Cryptococcal IRIS in Africa: clinical manifestations and pathogenesis

1. Cryptococcal IRIS in Africa: clinical manifestations and pathogenesis Paul R. Bohjanen, M.D., Ph.D. Associate Professor of Microbiology and Medicine University of Minnesota Minneapolis, MN, USA Professor in Residence Infectious Diseases Institute Makerere University Kampala, Uganda

2. Outline: • Role of immune activation in HIV pathogenesis. • Use of microarrays to assess immune activation in HIV-infected Ugandan patients before and after initiation of antiretroviral therapy. • Outcomes of cryptococcal meningitis in Uganda in the era of antiretroviral therapy (ART). • Clinical features of HIV Immune Reconstitution Inflammatory Syndrome (IRIS) in patients with recent cryptococcal meningitis after initiation of ART. • Use of microarrays to assess biomarkers of IRIS after initiation of ART in patients with or without recent cryptococcal meningitis.

3. Primary HIV Infection Viral Dissemination Partial Immune Containment Chronic Immune Activation Destruction of Lymphoid Tissue Decreased Circulating CD4 T Cells AIDS

4. Immune Reconstitution • Potent ARV therapy blocks viral replication and prevents the destruction of CD4 T cells. • This shifts the balance toward CD4 T cell regeneration and improvement in immune function. • CD4 T cell counts increase. • Antigen-specific immune responses are restored.

5. Immune Reconstitution • The bottom line is that patients who respond to ART have improved immune function and are at lower risk for the development of opportunistic infections.

6. Is immune reconstitution always beneficial?

7. Immune Reconstitution Inflammatory Syndromes (IRIS): • Immune Reconstitution Paradox: Recovery in the function of the immune system with ART can promote an inflammatory reaction to antigens that were previously not recognized by the immune system. • This inflammatory reaction can sometimes lead to worsening of a current or latent opportunistic infection. • The onset of IRIS often occurs 2-8 weeks after initiation of ARV therapy but can occur earlier or later.

8. IRIS: Triggering Antigens • In IRIS, the flaring of specific immune responses to microbial antigens occurs in the setting of improving immunity after the initiation of effective ART. • IRIS may represent either an appropriate inflammatory response that was previously masked by severe immune deficiency or a pathological exaggerated inflammatory reaction. • Inciting antigens may be from an active infection (replicating microorganisms) or from the remnants of a treated infection (microbial debris) or latent infection. • IRIS is also associated with autoimmune disorders or malignancies such as Kaposi’s sarcoma and lymphoma.

9. Pathogens associated with IRIS Mycobacterium avium Mycobacterium tuberculosis Mycobacterium leprae Cryptococcus neoformans Pneumocystis jiroveci Histoplasma capsulatum Hepatitis B virus Hepatitis C virus Varicella-zoster virus Cytomegalovirus BK Virus Parvovirus B19 JC virus Papilloma virus HHV-8 (KS)

10. Clinical Presentation • IRIS can occur as early as a few days after starting ARV therapy. In patients with baseline CD4+ T-cell counts below 50 cells/mm3, most events will happen within the first 8 weeks of therapy. Late IRIS with symptom onset after more than 1 year of ARVs have been described. • Patients typically become ill in the setting of improving virologic and immunological measures. • IRIS may be mistaken for a new opportunistic infection, but it can sometimes be distinguished by an atypical manifestation, such as localized inflammation where one would expect disseminated disease. • IRIS may also present as paradoxical worsening of a known opportunistic infection. • Depending on the site and activity of the immunologic response, the severity of clinical symptoms can vary widely from mild to life-threatening events.

11. IRIS Case Definition • Evidence of clinical response to ART with: • On ART • >1 log10 copies/mL decrease in HIV RNA (if available) • Infectious or Inflammatory condition within 6 months of ART initiation • Symptoms can not be explained by either: • Newly acquired infection • Expected clinical course of a previously recognized and successfully treated infectious agent • Treatment failure • Side effects of ART. • Complete ART non-compliance

12. IRIS Associated with Crytococcal Meningitis • IRIS may be associated with cryptococcal meningitis following initiation of ART. • Upon initiation of ART, ≈25% of CM patients experience IRIS with increases in headache, intracranial pressure, signs of inflammation, and in ≈25%, serious complications include loss of vision, cranial nerve palsies, reduced cognition and death.

13. IRIS Management • Evidence-based treatment recommendations are lacking. • Identify the inciting pathogen and treat it. • Most cases of IRIS are managed without stopping ARVs. • In severe cases, treatment options include stopping ARVs, steroids, NSAIDS, and surgical treatment (for example drainage of abscesses).

14. Why is so much IRIS seen in Africa? • IRIS occurs most often in patients with advanced HIV disease and severe immunosuppression. Because of limited availability of ART in Africa, treatment is often reserved only for patients with advanced disease. • Opportunistic infections associated with IRIS, such as tuberculosis and cryptococcal meningitis, occur frequently in Africa. • Limited diagnostic capabilities in resource poor regions may impair the diagnosis of alternative etiologies, such as a second opportunistic infection. The diagnosis of IRIS is often invoked when no other definitive diagnosis is found.

15. Research Questions • What is the incidence of IRIS? • How often is IRIS associated with significant morbidity and mortality? • Who is at greatest risk? • How should IRIS be treated? • Can the immune system be modulated to prevent IRIS? • In patients with IRIS-associated infections, such as TB or CM, is it better to treat the OI first and then start ART?

16. IRIS Study Design

17. Development of two pilot cohorts: 24 patients in each

18. Timing of IRIS after ART Incidence Rate: 35%

19. Response to ART in Cohort 1 (24 patients): Baseline: CD4: 58 ± 60 cells/uL viral load: 5.5 ± 5.4 log At 3 months: CD4: 192 ±133 cells (P < .001) with 23 of 24 individuals having a >50 CD4 cell/uL increase. viral load: undetectable among 19 of 24 patients (Range of five detectable subjects: 431-1,657 HIV RNA copies/mL). 20 of 24 patients have been followed for > 6 months with 8 study visits, and 100% follow up has occurred.

20. IRIS Events (Cohort 1)

21. A patient without prior cryptococcal disease and negative serum CRAG at enrollment, developed progressive tongue swelling. Biopsy revealed encapsulated yeast consistent with a cryptococcoma which regressed with two weeks of fluconazole therapy.

22. Cryptococcal Meningitis Outcomes Suspected Meningitis N=71 Non-CM meningitis N=22 Cryptococcal Meningitis N=49 On ART, IRIS Event N=5 Survived Hospitalization N=36 Died during hospitalization N=8 Started ART N=24 Lost prior to ART N=5 Died prior to ART N=7 Alive at 6 months N=18 Died after ART start N=6 37%

23. Response to ART in Cohort 2 (24 patients): Baseline: CD4: 29 ± 27 cells/uL viral load: 350,000 ± 258,000copies/mL At 3 months: CD4: 80 ± 63 cells viral load: 846 ± 1340copies/mL

24. IRIS Events in Cohort 2 Six patients have died since enrollment.

25. Immune activation in peripheral blood of HIV-infected patients in Uganda before and after initiation of ART

27. Most of the transcripts that were down-regulated after ART are T cell activation genes.

28. Multiple components of TNF and Interferon response pathways were down-regulated following initiation of ART

29. Immune activation in peripheral blood of HIV-infected patients in Uganda who do or do not develop IRIS after initiation of ART

30. Expression patterns of hundreds of genes are altered in patients with CM

31. Baseline Gene Expression that Predicts IRIS P=0.005

32. Gene Expression Predictors of IRIS (Aim 1) • Cell Signaling Pathways • Chemokine Signaling • PI3Kinase/AKT Signaling • Cell Death Pathways • Glycolysis/Gluconeogenesis Pathways • Drug Metabolism Pathways • PGK1; metabolism of lamivudine

33. Gene Expression Predictors of CM IRIS (Aim 2) • Cell Signaling Pathways • NF-B Signaling • Toll-like Receptor Signaling • Death Receptor Signaling • Cell Death Pathways • Cell Cycle Pathways • Transcription Factors

34. Biomarkers (signatures) of IRIS and CM IRIS

35. Biomarkers (signatures) of IRIS

36. Biomarkers (signatures) of CM and CM IRIS * IRIS, non-CM events ** TB at 6½ months * * ** No OI Controls CM IRIS CM Controls

37. Gene Expression Associated with CM IRIS

38. Conclusions HIV IRIS occurs frequently in sub-Saharan Africa, appearing in more than 1/3 of the patients in our study. IRIS has a diverse spectrum of clinical presentations and a wide range of severity. Mortality is high among patients with CM both prior to initiation of ART and after ART is initiated. IRIS occurs frequently in patients with CM with manifestations related to CM as well as other OIs that may be present. The normal response to ART is characterized by a decrease in immune activation that can be measured in peripheral blood using microarrays. Immune activation in IRIS can be measured in peripheral blood using microarrays and specific biomarkers or patterns of biomarkers (signatures) may be useful to diagnose IRIS. Even prior to initiation of ART, the expression of biomarkers in peripheral blood may be useful for predicting patients’ risk for subsequent development of IRIS.

39. Research Trainees Fellows: David Meya MBChB (ID-IDI) David Boulware, MD (ID-U of MN) Irina Vlasova MD,PhD (Postdoc- U of MN) Students: Joshua Rhein (4th year Med) Sam Goblirsch (4th year Med) Jack Staddon (Combined MD/PhD) Darlisha Williams (MPH student) Sarah Lee (Combined MD/MPH) Residents: Brett Handel-Paterson, MD (Med-Peds) Erin Huiras, MD (Dermatology)

40. Faculty Collaborators IDI/Makerere Univ:Univ of Manitoba: Andrew Kambugu (co-PI) Allan Ronald Keith McAdam Harriet Mayanja-Kizza Duke Univ: Moses Kamya John Perfect Univ of CO:Univ of WA: Edward Janoff (co-PI) Merle Sande Univ of MN:Institute of Tropical Paul Bohjanen (co-PI) Medicine, Antwerp: Tim Behrens Bob Colebunders Phil Peterson Luc Kestens James Neaton Tracy Bergemann