A Murine Viral Outgrowth Assay to Detect Residual HIV Type 1 in Patients With Undetectable Viral Loads

Abstract

Background: Sensitive assays are needed for detection of residual human immunodeficiency virus (HIV) in patients with undetectable plasma viral loads to determine whether eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir. We sought to determine whether xenograft of leukocytes from HIV type 1 (HIV)-infected patients with undetectable plasma viral loads into immunocompromised mice would result in viral amplification.

Methods: Peripheral blood mononuclear cells or purified CD4(+) T cells from HIV or simian immunodeficiency virus (SIV)-infected subjects with undetectable plasma viral loads were adoptively transferred into NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. The mice were monitored for viremia following depletion of human CD8(+) T cells to minimize antiviral activity. In some cases, humanized mice were also treated with activating anti-CD3 antibody.

Results: With this murine viral outgrowth assay (MVOA), we successfully amplified replication-competent HIV or SIV from all subjects tested, including 5 HIV-positive patients receiving suppressive antiretroviral therapy (ART) and 6 elite controllers or suppressors who were maintaining undetectable viral loads without ART, including an elite suppressor from whom we were unable to recover virus by QVOA.

Conclusions: Our results suggest that the MVOA has the potential to serve as a powerful tool to identify residual HIV in patients with undetectable viral loads.

Keywords: HIV; SIV; cure; humanized mouse; quantitative viral outgrowth assay (QVOA).

Figure 1.

Adoptive transfer of peripheral blood mononuclear cells (PBMCs) or CD4⁺ T cells from simian immunodeficiency virus (SIV)–infected macaques that were receiving antiretroviral therapy (ART) and had a history of undetectable viral loads into NSG mice results in amplification of SIV. Detection of virus with a murine viral outgrowth assay xenografted with PBMCs (A) or CD4⁺ T cells (B) from SIV-positive macaques over time following xenograft. Xenografted mouse plasma SIV loads are denoted by red circles, and circulating macaque CD4⁺ T cells in mouse are denoted by blue diamonds. Open circles represent the limit of detection for each sample in which no virus was detected.

Figure 2.

Adoptive transfer of peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)–infected patients who were receiving antiretroviral therapy (ART) and had a history of undetectable viral loads into NSG mice results in amplification of HIV type 1. Shown are viral loads and percentages of circulating human CD4⁺ T cells in representative mice with xenografted mouse plasma viral load (red circles) and circulating human CD4⁺ T cells in mouse blood (blue diamonds). Human CD8⁺ T cells were depleted by the murine viral outgrowth assay in all humanized mice as indicated (black arrows). Open circles represent the limit of detection for each sample in which no virus was detected.

Figure 3.

Adoptive transfer of peripheral blood mononuclear cells (PBMCs) or CD4⁺ T cells from human immunodeficiency virus (HIV)–infected elite suppressors with undetectable viral loads into NSG mice results in amplification of HIV type 1. Shown are viral loads and percentages of circulating human CD4⁺ T cells over time, following adoptive transfer, of CD4⁺ T cells (dashed lines) or PBMCs (solid lines) in representative mice with xenografted mouse plasma viral loads (red circles) and circulating human CD4⁺ T cells in mouse blood (blue diamonds). Human CD8⁺ T cells were depleted by the murine viral outgrowth assay as needed (black arrows). Three of 9 mice from which we were able to amplify virus from elite suppressors required anti-CD3 monoclonal antibody stimulation (yellow arrows) to amplify virus to detectable levels. Open circles represent the limit of detection for each sample in which no virus was detected. Abbreviations: IUPM, infectious units per million cells; QVOA, quantitative viral outgrowth assay.

Figure 4.

The murine viral outgrowth assay (MVOA) amplifies replication-competent human immunodeficiency virus (HIV) from peripheral blood mononuclear cells (PBMCs) or CD4⁺ T cells from HIV-positive patients with undetectable plasma viral loads. Schematic representation of xenograft of NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice with patient cells and subsequent detection of HIV in the murine plasma. Abbreviation: qRT-PCR, quantitative reverse transcription–polymerase chain reaction.