2 Experiences with BCG vaccination in the UK and Malawi Hazel M Dockrell London School of Hygiene & Tropical Medicine
3 We need correlates of protection to use in trials of new TB vaccines in man Can we exploit the observations that BCG vaccination can induce protection against pulmonary TB when given to adolescents in the UK, but not when given to young adults in Malawi?
4 BCG protection in Malawi and the UKIn Malawi, a single BCG vaccination gives 50% or more protection against leprosy but no protection against pulmonary tuberculosis*In the UK, vaccination of schoolchildren with BCG provides 77% protection against tuberculosis*Lancet, 348,: 17 (1996)Malawi because paul Fine had been working there forabout 15 years and had shown that BCG vaccination of previously unvaccinated young adults did not protect them against TB, wheras vacination of schoolchildren in the UK did. So paul and I decided to directly compare the immune responses in these two populations
5 Immunity to Tuberculosis Cell mediated immunity is importantInteraction between activated m, CD4+, CD8+, T cellsType 1 cytokines such as IFN/IL-12 play a role in protection; also role for TNFaCould whole blood assays measuring such cytokines be used as a correlate of protection?
6 Diluted whole blood assays to measure antigen-specific interferon-g responses Heparinised blood just diluted in tissue culture medium and antigens addedCytokines measured in culture supernatantsAssay proved easy, reliable and reproducible for field use when used with M.leprae antigens in NepalIt has now been used in studies in Malawi, Pakistan, The Philippines, South Africa, Uganda, China….Weir et al J Immunol Meth 176: 93 (1994)The assay that rosemay developed in very simple, you just dilute the heparinised blood and put it into culutre with the stimuli or antigens you want to use, and then measure a secreted product, like IFNg, in the supernatant, about 6 days later
7 Interferon-g responses measured in diluted whole blood cultures All the lab work in malawi was coordinated and performed by gill black, and again we were measuring the same product IFNg, but this time the results are expressed slightly differently, with the proportion of people making responses of increasing magnitude -so if the results are neagative they are down here, and if they are all positive they shift to the right – thisBlack et al, Int J Tuberc Lung Dis 5: 664 (2001)
8 Use of positive control supernatants to control for assay variation
10 Field work in Malawi recruited (age 10-28) (HIV-ve, BCG-ve) eligible (Mantoux <10mm) 2/3 BCG vaccinated 80% follow-up at 1 yearThe study design was to recruit adolescents and young adults who had not previously been BCG vaccinated, to skin test them, and unless they were strongly positive, to give them BCG vaccination, wait a year and then re-test them
11 and in Essex, UK…. 435 recruited (age 12-15, BCG scar -ve), In the UK, we adolescants were recruited through the Schools BCG vacination programme, using a virtually identical protocol, except that we didn’t test the UK school children for HIV or for pregnancy, and that they use a slightly different skin test to measure expose to mycobacterial antigens, the heaf test – you can see it’s a slightly different environment too435 recruited (age 12-15, BCG scar -ve),Heaf test grade >2 excluded2/3 given BCG vaccination80% followed up at 1 year Rosemary Weir
12 Does IFNg production increase in both UK and Malawian vaccinees? IFNg responses to M.tuberculosis PPD testedpre vaccination and 1 year post vaccination
13 Pre- and post- BCG vaccination IFNg responses to PPD in UK So in the UK, where the studies were coordinated and carried out by Rosemary Weir, most subjects wereneagtive before vaccination, stayed the same in the control group over the year, but increased dramatically in the vaccinees post vaccination - only 20% made a positive response of before vaccination and this increased to 80% postvaccinationIncrease in % respondersControls 19% to 21%Vaccinees 23% to 83%Black, Weir et al, Lancet 139: 1393 (2002)
14 Pre- and post- BCG vaccination IFNg responses to PPD in Malawi It was quite different in malawi there were a far higher number of responders before vaccination, and only a minimal increase following vaccination. We think that the greater responses we see pre vaccination are due to environemtnal mycobacteria in the envrinoment – i have time to show you that data - and also that although the malawians are often infected with parasites and worms, the majority havent switched to making a Type 2 response to mycobacterial antigensIncrease in % respondersPlacebo % to 76%Vaccinees % to 78%Black, Weir et al, Lancet 139: 1393 (2002)
16 IFNg responses to PPD: % changes over 1 year in placebo/control groups IFNg responses detected in 6 day whole blood assays are more variable in a tropical setting such as Malawi, than in the UKIFNg responses to PPD: % changes over 1 year in placebo/control groupsdecrease no change increaseMalawiUK
17 Distribution of IFNg responses to MTb PPD 1 year after BCG vaccination in Malawi &UK The increase in IFNg is associated with protection in the UKThere are conflicting views about whether exposure tomycobacteria confers protection or blocks its induction
18 Controlling for effective vaccination: Comparison of BCG scar size of vaccinees in Malawi and UK Malawi: 98.5% scar +veUK: 96.7% scar +ve
19 Choice of time point: maximal responses will be different in naive and sensitised subjects A small group of subjects were tested weeks after BCG vaccination in MalawiResults did indicate that BCG vaccination boosted the IFNg response, but this increase was transientIn our current study we are testing at both 3 and 12 months….
20 Frequency distributions of IFNγ responses to M tuberculosis PPD among 13-year olds in the UK, prior to and 3 months after receiving BCG vaccine or placeboPre-vaccination, placebo(n=27)proportion.18.104.22.168.22.214.171.124.91Pre-vaccination, vaccine(n=34)Post-vaccination, placeboresponse (pg/ml)3162125250500100020004000Post-vaccination, vaccineChange:1.3x vs16.9x
21 The timing of testing is critical: 8-10 weeks after BCG, IFNg responses are boosted in Malawian vaccineesPlacebo group (n=8)Vaccine group (n=17)4000400030003000IFN-g pg/mlIFN-g pg/ml20002000Its not that the Malawians are unable to increase their responses either- if you look at an earlier time point after vaccination they do show an increase, but this is largely lost by 1 year10001000PrePostPrePostData: Gill Black
22 IFNg responses to environmental mycobacterial PPDs one year after BCG vaccinationin MalawiExposure to non-tuberculous, environmental mycobacteria appearscommon in Malawians, and is responsible for sensitisation to MTb PPD
23 BCG vaccination is given to infants at brith in most low income countries If exposure to environmental mycobacteria accounts for the differences to response in adolescents to BCG vaccination in Malawi and the UK, would infants in the two settings behave similarly?
25 BCG vaccination is inducing good IFNg responses at 3 months in Malawi in infants
26 Frequency distributions of IFNγ responses to M tuberculosis PPD in Malawian and UK infants, three months after BCG vaccinationMalawian infants vaccinated at birth give good IFNg responses3 months laterResponses seem higher in the UK infants- is this real, is it becausethey are older (4 months vs 3 months), or ????
27 Before and after…...Before vaccination of adolescents/young adults, only 20% were IFNg responders to PPD in UK, compared to 60% in MalawiAfter vaccination, approx 80% of both the UK and Malawi populations respondedAre both populations equally protected?What else is different?
28 In Malawi, 55% of the young adults tested were infected with hookworm, 40% with Schistosoma mansoni and 25% with Schistosoma haematobium; filarial infection is also found in the north of the district
29 Effect of helminth infections on immune responses Odds ratios of having a strong IFNγ response (>250 pg/ml) (a) and a (“positive”) IL-5 response (>62 pg/ml) (b) to M tuberculosis PPD, streptokinase/streptodornase (SK/SD) or phytohaemagglutinin (PHA) by numbers of helminth infections (hookworm, S mansoni, S haematobium, W bancrofti). Baseline is group not infected with any helminths.
30 IL-5 response to M. tuberculosis PPD post-vaccination, Malawi and UK BCG vaccination does not induce increased IL-5 production in response to PPD, in Malawi or the UKIL-5 responders are high, not low, IFNg producers
31 Differences in innate immunity: Malawians also make more IL-10 and TNFa than UK subjects
32 Association between IFNg responses and Mantoux skin test response to PPD ESAT-6
33 Assays for IFNg as an alternative to skin testing Assays are robust and perform well in field settingsThey identify more T cell responders than skin testing- are they more sensitive?Two responses are associated although there are discordant individualsResponses are more variable in a tropical setting (Malawi vs UK)Assays can detect responses to individual recombinant antigens
34 Overnight and 6 day whole blood assays compared Overnight assays require more blood, and may need to be put into culture quickly6 day assays are similar to traditional PBMC assays, and involve cell proliferation6 days assays require access to a tissue culture hood and CO2 incubatorIn both assays cytokine can be measured by ELISA (multiplex assay, dipstick)
35 ConclusionsIn adolescents/young adults, the increase in the amount of IFNg produced in cultures stimulated with PPD correlates with the protection BCG vaccination gives against TB in the UK: just measuring absolute amounts of IFNg insufficientIn Malawi, BCG vaccination only induces a temporary boosting in IFNg responses: timing of testing criticalPrior sensitivity to mycobacterial antigens in Malawi seems to result from exposure to environmental mycobacteria: implications for vaccine design and timingOther infections may influence the status of the immune system in low income countries
36 Whether IFNg is a correlate of protection depends on whether the Malawians have protective immunity to TB equivalent to that given by BCG vaccination in the UK…..Does the immunity induced by environmental mycobacteria kill the BCG before it can induce real protection? Test mycobacterial killing…Does protection need IFNg together with another key cytokine (TNFa, ??) or without another cytokine (IL-4, IL-10, TGFb)?Does protection need CD8 T cells as well as CD4 T cells?One of the big questions this raises is wther the immunity the malawians have before vaccination gives them any protection against TB? It clearly doesn’t give them full protection as there is a lot of Tb in malawi.
37 Some implications and questions .... Diluted whole blood assays have the potential to be a useful tool for new TB vaccine trialsIf a new TB vaccine contains antigens cross-reactive with those in environmental mycobacteria, it may need to be given to infantsThere may be differences in the proportion of naïve and memory T cells, and in the maintenance of T cell memory, in tropical and non-tropical settings
38 BCG vaccination Malawi/UK Paul FineRosemary Weir, Patricia Gorak-Stolinska (UK)Gill Black, Anne Ben-Smith (Malawi)Steven Chaguluka, Huxley KanyongolokaMia Crampin, David WarndorffLifted Sichali, Lorren MwaunguluBernadette NazarethSian Floyd, Lyn Bliss, Jacky Saul, Keith BransonFunded by the Wellcome Trust, LEPRA and WHO