[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis is responsible for almost 2 million deaths annually. Mycobacterium bovis bacillus Calmette-Guérin, the only vaccine available against tuberculosis (TB), induces highly variable protection against TB, and better TB vaccines are urgently needed. A prerequisite for candidate vaccine Ags is that they are immunogenic and expressed by M. tuberculosis during infection of the primary target organ, that is, the lungs of susceptible individuals. In search of new TB vaccine candidate Ags, we have used a genome-wide, unbiased Ag discovery approach to investigate the in vivo expression of 2170 M. tuberculosis genes during M. tuberculosis infection in the lungs of mice. Four genetically related but distinct mouse strains were studied, representing a spectrum of TB susceptibility controlled by the supersusceptibility to TB 1 locus. We used stringent selection approaches to select in vivo-expressed M. tuberculosis (IVE-TB) genes and analyzed their expression patterns in distinct disease phenotypes such as necrosis and granuloma formation. To study the vaccine potential of these proteins, we analyzed their immunogenicity. Several M. tuberculosis proteins were recognized by immune cells from tuberculin skin test-positive, ESAT6/CFP10-responsive individuals, indicating that these Ags are presented during natural M. tuberculosis infection. Furthermore, TB patients also showed responses toward IVE-TB Ags, albeit lower than tuberculin skin test-positive, ESAT6/CFP10-responsive individuals. Finally, IVE-TB Ags induced strong IFN-γ(+)/TNF-α(+) CD8(+) and TNF-α(+)/IL-2(+) CD154(+)/CD4(+) T cell responses in PBMC from long-term latently M. tuberculosis-infected individuals. In conclusion, these IVE-TB Ags are expressed during pulmonary infection in vivo, are immunogenic, induce strong T cell responses in long-term latently M. tuberculosis-infected individuals, and may therefore represent attractive Ags for new TB vaccines.
The Journal of Immunology 01/2013; 190(4). DOI:10.4049/jimmunol.1201593 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than 2 billion individuals are latently infected with Mycobacterium tuberculosis (Mtb). Knowledge of the key Mtb antigens and responding T-cell subsets mediating protection against Mtb is critical for developing improved tuberculosis (TB) vaccines. We previously reported that Mtb DosR-regulon-encoded antigens are recognized well by human T cells in association with control of Mtb infection. The characteristics of the responding T-cell subsets, however, remained unidentified. We have therefore studied the cytokine production and memory phenotypes of Mtb DosR-regulon-encoded antigen-specific T cells from individuals who had been infected with Mtb decades ago, yet never developed TB (long-term latent Mtb-infected individuals). Using multi-parameter flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α and IL-2, we found double and single cytokine-producing CD4(+) as well as CD8(+) T cells to be the most prominent subsets, particularly IFN-γ(+) TNF-α(+) CD8(+) T cells. The majority of these T cells comprised effector memory and effector T cells. Furthermore, CFSE labeling revealed strong CD4(+) and CD8(+) T-cell proliferative responses induced by several "immunodominant" Mtb DosR antigens and their specific peptide epitopes. These findings demonstrate the prominent presence of double- and monofunctional CD4(+) and CD8(+) T-cell responses in naturally protected individuals and support the possibility of designing Mtb DosR antigen-based TB vaccines.
European Journal of Immunology 10/2011; 41(10):2925-36. DOI:10.1002/eji.201141602 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Mycobacterium bovis BCG vaccine is the only tuberculosis (TB) vaccine available, yet it provides limited protection against pulmonary TB in adults
and fails to protect against TB reactivation. We hypothesized that immunity against Mycobacterium tuberculosis “resuscitation-promoting factors” (Rpfs), which are small bacterial proteins that promote proliferation of dormant mycobacteria,
may be relevant in the human immune response to M. tuberculosis. In previous unpublished work, we found that Rpfs Rv0867c and Rv2389c induced gamma interferon (IFN-γ) production in the
blood of TB patients' healthy household contacts in several different African populations. Here we examine these two dominant
Rpf antigens in more detail and define the nature of the responding T-cell subsets. Multiparameter cytokine profiling showed
that Rv2389c and, to a lesser extent, Rv0867c were recognized by mycobacterium-responsive healthy Dutch individuals; peptide-scanning
revealed several epitopes, including a single immunodominant epitope in Rv2389c. Rv0867c and, to a lesser extent, Rv2389c
Rpf-specific T-cell responses were maintained for decades in long-term M. tuberculosis nonprogressors. Prominent Rv0867c-specific double- and single-cytokine-producing CD8+ T-cell subset responses were found, including a large population of CD8+ effector memory and effector T-cell subsets. We conclude that M. tuberculosis Rpf antigens are important targets in the human immune response to M. tuberculosis and represent interesting TB vaccine candidate antigens.
[Show abstract][Hide abstract] ABSTRACT: Influenza vaccine efficacy does not always correlate with humoral immune responses. Recent reports indicate that the cellular immune response also contributes to protection, however robust assays are lacking. We standardized and validated assays for detection of human influenza-specific cellular responses in four international laboratories. The production of granzyme B as marker of T cell-mediated cytotoxicity and release of Th1 and Th2 cytokines were evaluated. The granzyme B and cytokine assays were specific, accurate, precise, and robust. Replicate stimulations with PBMC from the same donors showed an intra-laboratory robustness (coefficient of variation) for quantitation of granzyme B of 33% and for cytokines - including IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4, IL-13, GM-CSF and including the log IFN-gamma/IL-10 ratio - of 52%. The inter-laboratory robustness for detection of granzyme B was 29% and for detection of all cytokines was 49%. The assays can now be used for determining cell-mediated immunity and explored as correlates of protection. Moreover, the precision and robustness of these cellular assays allow the reliable detection of cellular responses even in small study populations.
[Show abstract][Hide abstract] ABSTRACT: We intended to assess the risk for health care workers (HCWs) of acquiring M. tuberculosis infection after exposure to patients with sputum-smear positive pulmonary tuberculosis at three University Hospitals (Ullevål, Akershus, and Haukeland) in Norway.
We tested 155 exposed health care workers and 48 healthy controls both with a tuberculin skin test (Mantoux) and the T-SPOT.TB test, a recently developed interferon-gamma release assays based on the M. tuberculosis-specific ESAT-6 and CFP10 antigens, to investigate if this test might improve infection control measures.
Among the 155 exposed HCWs tested in this study, 27 individuals were defined as newly infected cases by TST after recent exposure, while only 3 of these had a positive T-SPOT.TB test. The number of T-SPOT.TB positives represents 11% of the individuals defined as recently infected by TST after exposure (3/27) and 2% of the total number of exposed people tested (3/155). In addition, 15 individuals had been previously defined as infected by TST before exposure of whom 2 subjects were T-SPOT.TB positive. All individuals detected as T-SPOT.TB positive belonged to the TST positive group (> 15 mm), and the percentage concordance between T-SPOT.TB and TST, including both previously and newly infected subjects, was 12% (5/42). The 48 control participants used in the study were all T-SPOT.TB negative, but 3 of these subjects were TST positive.
Our data indicate that the frequency of latent TB in the total cohort of HCWs is 3%, whereas the rate of transmission of TB to exposed individuals is approximately 2% and occurs through exposure periods of short duration. Thus, the risk of TB transmission to HCWs following TB exposure in a hospital setting in Norway is low, and improved screening approaches will benefit from the application of specific interferon-gamma release assays.
[Show abstract][Hide abstract] ABSTRACT: In Norway, screening for tuberculosis infection by tuberculin skin test (TST) has been offered for several decades to all children in 9th grade of school, prior to BCG-vaccination. The incidence of tuberculosis in Norway is low and infection with M. tuberculosis is considered rare. QuantiFERONTB Gold (QFT) is a new and specific blood test for tuberculosis infection. So far, there have been few reports of QFT used in screening of predominantly unexposed, healthy, TST-positive children, including first and second generation immigrants. In order to evaluate the current TST screening and BCG-vaccination programme we aimed to (1) measure the prevalence of QFT positivity among TST positive children identified in the school based screening, and (2) measure the association between demographic and clinical risk factors for tuberculosis infection and QFT positivity.
This cross-sectional multi-centre study was conducted during the school year 2005-6 and the TST positive children were recruited from seven public hospitals covering rural and urban areas in Norway. Participation included a QFT test and a questionnaire regarding demographic and clinical risk factors for latent infection. All positive QFT results were confirmed by re-analysis of the same plasma sample. If the confirmatory test was negative the result was reported as non-conclusive and the participant was offered a new test.
Among 511 TST positive children only 9% (44) had a confirmed positive QFT result. QFT positivity was associated with larger TST induration, origin outside Western countries and known exposure to tuberculosis. Most children (79%) had TST reactions in the range of 6-14 mm; 5% of these were QFT positive. Discrepant results between the tests were common even for TST reactions above 15 mm, as only 22 % had a positive QFT.
The results support the assumption that factors other than tuberculosis infection are widely contributing to positive TST results in this group and indicate the improved specificity of QFT for latent tuberculosis. Our study suggests a very low prevalence of latent tuberculosis infection among 9th grade school children in Norway. The result will inform the discussion in Norway of the usefulness of the current TST screening and BCG-policy.
[Show abstract][Hide abstract] ABSTRACT: QuantiFERONTB Gold (QFT) is a promising blood test for tuberculosis infection but with few data so far from immigrant screening. The aim of this study was to compare results of QFT and tuberculin skin test (TST) among newly arrived asylum seekers in Norway and to assess the role of QFT in routine diagnostic screening for latent tuberculosis infection.
The 1000 asylum seekers (age > or = 18 years) enrolled in the study were voluntarily recruited from 2813 consecutive asylum seekers arriving at the national reception centre from September 2005 to June 2006. Participation included a QFT test and a questionnaire in addition to the mandatory TST and chest X-ray.
Among 912 asylum seekers with valid test results, 29% (264) had a positive QFT test whereas 50% (460) tested positive with TST (indurations > or = 6 mm), indicating a high proportion of latent infection within this group. Among the TST positive participants 50% were QFT negative, whereas 7% of the TST negative participants were QFT positive. There was a significant association between increase in size of TST result and the likelihood of being QFT positive. Agreement between the tests was 71-79% depending on the chosen TST cut-off and it was higher for non-vaccinated individuals.
By using QFT in routine screening, further follow-up could be avoided in 43% of the asylum seekers who would have been referred if based only on a positive TST (> or = 6 mm). The proportion of individuals referred will be the same whether QFT replaces TST or is used as a supplement to confirm a positive TST, but the number tested will vary greatly. All three screening approaches would identify the same proportion (88-89%) of asylum seekers with a positive QFT and/or a TST > or = 15 mm, but different groups will be missed.
[Show abstract][Hide abstract] ABSTRACT: Sixty-five healthy adult volunteers were immunized four times at 1-week intervals with an inactivated whole-virus influenza vaccine based on the strain A/New Caledonia/20/99 (H1N1) without adjuvant. The vaccine was administered as nasal spray with a newly developed device to secure intranasal delivery (OptiMist, OptiNose AS, Oslo, Norway), as regular nasal spray, nasal drops or as an oral spray. Significant IgA-antibody responses in nasal secretions were induced in volunteers immunized intranasally but not after oral spray immunization. In saliva, IgA antibodies were only marginally amplified even after oral spray immunizations. At least 73% of the volunteers belonging to any group of vaccine delivery reached serum haemagglutination inhibition titres of 40 or higher, considered protective against influenza, after only two vaccine doses. Those who had the vaccine delivered intranasally also showed evidence from in vitro secretion of granzyme B that cytotoxic T cells had been stimulated. Although immunization with the breath-actuated OptiMist device and nasal drops were superior with respect to both mucosal and systemic immune responses, oral spray immunization might still be considered for studies of mucosal adjuvants that are not yet acceptable for intranasal use.
[Show abstract][Hide abstract] ABSTRACT: Twenty-eight healthy adult volunteers were immunized intranasally with an inactivated whole-virus influenza vaccine based on the strain A/New Caledonia/20/99 (H1N1), either in saline or mixed with formaldehyde-inactivated Bordetella pertussis as a mucosal adjuvant, or in a thixotropic vehicle with mucoadhesive properties. After four doses, all groups of vaccinees developed significant IgG- and IgA-antibody responses, measured by ELISA, in respectively serum and nasal secretions. None of the volunteers had demonstrable hemagglutination inhibition (HAI) antibodies in serum before being immunized, whereas more than 80% of them reached HAI titers>or=40, considered protective, after immunizations. In addition, cellular immune responses, measured as significant increases in CD4+ T-cell proliferation and granzyme B-producing cytotoxic T-cells, were detected against the vaccine strain as well as against heterologous virus strains (H3N2). However, no additive effect on these responses could be demonstrated with use of B. pertussis or the thixotropic substance in the present vaccines. It appeared, actually, that the mucoadhesive vehicle containing the thixotropic substance was less efficient than were the two other formulations. An influenza vaccine made as a simple particulate formulation of inactivated virus, and given repeatedly onto the nasal mucosa, may thus be an attractive alternative to currently available vaccines.
Human vaccines 03/2005; 1(2):85-90. DOI:10.4161/hv.1.2.1718 · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the ability of outer membrane vesicle (OMV) vaccines from Neisseria meningitidis serogroup B to induce vaccine-specific antibody and spleen cell proliferative responses in mice after being administered intranasally (i.n.) and/or subcutaneously (s.c.). A series of four weekly i.n. doses (25 microg) without adjuvant or a single s.c. dose (2.5 microg) with aluminum hydroxide was followed 2 months later by secondary i.n. or s.c. immunizations. After i.n. priming, both immunoglobulin G (IgG) antibody responses in serum, measured by enzyme-linked immunosorbent assay, and IgA antibodies in saliva and extracts of feces were significantly boosted by later i.n. immunizations. The IgG antibody responses in serum were also significantly augmented by secondary s.c. immunization after i.n. as well as s.c. priming. Sera from mice immunized i.n. reached the same level of bactericidal activity as after s.c. immunizations. The s.c. immunizations alone, however, had no effect on mucosal IgA antibody responses, but could prime for booster antibody responses in secretions to later i.n. immunizations. The i.n. immunizations also led to marked OMV-specific spleen cell proliferation in vitro. Both serum antibody responses and spleen cell proliferation were higher after i.n. priming and later s.c. immunizations than after s.c. immunizations alone. There was thus no evidence that i.n. priming had induced immunological tolerance within the B- or T-cell system. Our results indicate that a nonproliferating meningococcal OMV vaccine given i.n. can induce immunological memory and that it may be favorably combined with similar vaccines for injections.
Infection and Immunity 09/2001; 69(8):5010-5. DOI:10.1128/IAI.69.8.5010-5015.2001 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have studied the ability of an intranasally administered whole-cell pertussis vaccine (WCP) without adjuvant to induce antigen-specific T cell responses in humans. Six adult volunteers were given a vaccine dose (corresponding to 250 microg protein) by nasal spray four times at weekly intervals, and peripheral blood mononuclear cells were assayed for antigen-specific proliferative T cell responses. All six vaccinees had a WCP-specific response, which in four of them remained elevated throughout the 2 month study. All participants also responded to the filamentous haemagglutinin (FHA) antigen, and four of them responded to inactivated pertussis toxin (PTd). A significant correlation between T cell proliferation against WCP and WCP-specific IgA antibody levels in nasal secretions was observed. This demonstrates that intranasal administration of a non-proliferating bacterial vaccine without any additional mucosal adjuvant can induce vaccine-specific T cell responses related to mucosal IgA secretion.
[Show abstract][Hide abstract] ABSTRACT: We have studied the ability of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine, when administered intranasally without adjuvant, to induce T-cell responses in humans. A group of 12 vaccinees was immunized with four doses of OMVs (250 micrograms of protein/dose) at weekly intervals, and a single booster dose was given 5 months later. In vitro T-cell proliferation in response to the OMV vaccine, purified PorA (class 1) protein, PorB (class 3) protein, and one unrelated control antigen (Mycobacterium bovis BCG) was measured by [3H]thymidine incorporation into peripheral blood mononuclear cells obtained from the vaccinees before and after the immunizations. The nasal OMV immunizations induced antigen-specific T-cell responses in the majority of the vaccinees when tested against OMVs (7 of 12) and the PorA antigen (11 of 12). None of the vaccinees showed a vaccine-induced T-cell response to the PorB antigen after the initial four doses. Although some individuals responded to all the vaccine antigens after the booster dose, this response was not significant when the vaccinees were analyzed as a group. We have also demonstrated that the PorA antigen-specific T-cell responses correlated with anti-OMV immunoglobulin A (IgA) levels in nasal secretions, with anti-OMV IgG levels in serum, and with serum bactericidal activity. In conclusion, we have shown that it is possible to induce antigen-specific T-cell responses in humans by intranasal administration of a meningococcal OMV vaccine without adjuvant.
Infection and Immunity 03/1999; 67(2):921-7. · 3.73 Impact Factor