Bioluminescence Imaging Reveals Enhanced SARS-CoV-2 Clearance in Mice with Combinatorial Regimens
Irfan Ullah, Fanny Escudie, Ivan Scandale, Zoela Gilani, Gabrielle Gendron-Lepage, Fleur Gaudette, Charles Mowbray, Laurent Fraisse, Renée Bazin, Andrés Finzi, Walther Mothes, Priti Kumar, Eric Chatelain, Pradeep D Uchil
iScience, doi:10.1016/j.isci.2024.109049
Direct acting antivirals (DAAs) represent critical tools for combating SARS-CoV-2 variants of concern (VOCs) that have escaped vaccine-elicited spike-based immunity and future coronaviruses with pandemic potential. Here, we used bioluminescence imaging to evaluate therapeutic efficacy of DAAs that target SARS-CoV-2 RNA-dependent RNA polymerase (favipiravir, molnupiravir) or Main protease (nirmatrelvir) against Delta or Omicron VOCs in K18-hACE2 mice. Nirmatrelvir displayed the best efficacy followed by molnupiravir and favipiravir in suppressing viral loads in the lung. Unlike neutralizing antibody treatment, DAA monotherapy regimens did not eradicate SARS-CoV-2 in mice but combining molnupiravir with nirmatrelvir exhibited superior additive efficacy and led to virus clearance. Furthermore, combining molnupiravir with Caspase-1/4 inhibitor mitigated inflammation and lung pathology whereas combining molnupiravir with COVID-19 convalescent plasma demonstrated synergy, rapid virus clearance and 100% survival. Thus, our study provides insights into in vivo treatment efficacies of DAAs and other effective combinations to bolster COVID-19 therapeutic arsenal.
Competing interest The authors declare no competing interests.
J o u r n a l P r e -p r o o f Brain and lung samples were collected from mice at the time of necropsy. Approximately, 20 mg of tissue was suspended in 500 µL of RLT lysis buffer, and RNA was extracted using RNeasy plus Mini kit (Qiagen Cat # 74136), reverse transcribed with iScript advanced cDNA kit (Bio-Rad Cat #1725036). To determine mRNA copy numbers of signature inflammatory cytokines, multiplex qPCR was conducted using iQ Multiplex Powermix (Bio Rad Cat # 1725848) and PrimePCR Probe Assay mouse primers FAM-GAPDH, HEX-IL6, TEX615-CCL2, Cy5-CXCL10, Cy5.5-IFNgamma and HEX-IL1B. The reaction plate was analyzed using CFX96 touch real time PCR detection system. Scan mode was set to all channels. The PCR conditions were 95 °C 2 min, 40 cycles of 95 °C for 10 s and 60 °C for 45 s, followed by a melting curve analysis to ensure that each primer pair resulted in amplification of a single PCR product. mRNA copy numbers of Il6, Ccl2, Cxcl10, Ifng and Il1b in the cDNA samples of infected mice were normalized to Gapdh mRNA with the formula ΔC t (target gene)=C t (target gene)-C t (Gapdh). The fold increase was determined using 2 -ΔΔCt method comparing treated mice to uninfected controls.
Cryo-immunohistology of lung tissue Lung tissues were isolated after necropsy and fixed in 1X PBS containing freshly prepared 4% PFA for 12 h at RT or 4 C. They were then washed with PBS, cryoprotected with 10, 20 and 30% ascending..
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