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dc.contributor.advisorSiliciano, Robert F.en_US
dc.contributor.authorBullen, Cynthia K.en_US
dc.date.accessioned2015-02-11T04:05:24Z
dc.date.available2015-02-11T04:05:24Z
dc.date.created2014-12en_US
dc.date.issued2014-09-18en_US
dc.date.submittedDecember 2014en_US
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/37200
dc.description.abstractHighly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels in infected individuals to below the limit of detection of clinical assays (<50 copies HIV-1 RNA per ml of plasma). Despite this, antiretroviral therapy (ART) is not curative as HIV-1 establishes a state of latent infection in resting memory CD4+ T cells. This small but stable reservoir is a major barrier to HIV-1 eradication. Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects. Initial strategies to reactivate latent HIV-1 through nonspecific global T cell activation proved to have unacceptable toxicity, precluding its clinical use. This has fueled the search for small molecule latency–reversing agents (LRAs) that do not induce functional T cell activation and cytokine release. Using an in vitro model of HIV-1 latency in human primary CD4+ T cells, we demonstrate that the FDA-approved drug disulfiram reactivates latent HIV-1 without causing global T cell activation. However, the effects of disulfiram and other putative latency reversing agents on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo. Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually. In a broad comparative analysis using CD4+ T cells from infected individuals, we have shown for the first time that selected synergistic drug combinations can reverse latency ex vivo to levels approaching that seen with T cell activation. Protein kinase C (PKC) agonists combined with histone deacetylase (HDAC) inhibitors or JQ1 robustly induce HIV-1 transcription and virus production with efficacy similar to the maximum of T cell activation without causing the release of proinflammatory cytokines. We then apply a mathematical model that estimates in vivo viral load changes from ex vivo measurements of virus production. Our study reconciles previous in vitro and clinical studies and provides a framework to identify effective drug combinations that achieve the high levels of HIV-1 latency reversal likely required for a cure.en_US
dc.format.mimetypeapplication/pdfen_US
dc.languageen
dc.publisherJohns Hopkins University
dc.subjectHIV Cureen_US
dc.subjectLatent HIVen_US
dc.subjectHIV Persistenceen_US
dc.subjectLatency-Reversing Agentsen_US
dc.subjectShock and Killen_US
dc.titlePharmacologic Stratagies Toward Curing HIV-1 Infection: Identification and Evaluation of Small-Molecule HIV-1 Latency Reversing Agentsen_US
dc.typeThesisen_US
thesis.degree.disciplineBiologyen_US
thesis.degree.grantorJohns Hopkins Universityen_US
thesis.degree.grantorSchool of Medicineen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePh.D.en_US
dc.type.materialtexten_US
thesis.degree.departmentBiochemistry, Cellular and Molecular Biologyen_US
dc.contributor.committeeMemberBlankson, Joel N.en_US
dc.contributor.committeeMemberZachara, Natasha E.en_US
dc.contributor.committeeMemberLiu, Jun O.en_US


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