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Respiratory syncytial virus (RSV) is an RNA virus infecting the upper and lower respiratory tract and is recognized as a major respiratory health threat, particularly to older adults, immunocompromised individuals, and young children. Around 64 million children and adults are infected every year worldwide. Despite two vaccines and a new generation monoclonal antibody recently approved, no effective antiviral treatment is available. In this manuscript, we present the medicinal chemistry efforts resulting in the identification of compound 28 (JNJ-8003), a novel RSV non-nucleoside inhibitor displaying subnanomolar activity in vitro as well as prominent efficacy in mice and a neonatal lamb models.

Despite the availability of medicines preventing respiratory syncytial virus (RSV) infection, post-exposure treatment options are needed for addressing patient's needs. RSV non-nucleoside polymerase inhibitors (NNI) have emerged as a promising asset for which our group previously disclosed JNJ-8003 with potent in vitro antiviral activity and pronounced in vivo efficacy. In this work, a structural-guided design to modify the linker vector of JNJ-8003 resulted in the identification of 2-oxacyclo pyridine-containing derivatives whose various ring closing strategies are described. In addition, bioisosteric replacement of an amide bond with triazole retained potency, and cryo-electron microscopy (cryo-EM) confirmed binding in the capping domain. Subsequent NMR conformational analysis suggested a correlation between the potency and conformations. Our efforts have fulfilled the aim of identifying linker modifications with maintained biological activity while enriching structural diversity and allowing modulations of other parameters., Competing Interests: The authors declare the following competing financial interest(s): All authors are or were previously employed by Johnson and Johnson Innovative Medicine and are possible shareholders., (© 2024 American Chemical Society.)

Respiratory syncytial virus (RSV) is a major cause of hospitalization in infants, the elderly, and immune-compromised patients. While a half-life extended monoclonal antibody and 2 vaccines have recently been approved for infants and the elderly, respectively, options to prevent disease in immune-compromised patients are still needed. Here, we describe spiro-azetidine oxindoles as small molecule RSV entry inhibitors displaying favorable potency, developability attributes, and long-acting PK when injected as an aqueous suspension, suggesting their potential to prevent complications following RSV infection over a period of 3 to 6 months with 1 or 2 long-acting intramuscular (IM) or subcutaneous (SC) injections in these immune-compromised patients.

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., Competing Interests: 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., (Copyright © 2024 Janssen Pharmaceutica NV. Published by Elsevier B.V. All rights reserved.)

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze-thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log 10 TCID 50 /ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze-thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities., (© 2024. The Author(s).)

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs., (© 2023. Springer Nature Limited.)

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at μM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: P.R., N.Y., P.A, D.R are employees and possible shareholders of Janssen Pharmaceutical Companies of Johnson & Johnson.

Respiratory syncytial virus (RSV) is a seasonal virus that infects the lungs and airways of 64 million children and adults every year. It is a major cause of acute lower respiratory tract infection and is associated with significant morbidity and mortality. Despite the large medical and economic burden, treatment options for RSV-associated bronchiolitis and pneumonia are limited and mainly consist of supportive care. This publication covers the medicinal chemistry efforts resulting in the identification of JNJ-53718678, an orally bioavailable RSV inhibitor that was shown to be efficacious in a phase 2a challenge study in healthy adult subjects and that is currently being evaluated in hospitalized infants and adults. Cocrystal structures of several new derivatives helped in rationalizing some of the structure-activity relationship (SAR) trends observed.

Currently, there is an increasing interest to apply pre-fusion (pre-F) protein of respiratory syncytial virus (RSV) as antigen for the development of a subunit vaccine. A pre-F-containing powder would increase the flexibility regarding the route of administration. For instance, a pre-F-containing powder could be incorporated into a single-injection system releasing a primer, and after a lag time, a booster. The most challenging aspect, obtaining the booster after a lag time, may be achieved by incorporating the powder into a core encapsulated by a nonporous poly(dl-lactic- co -glycolic acid) (PLGA) shell. We intended to develop a stable freeze-dried pre-F-containing powder. Furthermore, we investigated whether incorporation of this powder into the core-shell implant was feasible and whether this system would induce a delayed RSV virus-neutralizing antibody (VNA) response in mice. The developed pre-F-containing powder, consisting of pre-F in a matrix of inulin, HEPES, sodium chloride, and Tween 80, was stable during freeze-drying and storage for at least 28 days at 60 °C. Incorporation of this powder into the core-shell implant was feasible and the core-shell production process did not affect the stability of pre-F. An in vitro release study showed that pre-F was incompletely released from the core-shell implant after a lag time of 4 weeks. The incomplete release may be the result of pre-F instability within the core-shell implant during the lag time and requires further research. Mice subcutaneously immunized with a pre-F-containing core-shell implant showed a delayed RSV VNA response that corresponded with pre-F release from the core-shell implant after a lag time of approximately 4 weeks. Moreover, pre-F-containing core-shell implants were able to boost RSV VNA titers of primed mice after a lag time of 4 weeks. These findings could contribute to the development of a single-injection pre-F-based vaccine containing a primer and a booster., Competing Interests: The authors declare no conflict of interest, except for the fact that K.A., F.C., B.D.C., E.V.G., G.K. and D.R. are employed by Janssen. K.A., F.C., B.D.C., E.V.G., G.K. and D.R. participated in the design of the study; in the collection, analyses and interpretation of the data; in the writing of the manuscript and the decision to publish the results.

Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Respiratory syncytial virus (RSV) disease has no effective treatment. JNJ-53718678 is a fusion inhibitor with selective activity against RSV., Methods: After confirmation of RSV infection or 5 days after inoculation with RSV, participants (n = 69) were randomized to JNJ-53718678 75 mg (n = 15), 200 mg (n = 17), 500 mg (n = 18), or placebo (n = 17) orally once daily for 7 days. Antiviral effects were evaluated by assessing RSV RNA viral load (VL) area under the curve (AUC) from baseline (before the first dose) until discharge, time-to-peak VL, duration of viral shedding, clinical symptoms, and quantity of nasal secretions., Results: Mean VL AUC was lower for individuals treated with different doses of JNJ-53718678 versus placebo (203.8-253.8 vs 432.8 log10 PFUe.hour/mL). Also, mean peak VL, time to peak VL, duration of viral shedding, mean overall symptom score, and nasal secretion weight were lower in each JNJ-53718678-treated group versus placebo. No clear exposure-response relationship was observed. Three participants discontinued due to treatment-emergent adverse events of grade 2 and 1 electrocardiogram change (JNJ-53718678 75 mg and 200 mg, respectively) and grade 2 urticaria (placebo)., Conclusions: JNJ-53718678 at all 3 doses substantially reduced VL and clinical disease severity, thus establishing clinical proof of concept and the compound's potential as a novel RSV treatment., Clinical Trials Registration: ClinicalTrials.gov: NCT02387606; EudraCT number: 2014-005041-41.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.

Influenza and respiratory syncytial virus (RSV) cause acute infections of the respiratory tract. Since the viruses both cause illnesses with similar symptoms, researchers often try to apply knowledge gleaned from study of one virus to the other virus. This can be an effective and efficient strategy for understanding viral dynamics or developing treatment strategies, but only if we have a full understanding of the similarities and differences between the two viruses. This study used mathematical modeling to quantitatively compare the viral kinetics of in vitro RSV and influenza virus infections. Specifically, we determined the viral kinetics parameters for RSV A2 and three strains of influenza virus, A/WSN/33 (H1N1), A/Puerto Rico/8/1934 (H1N1), and pandemic H1N1 influenza virus. We found that RSV viral titer increases at a slower rate and reaches its peak value later than influenza virus. Our analysis indicated that the slower increase of RSV viral titer is caused by slower spreading of the virus from one cell to another. These results provide estimates of dynamical differences between influenza virus and RSV and help provide insight into the virus-host interactions that cause observed differences in the time courses of the two illnesses in patients.

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.

Background: The study assessed the antiviral activity of TMC353121, a respiratory syncytial virus (RSV) fusion inhibitor, in a preclinical non-human primate challenge model with a viral shedding pattern similar to that seen in humans, following continuous infusion (CI)., Methods: African green monkeys were administered TMC353121 through CI, in 2 studies. Study 1 evaluated the prophylactic and therapeutic efficacy of TMC353121 at a target plasma level of 50 ng/mL (n=15; Group 1: prophylactic arm [Px50], 0.033 mg/mL TMC353121, flow rate 2.5 mL/kg/h from 24 hours pre-infection to 10 days; Group 2: therapeutic arm [Tx50], 0.033 mg/mL TMC353121 from 24 hours postinfection to 8 days; Group 3: control [Vh1] vehicle, 24 hours post-infection to 8 days). Study 2 evaluated the prophylactic efficacy of TMC353121 at target plasma levels of 5 and 500 ng/mL (n=12; Group 1: prophylactic 5 arm [Px5], 0.0033 mg/mL TMC353121, flow rate 2.5 mL/kg/h from 72 hours pre-infection to 14 days; Group 2: prophylactic 500 arm [Px500], 0.33 mg/mL TMC353121; Group 3: control [Vh2] vehicle, 14 days). Bronchoalveolar lavage fluid and plasma were collected every 2 days from day 1 postinfection for pharmacokinetics and safety analysis., Findings: TMC353121 showed a dose-dependent antiviral activity, varying from 1 log10 reduction of peak viral load to complete inhibition of the RSV replication. Complete inhibition of RSV shedding was observed for a relatively low plasma exposure (0.39 μg/mL) and was associated with a dose-dependent reduction in INFγ, IL6 and MIP1α. TMC353121 administered as CI for 16 days was generally well-tolerated., Conclusion: TMC353121 exerted dose-dependent antiviral effect ranging from full inhibition to absence of antiviral activity, in a preclinical model highly permissive for RSV replication. No new safety findings emerged from the study.