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Residual Viremia Caused by Clonally Expanded Tumor-Infiltrating CD4+ Cells
Francesco R. Simonetti1, Michele D. Sobolowski2, Shawn Hill1, Wei Shao3, Elizabeth Fyne2, Xiaolin Wu1, John M. Coffin4, Stephen H. Hughes1, John W. Mellors2, Frank Maldarelli1
1 HIV Drug Resistance Program, National Cancer Institute (NCI), Frederick, MD, United States. 2 Division of Infectious Diseases, University of Pittsburgh, Pittsburg, PA, United States. 3 Advanced BIomedical Computing Center, Frederick National Laboratory for Cancer Research, Frederick, MD, United States. 4 Department of Molecular Biology and Microbiology, Tufts University, Boston, MA, United States.
Background: Clonal expansion of infected cells can contribute to HIV-1 persistence. We recently reported that a clonally-expanded population of HIV-infected cells was responsible for persistent viremia despite ART in a patient with metastatic squamous cell carcinoma (Maldarelli, et al., 2014). We conducted extensive ante- and post-mortem analysis of HIV-infected cells from this patient to investigate the organ and tumor distribution of this clonal variant.
Methods: Plasma and peripheral blood mononuclear cells (PBMC) were obtained ante mortem, and samples from spleen, ileum, rectum, 5 lymph nodes, and 5 tumor metastases were obtained at autopsy. Tissues were characterized by histo- and immunohistochemistry and qPCR of the genomic DNA was used to measure the levels of HIV gag sequences. A new single genome sequencing assay was performed to amplify the 800bp nef-U3 region from blood and tissues. Integration site flanking sequences were used to PCR amplify the full length of expanded proviruses. An ex-vivo infectious virus recovery assay was developed to characterize inducible HIV variants from PBMC-derived CD4+ cells. Sequences were subjected to phylogenetic and statistical analyses.
Results: HIV sequences (N=317) were recovered from plasma, PBMC, spleen, lymph nodes, and tumor tissues, which were infiltrated with both CD4+ and CD8+ T cells. In general, HIV variants were well mixed across blood and tissues and there was no evidence of localized replication. One provirus from a highly amplified clone (AMB-1), known to produce the majority of the HIV RNA detected in plasma, was enriched in tumor tissues. AMB-1 proviruses were present in each tumor metastasis, with an overall abundance in tumor tissue significantly (3.5-fold) greater than that detected in lymphoid tissues (p=0.0005). The full length AMB-1 sequence revealed an intact provirus and no drug resistance mutations. AMB-1 RNA was recovered in day 7 culture supernatants after stimulation (PHA, irradiated blasts) and co-culture of CD4+ T-cells with CD8-depleted allogeneic blasts but declined with continued culture associated temporally with outgrowth of other variants.
Conclusions: This study is the first report of residual viremia caused by an expanded cell carrying a specific intact HIV provirus. Cells from this clone accumulated specifically in cancer metastases. These observations suggest that immune stimuli, like tumor antigens, can contribute to cell expansion, and perhaps to the activation of the provirus and release of virions into plasma.