IL-4, IL-13 or IL-4 have similar effects on HIV-1 replication in MDM when added at different times after infection, with the ability to decrease infection virus release when added for up to 7 days after infection. Removal of IL-13 from MDM revealed a reduction of infection by 16- to 81-fold based on the absence of viral re-emergence from lower multiplicity of infection (m.o.i.). The reduction of HIV-1 infectivity in MDM caused by IL-13 was further characterized by studies on the formation of viral DNA over a range of m.o.i. IL-13 increased the formation of LTR DNA at the lowest m.o.i. of 0.007 while concurrently inhibiting the formation of gag DNA, a later reverse transcription product, at the highest m.o.i. tested, 0.62. Overall, our data indicate that IL-13 can act on macrophages before and after HIV-1 infection by blocking the completion of reverse transcription, decreasing virus production, and reducing the infectivity of the progeny virions.

HIV-1 Vpr regulates expression of beta chemokines in human primary lymphocytes and macrophages.

The HIV-1 vpr gene encodes a 14-kDa virion-packaged protein that has been implicated in viral pathogenesis. Vpr exhibits profound effects on human primary cells influencing proliferation, differentiation, apoptosis, and cytokine production, in part through NF-kappaB-mediated transcription. NF-kappaB, a potent transcription factor, activates many proinflammatory cytokines/chemokines upon infection. Here, we analyzed the effect of extracellular Vpr as well as the virion-associated Vpr on beta chemokines (MIP-1alpha, MIP-1beta, and RANTES) production in human macrophages and primary lymphocytes (PBLs). Macrophages and PBLs exposed to HIV-1 vpr+ viruses or to recombinant Vpr protein produced significantly less beta chemokines compared with cells infected with HIV-1 vpr-viruses or irrelevant control protein (Gag)-exposed cells. These results suggest that a Vpr-mediated increase in virus replication could be in part through down-regulation of chemokine production.

CCR5 and CXCR4 expression correlated with X4 and R5 HIV-1 infection yet not sustained replication in Th1 and Th2 cells.

OBJECTIVE: To investigate the infectivity of T-helper (Th)1 and Th2 cells (derived from ccr5 wild-type and homozygous ccr5 Delta 32) to R5 and X4 HIV-1. DESIGN: It remains unclear whether infection of Th1 and Th2 CD4 cells by R5 and X4 viruses mirrors their co-receptor expression profile as no direct quantitation of coreceptor levels and infection has been performed. In addition, it is unknown whether the lack of CCR5 expression affects the degree of Th1/Th2 polarization. METHODS: Surface expression of CCR5 and CXCR4 was determined by quantitative fluorescence activated cell sorter analysis on in vitro differentiated Th1 and Th2 cells. R5 (Ba-L) and X4 (IIIB) HIV-1 isolates were used for infection studies and the efficiency of viral entry was determined by quantitative real time polymerase chain reaction detection of reverse transcribed proviral DNA. RESULTS: Cell surface density of CCR5 molecules was eight-fold higher in Th1 versus Th2 subsets (P = 0.005) whereas CXCR4 surface density was four-fold higher in Th2 versus Th1 subsets (P = 0.006). Preferential infection and entry of Th1 cells by R5 HIV-1 was not associated with preferential replication, as eventually the R5-virus replicated to a higher level in Th2 cells in spite of lower initial viral infection/entry. By contrast, Th2 cells preferentially supported X4-virus infection and replication. High beta chemokine secretion by Th1 cells was associated with a lower R5 replication rate. CONCLUSIONS: Th1 and Th2 cells differ in their infection efficiency for R5 and X4 HIV-1. ccr5 Delta 32-homozygous individuals maintain the ability for Th1/Th2 polarization, i.e., the expression of CCR5 is not required for Th1/Th2 polarization.

Vpr-GFP virion particle identifies HIV-infected targets and preserves HIV-1Vpr function in macrophages and T-cells.

Human immunodeficiency virus type 1 (HIV-1) is known for its ability to infect immune cells, including T-cells and macrophages. The 96-amino acid Vpr, a virion-associated protein, is essential for viral replication in monocytes/macrophages and increases viral replication in primary and established T-cell lines. The Vpr protein regulates a number of host cellular events, including proliferation, differentiation, apoptosis, cytokine production, and NF-kappaB-mediated transcription. Most of these functions have been analyzed using either endogenous Vpr protein or cells transfected with a Vpr expression plasmid. We developed a lentiviral vector complemented with a Vpr expression plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles containing Vpr, we fused green fluorescent protein (GFP) with the Vpr open reading frame and analyzed the biology of this novel particle. Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-GFP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in G2 as does the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by itself or with the reporter GFP showed a perinuclear staining pattern. Replication kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay. A flow cytometry analysis of peripheral blood lymphocytes and macrophages infected with Vpr-GFP-packaged virions and selected by GFP showed 56.7% infectivity for lymphocytes and 84.6% infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4+ cells, macrophages, and, importantly, dendritic cells. This system will allow us to identify specific cell populations including antigen-presenting cells, and allow quantitative analysis of the precise effect of Vpr on both target and bystander cells in vitro as well as in vivo.

Comparison of p24 measurement by ELISA versus indicator cells for detecting residual HIV infectivity in vitro.

Inactivation of HIV-1 contaminated materials or biological samples is of great importance and requires the use of a reliable assay to detect residual infectivity. In this study we treated cell-free or cell-associated (monocyte-derived macrophages) HIV-1 with two chemicals known for their antiviral activities, beta-propiolactone (beta PL) and formaldehyde (FO), and tested it for the presence of residual infectivity. HIV-1 infected primary monocyte-derived macrophages (MDM) or cell-free HIV-1 were fixed with increasing concentrations of either beta PL or FO for 1 day at 4 degrees C. Then either fresh primary MDM or fresh medium was added, and the supernatant p24 levels were assayed up to 12 days after infection. All the supernatants harvested were added to indicator cells, fresh primary MDM, to assess for residual infectivity. The results show that p24 measurement is not a reliable assay for the detection of residual infectious virions after chemical fixation of HIV-infected primary MDM. In contrast, the use of indicator primary cells (MDM) is a much more sensitive and reliable assay. By performing an indicator cell assay we showed that FO efficiently inactivates cell-associated and cell-free HIV-1 at concentrations as low as 1% v/v. In contrast beta PL is more efficient in inactivating cell-free than cell-associated virus and does not inactivate cell-associated HIV-1 at concentrations as high as 1% v/v.

Phase II trial of recombinant interferon-alpha2b in patients with advanced intractable multidrug-resistant pulmonary tuberculosis: long-term follow-up.

SETTING: Multidrug-resistant tuberculosis patients without human immunodeficiency virus (HIV) infection, with Mycobacterium tuberculosis resistant to almost all of the available drugs. OBJECTIVE: Limited phase II trial with recombinant interferon-alpha2b in five chronic multidrug-resistant tuberculosis patients. METHODS: Three million units of r-IFN-alpha2b were administered subcutaneously every week for 12 weeks. Before and after treatment, and during a 30-month follow-up period, the patients underwent clinical and radiological examination, together with bacteriological, immunological and routine laboratory testing. RESULTS: Two of the five patients became long-term sputum smear and culture negative after r-IFN-alpha2b therapy; one of the patients showed clinical improvement and negative smear after therapy, but remained culture positive. The other two patients showed no response. CONCLUSION: The results of this trial suggest that r-IFN-alpha2b should be evaluated further in multidrug-resistant tuberculosis in prospective controlled trials.

[Human immunodeficiency virus infection associated with tuberculosis]. / Asociación de la infección por el virus de la inmunodeficiencia humana (HIV) y tuberculosis.

In order to detect an association between HIV infection and tuberculosis (TB), 130 TB inpatients were studied one of whom presented a pulmonary disease due to Mycobacterium avium intracellulare. All had advanced TB, 95.4%, with pulmonary localization. Serum anti-HIV antibodies were detected by ELISA and their presence confirmed by immunoblotting in 4 (3.1%) individuals, three males and one female, with different degrees of pulmonary TB. Of the males, 1 was bisexual, 2 were promiscuous, and the female was the sexual partner of a non symptomatic HIV-infected man. No immunological disturbances or other AIDS related alterations were observed. There was one case of miliary TB, but neither atypical X-ray abnormalities nor extrapulmonary involvement were found. Tuberculin reaction was positive in three of the four HIV infected patients. Clinical, radiological and bacteriological evolution were favorable. Adverse drug reaction occurred in two cases, one of them presenting serious toxidermia caused by isoniazid. Of the 130 individuals, 12 presented risk factors for HIV infection so that the prevalence of anti-HIV antibodies presented here, 4 cases out of 12, is consistent with data from previous reports for high risk populations.

[Evaluation of an enzyme immunoassay for the rapid diagnosis of paucibacillary tuberculosis in adults]. / Evaluación del enzimoinmunoensayo para el diagnóstico rápido de la tuberculosis paucibacilar del adulto.

The ELISA has been extensively evaluated as a serodiagnostic method for tuberculosis. However, there is scarce information about its application to cases that cannot be diagnosed by microscopic examination: those with closed lesions or undergoing early stages of the disease. Since a reliable serological test might substantially contribute to their prompt detection, the objective of the present study was to determine the diagnostic value of an ELISA applied to adult smear-negative cases of tuberculosis. Sera from 235 patients with active tuberculosis--176 pulmonary and 59 extrapulmonary cases--and 181 control subjects were tested for IgG antibodies to PPD by ELISA. Eleven cases of non tuberculous mycobacterial (MOTT) disease and 33 cases of mycosis were also included in this group. With the adopted cut-off value, 73.9% (105/142) of smear positive and 52.7% (49/93) of smear negative tuberculosis cases, were correctly classified. Particularly in the latter, the test was positive in 55.2% (32/58) of patients with positive cultures for Mycobacterium tuberculosis and in 48.6% (17/35) of patients diagnosed by clinical, radiological and or histopathological findings. No antibody activity was demonstrated in 92.7% of sera from the control population which included 92 healthy volunteers, 32 non tuberculous diseased subjects and 13 household contacts of smear-positive cases. Among those control subjects who were skin tested, ELISA results were not related to the tuberculin reactivity: 93.7% (30/32) of tuberculin negative and 95.2% (40/42) of tuberculin positive healthy individuals had no detectable antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Multidrug-resistant tuberculosis associated with AIDS (kinetics of nosocomial epidemics of multidrug-resistant tuberculosis associated with AIDS. Possible transformation into endemic disease]. / Tuberculose multi-résistante associée au SIDA (cinétique d'une épidémie nosocomiale de tuberculose multi-résistante associée au SIDA. Transformation possible en endémie).

The increase in the incidence of AIDS-related tuberculosis over the last decades has fueled the dissemination of multiple drug resistance tuberculosis (including resistant strains to INH and rifampin). This has now been recognized in a variety of settings including hospitals, prisons and shelters. We have identified a nosocomial epidemic at the Muñiz Hospital in the city of Buenos Aires, Argentina. This has evolved as one of the largest institutional outbreaks yet to be recognized. The purpose of this paper is to characterize the evolution of this outbreak which at the end of 1997 had involved in excess of 500 cases. Among the 3,322 patients discharged at the Muñiz Hospital during the years 1996-1997 with the diagnosis of tuberculosis, 440 (13.24%) were discharged with the diagnosis of multiple drug resistance tuberculosis. The immediate mortality (during the ensuing four months following the bacteriological diagnosis) was of 91.3% of cases in 1995 and decreased progressively to 65.9% in 1996 and 55.9% in 1997. The bacteriological confirmation of the diagnosis was made after the patients death in a decreasing number of cases, going from 72.5% of the cases in 1995 to 28.3% of the cases in 1997. Despite the significant progress achieved with regard to the diagnosis and treatment of multiple drug resistance tuberculosis, the measures undertaken to decrease the spread of the cases have had limited success. This is chiefly attributable to the inability to isolate cases. This has continued to promote nosocomial spread of multiple drug resistance tuberculosis in our environment.

Prediction based classification for longitudinal biomarkers.

Assessment of circulating CD4 count change over time in HIV-infected subjects on antiretroviral therapy (ART) is a central component of disease monitoring. The increasing number of HIV-infected subjects starting therapy and the limited capacity to support CD4 count testing within resource-limited settings have fueled interest in identifying correlates of CD4 count change such as total lymphocyte count, among others. The application of modeling techniques will be essential to this endeavor due to the typically non-linear CD4 trajectory over time and the multiple input variables necessary for capturing CD4 variability. We propose a prediction based classification approach that involves first stage modeling and subsequent classification based on clinically meaningful thresholds. This approach draws on existing analytical methods described in the receiver operating characteristic curve literature while presenting an extension for handling a continuous outcome. Application of this method to an independent test sample results in greater than 98% positive predictive value for CD4 count change. The prediction algorithm is derived based on a cohort of n = 270 HIV-1 infected individuals from the Royal Free Hospital, London who were followed for up to three years from initiation of ART. A test sample comprised of n = 72 individuals from Philadelphia and followed for a similar length of time is used for validation. Results suggest that this approach may be a useful tool for prioritizing limited laboratory resources for CD4 testing after subjects start antiretroviral therapy.