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Avances en investigación básica

Avances en investigación básica. Javier Martinez -Picado 19th Conference on Retroviruses and Opportunistic Infections Seattle, March 5-8, 2012. Highligthed Topics. Restrictions Factors Towards a HIV-1 Cure. 1. Restriction Factors.

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Avances en investigación básica

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  1. Avances en investigación básica Javier Martinez-Picado 19th ConferenceonRetrovirusesandOpportunisticInfections Seattle, March 5-8, 2012

  2. Highligthed Topics Restrictions Factors Towards a HIV-1 Cure

  3. 1 RestrictionFactors Restrictionfactors: a defenseagainst retroviral infectionSAMHD1 is a myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx Benkirane #63 Lieberman #64 Luban #65 Kewal Ramani #66

  4. Host defensesagainstretroviruses Modifiedfrom Kirchhoff, 2010 Host CellMicrob

  5. Laguette & Benkirane, 2012. Trends in Immunology

  6. Dendritic Cells • DCscoordinateimmune responses toinvadingpathogens • DCs can carry HIV-1 totarget CD4+ T cells, amplifyingthecytopathiceffectsofthe virus • SAMHD1 isresponsibleforblockingreplicationof HIV within dendritic cells, althoughitfunctions in othermyeloidcells as well • SAMHD1 can be antagonized by vpx Benkirane #63

  7. Whatdoesmake DC refractoryto HIV-1 ? RESTRICTIVE INFECTION PERMISSIVE INFECTION REVERSE TRANSCRIPTION ProteosomalDegradation emulenews Modifiedfromemulenews

  8. Whatdoesmake DC refractoryto HIV-1 ? • VpxadditionorSilencingof SAMHD1 increasesthesusceptibilityofmonocytic-derivedDCstoinfection MDDC THP-1 VLP-Vpx VLP-vpx siRNA Laguette et al. Nature 2011

  9. SAMHD1 • SAM domainand HD domainform a dimerthat in humansisencoded by the SAMHD1 gene. sterile alpha motif Chr 20 • Itbelongsto a familyofproteinsthathavebeeninvolved in a raregeneticdisorder, theAicardi-GoutièresSyndrome, aninflammatoryencephalopathythatresemblescongenital viral infection, such as verticallyacquired HIV. Laguette & Benkirane, 2012. Trends in Immunology

  10. SAMHD1 cutsthepowerfor 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 • Themoleculecuts off thesupplylineofdNTPavailableforcDNAsynthesis by RT and so prevents viral replication • SAMHD1 istheonly HIV-1 restriction factor againstwhichthe virus has notevolved a neutralizingstrategy. Itsdiscoveryopensnewpossibilitiestounderstandtheintricaciesoftherelationbetweenlentiviralevolutionand primate hosts. Lahouassa et 1l 2012 Nat Immunol Jermi 2012 Nat Rev Microbiol Laguette et al. 2012 Hist Cell Microb

  11. Implications • SAMHD1 is a restriction factor thatblocks HIV-1 replication in myeloidcells. • SAMHD1 isantagonized by vpx, a moleculepresent in HIV-2 and SIV butnot in HIV-1. • Modulating SAMHD1 functioncould render human hosts more pronetodevelopappropriateinnateandadaptiveimmune responses. • Therapiesthatmimictheeffectof SAMHD1 orotherwisedepletetheintracellularsupplyofdNTPscouldrepresent a newapproachto HIV treatmentand cure. • Potential role in thedevelopmentof dendritic-cell-targetedvaccinesagainst HIV/AIDS.

  12. Paradox If HIV-1 doesnothavevpxbut HIV-2 does, why HIV-1 is more pathogenicthan HIV-2? vpx

  13. 2 HIV Cure HIV Reservoirsand Cure Research Pathways toward a Cure: Viral Latency and Reservoirs

  14. HAART iseffective • Suppression to below detectable HIV-1 RNA levels is rapid and durable.

  15. However, despiteprolonged 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. Whatsustains 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. Newmarkers • 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. Viral replication in pharmacologicsanctuaries • Quantitationofintracellular ARV concentrations, including NRTI-triphosphates in PMBC, LN and GALT. • Pharmacokinetic data suggest drug-specificcompartmentalization 105 104 104 103 103 EFV (ng/mL) 102 ATV (ng/mL) 102 101 101 1 1 PBMC LN Ileal Rectal PBMC LN Ileal Rectal Fletcher #108

  19. Viral replication in pharmacologicsanctuaries • Hypothesis: inadequate drug penetration may contributetocrypticreplicationof HIV, whichsustains HIV infection. • Mechanisms: • Physiochemicalcharacteristics • Susceptibilityforanddistributionofinfluxandeffluxtransporters(Minuesa #590; P-gp in primary CD4+ T cellsand raltegravir intracellularconcentrations) • Drug metabolizingenzymes • Expressionofphosphorylatingenzymes • Activationstateofthecompartment • Host genetictraits. Fletcher #108

  20. Reservoirs, does time oftreatmentinitiationmatter? • Treatmentofearly HIV infection reduces viral reservoirtolevelsfound in elite controllers Chronic Total HIV-1 DNA Acute Elite C Acute Chronic Buzon et al. #151

  21. Newchemotherapyagainstlatency • Transcriptional activators: reactivationofthelatent HIV-1 withoutinducing T cellactivation

  22. ChromatinReconfigurationforPurging HIV-1 fromtheLatentReservoir Johnstone, Nat Rev Drug Discov, 2002

  23. VorinostatDisrupts HIV-1 Latency in Patientson ART • n=5; CD4 count 562 cells/µL; 4 yearsof ART; 400 mg single doseofvoronistat; no AE • 👍 PBMC cellularhistoneacetylation • 👍HIV RNA levelsincreasedsignificantly (5-fold) in poolsofresting CD4+ cellsobtainedafter SAHA dosingcomparedto BL • Proof-of-conceptfor HDAC inhibitors as a therapeuticclasstodirectlyattackandpotentiallyeradicatelatent HIV infection • No demonstrationof viral production • Similar studysuggested in NHP without data (Lifson #107) Archin #157LB

  24. Autologous ZFN CCR5-disrupted CD4+ T cells June #155

  25. 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. Summary

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