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

Brecht Bonneux, Afzaal Shareef, Sergey Tcherniuk, Brandon Anson, Suzanne de Bruyn, Nick Verheyen, Kim Thys, Nádia Conceição-Neto, Marcia van Ginderen, Leen Kwanten, et al.

► To cite this version:

Brecht Bonneux, Afzaal Shareef, Sergey Tcherniuk, Brandon Anson, Suzanne de Bruyn, et al., JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase. Antiviral Research, 2024, 227, pp.105907. 10.1016/j.antiviral.2024.105907. hal-04633274

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

Brecht Bonneux<sup>a,b,1</sup>, Afzaal Shareef<sup>c,1,2</sup>, Sergey Tcherniuk<sup>d,1,3</sup>, Brandon Anson<sup>e,4</sup>, Suzanne de Bruyn<sup>b,5</sup>, Nick Verheyen<sup>b,3</sup>, Kim Thys<sup>b</sup>, Nádia Conceição-Neto<sup>b</sup>, Marcia Van Ginderen<sup>b,3</sup>, Leen Kwanten<sup>b</sup>, Nina Ysebaert<sup>b</sup>, Luc Vranckx<sup>b,3</sup>, Elien Peeters<sup>b</sup>, Ellen Lanckacker<sup>b,6</sup>, Jack M. Gallup<sup>f,7</sup>, Panchan Sitthicharoenchai<sup>f,8</sup>, Sarhad Alnajjar<sup>f,9</sup>, Mark R. Ackermann<sup>f,10</sup>, Suraj Adhikary<sup>e</sup>, Anusarka Bhaumik<sup>e</sup>, Aaron Patrick<sup>e</sup>, Amy Fung<sup>e,3</sup>, Priscila Sutto-Ortiz<sup>g</sup>, Etienne Decroly<sup>g</sup>, Stephen W. Mason<sup>e,3</sup>, David Lançois<sup>h,11</sup>, Jerome Deval<sup>e,12</sup>, Zhinan Jin<sup>e,3</sup>, Jean-François Eléouët<sup>d</sup>, Rachel Fearns<sup>c</sup>, Anil Koul<sup>b,**</sup>, Dirk Roymans<sup>b,13</sup>, Peter Rigaux<sup>b,3</sup>, Florence Herschke<sup>b,5,*</sup>

## ARTICLE INFO

### Keywords:

Respiratory syncytial virus

Direct anti-viral

Non-nucleos(t)ide inhibitor

Viral polymerase

Connector domain

## ABSTRACT

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

<footnote><sup>a</sup> Laboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Universiteitsbaan 1, 2610 Wilrijk, Belgium</footnote>

<footnote><sup>b</sup> Janssen Pharmaceutica NV, Turnhoutseweg 30, 2340 Beerse, Belgium</footnote>

<footnote><sup>c</sup> Department of Virology, Immunology & Microbiology, National Emerging Infectious Diseases Laboratories, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA</footnote>

<footnote><sup>d</sup> Unité de Virologie et Immunologie Moléculaires (VIM, UMR892), INRAE, Université Paris-Saclay, 78352 Jouy-en-Josas, France</footnote>

<footnote><sup>e</sup> Janssen Research & Development LLC, Spring House (PA 19477) And Brisbane (CA 94005), USA</footnote>

<footnote><sup>f</sup> Iowa State University, Ames, IA 50011, USA</footnote>

<footnote><sup>g</sup> AFMB, Aix-Marseille University, CNRS UMR 7257, 163 Avenue de Luminy, Marseille, France</footnote>

<footnote><sup>h</sup> Janssen France, 27106 Val de Reuil, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.
E-mail addresses: akoul@its.jnj.com (A. Koul), fherschk@its.jnj.com (F. Herschke).</footnote>

<footnote n="1">1 These authors contributed equally.</footnote>

<footnote n="2">2 Current address: Tulane University, LA, USA.</footnote>

<footnote n="3">3 Independent researcher.</footnote>

<footnote n="4">4 Current address: Ambry Genetics, CA, USA.</footnote>

<footnote n="5">5 Current address: Exevir Bio, Belgium.</footnote>

<footnote n="6">6 Current address: Galapagos NV, Belgium.</footnote>

<footnote n="7">7 Current address: Vaxxinova US at Worthington, MN, USA.</footnote>

<footnote n="8">8 Current address: North Carolina State University, NC, USA.</footnote>

<footnote n="9">9 Current address: Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7 A L UK.</footnote>

<footnote n="10">10 Current address: National Animal Disease Center, IA, USA.</footnote>

<footnote n="11">11 Current address: NovAliX, France.</footnote>

<footnote n="12">12 Current address: Bluejay Therapeutics, CA, USA.</footnote>

<footnote n="13">13 Current address: DNS Life Science Consulting BV, Belgium.</footnote>
transcription by inhibiting initiation or early elongation. JN7-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Abbreviations

<table><tr><td>ATCC</td><td>American Tissue Culture Collection</td></tr><tr><td>BALF</td><td>broncho-alveolar lavage fluid</td></tr><tr><td>BEH</td><td>ethylene bridged hybrid</td></tr><tr><td>Cap</td><td>polyribonucleotidyltransferase, capping domain of RSV-L</td></tr><tr><td>CC50</td><td>cytotoxicity concentration 50%</td></tr><tr><td>CD</td><td>connector domain</td></tr><tr><td>(c)DNA</td><td>(complementary) deoxyribonucleic acid</td></tr><tr><td>CMV</td><td>Cytomegalovirus (species name: Cytomegalovirus humanbeta5)</td></tr><tr><td>CPE</td><td>cytopathic effect</td></tr><tr><td>cryoEM</td><td>cryogenic electron microscopy</td></tr><tr><td>CTD</td><td>C-terminal domain</td></tr><tr><td>DENV</td><td>Dengue virus (species name: Orthoflavivirus dengue)</td></tr><tr><td>DMEM</td><td>Dulbecco's Modified Eagle Medium</td></tr><tr><td>DMSO</td><td>dimethylsulfoxide</td></tr><tr><td>dNTP</td><td>deoxyribonucleotide triphosphate</td></tr><tr><td>EC<sub>50/90</sub></td><td>effective concentration 50% or 90%</td></tr><tr><td>EDTA</td><td>ethylenediaminetetraacetic acid</td></tr><tr><td>eGFP</td><td>enhanced green fluorescent protein</td></tr><tr><td>FCS</td><td>fetal calf serum</td></tr><tr><td>FFPE</td><td>formalin-fixed paraffin-embedded</td></tr><tr><td>HBSS</td><td>Hank's buffered salt solution</td></tr><tr><td>HCI</td><td>high content imaging</td></tr><tr><td>HDX</td><td>hydrogen-deuterium exchange</td></tr><tr><td>H&E</td><td>hematoxylin & eosin</td></tr><tr><td>HDMSE</td><td>high-definition mass spectrometry</td></tr><tr><td>HEPES</td><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td></tr><tr><td>HIV</td><td>Human immunodeficiency virus</td></tr><tr><td>HMPV</td><td>Human metapneumovirus (species name: Metapneumovirus hominis)</td></tr><tr><td>hr(s)</td><td>hour(s)</td></tr><tr><td>HP-β-CD</td><td>hydroxypropyl-b-cyclodextrin</td></tr><tr><td>IACUC</td><td>institutional animal care and use committee</td></tr><tr><td>IC<sub>50</sub></td><td>inhibitory concentration 50%</td></tr></table>

<table><tr><td>IFFU</td><td>infectious focus-forming units</td></tr><tr><td>IHC</td><td>immunohistochemistry</td></tr><tr><td>IMS</td><td>ion mobility spectrometry</td></tr><tr><td>IN</td><td>intranasal</td></tr><tr><td>IVRS</td><td>in vitro resistance selection</td></tr><tr><td>L</td><td>large protein of RSV</td></tr><tr><td>Le</td><td>leader</td></tr><tr><td>M</td><td>matrix protein of RSV</td></tr><tr><td>MOI</td><td>multiplicity of infection</td></tr><tr><td>MTase</td><td>methyltransferase</td></tr><tr><td>N</td><td>nucleoprotein of RSV</td></tr><tr><td>NA</td><td>nucleic acid</td></tr><tr><td>NIH</td><td>National Institutes of Health</td></tr><tr><td>NNI</td><td>non-nucleoside inhibitor</td></tr><tr><td>nt</td><td>nucleotide</td></tr><tr><td>P</td><td>phosphoprotein of RSV</td></tr><tr><td>PBS</td><td>phosphate-buffered saline</td></tr><tr><td>(q)PCR</td><td>(quantitative) polymerase chain reaction</td></tr><tr><td>PFU</td><td>Plaque forming unit</td></tr><tr><td>PLGS</td><td>ProteinLynx Global Server</td></tr><tr><td>RdRp</td><td>RNA-dependent RNA polymerase</td></tr><tr><td>(m)RNA</td><td>(messenger) ribonucleic acid</td></tr><tr><td>RNase</td><td>ribonuclease</td></tr><tr><td>RPMI</td><td>Roswell Park Memorial Institute</td></tr><tr><td>RSV</td><td>Respiratory syncytial virus (species name: Orthopneumovirus hominis)</td></tr><tr><td>RT</td><td>reverse transcription</td></tr><tr><td>SD</td><td>standard deviation</td></tr><tr><td>SI</td><td>selectivity index</td></tr><tr><td>TCEP</td><td>tris (2-carboxyethyl) phosphine</td></tr><tr><td>Tr</td><td>trailer</td></tr><tr><td>UPLC</td><td>ultra performance liquid chromatography</td></tr><tr><td>VL</td><td>viral load</td></tr><tr><td>vRNP</td><td>viral ribonucleoprotein</td></tr><tr><td>VSV</td><td>Vesicular stomatitis virus (species name: Vesiculovirus indiana)</td></tr></table>

1. Introduction

RSV is a leading cause of lower respiratory tract infections in infants (Leader and Kohlhase, 2002) and in long-term care facilities. In the latter context, RSV is estimated to infect 5%–10% of the residents per year with significant rates of pneumonia (i.e., 10%–20%) and death (i.e., 2%–5%) (Falsey and Walsh, 2005). It is also a common cause of respiratory complications in populations with underlying chronic cardiopulmonary conditions (Buchholz et al., 2022). To date, the monoclonal antibodies palivizumab and nirsevimab are approved for RSV prevention in high-risk infants and demonstrated an efficacy of 40%–80% in prevention of hospitalization depending on the underlying risk condition (Wegzyn et al., 2014; Jones et al., 2023). Two prophylactic vaccines have been approved for either older adults or women in the 3rd trimester of pregnancy, and a third vaccine is under regulatory review (See, 2023). However, no effective treatment options are currently available.

The RSV entry inhibitors ziresovir and sisunatovir, targeting the RSV fusion protein, are in clinical development, and ziresovir has

demonstrated some efficacy in a phase IIb/III trial in children (https://www.pfizer.com/science/drug-product-pipeline, as of 2 April 2024), (Huang et al., 2023). Other RSV fusion inhibitors have been stopped for lack of efficacy/reprioritization (e.g., presatovir, rilematovir) (Marty et al., 2020), (Chemaly et al., 2020), (https://clinicaltrials.gov/ct2/show/NCT04583280?term=rilematovir&draw=2&rank=2, as of 2 April 2024), (https://clinicaltrials.gov/ct2/-show/NCT04978337?term=rilematovir&draw=2&rank=1, as of 2 April 2024). Post-entry inhibitors currently in early clinical development target the viral nucleoprotein (https://ir.enanta.com/news-releases/news-release-details/enanta-pharmaceuticals-initiates-phase-2b-clinical-study-edp-938, published on 3 October 2022) or the viral RNA polymerase (RSV-L) (https://www.pfizer.com/science/drug-product-pipeline, as of 2 April 2024), (DeVincenzo et al., 2022), a multidomain enzyme bearing RNA-dependent RNA polymerase (RdRp) activity, polyribonucleotidyltransferase (PRNTase) and nucleotide triphosphatase (NTPase) activities for cap addition, and methyltransferase (MTase) activity (Gilman et al., 2019), (Noton et al., 2019), (Sutto-Ortiz et al., 2021). Current inhibitors of RSV-L target the RdRp and capping activities, but do not exploit the MTase activity of the viral polymerase. Here, we describe the antiviral profile
of JNJ-7184, a non-nucleoside inhibitor interacting with the connector domain (CD) located between the PRNTase and MTase domains of RSV-L.

## 2. Material and methods

### 2.1. RSV-A2-eGFP reporter assay in HeLa cells

Assay was performed in HeLa cells as in ref. (Roymans et al., 2017), using rgRSV224 (RSV-A2-eGFP strain, Dr. ME Peeples, NIH/Nationwide Children's Hospital, US; wild-type or mutants).

### 2.2. In vitro resistance selection

HeLa cells were maintained in culture medium as in 2.1. They were infected with rgRSV224 (see 2.1) at a starting MOI of 0.01, in triplicate with ~10 × EC₉₀ (0.45 µM) or in singlicate without compound. A viral passage was defined as maximal eGFP fluorescence and 80–90% CPE. At each passage, 1:100 clarified supernatant (by centrifugation for 10 min at 3000 rpm) was used to initiate subsequent passage, for a total of 10 passages. At each passage, RNA was extracted from 100 µL clarified supernatant, on a MagNApure instrument, with the DNA and Viral NA small volume kit, and the Pathogen Universal 300 protocol (all from Roche). cDNA libraries were prepared from 1 ng RNA using a Revelo RNA-Seq High Sensitivity library preparation kit (Tecan) and sequencing was performed on an Illumina MiSeq platform (Pair-end sequencing, 300 cycles), targeting 5 M reads per sample). Mutations were compared to the parental virus and minus inhibitor control, using a 15% cut-off for the viral RNA population frequency.

### 2.3. HDX-MS

Coverage maps were obtained from duplicate undeuterated controls by diluting 10 µL 1.2 µM sample in Buffer B (20 mM Hepes, pH 7.5, 500 mM NaCl, 5% glycerol, 1 mM TCEP) with 60 µL ice-cold quench (100 mM Glycine, 7.04 M Guanidine-HCl, 20 mM TCEP, pH 2.4) for 1 min, then adding 180 µL dilution buffer (100 mM Glycine, 20 mM TCEP, pH 2.4). A single repeat of undeuterated control was performed with 10 µL of 1.2 µM sample in Buffer B diluted with 40 µL of Buffer B, quenched with 50 µL quench for 1 min then adding 150 µL dilution buffer. Samples were injected into a Waters HDX nanoAcquity UPLC (Waters, Milford, MA) with in-line protease XIII/pepsin digestion (NovaBioAssays). Fragments were trapped on an Acquity UPLC BEH C18 peptide trap and separated on an Acquity UPLC BEH C18 column. A 10 min, 5%–35% acetonitrile (0.1% formic acid) gradient at 60 µL/min was used to elute peptides directly into a Waters Synapt G2-Si mass spectrometer (Waters, Milford, MA). HDMSE data were acquired with an IMS wave velocity of 600 m/s. High energy acquisition of product ions was performed with 20–30 V ramp trap CE and continuous lock mass (Leu-Enk) acquired for mass accuracy correction. Peptides were identified using ProteinLynx Global Server 3.0.3 (PLGS) (Waters). Filtering of 0.3 fragments per residue was applied in DynamX 3.0. For the apo and ligand bound states, HDX reactions and controls were acquired in duplicate using a LEAP-autosampler controlled by Chronos software, by incubating 10 µL of 1.2 µM protein in 40 µL of Buffer B, 99.99% D₂O, pD 7.5, at 25 °C. Deuteration reactions were quenched at 10, 100 and 1000 s with 50 µL quench, incubated for 1 min and adding 150 µL of dilution buffer. The deuterium uptake for all identified peptides as a function of increasing deuteration time was determined using Waters DynamX 3.0 software. Deuterium uptake difference plots ΔDt (Apo–Ligand bound) were generated and 95% confidence intervals used to assign statistically significant differences.

### 2.4. Transient replicon assay

$5 \times 10^4$ BSR-T7/5 per 96-well were seeded and co-transfected the

next day using lipofectamine 2000 (Thermo Fisher) with RSV-L, P, N and M2-1 expressing plasmids at 30, 60, 60 and 15 ng, respectively, 60 ng pM/Luc subgenomic minireplicon plasmid (containing a *wild-type* or *mutant* RSV-L) (Blondot et al., 2012) and 15 ng β-Gal normalization plasmid, in 100 µL DMEM, 10% FCS (all plasmids synthetized at Gen-Script). After 4 h, JNJ-7184 was added (100 µL final volume; 0.64% DMSO). After 24 h, cells were lysed with 30 mM Tris pH 7.9, 10 mM MgCl₂, 1 mM DTT, 1% Triton X-1000, 15% glycerol. Luciferase and β-Gal activities were measured using a Tecan Infinite 200 PRO (Tecan) as previously described (Sourimant et al., 2015). Compound cytotoxicity towards BSR-T7/5 cells was assessed using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).

### 2.5. Virus growth kinetics

pM/RSV-A2 genomic plasmids, containing an mCherry reporter gene between the P and M genes, and wild-type or mutant RSV-L, were designed by JFE and synthetized at GenScript. They were used to rescue wild-type or mutant reporter viruses, as described in section 2.4.

2000 $A^{549}$ cells (ATCC)/well were seeded in 384-well plates and infected at MOI 5. They were live-stained with CellTracker™ Green CMFDA Dye (0.1 µM) and NucBlue® Live ReadyProbes® Reagent for 1 h (both Invitrogen). Plates were imaged every hr for 48 h, at 37 °C, on a Yokogawa CV7000 high content imager.

### 2.6. Minigenome assay

RSV transcription and RNA replication were analyzed using a replication-deficient minigenome, described previously (Tremaglio et al., 2013). The minigenome contained two reporter genes, each flanked with a *gene start* and *gene end* sequence. The 3' and 5' ends of the minigenome contained the *leader* and *trailer* regions respectively. The *trailer* region contained a single nucleotide substitution at position 2 relative to the 5' end, limiting it to the antigenome step of RNA replication and uncoupling transcription from replication (Tremaglio et al., 2013). RNA replication from the *trailer* promoter was analyzed using a minigenome that contained nucleotides 1–36 of the *trailer* promoter in place of the *leader* region, first *gene start* signal, and *NS1* non-translated region. This minigenome was limited to a single step of replication by a 22 nt deletion at the 5' end of the *trailer* region (Noton et al., 2015). RSV transcription and replication was reconstituted by transfecting BSRT7/5 cells (kindly provided by Dr. Karl-Klaus Conzelmann, Ludwig Maximilians University) with the minigenome reporter plasmid and pTM1 plasmids expressing the RSV viral proteins N, P, M2-1 and L (note that a plasmid containing a codon-optimized L open reading frame was used rather than native sequence). JNJ-7184, dissolved in DMSO, was included in the cell culture media at concentrations of 5, 25, and 125 nM. Cells were harvested 48 h post transfection in TRIzol (Thermo Fisher) and RNA was extracted according to the manufacturer's instructions, followed by an additional phenol chloroform extraction step. Total intracellular RNA was analyzed by Northern blot analysis using minigenome positive and negative sense specific probes, as described previously (McGivern et al., 2005). Primer extension analysis to detect the RNAs initiated at +1U and +3C was performed by reverse transcription at 37 °C using Sensiscript reverse transcriptase (Qiagen) and $^{32}$P-end-labeled primers corresponding to nucleotides 15–39 of the *leader* region or nucleotides 13–35 of the *trailer* region, as described previously (Kleiner et al., 2023). Primer extension analysis to detect RNAs initiated at the first *gene start* signal was performed by reverse transcription using Molony murine leukemia virus reverse transcriptase (Promega) and a $^{32}$P-end-labeled primer corresponding to nucleotides 12–31 of the mRNA, as described previously (Kleiner et al., 2023). RNA products were detected and quantified by phosphorimager analysis. The value of the minus L lane was subtracted from each other value as background. The value for each sample with inhibitor was normalized to the mean of the two DMSO treated controls which was set to 1.
## 2.7. Primer extension assay using recombinant RSV-L/P

Recombinant RSV-L/P polymerase from RSV-A2 strain (0.01 µM, produced in insect cells, wild-type or mutant (Buchholz et al., 1999), in 200 mM Tris pH 7.5, 100 mM KCl, 2 mM dithiothreitol, 0.1% Triton, 10% v/v DMSO, was added to compound and 6 mM MgCl2. At 30 °C, a template derived from the RSV leader promoter (5'-UUUGUUCGCGU-3', 0.2 µM), biotinylated primer (5'-bi-ACGC, 4 µM), GTP (10 µM), ATP (10 µM) and [α-33 P] CTP (0.025 µM) were added in a 10 µL volume. After 120 min, 90 µL EDTA (0.1 M) was added. Samples were transferred to Flashplates for 1 h at RT, washed twice with 0.1% Tween 20, sealed (Topseal) and read on a Trilux instrument (PerkinElmer).

## 2.8. De novo initiation assay to differentiate initiation from +1U or +3C initiation sites from the trailer promoter

Reactions were performed with a tr14 template and [α³²P]-UTP tracer in 50 µL with the following conditions: 2 µM tr14 RNA template (Dharmacon), 50 mM Tris pH 7.4; 8 mM MgCl₂; 5 mM DTT; 10% glycerol, 1 mM ATP, 1 mM CTP, and 1 mM GTP with 10 µCi [α³²P]-UTP (6000 Ci/mmol). JNJ-7184 in DMSO was added to each respective reaction at a concentration of 25, 125, and 625 nM. Reactions were heated to 30 °C for 5 min before the addition of purified RSV-L/P complexes containing 100 ng of L protein. Reactions were incubated at 30 °C for 1 h and after the reaction was complete, the L protein was inactivated by incubating the reactions at 95 °C for 5 min. After the heat inactivation, reactions were treated with calf intestinal phosphatase (NEB) at 37 °C for 1 h to reduce the background signal from unincorporated [α³²P]-UTP. The final RNA products were subject to a phenol chloroform extraction and precipitated with ethanol. RNA pellets were resuspended in RNase free water and an equal volume of stop buffer (xylene cyanol, bromophenol blue, deionized formamide containing 20 mM EDTA). The reactions were analyzed on a 20% polyacrylamide gel containing 7 M urea in tris-borate-EDTA buffer followed by autoradiography. The phosphorimager analysis was used for quantification. The value of the minus L lane was subtracted from each other value as background. The value for each sample with inhibitor was normalized to the mean of the two DMSO treated controls which was set to 1.

## 2.9. De novo initiation from the +3 initiation site of the trailer promoter, visualizing all extension products

The reactions were performed as described by Cressey et al. (2022). The reactions contained 10 nM RSV-L/P, 2 µM *trailer* promoter template (Tr14, 3'-UGCUCUUUUUUUCA-5'), 500 µM GTP, 10 µM ATP, 170 nM [α³³P]ATP (3000 Ci/mmol), and 5% DMSO or JNJ-7184 at various concentrations, in a buffer consisted of 50 mM Tris pH7.4, 8 mM MgCl₂, 6 mM dithiothreitol, and 10% glycerol. The reactions were incubated at 30 °C for 1 h and then stopped by adding gel loading buffer. Samples were denatured at 95 °C for 5 min and run on a 22.5% polyacrylamide urea sequencing gel at 80 W with radiolabeled RNA markers generated via the T7 RNA polymerase. The gel was dried, exposed to a phosphor-screen, scanned on a Typhoon scanner (GE Healthcare), and quantified using ImageQuant (GE Healthcare). Bands were identified as extension products or initiation products by the 11 nucleotide ppGA-GAAAAAAAG and dinucleotide ppGA RNA markers, respectively. The markers were produced as described previously (Milligan and Uhlenbeck, 1989).

## 2.10. In vivo efficacy in the neonatal lamb model

The study was approved by the IACUC and by the Institutional Biosafety Committee at Iowa State University and performed as in ref. (Roymans et al., 2017). Briefly, lambs were nebulized with 6 mL hRSV strain M37 (1.27 × 10⁷ Infectious Forming Unit (IFFU)/mL in media containing 20% w/v sucrose) on day 0. Control negative lambs

nebulized with 6 mL virus free Mock media. One day post infection lambs in the treatment groups were treated with either 16, 4 or 1 mg/kg JNJ-184 in 20 % HP-β-CD, pH = 7.4 orally, once daily, for 5 days. Control Positive group treated with Vehicle only (20 % HP-β-CD, pH = 7.4). Lambs were monitored daily for the presence of clinical signs, and all lambs were humanely euthanized at day 6 post nebulization. Lungs were evaluated for gross pathology, BALF, and lung samples were taken immediately for other assays (FFU, histopathology, IHC, RNAscope and qPCR). Gross RSV lesions were assessed as the percentage of lung with RSV lesions. Histopathology was performed on H&E-stained FFPE sections as in ref. 28,29 and RSV lesions were scored on a scale of 0–4 (Table S3). These changes included bronchiolitis, syncytial cells, epithelial cells necrosis, epithelial cells hyperplasia, neutrophils, bronchiolar nodules, and perivascular nodules. Then an accumulative histological lesion score was determined by adding up the individualized scores, resulting in a score of 0–28 that represented the overall pathological changes in each tissue section. RSV infection was evaluated with immunohistochemical staining of RSV and quantification of immunopositive cells was performed as in ref. (Derscheid et al., 2014; Olivier et al., 2009) and scored as follows: 0 = 0, 1 = 1–10, 2 = 11–39, 3 = 40–99, 4 = >100 positive cells. Similar scorings were applied to tissue section stained for positive *in situ* RSV mRNA using RNAscope technique (Roymans et al., 2017). Of note, 4/6 and 2/6 lambs reached the final endpoint in the 4 and 16 mg/kg dosing groups, respectively. This was not linked to toxicological effects of JNJ-7184. Indeed, JNJ-7184 was extensively characterized for safety pharmacology, and no effect was observed at the doses tested here. Neonatal lambs were not fed colostrom, to enable RSV infection, and were not preventatively administered antibiotics. This means that they were very sensitive to opportunistic infections. The animals were lost to bacterial infection during the experiment, as attested by histopathology at autopsy. This phenomenon was also observed in other neonatal lamb studies, testing the effect of other RSV inhibitors, and was entirely resolved once antibiotics were administered preventatively.

# 3. Results

## 3.1. JNJ-7184 inhibits RSV-A and -B strains and directly inhibits the RSV RdRp

Derived from a hit discovered during a cell-based high-throughput screening, JNJ-7184 displayed an EC₅₀ and EC₉₀ of 14 and 53 nM, respectively (6.6 and 25 ng/mL) in a HeLa cell-based infection assay based on RSV-A2 reporter strain (Fig. 1A). It had a CC₅₀ of 52 µM (Fig. 1A), giving a therapeutic index (TI) of >3700-fold. JNJ-7184 inhibited RSV-A subgroup strains with a 5-fold higher potency than RSV-B subgroup strains, in a discrete panel of clinical isolates and laboratory strains (EC₅₀ median ± SD: 13 ± 2 nM on 11 RSV-A strains and 72 ± 2 nM on 13 RSV-B strains, respectively; Fig. 1B–Table S1), demonstrating a good breadth of strain coverage.

JNJ-7184 selectivity was examined against multiple viruses. Apart from weak activity against VSV, DENV-2, HIV-1 and CMV (EC₅₀ of 3.8–79 µM) (Table S4), no significant antiviral activity was observed against other viruses. Therefore, JNJ-7184 demonstrated a >270-fold selectivity index (SI) for RSV over other viruses.

In primary human nasal/bronchial epithelial cells grown at the air-liquid interface, prophylactic and therapeutic treatments with JNJ-7184 (i.e. initiated -1 h, +6 h or +24 h after infection) resulted in potent inhibition of RSV-A2. Similar EC₅₀ values of 10, 9, and 20 nM, respectively, as determined by reporter virus activity or RNA production via RT-qPCR (Fig. 1C–H). These results show that JNJ-7184 efficiently inhibits the virus in a system mimicking the physiological infection.

JNJ-7184 inhibited a stable RSV replicon at a similar EC₅₀ to that obtained in antiviral assays (EC₅₀: 19 nM; CC₅₀ > 25 µM; Table S5), as well as a transient RSV replicon (Fig. 1I), indicating that JNJ-7184 possessed a post-entry mode of action. The RSV RNA-dependent RNA
A

<figure><img src="image_4.png" /></figure>

<table><thead><tr><th colspan="3">pEC50</th><th colspan="3">pCC50</th></tr><tr><th>Median</th><th>SD</th><th>n</th><th>Median</th><th>SD</th><th>n</th></tr></thead><tbody><tr><td>7.86</td><td>0.21</td><td>24</td><td>4.29</td><td>0.16</td><td>25</td></tr></tbody></table>

B

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C

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D

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E

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F

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G

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H

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I

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J

<figure><img src="image_13.png" /><figcaption>Fig. 1. Antiviral efficacy of JNJ-7184</figcaption></figure>

A. **top**: JNJ-7184 chemical structure; **bottom**: antiviral activity against RSV-A2-eGFP reporter virus, and corresponding cytotoxicity in HeLa cells. Median and standard deviation are shown. N: number of independent experiments.

B. Efficacy against RSV-A and -B laboratory strains and clinical isolates, measured in HeLa cells by RT-qPCR; EC₅₀ from at least 2 independent experiments (except for the last 4 RSV-B strains where a single experiment was performed).

C-H. Efficacy against RSV-A2-eGFP reporter laboratory strain in primary human nasal epithelial cells grown at the air-liquid interface. Compound was administered 1 h prior to (C and F), 6 h after (D and G) or 24 h after (E and H) infection. Mean ± standard error of the mean (SEM) (n = 4) is shown; C to E: eGFP fluorescence; F to H: quantification of viral RNA in apical washes by RT-qPCR. LLOD = 10 RFU/well.

I. Efficacy in a transient RSV replicon assay. Mean and SD of at least 3 independent experiments are presented.

J. Efficacy in a RdRp primer extension enzymatic assay.
polymerase (RdRp) activity was inhibited in a primer extension assay based on the purified L and P proteins (EC50: 21 nM; Fig. 1J–Table S7), indicating that RSV polymerase activity was the primary target of JNJ-7184. JNJ-7184 displayed little to no detectable inhibition of human DNA polymerases α, β, and γ, human mitochondrial RNA polymerase, and human RNA polymerase II, showing its specificity for RSV (EC50: >40 µM; Fig. S1).

## 3.2. JNJ-7184 targets the connector domain of the RSV polymerase

During *in vitro* resistance selection (IVRS) experiments, constant compound pressure at ~10x EC90 resulted in the consistent appearance of substitutions in the CD of the RSV-L gene: in one replicate, A1479E (433-fold shift in EC50) appeared at passage 2, followed by appearance of I23L in the N-term domain and L2007I in the C-term domain at passage 8. In a second replicate, A1479T (71-fold shift in EC50) appeared at passage 2. In a third replicate, M1636T/S840G double substitution (28-fold shift in EC50) emerged at passages 1–2, then was replaced by M1636P/S840G (760-fold shift in EC50) at passages 3–10 (Fig. 2A–S2, Table 1). None of the resistance mutations selected were observed in the currently circulating RSV strains, whose sequence was deposited on the GISAID server.

Substitutions A1479E, Y1631H, H1632Q, A1479 S/H1632R or T1684A in the CD of RSV-L, but not elsewhere, also induced a potency shift in transient replicon, primer extension and cell-based virus infection assays (Fig. 2B–C, Tables S6–8).

Virus growth kinetics of the mutants were further tested (in the absence of test compound): substitution of A1479 to E or T slightly decreased virus growth, as measured using a high-content imaging assay (Fig. 2D). Double substitution I23L/L2007I possibly improved the growth of the virus containing the A1479E mutant (Fig. 2D). Mutations giving rise to M1636 T/P alone did not affect viral kinetics, however S840G, located close to the RdRp active site (in the proximity of the two β-sheets positioning the active site), clearly improved viral growth kinetics (Fig. 2D–S2). These observations suggest a potential interplay between the connector domain, the RdRp and other domains of RSV-L.

To further define the binding site for JNJ-7184 within RSV-L, we performed hydrogen-deuterium exchange (HDX) experiment. Proteolytic hydrolysis in the presence or absence of JNJ-7184 resulted in the protection of a single peptide (1628–1640) within the CD, confirming this region of RSV-L as the primary target of JNJ-7184 (Fig. 2E). The CD has not been resolved in cryo-EM structures of the apo-RSV-L/P complex (PDB: 6PZK and 6UEN). Surface plasmon resonance assays with close analogs of JNJ-7184, demonstrated that they bind to a recombinant RSV-L/P complex (Fig. S3). Thermal shift assays showed that JNJ-7184 analogs further destabilized this complex at high compound concentrations (Fig. S4). This observation may explain why we were unsuccessful in our attempts to obtain a cryo-EM co-structure with JNJ-7184.

## 3.3. JNJ-7184 differentially inhibits multiple activities of the RSV polymerase

The mechanism of action of JNJ-7184 was further studied. In a minigenome assay where the *trailer* promoter is inactivated, JNJ-7184 inhibited both replication (formation of the antigenome) and transcription by the RSV polymerase, in a dose-dependent fashion from 25 nM (Fig. 3A). Primer extension analysis of RNA produced in minigenome assays showed JNJ-7184 inhibited initiation and/or early elongation from the +1U initiation site of the *leader* promoter (i.e., antigenome formation) as well as mRNA formation from the *gene start* sequences (Fig. 3B–C). However, initiation and/or early elongation from the +3C initiation site (i.e., the production of the ~25 nucleotides (nt) abortive *leader* RNA transcript) was not inhibited (Fig. 3B). The RSV *leader* and *trailer* promoters differ at position 4, meaning that the +3C initiation dinucleotide is different in each case. A previous study showed that initiation at position +3C of the *trailer* promoter is more sensitive to perturbation than initiation at position +3C of the *leader* promoter, likely because the *trailer* +3C initiation complex is less stable (Cressey et al., 2022). Therefore, a similar experiment was performed using a minireplicon with a *trailer* promoter at the 3' end in place of the *leader* and first *gene start* signal. In this case production of RNAs from both the +1U and +3C sites were inhibited by JNJ-7184 (Fig. 3D). This result was confirmed using an *in vitro* de novo RNA synthesis assay which does not include viral N protein (Fig. 3E), indicating that JNJ-7184 inhibits an RNA synthesis activity of the RSV polymerase, rather than encapsidation. Altogether, these results suggest that JNJ-7184 specifically inhibits the formation of the first dinucleotide from the +1U site of the *leader* and *trailer* promoters, and from the +3C initiation site of the *trailer* promoter, and/or inhibits the early elongation activity of the RSV polymerase.

To tease apart the two hypotheses, dinucleotide formation assays with each specific initiation site should be conducted. However, these proved difficult to establish *in vitro* in presence of the physiological salt Mg2+ 26 and we were only successful with the +3C initiation site of the *trailer* promoter. This assay also allowed the visualization of all synthesized products and thus the study of early elongation in this context. Dinucleotide formation was only partially inhibited (up to ~60% at 1 µM JNJ-7184), while formation of the 11-mer product (i.e. early elongation) was inhibited up to ~95% at 1 µM and with an IC50 of ~20 nM (Fig. 3F), similar to the EC50 in cell-based virus inhibition assays (14–20 nM, see 3.1).

To evaluate the impact on late elongation of transcripts from pre-assembled RSV polymerase complexes, crude extracts of RSV-infected cells were incubated with JNJ-7184. In contrast to an analog of nucleoside ALS-002230 (Deval et al., 2015), JNJ-7184 did not substantially inhibit the extension of long viral RNA transcripts (17% at 10 µM, Fig. 3G). Overall, these results suggest that JNJ-7184 mainly inhibits early elongation of RSV-L or is unable to disrupt pre-assembled replication and/or transcription complexes.

<table><caption>**Table 1**

*In vitro* resistance mutations in RSV L protein upon drug pressure with JNJ-7184, in an RSV-A2-eGFP background.</caption><thead><tr><th colspan="4">Replicates</th></tr><tr><th colspan="3">1</th><th colspan="3">2</th><th colspan="3">3</th></tr><tr><th>Mutation (s)</th><th>Passages of appearance (days of selection)</th><th>Fold change in EC50 versus wt</th><th>Mutation (s)</th><th>Passages of appearance (days of selection)</th><th>Fold change in EC50 versus wt</th><th>Mutation (s)</th><th>Passages of appearance (days of selection)</th><th>Fold change in EC50 versus wt</th></tr></thead><tbody><tr><td>S840G/ M1636T</td><td>1 (26) - 2 (43)</td><td>28</td><td>A1479T</td><td>2 (22) - 8 (72)</td><td>71</td><td>A1479E</td><td>2 (18) - 8 (62)</td><td>433</td></tr><tr><td>S840G/ M1636P</td><td>3 (54) - 10 (102)</td><td>760</td><td></td><td></td><td></td><td>A1479E/ I23L</td><td>8 (62) - 10 (77)</td><td>111</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td>A1479E/ L2007I</td><td>8 (62) - 10 (77)</td><td>316</td></tr></tbody></table>

Passages 1–10 of Replicate 1; 2, 4, 6, and 8 of Replicate 2; 2, 4, 6, 8, and 10 of Replicate 3 were analyzed by full genome sequencing. Only dominant (>50% frequency) mutations in RSV-L protein are shown.
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<figure><img src="image_18.png" /><figcaption>**Fig. 2.** JNJ-7184 targets the connector domain of the RSV large protein</figcaption></figure>

A. Schematic representation of RSV-L protein (not to scale). Conserved domains I to VI (denoted I, II, III, IV, V and VI) are represented according to data published in Gilman et al. (Gilman et al., 2019). Functional domains are represented in different colors on the top. The position of amino acid substitutions, selected during IVRS with JNJ-7184, is indicated below.

B. Efficacy in a transient RSV replicon assay. Wild-type or mutant L proteins were evaluated. Mean and SD of at least 3 independent experiments are presented.

C. Efficacy in a RdRp primer extension enzymatic assay. Wild-type or mutant L proteins were assayed.

D. Fitness of RSV-A2-mCherry viruses with amino acid substitutions obtained during IVRS with JNJ-7184, compared to their wild-type counterpart derived during a time course of viral growth detected on live virus by HCl. N = 4 independent experiments and SEM is presented.

E. RSV-L peptide protected by JNJ-7184 in an HDX experiment. ΔDt plot is presented.

As JNJ-7184 interacts with the connector domain, which links the capping and the methyltransferase domains of the RSV polymerase, both activities could potentially be inhibited. Capping activity was evaluated in the minigenome assays described above, and the ratio of capped versus uncapped mRNA remained unchanged (Fig. 3C). Methyltransferase activity was examined both in a methyltransferase assay using either the methyltransferase-C-terminal domain (MTase-CTD) of RSV-L and on the full-length RSV-L/P. Although inhibition of methyltransferase activity was observed, it was much weaker than described earlier for RNA synthesis (IC₅₀ ~110 µM vs 14–20 nM), suggesting that this is only a minor effect of JNJ-7184 (Fig. S5).

### 3.4. JNJ-7184 is highly efficacious in vivo in neonatal lambs

RSV infection of neonatal lambs has been proposed to be a more translational therapeutic model than cotton rats or mice. Therefore,

neonatal lambs were infected with RSV-A/M37 and treated once daily, from day 1–5 post-infection (Fig. S6) (Derscheid et al., 2014). On day 6, the viral load (VL) in BALF was significantly decreased by 1.5, 4.2 and 4.5 log₁₀ reduction at 1, 4 and 16 mg/kg JNJ-7184, respectively (Fig. 4A). Concomitantly, peak lung VL was drastically reduced by 2.0 log₁₀ at 1 mg/kg, and by 2.2 log₁₀ at 4 and 16 mg/kg (Fig. 4B). Results were corroborated by RT-qPCR on viral RNA levels from the BALF and lung (Fig. 4C and D). The frequency of RSV⁺ cells in the lung was completely inhibited at 4 and 16 mg/kg (Fig. 4E, S7-8). Lung gross lesions were decreased from 32% to 0% of consolidated lung surface at all doses (Fig. 4F–S9). Additionally, JNJ-7184 drastically reduced the total parenchymal lung consolidation, bronchiolitis, presence of syncytial cells, necrotic and hyperplastic epithelial cells in the peribronchial epithelium, neutrophil influx in the lungs and presence of peribronchiolar lymphocytic infiltrates and lymphocytic infiltrates around blood vessels (Fig. 4G–S10).
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<figure><img src="image_34.png" /><figcaption>**Fig. 3.** Efficacy of JNJ-7184 against the different enzymatic activities of RSV-L</figcaption></figure>

**A-C. top:** diagram of experimental design for analysis of RNA synthesis from the *leader* promoter of a minigenome; **middle:** Radiographic image of northern blot (A) or primer extension analysis (B, C) of minigenome assay with position of various bands (A: input minigenome, antigenome, mRNA 1 and 2; B: +1 and +3 primer extension products; C: Capped and uncapped mRNA products indicated on the right of the figure); **bottom:** quantification of images on the lower left of each panel showing the mean and range of two independent experiments.

**D. top:** diagram of experimental design for analysis of RNA synthesis from the *trailer* promoter of a minigenome; **middle:** radiographic image of primer extension analysis of minigenome generated RNAs, showing +1U and +3C primer extension products; **bottom:** quantification of primer extension products showing the mean and range of two independent experiments.

**E. top:** diagram of experimental design for *in vitro* transcription assay using purified RSV-L/P complex and tr 1–14 template, as indicated at top, nucleotide mix and product; **middle:** radiographic image of RNA synthesis products directly labeled with α-³²P-UTP; **bottom:** quantification of RNA synthesis products.

**F. top:** diagram of experimental design for *de novo* initiation assay from +3 initiation site of the *trailer* promoter. **middle:** radiographic image of RNA synthesis products directly labeled with α-³²P-ATP; **bottom:** quantification of RNA synthesis products. Mean ± SE of 3 independent experiments is shown.

**G.** Efficacy in a vRNP elongation assay.
<figure><img src="image_35.png" /><figcaption>Fig. 4. Therapeutic efficacy of JNJ-7184 in the neonatal lamb model</figcaption></figure>

A-D. VL in BAL fluid (red dots) and lavaged lung tissue (blue dots) via fluorescence-forming unit (FFU) assay (A, B) or vRNA levels by qRT-PCR (C, D). Bars: mean. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001. n. s.: not significative. 0 = vehicle treated group; 1, 4 and 16 = groups treated with 1, 4 and 16 mg/kg JNJ-7184, respectively.

E. RSV + cells by IHC with a goat polyclonal anti-RSV antibody, in the bronchiolar areas (bronchi, blue) or in the alveolar regions (alveoli, pink) of lung FFPE sections. The number of positive cells per area was scored, with the scale: 0 = no positive cells, 1 = 1-10 positive cells, 2 = 11-39 positive cells, 3 = 40-99 positive cells, 4 = >100 positive cells. Group mean ± standard error of the mean is displayed. \* p-value<0.05; \*\* p-value<0.01 (Kruskal-Wallis nonparametric test).

F. Number of gross legion foci per lung surface area. Individual values, mean and SD are shown. \*p < 0.05, \*\*p < 0.01.

G. Histopathology of FFPE lungs. Severity scores (see Table S7) are shown for the following endpoints (as labeled on the y-axis of each graph): consolidation, bronchiolitis, syncytia formation, epithelium necrosis, epithelium hyperplasia, neutrophilia, peribronchial lymphocytic infiltrates, and perivascular lymphocytic infiltrates. Group mean ± standard error of the mean. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001.

## 4. Discussion

We have shown that JNJ-7184 is a potent inhibitor of RSV-A and -B strains. Several lines of evidence suggest that this inhibitor is binding directly to RSV-L protein. IVRS studies and HDX experiments show that JNJ-7184 likely binds to the CD. Additionally, its mechanism of action involves inhibition of early elongation during viral transcription/elongation, as well as partial inhibition of dinucleotide formation from the +3C initiation site at the *trailer* promoter.

Several other RSV inhibitors have been identified with resistance mutations located in the CD: YM-53403 (Sudo et al., 2005), AZ-27 (Noton et al., 2015), Compound 1 (Laganas et al., 2015) and PC786 (Coates et al., 2017) are all sensitive to the substitution Y1631 H/C,

while the recently described AVG-233 (Sourimant et al., 2022; Cox et al., 2018) mainly loses its potency with substitution L1502Q (and partially H1632Q). The chemotype of JNJ-7184 (pyrazolopyrimidine) is unique among these inhibitors, with YM-53403, AZ-27 and PC786 forming a second chemotype (dihydrobenzothienoazepine), and AVG-233 and Compound 1 belonging to a third chemotype (dihydropyrazolopyridinedione) (Fig. S11). With reported EC₅₀ values of 200 nM and 1.9 µM, respectively, YM-53403 and Compound 1 are much less potent than JNJ-7184. While AZ-27 and PC786 are active against RSV-A strains, they lose ~50 to 100-fold potency on RSV-B strains. Therefore, in terms of antiviral potency, JNJ-7184 is one of the most potent inhibitors binding the CD to date.

On average, JNJ-7184 was 5-fold more potent on the RSV-A than on
the RSV-B strains we tested. This difference was even more pronounced for some of the previously reported CD inhibitors, AZ-27 and PC786. In the absence of a 3D structure of JNJ-7184 in complex with the RSV-L/P, aligning the CD sequence of 2160 RSV-A and 1158 RSV-B strains from the GISAID server, reveals that RSV-A and -B CD are 83.3% identical and 92.3% similar at the amino acid level. Of the peptide protected in the HDX experiment, the resistance mutations and the mutants displaying a decreased potency, only amino acid 1479 differs: an alanine in RSV-A and a valine in RSV-B strains. This variation may explain differences in potency for JNJ-7184. However, aa1479 was not reported as a site of resistance mutation for AZ-27 and PC786. Therefore, additional efforts are needed to further explore this question.

In terms of mechanism of action, only AZ-27, AVG-233 and JNJ-7184 have been studied in detail. Noton et al. (2015) described that AZ-27 inhibited initiation of both transcription and replication (Noton et al., 2015). It inhibited RNA synthesis from the +3C initiation site on the *trailer* promoter, though not when the polymerase was pre-assembled on the nucleocapsid template from elongating mRNAs or reinitiating at downstream gene start signals (Noton et al., 2015). Sourimant et al. (2022) showed that AVG-233 inhibited nucleotide addition from the +5 to the +12 position in a primer extension assay from the +1 site of the *leader* promoter, with a clear drop in early elongation from the +9 nucleotide (though still at concentrations 10-fold higher than the EC₅₀ reported in cell-based antiviral assays) (Sourimant et al., 2022). JNJ-7184 seems to act more like AVG-233, inhibiting prominently early elongation. Overall, it is striking that compounds from different chemotypes, with different RSV-A/B clade coverage, all target the same region of RSV-L but inhibit somewhat different enzymatic activities.

Of note, a similar phenomenon is observed for inhibitors BI-D (Liuzzi et al., 2005; Braun et al., 2017), MRK-1 (Kleiner et al., 2023), and JNJ-8003³⁸, targeting the capping domain of RSV-L. Indeed, compound BI-D (imidazoisoquinolinedione) induced selection of resistance mutation at position I1381 and inhibited the capping activity of RSV-L and initiation from the first *gene start* signal, while enhancing RNA elongation from the *leader* promoter past the first *gene start* signal (Braun et al., 2017). On the other hand, MRK-1 and JNJ-8003 (F-phenyl-pyridyl derivatives) also induced selection of resistance mutation at position I1381 but inhibited early elongation rather than capping (Kleiner et al., 2023; Yu et al., 2023). Overall, these observations highlight the plasticity of the RSV polymerase and potential to adopt different conformations for its different enzymatic activities.

While our results suggest that JNJ-7184 binds to the CD, it did not have a detectable effect on capping activity, and it inhibited MTase activity only at very high concentrations. This suggests that the CD, or at least the conformation in which JNJ-7184 binds/locks it, does not primarily contribute to viral mRNA capping or methylation, and barely ties these domains to the RdRp portion of the RSV-L. Rather, our results and that of others (Noton et al., 2015; Sourimant et al., 2022) suggest that the connector domain plays a role in viral RNA early elongation and possibly transcription/replication initiation.

Obtaining a cryoEM structure of the full-length RSV-L complexed with these different compounds, possibly in presence of the RNA template/product, would shed a welcome light on these paradoxes. So far, only two cryoEM structures of RSV-L/P in complex with Cap domain inhibitors were successfully reported (Kleiner et al., 2023; Yu et al., 2023). In the case of JNJ-7184, obtaining a structure in complex with RSV-L might prove difficult, as JNJ-7184 appears to destabilize the RSV-L/P complex. Additional cryoEM/crystallography techniques will probably be needed, such as stabilization techniques of the RSV-L/P. Similarly, novel techniques to reliably dissect dinucleotide formation under physiological conditions will be needed to dissect in further detail the mechanism of action of JNJ-7184 and other compounds.

Lastly, JNJ-7184 showed potent *in vivo* activity in the neonatal lamb model of RSV infection. Lambs display a similar lung development, respiratory tract architecture, body weight and develop similar symptoms upon RSV infection (e.g., coughing), histopathology (e.g.,

bronchiolitis) and neutrophil infiltration to infants (Sitticharoenchai et al., 2020). The model uses the clinical isolate RSV-A/M37, also used in human challenge studies (DeVincenzo et al., 2010). The potent *in vivo* activity of JNJ-7184 in this model, initiating treatment at 1 day post-infection (such as 3 days post infection, at peak of viral load (McSweeney et al., 2024), is highly encouraging. It will then be important to test its activity when starting treatment beyond 1 day post-infection, as patients seek medical care at multiple timepoints after symptom onset.

## CRediT authorship contribution statement

**Brecht Bonneux:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Afzaal Shareef:** Writing – review & editing, Visualization, Investigation. **Sergey Tcherniuk:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Brandon Anson:** Writing – review & editing, Visualization, Investigation, Formal analysis, Conceptualization. **Suzanne de Bruyn:** Writing – review & editing, Validation, Methodology, Investigation. **Nick Verheyen:** Writing – review & editing, Methodology, Investigation. **Kim Thys:** Writing – review & editing, Visualization, Methodology, Investigation. **Nádia Conceição-Neto:** Writing – review & editing, Methodology, Investigation. **Marcia Van Ginderen:** Writing – review & editing, Visualization, Investigation. **Leen Kwanten:** Writing – review & editing, Visualization, Validation, Investigation. **Nina Ysebaert:** Writing – review & editing, Visualization, Supervision, Methodology, Investigation. **Luc Vranckx:** Writing – review & editing, Supervision, Investigation. **Elien Peeters:** Writing – review & editing, Investigation. **Ellen Lanckacker:** Writing – review & editing, Visualization, Validation, Methodology, Investigation. **Jack M. Gallup:** Writing – review & editing, Visualization, Investigation, Formal analysis. **Panchan Sitthicharoenchai:** Writing – review & editing, Visualization, Investigation, Formal analysis. **Sarhad Alnajjar:** Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization. **Mark R. Ackermann:** Writing – review & editing, Visualization, Validation, Investigation, Formal analysis, Conceptualization. **Suraj Adhikary:** Writing – review & editing, Visualization, Investigation, Formal analysis. **Anusarka Bhaumik:** Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis. **Aaron Patrick:** Writing – review & editing, Supervision, Methodology, Formal analysis. **Amy Fung:** Writing – review & editing, Investigation, Formal analysis. **Priscila Sutto-Ortiz:** Writing – review & editing, Visualization, Investigation. **Etienne Decroly:** Writing – review & editing, Visualization, Supervision, Methodology, Formal analysis, Conceptualization. **Stephen W. Mason:** Writing – review & editing, Writing – original draft, Supervision, Formal analysis. **David Lançois:** Writing – review & editing, Conceptualization. **Jerome Deval:** Writing – review & editing, Methodology, Conceptualization. **Zhinan Jin:** Writing – review & editing, Visualization, Supervision, Methodology, Formal analysis. **Jean-François Eléouët:** Writing – review & editing, Supervision, Formal analysis, Conceptualization. **Rachel Fearnés:** Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Formal analysis, Conceptualization. **Anil Koul:** Writing – review & editing, Funding acquisition. **Dirk Roymans:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Peter Rigaux:** Writing – review & editing, Visualization, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Florence Herschke:** Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Formal analysis, Data curation.

## Declaration of competing interest

The following authors were employees of Johnson & Johnson (the sponsor of the work) at the time when the described work was
performed, and may hold Johnson & Johnson shares: BA, SdB, NV, KT, NCN, MVG, LK, NY, LV, EP, EL, AB, AP, AF, SWM, DL, JD, ZJ, AK, DR, PR, FH. The work of the other authors on this project was funded by Johnson & Johnson. BB is an employee of Antwerpen University and received funding from Johnson & Johnson and from Flanders Innovation & Entrepreneurship organization (VLAIO, HBC.2019.2157) for this work.RF has a sponsored research agreement with Merck & Co unrelated to the work presented here.

## Data availability

Data will be made available on request.

## Acknowledgements

The authors thank Carl Van Hove and Yves Wetzels for NGS help, Pravien Abeywickrema, Madison Piassek and Robyn Miller for protein production, Sarah Stevens for technical help for the vRNP assay, Jean-Marc Neefs for sequence alignments, the JNJ-7184 development team and Professor Peter Delputte for helpful discussions.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.antiviral.2024.105907.

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