Respiratory Syncytial Virus (RSV) Diseases

The respiratory syncytial virus (RSV) is an RNA virus that causes annual ARI outbreaks during winter with mild URTI in the general population, but with severe LRTI particularly among young children (bronchiolitis), patients with underlying diseases and people >65 years of age. RSV does not induce a long-lasting protective immunity and repeated infections throughout life are the norm. Basically, all children have been infected by 2 years of age and of those hospitalized, >50% are <3 months and 75% are <6 months of age. The overall CFR is 1/500. For adults ≥65 years, RSV hospitalization rates are 90–250/105. There is no specific therapy, general preventive measures include general hygiene and isolation/separation of patients. A monoclonal anti-F-protein antibody is available for passive immunization of selected high-risk children. It requires monthly injections, comes at a high cost and has limited efficacy (50% against RSV hospitalization). Active immunization failed in the past, probably as the post-fusion conformation of the F-protein was used. Long-acting monoclonal antibodies (for infants) as well as stabilized pre-fusion F-protein vaccines (for immunization of pregnant women, children, older adults) produced on various platforms are in late stages of clinical development.

YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity.

Detailed Evolutionary Analyses of the F Gene in the Respiratory Syncytial Virus Subgroup A

We performed evolution, phylodynamics, and reinfection-related antigenicity analyses of respiratory syncytial virus subgroup A (RSV-A) fusion (F) gene in globally collected strains (1465 strains) using authentic bioinformatics methods. The time-scaled evolutionary tree using the Bayesian Markov chain Monte Carlo method estimated that a common ancestor of the RSV-A, RSV-B, and bovine-RSV diverged at around 450 years ago, and RSV-A and RSV-B diverged around 250 years ago. Finally, the RSV-A F gene formed eight genotypes (GA1‑GA7 and NA1) over the last 80 years. Phylodynamics of RSV-A F gene, including all genotype strains, increased twice in the 1990s and 2010s, while patterns of each RSV-A genotype were different. Phylogenetic distance analysis suggested that the genetic distances of the strains were relatively short (less than 0.05). No positive selection sites were estimated, while many negative selection sites were found. Moreover, the F protein 3D structure mapping and conformational epitope analysis implied that the conformational epitopes did not correspond to the neutralizing antibody binding sites of the F protein. These results suggested that the RSV-A F gene is relatively conserved, and mismatches between conformational epitopes and neutralizing antibody binding sites of the F protein are responsible for the virus reinfection.

Probing In Silico the Benzimidazole Privileged Scaffold for the Development of Drug-Like Anti-RSV Agents

Targeting the fusion (F) protein has been recognized as a fruitful strategy for the development of anti-RSV agents. Despite the considerable efforts so far put into the development of RSV F protein inhibitors, the discovery of adequate therapeutics for the treatment of RSV infections is still awaiting a positive breakthrough. Several benzimidazole-containing derivatives have been discovered and evaluated in clinical trials, with only some of them being endowed with a promising pharmacokinetic profile. In this context, we applied a computational study based on a careful analysis of a number of X-ray crystallographic data of the RSV F protein, in the presence of different clinical candidates. A deepen comparison of the related electrostatic features and H-bonding motifs allowed us to pave the way for the following molecular dynamic simulation of JNJ-53718678 and then to perform docking studies of the in-house library of potent benzimidazole-containing anti-RSV agents. The results revealed not only the deep flexibility of the biological target but also the most relevant and recurring key contacts supporting the benzimidazole F protein inhibitor ability. Among them, several hydrophobic interactions and π-π stacking involving F140 and F488 proved to be mandatory, as well as H-bonding to D486. Specific requirements turning in RSV F protein binding ability were also explored thanks to structure-based pharmacophore analysis. Along with this, in silico prediction of absorption, distribution, metabolism, excretion (ADME) properties, and also of possible off-target events was performed. The results highlighted once more that the benzimidazole ring represents a privileged scaffold whose properties deserve to be further investigated for the rational design of novel and orally bioavailable anti-RSV agents.

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

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

Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the activity of the streptococcal ARE ABC-F protein MsrD on the ribosome and its regulation in response to macrolide exposure. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into a conserved crevice of the ribosomal exit tunnel, which remained thus far undocumented, concomitantly with 23S rRNA rearrangements that preclude proper accommodation of release factors and inhibits termination. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD powered by its two functionally asymmetric ATPase sites, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of β-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487–506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

Short-stalk isoforms of CADM1 and CADM2 trigger neuropathogenic measles virus-mediated membrane fusion by interacting with the viral hemagglutinin

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae , usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). MeV bears two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. The H protein possesses a head domain that initially mediates receptor binding and a stalk domain that subsequently transmits the fusion-triggering signal to the F protein. We have recently shown that cell adhesion molecule 1 (CADM1, also known as IGSF4A, Necl-2, SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors enabling cell-cell membrane fusion mediated by hyperfusogenic F proteins of neuropathogenic MeVs as well as MeV spread between neurons lacking the known receptors. CADM1 and CADM2 interact in cis with the H protein on the same cell membrane, triggering hyperfusogenic F protein-mediated membrane fusion. Multiple isoforms of CADM1 and CADM2 containing various lengths of their stalk regions are generated by alternative splicing. Here we show that only short-stalk isoforms of CADM1 and CADM2 predominantly expressed in the brain induce hyperfusogenic F protein-mediated membrane fusion. While the known receptors interact in trans with the H protein through its head domain, these isoforms can interact in cis even with the H protein lacking the head domain and trigger membrane fusion, presumably through its stalk domain. Thus, our results unveil a new mechanism of viral fusion triggering by host factors. Importance Measles, an acute febrile illness with skin rash, is still an important cause of childhood morbidity and mortality worldwide. Measles virus (MeV), the causative agent of measles, may also cause a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. The disease is fatal, and no effective therapy is available. Recently, we have reported that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV cell-to-cell spread in neurons. These molecules interact in cis with the MeV attachment protein on the same cell membrane, triggering the fusion protein and causing membrane fusion. CADM1 and CADM2 are known to exist in multiple splice isoforms. In this study, we report that their short-stalk isoforms can induce membrane fusion by interacting in cis with the viral attachment protein independently of its receptor-binding head domain. This finding may have important implications for cis -acting fusion triggering by host factors.

127. Development of a Kinetic ELISA (kELISA) and Reactive B-cell Frequency (RBF) Assay to Detect Respiratory Syncytial Virus (RSV) Pre-Fusion F Protein-Specific Immune Responses in Infants

Background RSV is a major cause of pediatric respiratory disease. Antibodies to the prefusion conformation of the RSV fusion (pre-F) protein are needed for virus neutralization. Methods We measured RSV-specific responses in two groups of children &lt; 3 years of age; subjects with laboratory-confirmed RSV (RSV-infected) or infants born in the period May to September and enrolled prior to their first RSV season (RSV-uninfected). RSV-infected infants had blood samples obtained at 1, 6, 9, and 12 months after infection. RSV-uninfected infants had blood samples obtained at enrollment, at the end of their first RSV season, and 6 months later. A kELISA to measure RSV pre-F-specific antibodies and an RBF assay to identify RSV F-specific B cells were developed. Results 102 subjects were enrolled; 11 were excluded due to missed visits or withdrawal. Of the 65 subjects in the RSV-uninfected group, all were kELISA positive at enrollment, consistent with maternal antibody transfer. 53 subjects had sufficient samples for analysis at multiple time points; 29 became seronegative and 24 remained seropositive. In the seronegative group, the kELISA value decreased rapidly to &lt; 0.25 by 6 months after the RSV season in 27/29 (93%), (Figure 1a). In the persistently seropositive group, all 24 subjects maintained a positive kELISA value, with some developing higher values over time, consistent with asymptomatic infection (Figure 1b). An RBF assay was used to determine whether antibodies were due to persistent maternal antibodies or endogenous production (Figure 2). In the seronegative group, 24/29 (80%) had a negative RBF; in the seropositive group, 23/24 (96%) had a positive RBF during follow-up. There were 26 subjects in the RSV-infected group; 22 had sufficient samples for analysis at multiple time points. All were seropositive by kELISA at one month post-infection with variable kELISA values during follow-up (Figure 3). 17/22 (77%) had a positive RBF, although 4 of the subjects without a positive RBF had indeterminate results at ≥ 1 visit. Figure 1. kELISA values of baseline RSV-negative subjects, by subject age at time of sample. Panel A: Subjects classified as seronegative (n=29). Panel B: Subjects without known RSV classified as persistently seropositive (n=24). Figure 2. Reactive B-cell frequency assay. The first step in the RBF assay is growth of Lymphoblastoid Cell Lines (LCLs), as shown over days 1-3 (Left-Day 1, Middle-Day 2, Right-Day 3, magnification 200X). The cells circled in the figure indicate a single LCL’s growth over time. LCL supernatant is used to detect RSV F-protein specific antibodies using traditional ELISA, resulting in a positive, indeterminate, or negative result. Indeterminate results occur due to a lack of cell viability and/or failure to form LCLs, resulting in failure to exceed an optical density of 5x background. Figure 3. kELISA values of RSV-infected subjects, by subject age at time of sample. First sample was obtained at approximately one month after laboratory-confirmed RSV. Conclusion Assays measuring F-specific immune responses in infants will be critical for RSV vaccine development. A kELISA targeting RSV pre-F epitopes, with an RBF assay targeting RSV F-specific B cells, may allow discrimination for maternal and infant-derived antibodies. Disclosures Isaac Thomsen, MD, MSCI, Horizon Therapeutics (Individual(s) Involved: Self): Consultant James E. Crowe, Jr., MD, Astra Zeneca (Grant/Research Support)IDBiologics (Board Member, Grant/Research Support, Shareholder)Luna Biologics (Consultant)Meissa Vaccines (Advisor or Review Panel member)Takeda Vaccines (Grant/Research Support) Kathryn M. Edwards, MD, Bionet (Individual(s) Involved: Self): Consultant; CDC (Individual(s) Involved: Self): Research Grant or Support; IBM (Individual(s) Involved: Self): Consultant; Merck (Individual(s) Involved: Self): member DSMC, Other Financial or Material Support; Moderna (Individual(s) Involved: Self): member DSMC, Other Financial or Material Support; NIH (Individual(s) Involved: Self): Research Grant or Support; Pfizer (Individual(s) Involved: Self): member DSMC, Other Financial or Material Support; Roche (Individual(s) Involved: Self): member of DSMB, Other Financial or Material Support; Sanofi Pasteur (Individual(s) Involved: Self): member DSMB, Other Financial or Material Support; Sequiras (Individual(s) Involved: Self): Member DSMB, Other Financial or Material Support; X4 Pharmaceuticals (Individual(s) Involved: Self): Consultant Buddy Creech, MD, MPH, Altimmune (Consultant)Astellas (Other Financial or Material Support, Data and Safety Monitoring Committee)Diotheris (Consultant)GSK (Consultant)Horizon (Consultant)Merck (Scientific Research Study Investigator)Premier Healthcare (Advisor or Review Panel member)Vir (Consultant)