Rosemary E. Weir

BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies

BACKGROUND The efficacy of BCG vaccines against pulmonary tuberculosis varies between populations, showing no protection in Malawi but 50-80% protection in the UK. To investigate the mechanism underlying these differences, randomised controlled studies were set up to measure vaccine-induced immune responsiveness to mycobacterial antigens in both populations. METHODS 483 adolescents and young adults in Malawi and 180 adolescents in the UK were tested for interferon-gamma (IFN-gamma) response to M tuberculosis purified protein derivative (PPD) in a whole blood assay, and for delayed type hypersensitivity (DTH) skin test response to tuberculin PPD, before and 1 year after receiving BCG (Glaxo 1077) vaccination or placebo or no vaccine. FINDINGS The percentages of the randomised individuals who showed IFN-gamma and DTH responses were higher in Malawi than in the UK pre-vaccination-ie, 61% (331/546) versus 22% (47/213) for IFN-gamma and 46% (236/517) versus 13% (27/211) for DTH. IFN-gamma responses increased more in the UK than in Malawi, with 83% (101/122) and 78% (251/321) respectively of the vaccinated groups responding, with similar distributions in the two populations 1 year post-vaccination. The DTH response increased following vaccination in both locations, but to a greater extent in the UK than Malawi. The IFN-gamma and DTH responses were strongly associated, except among vaccinees in Malawi. INTERPRETATION The magnitude of the BCG-attributable increase in IFN-gamma responsiveness to M tuberculosis PPD, from before to 1 year post-vaccination, correlates better with the known levels of protection induced by immunisation with BCG than does the absolute value of the IFN-gamma or DTH response after vaccination. It is likely that differential sensitisation due to exposure to environmental mycobacteria is the most important determinant of the observed differences in protection by BCG between populations.

Patterns and Implications of Naturally Acquired Immune Responses to Environmental and Tuberculous Mycobacterial Antigens in Northern Malawi

Interferon (IFN)-gamma responsiveness to 12 purified protein derivative (PPD) and new tuberculin antigens from 9 species of mycobacteria was assessed, using a whole blood assay, in 616 young adults living in northern Malawi, where Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination provides no protection against pulmonary tuberculosis. The prevalence of IFN-gamma responsiveness was highest for PPDs of M. avium, M. intracellulare, and M. scrofulaceum (the MAIS complex). Correlations between responsiveness paralleled genetic relatedness of the mycobacterial species. A randomized, controlled trial was carried out, to assess the increase in IFN-gamma responsiveness to M. tuberculosis PPD that can be attributed to M. bovis BCG vaccination. The BCG-attributable increase in IFN-gamma response to M. tuberculosis PPD was greater for individuals with low initial responsiveness to MAIS antigens than for those with high initial responsiveness. Although not statistically significant, the trend is consistent with the hypothesis that prior exposure to environmental mycobacteria interferes with immune responses to BCG vaccination.

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.

The influence of previous exposure to environmental mycobacteria on the interferon-gamma response to bacille Calmette-Guérin vaccination in southern England and northern Malawi.

We report a large study of the effect of BCG vaccination on the in vitro 6‐day whole blood interferon‐gamma (IFN‐γ) response to antigens from eight species of mycobacteria among schoolchildren in south‐eastern England, where bacille Calmette–Guérin (BCG) vaccination is highly protective against pulmonary tuberculosis, and among young adults in northern Malawi, where BCG vaccination is not protective. In the UK children, BCG induced an appreciable increase in IFN‐γ response to antigens from most species of mycobacteria. The degree of change was linked to the relatedness of the species to Mycobacterium bovis BCG, and provides further evidence of the cross‐reactivity of mycobacterial species in priming of the immune system. IFN‐γ responses to purified protein derivatives (PPDs) from M. tuberculosis and environmental mycobacteria were more prevalent in the Malawian than the UK group prior to vaccination; BCG vaccination increased the prevalence of responses to these PPDs in the UK group to a level similar to that in Malawi. There was no evidence that the vaccine‐induced change in IFN‐γ response was dependent upon the magnitude of the initial response of the individual to environmental mycobacteria in the United Kingdom or in Malawi. These observations should assist the development and interpretation of human clinical trials of new vaccines against M. tuberculosis in areas of both low and high exposure to environmental mycobacteria.

Development of a whole blood assay to measure T cell responses to leprosy: a new tool for immuno-epidemiological field studies of leprosy immunity

A whole blood assay is described to measure T cell mediated immune responses to leprosy and provide an alternative to the conventional lymphocyte transformation test. Optimal conditions were defined for the whole blood assay, and interferon-gamma measurement was found to be a more sensitive way of measuring responses than tritiated thymidine incorporation. The assay was shown to be useful for investigating responses to a range of leprosy antigens. A whole blood assay has the advantages of being quick, simple and requiring only a small volume of blood, making it more appropriate as an immuno-epidemiological field test in leprosy endemic areas.

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.

Persistence of the immune response induced by BCG vaccination

BackgroundAlthough 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.MethodsA randomised control study of UK adolescents was carried out to investigate persistence of BCG immune responses. Adolescents were tested for interferon-gamma (IFN-γ) 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).ResultsA 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-γ 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-γ 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-γ response of > 500 pg/ml than unvaccinated teenagers.ConclusionBCG 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.

Complex cytokine profiles induced by BCG vaccination in UK infants.

IFNγ 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; IFNγ (median 1705 pg/ml vs. 1.6 pg/ml in vaccinated and unvaccinated infants, respectively), TNFα (median 226 pg/ml vs. 18 pg/ml), as well as IL-2, IL-1α 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-1α 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 IFNγ. While responses for many cytokines were correlated with the IFNγ 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.

Interferon-γ and skin test responses of schoolchildren in southeast England to purified protein derivatives from Mycobacterium tuberculosis and other species of mycobacteria

The immune responses of schoolchildren in southeast England to Mycobacterium tuberculosis and other species of mycobacteria were studied prior to vaccination with bacille Calmette‐Guérin (BCG). Data are presented for tuberculin (Heaf) skin test and interferon‐γ (IFN‐γ) responses to M. tuberculosis purified protein derivative (PPD), and IFN‐γ responses to PPDs from eight other environmental mycobacteria, measured in 424 schoolchildren (13–15 years of age). Responses to M. tuberculosis PPD were detected in 27% of schoolchildren by in vitro IFN‐γ response and in 20% by the Heaf test. IFN‐γ responses were more prevalent to PPDs from species of mycobacteria other than M. tuberculosis, predominantly those of the MAIS complex and M. marinum (45–60% responders). Heaf test and IFN‐γ responses were associated (P < 0·001) for M. tuberculosis, MAIS and M. marinum. These findings have implications for appropriate implementation of vaccination against tuberculosis.

Use of a whole blood assay to evaluate in vitro T cell responses to new leprosy skin test antigens in leprosy patients and healthy subjects

Development of an immunological tool to detect infection with Mycobacterium leprae would greatly benefit leprosy control programmes, as demonstrated by the contribution of the tuberculin test to tuberculosis control. In a new approach to develop a ‘tuberculin‐like’ reagent for use in leprosy, two new fractions of M. leprae depleted of cross‐reactive and immunomodulatory lipids— MLSA‐LAM (cytosol‐derived) and MLCwA (cell wall‐derived)—have been produced in a form suitable for use as skin test reagents. T cell responses (interferon‐gamma (IFN‐γ) and lymphoproliferation) to these two new fractions were evaluated in a leprosy‐endemic area of Nepal using a simple in vitro whole blood test. The two fractions were shown to be highly potent T cell antigens in subjects exposed to M. leprae—paucibacillary leprosy patients and household contacts. Responses to the fractions decreased towards the lepromatous pole of leprosy. Endemic control subjects also showed high responses to the fractions, indicating high exposure to M. leprae, or cross‐reactive mycobacterial antigens, in this Nepali population. The new fractions, depleted of lipids and lipoarabinomannan (LAM) gave enhanced responses compared with a standard M. leprae sonicate. The cell wall fraction appeared a more potent antigen than the cytosol fraction, which may be due to the predominance of the 65‐kD GroEL antigen in the cell wall. The whole blood assay proved a robust field tool and a useful way of evaluating such reagents prior to clinical trials.