Respiratory Syncytial Virus F-protein Research Articles

Identification of Respiratory Syncytial Virus Fusion Protein Inhibitor: In silico Screening and Molecular Docking Approach

In young children, immunocompromised individuals, and elderly people, the respiratory syncytial virus (RSV) is the primary source of acute lower respiratory tract infection. Intervention with RSV-specific small-molecule antivirals may provide significant therapeutic potential. For virus entry, the RSV fusion protein (F) is crucial as it facilitates viral and hosts membrane fusion. To date, no approved vaccine or drug molecule is available to treat RSV. With this purpose, in the present study, virtual screening of a library of natural compounds against the active site of F protein was performed, followed by an in-depth molecular docking study of top-scored compounds. Selected hits ZINC8740013, ZINC4029781 and ZINC898642were found to strongly bind with RSV F protein relative to the other compounds as well as the control. The binding energy (BE) and inhibition constant for ‘ZINC8740013-RSV F’, ‘ZINC4029781-RSV F’, and ‘ZINC898642-RSVF’ complexes were found as ‘-7.8 kcal/mol and 63.27 µM’, ‘-7.7 kcal/mol and 19.04 µM’, and ‘-7.5 kcal/mol and 3.31 µM’, respectively. However, BE and inhibition constant of control (JNJ-53718678) with RSV F protein was found as -6.1 kcal/mol and 563.26 µM, respectively. Phe140 and Phe488 are the main interacting residues of RSV F protein with JNJ-53718678 and selected hit compounds. The finding of this study suggests that these hits can be utilized as the RSV Fprotein inhibitor to prevent the fusion of the viral envelope with the host cells. Further, bench work experiments are required to optimize these hit compounds as RSV Fprotein inhibitors.

Respiratory syncytial virus immunisation overview

Respiratory syncytial virus (RSV) continues to be a significant source of morbidity and mortality in both adults and children. Natural infection confers incomplete protection, permitting recurrent episodes. Treatment remains limited to supportive care. Initial endeavours to develop a vaccine resulted in an unexpected enhancement of RSV disease and increased recipient mortality. Current proposed strategies to prevent RSV infection rely on the principles of active and passive immunisation and utilise the highly conserved RSV F-protein. Maternal vaccines administered in pregnancy may provide protection; trials are ongoing. Palivizumab, a monoclonal antibody, has a moderate preventative efficacy. A similar newer longer lasting formulation appears promising. A number of other novel options are being developed and are undergoing assessment. Progress has been made, with more vaccine candidates under consideration. We are edging closer to an effective solution to prevent RSV infection. If successful, the impact on paediatric morbidity, mortality, workload and cost will be substantial.

Development and properties of neutralizing monoclonal antibodies for fusion protein of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants and the elderly. The absence of a wide range of therapeutic drugs and vaccines indicates to the high relevance of the development of new effective drugs for the prevention and treatment of RSV infections. to obtain highly active and specific monoclonal antibodies (MAbs) capable of detecting RSV in infected cells and neutralizing the infectious activity of the virus in vitro. RSV reference strains of group A 2 subgroups (A2 and Long) were propagated in HEp-2 and MA-104 cell lines, respectively. Mice were immunized with purified RSV A2 virus. MAbs were obtained using hybridoma technology. A panel of 6 MAbs reacting with RSV strains А2 and Long has been obtained. Four MAbs were IgG (IgG2a or IgG2b subtype), two MAbs were IgM. All MAbs reacted with RSV F-protein in immunochemical tests. The MAbs actively reacted with RSV in ELISA, in immufluorescence and peroxidase staining of infected cells, and in immunodot test. Five out of 6 MAbs neutralized of RSV in cell culture. Different properties of MAbs suggest that they target different antigenic sites of F-protein. Comparative analysis suggests that the obtained MAbs can be used for the development of diagnostic preparations, for RSV detection in clinical materials and confirmation of infection etiology by rapid culture method. High activity and specificity of MAbs indicate that they can serve as a basis for development vaccines and preventive medicines.

Nitazoxanide inhibits paramyxovirus replication by targeting the Fusion protein folding: role of glycoprotein-specific thiol oxidoreductase ERp57

Paramyxoviridae, a large family of enveloped viruses harboring a nonsegmented negative-sense RNA genome, include important human pathogens as measles, mumps, respiratory syncytial virus (RSV), parainfluenza viruses, and henipaviruses, which cause some of the deadliest emerging zoonoses. There is no effective antiviral chemotherapy for most of these pathogens. Paramyxoviruses evolved a sophisticated membrane-fusion machine consisting of receptor-binding proteins and the fusion F-protein, critical for virus infectivity. Herein we identify the antiprotozoal/antimicrobial nitazoxanide as a potential anti-paramyxovirus drug targeting the F-protein. We show that nitazoxanide and its circulating-metabolite tizoxanide act at post-entry level by provoking Sendai virus and RSV F-protein aggregate formation, halting F-trafficking to the host plasma membrane. F-protein folding depends on ER-resident glycoprotein-specific thiol-oxidoreductase ERp57 for correct disulfide-bond architecture. We found that tizoxanide behaves as an ERp57 non-competitive inhibitor; the putative drug binding-site was located at the ERp57-b/b′ non-catalytic domains interface. ERp57-silencing mimicked thiazolide-induced F-protein alterations, suggesting an important role of this foldase in thiazolides anti-paramyxovirus activity. Nitazoxanide is used in the clinic as a safe and effective antiprotozoal/antimicrobial drug; its antiviral activity was shown in patients infected with hepatitis-C virus, rotavirus and influenza viruses. Our results now suggest that nitazoxanide may be effective also against paramyxovirus infection.

Modification of the respiratory syncytial virus f protein in virus-like particles impacts generation of B cell memory.

Immunization with virus-like particles (VLPs) containing the Newcastle disease virus (NDV) core proteins, NP and M, and two chimera proteins (F/F and H/G) containing the respiratory syncytial virus (RSV) F- and G-protein ectodomains fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins, respectively, stimulated durable RSV-neutralizing antibodies, F-protein-specific long-lived, bone marrow-associated plasma cells (LLPCs), and B cell memory, in striking contrast to RSV infection, which did not (M. R. Schmidt, L. W. McGinnes, S. A. Kenward, K. N. Willems, R. T. Woodland, and T. G. Morrison, J. Virol. 86:11654-11662, 2012). Here we report the characterization of a VLP with an RSV F-protein ectodomain fused to the NDV F-protein heptad repeat 2 (HR2), transmembrane, and cytoplasmic domain sequences, creating a chimera with two tandem HR2 domains, one from the RSV F protein and the other from the NDV F-protein ectodomain (F/HR2F). The F/HR2F chimera protein was efficiently assembled into VLPs along with the H/G chimera protein. This VLP (VLP-H/G+F/HR2F) stimulated anti-F-protein and anti-G-protein IgG, durable RSV-neutralizing antibodies, and anti-RSV F-protein-secreting LLPCs. However, the subtypes of anti-F-protein IgG induced were different from those elicited by VLPs containing the F/F chimera (VLP-H/G+F/F). Most importantly, VLP-H/G+F/HR2F did not induce RSV F-protein-specific B cell memory, as shown by the adoptive transfer of B cells from immunized animals to immunodeficient animals. The VLP did, however, induce B cell memory specific to the RSV G protein. Thus, the form of the F protein has a direct role in inducing anti-F-protein B cell memory. The development of vaccines for respiratory syncytial virus (RSV) is hampered by a lack of a clear understanding of the requirements for eliciting protective as well as durable human immune responses to virus antigens. The results of this study indicate that the form of the RSV F protein has a direct and significant impact on the type of anti-F-protein IgG antibodies induced and the generation of F-protein-specific memory. Identification of the conformation of the RSV F protein that most effectively stimulates not only LLPCs and but also memory B cells will be important in the future development of RSV vaccines.

Characterization of neutralizing affinity‐matured human respiratory syncytial virus F binding antibodies in the sub‐picomolar affinity range

In the human adaptation and optimization of a mouse anti-human respiratory syncytial virus neutralizing antibody, affinity assessment was crucial to distinguish among potential candidates and to evaluate whether this correlated with function in vitro and in vivo. This affinity assessment was complicated by the trimeric nature of the antigen target, respiratory syncytial virus F (RSV-F) glycoprotein. In the initial affinity screen, surface plasmon resonance was used to determine the intrinsic binding affinities of anti-RSV-F Fab and immunoglobulin G (IgG) to the extracellular domain of RSV-F. This assessment required minimal biotinylation of the RSV-F protein and design of a capture strategy to minimize avidity effects. Approximately 30 Fabs were selected from three optimization phage display libraries on the basis of an initial ELISA screen. Surface plasmon resonance analysis demonstrated the success of optimization with some candidates from the screened libraries having low picomolar dissociation constants, more than 700-fold tighter than the parental monoclonal antibody (B21M). The affinities of these antibodies were further evaluated by a kinetic exclusion assay, a solution binding technology. One IgG (monoclonal antibody 029) displayed a low picomolar K(D) comparable with that of motavizumab, an RSV antibody in clinical study. Kinetic exclusion assay showed that two other of the matured IgGs (011 and 019) had sub-picomolar dissociation constants that could not be resolved further. We discuss the relevance of these interaction analysis results in the light of recently published data on the mechanism of F-driven viral fusion during paramyxoviral infection and 101F epitope conservation revealed from the recent crystal structure of RSV-F in the post-fusion state.

Immunogenicity and efficacy of codon optimized DNA vaccines encoding the F-protein of respiratory syncytial virus

Respiratory syncytial virus F-protein (RSV-F) is poorly expressed from DNA expression plasmids containing the wild type RSV-F open reading frame. By codon optimization, premature polyadenylation signals were deleted and a striking enhancement of RSV-F expression levels was achieved. Therefore, the immunogenicity and efficacy of wild type DNA vaccines were compared to codon optimized expression plasmids encoding full-length RSV-F or its ectodomain. Mice were immunized twice with the different DNA vaccines followed by an RSV challenge. Only codon optimized DNA vaccines and in particular the one encoding the ectodomain of RSV-F induced substantial antibody levels and reduced viral load 13-170-fold. Thus, codon optimization enhances the immunogenicity and efficacy of RSV encoding DNA vaccines.

Amino Acid Variation within the Fusion Protein of Respiratory Syncytial Virus Subtype A and B Strains during Annual Epidemics in South Africa

Recent evidence of positive selection within the cytotoxic T-cell (CTL) epitopes of the highly conserved nucleoprotein of influenza virus raised the question of whether the CTL epitopes of Respiratory syncytial virus (RSV) are also affected by immune driven change over annual epidemics. The fusion protein (F-protein) of RSV is highly conserved within the two subtypes (A and B) and the most important target for the protective response. The position of various neutralizing epitopes has been mapped and characterized between RSV subtypes. CTL epitopes have also recently been mapped for the F-protein of subtype A, however variation within these epitopes between and within the subtypes has not been determined. To address this question, the F-proteins of 18 strains representative of all subgroup A and B genotypes identified in South Africa over a period of 5 years were sequenced. F-protein sequences were highly conserved within and between South African genotypes, with most variability occurring at the nucleotide level. Most of the amino acid differences identified within neutralizing and CTL epitopes were conserved within the subtypes, and therefore does not indicate immune selection. However, out of three CTL epitopes previously identified in subtype A, two (restricted to HLA B*57 and HLA A *01) were conserved only within subtype A, while the third (restricted to Cw*12) contained both subtype- and genotype-specific changes. These results suggest that most of the identified CTL epitopes are subtype A-specific and may not be recognized in subtype B viruses, while the HLA Cw*12 restricted epitope may also not be recognized efficiently in GA5 strains.

Parainfluenza virus type 3 expressing the native or soluble fusion (F) Protein of Respiratory Syncytial Virus (RSV) confers protection from RSV infection in African green monkeys.

Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.

Two Neutralizing Human Anti-RSV Antibodies: Cloning, Expression, and Characterization

Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies with the ability to neutralize RSV could have a major impact on the immunotherapy of the disease. The generation of human antibodies has been difficult because there exists no general way to activate B cells against an antigen of choice in vitro. Human spleen cells from individuals exposed to RSV were used to repopulate SCID mice. Hu-SCID mice were boosted with RSV fusion (F)-protein and subsequently developed B cell tumors. The tumors were removed and cultured and subcloned in vitro, using a feeder layer of CD154-expressing T cells. Two of these tumors produced the antibodies designated RF-1 and RF-2. VL genes were isolated by standard PCR techniques, however, it was necessary to use high-temperature reverse transcriptase to clone the VH genes. RF-1 and RF-2 VH genes were both found to be closely related members of the VH2 family. Vk genes originated from the VK III family. RF-1 and RF-2 recombinant antibodies expressed in CHO cells (cRF-1 and cRF-2) were found to have affinities for RSV F-protein of 0.1 nM and 0.07 nM, respectively, and both were able to neutralize several A and B subtypes of RSV. The technique of immortalizing human B lymphocytes, by passage in SCID mice and expression as recombinant antibodies in CHO cells, provides a method by which high-affinity human antibodies can be developed for immunotherapy of viral diseases.

Phase I/II, Double-blind, Placebo-controlled, Multi-dose Escalation Trial of a Humanized Respiratory Syncytial Virus (RSV) Monoclonal Antibody (MEDI-493) in Children Hospitalized with RSV • 906

MEDI-493, a humanized monoclonal antibody to the F-protein of RSV, is highly active against RSV. The objective of this study was to describe the safety, tolerance, immunogenicity, pharmacokinetics and clinical outcome of a single intravenous (IV) dose of MEDI-493 administered at 5 or 15 mg/kg in children hospitalized with acute RSV infection. Sixty previously healthy children ≤24 months of age were enrolled at 10 centers. The first 16 children were randomized to receive 5 mg/kg MEDI-493 or placebo while the next 44 were randomized to either 15 mg/kg MEDI-493 or placebo. Adverse events(AEs) MEDI-493 concentrations (CONC), and anti-MEDI-493 binding were monitored. Fifty-nine children received study drug as follows: placebo (n=29), 5 mg/kg MEDI-493 (n=8), or 15 mg/kg MEDI-493 (n=22). AEs were balanced between groups at each dose level and were generally those expected in children hospitalized with RSV infection. One child in the placebo group died from progressive RSV infection. Mean serum CONC in the 15 mg/kg group were 303μg/mL 60 minutes, 144 μg/mL 2 days, and 38 μg/mL 30 days. Non-specific, transient, low-level anti-MEDI-493 binding was observed in ten patients (5 each in the placebo group and MEDI-493 groups). There was no evidence of enhancement of RSV illness. Compared to the placebo group, the 15 mg/kg MEDI-493 group had fewer hospital days per 100 children (276 vs. 466), admissions to ICU (4.5% vs. 19.0%), days of ICU per 100 children (18 vs. 113), days of mechanical ventilation per 100 children (13 vs. 43), days of supplemental oxygen per 100 children (218 vs. 348), and days in hospital with lower respiratory illness (LRI) score ≥3 per 100 children (91 vs. 162), but the differences were not statistically significant. MEDI-493 appeared to be safe and well tolerated when given IV to children hospitalized with acute RSV disease. Serum MEDI-493 CONC well above those which result in greater than a 2-log reduction of pulmonary RSV in cotton rats were achieved with a 15 mg/kg dose and were maintained for 30 days in most children. CONC and AE profiles were similar to those previously reported in children without RSV infection. Trends toward shorter duration of hospital stay and lesser severity of illness were seen in children receiving 15 mg/kg MEDI-493.

Reduction of Respiratory Syncytial Virus (RSV) Hospitalization in Children with Prematurity or Bronchopulmonary Dysplasia (BPD) Using Intramuscular (IM) Humanized Monoclonal Antibody to the RSV F-Protein (MEDI-493, Palivizumab)• 823

Reduction of Respiratory Syncytial Virus (RSV) Hospitalization in Children with Prematurity or Bronchopulmonary Dysplasia (BPD) Using Intramuscular (IM) Humanized Monoclonal Antibody to the RSV F-Protein (MEDI-493, Palivizumab)• 823

Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G

Peptides deduced from the central hydrophobic region (residues 158–189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.