Respiratory Syncytial Virus Attachment Research Articles

Both chebulagic acid and punicalagin inhibit respiratory syncytial virus entry via multi-targeting glycoprotein and fusion protein.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections, with no currently available small-molecule drugs that are both safe and effective. A major obstacle in antiviral drug development is the rapid emergence of drug-resistant viral strains. Targeting multiple viral compounds may help mitigate the development of resistance. Herein, we conducted a drug screening using the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to identify compounds that simultaneously target the RSV fusion (F) protein, glycoprotein (G), and the host heparan sulfate proteoglycans (HSPGs). From this screening, 10 candidate compounds were identified for their ability to interact with all three targets. Among these 10 candidates, chebulagic acid (CHLA) and punicalagin (PUG) demonstrated the most potent inhibition of RSV replication. In vitro dose-response assays confirmed the antiviral efficacy of CHLA (IC50: 0.07864 µM) and PUG (IC50: 0.08065 µM). Further experiments revealed both CHLA and PUG disrupt RSV attachment and membrane fusion by targeting the RSV-F and G proteins, rather than HSPG. Notably, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with docking assays predicting their binding sites at cysteines 176 and 182. Additionally, CHLA enhanced the conformational stability of the RSV-F protein before fusion. In an in vivo study, both CHLA and PUG were shown to alleviate RSV-induced pulmonary pathology by reducing viral titers, mitigating lung injury, and suppressing the inflammatory responses in the lungs. Our findings suggest that CHLA and PUG hold potential as therapeutic agents for RSV infection.IMPORTANCEA significant challenge in developing anti-respiratory syncytial virus (RSV) agents is the rapid emergence of resistant viral strains. Designing drugs that target multiple viral components can effectively reduce the likelihood of developing resistant strains. In this study, we screened compounds from the Antiviral Traditional Chinese Medicine Active Compound Library, aiming to simultaneously targe the RSV fusion (F) protein, glycoprotein (G), and host heparan sulfate proteoglycans (HSPGs). Our findings revealed that chebulagic acid (CHLA) and punicalagin (PUG) significantly inhibited RSV replication both in vitro and in vivo and interacted with all three targets. Both CHLA and PUG were able to disrupt RSV attachment and membrane fusion. Mechanistically, CHLA and PUG were found to bind to the CX3C motif of the RSV-G protein, with CHLA also enhancing the conformational stability of the RSV-F protein before fusion. In conclusion, our study suggests that CHLA and PUG hold promise as therapeutic agents against RSV infection.

Monoclonal antibodies targeting sites in respiratory syncytial virus attachment G protein provide protection against RSV-A and RSV-B in mice

Currently, only Palivizumab and Nirsevimab that target the respiratory syncytical virus (RSV) fusion protein are licensed for pre-treatment of infants. Glycoprotein-targeting antibodies may also provide protection against RSV. In this study, we generate monoclonal antibodies from mice immunized with G proteins from RSV-A2 and RSV-B1 strains. These monoclonal antibodies recognize six unique antigenic classes (G0-G5). None of the anti-G monoclonal antibodies neutralize RSV-A2 or RSV-B1 in vitro. In mice challenged with either RSV-A2 line 19 F or RSV-B1, one day after treatment with anti-G monoclonal antibodies, all monoclonal antibodies reduce lung pathology and significantly reduce lung infectious viral titers by more than 2 logs on day 5 post-RSV challenge. RSV dissemination in the lungs was variable and correlated with lung pathology. We demonstrate new cross-protective anti-G monoclonal antibodies targeting multiple sites including conformation-dependent class G0 MAb 77D2, CCD-specific class G1 MAb 40D8, and carboxy terminus of CCD class G5 MAb 7H11, to support development of G-targeting monoclonal antibodies against RSV.

Antiviral effect of alkaloids-free Ephedra Herb extract on respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children. Despite decades of efforts, no effective therapies are available. We recently reported that extracts of Ephedra Herb and Cinnamon Bark interacted with the G attachment protein of RSV to inhibit infectivity. The present in vitro study aimed to investigate the antiviral effect of ephedrine alkaloids-free Ephedra Herb extract (EFE), which is characterized by free of harmful effects of ephedrine alkaloids in Ephedra Herb, on experimental RSV infection. Infection of RSV into A549 cells simultaneously with EFE resulted the significant reduction of RSV RNA, viral protein, and viral titers after the incubation of the cells. We found that RSV attachment to the cell surface was inhibited both in the presence of EFE and when RSV particles were pre-treated with EFE. We also found that EFE specifically interacted with the central conserved domain of RSV G protein by surface plasmon resonance, demonstrating that specific binding of G protein to the cellular receptor was inhibited by EFE. Another mechanism was found in which a higher concentration of EFE inhibited the viral load immediately after the viral entry into host cells, suggesting the inhibition of viral RNA replication. These results demonstrate that EFE worked against RSV infection through multiple antiviral mechanisms, a unique feature of this crude drug extract.

Immune Prophylaxis Targeting the Respiratory Syncytial Virus (RSV) G Protein.

The respiratory syncytial virus (RSV) causes significant respiratory disease in young infants and the elderly. Immune prophylaxis in infants is currently limited to palivizumab, an anti-RSV fusion (F) protein monoclonal antibody (mAb). While anti-F protein mAbs neutralize RSV, they are unable to prevent aberrant pathogenic responses provoked by the RSV attachment (G) protein. Recently, the co-crystal structures of two high-affinity anti-G protein mAbs that bind the central conserved domain (CCD) at distinct non-overlapping epitopes were solved. mAbs 3D3 and 2D10 are broadly neutralizing and block G protein CX3C-mediated chemotaxis by binding antigenic sites γ1 and γ2, respectively, which is known to reduce RSV disease. Previous studies have established 3D3 as a potential immunoprophylactic and therapeutic; however, there has been no similar evaluation of 2D10 available. Here, we sought to determine the differences in neutralization and immunity to RSV Line19F infection which recapitulates human RSV infection in mouse models making it useful for therapeutic antibody studies. Prophylactic (24 h prior to infection) or therapeutic (72 h post-infection) treatment of mice with 3D3, 2D10, or palivizumab were compared to isotype control antibody treatment. The results show that 2D10 can neutralize RSV Line19F both prophylactically and therapeutically, and can reduce disease-causing immune responses in a prophylactic but not therapeutic context. In contrast, 3D3 was able to significantly (p < 0.05) reduce lung virus titers and IL-13 in a prophylactic and therapeutic regimen suggesting subtle but important differences in immune responses to RSV infection with mAbs that bind distinct epitopes.

Amino acid variations of the immuno-dominant domain of respiratory syncytial virus attachment glycoprotein (G) affect the antibody responses In BALB/c mice

Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in ruminants and infants. The G glycoprotein of RSV serves as the viral attachment ligand. Despite currently available vaccines, RSV immunity is insufficient, and re-infections occur. Vaccine studies employing the G-protein's 174–187 amino acids, representing the immunodominant domain, have protected mice and calves against infections. To investigate the causes of vaccination failure, we designed four synthetic peptides for the ruminant RSV isolates (391–2, Maryland-BRSV, European-BRSV, and ORSV) using the immune-dominant sequence and vaccinated mice groups with them. The produced antibodies targeting each peptide were evaluated using ELISA and flow cytometry to determine their reactivity against the linear antigen and the native form of the G protein, respectively. Antibodies responded to homologous and heterologous peptides as determined by ELISA. Using flow cytometry-analysis targeting the natively folded protein, most generated antibodies reacted only with their homologous strain. However, antibodies raised to 391–2 peptide reacted with homologous and heterologous Maryland-BRSV viral epitopes. Accordingly, inadequate immunity and recurring RSV infections might be attributed to variations of antibodies targeting the immunodominant region of the G-protein.

Anti-G protein antibodies targeting the RSV G protein CX3C chemokine region improve the interferon response.

Respiratory syncytial virus (RSV) is a poor inducer of antiviral interferon (IFN) responses which result in incomplete immunity and RSV disease. Several RSV proteins alter antiviral responses, including the non-structural proteins (NS1, NS2) and the major viral surface proteins, that is, fusion (F) and attachment (G) proteins. The G protein modifies the host immune response to infection linked in part through a CX3 C chemokine motif. Anti-G protein monoclonal antibodies (mAbs), that is, clones 3D3 and 2D10 that target the G protein CX3C chemokine motif can neutralize RSV and inhibit G protein-CX3CR1 mediated chemotaxis. Determine how monoclonal antibodies against the RSV F and G proteins modify the type I and III IFN responses to RSV infection. As the G protein CX3 C motif is implicated in IFN antagonism, we evaluated two mAbs that block G protein CX3C-CX3CR1 interaction and compared responses to isotype mAb control using a functional cellular assay and mouse model. Mouse lung epithelial cells (MLE-15 cells) and BALB/c mice were infected with RSV Line19 F following prophylactic mAb treatment. Cell supernatant or bronchoalveolar lavage fluid (BALF) were assayed for types I and III IFNs. Cells were interrogated for changes in IFN-related gene expression. Treatment with an anti-G protein mAb (3D3) resulted in improved IFN responses compared with isotype control following infection with RSV, partially independently of neutralization, and this was linked to upregulated SOCS1 expression. These findings show that anti-G protein antibodies improve the protective early antiviral response, which has important implications for vaccine and therapeutic design. RSV is a leading cause of respiratory disease in infants and the elderly. The only Food and Drug Administration-approved prophylactic treatment is limited to an anti-F protein monoclonal antibody (mAb), that is, palivizumab which has modest efficacy against RSV disease. Accumulating evidence suggests that targeting the RSV attachment (G) protein may provide improved protection from RSV disease. It is known that the G protein is an IFN antagonist, and IFN has been shown to be protective against RSV disease. In this study, we compared IFN responses in mouse lung epithelial (MLE-15) cells and in mice infected with RSV Line19 F treated with anti-G protein or anti-F protein mAbs. The levels of type I and III IFNs were determined. Anti-G protein mAbs improved the levels of IFNs compared with isotype-treated controls. These findings support the concept that anti-G protein mAbs mediate improved IFN responses against RSV disease, which may enable improved treatment of RSV infections.

Effect of hot water extracts of Arthrospira maxima (spirulina) against respiratory syncytial virus

Background: Spirulina (Arthrospira maxima) hot water extracts such as calcium spirulan (Ca-SP) have demonstrated antiviral effects against herpes simplex virus (HSV), human immunodeficiency virus-1 (HIV-1), and influenza virus infections. There is no prior evidence suggesting the anti-viral activity of the spirulina hot water extract against respiratory syncytial virus (RSV).Purpose: There are currently no effective antivirals available to treat RSV infection. Therefore, the development of safe and novel anti-RSV drugs is urgent and necessary. The aim of this work was to demonstrate the anti-RSV activity of spirulina hot water extracts and determine the potential mechanism of action.Methods: Cytotoxicity and anti-RSV activity of spirulina hot water extracts were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutralization assays, respectively. Potential mechanisms and components were assessed using time of addition, attachment, internalization, pull-down assays, and composition analysis.Results: The polysaccharide-enriched high-molecular weight fraction (>100 kDa, SHD1) had a high total sugar content, with rhamnose accounting for approximately 60 mol% of total monosaccharides. The main glycosyl linkages included 3-, 4-, and 2,3-rhamnopyranosyl linkages. All spirulina hot water extracts showed no toxicity toward human epithelial type 2 (HEp-2) cells but demonstrated anti-RSV activity. The SHD1 had a half maximal effective concentration (EC50) of 0.0915 mg/ml and a selective index (SI) of >261.5 against RSV. SHD1 significantly reduced viral yield in a dose-dependent manner during the RSV attachment stage. SHD1 disrupted RSV internalization and inhibited RSV attachment (G) protein binding to heparan sulfate receptors on the host cell surface, thus preventing RSV attachment and entry.Conclusion: SHD1 serves as an effective candidate for novel drug development against RSV infection.

Discovery of N-Containing (-)-Borneol Esters as Respiratory Syncytial Virus Fusion Inhibitors.

Respiratory syncytial virus (RSV) causes acute respiratory infections, thus, posing a serious threat to the health of infants, children, and elderly people. In this study, we have discovered a series of potent RSV entry inhibitors with the (-)-borneol scaffold. The active compounds 3b, 5a, 5c, 7b, 9c, 10b, 10c, and 14b were found to exhibit activity against RSV A strain A2 in HEp-2 cells. The most active substances, 3b (IC50 = 8.9 μM, SI = 111) and 5a (IC50 = 5.0 μM, SI = 83), displayed more potency than the known antiviral agent Ribavirin (IC50 = 80.0 μM, SI = 50). Time-of-addition assay and temperature shift studies demonstrated that compounds 3b, 5a, and 6b inhibited RSV entry, probably by interacting with the viral F protein that mediated membrane fusion, while they neither bound to G protein nor inhibited RSV attachment to the target cells. Appling procedures of molecular modeling and molecular dynamics, the binding mode of compounds 3b and 5a was proposed. Taken together, the results of this study suggest (-)-borneol esters to be promising lead compounds for developing new anti-RSV agents.

Immunoinformatics-Aided Analysis of RSV Fusion and Attachment Glycoproteins to Design a Potent Multi-Epitope Vaccine.

Respiratory syncytial virus (RSV) usually causes respiratory tract infections of upper airways in infants and young children. Despite recent medical advances, no approved vaccine is available to control RSV infections. Therefore, we conducted an immunoinformatics study to design and evaluate a potential multi-epitope vaccine against RSV. Sequence-based analyses of the glycoproteins F and G revealed a total of eight CD8 T-cell and three CD4 T-cell epitopes after considering antigenicity, binding affinity and other parameters. Molecular docking analysis confirmed that these T-cell epitopes developed strong structural associations with HLA allele(s). By integrating these prioritized epitopes with linkers and a cholera toxin-derived adjuvant, a multi-epitope vaccine was designed. The developed vaccine was found to be stable, non-allergenic, flexible and antigenic. Molecular docking analysis revealed a striking mean HADDOCK score (−143.3) of top-ranked vaccine-TLR cluster and a Gibbs free energy change (ΔG) value of −11.3 kcal mol−1. As per computational immune simulation results, the vaccine generated a high titer of antibodies (especially IgM) and effector T-cells. Also, codon optimization and in silico cloning ensured the increased expression of vaccine in Escherichia coli. Altogether, we anticipate that the multi-epitope vaccine reported in this study will stimulate humoral and cellular responses against RSV infection, subject to follow-up experimental validation.

Ephedrae Herba and Cinnamomi Cortex interactions with G glycoprotein inhibit respiratory syncytial virus infectivity

Although respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children, no effective therapies are available. Recently, RSV G, the attachment glycoprotein, has become a major focus in the development of therapeutic strategies against RSV infection. Treatment of RSV-infected cultured cells with maoto, a traditional herbal medicine for acute febrile diseases, significantly reduced the viral RNA and titers. RSV attachment to the cell surface was inhibited both in the presence of maoto and when RSV particles were pre-treated with maoto. We demonstrated that maoto components, Ephedrae Herba (EH) and Cinnamomi Cortex (CC), specifically interacted with the central conserved domain (CCD) of G protein, and also found that this interaction blocked viral attachment to the cellular receptor CX3CR1. Genetic mutation of CX3C motif on the CCD, the epitope for CX3CR1, decreased the binding capacity to EH and CC, suggesting that CX3C motif was the target for EH and CC. Finally, oral administration of maoto for five days to RSV-infected mice significantly reduced the lung viral titers. These experiments clearly showed the anti-RSV activity of EH and CC mixed in maoto. Taken together, this study provides insights for the rational design of therapies against RSV infection.

Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model

Respiratory syncytial virus (RSV) is one of the most important causes of respiratory disease in infants, immunocompromised individuals, and the elderly. Natural infection does not result in long-term immunity, and there is no licensed vaccine. Vesicular stomatitis virus (VSV) is a commonly used vaccine vector platform against infectious diseases, and has been used as a vector for a licensed Ebola vaccine. In this study, we expressed the RSV fusion (F) protein, the RSV F protein stabilized in either a pre-fusion or a post-fusion configuration, the attachment glycoprotein (G), or the G and F proteins of RSV in combination in a VSV vector. Cotton rats were immunized with these recombinants intranasally or subcutaneously to test immunogenicity. RSV F stabilized in either a pre-fusion or a post-fusion configuration proved to be poorly immunogenic and protective when compared to unmodified F. RSV G provided partial protection and moderate levels of neutralizing antibody production, both of which improved with intranasal administration compared to subcutaneous inoculation. The most successful vaccine vector was VSV expressing both the G and F proteins after intranasal inoculation. Immunization with this recombinant induced neutralizing antibodies and provided protection from RSV challenge in the upper and lower respiratory tract for at least 80 days. Our results demonstrate that co-expression of F and G proteins in a VSV vector provides synergistic effects in inducing RSV-specific neutralizing antibodies and protection against RSV infection.

Comparative analysis of spatial-temporal patterns of human metapneumovirus and respiratory syncytial virus in Africa using genetic data, 2011\u20132014

BackgroundHuman metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are leading causes of viral severe acute respiratory illnesses in childhood. Both the two viruses belong to the Pneumoviridae family and show overlapping clinical, epidemiological and transmission features. However, it is unknown whether these two viruses have similar geographic spread patterns which may inform designing and evaluating their epidemic control measures.MethodsWe conducted comparative phylogenetic and phylogeographic analyses to explore the spatial-temporal patterns of HMPV and RSV across Africa using 232 HMPV and 842 RSV attachment (G) glycoprotein gene sequences obtained from 5 countries (The Gambia, Zambia, Mali, South Africa, and Kenya) between August 2011 and January 2014.ResultsPhylogeographic analyses found frequently similar patterns of spread of RSV and HMPV. Viral sequences commonly clustered by region, i.e., West Africa (Mali, Gambia), East Africa (Kenya) and Southern Africa (Zambia, South Africa), and similar genotype dominance patterns were observed between neighbouring countries. Both HMPV and RSV country epidemics were characterized by co-circulation of multiple genotypes. Sequences from different African sub-regions (East, West and Southern Africa) fell into separate clusters interspersed with sequences from other countries globally.ConclusionThe spatial clustering patterns of viral sequences and genotype dominance patterns observed in our analysis suggests strong regional links and predominant local transmission. The geographical clustering further suggests independent introduction of HMPV and RSV variants in Africa from the global pool, and local regional diversification.

The larger attachment glycoprotein of respiratory syncytial virus produced in primary human bronchial epithelial cultures reduces infectivity for cell lines.

Respiratory syncytial virus (RSV) infects the upper and lower respiratory tracts and can cause lower respiratory tract infections in children and elders. RSV has traditionally been isolated, grown, studied and quantified in immortalized cell lines, most frequently HEp-2 cells. However, in vivo RSV infection is modeled more accurately in primary well differentiated human bronchial epithelial (HBE) cultures where RSV targets the ciliated cells and where the putative RSV receptor differs from the receptor on HEp-2 cells. The RSV attachment (G) glycoprotein in virions produced by HEp-2 cells is a highly glycosylated 95 kDa protein with a 32 kDa peptide core. However, virions produced in HBE cultures, RSV (HBE), contain an even larger, 170 kDa, G protein (LgG). Here we show that LgG is found in virions from both subgroups A and B lab-adapted and clinical isolates. Unexpectedly, RSV (HBE) virions were approximately 100-fold more infectious for HBE cultures than for HEp-2 cells. Surprisingly, the cause of this differential infectivity, was reduced infectivity of RSV (HBE) on HEp-2 cells rather than enhanced infectivity on HBE cultures. The lower infectivity of RSV(HBE) for HEp-2 cells is caused by the reduced ability of LgG to interact with heparan sulfate proteoglycans (HSPG), the RSV receptor on HEp-2 cells. The discovery of different infectivity corresponding with the larger form of the RSV attachment protein when produced by HBE cultures highlights the importance of studying a virus produced by its native host cell and the potential impact on quantifying virus infectivity on cell lines where the virus entry mechanisms differ from their natural target cell.

Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies.

Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine.

Immunogenicity and inflammatory properties of respiratory syncytial virus attachment G protein in cotton rats

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and young children worldwide. The attachment (G) protein of RSV is synthesized by infected cells in both a membrane bound (mG) and secreted form (sG) and uses a CX3C motif for binding to its cellular receptor. Cell culture and mouse studies suggest that the G protein mimics the cytokine CX3CL1 by binding to CX3CR1 on immune cells, which is thought to cause increased pulmonary inflammation in vivo. However, because these studies have used RSV lacking its G protein gene or blockade of the G protein with a G protein specific monoclonal antibody, the observed reduction in inflammation may be due to reduced virus replication and spread, and not to a direct role for G protein as a viral chemokine. In order to more directly determine the influence of the soluble and the membrane-bound forms of G protein on the immune system independent of its attachment function for the virion, we expressed the G protein in cotton rat lungs using adeno-associated virus (AAV), a vector system which does not itself induce inflammation. We found no increase in pulmonary inflammation as determined by histology and bronchoalveolar lavage after inoculation of AAVs expressing the membrane bound G protein, the secreted G protein or the complete G protein gene which expresses both forms. The long-term low-level expression of AAV-G did, however, result in the induction of non-neutralizing antibodies, CD8 T cells and partial protection from challenge with RSV. Complete protection was accomplished through co-immunization with AAV-G and an AAV expressing cotton rat interferon α.

A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Disease severity of respiratory syncytial virus (RSV) infection correlate to a novel set of five amino acid substitutions in the RSV attachment glycoprotein (G) in China

Human respiratory syncytial virus (RSV) is one of the major viruses of acute respiratory tract disease among infants and young children. We performed molecular epidemiology analysis of RSV among inpatient children in Guangzhou, China. Phylogenetic and Bayesian analysis showed that genotype ON1 was the only subgroup A virus in this study. Interestingly, the majority of Guangzhou ON1 strains formed a well-supported cluster, and these strains shared a novel set of five amino acid substitutions that never illustrated before. Furthermore, the degree of disease severity was assessed using a severity scoring system. The patients carrying the novel RSV A strain were associated with milder respiratory symptoms compared to other RSV A positives. In conclusion, a specific set of five amino acid substitutions was found in China and further analysis showed that disease severity was associated with these alterations. These findings will provide valuable information for the pathogenic mechanism and vaccine development of RSV.

Characterization of the role of N-glycosylation sites in the respiratory syncytial virus fusion protein in virus replication, syncytium formation and antigenicity

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.

Effects of Alterations to the CX3C Motif and Secreted Form of Human Respiratory Syncytial Virus (RSV) G Protein on Immune Responses to a Parainfluenza Virus Vector Expressing the RSV G Protein.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised.IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

Respiratory syncytial virus F and G protein core fragments fused to HBsAg-binding protein (SBP) induce a Th1-dominant immune response without vaccine-enhanced disease.

The induction of a dominant Th2-type response is the main cause of harmful inflammation in respiratory syncytial virus (RSV) vaccine trials. A balanced Th1 versus Th2 immune response is needed for a safe and effective RSV vaccine. In this study, we evaluated the potential of a recombinant protein SBP-FG as a vaccine candidate with the main focus on shifting the harmful Th2 response to a Th1 response. SBP-FG consists of epitopes from RSV fusion (F) and attachment (G) proteins conjugated to the N-terminus of HBsAg-binding protein (SBP). SBP-FG induced significantly stronger immune responses assessed at the level of total IgG, IgA and neutralizing antibodies as compared with formalin-inactivated RSV (FI-RSV) and live RSV. Analysis of IgG isotypes, lung cytokines and T helper cells showed that SBP-FG induced a dominant Th1-type response. Further, SBP-FG immunized mice showed significantly reduced lung eosinophilia, reduced viral multiplication in lungs after challenge infection and provided protection against RSV infection. These results suggest that SBP-FG can be developed into a safe and effective vaccine against RSV. However, more studies are required to further evaluate SBP-FG as a potent vaccine candidate against RSV.