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.

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.

Early exposure of infants to GI nematodes induces Th2 dominant immune responses which are unaffected by periodic anthelminthic treatment.

We have previously shown a reduction in anaemia and wasting malnutrition in infants <3 years old in Pemba Island, Zanzibar, following repeated anthelminthic treatment for the endemic gastrointestinal (GI) nematodes Ascaris lumbricoides, hookworm and Trichuris trichiura. In view of the low intensity of worm infections in this age group, this was unexpected, and it was proposed that immune responses to the worms rather than their direct effects may play a significant role in morbidity in infants and that anthelminthic treatment may alleviate such effects. Therefore, the primary aims of this study were to characterise the immune response to initial/early GI nematode infections in infants and the effects of anthelminthic treatment on such immune responses. The frequency and levels of Th1/Th2 cytokines (IL-5, IL-13, IFN-gamma and IL-10) induced by the worms were evaluated in 666 infants aged 6-24 months using the Whole Blood Assay. Ascaris and hookworm antigens induced predominantly Th2 cytokine responses, and levels of IL-5 and IL-13 were significantly correlated. The frequencies and levels of responses were higher for both Ascaris positive and hookworm positive infants compared with worm negative individuals, but very few infants made Trichuris-specific cytokine responses. Infants treated every 3 months with mebendazole showed a significantly lower prevalence of infection compared with placebo-treated controls at one year following baseline. At follow-up, cytokine responses to Ascaris and hookworm antigens, which remained Th2 biased, were increased compared with baseline but were not significantly affected by treatment. However, blood eosinophil levels, which were elevated in worm-infected children, were significantly lower in treated children. Thus the effect of deworming in this age group on anaemia and wasting malnutrition, which were replicated in this study, could not be explained by modification of cytokine responses but may be related to eosinophil function.

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.

Differences between naive and memory T cell phenotype in Malawian and UK adolescents: a role for Cytomegalovirus?

BACKGROUND: Differences in degree of environmental exposure to antigens in early life have been hypothesized to lead to differences in immune status in individuals from different populations, which may have implications for immune responses in later years. METHODS: Venous blood from HIV-negative adolescents and blood from the umbilical cords of babies, born to HIV-negative women, post-delivery was collected and analysed using flow cytometry. T cell phenotype was determined from peripheral blood lymphocytes and cytomegalovirus (CMV) seropositivity was assessed by ELISA in adolescents. RESULTS: HIV-negative Malawian adolescents were shown to have a lower percentage of naïve T cells (CD45RO-CD62Lhi CD11alo), a higher proportion of memory T cells and a higher percentage of CD28- memory (CD28-CD45RO+) T cells compared to age-matched UK adolescents. Malawian adolescents also had a lower percentage of central memory (CD45RA-CCR7+) T cells and a higher percentage of stable memory (CD45RA+CCR7-) T cells than UK adolescents. All of the adolescents tested in Malawi were seropositive for CMV (59/59), compared to 21/58 (36%) of UK adolescents. CMV seropositivity in the UK was associated with a reduced percentage of naïve T cells and an increased percentage of CD28- memory T cells in the periphery. No differences in the proportions of naïve and memory T cell populations were observed in cord blood samples from the two sites. CONCLUSION: It is likely that these differences between Malawian and UK adolescents reflect a greater natural exposure to various infections, including CMV, in the African environment and may imply differences in the ability of these populations to induce and maintain immunological memory to vaccines and natural infections.

Comparison of IFN-gamma responses to mycobacterial antigens as markers of response to BCG vaccination.

An increase in interferon-gamma (IFN-gamma) production to Mycobacterium tuberculosis purified protein derivative (Mtb PPD), as measured in the cultured diluted whole blood assay, is one indicator of a protective immune response to BCG vaccine. We have explored the potential for this assay to be improved by measuring IFN-gamma responses to more defined antigens of M. tuberculosis (short-term and mid-term culture filtrates, ESAT-6, 38 kDa), Mycobacterium bovis (MPB70), M. bovis BCG (Antigen 85) and Mycobacterium leprae (35 kDa), in UK teenagers before and 1 year after BCG vaccination (or no vaccination as controls). There was a significant increase in response to the culture filtrates post-vaccination, but this was no greater than that to Mtb PPD. Many teenagers responded to the purified antigens, in particular to Antigen 85, prior to vaccination, and BCG vaccination could only augment this pre-existing response to a limited extent; prior exposure to environmental mycobacteria can thus induce cross-reactive responses to antigens which complicate interpretation of in vitro assays of vaccine response. In contrast, ESAT-6 was recognised by only one teenager prior to vaccination, and, as expected, responses were not boosted by BCG. We therefore conclude that Mtb PPD is the antigen preparation of choice for assessing the immunogenicity of BCG vaccination.

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.

The PPD-specific T-cell clonal response in UK and Malawian subjects following BCG vaccination: a new repertoire evolves over 12 months.

Mycobacterium bovis bacille Calmette Guerin vaccination protects against pulmonary tuberculosis in the United Kingdom but not in Malawi. We investigated whether a difference in the clonal T-cell response to BCG vaccination might account for this. The results of clonal analysis were compared to those obtained by skin testing and in a whole blood interferon gamma assay. Pre-vaccination antigen specific T-cell clones were detected, but the majority of clones present 12 months after vaccination were not present earlier. The magnitude of the clonal response did not correlate well with results of the other assays. These data indicate that single assays may not be reliable and that a stable memory T-cell repertoire is slow to develop.

Mycobacterial purified protein derivatives stimulate innate immunity: Malawians show enhanced tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses compared to those of adolescents in the United Kingdom.

To investigate the role of innate immunity in variable efficacy of Mycobacterium bovis BCG vaccination in Malawi and the United Kingdom, we examined 24-h tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-10 responses to mycobacterial purified protein derivatives (PPDs). The rank order in stimulatory potency for different PPDs was the same for all three cytokines. Before vaccination Malawians made higher pro- and anti-inflammatory responses than did United Kingdom subjects. Fewer than 5% of United Kingdom subjects made IL-10 in response to any PPD, compared to 19 to 57% responders among Malawians. Priming for regulatory IL-10 may contribute to the smaller increase in gamma interferon responses in Malawians compared to United Kingdom subjects following BCG vaccination.

Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy, non-mycobacterium bovis BCG-vaccinated Malawian young adults.

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.

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.