Research Article: An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

Date Published: December 26, 2013

Publisher: Public Library of Science

Author(s): Celsa A. Spina, Jenny Anderson, Nancie M. Archin, Alberto Bosque, Jonathan Chan, Marylinda Famiglietti, Warner C. Greene, Angela Kashuba, Sharon R. Lewin, David M. Margolis, Matthew Mau, Debbie Ruelas, Suha Saleh, Kotaro Shirakawa, Robert F. Siliciano, Akul Singhania, Paula C. Soto, Valeri H. Terry, Eric Verdin, Christopher Woelk, Stacey Wooden, Sifei Xing, Vicente Planelles, Michael Emerman.

http://doi.org/10.1371/journal.ppat.1003834

Abstract

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

Partial Text

The possibility to achieve HIV eradication has been limited, at least in part, by the existence of latently infected cellular reservoirs [1]–[3]. The major known cellular reservoir is established in quiescent memory CD4+ T cells, providing an extremely long-lived set of cells in which the virus can remain transcriptionally silent [1]–[3]. Reactivation of latent viruses followed by killing of the infected cells has been proposed as a possible strategy (“shock and kill”) to purge the latent reservoir [4].

Thirteen stimuli shown in Table 2 were chosen on the basis of their known or proposed activities in reactivating latent HIV-1 in various systems. For primary cell assays, experiments were performed with cells from three different donors, and replicate samples (duplicate or triplicate) were used for each treatment variable tested (refer to Methods Section for details). For J-Lat clones, experiments were performed in triplicate for each clone. Figure 1, panels A and B depict the average responses (mean +/− SEM) obtained with each of the cell models and the patient cell outgrowth assay. In all cases, the stimulus providing maximal reactivation response was used as a reference and assigned a 100% value, and the results from all other stimuli were normalized as a percentage of the maximal response (Figure 1). Within each individual experiment (e.g., donor cells), the untreated baseline value was first subtracted from each treatment response value, prior to the normalization step. The average relative response was then calculated across all experiments (donors) for each stimulus tested. While transformed and primary cell models could be tested at three concentrations of each stimulus, assays with patient cells/QVOA were only performed at a single drug concentration due to limiting cell numbers. Therefore, two comparisons were performed: one which included all concentration points for each drug, and did not include patient cell assay data; and a second one in which a single concentration point was considered, to provide an analysis that could include patient cell results.

This study represents the first experimental comparison among several broadly used HIV latency systems, including primary cell models, transformed cell lines and patient-derived cells. To establish these comparisons in an unbiased manner, we chose a panel of known stimuli that were tested in parallel in the selected cell models. The methodology was designed to circumvent variations due to batch, formulation or concentration differences in the compounds tested. To the extent possible, the duration of exposure to each stimulus, the inclusion of appropriate controls and the maximal-response stimulus were standardized as well.

 

Source:

http://doi.org/10.1371/journal.ppat.1003834

 

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