There is renewed interest in testing strategies to eradicate HIV. A preclinical model for testing potential eradication approaches avoids exposing patients to ineffective and potentially harmful agents. Moreover, an ex vivo model might allow us to characterize why the reservoir is resistant to clearance. We propose a new approach to evaluate the effect of potential therapies on CTL clearance of HIV ex vivo. This model represents an innovative approach to reservoir characterization because it uses CTL clearance as an indicator of reservoir protein expression, allowing us to take advantage of the sensitivity of the immune system to measure low-level protein expression. Our ex vivo model uses cells from ART patients to study the effectiveness of a candidate anti-reservoir therapy: interferon alpha (IFN-a). We find that patient-derived CD4+ T cells show a drop in levels of integrated HIV DNA after treatment with IFN-a and subsequent coculture with Gag- primed CD8+T cells. Using this system we will characterize the proviruses that are resistant to ex vivo clearance by sequence analysis (Aim 1) and expression studies (Aim 2). Using samples from an ongoing IFN-a trial as a proof of principal, we will test whether our ex vivo model can predict which patients respond to IFN-a therapy in vivo (Aim 3). Finally, we will study the proviral clones that are resistant to clearance to understand if lack of RNA or protein expression may explain lack of clearance (Aim 4). We also will determine whether repeat stimulation or combination stimulation leads to enhanced reservoir clearance in coculture. We hypothesize that the fraction of proviral DNA that can be induced to express HIV proteins (and is therefore susceptible to clearance) is higher than previously appreciated. If our hypothesis is correct, then replication defective viruses are often capable of expressing viral protein and both replication competent and defective viruses will be cleared in our assay. To test our hypothesis, we will determine whether the protein-expressing cells in our system contain replication competent proviruses. Notably, as long as replication competent viruses are cleared along with replication defective proviruses, then our approach will likely have utility in identifying effective therapies.

Public Health Relevance

HIV is incurable because a treatment resistant reservoir persists that is largely not expressed and invisible to the immune system. Herein, we propose an ex vivo model to test the effectiveness of therapies designed to enhance reservoir visibility. In this model we detect reservoir visibility by measuring clearance of cells with integrated HIV DNA as a sensitive marker of reservoir expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI116216-01
Application #
8842406
Study Section
Special Emphasis Panel (ZRG1-AARR-K (51))
Program Officer
Lawrence, Diane M
Project Start
2014-09-19
Project End
2016-08-31
Budget Start
2014-09-19
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
$220,000
Indirect Cost
$82,500
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104