Respiratory Syncytial Virus Inhibitors Research Articles

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Synthesis and biological evaluation of cleistocaltone A, an inhibitor of respiratory syncytial virus (RSV).

The first chemical synthesis of the phloroglucinol meroterpenoid cleistocaltone A (1) is presented. This compound, previously isolated from Cleistocalyx operculatus was reported to show promising antiviral properties. Based on a modified biosynthesis proposal, a synthetic strategy was devised featuring an intramolecular Diels-Alder reaction and an epoxidation/elimination sequence to generate the allyl alcohol handle in the side chain. The strategy was successfully executed and synthetic cleistcaltone A was evaluated against a contemporary RSV-A strain.

Inhibition of respiratory syncytial virus by Daclatasvir and its derivatives: synthesis of computational derivatives as a new drug development

The most common cause of respiratory tract illness in newborns and young children is the respiratory syncytial virus (RSV). There is no approved vaccination or specific antiviral medication for RSV infections. Here, an attempt has been made to explore the potential of currently marketed drugs as well as their probable derivatives to improve the possibility of developing stronger medications against RSV. From the 100 synthetic drug compounds library, the best drug molecule was identified through drug-likeness properties, toxicity, molecular docking and molecular dynamics simulations. Molecular Mechanics Generalized Born Surface Area (MM-GBSA) was also a method that was applied in this study. Daclatasvir showed the highest binding energy and appeared as the best drug to inhibit matrix protein and a fusion protein of RSV. Based on Daclatasvir, 40 computational derivatives were made. D28, D34 and D40 showed far better results than the actual drug. Changes in lipophilicity character increase the binding energy of derivatives. Molecular dynamic simulations showed their non-deviated, non-fluctuated and stable complex formation with target proteins. The high number of amino acid contacts throughout the trajectory increases the stability and effectiveness of derivatives. The key to producing a novel medicine to eradicate RSV is provided by derivatives. Daclatasvir will be employed as a potential RSV inhibitor up until that point. Communicated by Ramaswamy H. Sarma

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1–3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.

Identification and evaluation of a novel tribenzamide derivative as an inhibitor targeting the entry of the respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants, the elderly, and the immunocompromised, yet no licensed vaccine and only limited therapeutic options for prevention and treatment are available, which poses a global health challenge and emphasizes the urgent medical need for novel antiviral agents. In the current study, a novel potent small molecule inhibitor of RSV was identified by performing a screening and structure optimization campaign, wherein a naturally occurring dicaffeoylquinic acid (DCQA) compound served as a chemical starting point. The reported benzamide derivative inhibitor, designated as 2f, was selected for its improved stability and potent antiviral activity from a series of investigated structurally related compounds. 2f was well tolerated by cells and able to inhibit RSV infection with a half maximal inhibitory concentration (IC50) of 35 nM and a favorable selectivity index (SI) of 3742. Although the exact molecular target for 2f is not known, in vitro mechanism of action investigations revealed that the compound inhibits the early stage of infection by interacting with RSV virion and interferes primarily with the attachment and potentially with the virus-cell fusion step. Moreover, intranasal administration of 2f to mice simultaneously or prior to intranasal infection with RSV significantly decreased viral load in the lungs, pointing to the in vivo potential of the compound. Our results suggest that 2f is a viable candidate for further preclinical development and evaluation as an antiviral agent against RSV infections.

Immunity Cell Responses to RSV and the Role of Antiviral Inhibitors: A Systematic Review.

Antigen-presenting cells recognize respiratory syncytial virus antigens, and produce cytokines and chemokines that act on immune cells. Dendritic cells play the main role in inflammatory cytokine responses. Similarly, alveolar macrophages produce IFN-β, IFN-α, TNF-α, IL-6, CXCL10, and CCL3, while alternatively activated macrophages differentiate at the late phase, and require IL-13 or IL-4 cytokines. Furthermore, activated NKT cells secrete IL-13 and IL-4 that cause lung epithelial, endothelial and fibroblasts to secrete eotaxin that enhances the recruitment of eosinophil to the lung. CD8+ and CD4+T cells infection by the virus decreases the IFN-γ and IL-2 production. Despite this, both are involved in terminating virus replication. CD8+T cells produce a larger amount of IFN-γ than CD4+T cells, and CD8+T cells activated under type 2 conditions produce IL-4, down regulating CD8 expression, granzyme and IFN-γ production. Antiviral inhibitors inhibit biological functions of viral proteins. Some of them directly target the virus replication machinery and are effective at later stages of infection; while others inhibit F protein dependent fusion and syncytium formation. TMC353121 reduces inflammatory cytokines, TNF-α, IL-6, and IL-1β and chemokines, KC, IP-10, MCP and MIP1-α. EDP-938 inhibits viral nucleoprotein (N), while GRP-156784 blocks the activity of respiratory syncytial virus ribonucleic acid (RNA) polymerase. PC786 inhibits non-structural protein 1 (NS-1) gene, RANTES transcripts, virus-induced CCL5, IL-6, and mucin increase. In general, it is an immune reaction that is blamed for the disease severity and pathogenesis in respiratory syncytial virus infection. Anti-viral inhibitors not only inhibit viral entry and replication, but also may reduce inflammatory cytokines and chemokines. Many respiratory syncytial virus inhibitors are proposed; however, only palivizumab and ribavirin are approved for prophylaxis and treatment, respectively. Hence, this review is focused on immunity cell responses to respiratory syncytial virus and the role of antiviral inhibitors.

Identification of a βCD-Based Hyper-Branched Negatively Charged Polymer as HSV-2 and RSV Inhibitor

Cyclodextrins and cyclodextrin derivatives were demonstrated to improve the antiviral potency of numerous drugs, but also to be endowed with intrinsic antiviral action. They are suitable building blocks for the synthesis of functionalized polymer structures with potential antiviral activity. Accordingly, four water-soluble hyper-branched beta cyclodextrin (βCD)-based anionic polymers were screened against herpes simplex virus (HSV-2), respiratory syncytial virus (RSV), rotavirus (HRoV), and influenza virus (FluVA). They were characterized by FTIR-ATR, TGA, elemental analyses, zeta-potential measurements, and potentiometric titrations, while the antiviral activity was investigated with specific in vitro assays. The polymer with the highest negative charge, pyromellitic dianhydride-linked polymer (P_PMDA), showed significant antiviral action against RSV and HSV-2, by inactivating RSV free particles and by altering HSV-2 binding to the cell. The polymer fraction with the highest molecular weight showed the strongest antiviral activity and both P_PMDA and its active fractions were not toxic for cells. Our results suggest that the polymer virucidal activity against RSV can be exploited to produce new antiviral materials to counteract the virus dissemination through the air or direct contact. Additionally, the strong HSV-2 binding inhibition along with the water solubility of P_PMDA and the acyclovir complexation potential of βCD are attractive features for developing new therapeutic topical options against genital HSV-2 infection.

EDP-938 demonstrates efficacy against RSV

EDP-938 demonstrates efficacy against RSV

EDP-938, a Respiratory Syncytial Virus Inhibitor, in a Human Virus Challenge

BackgroundRespiratory syncytial virus (RSV) infection causes substantial morbidity and mortality among infants, older adults, and immunocompromised adults. EDP-938, a nonfusion replication inhibitor of RSV, acts by modulating the viral nucleoprotein.MethodsIn a two-part, phase 2a, randomized, double-blind, placebo-controlled challenge trial, we assigned participants who had been inoculated with RSV-A Memphis 37b to receive EDP-938 or placebo. Different doses of EDP-938 were assessed. Nasal-wash samples were obtained from day 2 until day 12 for assessments. Clinical symptoms were assessed by the participants, and pharmacokinetic profiles were obtained. The primary end point was the area under the curve (AUC) for the RSV viral load, as measured by reverse-transcriptase–quantitative polymerase-chain-reaction assay. The key secondary end point was the AUC for the total symptom score.ResultsIn part 1 of the trial, 115 participants were assigned to receive EDP-938 (600 mg once daily [600-mg once-daily group] or 300 mg twice daily after a 500-mg loading dose [300-mg twice-daily group]) or placebo. In part 2, a total of 63 participants were assigned to receive EDP-938 (300 mg once daily after a 600-mg loading dose [300-mg once-daily group] or 200 mg twice daily after a 400-mg loading dose [200-mg twice-daily group]) or placebo. In part 1, the AUC for the mean viral load (hours×log10 copies per milliliter) was 204.0 in the 600-mg once-daily group, 217.7 in the 300-mg twice-daily group, and 790.2 in the placebo group. The AUC for the mean total symptom score (hours×score, with higher values indicating greater severity) was 124.5 in the 600-mg once-daily group, 181.8 in the 300-mg twice-daily group, and 478.8 in the placebo group. The results in part 2 followed a pattern similar to that in part 1: the AUC for the mean viral load was 173.9 in the 300-mg once-daily group, 196.2 in the 200-mg twice-daily group, and 879.0 in the placebo group, and the AUC for the mean total symptom score was 99.3, 89.6, and 432.2, respectively. In both parts, mucus production was more than 70% lower in each EDP-938 group than in the placebo group. The four EDP-938 regimens had a safety profile similar to that of placebo. Across all dosing regimens, the EDP-938 median time to maximum concentration ranged from 4 to 5 hours, and the geometric mean half-life ranged from 13.7 to 14.5 hours.ConclusionsAll EDP-938 regimens were superior to placebo with regard to lowering of the viral load, total symptom scores, and mucus weight without apparent safety concerns. (ClinicalTrials.gov number, NCT03691623.)

Monoterpene-Containing Substituted Coumarins as Inhibitors of Respiratory Syncytial Virus (RSV) Replication.

Respiratory syncytial virus (RSV) is a critical cause of infant mortality. However, there are no vaccines and adequate drugs for its treatment. We showed, for the first time, that O-linked coumarin–monoterpene conjugates are effective RSV inhibitors. The most potent compounds are active against both RSV serotypes, A and B. According to the results of the time-of-addition experiment, the conjugates act at the early stages of virus cycle. Based on molecular modelling data, RSV F protein may be considered as a possible target.

Statin-mediated disruption of Rho GTPase prenylation and activity inhibits respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory tract infections in children. To uncover new antiviral therapies, we developed a live cell-based high content screening approach for rapid identification of RSV inhibitors and characterized five drug classes which inhibit the virus. Among the molecular targets for each hit, there was a strong functional enrichment in lipid metabolic pathways. Modulation of lipid metabolites by statins, a key hit from our screen, decreases the production of infectious virus through a combination of cholesterol and isoprenoid-mediated effects. Notably, RSV infection globally upregulates host protein prenylation, including the prenylation of Rho GTPases. Treatment by statins or perillyl alcohol, a geranylgeranyltransferase inhibitor, reduces infection in vitro. Of the Rho GTPases assayed in our study, a loss in Rac1 activity strongly inhibits the virus through a decrease in F protein surface expression. Our findings provide new insight into the importance of host lipid metabolism to RSV infection and highlight geranylgeranyltransferases as an antiviral target for therapeutic development.

Role of G2-S16 Polyanionic Carbosilane Dendrimer in the Prevention of Respiratory Syncytial Virus Infection In Vitro and In Vivo in Mice.

The respiratory syncytial virus (RSV) causes respiratory infection and bronchiolitis, requiring hospitalization mainly in infants. The interaction between RSV, envelope glycoproteins G and F, and cell surface heparan sulfate proteoglycans (HSPG) is required for binding and entry into the host cells. A G2-S16 polyanionic carbosilane dendrimer was identified as a possible RSV inhibitor. We speculated that the G2-S16 dendrimer adheres to the host cell-surface HSPG, acts through binding to HS receptors, and prevents further RSV infection. The G2-S16 dendrimer was non-toxic when applied intranasally to Balb/c mice, and interestingly enough, this G2-S16 dendrimer inhibits 85% RSV. Therefore, our G2-S16 dendrimer could be a candidate for developing a new possible therapy against RSV infection.

Discovery of a Novel Respiratory Syncytial Virus Replication Inhibitor.

ABSTRACTA high-throughput screen of a Roche internal chemical library based on inhibition of the respiratory syncytial virus (RSV)-induced cytopathic effect (CPE) on HEp-2 cells was performed to identify RSV inhibitors. Over 2,000 hits were identified and confirmed to be efficacious against RSV infection in vitro. Here, we report the discovery of a triazole-oxadiazole derivative, designated triazole-1, as an RSV replication inhibitor, and we characterize its mechanism of action. Triazole-1 inhibited the replication of both RSV A and B subtypes with 50% inhibitory concentration (IC50) values of approximately 1 μM, but it was not effective against other viruses, including influenza virus A, human enterovirus 71 (EV71), and vaccinia virus. Triazole-1 was shown to inhibit RSV replication when added at up to 8 h after viral entry, suggesting that it inhibits RSV after viral entry. In a minigenome reporter assay in which RSV transcription regulatory sequences flanking a luciferase gene were cotransfected with RSV N/P/L/M2-1 genes into HEp-2 cells, triazole-1 demonstrated specific and dose-dependent RSV transcription inhibitory effects. Consistent with these findings, deep sequencing of the genomes of triazole-1-resistant mutants revealed a single point mutation (A to G) at nucleotide 13546 of the RSV genome, leading to a T-to-A change at amino acid position 1684 of the L protein, which is the RSV RNA polymerase for both viral transcription and replication. The effect of triazole-1 on minigenome transcription, which was mediated by the L protein containing the T1684A mutation, was significantly reduced, suggesting that the T1684A mutation alone conferred viral resistance to triazole-1.

In silico virtual screening of lead compounds for major antigenic sites in respiratory syncytial virus fusion protein

Human respiratory syncytial virus (RSV) is a leading ubiquitous respiratory pathogen in newborn infants, young children, and the elderly, with no vaccine available to date. The viral fusion glycoprotein (RSV F) plays an essential role in the infection process, and it is a primary target of neutralizing antibodies, making it an attractive site for vaccine development. With this in view, there is a persistent need to identify selective antiviral drugs against RSV, targeting the major antigenic sites on the F protein. We aimed to conduct a robust in silico high-throughput drug screening of one million compounds to explore potential inhibitors that bind the major antigenic site Ø and site II on RSV F protein, which are the main target of neutralizing antibodies (NAb). We utilized the three-dimensional crystallographic structure of both antigenic site Ø on pre-F and antigenic II on post-F to screen for potential anti-RSV inhibitors. A library of one million small compounds was docked to explore lead binders in the major antigenic sites by using virtual lab bench CLC Drug Discovery. We also performed Quantitative Structure-Activity and Relationship (QSAR) for the lead best binders known for their antiviral activity. Among one million tested ligands, seven ligands (PubChem ID: 3714418, 24787350, 49828911, 24802036, 79824892, 49726463, and 3139884) were identified as the best binders to neutralizing epitopes site Ø and four ligands (PubChem ID: 865999, 17505357, 24802036, and 24285058) to neutralizing epitopes site II, respectively. These binders exhibited significant interactions with neutralizing epitopes on RSV F, with an average of six H bonds, docking energy of − 15.43 Kcal·mol−1, and minimum interaction energy of − 7.45 Kcal·mol−1. Using in silico virtual screening, we identified potential RSV inhibitors that bind two major antigenic sites on the RSV F protein. Using structure-based design and combination-based drug therapy, identified molecules could be modified to generate the next generation anti-RSV drugs.

Substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues potently inhibit respiratory syncytial virus (RSV) replication and RSV infection-associated inflammatory responses

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children, and specific treatment for RSV infections remains unavailable. We herein reported a series of substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as potent RSV inhibitors. Among them, six low cytotoxic compounds (11, 12, 15, 22, 26, and 28) have been identified and selected to study associated inhibitory mechanisms. All these compounds suppressed not only the viral replication but also RSV-induced IRF3 and NF-κB activation and associated production of cytokines/chemokines. The two most potent compounds (15 and 22) were selected for further molecular mechanism studies associated with their suppression effect on RSV-activated IRF3 and NF-κB. These two compounds decreased RSV-induced IRF3 phosphorylation at serine 396 and p65 phosphorylation at serine 536 at both early and late infection phases. In addition, compound 22 also inhibited RSV-induced p65 phosphorylation at serine 276 at the late phase of RSV infection.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

Discovery of Sisunatovir (RV521), an Inhibitor of Respiratory Syncytial Virus Fusion.

RV521 is an orally bioavailable inhibitor of respiratory syncytial virus (RSV) fusion that was identified after a lead optimization process based upon hits that originated from a physical property directed hit profiling exercise at Reviral. This exercise encompassed collaborations with a number of contract organizations with collaborative medicinal chemistry and virology during the optimization phase in addition to those utilized as the compound proceeded through preclinical and clinical evaluation. RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A and B laboratory strains and clinical isolates with antiviral efficacy in the Balb/C mouse model of RSV infection. Oral bioavailability in preclinical species ranged from 42 to >100% with evidence of highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a significant reduction in viral load and symptoms compared to placebo.

EDP-938, a novel nucleoprotein inhibitor of respiratory syncytial virus, demonstrates potent antiviral activities in vitro and in a non-human primate model.

EDP-938 is a novel non-fusion replication inhibitor of respiratory syncytial virus (RSV). It is highly active against all RSV-A and B laboratory strains and clinical isolates tested in vitro in various cell lines and assays, with half-maximal effective concentrations (EC50s) of 21, 23 and 64 nM against Long (A), M37 (A) and VR-955 (B) strains, respectively, in the primary human bronchial epithelial cells (HBECs). EDP-938 inhibits RSV at a post-entry replication step of the viral life cycle as confirmed by time-of-addition study, and the activity appears to be mediated by viral nucleoprotein (N). In vitro resistance studies suggest that EDP-938 presents a higher barrier to resistance compared to viral fusion or non-nucleoside L polymerase inhibitors with no cross-resistance observed. Combinations of EDP-938 with other classes of RSV inhibitors lead to synergistic antiviral activity in vitro. Finally, EDP-938 has also been shown to be efficacious in vivo in a non-human primate model of RSV infection.

Repurposing of antiparasitic niclosamide to inhibit respiratory syncytial virus (RSV) replication

Despite being an important health problem, there are only supportive care treatments for respiratory syncytial virus (RSV) infection. Thus, discovery of specific therapeutic drugs for RSV is still needed. Recently, an antiparasitic drug niclosamide has shown a broad-spectrum antiviral activity. Here, our in vitro model was used to study the antiviral effect of niclosamide on RSV and its related mechanism. Niclosamide inhibited RSV with time and dose-dependent manner. Pretreatment with submicromolar concentration of niclosamide for 6 h presented the highest anti-RSV activity of 94 % (50 % effective concentration; EC50 of 0.022 μM). Niclosamide efficiently blocked infection of laboratory strains and clinical isolates of both RSV-A and RSV-B in a bronchial epithelial cell line. Although a disruption of the mechanistic target of rapamycin complex 1 (mTORC1) pathway by niclosamide was previously hypothesized as a mechanism against pH-independent viruses like RSV, using a chemical mTORC1 inhibitor, temsirolimus, and a chemical mTORC1 agonist, MHY1485 (MHY), we show here that the mechanism of RSV inhibition by niclosamide was mTORC1 independent. Indeed, our data indicated that niclosamide hindered RSV infection via proapoptotic activity by a reduction of AKT prosurvival protein, activation of cleaved caspase-3 and PARP (poly ADP-ribose polymerase), and an early apoptosis induction.

Micro-fusion inhibition tests: quantifying antibody neutralization of virus-mediated cell-cell fusion.

Although enveloped viruses canonically mediate particle entry through virus–cell fusion, certain viruses can spread by cell–cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell–cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell–cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell–cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP–Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell–cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.