Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).

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The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continue to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including immune responses, therapeutic treatments, and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring spike protein mutations linked to immune evasion. In this study, we characterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the spike protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neutralizing the most recent Omicron subvariants, particularly after the emergence of JN.1 subvariant. Using human airway epithelial cells derived from healthy bronchiolar tissue (hBAEC), we established both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Assessments were conducted to compare viral infectivity and the production and release of immune mediators in the apical and basolateral compartments. Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its parental strain JN.1 and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-β, and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfection with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying important implications for therapeutic development and public health preparedness.

The persistent evolution of SARS-CoV-2 has led to the emergence of antigenically distinct Omicron subvariants exhibiting increased transmissibility, immune evasion, and altered pathogenicity. Among these, recent subvariants such as JN.1, KP.3.1.1, and LB.1 possess unique antigenic and virological features, underscoring the need for continued surveillance and therapeutic evaluation. As vaccines and commercial monoclonal antibodies show reduced effectiveness against these variants, the role of direct-acting antivirals, such as Nirmatrelvir, targeting conserved viral elements like the main protease inhibitor, becomes increasingly crucial. In this study, we investigated the replication kinetics, host immune responses, and therapeutic susceptibility of three recently circulating Omicron subvariants in the K18-hACE2 transgenic mouse model, using the SARS-CoV-2 parent WA1/2020 strain as a reference. Omicron subvariants exhibited a marked temporal shift in viral infection kinetics characterized by an early lung viral titer peak (~7–8 Log PFU) at 2 days post-infection (dpi), followed by a decline (1–3 Log PFU) by 4 dpi. Pulmonary cytokine and chemokine responses (GM-CSF, TNF-α, IL-1β, IL-6) showed an earlier increase in subvariant-infected mice compared to a gradual response in WA1/2020 infection. Notably, Nirmatrelvir treatment led to significant reductions in lung viral titers in subvariant-infected mice compared to WA1/2020, surpassing its efficacy against the parent strain. These findings highlight that infection with Omicron subvariants yields a broad dynamic range in viral burden with minimum variability, while retaining a prominent therapeutic response to Nirmatrelvir. This study provides insights into the emerging subvariants’ pathogenesis and therapeutic responsiveness, reinforcing the importance of continued variant monitoring and the development of effective countermeasures.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its impact on public health continues to demand attention as the virus continues to evolve, demonstrating a remarkable ability to adapt to diverse selective pressures including im-mune responses, therapeutic treatments and prophylactic interventions. The SARS-CoV-2 variant landscape remains dynamic, with new subvariants continuously emerging, many harboring Spike (S)-protein mutations linked to immune evasion. In this study, we char-acterized a panel of live SARS-CoV-2 strains, including those key subvariants implicated in recent waves of infection. Our findings revealed a significant variability in mutation patterns in the S-protein across the strains analyzed. Commercial antibodies and human convalescent plasma (HCoP) samples from unvaccinated donors were ineffective in neu-tralizing the most recent Omicron subvariants, particularly after the emergence of JN.1. Using human airway epithelial cells derived from bronchiolar tissue (hBAEC), we estab-lished both monoinfections and coinfections involving SARS-CoV-2, Influenza A virus H1N1 (IFAV_H1N1) and Respiratory Syncytial Virus (RSV). Notably, Omicron KP.3.1.1 subvariant induced a more pronounced cytopathic effect in hBAEC compared to its pa-rental strain JN.1, and even surpassed the impact observed with the ancestral wild-type virus (WA1/2020, Washington strain). Furthermore, the coinfection of KP.3.1.1 subvariant with IFAV_H1N1 or RSV did not attenuate SARS-CoV-2 infectivity; instead, it significantly exacerbated the pathogenic synergy in the lung epithelium. Our study demonstrated that pro-inflammatory cytokines IL-6, IFN-? and IL-10 were upregulated in hBAEC following SARS-CoV-2 monoinfection with recent Omicron subvariants as well as during coinfec-tion with IFAV_H1N1 and RSV. Taken together, our findings offer new insights into the immune evasion strategies and pathogenic potential of evolving SARS-CoV-2 Omicron subvariants, as well as their interactions with other respiratory viruses, carrying im-portant implications for therapeutic development and public health preparedness.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC) which first emerged in 2012, persists and continues to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction of viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).