Respiratory Syncytial Virus Fusion Research Articles

Mapping respiratory syncytial virus fusion protein interactions with the receptor IGF1R and the impact of alanine-scanning mutagenesis on viral infection.

Respiratory syncytial virus (RSV) has two main surface glycoproteins, the attachment glycoprotein (G) and the fusion (F) protein, which together mediate viral entry. Attachment is mediated by the RSV-G protein, while the RSV-F protein makes specific contact with the cellular insulin-like growth factor 1 receptor (IGF1R). This interaction leads to IGF1R activation and initiates a signalling cascade that calls the co-receptor, nucleolin, from the nucleus to the cell surface, where it can trigger viral fusion. We performed molecular docking analysis, which provided a potential set of 35 residues in IGF1R that may be important for interactions with RSV-F. We used alanine-scanning mutagenesis to generate IGF1R mutants and assessed their abundance and maturation, as well as the effect of mutation on RSV infection. We identified several mutations that appear to inhibit IGF1R maturation; but surprisingly, these mutations had no significant effect on RSV infection. This suggests that maturation of IGF1R may not be required for RSV infection. Additionally, we identified one residue, S788, that, when mutated, significantly reduced RSV infection. Further analysis revealed that this mutation disrupted a hydrogen bonding network that may be important for both IGF1R maturation and RSV infection.

Filling two needs with one deed: a combinatory mucosal vaccine against influenza A virus and respiratory syncytial virus.

Influenza A Virus (IAV) and Respiratory Syncytial Virus (RSV) are both responsible for millions of severe respiratory tract infections every year worldwide. Effective vaccines able to prevent transmission and severe disease, are important measures to reduce the burden for the global health system. Despite the strong systemic immune responses induced upon current parental immunizations, this vaccination strategy fails to promote a robust mucosal immune response. Here, we investigated the immunogenicity and efficacy of a mucosal adenoviral vector vaccine to tackle both pathogens simultaneously at their entry site. For this purpose, BALB/c mice were immunized intranasally with adenoviral vectors (Ad) encoding the influenza-derived proteins, hemagglutinin (HA) and nucleoprotein (NP), in combination with an Ad encoding for the RSV fusion (F) protein. The mucosal combinatory vaccine induced neutralizing antibodies as well as local IgA responses against both viruses. Moreover, the vaccine elicited pulmonary CD8+ and CD4+ tissue resident memory T cells (TRM) against the immunodominant epitopes of RSV-F and IAV-NP. Furthermore, the addition of Ad-TGFβ or Ad-CCL17 as mucosal adjuvant enhanced the formation of functional CD8+ TRM responses against the conserved IAV-NP. Consequently, the combinatory vaccine not only provided protection against subsequent infections with RSV, but also against heterosubtypic challenges with pH1N1 or H3N2 strains. In conclusion, we present here a potent combinatory vaccine for mucosal applications, which provides protection against two of the most relevant respiratory viruses.

Safety and Immunogenicity of Respiratory Syncytial Virus Prefusion Maternal Vaccine Coadministered With Diphtheria-Tetanus-Pertussis Vaccine: A Phase 2 Study.

Respiratory syncytial virus (RSV) fusion protein stabilized in the prefusion conformation (RSVPreF3) was under investigation as a maternal vaccine. This phase 2, randomized, placebo-controlled, single-dose, multicenter study enrolled healthy, nonpregnant women, randomized 1:1:1:1:1 to 5 parallel groups studying RSVPreF3 (60 or 120 µg) coadministered with diphtheria, tetanus, and acellular pertussis vaccine (dTpa) or placebo, and dTpa coadministered with placebo. Safety and humoral immune responses were assessed. An extension phase also assessed a RSVPreF3 120 μg vaccination 12-18 months after first vaccination. The safety profile of RSVPreF3 was unaffected by dose or dTpa coadministration. Solicited and unsolicited adverse events (AEs) were evenly distributed across study groups. Injection-site pain was higher following the second vaccination versus the first vaccination. Medically attended AEs were rare (<5% overall). Both RSVPreF3 dose levels (alone and with dTpa) were immunogenic, increasing levels of RSV-A neutralizing antibody ≥8-fold and anti-RSVPreF3 IgG antibody ≥11-fold at 1 month postvaccination, which persisted at 12-18 months postvaccination; modest 2-fold increases were observed with a second RSVPreF3 vaccination. This study indicates RSVPreF3 coadministration with dTpa induces robust immune responses and is well tolerated, regardless of the RSVPreF3 dose level used. NCT04138056.

Prevention of respiratory syncytial virus infection in infants. What has been done and where are we today?

Respiratory syncytial virus (RSV) infection is a frequent cause of morbidity and mortality in children. Recently, great advances have been made in the development of new monoclonal antibodies and vaccines thanks to the recognition of the structural conformation of virus proteins. The objective of this study was to review the advances related to the prevention of RSV infection in the first 6 months of life. Advances in structural biology have shown that the RSV fusion protein (F-Protein) in its prefusion state (Pre-F) is an excellent antigen for developing monoclonal antibodies and vaccines to prevent respiratory syncytial virus (RSV) infections. A new single-dose monoclonal antibody, Nirsevimab, has greater neutralizing power than currently available Palivizumab, and prolonged protection for 5 to 6 months. Nirsevimab has demonstrated an efficacy of 76.8% (95% CI, 49.4 to 89.4) in preventing lower respiratory infection 150 days after vaccination, decreasing the risk of ICU admission by 90.1% (95% CI: 16.4-98.8). Clesrovimab is another single-dose monoclonal antibody that has also shown promising results in phase 1b-2a trials. More recently, a bivalent vaccine against RSV A and B (Bivalent Prefusion F) has also been developed by replicating the F-protein stabilized in its Pre-F state as an antigen, using genetic engineering. This antigen, when administered to pregnant women between 24-36 weeks of gestation, induces high levels of antibodies in the mother with high transplacental transfer to the fetus. This vaccine has demonstrated an efficacy of 81.8% (95% CI: 40.6-96.3) at 90 days and 69.4% (95% CI: 44.3-84.1) at 180 days to prevent severe RSV disease (primary endpoint) without safety events detected so far. Nirsevimab and the Pre-F vaccine for pregnant women confer effective protection through passive immunity against RSV that lasts for the first 5 to 6 months of life and have already been approved for use in Europe by the EMA and in Canada and the United States by the FDA.

Characterization of a Panel of Monoclonal Antibodies Targeting the F-Protein of the Respiratory Syncytial Virus (RSV) for the Typing of Contemporary Circulating Strains.

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV-A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV.

Pre-Clinical Development of an Adenovirus Vector Based RSV and Shingles Vaccine Candidate.

Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4+ T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection.

Nirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection. Nirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV. Nirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model. Nirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization.

Immunogenicity of an AS01-adjuvanted respiratory syncytial virus prefusion F (RSVPreF3) vaccine in animal models

Respiratory syncytial virus (RSV) causes a high disease burden in older adults. An effective vaccine for this RSV-primed population may need to boost/elicit robust RSV-neutralizing antibody responses and recall/induce RSV-specific T cell responses. To inform the selection of the vaccine formulation for older adults, RSVPreF3 (RSV fusion glycoprotein engineered to maintain the prefusion conformation) with/without AS01 adjuvant was evaluated in mice and bovine RSV infection-primed cattle. In mice, RSVPreF3/AS01 elicited robust RSV-A/B-specific neutralization titers and RSV F-specific polyfunctional CD4+ T cell responses exceeding those induced by non-adjuvanted RSVPreF3. In primed bovines, RSVPreF3/AS01 tended to induce higher pre-/post-vaccination fold-increases in RSV-A/B-specific neutralization titers relative to non-adjuvanted and Alum-adjuvanted RSVPreF3 formulations, and elicited higher RSV F-specific CD4+ T cell frequencies relative to the non-adjuvanted vaccine. Though AS01 adjuvanticity varied by animal species and priming status, RSVPreF3/AS01 elicited/boosted RSV-A/B-specific neutralization titers and RSV F-specific CD4+ T cell responses in both animal models, which supported its further clinical evaluation as prophylactic candidate vaccine for older adults.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.

Extensive Multiple 2D-/3D-QSAR Modeling, Molecular Docking and Pharmacophoric Approaches for Piperazinylquinoline Derivatives as Respiratory Syncytial Virus Fusion Inhibitors.

The human respiratory syncytial virus (RSV) is responsible for causing upper and lower respiratory tract infections in young children. RSV Fusion (F) protein is a surface glycoprotein that facilitates virus entry into host cells. Thus, newer designing of RSV Fusion (F) protein inhibitors is required on an urgent basis. In the present study, we have developed statistically robust. Quantitative structure-activity relationship (QSAR) models for the effective designing of newer analogues of piperazinylquinoline derivatives (H1-H12). Our developed models were retained with high statistical parameters (R2 > 0.6 and Q2 > 0.5). Our developed pharmacophore, model (AADHRR_2) (indicating that two hydrogen bond acceptors, one hydrogen bond donor, one hydrophobic group, and two aromatic rings) is crucial for retaining the activities of piperazinylquinoline derivatives against RSV. Moreover, docking analysis of 12 new analogues on RSV pre-F in complex with 5C4 Fab (PDB ID: 5W23) and post-F trimeric protein (PDB ID: 3RRR) suggested higher affinities of these molecules against studied targets with good docking scores. Thus, one can implement developed QSAR models, docking analogy and Pharmacophore models for identifications of potent leads for designed molecules as RSV Fusion (F) protein inhibitors.

Molecular and phenotypic characteristics of RSV infections in infants during two nirsevimab randomized clinical trials

Nirsevimab is a monoclonal antibody that binds to the respiratory syncytial virus (RSV) fusion protein. During the Phase 2b (NCT02878330) and MELODY (NCT03979313) clinical trials, infants received one dose of nirsevimab or placebo before their first RSV season. In this pre-specified analysis, isolates from RSV infections were subtyped, sequenced and analyzed for nirsevimab binding site substitutions; subsequently, recombinant RSVs were engineered for microneutralization susceptibility testing. Here we show that the frequency of infections caused by subtypes A and B is similar across and within the two trials. In addition, RSV A had one and RSV B had 10 fusion protein substitutions occurring at >5% frequency. Notably, RSV B binding site substitutions were rare, except for the highly prevalent I206M:Q209R, which increases nirsevimab susceptibility; RSV B isolates from two participants had binding site substitutions that reduce nirsevimab susceptibility. Overall, >99% of isolates from the Phase 2b and MELODY trials retained susceptibility to nirsevimab.

A pilot phase 2a, randomized, double-blind, placebo-controlled study to explore the antiviral activity, clinical outcomes, safety, and tolerability of rilematovir at two dose levels in non-hospitalized adults with respiratory syncytial virus infection

ObjectivesTo assess the antiviral effect, clinical outcomes, and safety of the respiratory syncytial virus (RSV) fusion inhibitor rilematovir in non-hospitalized RSV-infected adults. MethodsThis phase 2a, double-blind, multicentre study randomly assigned RSV-positive adult outpatients ≤5 days from symptom onset 1:1:1 to receive rilematovir 500 mg, 80 mg, or placebo once-daily for 7 days. Antiviral effect was assessed by RSV RNA viral load (VL), measured by quantitative RT-PCR, and Kaplan-Meier (KM) estimates of time to undetectable VL. Clinical course was assessed by KM estimates of median time to resolution of key RSV symptoms assessed through patient-reported outcomes. ResultsRSV-positive patients (n = 72) were randomly assigned; 66 had confirmed RSV infection and received rilematovir 500 mg (n = 23), 80 mg (n = 21) or placebo (n = 22). Differences versus placebo in mean RSV RNA VL area under the curve (90% CI) through days 3, 5 and 8, respectively, were 0.09 (−0.837; 1.011), −0.10 (−2.171; 1.963), and −1.03 (−4.746; 2.682) log10 copies.day/mL for rilematovir 500 mg, and 1.25 (0.291; 2.204), 2.53 (0.430; 4.634), and 3.85 (0.097; 7.599) log10 copies.day/mL for rilematovir 80 mg. KM estimates of median (90% CI) time-to-first confirmed undetectable VL were 5.9 (3.85; 6.90), 8.0 (6.86; 12.80) and 7.0 (6.62; 10.88) days and 5.7 (2.93; 7.01), 8.1 (6.74; 12.80) and 7.9 (6.62; 11.74) days in patients with symptom onset ≤3 days, for rilematovir 500 mg, 80 mg, and placebo, respectively. KM estimates of median (90% CI) time to resolution of key RSV symptoms were 7.1 (5.03; 11.43), 7.6 (5.93; 8.32), and 9.6 (5.95; 14.00) days for rilematovir 500 mg, 80 mg, and placebo, respectively; and in patients with symptom onset ≤3 days, median 8.0, 7.6, and 11.8 days, respectively. DiscussionRilematovir use, initiated early, suggests a potential clinical benefit in RSV-infected adults, with data supporting development of RSV therapeutic options. Trial registrationThis study is registered with clinicaltrials.gov (NCT03379675).

The Efficiency of p27 Cleavage during In Vitro Respiratory Syncytial Virus (RSV) Infection Is Cell Line and RSV Subtype Dependent.

Respiratory syncytial virus (RSV) fusion protein (F) is highly conserved between subtypes A and B (RSV/A and RSV/B). To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and F2 subunits and releases a 27-amino-acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous data show that p27 is detected on RSV F, but questions remain regarding if and how p27 affects the conformation of mature RSV F. Monoclonal antibodies against p27, site Ø (pre-F specific), and site II were used to monitor RSV F conformation by enzyme-linked immunosorbent assay (ELISA) and imaging flow cytometry. Pre-F to post-F conformational change was induced by a temperature stress test. We found that p27 cleavage efficiency was lower on sucrose-purified RSV/A (spRSV/A) than on spRSV/B. In addition, cleavage of RSV F was cell line dependent: HEp-2 cells had higher retention of p27 than did A549 cells when infected with RSV. Higher levels of p27 were also found on RSV/A-infected cells than on RSV/B-infected cells. We observed that RSV/A F with higher p27 levels could better sustain the pre-F conformation during the temperature stress challenge in both spRSV- and RSV-infected cell lines. Our findings suggest that despite F sequence similarity, p27 of RSV subtypes was cleaved with different efficiencies, which were also dependent on the cell lines used for infection. Importantly, the presence of p27 was associated with greater stability of the pre-F conformation, supporting the possibility that RSV has more than one mechanism for fusion to the host cell. IMPORTANCE RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F undergoes proteolytic cleavages releasing a 27-amino-acid peptide (p27) to become fully functional. The role of p27 in viral entry and the function of the partially cleaved F containing p27 has been overlooked. p27 is thought to destabilize the F trimers, and thus, there is need for a fully cleaved F. In this study, we detected p27 on purified RSV virions and on the surface of virus-infected HEp-2 and A549 cells for circulating RSV strains of both subtypes. Higher levels of partially cleaved F containing p27 better sustained the pre-F conformation during the temperature stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 contributes to the stability of the pre-F conformation.

Structural interrogation of a trimeric prefusion RSV fusion protein vaccine candidate by a camelid nanobody

In the past decade, camelid nanobodies have been developed for multiple applications, including immuno-imaging, cancer immunotherapy, and antiviral therapeutics. Despite the prevalence of these approaches, nanobodies have rarely been used to assess the potency of vaccine antigen candidates, which are primarily based on mAb binding approaches. In this work, we demonstrate that a nanobody-based ELISA method is suitable for characterization of a leading respiratory syncytial virus (RSV) vaccine candidate, RSVPreF3. This nanobody, F-VHH-L66, compares similarly with AM14, an antibody well-known to be specific for the prefusion form of the RSV surface fusion glycoprotein, RSV F. ELISA assays based on F-VHH-L66 were specific for the trimeric, prefusion form of RSV F, the antigen conformation that best generates neutralizing antibodies. Additionally, the F-VHH-L66-based ELISA proved accurate, linear, and stability-indicating. Statistical analysis of 65 independent F-VHH-L66-based ELISA experiments indicated assay performance similar to that of ELISA assays based on AM14. Moreover, the binding kinetics of F-VHH-L66 to RSVPreF3 are comparable to those of AM14 when measured by surface plasmon resonance (SPR). Finally, F-VHH-L66 neutralized RSV(A) with similar efficacy as AM14; this bioactivity data further supports its use as an alternative to AM14 for pre-fusion specific structural characterization of RSVPreF3.

Dissolving Microneedles Loaded with Nanoparticle Formulation of Respiratory Syncytial Virus Fusion Protein Virus-like Particles (F-VLPs) Elicits Cellular and Humoral Immune Responses.

Respiratory syncytial virus (RSV) is one of the leading causes of bronchiolitis and pneumonia in children ages five years and below. Recent outbreaks of the virus have proven that RSV remains a severe burden on healthcare services. Thus, a vaccine for RSV is a need of the hour. Research on novel vaccine delivery systems for infectious diseases such as RSV can pave the road to more vaccine candidates. Among many novel vaccine delivery systems, a combined system with polymeric nanoparticles loaded in dissolving microneedles holds a lot of potential. In this study, the virus-like particles of the RSV fusion protein (F-VLP) were encapsulated in poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). These NPs were then loaded into dissolving microneedles (MNs) composed of hyaluronic acid and trehalose. To test the in vivo immunogenicity of the nanoparticle-loaded microneedles, Swiss Webster mice were immunized with the F-VLP NPs, both with and without adjuvant monophosphoryl lipid A (MPL) NPs loaded in the MN. The mice immunized with the F-VLP NP + MPL NP MN showed high immunoglobulin (IgG and IgG2a) levels both in the serum and lung homogenates. A subsequent analysis of lung homogenates post-RSV challenge revealed high IgA, indicating the generation of a mucosal immune response upon intradermal immunization. A flowcytometry analysis showed high CD8+ and CD4+ expression in the lymph nodes and spleens of the F-VLP NP + MPL NP MN-immunized mice. Thus, our vaccine elicited a robust humoral and cellular immune response in vivo. Therefore, PLGA nanoparticles loaded in dissolving microneedles could be a suitable novel delivery system for RSV vaccines.

Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine.

Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F.We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B.Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.

Nirsevimab binding-site conservation in respiratory syncytial virus fusion glycoprotein worldwide between 1956 and 2021: an analysis of observational study sequencing data

Nirsevimab is an extended half-life monoclonal antibody to the respiratory syncytial virus (RSV) fusion protein that has been developed to protect infants for an entire RSV season. Previous studies have shown that the nirsevimab binding site is highly conserved. However, investigations of the geotemporal evolution of potential escape variants in recent (ie, 2015-2021) RSV seasons have been minimal. Here, we examine prospective RSV surveillance data to assess the geotemporal prevalence of RSV A and B, and functionally characterise the effect of the nirsevimab binding-site substitutions identified between 2015 and 2021. We assessed the geotemporal prevalence of RSV A and B and nirsevimab binding-site conservation between 2015 and 2021 from three prospective RSV molecular surveillance studies (the US-based OUTSMART-RSV, the global INFORM-RSV, and a pilot study in South Africa). Nirsevimab binding-site substitutions were assessed in an RSV microneutralisation susceptibility assay. We contextualised our findings by assessing fusion-protein sequence diversity from 1956 to 2021 relative to other respiratory-virus envelope glycoproteins using RSV fusion protein sequences published in NCBI GenBank. We identified 5675 RSV A and RSV B fusion protein sequences (2875 RSV A and 2800 RSV B) from the three surveillance studies (2015-2021). Nearly all (25 [100%] of 25 positions of RSV A fusion proteins and 22 [88%] of 25 positions of RSV B fusion proteins) amino acids within the nirsevimab binding site remained highly conserved between 2015 and 2021. A highly prevalent (ie, >40·0% of all sequences) nirsevimab binding-site Ile206Met:Gln209Arg RSV B polymorphism arose between 2016 and 2021. Nirsevimab neutralised a diverse set of recombinant RSV viruses, including new variants containing binding-site substitutions. RSV B variants with reduced susceptibility to nirsevimab neutralisation were detected at low frequencies (ie, prevalence <1·0%) between 2015 and 2021. We used 3626 RSV fusion-protein sequences published in NCBI GenBank between 1956 and 2021 (2024 RSV and 1602 RSV B) to show that the RSV fusion protein had lower genetic diversity than influenza haemagglutinin and SARS-CoV-2 spike proteins. The nirsevimab binding site was highly conserved between 1956 and 2021. Nirsevimab escape variants were rare and have not increased over time. AstraZeneca and Sanofi.

Nirsevimab: review of pharmacology, antiviral activity and emerging clinical experience for respiratory syncytial virus infection in infants.

Respiratory syncytial virus (RSV) is a leading cause of hospitalization and infant mortality worldwide. There are currently no approved vaccines against RSV, and immunoprophylaxis with the mAb palivizumab is limited to extremely vulnerable infants in resource-rich settings due to its high cost and the need for monthly injections throughout the RSV season. Nirsevimab (formerly MEDI8897) is a highly potent, long-acting, human, recombinant mAb that received approval for the prevention of RSV infection in newborns and infants during their first RSV season from the EMA and the UK's Medicines and Healthcare products Regulatory Agency in November 2022 based on positive results in Phase 2b and 3 clinical trials. Nirsevimab targets the highly conserved site Ø of the prefusion conformation of the RSV fusion (F) protein and contains a triple amino acid substitution in the Fc domain that extends its half-life, allowing for a single dose to cover a typical RSV season in regions with temperate climates. In this article I review key attributes of nirsevimab with an emphasis on pharmacology, pharmacokinetics, antiviral activity, and the potential for resistance and escape variants. I also summarize current progress in clinical trials and consider future research priorities.

A human antibody potently neutralizes RSV by targeting the conserved hydrophobic region of prefusion F.

Respiratory syncytial virus (RSV) continues to pose serious threats to pediatric populations due to the lack of a vaccine and effective antiviral drugs. RSV fusion (F) glycoprotein mediates viral-host membrane fusion and is a key target for neutralizing antibodies. We generated 23 full-human monoclonal antibodies (hmAbs) against prefusion F protein (pre-F) from a healthy adult with natural RSV infection by single B cell cloning technique. A highly potent RSV-neutralizing hmAb, named as 25-20, is selected, which targets a new site Ø-specific epitope. Site-directed mutagenesis and structural modelling analysis demonstrated that 25-20 mainly targets a highly conserved hydrophobic region located at the a4 helix and a1 helix of pre-F, indicating a site of vulnerability for drug and vaccine design. It is worth noting that 25-20 uses an unreported inferred germline (iGL) that binds very poorly to pre-F, thus high levels of somatic mutations are needed to gain high binding affinity with pre-F. Our observation helps to understand the evolution of RSV antibody during natural infection. Furthermore, by in silico prediction and experimental verification, we optimized 25-20 with KD values as low as picomolar range. Therefore, the optimized 25-20 represents an excellent candidate for passive protection against RSV infection.

Identification of a DRB3*011:01-restricted CD4+ T cell response against bovine respiratory syncytial virus fusion protein.

Although Human Respiratory Syncytial Virus (HRSV) is a significant cause of severe respiratory disease with high morbidity and mortality in pediatric and elderly populations worldwide there is no licensed vaccine. Bovine Respiratory Syncytial Virus (BRSV) is a closely related orthopneumovirus with similar genome structure and high homology between structural and nonstructural proteins. Like HRSV in children, BRSV is highly prevalent in dairy and beef calves and known to be involved in the etiology of bovine respiratory disease, in addition to being considered an excellent model for HRSV. Commercial vaccines are currently available for BRSV, though improvements in efficacy are needed. The aims of this study were to identify CD4+ T cell epitopes present in the fusion glycoprotein of BRSV, an immunogenic surface glycoprotein that mediates membrane fusion and a major target of neutralizing antibodies. Overlapping peptides representing three regions of the BRSV F protein were used to stimulate autologous CD4+ T cells in ELISpot assays. T cell activation was observed only in cells from cattle with the DRB3*011:01 allele by peptides from AA249-296 of the BRSV F protein. Antigen presentation studies with C-terminal truncated peptides further defined the minimum peptide recognized by the DRB3*011:01 allele. Computationally predicted peptides presented by artificial antigen presenting cells further confirmed the amino acid sequence of a DRB3*011:01 restricted class II epitope on the BRSV F protein. These studies are the first to identify the minimum peptide length of a BoLA-DRB3 class II-restricted epitope in BRSV F protein.