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 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

5. 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?

6. 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

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 – this
Black 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 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

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 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

12. Does IFNg production increase in both UK and Malawian vaccinees?
IFNg responses to M.tuberculosis PPD tested
pre 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% postvaccination
Increase in % responders
Controls 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 antigens
Increase in % responders
Placebo % to 76%
Vaccinees % to 78%
Black, Weir et al, Lancet 139: 1393 (2002)

15. Increase
1.4 vs 2.4
1.05 vs 8.3

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 UK
IFNg responses to PPD: % changes over 1 year in placebo/control groups
decrease no change increase
Malawi UK

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 UK
There are conflicting views about whether exposure to
mycobacteria 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 +ve
UK: 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 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….

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 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

21. 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

22. 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

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?

24. Vaccination schedule in Malawi

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 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 ????

27. 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?

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 UK
IL-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 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

34. 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)

35. 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

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 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

38. 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