Conflict of Interest Disclosure

1. Effect of intensification of long-term highly active antiretroviral therapy (HAART) with raltegravir on proviral HIV-1 DNA in blood and gut associated lymphoid tissue (GALT): a randomized, placebo controlled trial Jason Brunetta1, Colin Kovacs1,2,Tae Wook-Chun3, Janet Raboud2,4, Desheng Su4, Mario Ostrowski2,5,Gabor Kandel 2,5, Graham Smith 1, Rupert Kaul 2,4, Roberta Halpenny 1, Duncan Chege2, Mona Loutfy 1,2,5 1Maple Leaf Medical Clinic, Toronto, Ontario, Canada; 2University of Toronto, Toronto, Ontario, Canada; 3Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA; 4Division of Infectious Diseases, University Health Network, Toronto, Ontario, Canada; 5 St. Michael’s Hospital, Toronto, Ontario, Canada

2. Conflict of Interest Disclosure • Funding for this project was provided by a Research Grant from Merck Frosst Canada Ltd.

3. Background • Eradication of HIV remains elusive due to the persistence of viral reservoirs. • The gut-mucosal compartment is an important viral reservoir • Viral reservoirs mainly consist of long-lived and latently infected CD4+ T cells • New HAART drug classes may help target these latently infected reservoirs. E.g; • Integrase inhibitors (e.g. raltegravir) • Entry/fusion inhibitors

4. Background • Recent studies examining raltegravir intensification therapy failed to show a reduction in plasma HIV RNA[Reviewed in Schulze, et al JID 2011] • However, changes in plasma viremia may not reflect changes in the mucosal reservoir. • 1 recent study suggested no reduction in proviral DNA in the gut[Yukl et al, AIDS 2010]. However, this pilot study: • was an open-label study without controls • had a relatively small sample size (n=7) • intensified participants with raltegravir for a brief period (12 weeks).

5. HYPOTHESIS • Raltegravir intensification in long-term suppressed individuals will decrease blood and sigmoid CD4+ T cell HIV proviral levels.

6. Methods • Study design • A prospective, double-blind, placebo-controlled randomized controlled trial (ClinicalTrials.gov # NCT00520897) • Enrolled participants • HIV-infected individuals recruited from the Maple Leaf Medical Clinic • Inclusion criteria • Sustained virologic suppression (<50 viral copies/ml) for over 4years • Participant must be on first standard HAART with 2-3 NRTIs and 1-2 PIs or an NNRTI for at least four years • Exclusion criteria • Active AIDS-defining illness in past six months • Abnormal clinical laboratory test results at screening

7. Methods – Study schematic Blood phlebotomy 4w 8w 12w 16w 28w 40w 48w Raltegravir-intensification (400mg twice/day) 0w 48w Sigmoid biopsy n=12 Primary analysis at week 48 24 HIV+ patients fully suppressed on HAART Randomize Blood phlebotomy n=12 4w 8w 12w 16w 28w 40w 48w Placebo 0w 48w Sigmoid biopsy • Measured Outcomes: • - Blood & sigmoid HIV-1 proviral DNA in CD4+ T cells • - Blood CD4+ T cell counts

8. Study Endpoints • Primary endpoint Determine if 48 weeks of raltegravir intensification in long-term virologically suppressed participants on HAART is associated with a change in HIV-1 proviral DNA in blood and sigmoid CD4+ T cells • Secondary endpoint Determine effect of raltegravir intensification on blood CD4+ T cell populations

9. Laboratory methods Blood Phlebotomy PBMC Real time PCR: HIV-1 Proviral DNA amplification & quantitation Ficoll density separation PBMC/Sigmoid CD8 Depletion HIV-1 DNA copy number per 1x106 CD4+ T cells reported (LOD: 2.6 copies) GALT Sigmoidoscopy 0.5 & 1.0 ug/ml Collagenase-II tissue digestion (30 min each) • CD4+ T cell counts done on whole blood (counts/mm3) using FACS

10. Results – Table 1 • Baseline clinical and demographic characteristics were similar between the groups.

11. Results • No difference in blood HIV DNA proviral load between groups at week 48 (p=0.62) p from ANCOVA

12. Results • No difference in blood CD4+ T cell counts between groups at week 48 (p=0.25) p from ANCOVA

13. Results • No difference in sigmoid HIV DNA proviral load between groups at week 48 (p=0.74) p from ANCOVA

14. Summary • In virologically suppressed patients on HAART, 48 weeks of raltegravir-intensified therapy, as compared to placebo, • did NOT result in decay of blood or sigmoid HIV DNA in CD4+ T-lymphocytes • had NO impact on blood CD4+ T cell populations • Extending raltegravir intensification out to 96 weeks also did NOT result in any significant decrease in HIV DNA in blood or sigmoid CD4+ T lymphocytes (data not shown) • Additional novel approaches are required to help reduce the latent viral reservoir.

15. Acknowledgments • All the patients • Research staff at Maple Leaf Medical Clinic • For working on this project • Dr. Kandel for sigmoid biopsies • HIV Statistical Analytical Group at UHN • Dr. Janet Raboud’s team • Duncan Chege for analytical work, work on presentation & slides • Funders