Experiences with BCG vaccination in the UK and Malawi Hazel M Dockrell London School of Hygiene & Tropical Medicine

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?

BCG protection in Malawi and the UK In 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

Immunity to Tuberculosis Cell mediated immunity is important Interaction between activated m, CD4+, CD8+,  T cells Type 1 cytokines such as IFN/IL-12 play a role in protection; also role for TNFa Could whole blood assays measuring such cytokines be used as a correlate of protection?

Diluted whole blood assays to measure antigen-specific interferon-g responses Heparinised blood just diluted in tissue culture medium and antigens added Cytokines measured in culture supernatants Assay proved easy, reliable and reproducible for field use when used with M.leprae antigens in Nepal It 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

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 – this Black et al, Int J Tuberc Lung Dis 5: 664 (2001)

Use of positive control supernatants to control for assay variation

Field work in Malawi 633 recruited (age 10-28) (HIV-ve, BCG-ve) 562 eligible (Mantoux <10mm) 2/3 BCG vaccinated 80% follow-up at 1 year The 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

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 too 435 recruited (age 12-15, BCG scar -ve), Heaf test grade >2 excluded 2/3 given BCG vaccination 80% followed up at 1 year Rosemary Weir

Does IFNg production increase in both UK and Malawian vaccinees? IFNg responses to M.tuberculosis PPD tested pre vaccination and 1 year post vaccination

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% postvaccination Increase in % responders Controls 19% to 21% Vaccinees 23% to 83% Black, Weir et al, Lancet 139: 1393 (2002)

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 antigens Increase in % responders Placebo 60% to 76% Vaccinees 61% to 78% Black, Weir et al, Lancet 139: 1393 (2002)

Increase 1.4 vs 2.4 1.05 vs 8.3

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 UK IFNg responses to PPD: % changes over 1 year in placebo/control groups decrease no change increase Malawi 38 7 55 UK 25 43 32

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 UK There are conflicting views about whether exposure to mycobacteria confers protection or blocks its induction

Controlling for effective vaccination: Comparison of BCG scar size of vaccinees in Malawi and UK Malawi: 98.5% scar +ve UK: 96.7% scar +ve

Choice of time point: maximal responses will be different in naive and sensitised subjects A small group of subjects were tested 10-12 weeks after BCG vaccination in Malawi Results did indicate that BCG vaccination boosted the IFNg response, but this increase was transient In our current study we are testing at both 3 and 12 months….

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 placebo Pre-vaccination, placebo (n=27) proportion .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Pre-vaccination, vaccine (n=34) Post-vaccination, placebo response (pg/ml) 31 62 125 250 500 1000 2000 4000 Post-vaccination, vaccine Change: 1.3x vs 16.9x

The timing of testing is critical: 8-10 weeks after BCG, IFNg responses are boosted in Malawian vaccinees Placebo group (n=8) Vaccine group (n=17) 4000 4000 3000 3000 IFN-g pg/ml IFN-g pg/ml 2000 2000 Its 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 year 1000 1000 Pre Post Pre Post Data: Gill Black

IFNg responses to environmental mycobacterial PPDs one year after BCG vaccination in Malawi Exposure to non-tuberculous, environmental mycobacteria appears common in Malawians, and is responsible for sensitisation to MTb PPD

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?

Vaccination schedule in Malawi

BCG vaccination is inducing good IFNg responses at 3 months in Malawi in infants

Frequency distributions of IFNγ responses to M tuberculosis PPD in Malawian and UK infants, three months after BCG vaccination Malawian infants vaccinated at birth give good IFNg responses 3 months later Responses seem higher in the UK infants- is this real, is it because they are older (4 months vs 3 months), or ????

Before and after…... Before vaccination of adolescents/young adults, only 20% were IFNg responders to PPD in UK, compared to 60% in Malawi After vaccination, approx 80% of both the UK and Malawi populations responded Are both populations equally protected? What else is different?

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

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.

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 UK IL-5 responders are high, not low, IFNg producers

Differences in innate immunity: Malawians also make more IL-10 and TNFa than UK subjects

Association between IFNg responses and Mantoux skin test response to PPD ESAT-6

Assays for IFNg as an alternative to skin testing Assays are robust and perform well in field settings They identify more T cell responders than skin testing- are they more sensitive? Two responses are associated although there are discordant individuals Responses are more variable in a tropical setting (Malawi vs UK) Assays can detect responses to individual recombinant antigens

Overnight and 6 day whole blood assays compared Overnight assays require more blood, and may need to be put into culture quickly 6 day assays are similar to traditional PBMC assays, and involve cell proliferation 6 days assays require access to a tissue culture hood and CO2 incubator In both assays cytokine can be measured by ELISA (multiplex assay, dipstick)

Conclusions In 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 insufficient In Malawi, BCG vaccination only induces a temporary boosting in IFNg responses: timing of testing critical Prior sensitivity to mycobacterial antigens in Malawi seems to result from exposure to environmental mycobacteria: implications for vaccine design and timing Other infections may influence the status of the immune system in low income countries

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.

Some implications and questions .... Diluted whole blood assays have the potential to be a useful tool for new TB vaccine trials If a new TB vaccine contains antigens cross-reactive with those in environmental mycobacteria, it may need to be given to infants There 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

BCG vaccination Malawi/UK Paul Fine Rosemary Weir, Patricia Gorak-Stolinska (UK) Gill Black, Anne Ben-Smith (Malawi) Steven Chaguluka, Huxley Kanyongoloka Mia Crampin, David Warndorff Lifted Sichali, Lorren Mwaungulu Bernadette Nazareth Sian Floyd, Lyn Bliss, Jacky Saul, Keith Branson Funded by the Wellcome Trust, LEPRA and WHO