Timothy Lahey

Anti-HIV Activity in Cervical-Vaginal Secretions from HIV-Positive and -Negative Women Correlate with Innate Antimicrobial Levels and IgG Antibodies

Background We investigated the impact of antimicrobials in cervicovaginal lavage (CVL) from HIV(+) and HIV(−) women on target cell infection with HIV. Since female reproductive tract (FRT) secretions contain a spectrum of antimicrobials, we hypothesized that CVL from healthy HIV(+) and (−) women inhibit HIV infection. Methodology/Principal Findings CVL from 32 HIV(+) healthy women with high CD4 counts and 15 healthy HIV(−) women were collected by gently washing the cervicovaginal area with 10 ml of sterile normal saline. Following centrifugation, anti-HIV activity in CVL was determined by incubating CVL with HIV prior to addition to TZM-bl cells. Antimicrobials and anti-gp160 HIV IgG antibodies were measured by ELISA. When CXCR4 and CCR5 tropic HIV-1 were incubated with CVL from HIV(+) women prior to addition to TZM-bl cells, anti-HIV activity in CVL ranged from none to 100% inhibition depending on the viral strains used. CVL from HIV(−) controls showed comparable anti-HIV activity. Analysis of CH077.c (clone of an R5-tropic, mucosally-transmitted founder virus) viral inhibition by CVL was comparable to laboratory strains. Measurement of CVL for antimicrobials HBD2, trappin-2/elafin, SLPI and MIP3α indicated that each was present in CVL from HIV(+) and HIV(−) women. HBD2 and MIP3α correlated with anti-HIV activity as did anti-gp160 HIV IgG antibodies in CVL from HIV(+) women. Conclusions/Significance These findings indicate that CVL from healthy HIV(+) and HIV(−) women contain innate and adaptive defense mechanisms that inhibit HIV infection. Our data suggest that innate endogenous antimicrobials and HIV-specific IgG in the FRT can act in concert to contribute toward the anti-HIV activity of the CVL and may play a role in inhibition of HIV transmission to women.

Expansion and Contraction of HIV-Specific CD4 T Cells with Short Bursts of Viremia, but Physical Loss of the Majority of These Cells with Sustained Viral Replication

Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-γ-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7−CD45RA−) and functional properties (IFN-γ+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.

Test characteristics of urinary lipoarabinomannan and predictors of mortality among hospitalized HIV-infected tuberculosis suspects in Tanzania.

Background Tuberculosis is the most common cause of death among patients with HIV infection living in tuberculosis endemic countries, but many cases are not diagnosed pre-mortem. We assessed the test characteristics of urinary lipoarabinomannan (LAM) and predictors of mortality among HIV-associated tuberculosis suspects in Tanzania. Methods We prospectively enrolled hospitalized HIV-infected patients in Dar es Salaam, with ≥2 weeks of cough or fever, or weight loss. Subjects gave 2 mLs of urine to test for LAM using a commercially available ELISA, ≥2 sputum specimens for concentrated AFB smear and solid media culture, and 40 mLs of blood for culture. Results Among 212 evaluable subjects, 143 (68%) were female; mean age was 36 years; and the median CD4 count 86 cells/mm3. 69 subjects (33%) had culture confirmation of tuberculosis and 65 (31%) were LAM positive. For 69 cases of sputum or blood culture-confirmed tuberculosis, LAM sensitivity was 65% and specificity 86% compared to 36% and 98% for sputum smear. LAM test characteristics were not different in patients with bacteremia but showed higher sensitivity and lower specificity with decreasing CD4 cell count. Two month mortality was 64 (53%) of 121 with outcomes available. In multivariate analysis there was significant association of mortality with absence of anti-retroviral therapy (p = 0.004) and a trend toward association with a positive urine LAM (p = 0.16). Among culture-negative patients mortality was 9 (75%) of 12 in LAM positive patients and 27 (38%) of 71 in LAM negative patients (p = 0.02). Conclusions Urine LAM is more sensitive than sputum smear and has utility for the rapid diagnosis of culture-confirmed tuberculosis in this high-risk population. Mortality data raise the possibility that urine LAM may also be a marker for culture-negative tuberculosis.

Disseminated tuberculosis in human immunodeficiency virus infection: ineffective immunity, polyclonal disease and high mortality.

BACKGROUND Disseminated tuberculosis (TB) is a major cause of death in patients with the acquired immune-deficiency syndrome (AIDS), but its pathogenesis and clinical features have not been defined prospectively. METHODS Human immunodeficiency virus (HIV) infected adults with a CD4 count ≥ 200 cells/μl and bacille Calmette-Guérin scar underwent immunologic evaluation and subsequent follow-up. RESULTS Among 20 subjects who developed disseminated TB, baseline tuberculin skin tests were ≥15 mm in 14 (70%) and lymphocyte proliferative responses to Mycobacterium tuberculosis were positive in 14 (70%). At the time of diagnosis, fever ≥2 weeks plus ≥5 kg weight loss was reported in 16 (80%) patients, abnormal chest X-rays in 7/17 (41%), and positive sputum cultures in 10 (50%); median CD4 count was 30 cells/μl (range 1-122). By insertion sequence (IS) 6110 analysis, 14 (70%) blood isolates were clustered and 3/8 (37%) concurrent sputum isolates represented a different strain (polyclonal disease). Empiric TB treatment was given to eight (40%) patients; 11 (55%) died within a month. CONCLUSIONS Disseminated TB in HIV occurs with cellular immune responses indicating prior mycobacterial infection, and IS6110 analysis suggests an often lethal combination of reactivation and newly acquired infection. Control will require effective prevention of both remotely and recently acquired infection, and wider use of empiric therapy in patients with advanced AIDS and prolonged fever.

Semen Protects CD4+ Target Cells from HIV Infection but Promotes the Preferential Transmission of R5 Tropic HIV

Sexual intercourse is the major means of HIV transmission, yet the impact of semen on HIV infection of CD4+ T cells remains unclear. To resolve this conundrum, we measured CD4+ target cell infection with X4 tropic HIV IIIB and HC4 and R5 tropic HIV BaL and SF162 after incubation with centrifuged seminal plasma (SP) from HIV-negative donors and assessed the impact of SP on critical determinants of target cell susceptibility to HIV infection. We found that SP potently protects CD4+ T cells from infection with X4 and R5 tropic HIV in a dose- and time-dependent manner. SP caused a diminution in CD4+ T cell surface expression of the HIVR CD4 and enhanced surface expression of the HIV coreceptor CCR5. Consequently, SP protected CD4+ T cells from infection with R5 tropic HIV less potently than it protected CD4+ T cells from infection with X4 tropic HIV. SP also reduced CD4+ T cell activation and proliferation, and the magnitude of SP-mediated suppression of target cell CD4 expression, activation, and proliferation correlated closely with the magnitude of the protection of CD4+ T cells from infection with HIV. Taken together, these data show that semen protects CD4+ T cells from HIV infection by restricting critical determinants of CD4+ target cell susceptibility to HIV infection. Further, semen contributes to the selective transmission of R5 tropic HIV to CD4+ target cells.

A Locus at 5q33.3 Confers Resistance to Tuberculosis in Highly Susceptible Individuals

Immunosuppression resulting from HIV infection increases the risk of progression to active tuberculosis (TB) both in individuals newly exposed to Mycobacterium tuberculosis (MTB) and in those with latent infections. We hypothesized that HIV-positive individuals who do not develop TB, despite living in areas where it is hyperendemic, provide a model of natural resistance. We performed a genome-wide association study of TB resistance by using 581 HIV-positive Ugandans and Tanzanians enrolled in prospective cohort studies of TB; 267 of these individuals developed active TB, and 314 did not. A common variant, rs4921437 at 5q33.3, was significantly associated with TB (odds ratio = 0.37, p = 2.11 × 10(-8)). This variant lies within a genomic region that includes IL12B and is embedded in an H3K27Ac histone mark. The locus also displays consistent patterns of linkage disequilibrium across African populations and has signals of strong selection in populations from equatorial Africa. Along with prior studies demonstrating that therapy with IL-12 (the cytokine encoded in part by IL12B, associated with longer survival following MTB infection in mice deficient in CD4 T cells), our results suggest that this pathway might be an excellent target for the development of new modalities for treating TB, especially for HIV-positive individuals. Our results also indicate that studying extreme disease resistance in the face of extensive exposure can increase the power to detect associations in complex infectious disease.

Immunogenicity of a protective whole cell mycobacterial vaccine in HIV-infected adults: A phase III study in Tanzania ,

Preventive immunization with whole inactivated Mycobacterium vaccae (MV) confers protection against HIV-associated tuberculosis (TB) in BCG-immunized adults with CD4 counts ≥200 cells/μl. We evaluated the immunogenicity of MV in the 2013 subjects of the phase III DarDarTrial using an interferon gamma (IFN-γ) enzyme linked immunosorbent assay (ELISA), tritiated thymidine lymphocyte proliferation assay (LPA) and an ELISA for antibodies to the TB glycolipid lipoarabinomannan (LAM). MV immunization boosts IFN-γ and LPA responses to MV sonicate, and antibody responses to LAM. Post-immunization immune responses to MV correlated with baseline clinical factors, but the responses did not predict protection from HIV-associated TB.

Children's medicines in Tanzania: a national survey of administration practices and preferences.

Objective The dearth of age-appropriate formulations of many medicines for children poses a major challenge to pediatric therapeutic practice, adherence, and health care delivery worldwide. We provide information on current administration practices of pediatric medicines and describe key stakeholder preferences for new formulation characteristics. Patients and Methods We surveyed children aged 6–12 years, parents/caregivers over age 18 with children under age 12, and healthcare workers in 10 regions of Tanzania to determine current pediatric medicine prescription and administration practices as well as preferences for new formulations. Analyses were stratified by setting, pediatric age group, parent/caregiver education, and healthcare worker cadre. Results Complete data were available for 206 children, 202 parents/caregivers, and 202 healthcare workers. Swallowing oral solid dosage forms whole or crushing/dissolving them and mixing with water were the two most frequently reported methods of administration. Children frequently reported disliking medication taste, and many had vomited doses. Healthcare workers reported medicine availability most significantly influences prescribing practices. Most parents/caregivers and children prefer sweet-tasting medicine. Parents/caregivers and healthcare workers prefer oral liquid dosage forms for young children, and had similar thresholds for the maximum number of oral solid dosage forms children at different ages can take. Conclusions There are many impediments to acceptable and accurate administration of medicines to children. Current practices are associated with poor tolerability and the potential for under- or over-dosing. Children, parents/caregivers, and healthcare workers in Tanzania have clear preferences for tastes and formulations, which should inform the development, manufacturing, and marketing of pediatric medications for resource-limited settings.

Interferon γ Responses to Mycobacterial Antigens Protect against Subsequent HIV-Associated Tuberculosis

BACKGROUND The cellular immune responses that protect against tuberculosis have not been identified. METHODS We assessed baseline interferon γ (IFN‐γ) and lymphocyte proliferation assay (LPA) responses to antigen 85 (Ag85), early secretory antigenic target 6 (ESAT‐6), and Mycobacterium tuberculosis whole cell lysate (WCL) in human immunodeficiency virus (HIV)-infected and bacille Calmette‐Guérin (BCG)-immunized adults with CD4 cell counts of >or= 200 cells/μL who received placebo in the DarDar tuberculosis vaccine trial in Tanzania. Subjects were followed prospectively to diagnose definite or probable tuberculosis. RESULTS Tuberculosis was diagnosed in 92 of 979 subjects during a mean follow‐up of 3.2 years. The relative risk of tuberculosis among subjects with positive IFN‐γ responses to Ag85 was 0.51 (95% confidence interval [CI], 0.26-0.99; P = .049), to ESAT‐6 was 0.44 (95% CI, 0.23-0.85; P = .004), and to WCL was 0.67 (95% CI, 0.49-0.88; P = .002). The relative risk of tuberculosis was not significantly associated with baseline LPA responses. In a multivariate Cox regression model, subjects with IFN‐γ responses to ESAT‐6 and WCL had a lower hazard of developing tuberculosis, with a hazard ratio for ESAT‐6 of 0.35 (95% CI, 0.16–0.77; P = .009) and a hazard ratio for WCL of 0.30 (95% CI, 0.16-0.56; P < .001). CONCLUSIONS Baseline IFN‐γ responses to ESAT-6 and WCL were associated with protection from subsequent tuberculosis among HIV-infected subjects with childhood BCG immunization in a region of high tuberculosis prevalence. Trial registration. ClinicalTrials.gov identifier: NCT00052195.

Recurrent Tuberculosis Risk Among HIV-Infected Adults in Tanzania With Prior Active Tuberculosis

BACKGROUND Active tuberculosis is common among human immunodeficiency virus (HIV)-infected persons living in tuberculosis-endemic areas, but the hazard of subsequent tuberculosis disease has not been quantified in a single prospective cohort. METHODS Among HIV-infected, BCG-immunized adults with CD4 counts ≥200 cells/μL who received placebo in the DarDar tuberculosis vaccine trial in Tanzania, we compared the prospective risk of active tuberculosis between subjects who did and who did not report prior active tuberculosis. All subjects with a positive tuberculin skin test without prior active tuberculosis were offered isoniazid preventive treatment. Definite or probable tuberculosis was diagnosed during active follow-up using rigorous published criteria. RESULTS We diagnosed 52 cases of definite and 92 cases of definite/probable tuberculosis among 979 subjects during a median follow-up of 3.2 years. Among the 80 subjects who reported prior active tuberculosis, 11 (13.8%) subsequently developed definite tuberculosis and 17 (21.3%) developed definite/probable tuberculosis, compared with 41 (4.6%) and 75 (8.3%), respectively, of 899 subjects without prior active tuberculosis (definite tuberculosis risk ratio [RR], 3.01; 95% confidence interval [CI], 1.61-5.63, P < .001; definite/probable tuberculosis RR, 2.55; 95% CI, 1.59-4.09, P < .001). In a Cox regression model adjusting for age, CD4 count, and isoniazid receipt, subjects with prior active tuberculosis had substantially greater hazard of subsequent definite tuberculosis (hazard radio [HR], 3.69; 95% CI, 1.79-7.63, P < .001) and definite/probable tuberculosis (HR, 2.78; 95% CI, 1.58-4.87, P < .001). CONCLUSIONS Compared to subjects without prior tuberculosis, the hazard of active tuberculosis is increased 3-fold among HIV-infected adults with prior active tuberculosis. Clinical Trials Registration. NCT0052195.