The Design of Vaccines Based on the Shielding of Antigenic Site Ø of a Respiratory Syncytial Virus Fusion Protein Immunogen.

Respiratory syncytial virus (RSV), for which there is currently no licensed vaccine, displays a fusion (F) protein that is considered a vaccine target. This protein has an antigenic site called site Ø, which has been shown to elicit potent, neutralizing antibodies and has therefore been considered important in the formulation of RSV vaccines. However, this site is also the least conserved region on the F protein across RSV subtypes. Therefore, directing the immune response away from site Ø and refocusing it toward more conserved parts of the RSV F protein might serve to better elicit broadly neutralizing antibodies. To demonstrate that directing the immune response away from site Ø is a viable approach, a prefusion F-based vaccine based on an F protein with a shielded site Ø is generated. Sera from mice immunized with multivalent scaffolds presenting this immunogen is capable of neutralizing RSV of both subtypes. This result may have application in the development of an effective and broadly protective RSV vaccine.

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.

EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein.

UNLABELLED: Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. IMPORTANCE: RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.

Identification of residues in the human respiratory syncytial virus fusion protein that modulate fusion activity and pathogenesis.

UNLABELLED: Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein. IMPORTANCE: Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.

The respiratory syncytial virus fusion protein and neutrophils mediate the airway mucin response to pathogenic respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the leading cause of death due to a viral etiology in infants. RSV disease is characterized by epithelial desquamation, neutrophilic bronchiolitis and pneumonia, and obstructive pulmonary mucus. It has been shown that infection of BALB/cJ mice with RSV clinical isolate A2001/2-20 (2-20) results in a higher early viral load, greater airway necrosis, and higher levels of interleukin-13 (IL-13) and airway mucin expression than infection with RSV laboratory strain A2. We hypothesized that the fusion (F) protein of RSV 2-20 is a mucus-inducing viral factor. In vitro, the fusion activity of 2-20 F but not that of A2 F was enhanced by expression of RSV G. We generated a recombinant F-chimeric RSV by replacing the F gene of A2 with the F gene of 2-20, generating A2-2-20F. Similar to the results obtained with the parent 2-20 strain, infection of BALB/cJ mice with A2-2-20F resulted in a higher early viral load and higher levels of subsequent pulmonary mucin expression than infection with the A2 strain. A2-2-20F infection induced greater necrotic airway damage and neutrophil infiltration than A2 infection. We hypothesized that the neutrophil response to A2-2-20F infection is involved in mucin expression. Antibody-mediated depletion of neutrophils in RSV-infected mice resulted in lower tumor necrosis factor alpha levels, fewer IL-13-expressing CD4 T cells, and less airway mucin production in the lung. Our data are consistent with a model in which the F and attachment (G) glycoprotein functional interaction leads to enhanced fusion and F is a key factor in airway epithelium infection, pathogenesis, and subsequent airway mucin expression.

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice.

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.