A murine viral outgrowth assay to detect HIV in patients with undetectable viral loads
Kelly Metcalf Pate1; Chris Pohlmeyer2; Victoria Walker-Sperling2;Jeremy Foote1; Kevin Najarro1; Catherine Cryer1,3; Maria Salgado2,4;Lucio Gama1; Elizabeth Engle1; Erin Shirk1; Suzanne Queen1;Stanley Chioma2; Meghan Vermillion1; Brandon Bullock1; Ming Li1;Claire Lyons1,5; Robert Adams1; Chris Zink1; Janice Clements1;Joseph Mankowski1and Joel Blankson21Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, United States.2Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, United States.3School of Veterinary Medicine, University of Pennsylvania, Philadelphia, United States.4InstitueIrsiCaixa, Universitat Auto`noma de Barcelona, Badalona, Spain.5Cummings School of Veterinary Medicine, Tufts University, North Grafton, United States.
Presenting author Kelly Metcalf Pate email:
Background: Sensitive assays are needed for detection of residual HIV in patients with undetectable plasma viral loads to determine if eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir, while sensitive PCR-based assays lack the ability to distinguish replication-competent from the defective virus. We sought to determine whether xenograft of leukocytes from HIV-1 infected patients with undetectable plasma viral loads into severely immuno-compromised mice would result in viral amplification and measurable viral loads within the aberrant murine host.
Methods: We evaluated whether xenograft of 1) peripheral blood mononuclear cells (PBMCs) from five HIV-1patients on suppressive antiretroviral therapy (ART), 2) PBMCs or purified resting CD4T cells from 5 HIV-1elite suppressors (ES), or 3) PBMCs from a Simian Immunodeficiency Virus (SIV)pigtailed macaque on suppressive ART, all with undetectable plasma viral loads, into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice resulted in viral amplification in the mouse. Successful xenograft of mice was confirmed by flow cytometry. Human CD8T cells were depleted in humanized mice with depleting antibody, and CD4T cells were activated in a subset of mice with activating anti-CD3. Plasma viral loads in xenografted mice were quantified using qRT-PCR, and compared to plasma viral load and QVOA results from the human or macaque donor.
Results: With this murine viral outgrowth assay (MVOA), we amplifiedHIV-1 from all 10 HIV subjects with undetectable plasma viral load, including an ES from whom we were unable to recover virus by QVOA. We detected HIV in mice an average of 20 days after xenograft with PBMCs from patients on suppressive ART, and an average of 28 days after xenograft with PBMCs or resting CD4T cells from ES. For two of the mice xenografted with CD4T cells from ES, we detected HIV only after activation with anti-CD3. We similarly detected SIV inmacaquized mice by seven days post-xenograft.
Conclusions: The MVOA has the potential to serve as a powerful tool to identify residual HIV-1 in patients with undetectable viral loads, such as those who have undergone promising cure therapies