The Evolution of Antiviral Strategies Against SARS-CoV-2: From Broad-Spectrum Hope to Targeted Therapies and Emerging Resistance


Since the emergence of the SARS-CoV-2 pandemic in 2020, the scientific community has embarked on an urgent quest to discover effective antiviral therapeutics. This journey reflects a pattern seen in previous battles against formidable viral foes such as Influenza and HIV, where the development of effective treatments marked significant turning points in managing health crises.

Initially, the repurposing of broad-spectrum antivirals, which target the viral replication process, was met with high hopes. Studies by Wang et al. (2020) and Williamson et al. (2020) pointed to the potential of these antivirals in tackling SARS-CoV-2. Unfortunately, these expectations were not met, as clinical trials yielded underwhelming results, a reality underscored by Beigel et al. (2020).

The focus then shifted to therapeutic antibodies, which showed promise in early studies (Pinto et al., 2020; Starr et al., 2021). However, these therapies struggled to keep pace with the virus’s ability to mutate, leading to variants that could evade the humoral immune response, as noted by Touret et al. (2023).

Amidst these challenges, a breakthrough was achieved with the development of the protease inhibitor nirmatrelvir (PF-07321332, Paxlovid®). Originally developed against SARS-CoV-1 (Boras et al., 2021), nirmatrelvir targets the 3CLpro, a coronavirus protease. Its efficacy against SARS-CoV-2, as demonstrated in clinical trials (Hammond et al., 2022), lies in its ability to inhibit the cleavage of polyproteins PP1a and PP1ab, thus blocking the production of essential non-structural proteins for viral replication (Jin et al., 2020).

The pharmacokinetics of nirmatrelvir, particularly its metabolism predominantly through the CYP3A4 pathway, has been enhanced by combining it with ritonavir, an exposure booster (Owen et al., 2021). However, this combination poses challenges in patients concurrently on medications affected by ritonavir, a complexity highlighted by Hoertel et al. (2022).

In parallel, a second 3CLpro inhibitor, ensitrelvir (S-217622), was developed by Shionogi, a Japanese pharmaceutical company. This molecule, identified through a blend of virtual and biological screenings and optimized using a structure-based drug design strategy (Unoh et al., 2022), is proposed for use without ritonavir.

After showing promise in Phase 2 clinical trials, it has progressed to Phase 3 testing (Shionogi, 2023; University of Minnesota, 2023). Notably, ensitrelvir (Xocova®) received emergency regulatory approval in Japan in November 2022 and has been prescribed since March 31, 2023.

A major challenge in the use of antiviral mono-therapies is the development of resistance mutations. These mutations can occur naturally in circulating strains, as indicated by studies from Bloom et al. (2010), Ip et al. (2023), and Kawashima et al. (2023), or emerge in patients under conditions conducive to sub-optimal drug efficacy. This was exemplified during the Delta wave of SARS-CoV-2, where resistance to the monoclonal antibody sotrovimab was observed (Rockett et al., 2022).

To understand the resistance mechanism further, researchers experimentally generated resistance mutants for both nirmatrelvir and ensitrelvir. The mutants exhibited reduced sensitivity to these drugs in vitro. Additionally, using a Syrian golden hamster model, it was shown that the ensitrelvir resistance mutation M49L results in significant in vivo resistance. Alarmingly, a recent rise in the prevalence of M49L-carrying sequences has been observed, especially in Japan, which may correlate with the drug’s commercialization since April 2023.

This ongoing battle against SARS-CoV-2 underscores the dynamic nature of viral evolution and the need for continual adaptation in our therapeutic strategies. As the virus evolves, so must our approach to combating it, blending the lessons of the past with the innovations of the present.


In a pivotal study, researchers have successfully generated six SARS-CoV-2 viruses carrying resistance mutations against the protease inhibitors nirmatrelvir and ensitrelvir, currently in clinical stages for COVID-19 treatment. This research sheds light on the intricacies of viral resistance, a critical factor in the ongoing battle against COVID-19 and future pandemics.

The team developed three different strains resistant to nirmatrelvir, each with distinct mutation sets. Interestingly, all three ensitrelvir-resistant strains harbored the same mutation, M49L. The in vitro characterization revealed that while nirmatrelvir-resistant strains showed significant resistance to nirmatrelvir, they exhibited low resistance to ensitrelvir. Conversely, ensitrelvir-resistant strains displayed a similar pattern, being more resistant to ensitrelvir and less so to nirmatrelvir.

A critical aspect of this research involved generating resistance mutations through repeated exposure to increasing concentrations of nirmatrelvir. This method mirrors the approach by Iketani et al. (2023), who also reported similar mutations. In this context, the T21I mutation emerged as a key player, occurring alone or in combination with E166V or T304I mutations. Notably, a mutation at residue 166, previously thought to be cell line-specific, was also observed in this study, challenging earlier assumptions and indicating a broader applicability.

The study delved into the complex interactions between these mutations and viral replicative fitness. For example, the combination of T21I and E166A mutations maintained replication capacity at levels comparable to the wild-type virus. This finding is significant, as it shows how specific mutations can compensate for fitness costs incurred by others.

Turning to ensitrelvir, the consistent emergence of the M49L mutation across different experiments and studies underscores its importance. This mutation, already present in circulating SARS-CoV-2 isolates, raises concerns about the potential for widespread resistance as ensitrelvir usage increases.

The study conducted an in-depth analysis of the M49L mutation’s impact on ensitrelvir’s efficacy. The results revealed a substantial decrease in susceptibility, aligning with previous findings from enzymatic assays and cleavage system studies. This consistency across various methods and virus strains solidifies the conclusion that the M49L mutation significantly reduces ensitrelvir’s effectiveness.

A novel aspect of this research was the exploration of the M49L mutation’s effect on ensitrelvir sensitivity in vivo. Using animal models, the researchers demonstrated that the M49L mutation alone could confer complete resistance to ensitrelvir, a finding that was not observed in previous studies focusing only on the mutation’s impact on replication fitness and pathogenicity.

The study’s findings also have geographical implications. A recent increase in M49L-carrying sequences was observed in Japan, coinciding with the use of ensitrelvir in the country. This correlation suggests a direct link between drug use and the emergence of resistance. It highlights the importance of genomic surveillance, especially in regions where the drug is widely used or being considered for emergency approval.

One significant revelation was the discovery of M49L in early sequences dating back to August 2020, suggesting a natural occurrence of this mutation. This natural presence could facilitate the emergence of ensitrelvir resistance, a concern that extends to other variations in the NSP5 protein, like the M49I mutation.

The study’s comprehensive approach, including in vitro and in vivo analyses, highlights the dynamic nature of SARS-CoV-2 and its ability to develop resistance to targeted therapies. It underscores the need for continuous monitoring and adaptation of treatment strategies, including the potential for dual therapies and the development of antivirals targeting other viral proteins.

The research team, comprising Hawa Sophia Bouzidi, Jean-Sélim Driouich, Raphaëlle Klitting, Ornéllie Bernadin, Géraldine Piorkowski, Rayane Amaral, Laurent Fraisse, Charles E. Mowbray, Ivan Scandale, Fanny Escudié, Eric Chatelain, Xavier de Lamballerie, Antoine Nougairède, and Franck Touret, contributed significantly across various aspects of the study.

Their collaborative efforts in writing, editing, visualization, methodology, investigation, and formal analysis have culminated in a seminal work that deepens our understanding of viral resistance mechanisms and guides future therapeutic strategies.

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