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1.  Inflammatory and myeloid-associated gene expression before and one day after infant vaccination with MVA85A correlates with induction of a T cell response 
BMC Infectious Diseases  2014;14:314.
Background
Tuberculosis (TB) remains a global health problem, with vaccination likely to be a necessary part of a successful control strategy. Results of the first Phase 2b efficacy trial of a candidate vaccine, MVA85A, evaluated in BCG-vaccinated infants were published last year. Although no improvement in efficacy above BCG alone was seen, cryopreserved samples from this trial provide an opportunity to study the immune response to vaccination in this population.
Methods
We investigated blood samples taken before vaccination (baseline) and one and 28 days post-vaccination with MVA85A or placebo (Candin). The IFN-γ ELISpot assay was performed at baseline and on day 28 to quantify the adaptive response to Ag85A peptides. Gene expression analysis was performed at all three timepoints to identify early gene signatures predictive of the magnitude of the subsequent adaptive T cell response using the significance analysis of microarrays (SAM) statistical package and gene set enrichment analysis.
Results
One day post-MVA85A, there is an induction of inflammatory pathways compared to placebo samples. Modules associated with myeloid cells and inflammation pre- and one day post-MVA85A correlate with a higher IFN-γ ELISpot response post-vaccination. By contrast, previous work done in UK adults shows early inflammation in this population is not associated with a strong T cell response but that induction of regulatory pathways inversely correlates with the magnitude of the T cell response. This may be indicative of important mechanistic differences in how T cell responses develop in these two populations following vaccination with MVA85A.
Conclusion
The results suggest the capacity of MVA85A to induce a strong innate response is key to the initiation of an adaptive immune response in South African infants but induction of regulatory pathways may be more important in UK adults. Understanding differences in immune response to vaccination between populations is likely to be an important aspect of developing successful vaccines and vaccination strategies.
Trial registration
ClinicalTrials.gov number NCT00953927
doi:10.1186/1471-2334-14-314
PMCID: PMC4061512  PMID: 24912498
Tuberculosis; Vaccine; Innate immunity; Transcriptomics; MVA85A
2.  A strategy to determine HLA class II restriction broadly covering the DR, DP and DQ allelic variants most commonly expressed in the general population 
Immunogenetics  2013;65(5):357-370.
Classic ways to determine MHC restriction involve inhibition with locus specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T-cell epitope degeneracy and promiscuity. We describe selection of 46 HLA DR, DQ and DP specificities that provide worldwide population (phenotypic) coverage of almost 90% at each locus, and account for over 66% of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95% of the individuals in four study populations of diverse ethnicity from the US and South Africa. Next, a panel of single HLA class II transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T-cell lines of known restriction. We also show that these HLA class II transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T-cell responses and facilitating the development of HLA tetrameric staining reagents.
doi:10.1007/s00251-013-0684-y
PMCID: PMC3633633  PMID: 23392739
HLA Class II; restriction; transfectants; epitopes; population coverage; polymorphism
3.  Patients with Tuberculosis Disease Have Mycobacterium tuberculosis-Specific CD8 T Cells with a Pro-Apoptotic Phenotype and Impaired Proliferative Capacity, Which Is Not Restored following Treatment 
PLoS ONE  2014;9(4):e94949.
CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ+ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ+ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.
doi:10.1371/journal.pone.0094949
PMCID: PMC3989259  PMID: 24740417
4.  Longitudinal Changes in CD4+ T-Cell Memory Responses Induced by BCG Vaccination of Newborns 
The Journal of Infectious Diseases  2013;207(7):1084-1094.
Background. Improved vaccination strategies against tuberculosis are needed, such as approaches to boost immunity induced by the current vaccine, BCG. Design of these strategies has been hampered by a lack of knowledge of the kinetics of the human host response induced by neonatal BCG vaccination. Furthermore, the functional and phenotypic attributes of BCG-induced long-lived memory T-cell responses remain unclear.
Methods. We assessed the longitudinal CD4+ T-cell response following BCG vaccination of human newborns. The kinetics, function, and phenotype of these cells were measured using flow cytometric whole-blood assays.
Results. We showed that the BCG-specific CD4+ T-cell response peaked 6–10 weeks after vaccination and gradually waned over the first year of life. Highly activated T-helper 1 cells, predominantly expressing interferon γ, tumor necrosis factor α, and/or interleukin 2, were present at the peak response. Following contraction, BCG-specific CD4+ T cells expressed high levels of Bcl-2 and displayed a predominant CD45RA–CCR7+ central memory phenotype. However, cytokine and cytotoxic marker expression by these cells was more characteristic of effector memory cells.
Conclusions. Our findings suggest that boosting of BCG-primed CD4+ T cells with heterologous tuberculosis vaccines may be best after 14 weeks of age, once an established memory response has developed.
doi:10.1093/infdis/jis941
PMCID: PMC3583271  PMID: 23293360
Bacille Calmette-Guérin; Vaccination; Newborns; Memory T cells; T cell kinetics
5.  The Candidate TB Vaccine, MVA85A, Induces Highly Durable Th1 Responses 
PLoS ONE  2014;9(2):e87340.
Background
Vaccination against tuberculosis (TB) should provide long-term protective immunity against Mycobacterium tuberculosis (M.tb). The current TB vaccine, Bacille Calmette-Guerin (BCG), protects against disseminated childhood TB, but protection against lung TB in adolescents and adults is variable and mostly poor. One potential reason for the limited durability of protection may be waning of immunity through gradual attrition of BCG-induced T cells. We determined if a MVA85A viral-vector boost could enhance the durability of mycobacteria-specific T cell responses above those induced by BCG alone.
Methods
We describe a long-term follow-up study of persons previously vaccinated with MVA85A. We performed a medical history and clinical examination, a tuberculin skin test and measured vaccine-specific T cell responses in persons previously enrolled as adults, adolescents, children or infants into three different Phase II trials, between 2005 and 2011.
Results
Of 252 potential participants, 183 (72.6%) consented and completed the study visit. Vaccine-induced Ag85A-specific CD4+ T cell responses were remarkably persistent in healthy, HIV-uninfected adults, adolescents, children and infants, up to 6 years after MVA85A vaccination. Specific CD4+ T cells expressed surface markers consistent with either CD45RA−CCR7+ central memory or CD45RA−CCR7− effector memory T cells. Similarly durable Ag85A-specific CD4+ T cell responses were detected in HIV-infected persons who were on successful antiretroviral therapy when MVA85A was administered. By contrast, Ag85A-specific CD4+ T cell frequencies in untreated MVA85A-vaccinated HIV-infected persons were mostly undetectable 3–5 years after vaccination.
Conclusion
MVA85A induces remarkably durable T cell responses in immunocompetent persons. However, results from a recent phase IIb trial of MVA85A, conducted in infants from the same geographic area and study population, showed no vaccine efficacy, suggesting that these durable T cell responses do not enhance BCG-induced protection against TB in infants.
doi:10.1371/journal.pone.0087340
PMCID: PMC3911992  PMID: 24498312
6.  Relationship between chemokine receptor expression, chemokine levels and HIV-1 replication in the lungs of persons exposed to Mycobacterium tuberculosis 
European journal of immunology  2012;43(2):540-549.
Increased susceptibility to tuberculosis following HIV-1 seroconversion contributes significantly to the tuberculosis epidemic in sub-saharan Africa. Lung specific mechanisms underlying the interaction between HIV-1 and Mycobacterium (M.) tuberculosis infection are incompletely understood. This study addressed the effect of HIV-1 and latent M. tuberculosis infection on viral-entry receptors and ligands in bronchoalveolar lavage (BAL). Median fluorescence intensity (MFI) of entry receptor expression was measured by multiparameter flow cytometry and chemokine expression by multiplex bead array.
Irrespective of HIV-1 status, BAL T-cells expressed higher MFI for the beta-chemokine receptor (CCR)5 than peripheral blood T-cells (p<0.001), in particular the CD8+ T-cells of HIV-1 infected persons showed elevated CCR5 expression (p=0.026). The concentration of BAL CCR5 ligands, regulated upon activation normal T-cell expressed and secreted (RANTES; p<0.001) and macrophage inflammatory protein (MIP)-1β (p=0.004) were elevated in the BAL of HIV-1 infected persons compared to controls. CCR5 expression and RANTES concentration correlated strongly with HIV-1 viral load in BAL. By contrast these alterations were not associated with M. tuberculosis sensitization in vivo nor did M. tuberculosis infection of BAL cells ex vivo change RANTES expression.
These data suggest ongoing HIV-1 replication predominantly drives local pulmonary CCR5+ T-cell activation in HIV/latent M. tuberculosis co-infection.
doi:10.1002/eji.201242804
PMCID: PMC3791514  PMID: 23147374
BAL; CCR5; RANTES; TB; viral load
7.  Analysis of Host Responses to Mycobacterium tuberculosis Antigens in a Multi-Site Study of Subjects with Different TB and HIV Infection States in Sub-Saharan Africa 
PLoS ONE  2013;8(9):e74080.
Background
Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas.
Methods
We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens.
Results
There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST- and TST+ contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST+ contacts (LTBI) compared to TB and TST- contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen.
Conclusions
Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.
doi:10.1371/journal.pone.0074080
PMCID: PMC3769366  PMID: 24040170
8.  TB Incidence in an Adolescent Cohort in South Africa 
PLoS ONE  2013;8(3):e59652.
Background
Tuberculosis (TB) is a major public health problem globally. Little is known about TB incidence in adolescents who are a proposed target group for new TB vaccines. We conducted a study to determine the TB incidence rates and risk factors for TB disease in a cohort of school-going adolescents in a high TB burden area in South Africa.
Methods
We recruited adolescents aged 12 to 18 years from high schools in Worcester, South Africa. Demographic and clinical information was collected, a tuberculin skin test (TST) performed and blood drawn for a QuantiFERON TB Gold assay at baseline. Screening for TB cases occurred at follow up visits and by surveillance of registers at public sector TB clinics over a period of up to 3.8 years after enrolment.
Results
A total of 6,363 adolescents were enrolled (58% of the school population targeted). During follow up, 67 cases of bacteriologically confirmed TB were detected giving an overall incidence rate of 0.45 per 100 person years (95% confidence interval 0.29–0.72). Black or mixed race, maternal education of primary school or less or unknown, a positive baseline QuantiFERON assay and a positive baseline TST were significant predictors of TB disease on adjusted analysis.
Conclusion
The adolescent TB incidence found in a high burden setting will help TB vaccine developers plan clinical trials in this population. Latent TB infection and low socio-economic status were predictors of TB disease.
doi:10.1371/journal.pone.0059652
PMCID: PMC3606161  PMID: 23533639
9.  Lessons learnt from the first efficacy trial of a new infant tuberculosis vaccine since BCG 
Summary
Background
New tuberculosis (TB) vaccines are being developed to combat the global epidemic. A phase IIb trial of a candidate vaccine, MVA85A, was conducted in a high burden setting in South Africa to evaluate proof-of-concept efficacy for prevention of TB in infants.
Objective
To describe the study design and implementation lessons from an infant TB vaccine efficacy trial.
Methods
This was a randomised, controlled, double-blind clinical trial comparing the safety and efficacy of MVA85A to Candin control administered to 4–6-month-old, BCG-vaccinated, HIV-negative infants at a rural site in South Africa. Infants were followed up for 15–39 months for incident TB disease based on pre-specified endpoints.
Results
2797 infants were enrolled over 22 months. Factors adversely affecting recruitment and the solutions that were implemented are discussed. Slow case accrual led to six months extension of trial follow up.
Conclusion
The clinical, regulatory and research environment for modern efficacy trials of new TB vaccines are substantially different to that when BCG vaccine was first evaluated in infants. Future infant TB vaccine trials will need to allocate sufficient resources and optimise operational efficiency. A stringent TB case definition is necessary to maximize specificity, and TB case accrual must be monitored closely.
doi:10.1016/j.tube.2013.01.003
PMCID: PMC3608032  PMID: 23410889
BCG; Vaccine; Tuberculosis; Lessons learnt; Implementation
10.  Optimization of a whole blood intracellular cytokine assay for measuring innate cell responses to mycobacteria 
Journal of Immunological Methods  2011;376(1-2):79-88.
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 mDCs 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 mDCs 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 200µL per condition, making the assay particularly suitable for pediatric studies.
doi:10.1016/j.jim.2011.11.011
PMCID: PMC3273614  PMID: 22155193
Mycobacteria; flow cytometry; monocytes; dendritic cells; granulocytes; innate cytokines
11.  Moxifloxacin Population Pharmacokinetics in Patients with Pulmonary Tuberculosis and the Effect of Intermittent High-Dose Rifapentine 
We described the population pharmacokinetics of moxifloxacin and the effect of high-dose intermittent rifapentine in patients with pulmonary tuberculosis who were randomized to a continuation-phase regimen of 400 mg moxifloxacin and 900 mg rifapentine twice weekly or 400 mg moxifloxacin and 1,200 mg rifapentine once weekly. A two-compartment model with transit absorption best described moxifloxacin pharmacokinetics. Although rifapentine increased the clearance of moxifloxacin by 8% during antituberculosis treatment compared to that after treatment completion without rifapentine, it did not result in a clinically significant change in moxifloxacin exposure.
doi:10.1128/AAC.00404-12
PMCID: PMC3421597  PMID: 22585223
12.  Single nucleotide polymorphisms in toll-like receptor 6 are associated with altered lipopeptide- and mycobacteria-induced IL-6 secretion 
Genes and immunity  2010;11(7):561-572.
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 (SNPs) 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 IL-6 production in whole blood. We found 2 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, P = 0.018) and 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 a 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.
doi:10.1038/gene.2010.14
PMCID: PMC3518443  PMID: 20445564
Toll-like receptor 6; polymorphism; interleukin 6; tuberculosis; immune response
13.  HIV-Specific Gag Responses in Early Infancy Correlate with Clinical Outcome and Inversely with Viral Load 
AIDS Research and Human Retroviruses  2011;27(12):1311-1316.
Abstract
Many HIV-infected infants progress to AIDS during the first year of life when antiretroviral therapy (ART) is not given. The immune determinants of progression to AIDS are not known. We hypothesized that distinct HIV-specific T cell responses correlate with viral load and survival over the first year of life. Whole blood of infants at 3, 6, 9, and 12 months of age was incubated with HIV antigens Gag and Env. The frequency of specific T cells producing interferon (IFN)-γ was then measured by flow cytometry. Viral load and CD4% in HIV+ infants were determined at each time point. ART was not available for this population at the time of sample collection. Those infants who survived to 12 months of age (n=12) had lower viral loads and higher Gag-specific CD8+ T cell responses at 3 months, compared with infants who died (n=8). Furthermore, the frequency of Gag-specific CD4+ T cells correlated inversely with viral load at 3 and 6 months of age. Together these data indicate that the early presence of quantitatively higher Gag-specific T cell responses in HIV-infected infants is associated with lower viral loads and decreased mortality in the first year of life. Our data support the design of a vaccine that preferentially elicits Gag responses, which may result in lower levels of viremia and possibly improve outcome.
doi:10.1089/aid.2011.0081
PMCID: PMC3227240  PMID: 21476948
14.  Immune Activation in the Female Genital Tract During HIV Infection Predicts Mucosal CD4 Depletion and HIV Shedding 
The Journal of Infectious Diseases  2011;204(10):1550-1556.
Plasma viral load predicts genital tract human immunodeficiency virus (HIV) shedding in HIV-infected women. We investigated whether local mucosal T-cell activation (HLA-DR, CD38, CCR5, and Ki67) contributed to HIV shedding in the genital tracts of HIV-infected women. We showed that cervical cytobrush-derived T cells expressed higher frequencies of T-cell activation markers (CD38+ and HLA-DR+) than blood-derived T cells. Expression was significantly higher in HIV-infected women than in uninfected women. We found that the frequency of activated proliferating cervical T cells (Ki67+; Ki67+CCR5+) broadly predicted HIV shedding in the genital tract in HIV-infected women, independently of plasma viral loads. Furthermore, activated cervical T cells (HLA-DR+CD38+ and HLA-DR+CCR5+) and local HIV shedding were independently associated with CD4 depletion in the genital tract. These data suggest that the presence of high frequencies of activated T cells in the female genital mucosa during HIV infection facilitates both local HIV shedding and CD4 T-cell depletion.
doi:10.1093/infdis/jir591
PMCID: PMC3192190  PMID: 21940422
15.  Genetic Variation in TLR Genes in Ugandan and South African Populations and Comparison with HapMap Data 
PLoS ONE  2012;7(10):e47597.
Genetic epidemiological studies of complex diseases often rely on data from the International HapMap Consortium for identification of single nucleotide polymorphisms (SNPs), particularly those that tag haplotypes. However, little is known about the relevance of the African populations used to collect HapMap data for study populations conducted elsewhere in Africa. Toll-like receptor (TLR) genes play a key role in susceptibility to various infectious diseases, including tuberculosis. We conducted full-exon sequencing in samples obtained from Uganda (n = 48) and South Africa (n = 48), in four genes in the TLR pathway: TLR2, TLR4, TLR6, and TIRAP. We identified one novel TIRAP SNP (with minor allele frequency [MAF] 3.2%) and a novel TLR6 SNP (MAF 8%) in the Ugandan population, and a TLR6 SNP that is unique to the South African population (MAF 14%). These SNPs were also not present in the 1000 Genomes data. Genotype and haplotype frequencies and linkage disequilibrium patterns in Uganda and South Africa were similar to African populations in the HapMap datasets. Multidimensional scaling analysis of polymorphisms in all four genes suggested broad overlap of all of the examined African populations. Based on these data, we propose that there is enough similarity among African populations represented in the HapMap database to justify initial SNP selection for genetic epidemiological studies in Uganda and South Africa. We also discovered three novel polymorphisms that appear to be population-specific and would only be detected by sequencing efforts.
doi:10.1371/journal.pone.0047597
PMCID: PMC3480404  PMID: 23112821
16.  A Phase IIa Trial of the New Tuberculosis Vaccine, MVA85A, in HIV- and/or Mycobacterium tuberculosis–infected Adults 
Rationale: Novel tuberculosis (TB) vaccines should be safe and effective in populations infected with Mycobacterium tuberculosis (M.tb) and/or HIV for effective TB control.
Objective: To determine the safety and immunogenicity of MVA85A, a novel TB vaccine, among M.tb- and/or HIV-infected persons in a setting where TB and HIV are endemic.
Methods: An open-label, phase IIa trial was conducted in 48 adults with M.tb and/or HIV infection. Safety and immunogenicity were analyzed up to 52 weeks after intradermal vaccination with 5 × 107 plaque-forming units of MVA85A. Specific T-cell responses were characterized by IFN-γ enzyme-linked immunospot and whole blood intracellular cytokine staining assays.
Measurements and Main Results: MVA85A was well tolerated and no vaccine-related serious adverse events were recorded. MVA85A induced robust and durable response of mostly polyfunctional CD4+ T cells, coexpressing IFN-γ, tumor necrosis factor-α, and IL-2. Magnitudes of pre- and postvaccination T-cell responses were lower in HIV-infected, compared with HIV-uninfected, vaccinees. No significant effect of antiretroviral therapy on immunogenicity of MVA85A was observed.
Conclusions: MVA85A was safe and immunogenic in persons with HIV and/or M.tb infection. These results support further evaluation of safety and efficacy of this vaccine for prevention of TB in these target populations.
doi:10.1164/rccm.201108-1548OC
PMCID: PMC3326425  PMID: 22281831
tuberculosis; HIV-1; vaccine; MVA85A; clinical trial
17.  A Comparative Analysis of Polyfunctional T Cells and Secreted Cytokines Induced by Bacille Calmette-Guérin Immunisation in Children and Adults 
PLoS ONE  2012;7(7):e37535.
BCG vaccine is one of the most commonly-administered vaccines worldwide. Studies suggest the protective efficacy of BCG against TB is better for children than for adults. One potential explanation is that BCG induces a better protective immune response in children. Twenty six children and adults were immunised with BCG. The proportion of Th1-cytokine-producing mycobacterial-specific T cells, and the concentrations of secreted cytokines, were measured before and 10 weeks after BCG immunisation. A significant increase in the proportion of mycobacterial-specific cytokine-producing T cells was observed in both age groups. After BCG immunisation, children and adults had comparable proportions of mycobacterial-specific polyfunctional CD4 T cells when measured relative to the total number of CD4 T cells. However, relative to the subset of Th-1-cytokine-producing CD4 T cells, the proportion of polyfunctional cells was greater in children. Concentrations of secreted cytokines were comparable in children and adults. These findings suggest that the mycobacterial-specific cell-mediated immune response induced by BCG immunisation in children and adults is similar. The implication of a shift to a more polyfunctional immune response within the Th1-cytokine-producing CD4 T cells in children is uncertain as this aspect of the immune response has not been assessed as a potential correlate of protection against TB.
doi:10.1371/journal.pone.0037535
PMCID: PMC3400612  PMID: 22829867
18.  Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease 
Vaccine  2010;29(1):51-57.
One third of the world’s population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4+ T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.
doi:10.1016/j.vaccine.2010.10.022
PMCID: PMC3376751  PMID: 20974305
Mycobacterium tuberculosis; LTBI; TB disease; latency antigens; post-infection vaccine
19.  Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load1 
High antigen load in chronic viral infections has been associated with impairment of antigen-specific T cell responses; however, the relationship between antigen load in chronic Mycobacterium tuberculosis (Mtb) infection and functional capacity of Mtb-specific T cells in humans is not clear. We compared Mtb-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads, i.e., persons with latent Mtb infection (LTBI), with smear − pulmonary tuberculosis (TB), and with smear+ TB. Patients with smear+ TB had decreased polyfunctional IFN-γ+IL-2+TNF-α+ and IL-2-producing specific CD4 T cells and increased TNF-α-single positive cells, when compared with smear − TB and LTBI. TB patients also had increased frequencies of Mtb-specific CD8 T cells, compared with LTBI. Mtb-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear+ TB, and correlated positively with ex vivo IFN-γ+IL-2+TNF-α+ CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 months of anti-TB treatment, specific IFN-γ+IL-2+TNF-α+ CD4 and CD8 T cells increased, whereas TNF-α- and IFN-γ-single positive T cells decreased. These results suggest progressive impairment of Mtb-specific T cell responses with increasing mycobacterial load, and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of Mtb-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. Taken together, these data have potentially significant applications for diagnosis of TB and for identification of T cell correlates of TB disease progression.
doi:10.4049/jimmunol.1101122
PMCID: PMC3159795  PMID: 21775682
20.  Predominance of interleukin-22 over interleukin-17 at the site of disease in human tuberculosis 
Tuberculosis (Edinburgh, Scotland)  2011;91(6-3):587-593.
Summary
The inflammatory response to Mycobacterium tuberculosis (M.tb) at the site of disease is Th1 driven. Whether the Th17 cytokines, IL-17 and IL-22, contribute to this response in humans is unknown. We hypothesized that IL-17 and IL-22 contribute to the inflammatory response in pleural and pericardial disease sites of human tuberculosis (TB).
We studied pleural and pericardial effusions, established TB disease sites, from HIV-uninfected TB patients. Levels of soluble cytokines were measured by ELISA and MMP-9 by luminex. Bronchoalveolar lavage or pericardial mycobacteria-specific T cell cytokine expression was analyzed by intracellular cytokine staining.
IL-17 was not abundant in pleural or pericardial fluid. IL-17 expression by mycobacteria-specific disease site T cells was not detected in healthy, M.tb-infected persons, or patients with TB pericarditis. These data do not support a major role for IL-17 at established TB disease sites in humans.
IL-22 was readily detected in fluid from both disease sites. These IL-22 levels exceeded matching peripheral blood levels. Further, IL-22 levels in pericardial fluid correlated positively with MMP-9, an enzyme known to degrade the pulmonary extracellular matrix. We propose that our findings support a role for IL-22 in TB-induced pathology or the resulting repair process.
doi:10.1016/j.tube.2011.06.009
PMCID: PMC3220762  PMID: 21767990
Pleural tuberculosis; Pericardial tuberculosis; IL-17; IL-22; Inflammation
21.  Identification of Antigens Specific to Non-Tuberculous Mycobacteria: The Mce Family of Proteins as a Target of T Cell Immune Responses 
PLoS ONE  2011;6(10):e26434.
The lack of an effective TB vaccine hinders current efforts in combating the TB pandemic. One theory as to why BCG is less protective in tropical countries is that exposure to non-tuberculous mycobacteria (NTM) reduces BCG efficacy. There are currently several new TB vaccines in clinical trials, and NTM exposure may also be relevant in this context. NTM exposure cannot be accurately evaluated in the absence of specific antigens; those which are known to be present in NTM and absent from M. tuberculosis and BCG. We therefore used a bioinformatic pipeline to define proteins which are present in common NTM and absent from the M. tuberculosis complex, using protein BLAST, TBLASTN and a short sequence protein BLAST to ensure the specificity of this process. We then assessed immune responses to these proteins, in healthy South Africans and in patients from the United Kingdom and United States with documented exposure to NTM. Low level responses were detected to a cluster of proteins from the mammalian cell entry family, and to a cluster of hypothetical proteins, using ex vivo ELISpot and a 6 day proliferation assay. These early findings may provide a basis for characterising exposure to NTM at a population level, which has applications in the field of TB vaccine design as well as in the development of diagnostic tests.
doi:10.1371/journal.pone.0026434
PMCID: PMC3201954  PMID: 22046285
22.  Specific T Cell Frequency and Cytokine Expression Profile Do Not Correlate with Protection against Tuberculosis after Bacillus Calmette-Guérin Vaccination of Newborns 
Rationale: Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells.
Objectives: We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG).
Methods: Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB—these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified—these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell–specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry.
Measurements and Main Results: A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants.
Conclusions: The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.
doi:10.1164/rccm.201003-0334OC
PMCID: PMC2970848  PMID: 20558627
mycobacteria immunity; pediatric settings
23.  Association of Human TLR1 and TLR6 Deficiency with Altered Immune Responses to BCG Vaccination in South African Infants 
PLoS Pathogens  2011;7(8):e1002174.
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 BCG's 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.
Author Summary
Tuberculosis (TB) is one of the leading infectious causes of death worldwide. The current vaccine for TB, BCG, is widely used but it is not highly effective in preventing disease. We investigated the role of host genetics in the immune response to BCG vaccination. We found that variants of innate immunity genes (TLR1 and TLR6) were associated with BCG-induced immune responses after vaccination. These findings may guide new strategies for vaccine development as well as diagnosis of TB.
doi:10.1371/journal.ppat.1002174
PMCID: PMC3154845  PMID: 21852947
25.  The Novel Tuberculosis Vaccine, AERAS-402, Induces Robust and Polyfunctional CD4+ and CD8+ T Cells in Adults 
Rationale: AERAS-402 is a novel tuberculosis vaccine designed to boost immunity primed by bacillus Calmette-Guérin (BCG), the only licensed vaccine.
Objectives: We investigated the safety and immunogenicity of AERAS-402 in healthy Mycobacterium tuberculosis–uninfected BCG-vaccinated adults from a tuberculosis-endemic region of South Africa.
Methods: Escalating doses of AERAS-402 vaccine were administered intramuscularly to each of three groups of healthy South African BCG-vaccinated adults, and a fourth group received two injections of the maximal dose. Participants were monitored for 6 months, with all adverse effects documented. Vaccine-induced CD4+ and CD8+ T-cell immunity was characterized by an intracellular cytokine staining assay of whole blood and peripheral blood mononuclear cells.
Measurements and Main Results: AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine induced a robust CD4+ T-cell response dominated by cells coexpressing IFN-γ, tumor necrosis factor-α, and IL-2 (“polyfunctional” cells). AERAS-402 also induced a potent CD8+ T-cell response, characterized by cells expressing IFN-γ and/or tumor necrosis factor-α, which persisted for the duration of the study.
Conclusions: Vaccination with AERAS-402 is safe and immunogenic in healthy adults. The immunity induced by the vaccine appears promising: polyfunctional T cells are thought to be important for protection against intracellular pathogens such as Mycobacterium tuberculosis, and evidence is accumulating that CD8+ T cells are also important. AERAS-402 induced a robust and durable CD8+ T-cell response, which appears extremely promising.
Clinical trial registered with www.sanctr.gov.za (NHREC no. 1381).
doi:10.1164/rccm.200910-1484OC
PMCID: PMC2894413  PMID: 20167847
tuberculosis; vaccine; immunity; CD4; CD8

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