Highly active antiretroviral therapy (HAART) effectively reduces HIV-1 replication, but the early optimism for viral eradication through prolonged treatment with these drugs has not been met. This is largely due to the fact that HIV-1 can cause a latent infection of host cells that develops when infected, activated cells revert back to a quiescent state. These latently infected cells represent a transcriptionally silent reservoir for HIV-1 infection that is resistant to currently available antiretroviral drugs and HIV-1-specific immune responses. The prolonged half life of latently infected cells (approximately 44 months), combined with their ability to rapidly initiate virological rebound after discontinuation of HAART, are the main factors responsible for viral long-term persistence. These characteristics are in sharp contrast to lytically infected cells, which represent a highly activated, short-lived cell population with high viral production that can be effectively suppressed by antiretroviral drugs and HIV-1-specific CD8+ T cells. Understanding the molecular mechanisms that govern the maintenance, survival, turnover and HIV-1 gene expression in latently and lytically infected cells would open up novel perspectives for developing specific therapeutic options to target cells with persistent viral replication or latent HIV-1 infection, and therefore represent one of the highest priority topics in current HIV-1 research. The major technological obstacle to characterizing latently infected cells in sufficient detail is our current inability to identify and physically isolate these cells in an efficient and accurate manner. In this proposal, we address this key issue by proposing a novel microengraving technology that allows unprecedented high-throughput testing of the infection status of individual primary cells. For this purpose, we will use a unique biochip that allows for the physical separation of individual mononuclear cells into more than ~105-106 wells with subnanoliter volumes. Protein microarrays printed from the supernatant of each individual cell and their subsequent interrogation with gp120 antibodies will then be used to detect cells with active viral production on a single cell level. By selectively subjecting primary activated cells or quiescent cells following ex vivo activation to this assay, we will be able to identify, physically isolate, and expand lytically and latently HIV-1 infected cells, and to study their key biological properties, including their TCR repertoire and clonotypic composition, their phenotypic characteristics, and the molecular mechanisms that determine their growth, proliferation and long-term maintenance. Moreover, applying this novel, multiplexed technology to the analysis of mononuclear cells from peripheral blood and tissue specimens in individuals with different rates of HIV-1 disease progression will allow us to determine specific quantitative or qualitative characteristics of the lytic or latent HIV-1 reservoir associated with distinct clinical outcomes of HIV-1 infection. This project is a collaboration between the Love lab (MIT) with expertise in engineering of single-cell biochip assays and the Yu lab (Ragon Institute/MGH) with expertise in the immunology and virology of HIV. This interdisciplinary approach to one of the most significant problems in HIV-1 research has the potential to result in novel strategies to target latently infected cells, and thus to cure HIV-1 entirely.

Public Health Relevance

Quiescent CD4 T cells with latent HIV-1 infection represent the main reason for viral persistence and our inability to cure HIV-1 infection. In this proposal, we will use a novel, multiplexed bio-chip analysis system that will allow to identify, isolate and characterize these latently infected cells, and to contribute to the development of specific therapeutic strategies to target them.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI089339-01A1
Application #
8012425
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Salzwedel, Karl D
Project Start
2010-09-15
Project End
2014-08-30
Budget Start
2010-09-15
Budget End
2011-08-31
Support Year
1
Fiscal Year
2010
Total Cost
$426,909
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Martin-Gayo, Enrique; Cole, Michael B; Kolb, Kellie E et al. (2018) A Reproducibility-Based Computational Framework Identifies an Inducible, Enhanced Antiviral State in Dendritic Cells from HIV-1 Elite Controllers. Genome Biol 19:10
Sun, Xiaoming; Hua, Stephane; Chen, Hsiao-Rong et al. (2017) Transcriptional Changes during Naturally Acquired Zika Virus Infection Render Dendritic Cells Highly Conducive to Viral Replication. Cell Rep 21:3471-3482
Viganò, Selena; Negrón, Jordi J; Tse, Samantha et al. (2017) HLA-G+ HIV-1-specific CD8+ T cells are associated with HIV-1 immune control. AIDS 31:207-212
Sun, Hong; Kim, Dhohyung; Li, Xiaodong et al. (2015) Th1/17 Polarization of CD4 T Cells Supports HIV-1 Persistence during Antiretroviral Therapy. J Virol 89:11284-93
Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu et al. (2015) Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers. PLoS Pathog 11:e1004930
Vigano, Selena; Negron, Jordi; Ouyang, Zhengyu et al. (2015) Prolonged Antiretroviral Therapy Preserves HIV-1-Specific CD8 T Cells with Stem Cell-Like Properties. J Virol 89:7829-40
Leng, Jin; Ho, Hsin-Pin; Buzon, Maria J et al. (2014) A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4(+) T cells from elite controllers. Cell Host Microbe 15:717-728
Bashirova, Arman A; Martin-Gayo, Enrique; Jones, Des C et al. (2014) LILRB2 interaction with HLA class I correlates with control of HIV-1 infection. PLoS Genet 10:e1004196
Sun, Hong; Buzon, Maria J; Shaw, Amy et al. (2014) Hepatitis C therapy with interferon-? and ribavirin reduces CD4 T-cell-associated HIV-1 DNA in HIV-1/hepatitis C virus-coinfected patients. J Infect Dis 209:1315-20
Buzon, Maria J; Martin-Gayo, Enrique; Pereyra, Florencia et al. (2014) Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells. J Virol 88:10056-65

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