1. Pathogenesis of the HIV-TB associated immune reconstitution inflammatory syndrome 4th IAS, Sydney, July 23rd 2007 Robert J Wilkinson Wellcome Senior Fellow, Imperial College London and University of Cape Town Programme Leader, MRC National Institute for Medical Research London

2. MÉDECINS SANS FRONTIÈRES SOUTH AFRICA Contributors Graeme Meintjes Priscilla Mouton Keira Skolimowska Kerryn van Veen Mark Nicol Molebogeng Rangaka Musaed Abrahams Gary Maartens Kevin Rebe Anne O’Garra Chelsea Morroni Katalin Wilkinson Ronnett Seldon David Stead Dominique Pepper Adrian Martineau Gilles van Cutsem Eric Goemaere Steven Lawn

3. The Western Cape is the richest part of Africa but has the worst TB problem in the world Courtesy Dr Keith Cloete, Provincial administration 27509 28843 31536 33665 40144 42123 44414 46256 47603 199719981999200020012002200320042005 0 10000 20000 30000 40000 50000 Cases Registered Entire US UK

4. Evolution of HIV prevalence rate in Cape Metropole (antenatal VCT results) Metropole Health areas HIV Prevalence (95% CI) 200020012002200320042005 Blaauwberg 0.6  1.1 8.2±64.4±3.01.2±17.32 ±3.6 CapeTown Central 3.7  3.6 11.9±611.6±513.7 ±4.711.5±3.3 Greater Athlone 6.8  4.6 8.9±410.1±4.416.4 ±3.617.7±3.5 Helderberg 19  6 19.1±4.519.1±4.218.8 ±3.312.8±3.0 Khayelitsha 22  5 24.9±4.227.2±4.233.0 ±3.533 ±3.9 Mitchells Plain 5.4  0.10.7  1.3 4± 4.06.3±412.9 ±3.55.1±2.1 Gugulethu/Nyanga 16.1  6.5 27.8±5.228.1±4.229.1 ±2.829.0±3.9 Oostenberg 5.7  3.3 14.5± 6.016.1±4.314.8 ±3.316.2±3.5 South Peninsula 5.9  3.9 6± 4.19.3±3.810.8 ±3.212.4±3.1 Tygerberg Eastern 5.1  3.76.1  3.4 10.4±58.0±3.912.7 ±3.615.2±3.5 Tygerberg Western 7.9  3.9 12.7±58.1±3.315.1 ±415.0±3.1 Courtesy of Dr Keith Cloete, PAWC

5. Tuberculosis in Khayelitsha, South Africa Population: ~350,000 HIV prevalence 33 ± 3.9% (2005) TB incidence rate (2006) ~1600/100,000 ~70% TB is HIV associated 5745 cases found in 2006 645 of 3124 reported deaths in 2006 due to TB (21%)

6. GF Jooste Referral Area

7. GF Jooste Hospital serves a population of 1.2 million 8244 people are prescribed antiretroviral treatment in catchment area

8. Little information on Cause Diagnosis Management An ‘explosion’ of TB-IRIS

9. TB-IRIS 1 Spectrum and practical case definition 3 Severe TB-IRIS can complicate drug resistant tuberculosis 2 Management with steroids 4 Immunology of TB-IRIS

10. Nodal enlargement

11. New pulmonary infiltrate

12. Cold abscess

13. Serous effusions

14. Neurological deterioration

15. Design of studies Referral to study Excluded or refused consent Severe 1)Resp failure 2)Vital structure compressed 3) TBM Open-label steroids Observational cohort Effusions Pulmonary infiltrate LN Cold abscess Other IRIS manifestations e.g. Bone marrow infiltration RCT 100 patients Observational cohort Steroids at discretion of clinician Immunology and genetic studies Non-IRIS Comparison cohort ‘TB-ART’

16. Immunological studies Cross-sectional (compare IRIS with non-IRIS groups) Longitudinal within IRIS cohort irrespective of treatment allocation (as IRIS progresses) within TB-ART cohort to document changes should IRIS occur Assays Interferon-gamma ELISpot analysis of PBMC using various antigens 6 and 24 hour restimulation with H37Rv with collection of supernatant and harvesting of RNA for microarray and qRT-PCR 4- and 13- colour FACS (activation status, regulatory and Th17 subsets) Serum cytokines DNA stored. Consent for genetic testing obtained

17. PPD specificTh1 expansions Granuloma expansion release of inflammatory cytokines TB-IRIS

18. Antigen selection ESAT-6 38 kDa Acr interim analysis of 184 subjects, data censored 27 May 2007

19. Cases and controls

20. Untreated n = 31 p = 0.004 TB Rx n = 27 p = 0.002 PPD 0 1000 2000 3000 4000 5000 HIV-TB patient category ART gives rise to large expansions of PPD specific T cells irrespective of whether IRIS occurs IFN-gamma SFC/10 6 PBMC

21. Responses are directed to several categories of antigen p = 0.0003 p = 0.05 IFN-gamma SFC/10 6 PBMC 38 kDa IRISTB Rx 0 1000 2000 3000 4000 HIV-TB patient category H37Rv IRISTB Rx 0 1000 2000 3000 HIV-TB patient category IFN-gamma SFC/10 6 PBMC ESAT-6 IRISUntreatedTB Rx 0 1000 2000 3000 4000 5000 HIV-TB patient category Acr 1 IRISUntreatedTB Rx 0 500 1000 1500 2000 2500 3000 3500 4000 HIV-TB patient category

22. Longitudinal analysis of TB patients starting ART n=55 Proportion responding to H37Rv week 0week 2week 4week 8 IRIS 30 40 50 60 70 80 90 Percentage responders Non-IRIS Proportion responding to PPD 50 60 70 80 90 Percentage responders week 0week 2week 4week 8 IRIS Non-IRIS

23. 0 50 100 150 2500 Longitudinal analysis of TB patients starting ART week 0At diagnosisweek 0week 2 IRISNon-IRIS Interferon-  SFC/10 6 PBMC p = 0.01 p = 0.34

24. M. Tuberculosis induced proliferation, activation and regulation 0 10 20 30 40 50 60 70 80 CD4 + CD71 + Marker Percentage CD3 cells positive CD4 + DR + CD8 + CD71 + CD8 + DR + CD4 + FoxP3 + unstimulated CD4 + FoxP3 + stimulated TB-IRIS n=10 Day 14 non-IRIS n=10

25. Conclusions ART mediated immune restoration during therapy of active TB is associated with a substantial early expansion of TB antigen specific IFN-  secreting T cells TB therapy in the absence of ART is also associated with restoration of responses to some antigens An increased response to heat killed bacilli best associates with IRIS The numbers of regulatory T cells did not differ between IRIS and non- IRIS subjects IRIS is clinically significant and very heterogenous. Studies need to be adequately powered.

26. Generalisability: Protective and pathogenic immune responses in tuberculosis Robert Koch Thinking hard about IRIS

27. Thank you

28. Corticosteroids have little effect on T cell expansions 0 500 1000 1500 2000 2500 3000 3500 0102030405060708090 ESAT-6 PPD Days since onset IFN gamma SFC/million PBMC New cervical LN 21 days after starting HAART in lady with Cult. +ve PTB. Prescribed study drug Neurological deterioration Open label Prednisone Resolution of symptoms

29. Too much killing of M. tuberculosis? Serum HNP1-3 ng/ml Martineau et al. J. Clin. Invest. in press RLU/ml

30. Who gets IRIS?

31. Why the ELISpot results differ from Bourgarit et al. PPD Week 0Week 2Week 4Week 8IRIS 0 1000 2000 3000 4000 IFN-gamma SFC/10 6 PBMC

32. When and what form of IRIS?

33. Challenges in diagnosis No diagnostic test; diagnosis of exclusion ALTERNATIVE DIAGNOSIS Bacterial infections Fungal infection PCP NTM Lymphoma Kaposi’s sarcoma Drug resistance 14/141 in Cape Town cohort of TB-IRIS suspects had MDR or Rifampicin monoresistance DRUG REACTION especially if hepatic involvement

34. MDR-TB-IRIS overlap syndrome Drug resistance and TB-IRIS 14/141 had drug resistance (10%) –3 known to have MDR –2 known to have Rif mono-resistance –1 known to have INH mono-resistance –6 presented with TB IRIS then MDR diagnosed* –2 presented with TB IRIS then Rif mono-resistance diagnosed* * Most of these patients reported some (or complete) improvement on TB Rx and all reported symptomatic deterioration after HAART, some required steroids Fastplaque Rif resistance assay being used to expedite diagnosis 5 died