Understanding HIV persistence: The role of animal models Andrea Savarino, M.D.

Obstacles to development of a widely accepted model for HIV persistence during therapy: Skepticism for small animal models (humanized SCID mice, FIV-infected cats) 1)insensitive to NNRTIs, 2) different profiles of susceptibility to PIs Different susceptibility of non/human primate lentiviruses to antiretroviral drugs adopted in humans

Deere et al., 2010 RT-SHIV viral decay kinetics of ART-treated macaques RT-SHIV viral decay of macaques treated with ART (tenofovir emtricitabine and efavirenz) North et al., 2010 Low-level viral replication Virus: SIVmac239 (HIV-1 RT )

North, T. W. et al J. Virol. 84(6): Persistence of viral DNA and viral RNA detected in multiple samples of the same tissues Low-level viral replication

Dinoso, J. B. et al J. Virol. 83(18): Suppression of SIV viremia with ART (tenofovir; integrase inhibitor L-870,812; saquinavir; atazanavir) Low-level viral replication Viruses: SIV/17E-Fr (neurovirulent) SIV/Delta B670 (immunosuppressive)

Bourry et al. Retrovirology :78 doi: / Viral dynamics in SIV-infected macaques receiving a short-term ART (AZT/3TC/indinavir) during early chronic infection. Virus: SIVmac251 Low-level viral replication

Skepticism toward animal models “Given the absence of a robust animal model of suppressive cART, it is currently unclear whether there is a real need for all interventions to first be trialled in macaques to determine efficacy. To date there have been a limited number of antiretroviral agents that are active or can be administered to SIV-infected macaques allowing for durable control of SIV RNA to < 50 copies/ml for a prolonged period of time”. Lewin et al. 2011

Vahey M T et al. J Infect Dis. 2007;196: Tenofovir (PMPA) Emtricitabine (FTC) Characteristics of infection with SIVmac251 in 7 cynomolgus macaques. A, Plasma viral load during the course of the study. Peak seroconversion occurred at 2 weeks after infection, and the viral set point occurred at 20 weeks after infection. All macaques cleared virus from plasma by 2 weeks after successful antiretroviral treatment. B, Levels of CD4+T cells during the course of the study. The arrow indicates initiation of antiretroviral treatment, at 24 weeks after infection. PMPA FTC Virus: SIVmac251

In our study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates In-silico analyses (sequence alignments, docking simulations of drug/target interactions). Rational approach: In-vitro analyses of SIVmac251 susceptibility to antiretroviral drugs adopted in humans. In-vivo testing of the effects of antiretroviral drug combinations in SIVmac251-infected macaques.

3’ 5’ Mg 2+ DNA Catalytic core domain Lewis et al., 2010

Response of SIVmac251 to raltegravir in tissue culture RESULTS CEM x174 (syncytium formation ) Lewis et al., 2010

Response of SIVmac251-infected macaques to raltegravir (viral load) challenge RAL RAL + tenofovir / emtricitabine RAL ART Response of SIVmac251-infected macaques to raltegravir (CD4 counts) (Macaca mulatta) ART Threshold 40 RNA copies ml -1 Lewis et al., 2010

Persistence of viral DNA during therapy during ART treatment PBMCs Lewis et al., 2010

Darunavir (DRV) complexed with HIV-2 proteaseDRV complexed with SIVmac251 protease Barreca ML, Norelli S, and Savarino A, unpublished Kovalevsky et al., 2008 Darunavir as a tool for ART intensification in SIV + macaques

Lewis et al., 2011 Auranofin as an “antimemory” drug

Effect of intensified ART (iART) plus the anti-reservoir drug auranofin on viral DNA in PBMCs Lewis et al., 2011

Viral dynamics in monkeys following suspension of a drug regimen consisting of intensified ART (iART) plus auranofin

Benlhassan-Chahour, K. et al J. Virol. 77(23): FIG. Effect of HAART on plasma viral load (top panels) and on proliferation of NK cells and CD4+ and CD8+ T lymphocytes (middle and bottom panels) in macaques treated with HAART after SIV exposure Hocqueloux L, et al. AIDS Jun 19;24(10): Ananworanich J, et al. CROI ) Remission of acute infection in macaques induced by ART (AZT, ddI, indinavir) 2) Remission in humans treated with ART (optimized background therapy) at acute infection 3) Reduction of the viral reservoir in humans treated with mega ART (tenofovir emtricitabine/efavirenz/raltegravir/ maraviroc) at acute infection

P < viral set point Lewis et al., 2011 Correlation between the area under curve (AUC) at peak and the viral set point in monkeys following suspension of a drug regimen consisting of intensified ART (iART) plus auranofin

IMPACT OF MEGA-ART ON RE-ESTABLISHMENT OF VIRAL LOAD MONKEY P252 (tenofovir, emtricitabine darunavir/r, maraviroc) (98% sequence identity!) Maraviroc binding site Differences between H. sapiens and M. mulatta

Lymph node and rectal tissue levels of viral DNA and viral RNA were undetectable tenofovir + emtricitabine + raltegravir + darunavir + maraviroc

Sooty mangabay vs. rhesus macaque (nonprogressor to AIDS)(disease progressor)

Mathematical modeling of the latently infected cell pool decay Rong et al., 2009

Mathematical modeling of the latently infected cell pool decay

Conclusions We have reached the goal of obtaining fully suppressed viral replication and disappearance of viral RNA and DNA from reservoirs such as the gut and lymph nodes. Finally, our results are supported by mathematical modelling and are mathematically predictable. Reduction of the viral set point following therapy suspension has so far been reported only when treating during early infection but not in the chronic phase.

Lentiviral eradication: a Herculean task? Margolis DM, 2011 “The clearance of a retroviral infection in patients on ART is therefore a herculean task. While much is known about HIV persistence despite ART, many puzzles remain. Of even greater significance, while the clinical development path for antiviral therapies is well trodden, and paradigms for studying preventive microbicides or vaccines are being developed, a validated framework for inventing and testing eradication therapies does not exist”.

Acknowledgments Bioqual Mark G. Lewis Matt Collins Jake Yalley-OgunroUniversity of Rome “Tor Vergata” Jack Greenhouse Enrico Garaci Wendy Wagner University of Perugia, Italy Istituto Superiore di Sanità, RomeNunzio Iraci Barbara Chirullo Maria Letizia Barreca Iart Luca Shytaj Sandro Norelli Marco Sgarbanti Andrea Savarino University of Rome, La Sapienza Rossella Sgarbanti Anna Teresa Palamara VTGI, Florida Nicolas Chomont Sandrina DaFonseca