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

Abstract

Immunocompromised individuals are susceptible to severe coronavirus disease 2019 and potentially contribute to the emergence of variants with altered pathogenicity due to persistent infection. This study investigated the impact of immunosuppression on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in K18-hACE2 mice and the effectiveness of antiviral treatments in this context during the first 7 days of infection. Mice were immunosuppressed using cyclophosphamide and infected with a B lineage of SARS-CoV-2. Molnupiravir and nirmatrelvir, alone and in combination, were administered, and viral load and viral sequence diversity were assessed. Treatment of infected but immunocompromised 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 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 characteristics of variants of concern (VOCs). Further investigations are warranted to fully understand the role of immunocompromised individuals in VOC development, especially by taking persistence into consideration, and to inform optimized 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.

Importance: The central aim of this study was to risk-assess the impact of administering a mutagenic antiviral compound, molnupiravir, to patients believed to already be at risk of generating increased viral diversity, which could have severe implications for antiviral resistance development. Combination therapy has a long history of mitigating antiviral resistance risk and was used in this study to demonstrate its potential usefulness in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) context. Animals treated with molnupiravir showed an increase in transition/transversion ratios over time, consistent with the drug's mechanism of action and a recent UK-wide phase II clinical trial assessing the efficacy of molnupiravir in humans. The addition of nirmatrelvir increased viral clearance, which in turn reduces the probability of viral persistence and rapid intra-host evolution of SARS-CoV-2.

Keywords: COVID-19; Paxlovid; SARS-CoV-2; immunocompromised; intra-host evolution; molnupiravir; nirmatrelvir.

Fig 1

Schematic diagram of the experimental design for infection of immunocompromised 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 was used 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.

Fig 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). Asterisks below the curves represent *P < 0.05 and **P < 0.01 between the cyclophosphamide and vehicle groups. Brackets and asterisks at the side represent P < 0.05 for the vehicle/cycophosphamide groups and the drug-treated groups.

Fig 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 was determined using the log-rank (Mantel-Cox) test (n = 4).

Fig 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 analyzed for virus RNA load using qRT-PCR and primers specific for the SARS-CoV-2 N gene. Assays were normalized relative to levels of 18S RNA. Lung tissue was analyzed 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, and (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.

Fig 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).

Fig 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.

Fig 7

The mean Ts/Tv ratio per genome plotted as boxplots. The plot is faceted by day post-infection. Fewer 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).

Fig 8

C to U and G to A minor variation changes significantly increased between day 1 and day 3 post-infection in the molnupiravir-only group. A similar trend is observed between other groups, including molnupiravir treatment; however, the change is not reported as significant. * represents a P-value <0.05 (Mann-Whitney U-test).