Antibodies targeting a quaternary site on SARS-CoV-2 spike glycoprotein prevent viral receptor engagement by conformational locking.

SARS-CoV-2 continues to evolve, with many variants evading clinically authorized antibodies. To isolate monoclonal antibodies (mAbs) with broadly neutralizing capacities against the virus, we screened serum samples from convalescing COVID-19 patients. We isolated two mAbs, 12-16 and 12-19, which neutralized all SARS-CoV-2 variants tested, including the XBB subvariants, and prevented infection in hamsters challenged with Omicron BA.1 intranasally. Structurally, both antibodies targeted a conserved quaternary epitope located at the interface between the N-terminal domain and subdomain 1, uncovering a site of vulnerability on SARS-CoV-2 spike. These antibodies prevented viral receptor engagement by locking the receptor-binding domain (RBD) of spike in the down conformation, revealing a mechanism of virus neutralization for non-RBD antibodies. Deep mutational scanning showed that SARS-CoV-2 could mutate to escape 12-19, but such mutations are rarely found in circulating viruses. Antibodies 12-16 and 12-19 hold promise as prophylactic agents for immunocompromised persons who do not respond robustly to COVID-19 vaccines.

Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity.

While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 µg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1.

Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant, BA.2.86, has emerged and spread to numerous countries worldwide, raising alarm because its spike protein contains 34 additional mutations compared with its BA.2 predecessor1. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was no more resistant to human sera than the currently dominant XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from people who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. Although BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the 'inner face' of the RBD that is exposed only when this domain is in the 'up' position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.

Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2.

The B.1.1.529/Omicron variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings of our study. Here we found that B.1.1.529 is markedly resistant to neutralization by serum not only from patients who recovered from COVID-19, but also from individuals who were vaccinated with one of the four widely used COVID-19 vaccines. Even serum from individuals who were vaccinated and received a booster dose of mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies against all known epitope clusters on the spike protein, we noted that the activity of 17 out of the 19 antibodies tested were either abolished or impaired, including ones that are currently authorized or approved for use in patients. Moreover, we also identified four new spike mutations (S371L, N440K, G446S and Q493R) that confer greater antibody resistance on B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.

Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4 and BA.5.

SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 have surged notably to become dominant in the United States and South Africa, respectively1,2. These new subvariants carrying further mutations in their spike proteins raise concerns that they may further evade neutralizing antibodies, thereby further compromising the efficacy of COVID-19 vaccines and therapeutic monoclonals. We now report findings from a systematic antigenic analysis of these surging Omicron subvariants. BA.2.12.1 is only modestly (1.8-fold) more resistant to sera from vaccinated and boosted individuals than BA.2. However, BA.4/5 is substantially (4.2-fold) more resistant and thus more likely to lead to vaccine breakthrough infections. Mutation at spike residue L452 found in both BA.2.12.1 and BA.4/5 facilitates escape from some antibodies directed to the so-called class 2 and 3 regions of the receptor-binding domain3. The F486V mutation found in BA.4/5 facilitates escape from certain class 1 and 2 antibodies but compromises the spike affinity for the viral receptor. The R493Q reversion mutation, however, restores receptor affinity and consequently the fitness of BA.4/5. Among therapeutic antibodies authorized for clinical use, only bebtelovimab retains full potency against both BA.2.12.1 and BA.4/5. The Omicron lineage of SARS-CoV-2 continues to evolve, successively yielding subvariants that are not only more transmissible but also more evasive to antibodies.

Antibody evasion properties of SARS-CoV-2 Omicron sublineages.

The identification of the Omicron (B.1.1.529.1 or BA.1) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Botswana in November 20211 immediately caused concern owing to the number of alterations in the spike glycoprotein that could lead to antibody evasion. We2 and others3-6 recently reported results confirming such a concern. Continuing surveillance of the evolution of Omicron has since revealed the rise in prevalence of two sublineages, BA.1 with an R346K alteration (BA.1+R346K, also known as BA.1.1) and B.1.1.529.2 (BA.2), with the latter containing 8 unique spike alterations and lacking 13 spike alterations found in BA.1. Here we extended our studies to include antigenic characterization of these new sublineages. Polyclonal sera from patients infected by wild-type SARS-CoV-2 or recipients of current mRNA vaccines showed a substantial loss in neutralizing activity against both BA.1+R346K and BA.2, with drops comparable to that already reported for BA.1 (refs. 2,3,5,6). These findings indicate that these three sublineages of Omicron are antigenically equidistant from the wild-type SARS-CoV-2 and thus similarly threaten the efficacies of current vaccines. BA.2 also exhibited marked resistance to 17 of 19 neutralizing monoclonal antibodies tested, including S309 (sotrovimab)7, which had retained appreciable activity against BA.1 and BA.1+R346K (refs. 2-4,6). This finding shows that no authorized monoclonal antibody therapy could adequately cover all sublineages of the Omicron variant, except for the recently authorized LY-CoV1404 (bebtelovimab).

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Developing inhibitory peptides against SARS-CoV-2 envelope protein.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.

Emergence and expansion of SARS-CoV-2 B.1.526 after identification in New York.

SARS-CoV-2 infections have surged across the globe in recent months, concomitant with considerable viral evolution1-3. Extensive mutations in the spike protein may threaten the efficacy of vaccines and therapeutic monoclonal antibodies4. Two signature spike mutations of concern are E484K, which has a crucial role in the loss of neutralizing activity of antibodies, and N501Y, a driver of rapid worldwide transmission of the B.1.1.7 lineage. Here we report the emergence of the variant lineage B.1.526 (also known as the Iota variant5), which contains E484K, and its rise to dominance in New York City in early 2021. This variant is partially or completely resistant to two therapeutic monoclonal antibodies that are in clinical use and is less susceptible to neutralization by plasma from individuals who had recovered from SARS-CoV-2 infection or serum from vaccinated individuals, posing a modest antigenic challenge. The presence of the B.1.526 lineage has now been reported in all 50 states in the United States and in many other countries. B.1.526 rapidly replaced earlier lineages in New York, with an estimated transmission advantage of 35%. These transmission dynamics, together with the relative antibody resistance of its E484K sub-lineage, are likely to have contributed to the sharp rise and rapid spread of B.1.526. Although SARS-CoV-2 B.1.526 initially outpaced B.1.1.7 in the region, its growth subsequently slowed concurrently with the rise of B.1.1.7 and ensuing variants.

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7.

The COVID-19 pandemic has had widespread effects across the globe, and its causative agent, SARS-CoV-2, continues to spread. Effective interventions need to be developed to end this pandemic. Single and combination therapies with monoclonal antibodies have received emergency use authorization1-3, and more treatments are under development4-7. Furthermore, multiple vaccine constructs have shown promise8, including two that have an approximately 95% protective efficacy against COVID-199,10. However, these interventions were directed against the initial SARS-CoV-2 virus that emerged in 2019. The recent detection of SARS-CoV-2 variants B.1.1.7 in the UK11 and B.1.351 in South Africa12 is of concern because of their purported ease of transmission and extensive mutations in the spike protein. Here we show that B.1.1.7 is refractory to neutralization by most monoclonal antibodies against the N-terminal domain of the spike protein and is relatively resistant to a few monoclonal antibodies against the receptor-binding domain. It is not more resistant to plasma from individuals who have recovered from COVID-19 or sera from individuals who have been vaccinated against SARS-CoV-2. The B.1.351 variant is not only refractory to neutralization by most monoclonal antibodies against the N-terminal domain but also by multiple individual monoclonal antibodies against the receptor-binding motif of the receptor-binding domain, which is mostly due to a mutation causing an E484K substitution. Moreover, compared to wild-type SARS-CoV-2, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4-fold) and sera from individuals who have been vaccinated (10.3-12.4-fold). B.1.351 and emergent variants13,14 with similar mutations in the spike protein present new challenges for monoclonal antibody therapies and threaten the protective efficacy of current vaccines.

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1-3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

Identification of SARS-CoV-2 inhibitors using lung and colonic organoids.

There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml-1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.

Persistence of an infectious form of SARS-CoV-2 post protease inhibitor treatment of permissive cells in vitro.

Reports have described SARS-CoV-2 rebound in COVID-19 patients treated with nirmatrelvir, a 3CL protease inhibitor. The cause remains a mystery, although drug resistance, re-infection, and lack of adequate immune responses have been excluded. We now present virologic findings that provide a clue to the cause of viral rebound, which occurs in ∼20% of the treated cases. Persistence of infectious SARS-CoV-2 was experimentally documented in vitro after treatment with nirmatrelvir or another 3CL protease inhibitor, but not with a polymerase inhibitor, remdesivir. This infectious form decayed slowly with a half-life of ∼1 day, suggesting that its persistence could outlive the treatment course to re-ignite SARS-CoV-2 infection as the drug is eliminated. Notably, extending nirmatrelvir treatment beyond 8 days abolished viral rebound in vitro. Our findings point in a particular direction for future investigation of virus persistence and offer a specific treatment recommendation that should be tested clinically.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

Exploring the effect of Clostridium butyricum on lung injury associated with acute pancreatitis in mice by combined 16S rRNA and metabolomics analysis.

OBJECTIVES: Acute lung injury is a critical complication of severe acute pancreatitis (SAP). The gut microbiota and its metabolites play an important role in SAP development and may provide new targets for AP-associated lung injury. Based on the ability to reverse AP injury, we proposed that Clostridium butyricum may reduce the potential for AP-associated lung injury by modulating with intestinal microbiota and related metabolic pathways. METHODS: An AP disease model was established in mice and treated with C. butyricum. The structure and composition of the intestinal microbiota in mouse feces were analyzed by 16 S rRNA gene sequencing. Non-targeted metabolite analysis was used to quantify the microbiota derivatives. The histopathology of mouse pancreas and lung tissues was examined using hematoxylin-eosin staining. Pancreatic and lung tissues from mice were stained with immunohistochemistry and protein immunoblotting to detect inflammatory factors IL-6, IL-1ß, and MCP-1. RESULTS: C. butyricum ameliorated the dysregulation of microbiota diversity in a model of AP combined with lung injury and affected fatty acid metabolism by lowering triglyceride levels, which were closely related to the alteration in the relative abundance of Erysipelatoclostridium and Akkermansia. In addition, C. butyricum treatment attenuated pathological damage in the pancreatic and lung tissues and significantly suppressed the expression of inflammatory factors in mice. CONCLUSIONS: C. butyricum may alleviate lung injury associated with AP by interfering with the relevant intestinal microbiota and modulating relevant metabolic pathways.

An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts.

[Figure: see text].

Quantifying the contribution of Fc-mediated effector functions to the antiviral activity of anti-HIV-1 IgG1 antibodies in vivo.

In combating viral infections, the Fab portion of an antibody could mediate virus neutralization, whereas Fc engagement of Fc-γ receptors (FcγRs) could mediate an array of effector functions. Evidence abounds that effector functions are important in controlling infections by influenza, Ebola, or HIV-1 in animal models. However, the relative contribution of virus neutralization versus effector functions to the overall antiviral activity of an antibody remains unknown. To address this fundamental question in immunology, we utilized our knowledge of HIV-1 dynamics to compare the kinetics of the viral load decline (ΔVL) in infected animals given a wild-type (WT) anti-HIV-1 immunoglobulin G1 (IgG1) versus those given a Fc-Null variant of the same antibody. In three independent experiments in HIV-1-infected humanized mice and one pivotal experiment in simian-human immunodeficiency virus (SHIV)-infected rhesus macaques, an earlier and sharper decline in viral load was consistently detected for the WT antibody. Quantifications of the observed differences indicate that Fc-mediated effector functions accounted for 25-45% of the total antiviral activity in these separate experiments. In this study, Fc-mediated effector functions have been quantified in vivo relative to the contribution of virus neutralization mediated by the Fab.

Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity.

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.