HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy. The main challenge in achieving a cure for HIV-1 infection is the persistence of these latent viral reservoirs. Assays that allow for identification, characterization, and isolation of latently infected single cells fo downstream genomic sequencing are needed to efficiently and fully characterize HIV-1 reservoirs. However, existing assays that analyze latently-infected cells require burdensome and costly serial cell dilutions. Other proposed methods to identify and analyze HIV-infected cells require significant investment in costly equipment and reagents and have not been adapted for downstream characterization of latent reservoirs. We propose to develop and validate an innovative and novel assay using microfluidic methods with PCR for high-throughput identification, enumeration, and isolation and downstream characterization of viral genomes from latently-infected human cells. The application of our approach will be particular useful in the analysis of samples from patients on combination antiretroviral therapy and/or in studies of novel modalities of reservoir eradication.
Specific aims i nclude: 1) develop and test the efficacy of the proposed method to identify and enumerate latently-infected human PBMCs using microfluidic methods and PCR, and, 2) validate our assay to isolate single-cell droplets with integrated HIV-1 DNA for downstream sequencing and secondary target gene quantification. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs on the single-cell level in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our prior experiences in the detection and quantification of very low-levels of HIV-1 genetic material and in the development of microfluidic devices for viral and immune characterization will be crucial to the development of novel assays to identify and characterize HIV-infection at the single-cell level. The proposed method has the potential to be adapted for a wide variety of multidisciplinary research studies, such as single-cell characterization of viral and intracellular pathogens or analysis of stem cells and/or malignant tissues.

Public Health Relevance

We will design and devised a method that allows for high-throughput screening, enumeration and isolation of HIV-1 latently-infected human cells, followed by downstream characterization of viral genomes from these cells using innovative technological approaches. Our assay has the potential to significantly advance our understanding of the mechanisms of HIV-1 persistence and to be important for the development of novel HIV-1 curative strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
7R21AI110277-03
Application #
9129363
Study Section
Special Emphasis Panel (ZRG1-AARR-E (81))
Program Officer
Church, Elizabeth Stansell
Project Start
2014-06-01
Project End
2016-05-31
Budget Start
2015-09-01
Budget End
2016-05-31
Support Year
3
Fiscal Year
2015
Total Cost
$247,779
Indirect Cost
$52,404
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Liang, Li-Guo; Kong, Meng-Qi; Zhou, Sherry et al. (2017) An integrated double-filtration microfluidic device for isolation, enrichment and quantification of urinary extracellular vesicles for detection of bladder cancer. Sci Rep 7:46224
Liang, Li-Guo; Sheng, Ye-Feng; Zhou, Sherry et al. (2017) An Integrated Double-Filtration Microfluidic Device for Detection of Extracellular Vesicles from Urine for Bladder Cancer Diagnosis. Methods Mol Biol 1660:355-364
Yucha, Robert W; Hobbs, Kristen S; Hanhauser, Emily et al. (2017) High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay. EBioMedicine 20:217-229
Wang, ShuQi; Chinnasamy, Thiruppathiraja; Lifson, Mark A et al. (2016) Flexible Substrate-Based Devices for Point-of-Care Diagnostics. Trends Biotechnol 34:909-921
Lifson, Mark A; Ozen, Mehmet Ozgun; Inci, Fatih et al. (2016) Advances in biosensing strategies for HIV-1 detection, diagnosis, and therapeutic monitoring. Adv Drug Deliv Rev 103:90-104
El Assal, Rami; Gurkan, Umut A; Chen, Pu et al. (2016) 3-D Microwell Array System for Culturing Virus Infected Tumor Cells. Sci Rep 6:39144
Yildiz, U Hakan; Inci, Fatih; Wang, ShuQi et al. (2015) Recent advances in micro/nanotechnologies for global control of hepatitis B infection. Biotechnol Adv 33:178-190
Shafiee, Hadi; Wang, ShuQi; Inci, Fatih et al. (2015) Emerging technologies for point-of-care management of HIV infection. Annu Rev Med 66:387-405