AVE0991, a Mas receptor agonist, increases influenza and COVID-19 severity in vivo.

1. Problem Statement

Severe respiratory viral infections, including Influenza A Virus (IAV) and SARS-CoV-2, are often driven by dysregulated immune responses and tissue damage associated with the Renin-Angiotensin System (RAS).

• Theoretical Basis: The protective arm of the RAS involves the ACE2/Angiotensin-(1-7)/Mas receptor (MasR) axis, which counteracts the pro-inflammatory Angiotensin II pathway.
• The Gap: While recombinant Ang-(1-7) has shown promise in preclinical models, it suffers from poor pharmacokinetics (short half-life, need for IV administration). AVE0991, a non-peptide, orally bioavailable MasR agonist, mimics Ang-(1-7) and has proven effective in non-infectious models of lung injury and chronic inflammation.
• The Question: It was unknown whether AVE0991 would be beneficial or detrimental in the context of acute viral respiratory infections, where the timing and nature of immune modulation are critical.

2. Methodology

The study employed a dual approach using in vitro human cell models and in vivo murine models to evaluate the therapeutic potential of AVE0991.

• In Vitro Models:

  • Cell Line: Calu-3 human lung epithelial cells.
  • Infection: Infected with IAV (A/H1N1/Auckland/1/2009, MOI 0.1) or SARS-CoV-2 Delta variant (MOI 0.01).
  • Treatment: Cells were treated with AVE0991 (12.5 μM) or vehicle (0.125% DMSO) starting 24 hours post-infection for an additional 24 hours.
  • Assays: qPCR for cytokine gene expression (IL-6, TNF-α), plaque assays for viral titers, and cytotoxicity assays.

• In Vivo Models:

  • Subjects: Male C57BL/6J mice (13 weeks for IAV; 20 weeks for SARS-CoV-2).
  • Infection: Intranasal infection with IAV (2,500 PFU) or mouse-adapted SARS-CoV-2 (10⁴ PFU).
  • Treatment: Oral gavage of AVE0991 (10 mg/kg) twice daily, starting at 2 days post-infection (dpi) for IAV and 1.5 dpi for SARS-CoV-2.
  • Endpoints:
    • Clinical: Body weight loss, blood oxygen saturation (pulse oximetry).
    • Pathological: Lung histology (H&E staining) scored by a blinded pathologist.
    • Virological: Viral titers in lung homogenates.
    • Immunological: Flow cytometry of lung immune cells (macrophages, T cells) and multiplex cytokine assays on serum.

3. Key Contributions

• First Evaluation in Acute Viral Models: This is the first study to test the orally bioavailable MasR agonist AVE0991 in acute models of both IAV and SARS-CoV-2 infection.
• Demonstration of In Vitro/In Vivo Discordance: The study highlights a critical disconnect between cell culture results (suggesting benefit) and whole-organism outcomes (demonstrating harm).
• Context-Dependency of RAS Therapy: It provides evidence that MasR activation, while anti-inflammatory in chronic settings, may be deleterious during the acute phase of viral clearance.

4. Results

A. In Vitro Findings (Calu-3 Cells)

• Anti-inflammatory Effect: AVE0991 significantly suppressed the gene expression of pro-inflammatory cytokines IL-6 and TNF-α in cells infected with both IAV and SARS-CoV-2.
• Antiviral Effect: AVE0991 significantly reduced IAV viral titers. However, it had no effect on SARS-CoV-2 viral titers.
• Conclusion: In a controlled cellular environment, AVE0991 exhibited anti-inflammatory and specific antiviral properties against influenza.

B. In Vivo Findings (Murine Models)

Contrary to the in vitro data, AVE0991 treatment worsened disease severity in both mouse models:

• Clinical Severity: Treated mice experienced significantly greater weight loss compared to vehicle controls in both IAV and SARS-CoV-2 models.
• Viral Load:
  • IAV: Viral titers in the lungs were elevated in AVE0991-treated mice.
  • SARS-CoV-2: Viral titers remained unchanged, but disease severity still increased.
• Immune Profiling:
  • IAV: Treatment led to a significant reduction in alveolar macrophages (critical for early viral clearance) and reduced serum IFN-γ. T cell frequencies increased, but functional implications were unclear.
  • SARS-CoV-2: Serum levels of chemokines CXCL1 and CXCL10 were elevated.
• Histology: A trend toward increased tissue damage was observed in IAV-infected mice treated with AVE0991 (p=0.0558).

5. Significance and Discussion

• Paradoxical Outcome: The study challenges the conventional view that enhancing the ACE2/Ang-(1-7)/MasR axis is universally protective in respiratory viral infections.
• Mechanism of Harm: The authors hypothesize that premature suppression of pro-inflammatory and interferon responses (specifically IFN-γ and macrophage activity) by AVE0991 impairs the host's ability to clear the virus during the acute phase. This mirrors the known risks of early corticosteroid administration in viral pneumonia.
• Therapeutic Implications:
  • Timing is Critical: The beneficial effects of MasR agonists seen in chronic injury models may not translate to acute viral infections. The "window of opportunity" for such therapies likely lies in the late inflammatory phase (cytokine storm) rather than the early viral replication phase.
  • Caution in Repurposing: RAS-targeting therapies must be rigorously evaluated in the specific context of acute infection before clinical application.
  • Future Directions: Further research is needed to determine if delayed administration (post-peak viral replication) or different dosing strategies could yield protective effects, or if MasR antagonists might be required in specific phases of infection.

Conclusion: While AVE0991 successfully reduced inflammation and viral load in isolated cells, its administration in live animals during acute viral infection exacerbated disease, increased weight loss, and compromised immune clearance. This underscores the complexity of RAS modulation in viral pathogenesis and the necessity of in vivo validation for RAS-targeted therapeutics.