The Combination of Nirmatrelvir and Ivermectin Exerts Strongly Synergistic Antiviral and Anti-Inflammatory Effects Against Murine Coronavirus Infection of Macrophages

The emergence of SARS-CoV-2 variants and Antiviral resistance highlights the need for improved therapeutic strategies against COVID-19 and other coronavirus infections. By pairing direct-acting antivirals with repurposed host-modulating drugs, combination therapy approaches may enhance Antiviral efficacy while mitigating inflammation. In this study, we evaluated the effects of combining Nirmatrelvir (a SARS-CoV-2 main Protease inhibitor) with Ivermectin, Azithromycin or Doxycycline-using a murine hepatitis virus (MHV) Infection model of RAW264.7 Macrophages. Checkerboard assays demonstrated that the Nirmatrelvir-Ivermectin combination exhibited strongly synergistic Antiviral activity. This combination achieved potent inhibition of live virus titer of at least 5 to 6 log₁₀ and reduction in viral RNA load of 3 log₁₀, at drug concentrations much lower than the respective monotherapies. The Nirmatrelvir-Ivermectin combination treatment affected the coronavirus replication cycle at the same time-point of 8 h as Nirmatrelvir monotherapy. Moreover, multiplex cytokine protein profiling revealed that Nirmatrelvir-Ivermectin markedly suppressed key pro-inflammatory cytokines and chemokines associated with the cytokine storm, including IL-6, TNF-α, IL-1β, and MCP-1. Conversely, the combinations of Nirmatrelvir with Azithromycin or Doxycycline exhibited only additive or mildly additive effects, with alterations in certain cytokine levels. These findings support the potential of Nirmatrelvir-Ivermectin as a promising novel combination therapy with both Antiviral and anti-inflammatory benefits, warranting further in vivo validation and clinical investigation.