The 3C-like protease (3CL^pro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CL^pro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CL^pro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CL^pro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CL^pro and two luciferase fragments linked together by a 3CL^pro cleavage site. 3CL^pro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CL^pro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CL^pro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CL^pro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CL^pro. Of these, only five exhibited significant inhibition of 3CL^pro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CL^pro.

Viral diseases represent a major public health concerns and ever-present risks for developing into future pandemics. Antiviral antibody therapeutics, either alone or in combination with other therapies, emerged as valuable preventative and treatment options, including during global emergencies. Here we will discuss polyclonal and monoclonal antiviral antibody therapies, focusing on the unique biochemical and physiological properties that make them well-suited as therapeutic agents. We will describe the methods of antibody characterization and potency assessment throughout development, highlighting similarities and differences between polyclonal and monoclonal products as appropriate. In addition, we will consider the benefits and challenges of antiviral antibodies when used in combination with other antibodies or other types of antiviral therapeutics. Lastly, we will discuss novel approaches to the characterization and development of antiviral antibodies and identify areas that would benefit from additional research.

Background: PAXLOVID consists of nirmatrelvir, an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), copackaged with ritonavir, a pharmacokinetic enhancer. Nirmatrelvir/ritonavir received emergency use authorization in the United States in 2021 and was approved in 2023. However, there is limited published information on SARS-CoV-2 clinical resistance to nirmatrelvir/ritonavir. Methods: To investigate SARS-CoV-2 resistance development to nirmatrelvir/ritonavir in treated patients, we analyzed baseline and matching post-baseline SARS-CoV-2 next-generation sequencing data from 1862 participants (912 nirmatrelvir/ritonavir, 950 placebo) in Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients (EPIC-HR) and Evaluation of Protease Inhibition for COVID-19 in Standard-Risk Patients (EPIC-SR), which were Phase 2/3, randomized, double-blind, placebo-controlled trials in participants with mild-to-moderate coronavirus disease 2019 (COVID-19). Potential resistance-associated substitutions (RAS) were defined as those that were enriched in nirmatrelvir/ritonavir-treated participants or occurred at Mpro positions of interest, defined using nonclinical data. SARS-CoV-2 sequence databases were analyzed to characterize temporal frequencies of nirmatrelvir/ritonavir RAS in circulating viruses. Results: In EPIC-HR, nirmatrelvir/ritonavir RAS included Mpro T21I (n = 1), E166V (n = 3), A173T (n = 1), and T304I (n = 1), with E166V being the clearest RAS observed. In EPIC-SR, no RAS were detected. Nirmatrelvir/ritonavir RAS were not associated with hospitalization or death. Analyses of SARS-CoV-2 sequence databases did not reveal concerning increases in the frequencies of nirmatrelvir/ritonavir RAS over time. Conclusions: In clinical trials, emergence of SARS-CoV-2 resistance to nirmatrelvir/ritonavir was infrequent (<0.3%–1.1%). Surveillance data currently indicate a low frequency of circulating SARS-CoV-2 variants with nirmatrelvir/ritonavir RAS. Collectively, these results provide the most comprehensive analysis of SARS-CoV-2 resistance to nirmatrelvir/ritonavir in the clinical setting to date. Viral sequences should continue to be closely monitored to identify the potential emergence of nirmatrelvir/ritonavir-resistant variants.

Novel retroviruses can emerge from recombination between distantly related retroviruses. Most notably, HIV-1 originated from zoonotic transmission of a novel recombinant (SIV _cpz ) into humans.

Coronaviruses pose a global pandemic threat, making development of a pan-coronavirus inhibitor crucial for preparedness and containment in the event of a new coronavirus outbreak. The 3C-like protease (3CL^pro) is a key target for antiviral development, as it is essential for viral replication and conserved across human coronaviruses. We previously developed an assay to monitor SARS-CoV-2 3CL^pro activity in cells. This assay uses a single vector that coexpresses the 3CL^pro enzyme and the reporter, which consists of two luciferase fragments linked by a 3CL^pro cleavage site. Cleavage of this site by 3CL^pro decreases luciferase activity, whereas inhibition of 3CL^pro increases the luciferase activity. Here, we adapted this assay to examine 3CL^pro activity from six other human coronaviruses: SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. We further determined the effects of different cleavage sites to improve the signal-to-background ratio. The Nsp4-Nsp5 site and super-active substrate (SAS) resulted in the largest dynamic range for most coronaviruses in our assay. Using the broad-spectrum 3CL^pro inhibitor GC376, we observed increased reporter activity, indicating the assay’s efficacy for identifying inhibitors across multiple coronaviruses. The adaptation and improvement of the assay can facilitate the development of inhibitors against 3CL^pro from multiple or novel coronaviruses.

During retrovirus assembly, Gag packages unspliced viral RNA as the virion genome. Genome packaging is usually specific with occasional exceptions of cross-packaging RNA from distantly related retroviruses. For example, HIV-1 Gag can efficiently package HIV-2 RNA. To better understand how HIV-1 Gag selects packaging substrates, we defined elements in the HIV-2 5′ untranslated region (UTR) that are important for this process. Although sharing little homology, both HIV-1 and HIV-2 5′ UTRs have unpaired guanosines essential for packaging by their own Gag. Simultaneously substituting guanosines of nine sites in the HIV-2 5′ UTR caused severe defects in HIV-1 Gag-mediated packaging. Two of the nine sites are particularly important, mutating each one impaired HIV-1 Gag-mediated packaging, whereas the other sites required mutations in multiple sites to produce similar effects. Additionally, we identified one site that impacts HIV-1 Gag but is dispensable for HIV-2 Gag selective packaging. Furthermore, combining mutations has an additive effect on packaging defects for HIV-1 Gag, in contrast to the previously reported synergistic effects for HIV-2 Gag. Our study demonstrates that Gag proteins from two different retroviruses recognize and use mostly the same set of cis-acting elements to mediate RNA packaging and provide the mechanistic basis for genome cross-packaging.