Anti-retroviral therapy (ART) suppresses viral replication and restores immune function in HIV patients. However, cessation of treatment results in viral rebound from persistent proviruses in latently infected cells. A recent clinical trial investigated the ability of the latency-reversing agent romidepsin to increase HIV-1 transcription as part of an approach to clear persistent proviruses. The administration of romidepsin once weekly for three consecutive weeks to individuals on suppressive ART revealed quantifiable increases of intracellular and plasma HIV-1 RNA in 5 of 6 participants which coincided with the romidepsin infusions. However, the origin of the romidepsin-induced plasma HIV-1 RNA is unknown. To address this, we compared intracellular HIV-1 DNA and RNA sequences from peripheral blood CD4+ T cells to HIV-1 RNA sequences obtained from the plasma during romidepsin treatment. CD4+ T-cells were obtained at baseline, following the second and third romidepsin infusion, and 10 weeks after the final romidepsin treatment. Plasma was collected 24 and 72 h following each romidepsin infusion. Single-genome sequencing of the env region was used to genetically characterize the virus from intracellular proviral DNA, the transcribed intracellular HIV-1 RNA as well as the plasma RNA pool and MEGA 6.0 was used to perform phylogenetic analysis. The intracellular HIV-1 DNA and RNA sequences obtained during romidepsin therapy contained a mean of 13.5% and 36% defective sequences, respectively. However, 8% defective sequences were found in plasma-derived HIV-1 RNA. Plasma-derived RNA and intracellular HIV-1 DNA and RNA sequences intermingled throughout the phylogenetic tree. In one participant, we identified one plasma-derived HIV-1 RNA sequence identical to, and another highly similar (>99.7%) to, intracellular HIV-1 DNA sequences. Another participant had 16 plasma-derived HIV-1 RNA sequences that were >99.7% similar to intracellular HIV-1 RNA or DNA sequences. One of these plasma sequences was identical to both intracellular RNA and DNA sequences. The plasma-derived HIV-1 RNA sequences in this participant also contained three large clonal populations. Our findings demonstrate that romidepsin induced transcription from proviruses in peripheral blood cells, which contributed to viremia in patients on suppressive ART. Intermingling of plasma-derived HIV-1 RNA sequences with intracellular HIV-1 RNA and DNA sequences indicates activation of multiple infected cells. In one participant the clonal plasma HIV-1 RNA sequences indicated that a subset of transcriptionally activated proviruses contributed to the majority of viremia. Therefore, HIV-infected cells in the blood are important reservoirs of HIV-1 during effective therapy and harbor proviruses capable of contributing to viremia during romidepsin therapy.
Contributor Information
A. Winckelmann, Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark; The Westmead Institute for Medical Research, University of Sydney, Westmead, Australia.
K. Barton, The Westmead Institute for Medical Research, University of Sydney, Westmead, Australia.
B. Hiener, The Westmead Institute for Medical Research, University of Sydney, Westmead, Australia.
W. Shao, Leidos Biomedical Research, Inc., Advanced Biomedical Computing Center, Reston, VA, USA; Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
L. Østergaard, Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark.
T. Rasmussen, Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark.
O. Søgaard, Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark.
M. Tolstrup, Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark.
S. Palmer, The Westmead Institute for Medical Research, University of Sydney, Westmead, Australia.