Immune Responses Following BCG Immunization of Infants in Uganda and United Kingdom Are Similar for Purified Protein Derivative but Differ for Secretory Proteins of Mycobacterium tuberculosis.

Introduction: The immunogenicity of BCG vaccination in infants differs between populations. We hypothesized that prenatal exposure to mycobacterial antigens might explain the differences in immune responses to BCG seen in other studies of infants in Africa and the United Kingdom (UK) and we explored this in birth cohorts in Uganda and the UK. Materials and Methods: Blood samples were obtained from BCG-immunized infants of mothers with (n = 110) and without (n = 121) latent Mycobacterium tuberculosis infection (LTBI) in Uganda and BCG-immunized infants of mothers without LTBI (n = 25) in the UK at 10 and 52 weeks after birth. Cytokine and chemokine responses to PPD were measured to assess responses to BCG immunization, and to ESAT6/CFP10 to assess exposure to or infection with M. tuberculosis or non-tuberculous mycobacteria (NTM) in 6-day whole blood culture supernatants by a 17-plex Luminex assay. Median responses were compared between Ugandan infants (together, and separated by maternal LTBI status) and UK infants. Results: The IFN-γ response to BCG vaccination was similar between Ugandan and UK infants at 10 and 52 weeks. At week 52, TNF production was marginally higher in Ugandan infants, but after adjusting for multiple comparisons this difference was not significant. At weeks 10 and 52, stimulation of blood with ESAT6/CFP10 produced significantly higher IFN-γ, TNF, IL-12p40, IL-1α, IL-1ß, IL-1Ra, IP-10, MIP-1α, MIP-1ß, and GM-CSF in Ugandan compared to UK infants. Stimulation of blood with ESAT6/CFP10 produced significantly higher amounts of IL-8 (p = 0.0001), IL-10 (p = 0.0022), and IL-13 (p = 0.0020) in the UK than in Ugandan infants of mothers without LTBI at week 10, but not at week 52. Conclusions: Immune responses to mycobacterial antigens following BCG immunization are similar for PPD, but differ for ESAT6/CFP10, between infants in Uganda and the UK. Neither maternal LTBI nor infant exposure to or infection with mycobacteria impacts the response to BCG. The observed global differences in immune response to BCG immunization are likely to be due to other causes.

Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants.

BACKGROUND: Vaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition. METHODS: Blood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis (M. tb) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined. RESULTS: PPD-specific IFNγ+TNFα+IL-2+CD4+T-cells represented the dominant T-cell response at 4monthsand1yearafter infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+CD4+T-cells were significantly more frequent in vaccinated infants at 4monthsbut not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4monthspost-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNγ+TNFα+IL-2+) CD4+T-cells. CONCLUSIONS: These data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two.

Broad heparin-binding haemagglutinin-specific cytokine and chemokine response in infants following Mycobacterium bovis BCG vaccination.

Heparin-binding haemagglutinin (HBHA)-specific immune responses have been linked to protection against tuberculosis (TB). We investigated the hypothesis that BCG vaccination of human infants primes an HBHA-specific response, using multiplex to measure secreted cytokines and chemokines following HBHA and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation of diluted whole blood samples from BCG-vaccinated or -unvaccinated infants. Of 42 analytes measured, 24 and 32 significant, BCG-associated increases were detected in response to HBHA and PPD, respectively. Both response profiles included Th-1, Th-2, Th-17 and inflammatory cytokines and chemokines (e.g. IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17, MIP-1α and MIP-1ß). We also found that six of the seven responses most closely correlated with IFN-γ were common to both HBHA and PPD. Notably, all HBHA-specific secretion of cytokines and chemokines from infant samples was dependent on previous BCG vaccination. Also, long-term persistence of HBHA-specific responses was found in adolescents with evidence of infant BCG vaccination. This study demonstrates for the first time BCG priming of an HBHA-specific immune response in infants that is characterised by a broad cytokine and chemokine signature. It also suggests a number of BCG vaccination associated, HBHA-induced responses that should be useful for future studies of biomarkers of protection against TB.

BCG vaccination induces different cytokine profiles following infant BCG vaccination in the UK and Malawi.

BACKGROUND: BCG vaccination of infants is thought to provide good protection in all settings. This study investigated whether Malawian infants made weaker responses across a cytokine panel after BCG vaccination, compared with UK infants. METHODS: Diluted whole-blood samples were cultured with Mycobacterium tuberculosis purified protein derivative for 6 days from BCG-vaccinated infants 3 months (n = 40 Malawi, 28 UK) and 12 months (n = 34 Malawi, 26 UK) after vaccination, and also from UK unvaccinated infants (n = 9 at 3 months, n = 10 at 12 months). Forty-two cytokines were measured in supernatants using a multiplex bead array assay. Principal component analysis was used to summarize the overall patterns in cytokine responses. RESULTS: We found differences in median responses in 27 of the 42 cytokines: 7 higher in the UK and 20 higher in Malawi. The cytokines with higher responses in the UK were all T helper 1 related. The cytokines with higher responses in Malawi included innate proinflammatory cytokines, regulatory cytokines, interleukin 17, T helper 2 cytokines, chemokines, and growth factors. Principal component analysis separated the BCG-vaccinated infants from Malawi from the UK vaccinated infants and from the unvaccinated infants. CONCLUSIONS: Malawian infants make cytokine responses following BCG vaccination, but the cytokine profile is different from that in the UK. The different biosignatures following BCG vaccination in the 2 settings may indicate variability in the protective efficacy of infant BCG vaccination.

BCG vaccination: a role for vitamin D?

BACKGROUND: BCG vaccination is administered in infancy in most countries with the aim of providing protection against tuberculosis. There is increasing interest in the role of vitamin D in immunity to tuberculosis. This study objective was to determine if there was an association between circulating 25(OH)D concentrations and BCG vaccination status and cytokine responses following BCG vaccination in infants. METHODS: Blood samples were collected from UK infants who were vaccinated with BCG at 3 (n = 47) and 12 (n = 37) months post BCG vaccination. These two time-points are denoted as time-point 1 and time-point 2. Two blood samples were also collected from age-matched unvaccinated infants (n = 32 and 28 respectively), as a control group. Plasma vitamin D concentrations (25(OH)D) were measured by radio-immunoassay. The cytokine IFNγ was measured in supernatants from diluted whole blood stimulated with M.tuberculosis (M.tb) PPD for 6 days. RESULTS: 58% of infants had some level of hypovitaminosis (25(OH)D <30 ng/ml) at time-point 1, and this increased to 97% 9 months later. BCG vaccinated infants were almost 6 times (CI: 1.8-18.6) more likely to have sufficient vitamin D concentrations than unvaccinated infants at time-point 1, and the association remained strong after controlling for season of blood collection, ethnic group and sex. Among vaccinees, there was also a strong inverse association between IFNγ response to M.tb PPD and vitamin D concentration, with infants with higher vitamin D concentrations having lower IFNγ responses. CONCLUSIONS: Vitamin D may play an immuno-regulatory role following BCG vaccination. The increased vitamin D concentrations in BCG vaccinated infants could have important implications: vitamin D may play a role in immunity induced by BCG vaccination and may contribute to non-specific effects observed following BCG vaccination.

Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and intracellular cytokine staining.

BACKGROUND: The vaccine efficacy reported following Mycobacterium bovis Bacillus Calmette Guerin (BCG) administration to UK adolescents is 77% and defining the cellular immune response in this group can inform us as to the nature of effective immunity against tuberculosis. The aim of this study was to identify which cytokines and lymphocyte populations characterise the peripheral blood cellular immune response following BCG vaccination. RESULTS: Diluted blood from before and after vaccination was stimulated with Mycobacterium tuberculosis purified protein derivative for 6 days, after which soluble biomarkers in supernatants were assayed by multiplex bead array. Ten out of twenty biomarkers measured were significantly increased (p < 0.0025) 1 month after BCG vaccination when compared to paired samples (n = 12) taken prior to vaccination (IFNgamma, TNFalpha, IL-1alpha, IL-2, IL-6, IL-10, IL-17, GM-CSF, MIP1alpha, IP-10). All of these remained detectable by multiplex bead array in samples taken 12 months after BCG vaccination of a partially overlapping adolescent group (n = 12). Intracellular cytokine staining after 24 hour Mycobacterium tuberculosis purified protein derivative stimulation of PBMC samples from the 12 month group revealed that IFNgamma expression was detectable in CD4 and CD8 T-cells and natural killer cells. Polyfunctional flow cytometry analysis demonstrated that cells expressing IFNgamma alone formed the majority in each subpopulation of cells. Only in CD4 T-cells and NK cells were there a notable proportion of responding cells of a different phenotype and these were single positive, TNFalpha producers. No significant expression of the cytokines IL-2, IL-17 or IL-10 was seen in any population of cells. CONCLUSIONS: The broad array of biomarker responses detected by multiplex bead array suggests that BCG vaccination is capable, in this setting, of inducing a complex immune phenotype. Although polyfunctional T-cells have been proposed to play a role in protective immunity, they were not present in vaccinated adolescents who, based on earlier epidemiological studies, should have developed protection against pulmonary tuberculosis. This may be due to the later sampling time point available for testing or on the kinetics of the assays used.

Complex cytokine profiles induced by BCG vaccination in UK infants.

IFNgamma plays an important part in immunity to tuberculosis (TB), but although it is necessary, it is not on its own sufficient for protection against TB. To identify other cytokines that play a role in the protection against TB induced by BCG vaccination, immune responses were compared between vaccinated and unvaccinated infants from the UK where BCG is known to provide protection. Twenty-one cytokines and chemokines were tested in supernatants from diluted whole blood cultures that had been stimulated for 6 days with Mycobacterium tuberculosis PPD. For 15 out of 21 of the cytokines tested responses were much higher in BCG vaccinated infants than in unvaccinated infants. These included: pro-inflammatory cytokines; IFNgamma (median 1705 pg/ml vs. 1.6 pg/ml in vaccinated and unvaccinated infants, respectively), TNFalpha (median 226 pg/ml vs. 18 pg/ml), as well as IL-2, IL-1alpha and IL-6; TH2 cytokines: IL-4, IL-5 and IL-13 (median 104 pg/ml vs. 1.6 pg/ml); the regulatory cytokine IL-10 (median response 96 pg/ml vs. 8 pg/ml); the TH17 cytokine IL-17, chemokines (IP-10, MIP-1alpha and IL-8) and growth factors (GM-CSF and G-CSF). The greatest increase in cytokine production in BCG vaccinees compared to unvaccinated infants was seen with IFNgamma. While responses for many cytokines were correlated with the IFNgamma response, others including IL-17 and IL-10 were not. The pattern of cytokine induction following BCG vaccination is complex and measurement of one of two cytokines does not reveal the whole picture of vaccine-induced protection.

Population differences in immune responses to Bacille Calmette-Guérin vaccination in infancy.

Bacille Calmette-Guérin (BCG) vaccination induces a marked increase in the interferon (IFN)-gamma response to Mycobacterium tuberculosis purified protein derivative (Mtb PPD) in UK adolescents, but not in Malawian adolescents. We hypothesized that Mtb PPD-induced IFN-gamma after BCG vaccination would be similar in infants from these 2 countries. Infants were vaccinated with BCG during the first 3-13 weeks of life. Three months after BCG vaccination, 51 (100%) of 51 UK infants had an IFN-gamma response to Mtb PPD, compared to 41 (53%) of 78 of Malawian infants, in whom responses varied according to their season of birth. We conclude that population differences in immune responses after BCG vaccination are observed among infants, as well as among young adults.

Persistence of the immune response induced by BCG vaccination.

BACKGROUND: Although BCG vaccination is recommended in most countries of the world, little is known of the persistence of BCG-induced immune responses. As novel TB vaccines may be given to boost the immunity induced by neonatal BCG vaccination, evidence concerning the persistence of the BCG vaccine-induced response would help inform decisions about when such boosting would be most effective. METHODS: A randomised control study of UK adolescents was carried out to investigate persistence of BCG immune responses. Adolescents were tested for interferon-gamma (IFN-gamma) response to Mycobacterium tuberculosis purified protein derivative (M.tb PPD) in a whole blood assay before, 3 months, 12 months (n = 148) and 3 years (n = 19) after receiving teenage BCG vaccination or 14 years after receiving infant BCG vaccination (n = 16). RESULTS: A gradual reduction in magnitude of response was evident from 3 months to 1 year and from 1 year to 3 years following teenage vaccination, but responses 3 years after vaccination were still on average 6 times higher than before vaccination among vaccinees. Some individuals (11/86; 13%) failed to make a detectable antigen-specific response three months after vaccination, or lost the response after 1 (11/86; 13%) or 3 (3/19; 16%) years. IFN-gamma response to Ag85 was measured in a subgroup of adolescents and appeared to be better maintained with no decline from 3 to 12 months. A smaller group of adolescents were tested 14 years after receiving infant BCG vaccination and 13/16 (81%) made a detectable IFN-gamma response to M.tb PPD 14 years after infant vaccination as compared to 6/16 (38%) matched unvaccinated controls (p = 0.012); teenagers vaccinated in infancy were 19 times more likely to make an IFN-gamma response of > 500 pg/ml than unvaccinated teenagers. CONCLUSION: BCG vaccination in infancy and adolescence induces immunological memory to mycobacterial antigens that is still present and measurable for at least 14 years in the majority of vaccinees, although the magnitude of the peripheral blood response wanes from 3 months to 12 months and from 12 months to 3 years post vaccination. The data presented here suggest that because of such waning in the response there may be scope for boosting anti-tuberculous immunity in BCG vaccinated children anytime from 3 months post-vaccination. This supports the prime boost strategies being employed for some new TB vaccines currently under development.

Immunogenicity of Danish-SSI 1331 BCG vaccine in the UK: comparison with Glaxo-Evans 1077 BCG vaccine.

The immunogenicity and reactogenicity, in British schoolchildren, of the newly introduced Danish-SSI 1331 BCG vaccine was compared with that of the previously used Glaxo-Evans 1077 BCG vaccine. Interferon-gamma (IFN-gamma) response to M. tuberculosis purified protein derivative (M.tb PPD) in a 6-day whole blood assay and delayed type hypersensitivity (DTH) to tuberculin PPD were determined before and 1 year after receiving BCG or no vaccination. Scar size was measured 1 year after vaccination. There was no evidence of a difference in immunogenicity (IFN-gamma and DTH conversion rates) but evidence of lower reactogenicity (scar size) with Danish-SSI 1331 compared to Glaxo-Evans 1077 vaccines.