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Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) has variable efficacy in preventing tuberculosis. Both BCG strain and route of administration have been implicated in determining efficacy; however, these variables are not considered in current clinical recommendations for vaccine choice. We evaluated antigen-specific immunity after percutaneous or intradermal administration of Japanese BCG or intradermal administration of Danish BCG. Ten weeks after vaccination of neonates, percutaneous Japanese BCG had induced significantly higher frequencies of BCG-specific interferon-γ-producing CD4+ and CD8+ T cells in BCG-stimulated whole blood than did intradermal Danish BCG. Similarly, percutaneous vaccination with Japanese BCG resulted in significantly greater secretion of the T helper 1-type cytokines interferon-γ, tumor necrosis factor-α, and interleukin-2; significantly lower secretion of the T helper 2-type cytokine interleukin-4; and greater CD4+ and CD8+ T cell proliferation. Thus, BCG strain and route of neonatal vaccination confer different levels of immune activation, which may affect the efficacy of the vaccine.

Background. The efficacy of bacille Calmette-Guérin (BCG) may be enhanced by heterologous vaccination strategies that boost the BCG-primed immune response. One leading booster vaccine, MVA85A (where “MVA” denotes “modified vaccinia virus Ankara”), has shown promising safety and immunogenicity in human trials performed in the United Kingdom. We investigated the safety and immunogenicity of MVA85A in mycobacteria-exposed—but Mycobacterium tuberculosis-uninfected—healthy adults from a region of South Africa where TB is endemic. Methods. Twenty-four adults were vaccinated with MVA85A. All subjects were monitored for 1 year for adverse events and for immunological assessment. Results. MVA85A vaccination was well tolerated and induced potent T cell responses, as measured by interferon (IFN)-γ enzyme-linked immunospot assay, which exceeded prevaccination responses up to 364 days after vaccination. BCG-specific CD4+ T cells boosted by MVA85A were comprised of multiple populations expressing combinations of IFN-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-17, as measured by polychromatic flow cytometry. IFN-γ-expressing and polyfunctional IFN-γ+TNF-α+IL-2+ CD4+ T cells were boosted during the peak BCG-specific response, which occurred 7 days after vaccination. Conclusion. The excellent safety profile and quantitative and qualitative immunogenicity data strongly support further trials assessing the efficacy of MVA85A as a boosting vaccine in countries where TB is endemic. Trial registration. ClinicalTrials.gov identifier: NCT00460590.

Background Despite the recent innovations in tuberculosis (TB) and multi-drug resistant TB (MDR-TB) diagnosis, culture remains vital for difficult-to-diagnose patients, baseline and end-point determination for novel vaccines and drug trials. Herein, we share our experience of establishing a BSL-3 culture facility in Uganda as well as 3-years performance indicators and post-TB vaccine trials (pioneer) and funding experience of sustaining such a facility. Methods Between September 2008 and April 2009, the laboratory was set-up with financial support from external partners. After an initial procedure validation phase in parallel with the National TB Reference Laboratory (NTRL) and legal approvals, the laboratory registered for external quality assessment (EQA) from the NTRL, WHO, National Health Laboratories Services (NHLS), and the College of American Pathologists (CAP). The laboratory also instituted a functional quality management system (QMS). Pioneer funding ended in 2012 and the laboratory remained in self-sustainability mode. Results The laboratory achieved internationally acceptable standards in both structural and biosafety requirements. Of the 14 patient samples analyzed in the procedural validation phase, agreement for all tests with NTRL was 90% ( P <0.01). It started full operations in October 2009 performing smear microscopy, culture, identification, and drug susceptibility testing (DST). The annual culture workload was 7,636, 10,242, and 2,712 inoculations for the years 2010, 2011, and 2012, respectively. Other performance indicators of TB culture laboratories were also monitored. Scores from EQA panels included smear microscopy >80% in all years from NTRL, CAP, and NHLS, and culture was 100% for CAP panels and above regional average scores for all years with NHLS. Quarterly DST scores from WHO-EQA ranged from 78% to 100% in 2010, 80% to 100% in 2011, and 90 to 100% in 2012. Conclusions From our experience, it is feasible to set-up a BSL-3 TB culture laboratory with acceptable quality performance standards in resource-limited countries. With the demonstrated quality of work, the laboratory attracted more research groups and post-pioneer funding, which helped to ensure sustainability. The high skilled experts in this research laboratory also continue to provide an excellent resource for the needed national discussion of the laboratory and quality management systems.

Antigen-specific, MHC-restricted αβ T cells are necessary for protective immunity against Mycobacterium tuberculosis , but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis -specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis ( n  = 48) or controlled infection ( n  = 118). We found 24 T cell groups with similar TCR-β sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection ( n  = 17), and others that were associated with progression to disease ( n  = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development. Analysis of peripheral mycobacteria-reactive CD4 + T cell receptor sequences from individuals infected with Mycobacterium tuberculosis shows a high degree of overlap between progressors and controllers, but points to some distinct clonotypes that are enriched in either group.

Abstract 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.

Abstract Rationale Tuberculosis (TB) is a major cause of morbidity and mortality worldwide, thus there is an urgent need for novel TB vaccines. Objectives We investigated a novel TB vaccine candidate, M72/AS01, in a phase IIa trial of bacille Calmette-Guérin–vaccinated, HIV-uninfected, and Mycobacterium tuberculosis (Mtb)–infected and -uninfected adults in South Africa. Methods Two doses of M72/AS01 were administered to healthy adults, with and without latent Mtb infection. Participants were monitored for 7 months after the first dose; cytokine production profiles, cell cycling, and regulatory phenotypes of vaccine-induced T cells were measured by flow cytometry. Measurements and Main Results The vaccine had a clinically acceptable safety profile, and induced robust, long-lived M72-specific T-cell and antibody responses. M72-specific CD4 T cells produced multiple combinations of Th1 cytokines. Analysis of T-cell Ki67 expression showed that most vaccination-induced T cells did not express Th1 cytokines or IL-17; these cytokine-negative Ki67+ T cells included subsets of CD4 T cells with regulatory phenotypes. PD-1, a negative regulator of activated T cells, was transiently expressed on M72-specific CD4 T cells after vaccination. Specific T-cell subsets were present at significantly higher frequencies after vaccination of Mtb-infected versus -uninfected participants. Conclusions M72/AS01 is clinically well tolerated in Mtb-infected and -uninfected adults, induces high frequencies of multifunctional T cells, and boosts distinct T-cell responses primed by natural Mtb infection. Moreover, these results provide important novel insights into how this immunity may be appropriately regulated after novel TB vaccination of Mtb-infected and -uninfected individuals. Clinical trial registered with www.clinicaltrials.gov (NCT 00600782).

Abstract 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).

Abstract 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.

In most tuberculosis (TB) endemic countries, bacillus Calmette–Guérin (BCG) is usually given around birth to prevent severe TB in infants. The neonatal immune system is immature. Our hypothesis was that delaying BCG vaccination from birth to 10 weeks of age would enhance the vaccine-induced immune response. In a randomized clinical trial, BCG was administered intradermally either at birth ( n = 25) or at 10 weeks of age ( n = 21). Ten weeks after vaccination, and at 1 year of age, vaccine-specific CD4 and CD8 T cell responses were measured with a whole blood intracellular cytokine assay. Infants who received delayed BCG vaccination demonstrated higher frequencies of BCG-specific CD4 T cells, particularly polyfunctional T cells co-expressing IFN-γ, TNF-α and IL-2, and most strikingly at 1 year of age. Delaying BCG vaccination from birth to 10 weeks of age enhances the quantitative and qualitative BCG-specific T cell response, when measured at 1 year of age.

Background. BCG, the only licensed tuberculosis vaccine, affords poor protection against lung tuberculosis in infants and children. A new tuberculosis vaccine, which may enhance the BCG-induced immune response, is urgently needed. We assessed the safety of and characterized the T cell response induced by 3 doses of the candidate vaccine, MVA85A, in BCG-vaccinated infants from a setting where tuberculosis is endemic. Methods. Infants aged 5-12 months were vaccinated intradermally with either 2.5 X 10⁷, 5 X 10⁷, or 10 × 10⁷ plaque-forming units of MVA85A, or placebo. Adverse events were documented, and T-cell responses were assessed by interferon γ (IFN-γ) enzyme-linked immunospot assay and intracellular cytokine staining. Results. The 3 MVA85A doses were well tolerated, and no vaccine-related serious adverse events were recorded. MVA85A induced potent, durable T-cell responses, which exceeded prevaccination responses up to 168 d after vaccination. No dose-related differences in response magnitude were observed. Multiple CD4 T cell subsets were induced; polyfunctional CD4 T cells co-expressing T-helper cell 1 cytokines with or without granulocytemacrophage colony-stimulating factor predominated. IFN-γ-expressing CD8 T cells, which peaked later than CD4 T cells, were also detectable. Conclusions. MVA85A was safe and induced robust, polyfunctional, durable CD4 and CD8 T-cell responses in infants. These data support efficacy evaluation of MVA85A to prevent tuberculosis in infancy. Clinical Trials Registration. NCT00679159.