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 Brunetta 1, Colin Kovacs 1,2,Tae Wook-Chun 3, Janet Raboud 2,4, Desheng Su 4, Mario Ostrowski 2,5,Gabor Kandel 2,5, Graham Smith 1, Rupert Kaul 2,4, Roberta Halpenny 1, Duncan Chege 2, Mona Loutfy 1,2,5 1 Maple Leaf Medical Clinic, Toronto, Ontario, Canada; 2 University of Toronto, Toronto, Ontario, Canada; 3 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA; 4 Division 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 Raltegravir- intensification (400mg twice/day) Placebo Randomize n=12 24 HIV+ patients fully suppressed on HAART 4w 8w 12w 16w 28w 40w 48w Primary analysis at week 48 n=12 Measured Outcomes: - Blood & sigmoid HIV-1 proviral DNA in CD4+ T cells - Blood CD4+ T cell counts 48w 0w Sigmoid biopsy Blood phlebotomy 4w 8w 12w 16w 28w 40w 48w 48w 0w Sigmoid biopsy Blood phlebotomy

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 Ficoll density separation PBMC/Sigmoid CD8 Depletion Real time PCR: HIV-1 Proviral DNA amplification & quantitation Sigmoidoscopy 0.5 & 1.0 ug/ml Collagenase-II tissue digestion (30 min each)  CD4+ T cell counts done on whole blood (counts/mm 3 ) using FACS HIV-1 DNA copy number per 1x10 6 CD4+ T cells reported (LOD: 2.6 copies) PBMC GALT

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

11. Results Treatment Median Baseline proviral load (log 10 ) Median w48 proviral load (log 10 ) Raltegravir group3.053.11 Placebo group2.862.91  No difference in blood HIV DNA proviral load between groups at week 48 (p=0.62) p from ANCOVA

12. Results Treatment Median Baseline CD4 count (cells/mm 3 ) Median w48 CD4 count (cells/mm 3 ) Raltegravir group665690 Placebo group610700  No difference in blood CD4+ T cell counts between groups at week 48 (p=0.25) p from ANCOVA

13. Results Treatment Median Baseline proviral load (log 10 ) Median w48 proviral load (log 10 ) Raltegravir group3.102.84 Placebo group3.203.08  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