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BackgroundThere is an urgent need for fair and equitable access to safe and effective vaccines to end the COVID-19 pandemic. Shortages in reagents and vaccines are a major challenge, as well as limited knowledge on dose response relationship with mRNA COVID-19 vaccines. We explored intradermal fractional dose administration of a mRNA SARS-CoV-2/COVID-19 vaccine as a potential dose-sparing strategy.MethodsWe conducted a proof-of-concept, dose-escalation, open-label, randomised-controlled vaccine trial (IDSCOVA) in healthy adults aged 18-30 years. To test initial safety, ten participants received 10 µg mRNA-1273 vaccine through intradermal injection at day 1 and 29. Following a favourable safety review, thirty participants were 1:1 randomised to receive 20 µg mRNA-1273 either intradermally or intramuscularly. The primary endpoint was tolerability and safety. The secondary endpoint was seroconversion and specific IgG concentration against SARS-CoV-2 spike S1 and Receptor Binding Domain (RBD) after the second dose at day 43. We compared results to two historical cohorts of non-hospitalised COVID-19 patients and vaccinated individuals.FindingsThirty-eight of forty included participants (median age 25 years) completed the study. There were no serious adverse events. Self-reported local adverse reactions after intradermal delivery were mild, both in the 10 µg and the 20 µg group. In the higher dose group, systemic adverse reactions were more common, but still well tolerated. All 38 participants mounted substantially higher IgG-anti-S1 and IgG-anti-RBD concentrations at day 43 than COVID-19 controls. At day 43, anti-S1 (95% CI) was 1,696 (1,309-2,198) BAU/mL for the 10 µg intradermal group, 1,406 (953·5-2,074) BAU/mL for the 20 µg intramuscular group and 2,057 (1,421-2,975) BAU/mL for the 20 µg intradermal group. Anti-S1 was 107·2 (63-182·2) BAU/mL for the convalescent plasma control group and 1,558 (547·8-4,433) BAU/mL for the individuals vaccinated with 100 µg mRNA-1273.InterpretationIntradermal administration of 10 µg and 20 µg mRNA-1273 vaccine was well tolerated and safe, and resulted in a robust antibody response. Intradermal vaccination has the potential to be deployed for vaccine dose-sparing.FundingThe trial was supported by crowdfunding (Wake Up to Corona).

The immune system plays a major role in Coronavirus Disease 2019 (COVID-19) pathogenesis, viral clearance and protection against re-infection. Immune cell dynamics during COVID-19 have been extensively documented in peripheral blood, but remain elusive in the respiratory tract. We performed minimally-invasive nasal curettage and mass cytometry to characterize nasal immune cells of COVID-19 patients during and 5-6 weeks after hospitalization. Contrary to observations in blood, no general T cell depletion at the nasal mucosa could be detected. Instead, we observed increased numbers of nasal granulocytes, monocytes, CD11c+ NK cells and exhausted CD4+ T effector memory cells during acute COVID-19 compared to age-matched healthy controls. These pro-inflammatory responses were found associated with viral load, while neutrophils also negatively correlated with oxygen saturation levels. Cell numbers mostly normalized following convalescence, except for persisting CD127+ granulocytes and activated T cells, including CD38+ CD8+ tissue-resident memory T cells. Moreover, we identified SARS-CoV-2 specific CD8+ T cells in the nasal mucosa in convalescent patients. Thus, COVID-19 has both transient and long-term effects on the immune system in the upper airway.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates

Background: There is an urgent need for fair and equitable access to safe and effective vaccines to end the COVID-19 pandemic. Shortages in reagents and vaccines are a major challenge, as well as limited knowledge on dose response relationship with mRNA COVID-19 vaccines. We explored intradermal fractional dose administration of a mRNA SARS-CoV-2/COVID-19 vaccine as a potential dose-sparing strategy. Methods: We conducted a proof-of-concept, dose-escalation, open-label, randomised-controlled vaccine trial (IDSCOVA) in healthy adults aged 18-30 years. To test initial safety, ten participants received 10 µg mRNA-1273 vaccine through intradermal injection at day 1 and 29. Following a favourable safety review, thirty participants were 1:1 randomised to receive 20 µg mRNA-1273 either intradermally or intramuscularly. The primary endpoint was tolerability and safety. The secondary endpoint was seroconversion and specific IgG concentration against SARS-CoV-2 spike S1 and Receptor Binding Domain (RBD) after the second dose at day 43. We compared results to two historical cohorts of non-hospitalised COVID-19 patients and vaccinated individuals. Findings: Thirty-eight of forty included participants (median age 25 years) completed the study. There were no serious adverse events. Self-reported local adverse reactions after intradermal delivery were mild, both in the 10 µg and the 20 µg group. In the higher dose group, systemic adverse reactions were more common , but still well tolerated. All 38 participants mounted substantially higher IgG-anti-S1 and IgG-anti-RBD concentrations at day 43 than COVID-19 controls. At day 43, anti-S1 (95% CI) was 1,696 (1,309-2,198) BAU/mL for the 10 µg intradermal group, 1,406 (953·5-2,074) BAU/mL for the 20 µg intramuscular group and 2,057 (1,421-2,975) BAU/mL for the 20 µg intradermal group. Anti-S1 was 107·2 (63-182·2) BAU/mL for the convalescent plasma control group and 1,558 (547·8-4,433) BAU/mL for the individuals vaccinated with 100 µg mRNA-1273. Interpretation: Intradermal administration of 10 µg and 20 µg mRNA-1273 vaccine was well tolerated and safe, and resulted in a robust antibody response. Intradermal vaccination has the potential to be deployed for vaccine dose-sparing. Clinical Trial Registration Details: registered in the Netherlands Trial Register (NTR) (https://www.trialregister.nl/trial/9275). Funding Information: The trial was supported by crowdfunding (Wake Up to Corona). Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: The trial was performed in accordance with the ethical principles of the Declaration of Helsinki and Good Clinical Practice guidelines developed by the International Harmonisation Task Force. The protocol was approved by the Medical Ethical Committee Leiden, Den Haag, Delft (NL 76702.058.21). All participants provided written informed consent. The vaccine manufacturer was not involved in this trial.

Intravesical bacillus Calmette-Guerin (BCG) is widely used for high-risk, non-muscle-invasive bladder cancer. This report describes four cases that illustrate the spectrum of BCG-induced complications, varying from granulomatous prostatitis to sepsis. There is considerable debate regarding whether inflammation or infection is the predominant mechanism in the pathogenesis of BCG disease. In two patients with a systemic illness, the symptoms first resolved after adding prednisone, indicating a principal role for inflammation in systemic disease. In vitro testing of T-cell responses and a mycobacterial growth inhibition assay were performed for these patients with systemic disease. The patient with mild symptoms showed more effective in vitro growth reduction of BCG, while the patient with sepsis and organ involvement had high T-cell responses but ineffective killing. While these findings are preliminary, it is believed that immunological assays, as described in this report, may provide a better insight into the pathogenesis of BCG disease in individual patients, justifying further research. (C) 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background Recently, internet-based cognitive behavioral therapy (ICBT) and serious gaming interventions have been suggested to enhance accessibility to interventions and engagement in psychological interventions that aim to promote health outcomes. Few studies, however, have investigated their effectiveness in the context of simulated real-life challenges. Objective We aimed to examine the effectivity of a guided ICBT combined with a serious gaming intervention in improving self-reported psychophysiological and immunological health endpoints in response to psychophysiological and immune-related challenges. Methods Sixty-nine healthy men were randomly assigned to the intervention condition, receiving ICBT combined with serious gaming for 6 weeks, or the control condition, receiving no intervention. Self-reported vitality was the primary endpoint. Other self-reported psychophysiological and immunological endpoints were assessed following various challenges, including a bacillus Calmette-Guérin vaccination evoking pro-inflammatory responses, 1 and 4 weeks after the intervention period. Results Although the intervention did not affect vitality-associated parameters, self-reported sleep problems (P=.027) and bodily sensations (P=.042) were lower directly after the intervention compared with controls. Furthermore, wellbeing (P=.024) was higher in the intervention group after the psychophysiological challenges. Although no significant group differences were found for the psychophysiological and immunological endpoints, the data provided preliminary support for increased immunoglobulin antibody responses at the follow-up time points (P Conclusions The present study provides some support for improving health endpoints with an innovative ICBT intervention. Future research should replicate and further extend the present findings by consistently including challenges and a wide range of immune parameters into the study design. Trial Registration Nederlands Trial Register NTR5610; https://www.trialregister.nl/trial/5466

BACKGROUND Recently, internet-based cognitive behavioral therapy (ICBT) and serious gaming interventions have been suggested to enhance accessibility to interventions and engagement in psychological interventions that aim to promote health outcomes. Few studies, however, have investigated their effectiveness in the context of simulated real-life challenges. OBJECTIVE We aimed to examine the effectivity of a guided ICBT combined with a serious gaming intervention in improving self-reported psychophysiological and immunological health endpoints in response to psychophysiological and immune-related challenges. METHODS Sixty-nine healthy men were randomly assigned to the intervention condition, receiving ICBT combined with serious gaming for 6 weeks, or the control condition, receiving no intervention. Self-reported vitality was the primary endpoint. Other self-reported psychophysiological and immunological endpoints were assessed following various challenges, including a bacillus Calmette-Guérin vaccination evoking pro-inflammatory responses, 1 and 4 weeks after the intervention period. RESULTS Although the intervention did not affect vitality-associated parameters, self-reported sleep problems (P=.027) and bodily sensations (P=.042) were lower directly after the intervention compared with controls. Furthermore, wellbeing (P=.024) was higher in the intervention group after the psychophysiological challenges. Although no significant group differences were found for the psychophysiological and immunological endpoints, the data provided preliminary support for increased immunoglobulin antibody responses at the follow-up time points (P CONCLUSIONS The present study provides some support for improving health endpoints with an innovative ICBT intervention. Future research should replicate and further extend the present findings by consistently including challenges and a wide range of immune parameters into the study design. CLINICALTRIAL Nederlands Trial Register NTR5610; https://www.trialregister.nl/trial/5466

BackgroundSeveral radiological features have been reported in association with latent tuberculosis infection (LTBI) but it has not been studied which are specific. The aim of this study was to evaluate allegedly characteristic abnormalities on chest radiography (CXR) in patients with LTBI compared to uninfected controls.MethodsFrom 236 patients tested with QuantiFERON-TB Gold In-Tube (QFT), the CXR was re-evaluated in a blinded fashion for fibrotic scarring, (non-)calcified nodules and pleural thickening. LTBI was defined as presence of a positive QFT result and/or positive tuberculin skin test result stratified by Bacille Calmette-Guérin-vaccination status.ResultsAny predefined abnormality of LTBI was observed in 116/236 (49.2%) patients, the frequency not being different between groups. However, the specificity for LTBI of a fibrotic scar ≥ 2 cm2 was 100% [95% CI: 92.0%–100%] and of a calcified nodule ≥1.5 mm was 95.7% [95% CI: 85.2%–99.5%]. The frequency of non-calcified nodules and pleural thickening did not differ between groups.ConclusionOnly a fibrotic scar ≥ 2 cm2 and/or a calcified nodule ≥1.5 mm were significantly associated with LTBI. This finding is clinically relevant mainly in patients who are at significant risk of TB reactivation and in whom indirect diagnostic tests may be unreliable.

In cancer and chronic infectious diseases, immune checkpoint-blockade of inhibitory receptors can enhance T-cell immunity. In tuberculosis (TB), a chronic infectious disease, prolonged antigen exposure can potentially drive terminal T-cell differentiation towards functional 'exhaustion': in human TB T-cells express PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein-4). However, in murine TB not PD-1 but rather killer cell lectin-like receptor subfamily-G1 (KLRG1) was a superior indicator of terminal T-cell differentiation. We therefore compared expression of KLRG1, PD-1 and CTLA-4 on T-cells in different stages of human TB, and also analysed their induction following BCG-vaccination. KLRG1, PD-1 and CTLA-4-expression were highest on in vitro BCG-stimulated CD4(+) T-cells following recent TB-treatment; KLRG1 and PD-1-expression on CD4(+) T-cells in active - but not latent TB were only slightly increased compared to healthy donors. BCG-vaccination induced KLRG1-expression on BCG-stimulated CD8(+) but not CD4(+) T-cells, while neither PD-1 nor CTLA-4-expression increased. KLRG1-expressing CD8(+) T-cells exhibited markedly decreased proliferation, whereas PD-1(+) T-cells proliferated after in vitro BCG-stimulation. Thus, we demonstrate the presence of increased KLRG1-expressing T-cells in TB-treated individuals, and present KLRG1 as a marker of decreased human T-cell proliferation following BCG-vaccination. These results expand our understanding of cell-mediated immune control of mycobacterial infections. (c) 2015 Elsevier Ltd. All rights reserved.

The lack of defined correlates of protection hampers development of vaccines against tuberculosis (TB). In vitro mycobacterial outgrowth assays are thought to better capture the complexity of the human host/Mycobacterium tuberculosis (Mtb) interaction. Here, we used a mycobacterial growth inhibition assay (MGIA) based on peripheral blood mononuclear cells to investigate the capacity to control outgrowth of bacille Calmette-Guerin (BCG). Interestingly, strong control of BCG outgrowth was observed almost exclusively in individuals with recent exposure to Mtb, but not in (long-term) latent TB infection, and only modestly in BCG vaccinees. Mechanistically, control of mycobacterial outgrowth strongly correlated with the presence of a CD14dim monocyte population, but also required the presence of T cells. The nonclassical monocytes produced CXCL10, and CXCR3 receptor blockade inhibited the capacity to control BCG outgrowth. Expression of CXCR3 splice variants was altered in recently Mtb-exposed individuals. Cytokines previously associated with trained immunity were detected in MGIA supernatants, and CXCL9, CXCL10, and CXCL11 represent new markers of trained immunity. These data indicate that CXCR3 ligands are associated with trained immunity and are critical factors in controlling mycobacterial outgrowth. In conclusion, control of mycobacterial outgrowth early after exposure to Mtb is the result of trained immunity mediated by a CXCL10-producing nonclassical CD14dim monocyte subset.

The diagnosis of a latent tuberculosis infection (LTBI) is based on detection of immunity against Mycobacterium tuberculosis (Mtb). The tuberculin skin test (TST), the Quantiferon (QFT) and a prolonged lymphocyte stimulation test using either ESAT-6/CFP-10 (LST-EC) or PPD (LST-PPD) were evaluated in a cohort of 495 individuals, suspected to have LTBI, in a low endemic country. While the TST and LST-PPD were both positive in the majority (75%) of individuals, only one third responded in the LST-EC and in the QFT. The choice for LTBI treatment was significantly associated with ESAT6/CFP10 recognition, however the LST-EC detected considerably more individuals (21%) with immunity against Mtb, who might also be at risk for development of active TB, although none of them did during follow up. Follow-up for 2 years showed 7% conversions and 32% reversions for the QFT. The LST-EC showed higher conversion rates (∼45%), although the percentage of individuals positive in the LST-EC did not change significantly within the follow-up period. LTBI treatment did not alter immune recognition of Mtb antigens. In conclusion, the sensitivity of tests for detection of cellular immunity to Mtb specific antigens depends on test methodology and may vary considerably over time in a low endemic region.

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, IVETB 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.

Limited specificity of the tuberculin skin test incited the development of in vitro assays based on Mycobacterium tuberculosis-specific antigens such as ESAT-6 that are lacking in Bacillus Calmette Guérin (BCG). In animal studies, intradermal ESAT-6 was safe and induced specific skin test responses. The aim of the study was to assess the safety of intradermal recombinant dimer ESAT-6 (rdESAT-6) compared with tuberculin and to determine the human dose. The study design was a double-blind Phase I study with intra-subject randomization to the left and right forearm, comparing 2 Tuberculin Units (TU) intradermal tuberculin (RT23) with 0.01, 0.1, 1 or 10 microg rdESAT-6 in groups of five healthy controls or treated tuberculosis (TB) patients. The risk of sensitization after skin testing was assessed in healthy volunteers. All doses were tolerated well by healthy volunteers and responses to rdESAT-6 were limited to transient redness after 24 h only at the highest dose. No sensitization was observed. Because 1 microg rdESAT-6 induced large responses with local side effects in some TB patients, the 10 microg dose of rdESAT-6 was not tested. Mean responses to 0.01, 0.1 and 1 microg rdESAT-6 measured 14.0, 19.8 and 38.8 mm of redness, respectively, and 7.0, 13.4 and 14.6 mm of induration. The response to tuberculin was similar to the responses to 0.1 microg rdESAT-6. Mild local side effects due to tuberculin and rdESAT-6 were observed in 8/15, respectively, 6/15 patients, more pronounced at the highest rdESAT-6 dose. In conclusion, this pilot Phase I study of safety, feasibility and dose finding of intradermal rdESAT-6 provides proof of principle of a specific skin test for human use. No serious adverse events were observed but the study was not sufficiently powered to demonstrate complete safety. Intradermal rdESAT-6 did not seem to sensitize healthy volunteers. In treated TB patients, responses to rdESAT-6 were optimal at 0.1 microg. Further studies are needed to evaluate sensitization after repeated doses and to study the effect of additional CFP-10 on the sensitivity of a TB-specific skin test.

The tuberculin skin test (TST) was compared with QuantiFERON-TB Gold in-tube (QFT-GIT) test for the diagnosis of tuberculosis in non- Mycobacterium bovis BCG-vaccinated military personnel. Among subjects positive by TST, 44.4% of recruits were positive by QFT-GIT compared with 11.5% subjects tested after missions abroad, suggesting that most TST conversions in the latter group were caused by nontuberculous mycobacteria.

Mycobacterium bovisbacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ+) interleukin 2-positive (IL-2+) tumor necrosis factor α-positive (TNF-α+) polyfunctional CD4+T cells concurrent with CD4+IL-17A+and CD8+IFN-γ+T cells or, in contrast, virtually absent cytokine responses with induction of CD8+regulatory T cells. Significant induction of polyfunctional CD4+IFN-γ+IL-2+TNF-α+T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8+T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8+regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4+T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.

The dormancy (DosR) regulon of Mycobacterium tuberculosis is expressed in vitro during hypoxia and low-dose nitric oxide stimulation. Tubercle bacilli are thought to encounter these conditions in humans during latent infection. In this study, immune responses were evaluated to 25 most strongly induced DosR-regulon-encoded proteins, referred to as latency antigens. Proliferation assays were performed using M. tuberculosis-specific T-cell lines and peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients, tuberculin skin test positive (TST+) individuals and uninfected controls. All 25 latency antigens were able to induce production of interferon-gamma (IFN-gamma) by T-cell lines. Eighteen latency antigens were also recognized by PBMC of M. tuberculosis-infected individuals, which indicates expression of the DosR-regulon during natural infection. Differential analysis showed that TST+ individuals recognized more latency antigens and with a stronger cumulative IFN-gamma response than TB patients, while the opposite profile was found for culture filtrate protein-10. In particular Rv1733c, Rv2029c, Rv2627c and Rv2628 induced strong IFN-gamma responses in TST+ individuals, with 61%, 61%, 52% and 35% responders, respectively. In conclusion, several new M. tuberculosis antigens were identified within the DosR-regulon. Particularly strong IFN-gamma responses to latency antigens were observed in latently infected individuals, suggesting that immune responses against these antigens may contribute to controlling latent M. tuberculosis infection.

This report of an accidental exposure to Mycobacterium tuberculosis in a microbiological laboratory illustrates the value of gamma interferon enzyme-linked immunospot assay using peptides of ESAT-6, CFP-10, TB37.6, and TB7.7 for the diagnosis of latent infection. In particular, positive responses to peptides 2 to 6 of TB37.6 were observed exclusively in recently infected persons.

Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.

New TB vaccines are urgently needed because of the apparent lack of effect of the BCG vaccine on rates of adult contagious pulmonary tuberculosis and the risk of disseminated BCG disease in immunocompromised individuals. Since BCG appears to protect children, the primary target for vaccine development is a booster vaccine for adults but such vaccines ideally need to be able to efficiently prime mycobacterially naïve individuals as well as boost individuals previously vaccinated with BCG and those latently infected with TB. Protective immunity against Mycobacterium tuberculosis depends mainly on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-γ) production. In the present study, we monitored safety and IFN-γ responses in healthy BCG-vaccinated and prior or latently TB-infected individuals receiving a novel vaccine composed of the fusion protein Ag85B-ESAT-6 combined with the adjuvant IC31(®), administered at 0 and 2 months. Vaccination caused few local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against Ag85B-ESAT-6 and both the Ag85B and ESAT-6 components, that could be augmented by second vaccination. The strong responses persisted through 32 weeks of follow-up, indicating the induction of a persistent memory response in the vaccine recipients.

Though widely used, the BCG vaccine has had little apparent effect on rates of adult pulmonary tuberculosis. Moreover, the risk of disseminated BCG disease in immunocompromised individuals means that improved TB vaccines ideally need to be able to efficiently prime mycobacterially-naïve individuals as well as boost individuals previously vaccinated with BCG. Protective immunity against Mycobacterium tuberculosis is thought to depend on the generation of a Th1-type cellular immune response characterized by interferon-gamma (IFN-gamma) production. In the present study, we monitored safety and IFN-gamma responses in healthy TB-naïve humans receiving an entirely novel vaccine, composed of the fusion protein Ag85B-ESAT-6, administered at 0 and 2 months either as recombinant protein alone or combined with two concentrations of the novel adjuvant IC31. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site, but it elicited strong antigen-specific T cell responses against H1 and both the Ag85B and the ESAT-6 components. These strong responses persisted through 2.5 years of follow-up, indicating the induction of a substantial memory response in the vaccine recipients.

The sensitivities of various gamma interferon release assays (IGRAs) for the detection of past latent Mycobacterium tuberculosis infection are not known. In this study, we aimed to assess the effects of various IGRA formats and in vitro incubation periods on test outcome. The results of the tuberculin skin test (TST) were compared with those of the QuantiFERON-TB Gold in-tube (QFT-GIT) test, an overnight enzyme-linked immunospot assay (ELISPOT), and a 6-day lymphocyte stimulation test (LST) by using the same M. tuberculosis -specific peptides and samples from 27 TST-positive persons with a history of exposure to M. tuberculosis , 4 patients cured of tuberculosis (TB), and 9 TST-negative controls. Among the TST-positive persons, the LST was more frequently positive (92%; P < 0.01) than either the QFT-GIT test (33%) or ELISPOT (46%). While good agreement was observed between the QFT-GIT test and ELISPOT (κ = 0.71) and between TST and LST (κ = 0.78), the agreement between TST or LST, on the one hand, and the QFT-GIT test or ELISPOT, on the other, was poor. These data indicate that the QFT-GIT test and overnight ELISPOT are less sensitive for the detection of past latent TB than the 6-day LST. The observed discrepancies between these IGRAs are most likely related to differences in incubation periods. Whether TST-positive persons with positive LST results but negative QFT-GIT and ELISPOT results are at risk for the development of TB needs to be elucidated before short-incubation IGRAs can be used for the screening of individuals for latent TB before immunosuppressive treatment.

Recently, interferon-gamma release assays (IGRA) for specific diagnosis of Mycobacterium tuberculosis infection have become available. In recent UK tuberculosis (TB) guidelines, it has been advised to screen for latent M. tuberculosis infection using the tuberculin skin test (TST), followed by IGRA if the TST is positive. Since TST can boost immune responses to tuberculin, the present authors evaluated whether TST administration affects the result of QuantiFERON-TB Gold in-tube (QFT-GIT), a whole blood-based IGRA. QFT-GIT was performed on the day of TST administration and the day of reading in 15 TST-negative subjects, 46 TST-positive subjects with recent or remote exposure to M. tuberculosis and five cured TB patients. No systematic boosting of QFT-GIT responses from negative to positive was observed. Only in a few TST-positive persons did TST enhance pre-existing QFT-GIT responses. Screening for latent Mycobacterium tuberculosis infection using tuberculin skin testing followed by interferon-gamma release assays on the day of reading is a reliable approach, as the specificity of QuantiFERON-TB Gold in-tube is not affected by prior tuberculin skin test administration.

16 páginas, 8 figuras. Disponoble información suplementaria en: http://www.nature.com/srep, New strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection. By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10-positive donors by proliferation and multi-cytokine production. This was validated in an independent cohort of latently Mtb-infected individuals. Significant T-cell responses were observed in the absence of IFN-γ-production. Collectively, the results underscore the power of our novel antigen discovery approach in identifying Mtb antigens, including those that induce unconventional T-cell responses, which may provide important novel tools for TB vaccination and biomarker profiling. Our generic approach is applicable to other infectious diseases., We acknowledge funding by EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381); EC ITN FP7 VACTRAIN project (the text represents the authors’ views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information), The Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038); European Research Council (ERC TB-ACCELERATE Grant Agreement No. 638553). Funding was also supplied by the Ministerio de Economía y Competitividad (Spanish Government) research grant SAF2013-43521-R, and the European Research Council (ERC) (638553-TB-ACCELERATE) (to IC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.