Potent activity of the HIV-1 maturation inhibitor bevirimat in SCID-hu Thy/Liv mice

Abstract

Background: The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation.

Methods and findings: Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1.

Conclusions: These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness.

Figure 1. Bevirimat Potently Inhibits HIV-1 Replication and Protects Thymocytes from Virus-Mediated Depletion in Vivo.

SCID-hu Thy/Liv mice were treated with bevirimat by twice-daily oral gavage beginning 1 day before inoculation of implants with HIV-1 NL4-3, and dosing was continued until implant collection 21 days after inoculation. Antiviral efficacy was assessed by determining implant viral load (A) and protection from thymocyte depletion (B) for bevirimat-treated mice and mice treated with 10% hydroxypropyl-β-cyclodextrin vehicle alone. Mock-infected mice were not treated. Data are expressed as means±SEM; *p≤0.05 for bevirimat- or vehicle-treated mice versus untreated mice by the Mann-Whitney U test.

Figure 2. Mice have High Concentrations of Bevirimat in Plasma 0.5–1 h after Oral Administration.

Serial plasma samples were collected after the final dose from SCID-hu Thy/Liv mice that had been treated with 100 mg/kg per day for 21 days. Mean±SEM for four mice.

Figure 3. Postexposure Dosing of Bevirimat also Reduces Viral Load.

Mice were treated by twice-daily oral gavage with bevirimat at 100 mg/kg per day beginning 1 day before (−1), 1 day after (+1), or 3 days after (+3) virus inoculation of Thy/Liv implants with HIV-1 NL4-3 and JD. Dosing was continued until implant collection 14 days (JD) and 21 days (NL4-3) after inoculation. Antiviral efficacy was assessed by determining cell-associated HIV-1 RNA and p24. There was no reduction in viral load in bevirimat-treated mice inoculated with bevirimat-resistant SP1/A1V, whereas 3TC treatment (30 mg/kg per day by twice-daily oral gavage) was highly effective. Data are expressed as means±SEM; *p≤0.05 for treated mice versus untreated mice by the Mann-Whitney U test for 6–8 mice per group.

Figure 4. Bevirimat Inhibits p24 Release from the Capsid p25 Precursor in Human Thymocytes.

Thymocytes dispersed from NL4-3- and SP1/A1V-infected SCID-hu Thy/Liv implants were cultured in the presence of the indicated range of concentrations of bevirimat for 2 days. Purified virions was lysed and individual viral proteins were detected with HIV-1 Ig. Note the target-specific and dose-dependent reduction of CAp25 cleavage in the presence of bevirimat. The presence of the drug does not affect the stoichiometry of the precursor Pr55Gag polyprotein nor does it inhibit cleavage of the matrix protein, MAp17 (lower panel, longer exposure).

Figure 5. The A1V Mutation in SP1 Does Not Affect Kinetics of Viral Replication or Thymocyte Depletion.

Bevirimat-resistant SP1/A1V replicates and depletes thymocytes with kinetics comparable to wild-type NL4-3 in SCID-hu Thy/Liv mice. Viral replication was assessed by determining implant viral load (A), and thymocyte depletion was assessed by total implant cellularity, thymocyte viability, and CD4/CD8 ratio (B) for NL4-3-infected versus SP1/AIV-infected mice for 6 mice per group. Data are expressed as means±SEM; there were no statistically significant differences in viral load or thymocyte depletion at any of the three time points. The number in each graph is the proportion of SP1/A1V to NL4-3 in area under the curve.

Figure 6. In Vivo Competitions Reveal No Impairment of SP1/A1V Replication.

Sequencing of CA-SP1 RNA from three SCID-hu Thy/Liv mice coinfected with equivalent infectious units of NL4-3 and SP1/A1V reveal no impairment of SP1/A1V replication 28 days after implant inoculation. The A-to-V substitution in SP1 is conferred by a C-to-T mutation at nucleotide 1880.

Figure 7. Protease Inhibitor-Resistant HIV-1 has Impaired CA-SP1 Cleavage.

Wild-type NL4-3 and protease mutant NL4-3 PR_I54V+V82A virions were collected from transfected 293T cells grown in the presence (20 µM) or absence of bevirimat and analyzed for particle maturation by Western blot using a p24 monoclonal antibody. The protease mutant is deficient in CA-SP1 cleavage and is comparable to bevirimat-treated wild-type virus, however, the drug has a more dramatic effect on CA-SP1 cleavage in HIV-1 PR_I54V+V82A.

Figure 8. Protease Inhibitor-Resistant HIV-1 with Impaired CA-SP1 Cleavage Is Hypersensitive to Bevirimat In Vitro.

NL4-3 PR_I54V+V82A is hypersensitive to bevirimat in PHA-activated PBMCs with 50% inhibitory concentration (IC₅₀) >100 times lower than for wild-type NL4-3. CC₅₀ is the 50% cytotoxic concentration as assessed by MTT assay.