Synergistic Activity of Remdesivir-Nirmatrelvir Combination on a SARS-CoV-2 In Vitro Model and a Case Report

Abstract

Background: This study aims to investigate the activity of the remdesivir-nirmatrelvir combination against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and to report a case of Coronavirus Disease 2019 (COVID-19) cured with this combination.

Methods: A Vero E6 cell-based infection assay was used to investigate the in vitro activity of the remdesivir-nirmatrelvir combination. The SARS-CoV-2 strains tested were 20A.EU1, BA.1 and BA.5. After incubation, a viability assay was performed. The supernatants were collected and used for viral titration. The Highest Single Agent (HSA) reference model was calculated. An HSA score >10 is considered synergic.

Results: Remdesivir and nirmatrelvir showed synergistic activity at 48 and 72 h, with an HSA score of 52.8 and 28.6, respectively (p < 0.0001). These data were confirmed by performing supernatant titration and against the omicron variants: the combination reduced the viral titer better than the more active compound alone. An immunocompromised patient with prolonged and critical COVID-19 was successfully treated with remdesivir, nirmatrelvir/ritonavir, tixagevimab/cilgavimab and dexamethasone, with an excellent clinical-radiological response. However, she required further off-label prolonged therapy with nirmatrelvir/ritonavir until she tested negative.

Conclusions: Remdesivir-nirmatrelvir combination has synergic activity in vitro. This combination may have a role in immunosuppressed patients with severe COVID-19 and prolonged viral shedding.

Keywords: COVID-19; SARS-CoV-2; antiviral; immunosuppressed; nirmatrelvir; remdesivir; synergy; variant.

Figure 1

The interaction landscapes of the remdesivir–nirmatrelvir combination after 48 h (A) and 72 h (B) of incubation. A Vero E6 cell-based infection assay was used to investigate the in vitro activity of the combination. All experiments were performed using a 20A.EU1 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) strain. After the incubation, a viability assay was performed. To test whether the drug combinations act synergistically, the Highest Single Agent (HSA) reference model was calculated. An HSA score > 10 is considered synergic. Data are from 3 independent experiments performed in triplicate. Both panels (A,B) show, in order, numerical values across the matrices, synergy map and 3D plot.

Figure 2

Viral titration (plaque reduction assay) of remdesivir–nirmatrelvir combination. After synergy tests (A) or yield reduction assay (B,C), supernatants of SARS-CoV-2 20A.EU1 (A), BA.1 (case report strain) (B) and BA.5 (C) infected Vero E6 cells treated with the antiviral combination were frozen and tested for viral load. Data are expressed as plaque-forming units (PFU)/mL (mean ± standard deviation, SD) from 2 or 3 experiments performed in triplicate. * p < 0.05, antiviral combination vs more active single agent. Viral eradication was defined as <50 PFU/mL, the detection limit of the method. (A): The combination at the concentration of 3.1 + 4.5 μM eradicated the virus. (B,C): remdesivir at the concentration of 3.1 μM and the combination at the concentration of 3.1 + 4.5 μM eradicated the virus.

Figure 3

Case report timeline: A 50-year-old woman with a history of non-Hodgkin follicular lymphoma treated with anti-CD20 therapy was SARS-CoV-2 positive starting on 18 February 2022. Multiple nasopharyngeal swabs were collected and all tested positive. On 19 April, 4 May and 5 September, SARS-CoV-2 was isolated from nasopharyngeal swabs, and the viruses were identified as BA.1 variants. On 24 March, she underwent Computed Tomography (CT) that showed “multiple and diffuse areas of parenchymal opacities with a ground glass appearance, with a tendency to consolidation in sloping areas” (A). On hospital readmission, the clinical conditions immediately appeared critical with acute progressive respiratory failure due to extensive COVID-19 interstitial pneumonia, as shown in CT images (B): “Bilateral extensive consolidative foci, “crazy paving” and “ground glass” opacities are evident in the lung parenchyma””. The medical therapy was set as follows: antibiotic therapy, off-label antiviral therapy with remdesivir (10-day course) plus nirmatrelvir/ritonavir (5 day course), tixagevimab/cilgavimab, and dexamethasone (6 mg/die for 10 days). The patient presented an excellent clinical-radiological response with progressive reduction of oxygen requirement up to the suspension and improvement on control CT after 2 weeks (C). However, she required further off-label prolonged therapy with nirmatrelvir/ritonavir up to negativization obtained on 2 November 2022. NPS, nasopharyngeal swab; R, remdesivir; S, sotrovimab; D, dexamethasone; TC, tixagevimab/cilgavimab; N/R, nirmatrelvir/ritonavir; HFNC, high-flow nasocanula.