Characterization of HIV-1 RNA and DNA during long-term suppressive therapy

Human immunodeficiency virus (HIV) is the virus causing acquired immune deficiency syndrome (AIDS). Since the discovery of HIV/AIDS over three decades ago, this disease has claimed millions of lives. One of the major accomplishments of modern history came in 1996, when combination antiretroviral therapy (cART) was introduced. Treatment with cART caused the death rates from AIDS to decrease dramatically. Although current cART is effective in suppressing HIV type 1 (HIV-1) it is not curative and therefore meticulous lifelong therapy is necessary. To effectively target HIV-1 persistence with the goal of achieving a cure, it will be important to determine the source and dynamics of persistent viremia. In the work presented in this thesis we compare the different approaches for measuring the persistent HIV-1 reservoir. We also use highly sensitive assays to genetically characterize intracellular HIV-1 within a broad spectrum of cells sorted from unique tissue samples from patients on long-term suppressive cART. In paper I we compare eleven different approaches for quantifying persistent HIV-1. Results from this study showed major differences among the assays. The viral outgrowth assay, which is a culture-based assay that quantifies replication competent virus, resulted in measurements of replication competent virus that were at least 300-fold lower compared to PCR-based methods which measured total and/or integrated HIV-1 DNA. The differences between these methods may reflect the number of defective viral genomes in cells. Overall, the study reveals many difficulties in measuring the latent reservoir and shows that there is currently no assay that will accurately measure the latent reservoir during clinical trials of curative strategies. In papers II-IV we genetically characterized intracellular HIV-1 DNA within a broad spectrum of cells sorted from different anatomical compartments of eight patients on longterm cART. In paper II we investigated whether CD34 + hematopoietic progenitor cells (HPCs) from the bone marrow serve as an HIV-1 reservoir. In this study we did not detect HIV-1 DNA in CD34 + HPCs indicating that this cell type is not a source of persistent HIV-1. In papers III and IV we genetically characterized intracellular HIV-1 in different cell types from peripheral blood, gut-associated lymphoid tissue (GALT) and lymph node tissue. We found that the majority of HIV-1 DNA was detected in memory CD4 + T cells and that participants who initiate therapy during early infection have a lower intracellular HIV-1 infection frequency. These results imply that despite several years of therapy, memory CD4 + T cells serve as an important reservoir and that early initiation of therapy results in a smaller latent reservoir. In paper III we used phylogenetic analyses to study the genetic evolution of HIV-1 between samples isolated before initiation of therapy and several years after suppressive therapy. Our studies revealed a lack of substantial HIV-1 genetic evolution during cART which strongly suggests that ongoing replication is not a major cause of viral persistence in memory T cells. In paper IV we evaluated the longitudinal stability of the HIV-1 reservoir and the role of cellular proliferation in maintaining persistent HIV-1 during cART. Our results show that memory T cells retained a relatively constant HIV-1 DNA integrant pool that was genetically stable during long-term cART. These DNA integrants appear to be maintained by cellular proliferation and longevity of infected cells, rather than by ongoing viral replication. In conclusion, the work presented in this thesis has helped us to gain a fuller appreciation for the range of cells and tissues containing HIV-1 DNA in patients on long-term cART and a better understanding as to how intracellular HIV-1 DNA is maintained in different tissues. LIST OF SCIENTIFIC PAPERS I. S. Eriksson, E.H. Graf, V. Dahl, M.C. Strain, S.A. Yukl, E.S. Lysenko, R.J. Bosch, J. Lai, S. Chioma, F. Emad, M. Abdel-Mohsen, R. Hoh, F. Hecht, P. Hunt, M. Somsouk, J. Wong, R. Johnston, R.F. Siliciano, D.D. Richman, U. O'Doherty, S. Palmer, S.G. Deeks, J.D. Siliciano. Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies. PLoS Pathog. 2013;9(2)e1003174. II. L. Josefsson, S. Eriksson, E. Sinclair, T. Ho, M. Killian, L. Epling, W. Shao, B. Lewis, P. Bacchetti, L. Loeb, J. Custer, L. Poole, F.M. Hecht, S. Palmer. Hematopoietic Precursor Cells Isolated from Patients on Long Term Suppressive HIV Therapy Did Not Contain HIV-1 DNA. J Infect Dis. 2012;206:28-34. III. L. Josefsson, S. von Stockenstrom, NR. Faria, E. Sinclair, P. Bacchetti, T. Ho, M. Killian, L. Epling, A. Tan, P. Lemey, W. Shao, P. Hunt, M. Somsouk, W. Wylie, D. Douek, L. Loeb, J. Custer, L.Poole, S. Deeks, F. M. Hecht and S. Palmer. The HIV-1 Reservoir in Eight Patients on Long-term Suppressive Antiretroviral Therapy is Stable With Few Genetic Changes Over Time. Proc Natl Acad Sci U S A. 2013;17:110-151. IV. S. von Stockenstrom, L. Odevall, E. Lee, E. Sinclair, P. Bacchetti, M. Killian, L. Epling, W. Shao, R. Hoh, T. Ho, N.R. Faria, P. Lemey, J. Albert, P. Hunt, L. Loeb, C. Pilcher, L. Poole, H. Hatano, M. Somsouk, D. Douek, E. Boritz, S.G. Deeks, F.M. Hecht and S. Palmer. Longitudinal Genetic Characterization Reveals That Cell Proliferation Maintains a Persistent HIV Type 1 DNA Pool During Effective HIV Therapy. J Infect Dis. 2015;212:596607.