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HIV-1 Diversity and Tropism of Rebound Virus After Treatment Discontinuation
Maria M. Bednar1, Blake Hauser1, Jeffrey M. Jacobson2, Ian Frank4, Joseph J. Eron3, Ronald Swanstrom1
1 LCCC, University of North Carolina, Durham, NC, United States. 2 Drexel University College of Medicine, Philadelphia, PA, United States. 3 Division of Infectious Diseases, University of North Carolina, Chapel Hill, NC, United States. 4 University of Pennsylvania, Philadelphia, PA, United States.
Background: Suppressive antiviral therapy has not been successful in eliminating virus, as patients on suppressive therapy retain a persistent, measurable, stable low-level viremia (LLV), and people discontinuing therapy see a rebound of virus from a persistent reservoir, as seen in the ACTG study A5068. ACTG study A5068 was designed to determine the efficacy of structured treatment interruptions and/or vaccination with an ALVAC-HIV vector for controlling viral replication after therapy discontinuation. Arm A (n=24) received neither STI nor vaccination, virologic rebound was detectable 2-4 weeks after discontinuation of therapy, and reached a peak several weeks later. We have used single genome amplification (SGA) of the viral env gene to assess genetic diversity followed by cloning to examine the tropism of the rebound virus by performing tropism assays to determine if the virus was adapted to grow in T cells (requiring high levels of CD4) or adapted to grow in macrophages (able to enter cells with low levels of CD4).
Methods: Viral RNA was isolated from the first available blood plasma sample containing viral load >1000 copies/mL from subjects infected with subtype B HIV-1 who had stopped therapy (10 total). We performed SGA to isolate individual env gene amplicons, 51 of which were cloned (average of 4 amplicons per tree) and infectivity analyzed at varying CD4 levels using Affinofile cells, in order to determine the viral entry phenotype for CD4 usage.
Results: Phylogenetic analysis of the viral env genes showed low diversity in each subject, consistent with an initially clonal repopulation of the viral population during rebound. No evidence of macrophage-tropic virus was found meaning that all of the rebound env genes tested encoded proteins that required high levels of CD4 for efficient entry of pseudotyped virus into Affinofile cells.
Conclusions: The source of the rebound virus is poorly understood, with the current model assuming this virus comes from resting CD4+ T cells; however, other cell types could be the source of this virus. Our analysis further excludes a myeloid cell source, where virus was persistently infecting these cells prior to therapy. Such a virus would have adapted to be able to use low levels of CD4, and this type of virus was not detected as the rebound virus. While our analysis does not identify the cell type harboring the virus that initiated the rebound, it is consistent with a CD4+ T cell as the source of the virus.