ThelongtermgoalofthisapplicationistostudyHIV-1reservoirsthatpersistinCNSandtounderstand theroleofillicitdrugsinestablishingandreactivatingthelatentreservoirs.Persistenceoflatentlyinfectedcells inCNSposesamajorbarrierforHIV-1eradication.Currentstrategiestoeliminatethelatentreservoirsinclude a ?shock and kill? therapy and is aimed at peripheral blood, which constitutes only 1% of the total reservoirs. Whether it is possible to envision similar strategies for the eradication of HIV-infected CNS cells is currently unknown. In addition to the lack of knowledge about latency in CNS HIV+ cells and the effects of latency reversingagents,illicitdrugscommonwithintheHIV-infectedpopulationsconstituteafurthercomplexity.Many illicitdrugsareknowntostimulateHIV-1replication.Sincethecurrentmethodtomeasuretheperipheralblood HIVreservoirisnotapplicabletosolidtissuesortocellsthatreplicatepoorlysuchasmacrophages,microglia and astrocytes found in the brain. We have developed a novel, Single cell-single molecule, Multiplex, Immunofluorescence (IF) and RNA FISH-based Assay (SMIRA) to detect cells in which HIV-1 is actively replicating. Using automation, a large number of cells can be scanned. In this proposal, we will employ this novelmethodtofirstquantitateandcharacterizelatencyincelllinemodels,thenthelatentreservoirsinbrain derivedmicrogliaandastrocytesanddelineatetheeffectofthreedrugsofabuse(methamphetamine,cocaine and cannabis) on the efficiency of formation of latent cells, the rate of reactivation of latent cells, and establishmentoflatencyacrossblood-brain-barrier(BBB).
Three Aims areproposed.
In Aim1, wewillstudy theroleofillicitdrugsonthereactivationoflatentlyinfectedCNS-derivedcellsbyestablishinginvitroandex vivo models of latency. First, we will employ immortalized human monocyte and microglial cell lines to study the kinetics of reactivation and the effect of METH, Cocaine and cannabis on the reactivation using SMIRA. ThiswillbefollowedbysimilarstudiesusingaprimaryCNScells.
In Aim2, wewillstudytheroleofillicitdrugs ontheestablishmentofHIVreservoirsinCNScellsacrossBBB.UsingSMIRAtodetectHIV-1+cells,wewill determine the relative effect of drugs of abuse, antiretrovirals and the addition of LRAs to establish latency acrossBBB.
In Aim3, wewillstudytheeffectofillicitdrugsonCNSHIVreservoirsinHIV-infectedindividuals. Employing brain autopsy samples from HIV+ individuals with or without illicit drug use history (from the ManhattanBrainBank(NNTC),wewillcharacterizetherelativefrequencyofmicroglialandastrocytelatentcell reservoirs in which HIV-1 is actively replicating vs. those that are latent, using SMIRA and state-of-the-art, high-speed/resolutionwholeslidescanner,thePerkinElmerPannoramic250FlashII.Wewillstudytheeffect of drug abuse on the rates of reactivation. The proposed studies will provide valuable tools to study establishmentandreactivationofCNSlatencyinresponsetoillicitdrugsandprovidesadrugscreeningassay toeradicatetheselatentcellsinthefuture.

Public Health Relevance

ProjectRelevance AlthougheradicationofHIV/AIDSisanationalpriority,theabilityofHIVtohidelatently(forexamplein thebrain)posesaseverehurdle.Cellsinthebrainsuchasmicrogliaarepoorlystudiedandthereareno methods to study this reservoir. We have developed a novel single-??cell based, ultra-??sensitive assay that providesauniqueopportunitytostudyCNSreservoirsandeffectofillicitdrugsonthesereservoirs.Our studiesarelikelytohelpdevelopstrategiestoeradicateHIVreservoirsinthebrain.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA043169-02
Application #
9333307
Study Section
Special Emphasis Panel (ZDA1)
Program Officer
Lin, Yu
Project Start
2016-09-01
Project End
2021-07-31
Budget Start
2017-08-01
Budget End
2018-07-31
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine, Inc
Department
Type
DUNS #
079783367
City
Bronx
State
NY
Country
United States
Zip Code
10461