1. Avances en investigación básica Javier Martinez-Picado 19th Conference on Retroviruses and Opportunistic Infections Seattle, March 5-8, 2012 Javier Martinez-Picado 19th Conference on Retroviruses and Opportunistic Infections Seattle, March 5-8, 2012

2. Barcelona, 13/03/12 Restrictions Factors Towards a HIV-1 Cure Highligthed Topics

3. Restriction factors: a defense against retroviral infection SAMHD1 is a myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx Barcelona, 13/03/12 Restriction Factors Benkirane #63 Lieberman #64 Luban #65 Kewal Ramani #66

4. Barcelona, 13/03/12 Host defenses against retroviruses Modified from Kirchhoff, 2010 Host Cell Microb

5. Laguette & Benkirane, 2012. Trends in Immunology

6. Barcelona, 13/03/12 Dendritic Cells  DCs coordinate immune responses to invading pathogens  DCs can carry HIV-1 to target CD4+ T cells, amplifying the cytopathic effects of the virus  SAMHD1 is responsible for blocking replication of HIV within dendritic cells, although it functions in other myeloid cells as well  SAMHD1 can be antagonized by vpx Benkirane #63

7. Barcelona, 13/03/12 What does make DC refractory to HIV-1 ? Modified from emulenews emulenews Proteosomal Degradation REVERSE TRANSCRIPTION RESTRICTIVE INFECTIONPERMISSIVE INFECTION

8. Barcelona, 13/03/12 What does make DC refractory to HIV-1 ?  Vpx addition or Silencing of SAMHD1 increases the susceptibility of monocytic-derived DCs to infection Laguette et al. Nature 2011 MDDC siRNA VLP-Vpx THP-1 VLP-vpx

9. Barcelona, 13/03/12 SAMHD1  SAM domain and HD domain form a dimer that in humans is encoded by the SAMHD1 gene. Chr 20 sterile alpha motif  It belongs to a family of proteins that have been involved in a rare genetic disorder, the Aicardi-Goutières Syndrome, an inflammatory encephalopathy that resembles congenital viral infection, such as vertically acquired HIV. Laguette & Benkirane, 2012. Trends in Immunology

10. Barcelona, 13/03/12 SAMHD1 cuts the power for HIV-1  SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that restricts the availability of dNTPs, the building blocks of genetic material, in macrophages and dendritic cells. dNTP → dN + Pi  The molecule cuts off the supply line of dNTP available for cDNA synthesis by RT and so prevents viral replication  SAMHD1 is the only HIV-1 restriction factor against which the virus has not evolved a neutralizing strategy. Its discovery opens new possibilities to understand the intricacies of the relation between lentiviral evolution and primate hosts. Lahouassa et 1l 2012 Nat Immunol Jermi 2012 Nat Rev Microbiol Laguette et al. 2012 Hist Cell Microb

11. Barcelona, 13/03/12  SAMHD1 is a restriction factor that blocks HIV-1 replication in myeloid cells.  SAMHD1 is antagonized by vpx, a molecule present in HIV-2 and SIV but not in HIV-1.  Modulating SAMHD1 function could render human hosts more prone to develop appropriate innate and adaptive immune responses.  Therapies that mimic the effect of SAMHD1 or otherwise deplete the intracellular supply of dNTPs could represent a new approach to HIV treatment and cure.  Potential role in the development of dendritic-cell- targeted vaccines against HIV/AIDS. Implications

12. Barcelona, 13/03/12 If HIV-1 does not have vpx but HIV-2 does, why HIV-1 is more pathogenic than HIV-2? Paradox vpx

13. HIV Reservoirs and Cure Research Pathways toward a Cure: Viral Latency and Reservoirs Barcelona, 13/03/12 HIV Cure

14. Barcelona, 13/03/12 HAART is effective  Suppression to below detectable HIV-1 RNA levels is rapid and durable.

15. Barcelona, 13/03/12 However, despite prolonged HAART …  Replication competent viruses can be rescued ex vivo from resting CD4+ T cells  Low level plasma viremia (< 50 copies) persists  Immune activation diminishes but does not normalize  Plasma viremia rebounds after treatment interruption

16. Barcelona, 13/03/12 What sustains HIV-1 despite HAART?  Ongoing HIV-1 replication cycles in cells located in sanctuary sites where drug levels are suboptimal  Latently infected cells (resting central memory T cells):  Might produce virus upon reactivation.  Might proliferate Lewin #106

17. Barcelona, 13/03/12 New markers  Plasma:  HIV RNA  Ultrasensitive HIV RNA  Cell-associated:  Total HIV DNA  Integrated cell HIV DNA  Episomal HIV DNA  HIV RNA  Cell-activation markers  Inflammation Lewin #106

18. Barcelona, 13/03/12 Viral replication in pharmacologic sanctuaries  Quantitation of intracellular ARV concentrations, including NRTI- triphosphates in PMBC, LN and GALT.  Pharmacokinetic data suggest drug-specific compartmentalization Fletcher #108 10 4 10 3 10 2 10 1 1 ATV (ng/mL) PBMCLNIlealRectal EFV (ng/mL) 10 5 10 3 10 2 10 1 1 10 4 PBMCLNIlealRectal

19. Barcelona, 13/03/12 Viral replication in pharmacologic sanctuaries  Hypothesis: inadequate drug penetration may contribute to cryptic replication of HIV, which sustains HIV infection.  Mechanisms:  Physiochemical characteristics  Susceptibility for and distribution of influx and efflux transporters (Minuesa #590; P-gp in primary CD4+ T cells and raltegravir intracellular concentrations)  Drug metabolizing enzymes  Expression of phosphorylating enzymes  Activation state of the compartment  Host genetic traits. Fletcher #108

20. Barcelona, 13/03/12 Reservoirs, does time of treatment initiation matter? Reservoirs, does time of treatment initiation matter?  Treatment of early HIV infection reduces viral reservoir to levels found in elite controllers Buzon et al. #151 Total HIV-1 DNA Chronic Acute Elite CAcuteChronic

21. Barcelona, 13/03/12 New chemotherapy against latency  Transcriptional activators: reactivation of the latent HIV-1 without inducing T cell activation

22. Barcelona, 13/03/12 Chromatin Reconfiguration for Purging HIV-1 from the Latent Reservoir Johnstone, Nat Rev Drug Discov, 2002

23. Barcelona, 13/03/12 Vorinostat Disrupts HIV-1 Latency in Patients on ART  n=5; CD4 count 562 cells/µL; 4 years of ART; 400 mg single dose of voronistat; no AE   PBMC cellular histone acetylation   HIV RNA levels increased significantly (5-fold) in pools of resting CD4+ cells obtained after SAHA dosing compared to BL  Proof-of-concept for HDAC inhibitors as a therapeutic class to directly attack and potentially eradicate latent HIV infection  No demonstration of viral production  Similar study suggested in NHP without data (Lifson #107) Archin #157LB

24. Barcelona, 13/03/12 Autologous ZFN CCR5-disrupted CD4 + T cells June #155

25. Barcelona, 13/03/12 Autologous ZFN CCR5-disrupted CD4 + T cells  SB-728-T infusion increases CD4 counts that persist over time.  SB-728-T expands rapidly and home to the gut.  3/6 subjects with >11 months follow up had an ~1-log decrease in total HIV-1 DNA over time: SB-728-T may decrease proviral DNA.  In one subject with the highest level of CCR5 modification, pVL was controlled (< limit of detection) without HAART.  These data suggest that in addition to the previously documented increases in CD4 cell, SB-728-T may also suppress HIV replication June #155

26. Barcelona, 13/03/12 Summary