Muki Shey

Clinical, Immunological, and Epidemiological Importance of Antituberculosis T Cell Responses in HIV-Infected Africans

BACKGROUND Human immunodeficiency virus (HIV)-associated tuberculosis is a major cause of mortality in Africa. The assay of T cell interferon- gamma released in response to antigens of greater specificity than purified protein derivative is a useful improvement over the Mantoux tuberculin skin test, but few studies have evaluated interferon-gamma secretion in HIV-infected individuals. METHODS Mycobacterium tuberculosis antigen-specific interferon-gamma secretion was assessed by whole blood assay and enzyme-linked immunospot, which were compared with the Mantoux tuberculin skin test in HIV-infected and HIV-uninfected individuals without active tuberculosis and HIV-infected patients with pulmonary tuberculosis in Khayelitsha, South Africa. RESULTS The skin test and whole blood assay responses to purified protein derivative in HIV-positive subjects were decreased, compared with responses in HIV-negative subjects (P < .001). By contrast, the responses to M. tuberculosis antigens (early secreted antigenic target 6, culture filtrate protein 10, TB10.3, and alpha-crystallin 2) were less affected, indicating a high prevalence of latent tuberculosis (approximately 80%) in both HIV-negative and HIV-positive subject groups. Whole blood assay responses did not differ between the HIV-positive subjects without tuberculosis and HIV-positive subjects with tuberculosis, but the enzyme-linked immunospot method response to early secreted antigenic target 6 and culture filtrate protein 10 was higher in the group of HIV-infected subjects with tuberculosis (P < or = .04), although this group had lower CD4+ cell counts. A ratio of the combined enzyme-linked immunospot method response divided by the CD4+ cell count of > 1.0 had 88% sensitivity and 80% specificity for active pulmonary tuberculosis in HIV-infected individuals. CONCLUSIONS Interferon-gamma release appears to be less impaired than skin testing by HIV coinfection. The novel potential to relate the enzyme-linked immunospot method and CD4+ cell count to assist diagnosis of active tuberculosis in patients with HIV infection is important and deserves further evaluation.

Clinical characteristics and initial management of patients with tuberculous pericarditis in the HIV era: the Investigation of the Management of Pericarditis in Africa (IMPI Africa) registry

BackgroundThe incidence of tuberculous pericarditis has increased in Africa as a result of the human immunodeficiency virus (HIV) epidemic. However, the effect of HIV co-infection on clinical features and prognosis in tuberculous pericarditis is not well characterised. We have used baseline data of the Investigation of the Management of Pericarditis in Africa (IMPI Africa) registry to assess the impact of HIV co-infection on clinical presentation, diagnostic evaluation, and treatment of patients with suspected tuberculous pericarditis in sub-Saharan Africa.MethodsConsecutive adult patients in 15 hospitals in three countries in sub-Saharan Africa were recruited on commencement of treatment for tuberculous pericarditis, following informed consent. We recorded demographic, clinical, diagnostic and therapeutic information at baseline, and have used the chi-square test and analysis of variance to assess probabilities of significant differences (in these variables) between groups defined by HIV status.ResultsA total of 185 patients were enrolled from 01 March 2004 to 31 October 2004, 147 (79.5%) of whom had effusive, 28 (15.1%) effusive-constrictive, and 10 (5.4%) constrictive or acute dry pericarditis. Seventy-four (40%) had clinical features of HIV infection. Patients with clinical HIV disease were more likely to present with dyspnoea (odds ratio [OR] 3.2, 95% confidence interval [CI] 1.4 to 7.4, P = 0.005) and electrocardiographic features of myopericarditis (OR 2.8, 95% CI 1.1 to 6.9, P = 0.03). In addition to electrocardiographic features of myopericarditis, a positive HIV serological status was associated with greater cardiomegaly (OR 3.89, 95% CI 1.34 to 11.32, P = 0.01) and haemodynamic instability (OR 9.68, 95% CI 2.09 to 44.80, P = 0.0008). However, stage of pericardial disease at diagnosis and use of diagnostic tests were not related to clinical HIV status. Similar results were obtained for serological HIV status. Most patients were treated on clinical grounds, with microbiological evidence of tuberculosis obtained in only 13 (7.0%) patients. Adjunctive corticosteroids were used in 109 (58.9%) patients, with patients having clinical HIV disease less likely to be put on them (OR 0.37, 95% CI 0.20 to 0.68). Seven patients were on antiretroviral drugs.ConclusionPatients with suspected tuberculous pericarditis and HIV infection in Africa have greater evidence of myopericarditis, dyspnoea, and haemodynamic instability. These findings, if confirmed in other studies, may suggest more intensive management of the cardiac disease is warranted in patients with HIV-associated pericardial disease.

Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCGs variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB.

Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced interleukin-6 secretion

Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens. The influence of human TLR6 polymorphisms on susceptibility to infection is only partially understood. Most microbes contain lipopeptides recognized by TLR2/1 or TLR2/6 heterodimers. Our aim was to determine whether single nucleotide polymorphisms in TLR6 are associated with altered immune responses to lipopeptides and whole mycobacteria. We sequenced the TLR6 coding region in 100 healthy South African adults to assess genetic variation and determined associations between polymorphisms and lipopeptide- and mycobacteria-induced interleukin (IL)-6 production in whole blood. We found two polymorphisms, C745T and G1083C, that were associated with altered IL-6 secretion. G1083C was associated with altered IL-6 levels in response to lipopeptides, Mycobacterium tuberculosis lysate (Mtb lysate, P=0.018) and Bacille Calmette-Guerin (BCG P=0.039). The 745T allele was also associated with lower NF-κB signaling in response to di-acylated lipopeptide, PAM2 (P=0.019) or Mtb (P=0.026) in an HEK293 cell line reconstitution assay, compared with the 745C allele. We conclude that TLR6 polymorphisms may be associated with altered lipopeptide-induced cytokine responses and recognition of Mtb. These studies provide new insight into the role of TLR6 variation and the innate immune response to human infection.

Advances in childhood immunisation in South Africa: where to now? Programme managers’ views and evidence from systematic reviews

BackgroundThe Expanded Programme on Immunisation (EPI) is one of the most powerful and cost-effective public health programmes to improve child survival. We assessed challenges and enablers for the programme in South Africa, as we approach the 2015 deadline for the Millennium Development Goals.MethodsBetween September 2009 and September 2010 we requested national and provincial EPI managers in South Africa to identify key challenges facing EPI, and to propose appropriate solutions. We collated their responses and searched for systematic reviews on the effectiveness of the proposed solutions; in the Health Systems Evidence, Cochrane Library, and PubMed electronic databases. We screened the search outputs, selected systematic reviews, extracted data, and assessed the quality of included reviews (using AMSTAR) and the quality of the evidence (using GRADE) in duplicate; resolving disagreements by discussion and consensus.ResultsChallenges identified by EPI managers were linked to healthcare workers (insufficient knowledge of vaccines and immunisation), the public (anti-immunisation rumours and reluctance from parents), and health system (insufficient financial and human resources). Strategies proposed by managers to overcome the challenges include training, supervision, and audit and feedback; strengthening advocacy and social mobilisation; and sustainable EPI funding schemes, respectively. The findings from reliable systematic reviews indicate that interactive educational meetings, audit and feedback, and supportive supervision improve healthcare worker performance. Structured and interactive communication tools probably increase parents’ understanding of immunisation; and reminders and recall, use of community health workers, conditional cash transfers, and mass media interventions probably increase immunisation coverage. Finally, a national social health insurance scheme is a potential EPI financing mechanism; however, given the absence of high-quality evidence of effects, its implementation should be pilot-tested and the impacts and costs rigorously monitored.ConclusionIn line with the Millennium Development Goals, we have to ensure that our children’s right to health, development and survival is respected, protected and promoted. EPI is central to this vision. We found numerous promising strategies for improving EPI performance in South Africa. However, their implementation would need to be tailored to local circumstances and accompanied by high-quality monitoring and evaluation. The strength of our approach comes from having a strong framework for interventions before looking for systematic reviews. Without a framework, we would have been driven by what reviews have been done and what is easily researchable; rather than the values and preferences of key immunisation stakeholders.

A systematic review of randomized controlled trials of prenatal and postnatal vitamin A supplementation of HIV-infected women

We searched the Cochrane Library, MEDLINE, EMBASE, AIDSearch, and Gateway to assess the effect of prenatal and/or postnatal vitamin A supplementation on the risk of mother‐to‐child transmission (MTCT) of HIV and other pregnancy outcomes. We included 5 trials totaling 7528 women (4 trials of prenatal and 1 trial of postnatal supplementation). Overall, there was no evidence of an effect of prenatal and/or postnatal vitamin A supplementation on the risk of MTCT of HIV (Relative Risk [RR] 1.06, 95% Confidence Interval [CI] 0.89–1.26). However, prenatal vitamin A supplementation significantly improved birth weight (weighted mean difference 89.78; 95% CI, 84.73–94.83), but there was no evidence of an effect on stillbirths (RR 0.99; 95% CI, 0.68–1.43), preterm births (RR 0.88; 95% CI, 0.65–1.19), death before 24 months among live births (RR 1.08; 95% CI, 0.91–1.29), and maternal death (RR 0.83; 95% CI, 0.59–1.17). The available evidence does not support vitamin A supplementation of HIV‐infected pregnant and lactating women, despite improvement in birth weight.

Maturation of Innate Responses to Mycobacteria over the First Nine Months of Life

Newborns and young infants are particularly susceptible to infections, including Mycobacterium tuberculosis. Further, immunogenicity of vaccines against tuberculosis and other infectious diseases appears suboptimal early in life compared with later in life. We hypothesized that developmental changes in innate immunity would underlie these observations. To determine the evolution of innate responses to mycobacteria early in life, whole blood or PBMC from newborns, as well as 10- and 36-wk-old infants, was incubated with viable Mycobacterium bovis bacillus Calmette–Guérin or TLR ligands. Innate cell expression of cytokines and maturation markers was assessed, as well as activation of the proinflammatory NF-κB– and MAPK-signaling pathways. Bacillus Calmette–Guérin–induced production of the proinflammatory cytokines TNF-α, IL-6, and IL-12p40 increased from the newborn period to 9 mo of age in monocytes but not in myeloid dendritic cells. No changes in production of anti-inflammatory IL-10 were observed. CD40 expression increased with age in both cell populations. Older infants displayed substantial activation of all three signal transduction molecules: degradation of NF-κB inhibitor IκBα and phosphorylation of MAPK Erk and p38 upon TLR1/2 triggering, compared with predominant activation of only one of any of these molecules in newborns. Maturation of innate proinflammatory responses during the first 9 mo of life may underlie more effective control of mycobacteria and other pathogens observed later in infancy and age-related differential induction of Th1 responses by vaccination.

Enhanced Ex Vivo Stimulation of Mycobacterium tuberculosis-Specific T Cells in Human Immunodeficiency Virus-Infected Persons via Antigen Delivery by the Bordetella pertussis Adenylate Cyclase Vector

ABSTRACT The genetically detoxified Bordetella pertussis adenylate cyclase is a promising delivery system for immunodominant tuberculosis antigens in gamma interferon release assays. This system has not been evaluated in human immunodeficiency virus (HIV)-infected persons in high tuberculosis prevalence areas. A whole-blood gamma interferon release assay with Mycobacterium tuberculosis antigens (early-secreted antigenic target 6, culture filtrate protein 10, alpha-crystallin 2, and TB10.3) delivered by adenylate cyclase in addition to native tuberculosis antigens (without adenylate cyclase delivery) was evaluated in 119 adults in Khayelitsha Township, Cape Town, South Africa. Results were compared to tuberculin skin test results of 41 HIV-positive and 42 HIV-negative asymptomatic persons, in addition to 36 HIV-positive persons with recently diagnosed smear- or culture-positive pulmonary tuberculosis. Delivery of tuberculosis antigens by adenylate cyclase decreased by 10-fold the amount of antigen required to restimulate T cells. Furthermore, the responses of HIV-positive persons with a low response to native tuberculosis antigens were enhanced when these antigens were delivered by adenylate cyclase. When gamma interferon responses to the tuberculosis antigens (with or without delivery by adenylate cyclase) were combined, a significantly higher number of patients were scored positive than by tuberculin skin testing. Ex vivo responses to tuberculosis antigens delivered by adenylate cyclase are maintained in the context of HIV infection. Our findings suggest that the majority of those in this population are infected with tuberculosis, which is of significant public health importance.

Lower concentrations of chemotactic cytokines and soluble innate factors in the lower female genital tract associated with the use of injectable hormonal contraceptive

Progesterone-based injectable hormonal contraceptives (HCs) potentially modulate genital barrier integrity and regulate the innate immune environment in the female genital tract, thereby enhancing the risk of STIs or HIV infection. We investigated the effects of injectable HC use on concentrations of inflammatory cytokines and other soluble factors associated with genital epithelial repair and integrity. The concentrations of 42 inflammatory, regulatory, adaptive growth factors and hematopoietic cytokines, five matrix metalloproteinases (MMPs), and four tissue inhibitors of metalloproteinases (TIMPs) were measured in cervicovaginal lavages (CVLs) from 64 HIV-negative women using injectable HCs and 64 control women not using any HCs, in a matched case-control study. There were no differences between groups in the prevalence of bacterial vaginosis (BV; Nugent score ≥7), or common sexually transmitted infections (STIs). In multivariate analyses adjusting for condom use, sex work status, marital status, BV and STIs, median concentrations of chemokines (eotaxin, MCP-1, MDC), adaptive cytokines (IL-15), growth factors (PDGF-AA) and a metalloproteinase (TIMP-2) were significantly lower in CVLs from women using injectable HCs than controls. In addition, the pro-inflammatory cytokine IL-12p40 and the chemokine fractalkine were less likely to have detectable levels in women using injectable HCs compared with those not using HCs. We conclude that injectable HC use was broadly associated with an immunosuppressive female genital tract innate immune profile. While the relationship between injectable HC use and STI or HIV risk is yet to be resolved, our data suggest that the effects of injectable HCs were similar in STI-positive and STI-negative participants.

Optimization of a whole blood intracellular cytokine assay for measuring innate cell responses to mycobacteria

Innate cells are essential for host defense against invading pathogens, and the induction and direction of adaptive immune responses to infection. We developed and optimized a flow cytometric assay that allows measurement of intracellular cytokine expression by monocytes, dendritic cells (DC) and granulocytes, as well as cellular uptake of green-fluorescent protein (GFP)-expressing mycobacteria, in very small volumes of peripheral blood. We show that innate cell stimulation resulted in increased granularity of monocytes and mDC and decreased granulocyte granularity that precluded flow cytometric discernment of granulocytes from monocytes and myeloid DC by forward and side scatter gating. Anti-CD66a/c/e antibody staining allowed reliable identification and exclusion of granulocytes for subsequent delineation of monocytes and myeloid DC. Intracellular cytokine expression by granulocytes, monocytes and mDC was remarkably sensitive to the dose of mycobacterial inoculum. Moreover, activation of monocytes and mDC with live BCG reduced expression levels of CD14 and CD11c, respectively, necessitating optimization of staining conditions to reliably measure these lineage markers. Finally, we characterized expression of IL-12/23p40, TNF-α, IL-6, and IL-10, by GFP(+) and GFP(-) monocytes and mDC from 25 healthy adults. This assay may be applied to the study of innate cell responses to any GFP-expressing pathogen, and can be performed on blood volumes as low as 200 μL per condition, making the assay particularly suitable for pediatric studies.