255b Molecular Characterization, Reactivation and Depletion of Latent HIV D H Hamer*1, D G Brooks2, P A Arlen2, L Gao2, G Bristol2, E A Berger3, J A Zack2 1NCI, Bethesda, MD; 2UCLA, Los Angeles, CA; and 3NIAID, Bethesda, MD
Background: A serious problem facing HIV infected patients is the inability of antiretroviral therapy to completely eliminate infection. Rebound of viral load following the cessation of therapy has been attributed largely to the persistence of a long-lived population of latently infected T cells. The scarcity of latently infected cells in vivo makes it difficult to characterize this reservoir. We took advantage of the high frequency with which latent virus is generated in the SCID-hu (Thy/Liv) mouse to quantitatively analyze viral RNA and protein production and to test the ability of combined inducer and immunotoxin treatment to reduce the size of the latent reservoir.
Methods: SCID-hu (Thy/Liv) mice were infected with the pathogenic reporter virus HIVNL-r-HSAS and a population enriched in latently infected cells was isolated based on low expression of the reporter. RNA was analyzed in individual cells by a novel single-cell, multiplex, real-time, reverse transcription polymerase chain reaction before and after treatment with inducers. An immunotoxin containing a dsFv directed against the conserved CD4-binding site of gp120 linked was used to selectively kill reactivated cells.
Results: Single cell analysis demonstrated that HIV in the latent state is
transcriptionally dormant; however, following reactivation there is a dramatic increase in viral expression, rendering the cells susceptible to the anti-HIV immunotoxin. Treatment with the immunotoxin in conjunction with agents that activate virus expression without inducing cell division (IL-7 or the non-tumor promoting phorbol ester prostratin) depleted the bulk of the latent reservoir and left uninfected cells able to respond to subsequent costimulation.
Conclusion: Ourr results show that activation of latent virus is required for targeting by antiviral agents and provide the basis for future therapeutic strategies to decrease or eradicate the latent reservoir.
