Validation of the SCID-hu Thy/Liv mouse model with four classes of licensed antiretrovirals

Abstract

Background: The SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals.

Methodology/principal findings: Endpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug-resistance substitutions.

Conclusion: Given the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans.

Figure 1. Implant viral loads in untreated- and 3TC-treated SCID-hu Thy/Liv mice are highly reproducible, and 3TC treatment after HIV-1 inoculation is nearly as effective as prophylactic treatment.

A, HIV-1 RNA in Thy/Liv implants of untreated mice and mice treated by twice-daily oral gavage with 3TC at 30 mg/kg per day beginning 1 day before inoculation with NL4-3 (means±SEM) Each experiment was performed in a mouse cohort made with tissues from a single donor. B, Implant p24 for the same groups as in panel A. C, Implant HIV-1 RNA in mice treated with 3TC beginning on day −1, day +1, day +3, and day +7 with respect to inoculation with the indicated HIV-1 strains. D, Implant p24 for the same groups as in panel C. Implants were collected 21 days after inoculation for NL4-3, 14 days for JD, and 42 days for Ba-L. *p≤0.05, compared with untreated mice for 5–7 mice per group.

Figure 2. 3TC treatment of HIV-1 JD-infected SCID-hu Thy/Liv mice inhibits viral replication and protects implants from virus-mediated thymocyte depletion.

Mice were treated by twice-daily oral gavage with 3TC at 300 mg/kg per day beginning on day +1 after inoculation, and Thy/Liv implants were collected 14, 21, and 28 days after inoculation. Antiviral efficacy was assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24⁺ thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio (H) for 3TC-treated mice versus untreated mice (means±SEM). *p≤0.05, compared with untreated mice for 5–7 mice per group.

Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion.

Flow cytometric analysis of representative implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after inoculation).

Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in SCID-hu Thy/Liv mice.

Mice were treated by twice-daily oral gavage with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day –1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means±SEM). *p≤0.05, compared with untreated mice for the number of mice indicated under each bar.

Figure 5. Efavirenz is more potent than nevirapine against HIV-1 NL4-3 in SCID-hu Thy/Liv mice.

Mice were treated by twice-daily oral gavage with nevirapine or efavirenz at the indicated dosage levels beginning on day −1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means±SEM). *p≤0.05, compared with untreated mice for the number of mice indicated under each bar.

Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4-3 in SCID-hu Thy/Liv mice.

Mice were treated by twice-daily oral gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per day beginning on day −1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means±SEM). *p≤0.05, compared with untreated mice for the number of mice indicated under each bar.

Figure 7. T-20 causes dose-dependent reductions in viral load in HIV-1 NL4-3 D36G-infected SCID-hu Thy/Liv mice.

Mice were treated by twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg per day beginning on day −1. Antiviral efficacy was assessed by determining cell-associated HIV-1 RNA and p24. Data are expressed as means±SEM; *p≤0.05 for treated mice versus untreated mice by the Mann-Whitney U test for the number of mice indicated under each bar.

Figure 8. Multidrug-resistant (MDR) NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 in SCID-hu Thy/Liv mice.

Viral replication assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24⁺ thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio (H) for NL4-3 versus MDR NY index case HIV-1-infected mice (means±SEM). *p≤0.05, compared with untreated mice for 7 mice per group.