An independent review for the British government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospects for tuberculosis control in British herds. The development of complementary diagnostic tests to differentiate between vaccinated and infected animals is necessary to allow the continuation of test-and-slaughter-based control policies alongside vaccination. Vaccination with M. bovis bacillus Calmette-Guérin (BCG), the only available vaccine, results in tuberculin purified protein derivative sensitivity and has shown varying vaccine efficacies in cattle. Thus, identification of more-specific reagents to distinguish between vaccination and infection, as well as the identification of subunit vaccine candidates for improved tuberculosis vaccines, is a research priority. In the present study, we applied comparative genomics to identify M. bovis-Mycobacterium tuberculosis antigens whose genes had been deleted in BCG Pasteur. In total, 13 open reading frames (ORFs) from the RD1, RD2, and RD14 regions of the M. tuberculosis genome were selected. Pools of overlapping peptides spanning these ORFs were tested in M. bovis-infected (n = 22), BCG-vaccinated (n = 6), and unvaccinated (n = 10) control cattle. All were recognized in infected cattle, with responder frequencies varying between 16 and 86%. In particular, eight highly immunogenic antigens were identified whose potentials as diagnostic reagents or as subunit vaccines warrant further study (Rv1983, Rv1986, Rv3872, Rv3873, Rv3878, Rv3879c, Rv1979c, and Rv1769).

Amino Acid Sequence↗

PubMed2001

Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment.

The wide spectrum of clinical outcomes following infection with Mycobacterium tuberculosis is largely determined by the host immune response; therefore, we studied several clinically defined groups of individuals (n = 120) that differ in their ability to contain the bacillus. To quantitate M. tuberculosis-specific T cells directly ex vivo, we enumerated IFN-gamma-secreting CD4 T cells specific for ESAT-6, a secreted Ag that is highly specific for M. tuberculosis, and a target of protective immune responses in animal models. We found that frequencies of circulating ESAT-6 peptide-specific IFN-gamma-secreting CD4 T cells were higher in latently infected healthy contacts and subjects with minimal disease and low bacterial burdens than in patients with culture-positive active pulmonary tuberculosis (p = 0.009 and p = 0.002, respectively). Importantly, the frequency of these Ag-specific CD4 T cells fell progressively in all groups with treatment (p = 0.005), suggesting that the lower responses in patients with more extensive disease were not due to tuberculosis-induced immune suppression. This population of M. tuberculosis Ag-specific Th1-type CD4 T cells appears to correlate with clinical phenotype and declines during successful therapy; these features are consistent with a role for these T cells in the containment of M. tuberculosis in vivo. Such findings may assist in the design and evaluation of novel tuberculosis vaccine candidates.

Adult↗

PubMed2001

Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells.

BACKGROUND: Identification of individuals latently infected with Mycobacterium tuberculosis is an important part of tuberculosis control. The current method, the tuberculin skin test (TST), has poor specificity because of the antigenic cross-reactivity of purified protein derivative (PPD) with M bovis BCG vaccine and environmental mycobacteria. ESAT-6 is a secreted antigen that is highly specific for M tuberculosis complex, but is absent from M bovis BCG. With an enzyme-linked immunospot (ELISPOT) assay for interferon gamma, we have identified ESAT-6-specific T cells as an accurate marker of M tuberculosis infection. METHODS: We did a prospective, masked study of 50 healthy contacts, with varying but well defined degrees of exposure to M tuberculosis, who attended an urban contact-tracing clinic. We assessed and compared the efficacy of our assay and TST for detection of symptomless infected individuals by correlation of test results with the degree of exposure to an infectious index case. FINDINGS: The ESAT-6 ELISPOT assay results had a strong positive relation with increasing intensity of exposure (odds ratio=9.0 per unit increase in level of exposure [95% CI 2.6--31.6], p=0.001), whereas TST results had a weaker relation with exposure (1.9 [1.0--3.5], p=0.05). By contrast, ELISPOT results were not correlated with BCG vaccination status (p=0.7), whereas TST results were significantly more likely to be positive in BCG-vaccinated contacts (12.1 [1.3--115.7], p=0.03). INTERPRETATION: This new antigen-specific T cell-based assay could allow more accurate identification of symptom-free individuals recently exposed to M tuberculosis, and thereby help to improve tuberculosis control.

Adult↗

PubMed2001

Derivation and validation of guidelines for stool cultures for enteropathogenic bacteria other than Clostridium difficile in hospitalized adults.

CONTEXT: The yield of in-hospital stool cultures performed more than 72 hours after admission is low, and a commonly used policy dictates that laboratories reject these cultures to save costs. However, enteropathogenic bacteria other than Clostridium difficile (EPB) may cause nosocomial illness that would be missed by use of such a "3-day rule." OBJECTIVE: To develop guidelines for hospital use of stool cultures that are sensitive to clinically relevant cases of sporadic and epidemic nosocomial diarrhea. DESIGN: Five-part study that incorporated a derivation sample based on retrospective chart review and a prospective cohort study (including cost savings analysis), and a validation sample based on retrospective chart review. SETTING: Four European academic health care centers. PATIENTS: Derivation sample: 1735 adult inpatients from whom 3416 stool cultures were obtained during a 19-month period (1995-1997) and 68 adult inpatients for whom EPB were grown from stool cultures during a 10-year period (1988-1998); validation sample: 65 patients with sporadic isolation of EPB (1993-1998), 56 patients involved in 2 nosocomial Salmonella outbreaks (1992 and 1997), and 330 patients who had stool cultures performed (1998). MAIN OUTCOME MEASURE: Performance of derived criteria in detecting pathogenic bacteria and outbreaks and reducing total number of stool cultures performed. RESULTS: Stool cultures grew EPB in 3.3% of samples obtained </=72 hours after admission and 0.5% of samples obtained thereafter (P<.001). Isolation of EPB >72 hours after admission was not associated with clinical symptoms or signs but was associated with community-acquired diarrhea (24%), age 65 years or older with preexisting comorbid disease (25%), neutropenia (13%), HIV infection (10%), and nondiarrheal manifestations of enteric infections (16%). Twelve percent were asymptomatic carriers. These characteristics were used to create criteria for selecting patients for whom stool cultures would be indicated. These criteria were applied post hoc to a series of 1025 stool cultures; the number of stool cultures would have been reduced by 52% and no clinically significant cases would have been missed. Annual savings to a 355-bed institution would be approximately $7800 for reagent costs and 75 hours of technician time. In the validation samples, only 2 patients of 65 who had EPB would not have been identified, and neither required treatment. If the 3-day rule had been applied, 52 cases would not have been identified, 28 of which required antibiotic treatment. CONCLUSION: Our modified 3-day rule for use in selecting cases for stool culture is sensitive to sporadic and epidemic cases of nosocomial diarrhea in hospitalized adults.

Adult↗

PubMed2001

Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells.

There is no reliable means of detecting latent M. tuberculosis infection, and even in patients with active tuberculosis, infection is often unconfirmed. We hypothesized that M. tuberculosis antigen-specific T cells might reliably indicate infection. We enumerated peripheral blood-derived interferon gamma (IFN-gamma)-secreting T cells responding to epitopes from ESAT-6, an antigen that is highly specific for M. tuberculosis complex but absent from BCG, in four groups of individuals. Forty-five of 47 patients with bacteriologically confirmed tuberculosis had ESAT-6-specific IFN-gamma-secreting T cells, compared with four of 47 patients with nontuberculous illnesses, indicating that these T cells are an accurate marker of M. tuberculosis infection. This assay thus has a sensitivity of 96% (95% confidence interval [CI] 92-100) for detecting M. tuberculosis infection in this patient population. By comparison, of the 26 patients with tuberculosis who had a diagnostic tuberculin skin test (TST), only 18 (69%) were positive (p = 0.003). In addition, 22 of 26 (85%) TST-positive exposed household contacts had ESAT-6-specific T cells, whereas zero of 26 unexposed BCG-vaccinated subjects responded. This approach enables rapid detection of M. tuberculosis infection in patients with active tuberculosis and in exposed asymptomatic individuals at high risk of latent infection; it also successfully distinguishes between M. tuberculosis infection and BCG vaccination. This capability may facilitate tuberculosis control in nonendemic regions.

Adolescent↗

PubMed2000

Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians.

Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin. The M. tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M. bovis bacille Calmette-Guérin (BCG) and most environmental mycobacteria. We recently identified circulating ESAT-6-specific T cells as an accurate marker of M. tuberculosis infection. Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India. Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides. In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen. This study suggests an 80% prevalence of latent M. tuberculosis infection in urban India.

Adolescent↗

PubMed2000

Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study.

BACKGROUND: Susceptibility to disease after infection by Mycobacterium tuberculosis is influenced by environmental and host genetic factors. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of M. tuberculosis. This effect may be influenced by polymorphisms at three sites in the vitamin D receptor (VDR) gene. We investigated the interaction between serum vitamin D (25-hydroxycholecalciferol) concentrations and VDR genotype on susceptibility to tuberculosis. METHODS: This study was a hospital-based case-control analysis of Asians of Gujarati origin, a mainly vegetarian immigrant population with a high rate of tuberculosis. We typed three VDR polymorphisms (defined by the presence of restriction endonuclease sites for Taq1, Bsm1, and Fok1) in 91 of 126 untreated patients with tuberculosis and 116 healthy contacts who had been sensitised to tuberculosis. Serum 25-hydroxycholecalciferol was recorded in 42 contacts and 103 patients. FINDINGS: 25-hydroxycholecalciferol deficiency was associated with active tuberculosis (odds ratio 2.9 [95% CI 1.3-6.5], p=0.008), and undetectable serum 25-hydroxycholecalciferol (<7 nmol/L) carried a higher risk of tuberculosis (9.9 [1.3-76.2], p=0.009). Although there was no significant independent association between VDR genotype and tuberculosis, the combination of genotype TT/Tt and 25-hydroxycholecalciferol deficiency was associated with disease (2.8 [1.2-6.5]) and the presence of genotype ff or undetectable serum 25-hydroxycholecalciferol was strongly associated with disease (5.1 [1.4-18.4]). INTERPRETATION: 25-hydroxycholecalciferol deficiency may contribute to the high occurrence of tuberculosis in this population. Polymorphisms in the VDR gene also contribute to susceptibility when considered in combination with 25-hydroxycholecalciferol deficiency.

Calcifediol↗

PubMed2000

High frequencies of circulating IFN-gamma-secreting CD8 cytotoxic T cells specific for a novel MHC class I-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease.

MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Mycobacterium tuberculosis in animal models but their role in humans remains unclear. We therefore studied subjects who had successfully contained M. tuberculosis infection in vivo, i.e. exposed healthy household contacts and individuals with inactive self-healed pulmonary tuberculosis. Using the ELISPOT assay for IFN-gamma, we screened peptides from ESAT-6, a secreted antigen that is highly specific for M. tuberculosis. We identified a novel nonamer epitope: unstimulated peripheral blood-derived CD8 T cells displayed peptide-specific IFN-gamma release ex vivo while CD8 T cell lines and clones exhibited HLA-A68.02-restricted cytolytic activity and recognized endogenously processed antigen. The frequency of CD8 CTL specific for this single M. tuberculosis epitope, 1/2500 peripheral blood lymphocytes, was equivalent to the combined frequency of all IFN-gamma-secreting purified protein derivative-reactive T cells ex vivo. This highly focused CTL response was maintained in an asymptomatic contact over 2 years and is the most potent antigen-specific antimycobacterial CD8 CTL response hitherto described. Thus, human M. tuberculosis-specific CD8 CTL are not necessarily associated with active disease per se. Rather, our results are consistent with a protective role for these ESAT-6-specific CD8 T cells in the long-term control of M. tuberculosis in vivo in humans.

Amino Acid Sequence↗

PubMed2000

Cytotoxic T-lymphocyte epitopes for HLA-B53 and other HLA types in the malaria vaccine candidate liver-stage antigen 3.

The development of an effective preerythrocytic vaccine against Plasmodium falciparum malaria is likely to require inclusion of components from several preerythrocytic antigens. The association of HLA-B53 with resistance to severe malaria in West Africa provided evidence that HLA class I-restricted CD8(+) T-cell responses play a role in protective immunity in African children, supporting data from rodent models of malaria. Previously, a single epitope from liver-stage-specific antigen 1 (LSA-1) has been shown to be recognized by HLA-B53-specific cytotoxic T lymphocytes (CTL), but HLA-B53 epitopes were not found in four other antigens. In this study we measured CTL responses to peptides from the recently sequenced antigen liver-stage antigen 3 (LSA-3) and identified in it a new epitope restricted by HLA-B53. Several CTL epitopes restricted by other class I types were also identified within LSA-3 in studies in The Gambia and Tanzania. CTL were also identified to an additional P. falciparum antigen, exported protein 1 (Exp-1), the homologue of which is a protective antigen in a rodent model of malaria. These findings emphasize the diversity of P. falciparum antigens recognized by CD8(+) T cells in humans and support the inclusion of components from several antigens in new CTL-inducing vaccines against malaria.

Amino Acid Sequence↗

PubMed1999

Potent induction of focused Th1-type cellular and humoral immune responses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine.

The RTS,S/SBAS2 vaccine confers sterile protection against Plasmodium falciparum sporozoite challenge. The mechanisms underlying this are of great interest, yet little is known about the immune effector mechanisms induced by this vaccine. The immune responses induced by RTS,S/SBAS2 were characterized in 10 malaria-naive volunteers. Several epitopes in the circumsporozoite protein (CSP) were identified as targets of cultured interferon (IFN)-gamma-secreting CD4+ T cells. RTS,S-specific IFN-gamma-secreting effector T cells were induced in 8 subjects; this ex vivo response mapped to a single peptide in Th2R. CSP-specific CD8+ cytotoxic T lymphocytes were not detected. RTS, S-specific IFN-gamma production was universal, whereas interleukin-4 and -5 production was rare. RTS,S-specific lymphoproliferative responses and antibodies to CSP were strongly induced in all volunteers. Responses waned with time but were boostable. Thus, RTS, S/SBAS2 is a potent inducer of Th1-type cellular and humoral immunity. These results highlight possible immune mechanisms of protection and have important implications for vaccine design in general.

Adult↗

PubMed1999

Acute pulmonary schistosomiasis in travelers returning from Lake Malawi, sub-Saharan Africa.

We describe four cases of acute schistosomiasis presenting to the Infectious Diseases Unit of John Radcliffe Hospital (Oxford, England) during a 2-month period in autumn 1997. All four patients had swum in Lake Malawi, a freshwater lake in sub-Saharan Africa that is associated with Schistosoma haematobium and, less commonly, Schistosoma mansoni infections. All four patients had a severe acute illness and had prominent pulmonary involvement, both clinically and radiologically. This represents a change in the recognized pattern of presentation and could possibly reflect a new parasite variant in the lake.

Acute Disease↗

PubMed1998

Human cytolytic and interferon gamma-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis.

Protective immunity to Mycobacterium tuberculosis is poorly understood, but mounting evidence, at least in animal models, implicates major histocompatibility complex class I-restricted CD8+ T cells as an essential component. By using a highly sensitive assay for single cell interferon gamma release, we screened an array of M. tuberculosis antigen-derived peptides congruent with HLA class I allele-specific motifs. We identified CD8+ T cells specific for epitopes in the early secretory antigenic target 6 during active tuberculosis, after clinical recovery and in healthy contacts. Unrestimulated cells exhibited peptide-specific interferon gamma secretion, whereas lines or clones recognized endogenously processed antigen and showed cytolytic activity. These results provide direct evidence for the involvement of CD8+ cytotoxic T lymphocytes in host defense against M. tuberculosis in humans and support current attempts to generate protective cytotoxic T lymphocyte responses against M. tuberculosis by vaccination.

Adult↗

PubMed1998

Cytotoxic T-lymphocytes against malaria and tuberculosis: from natural immunity to vaccine design.

1. Mycobacterium tuberculosis and the liver stage of Plasmodium falciparum are intracellular pathogens which are potentially susceptible to cytotoxic T-lymphocytes, a crucial component of the protective immune response to viral infections. Evidence from animal models points to a protective role for cytotoxic T-lymphocytes against M. tuberculosis and P. falciparum, but cytotoxic T-lymphocytes specific for these pathogens have been difficult to identify in man.2. Using a reverse immunogenetic approach, candidate epitopes from selected antigens of P. falciparum and M. tuberculosis were used to detect peptide-specific cytotoxic T-lymphocyte responses in individuals exposed to these pathogens. Cytotoxic T-lymphocyte activity was detected by the 51Cr release cytotoxicity assay and a sensitive ELISPOT assay for single-cell interferon-gamma release.3. In naturally exposed, partially immune Africans in The Gambia, eight largely conserved cytotoxic T-lymphocyte epitopes in P. falciparum, restricted by several different HLA class I alleles, were identified. Several epitopes were also recognized in Tanzanians and cytotoxic T-lymphocytes recognized endogenously processed antigen.4. In tuberculosis patients with HLA-B52, a CD8+ cytotoxic T-lymphocyte epitope was identified in ESAT-6, a secreted antigen specific for M. tuberculosis complex but absent in BCG. Cytotoxic T-lymphocytes exhibited HLA-B52-restricted peptide-specific interferon-gamma release and lytic activity and recognized endogenously processed antigen.5. These studies demonstrate that CD8+ cytotoxic T-lymphocytes specific for mycobacterial and protozoal antigens are induced during natural infections in humans. The identification of these T-cells endorses current strategies to develop cytotoxic T-lymphocyte-inducing vaccines against P. falciparum and M. tuberculosis and highlights candidate antigens for inclusion in subunit vaccines.

Animals↗