A major hurdle in HIV cure research is the lack of robust high throughput assays to assess the character and size of the reservoir. This challenge is compounded by the need to distinguish between cells infected with replication competent and replication defective HIV. Objectives: We propose to distinguish replication competent from defective proviruses by counting the number of cells that contain HIV DNA that are capable of expressing HIV Gag+ and down regulating CD4. We achieve high throughput detection by adapting a new technology, called FAST (Fiber-optic Array Scanning Technology), to detect rare cells that express HIV Gag and down regulate CD4 (Gag+CD4dim cells). FAST is a proven technique that has been utilized to detect rare tumor cells in a high throughput fashion (e.g. ~1 tumor cell in 20 million normal cells in one minute). Our goal is to determine if FAST can be used to monitor therapies that target HIV reservoirs. Method: We first compare the FAST assay to a FACS sorting assay developed by Dr. O'Doherty to test if our FAST assay can specifically measure true Gag+CD4dim cells. In the FACS assay, Gag+CD4dim cells are sorted and the number of true positives is determined by measuring proviral DNA in the sorted population. Specificity is demonstrated by showing enrichment for HIV DNA only in the Gag+CD4dim and not in the Gagneg cells.
In Aim 1, we test the specificity of FAST to identify Gag+CD4dim by comparing it to the number quantified by FACs sorting in patients at baseline. FAST provides additional specificity by visualizing the characteristic punctate staining of internalized CD4.
In Aim 2, we compare FAST to FACS after stimulating negatively-selected CD4+ T cells.
In Aims 1 and 2, we will determine if the Gag+CD4dim cell phenotype distinguishes replication competent from defective HIV by sequencing provirus from the sorted cells after limiting dilution PCR. We expect that hyper mutated proviruses will not express Gag or any HIV proteins and proviruses with massive deletions will either be disabled to express Gag and/or the proteins responsible for CD4 down regulation. Thus, by selecting for Gag+CD4dim we expect to eliminate most proviruses that are defective.
In Aim 3, we correlate FAST measures of reservoir with QVOA and integration levels. Significance and relevance to public health: A high-throughput quantitative assay would allow more rapid evaluation of multiple therapies that target reservoirs. Our preliminary data suggest to us that the ability to express HIV Gag proteins and down regulate CD4 upon stimulation as in Aim 2 will be a strong correlate of replication competence. Moreover, we envision the utility of the assay will extend beyond reservoir measurement as it can be exploited to determine the dose response, efficacy and kinetics of multiple candidate therapies that are designed to induce HIV protein expression.

Public Health Relevance

In this application, we propose to develop a high throughput assay that can measure HIV reservoir expression and size by exploiting a new technology, called Fiber-optic Array Scanning Technology (or FAST), which is capable of scanning 20 million cells in 1 minute. We use this new tool to count rare cells that express HIV proteins after stimulating and staining patient cells ex vivo. We envision this new tool will allow more rigorous and rapid assessment of the efficacy of anti-latency drugs designed to induce reservoir expression and clearance.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZAI1-JRR-A (M1))
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Sharma, Opendra K
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University of Pennsylvania
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Lada, Steven M; Huang, Karissa; VanBelzen, D Jake et al. (2018) Quantitation of Integrated HIV Provirus by Pulsed-Field Gel Electrophoresis and Droplet Digital PCR. J Clin Microbiol 56:
Baxter, Amy E; O'Doherty, Una; Kaufmann, Daniel E (2018) Beyond the replication-competent HIV reservoir: transcription and translation-competent reservoirs. Retrovirology 15:18
Strongin, Zachary; Sharaf, Radwa; VanBelzen, D Jake et al. (2018) Effect of Short-Term Antiretroviral Therapy Interruption on Levels of Integrated HIV DNA. J Virol 92:
Natesampillai, Sekar; Cummins, Nathan W; Nie, Zilin et al. (2018) HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome. J Virol 92:
Villa, Carlos H; Porturas, Thomas; Sell, Mary et al. (2018) Rapid prediction of stem cell mobilization using volume and conductivity data from automated hematology analyzers. Transfusion 58:330-338
Pasternak, Alexander O; DeMaster, Laura K; Kootstra, Neeltje A et al. (2016) Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA. J Virol 90:1148-51
Imamichi, Hiromi; Dewar, Robin L; Adelsberger, Joseph W et al. (2016) Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy. Proc Natl Acad Sci U S A 113:8783-8
Pinzone, Marilia Rita; Graf, Erin; Lynch, Lindsay et al. (2016) Monitoring Integration over Time Supports a Role for Cytotoxic T Lymphocytes and Ongoing Replication as Determinants of Reservoir Size. J Virol 90:10436-10445
DeMaster, Laura K; Liu, Xiaohe; VanBelzen, D Jake et al. (2015) A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Therapy. J Virol 90:2165-79