The effect of molnupiravir and nirmatrelvir on SARS-CoV-2 genome diversity in severe models of COVID-19

Abstract

Objectives: Immunocompromised individuals are susceptible to severe COVID-19 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on SARS-CoV-2 infection in k18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection.

Methods: Mice were immunosuppressed using cyclophosphamide and infected with a B daughter lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered and viral load and viral sequence diversity was assessed.

Results: Treatment of infected but immune compromised mice with both compounds either singly or in combination resulted in decreased viral loads and pathological changes compared to untreated animals. Treatment also abrogated infection of neuronal tissue. However, no consistent changes in the viral consensus sequence were observed, except for the emergence of the S:H655Y mutation. Molnupiravir, but not nirmatrelvir or immunosuppression alone, increased the transition/transversion (Ts/Tv) ratio, representative of G>A and C>U mutations and this increase was not altered by the co-administration of nirmatrelvir with molnupiravir.Notably, immunosuppression itself did not appear to promote the emergence of mutational characteristic of variants of concern (VOCs).

Conclusions: Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimised public health strategies. It is more likely that immunodeficiency promotes viral persistence but does not necessarily lead to substantial consensus-level changes in the absence of antiviral selection pressure. Consistent with mechanisms of action, molnupiravir showed a stronger mutagenic effect than nirmatrelvir in this model.

Keywords: COVID-19; Molnupiravir; Nirmatrelvir; Paxlovid; SARS-CoV-2; immunocompromised; intra-host evolution.

Figure 1.

Schematic diagram of the experimental design for infection of immune compromised K18-hACE2 mice with SARS-CoV-2 and evaluation of two antiviral drugs given at a human equivalent dose; molnupiravir, a broad acting compound causing error catastrophe, or nirmatrelvir which specifically targets the viral 3C-like protease. Cyclophosphamide was used at 100 mg/kg via the intraperitoneal route to immunosuppress mice. Molnupiravir was used at 100 mg/kg and nirmatrelvir at 500 mg/kg both via the oral route. Effects of infection and treatment were evaluated by measuring the weight of the mice daily, determining viral loads in sequential oral/throat swabs and at day 7 post-infection, and examining nose, brain and lung at day 7 post infection for any histological changes and the expression of SARS-CoV-2 nucleoprotein.

Figure 2:. Treatment of SARS-CoV-2-infected mice leads to decreased weight loss.

K18-hACE2 mice were challenged intranasally with 10⁴ PFU SARS-CoV-2 Mice were monitored for weight at indicated time-points. (n = 4). Data represent the mean residual weight ± SEM. Comparisons were made using a repeated-measures two-way ANOVA (Bonferroni post-test). * on the represents P < 0.05. Asterisks below the curves represent * P < 0.05 and ** P < 0.01 between the cylophophamide and vehicle groups. Brackets and asterisk at the side represents P < 0.05 for the Vehicle/cycophosphamide groups and the drug treated groups.

Figure 3:. Treatment of SARS-CoV-2-infected mice leads to enhanced survival.

K18-hACE2 mice were challenged intranasally with 10⁴ PFU SARS-CoV-2. Survival was assessed at indicated time points and significance determined using log rank (Mantel-Cox) test (n = 4).

Figure 4.. Viral loads in swabs and tissues.

K18-hACE2 mice were challenged intranasally with 10⁴ PFU SARS-CoV-2 and treated as indicated (n = 4 per group). RNA extracted from oral/throat swabs and nasal tissue was analysed for virus RNA load using qRT-PCR and primers specific for the SARS-CoV-2 N gene. Assays were normalised relative to levels of 18S RNA. Lung tissue was analysed for live virus by plaque assay. Data for individual animals are shown with the median value represented by a black line. (A) Throat swabs; (B) nasal tissue; (C) lung tissue. Comparisons were made using two-way ANOVA (Bonferroni post-test) in panel A and Mann-Whitney U test (Panels B and C). * Represents p < 0.05.

Figure 5:

K18-hACE2 mice were challenged intranasally with 10⁴ PFU SARS-CoV-2 and treated as indicated below (n = 4 per group). Immunohistology for the detection of viral antigen in the lung at day 6 or 7 post infection. Sections from the formalin-fixed, paraffin embedded left lung lobe were stained using anti-SARS-CoV nucleoprotein and counterstained with hematoxylin. Representative images from the individual treatment groups are shown as follows: A. vehicle; B. cyclophosphamide; C. molnupiravir; D. cyclophosphamide and molnupiravir; E. cyclophosphamide and nirmatrelvir; F. cyclophosphamide, molnupiravir and nirmatrelvir. Viral antigen expression is restricted to pneumocytes in a few individual alveoli (higher magnifications in insets). Bars represent 2.5 mm (A-E), 1 mm (F) and 20 μm (F, insets).

Figure 6:

K18-hACE2 mice were challenged intranasally with 10⁴ PFU SARS-CoV-2 and treated as indicated below (n = 4 per group). Immunohistology for the detection of viral antigen in the brain and nose at day 6 or 7 post infection. Sections from formalin-fixed, decalcified and paraffin embedded heads after longitudinal sawing in the midline were stained using anti-SARS-CoV nucleoprotein, and counterstained with hematoxylin. Only small fragments of nasal mucosa were available for the examination, as the nasal turbinates had been sampled for PCR. Representative images from the individual treatment groups are shown as follows: A. Vehicle. There is widespread infection of the brain. The insets show infection of individual cells with the morphology of olfactory sensory neurons and epithelial cells in the olfactory epithelial layer (left inset) and individual respiratory epithelial cells in the nasal mucosa (arrowhead; right inset); B. Cyclophosphamide. There is widespread infection of the brain. The inset shows a group of positive epithelial cells/sensory neurons in the olfactory epithelial layer (arrowhead); C. Molnupiravir. There is no evidence of brain infection. D. Cyclophosphamide and molnupiravir. There is no evidence of brain infection. E. Cyclophosphamide and nirmatrelvir. There is no evidence of brain infection. F. Cyclophosphamide, molnupiravir and nirmatrelvir. There is no evidence of brain infection. Bars represent 2.5 mm (A-F) and 20 μm (A, B insets). FC – frontal cortex, HC – hippocampus, MO – medulla oblongata, OB – olfactory bulb, OSN - olfactory sensory neurons.

Figure 7:

The mean Ts/Tv ratio per genome plotted as boxplots. The plot is facetted by day post infection. Less genomes were recovered for cyclophosphamide and nirmatrelvir and cyclophosphamide, nirmatrelvir and molnupiravir, therefore statistical analysis returns the differences as non-significant. Trends can be concluded with caution. * Represents a P value <0.05 (Mann Whitney U test).