Live-attenuated Mycobacterium tuberculosis vaccine MTBVAC versus BCG in adults and neonates: a randomised controlled, double-blind dose-escalation trial

Background: Infants are a key target population for new tuberculosis vaccines. We assessed the safety and immunogenicity of the live-attenuated Mycobacterium tuberculosis vaccine candidate MTBVAC in adults and infants in a region where transmission of tuberculosis is very high. Methods: We did a randomised, double-blind, BCG-controlled, dose-escalation trial at the South African Tuberculosis Vaccine Initiative site near Cape Town, South Africa. Healthy adult community volunteers who were aged 18–50 years, had received BCG vaccination as infants, were HIV negative, had negative interferon-¿ release assay (IGRA) results, and had no personal history of tuberculosis or current household contact with someone with tuberculosis were enrolled in a safety cohort. Infants born to HIV-negative women with no personal history of tuberculosis or current household contact with a person with tuberculosis and who were 96 h old or younger, generally healthy, and had not yet received routine BCG vaccination were enrolled in a separate infant cohort. Eligible adults were randomly assigned (1:1) to receive either BCG Vaccine SSI (5 × 105 colony forming units [CFU] of Danish strain 1331 in 0·1 mL diluent) or MTBVAC (5 × 105 CFU in 0·1 mL) intradermally in the deltoid region of the arm. After favourable review of 28-day reactogenicity and safety data in the adult cohort, infants were randomly assigned (1:3) to receive either BCG Vaccine SSI (2·5 × 105 CFU in 0·05 mL diluent) or MTBVAC in three sequential cohorts of increasing MTBVAC dose (2·5 × 103 CFU, 2·5 × 104 CFU, and 2·5 × 105 CFU in 0·05 mL) intradermally in the deltoid region of the arm. QuantiFERON-TB Gold In-Tube IGRA was done on days 180 and 360. For both randomisations, a pre-prepared block randomisation schedule was used. Participants (and their parents or guardians in the case of infant participants), investigators, and other clinical and laboratory staff were masked to intervention allocation. The primary outcomes, which were all measured in the infant cohort, were solicited and unsolicited local adverse events and serious adverse events until day 360; non-serious systemic adverse events until day 28 and vaccine-specific CD4 and CD8 T-cell responses on days 7, 28, 70, 180, and 360. Secondary outcomes measured in adults were local injection-site and systemic reactions and haematology and biochemistry at study day 7 and 28. Safety analyses and immunogenicity analyses were done in all participants who received a dose of vaccine. This trial is registered with ClinicalTrials.gov, number NCT02729571. Findings: Between Sept 29, 2015, and Nov 16, 2015, 62 adults were screened and 18 were enrolled and randomly assigned, nine each to the BCG and MTBVAC groups. Between Feb 12, 2016, and Sept 21, 2016, 36 infants were randomly assigned—eight to the BCG group, nine to the 2·5 × 103 CFU MTBVAC group, nine to the 2·5 × 104 CFU group, and ten to the 2·5 × 105 CFU group. Mild injection-site reactions occurred only in infants in the BCG and the 2·5 × 105 CFU MTBVAC group, with no evidence of local or regional injection-site complications. Systemic adverse events were evenly distributed across BCG and MTBVAC dose groups, and were mostly mild in severity. Eight serious adverse events were reported in seven vaccine recipients (one adult MTBVAC recipient, one infant BCG recipient, one infant in the 2·5 × 103 CFU MTBVAC group, two in the 2·5 × 104 CFU MTBVAC group, and two in the 2·5 × 105 CFU MTBVAC group), including one infant in the 2·5 × 103 CFU MTBVAC group treated for unconfirmed tuberculosis and one in the 2·5 × 105 CFU MTBVAC group treated for unlikely tuberculosis. One infant died as a result of possible viral pneumonia. Vaccination with all MTBVAC doses induced durable antigen-specific T-helper-1 cytokine-expressing CD4 cell responses in infants that peaked 70 days after vaccination and were detectable 360 days after vaccination. For the highest MTBVAC dose (ie, 2·5 × 105 CFU), these responses exceeded responses induced by an equivalent dose of the BCG vaccine up to 360 days after vaccination. Dose-related IGRA conversion was noted in three (38%) of eight infants in the 2·5 × 103 CFU MTBVAC group, six (75%) of eight in the 2·5 × 104 CFU MTBVAC group, and seven (78%) of nine in the 2·5 × 105 CFU MTBVAC group at day 180, compared with none of seven infants in the BCG group. By day 360, IGRA reversion had occurred in all three infants (100%) in the 2·5 × 103 CFU MTBVAC group, four (67%) of the six in the 2·5 × 104 CFU MTBVAC group, and three (43%) of the seven in the 2·5 × 105 CFU MTBVAC group. Interpretation: MTBVAC had acceptable reactogenicity, and induced a durable CD4 cell response in infants. The evidence of immunogenicity supports progression of MTBVAC into larger safety and efficacy trials, but also confounds interpretation of tests for M tuberculosis infection, highlighting the need for stringent endpoint definition. Funding: Norwegian Agency for Development Cooperation, TuBerculosis Vaccine Initiative, UK Department for International Development, and Biofabri

Functional, Antigen-Specific Stem Cell Memory (TSCM) CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

BackgroundMaintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM) or effector (TEFF) T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology.MethodsWe studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette–Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.ResultsM. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE) profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN) and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4+ T cell proliferative potential after infant vaccination.ConclusionHuman infection with M. tb induced distinct, antigen-specific CD4+ TSCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4+ TSCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb

Detection of GFP-expressing BCG by innate cells and association between absolute numbers of innate cells and mycobacterial growth inhibition.

<p>(<b>A</b>) Representative flow cytometry plot of IL-6, IL-12 and TNF-α cytokine expression by myeloid dentritic cells (mDCs), monocytes and neutrophils, measured in whole blood stimulated for 6 hours with BCG, BCG-GFP (shown) or LPS, relative to an unstimulated control sample. (<b>B</b>) Representative histograms indicating proportions of innate cells that phagocytosed BCG-GFP (green). (<b>C</b>) Absolute numbers of innate cell subsets per milliliter of unstimulated whole blood plotted against <i>M</i>.<i>tb</i> H37Rv growth. R and p values were calculated using Spearman’s correlation. (<b>D</b>) Absolute numbers of BCG-GFP-positive mDCs, monocytes or neutrophils per mL of whole blood in adult individuals, stratified by QFT status. The inclusion of TruCount beads during the cell staining steps of the innate whole blood assay allowed determination of the absolute number of each subset of cells per mL of whole blood. The red and blue circles represent QFT+ and QFT- adults, respectively. Horizontal lines represent medians and whiskers, the interquartile range. Differences in absolute counts of BCG-GFP-positive innate cells between the groups were evaluated with the Mann-Whitney test (shown <i>P</i> values). The pie charts show relative proportions of BCG-GFP-positive cells among each innate cell subset.</p

Mycobacteria-specific T cells in whole blood from adults and mycobacterial growth inhibition.

<p>(<b>A</b>) Absolute numbers of BCG-specific CD4, CD8 and γδ T cell subsets co-expressing IL-2, IFN-γ, TNF-α and/or IL-17, in whole bood from QFT+ (red) and QFT- (blue) adults. Medians are represented by the horizontal lines, interquartile ranges by the boxes, and ranges by the whiskers. The Mann-Whitney test was used to assess differences between QFT+ and QFT- adults and none were found to be different. (<b>B</b>) <i>M</i>.<i>tb</i> H37Rv growth plotted against frequencies of BCG-specific CD4, CD8 or γδ T cells expressing IFN-γ, or CD4 T cells expressing IL-17 in adults. R and p values were calculated using Spearman’s correlation analysis.</p

The Novel Tuberculosis Vaccine, AERAS-402, Induces Robust and Polyfunctional CD4+ and CD8+ T Cells in Adults

Rationale: AERAS-402 is a novel tuberculosis vaccine designed to boost immunity primed by bacillus Calmette-Guérin (BCG), the only licensed vaccine

Mycobacteria-specific cytokine expression by innate cells in whole blood from adults and mycobacterial growth inhibition.

<p>(<b>A</b>) Absolute numbers of total cytokine expressing innate cells per mL of whole blood plotted against <i>M</i>.<i>tb</i> H37Rv growth. R and p values were calculated using Spearman’s correlation analysis. (<b>B</b>) Numbers of BCG-specific mDCs, monocytes and neutrophils co-expressing IL-6, IL-12 and/or TNF-α in whole blood from QFT+ (red) and QFT- (blue) adults. Medians are represented by the horizontal line, interquartile ranges by the boxes, and ranges by the whiskers. The Mann-Whitney test was used to assess differences between QFT+ and QFT- adults and none were found to be different.</p

<i>In vitro</i> mycobacterial growth inhibition in <i>M</i>.<i>tb</i>-infected (QFT+) and uninfected (QFT-) individuals.

<p>Inhibition of <i>M</i>.<i>tb</i> H37Rv growth by whole blood from QFT+ and QFT- adults (<b>A</b>), young adults (<b>B</b>) and children (<b>C</b>) assessed using a mycobacterial growth inhibition assay (MGIA). The growths of <i>M</i>. <i>bovis</i> BCG, <i>M</i>.<i>tb</i> H37Rv, <i>M</i>.<i>tb</i> isolate CDC1551 and <i>M</i>.<i>tb</i> isolate HN878 in the whole blood of adults (<b>D</b>) and young adults (<b>E</b>), respectively were compared. Spearman’s correlation analyses of the growth of different mycobacterial strains (<b>F</b> and <b>G</b>). The growth of BCG (<b>H</b>), HN878 and CDC1551 (<b>I</b> and <b>J</b>), was measured in the whole blood of adults and young adults, respectively. The red and blue circles represent QFT+ and QFT- individuals, respectively while the green and orange circles represent children and young adults respectively. The horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the range. Differences in mycobacterial growth inhibition between both groups of individuals were evaluated with the Mann-Whitney test (shown <i>P</i> values).</p

Application of a whole blood mycobacterial growth inhibition assay to study immunity against <i>Mycobacterium tuberculosis</i> in a high tuberculosis burden population

<div><p>The determinants of immunological protection against <i>Mycobacterium tuberculosis (M</i>.<i>tb)</i> infection in humans are not known. Mycobacterial growth inhibition assays have potential utility as <i>in vitro</i> surrogates of <i>in vivo</i> immunological control of <i>M</i>.<i>tb</i>. We evaluated a whole blood growth inhibition assay in a setting with high burden of TB and aimed to identify immune responses that correlate with control of mycobacterial growth. We hypothesized that individuals with underlying <i>M</i>.<i>tb</i> infection will exhibit greater <i>M</i>.<i>tb</i> growth inhibition than uninfected individuals and that children aged 4 to 12 years, an age during which TB incidence is curiously low, will also exhibit greater <i>M</i>.<i>tb</i> growth inhibition than adolescents or adults. Neither <i>M</i>.<i>tb</i> infection status, age of the study participants, nor <i>M</i>.<i>tb</i> strain was associated with differential control of mycobacterial growth. Abundance and function of innate or T cell responses were also not associated with mycobacterial growth. Our data suggest that this assay does not provide a useful measure of age-associated differential host control of <i>M</i>.<i>tb</i> infection in a high TB burden setting. We propose that universally high levels of mycobacterial sensitization (through environmental non-tuberculous mycobacteria and/or universal BCG vaccination) in persons from high TB burden settings may impart broad inhibition of mycobacterial growth, irrespective of <i>M</i>.<i>tb</i> infection status. This sensitization may mask the augmentative effects of mycobacterial sensitization on <i>M</i>.<i>tb</i> growth inhibition that is typical in low burden settings.</p></div

<i>In vitro</i> mycobacterial growth inhibition in children and young adults.

<p>Inhibition of <i>M</i>.<i>tb</i> H37Rv growth by whole blood from QFT- and QFT+ 8 and 18 year olds. Green and orange circles represent children and young adults, respectively. The horizontal line represents the median, the boxes represent the interquartile range, and the whiskers represent the range. Differences in mycobacterial growth inhibition between both groups of individuals were evaluated with the Mann-Whitney test (shown <i>P</i> values).</p