Enhancing the Thermostability and Immunogenicity of a Respiratory Syncytial Virus (RSV) Live-Attenuated Vaccine by Incorporating Unique RSV Line19F Protein Residues.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, and an effective vaccine is not yet available. We previously generated an RSV live-attenuated vaccine (LAV) candidate, DB1, which was attenuated by a low-fusion subgroup B F protein (BAF) and codon-deoptimized nonstructural protein genes. DB1 was immunogenic and protective in cotton rats but lacked thermostability and stability of the prefusion conformation of F compared to strains with the line19F gene. We hypothesized that substitution of unique residues from the thermostable A2-line19F strain could thermostabilize DB1 and boost its immunogenicity. We therefore substituted 4 unique line19F residues into the BAF protein of DB1 by site-directed mutagenesis and rescued the recombinant virus, DB1-QUAD. Compared to DB1, DB1-QUAD had improved thermostability at 4°C and higher levels of prefusion F as measured by enzyme-linked immunosorbent assays (ELISAs). DB1-QUAD was attenuated in normal human bronchial epithelial cells, in BALB/c mice, and in cotton rats but grew to wild-type titers in Vero cells. In mice, DB1-QUAD was highly immunogenic and generated significantly higher neutralizing antibody titers to a panel of RSV A and B strains than did DB1. DB1-QUAD was also efficacious against wild-type RSV challenge in mice and cotton rats. Thus, substitution of unique line19F residues into RSV LAV DB1 enhanced vaccine thermostability, incorporation of prefusion F, and immunogenicity and generated a promising vaccine candidate that merits further investigation.IMPORTANCE We boosted the thermostability and immunogenicity of an RSV live-attenuated vaccine candidate by substituting 4 unique residues from the RSV line19F protein into the F protein of the heterologous vaccine strain DB1. The resultant vaccine candidate, DB1-QUAD, was thermostable, attenuated in vivo, highly immunogenic, and protective against RSV challenge in mice and 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.

Prophylaxis with a respiratory syncytial virus (RSV) anti-G protein monoclonal antibody shifts the adaptive immune response to RSV rA2-line19F infection from Th2 to Th1 in BALB/c mice.

UNLABELLED: Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. In the present study, we investigated the effect of prophylactic treatment with the intact and F(ab')2 forms of an anti-G protein monoclonal antibody (MAb), 131-2G, on the humoral and cellular adaptive immune responses to RSV rA2-line19F (r19F) challenge in BALB/c mice. The F(ab')2 form of 131-2G does not decrease virus replication, but intact 131-2G does. The serum specimens for antibodies and spleen cells for memory T cell responses to RSV antigens were analyzed at 30, 45, 75, and 95 days postinfection (p.i.) with or without prior treatment with 131-2G. The ratios of Th2 to Th1 antibody isotypes at each time p.i indicated that both forms of MAb 131-2G shifted the subclass response from a Th2 (IgG1 and IgG2b) to a Th1 (IgG2A) bias. The ratio of IgG1 to IgG2A antibody titer was 3-fold to 10-fold higher for untreated than MAb-treated mice. There was also some increase in IgG (22% ± 13% increase) and neutralization (32% increase) in antibodies with MAb 131-2G prophylaxis at 75 days p.i. Treatment with 131-2G significantly (P ≤ 0.001) decreased the percentage of interleukin-4 (IL-4)-positive CD4 and CD8 cells in RSV-stimulated spleen cells at all times p.i., while the percentage of interferon gamma (IFN-γ) T cells significantly (P ≤ 0.001) increased ≥ 75 days p.i. The shift from a Th2- to a Th1-biased T cell response in treated compared to untreated mice likely was directed by the much higher levels of T-box transcription factor (T-bet) (≥ 45% versus <10%) in CD4 and CD8 T cells and lower levels of Gata-3 (≤ 2% versus ≥ 6%) in CD4 T cells in peptide-stimulated, day 75 p.i. spleen cells. These data show that the RSV G protein affects both humoral and cellular adaptive immune responses, and induction of 131-2G-like antibodies might improve the safety and long-term efficacy of an RSV vaccine. IMPORTANCE: The data in this report suggest that the RSV G protein not only contributes to disease but also dampens the host immune response to infection. Both effects of G likely contribute to difficulties in achieving an effective vaccine. The ability of MAb 131-2G to block these effects of G suggests that inducing antibodies similar to 131-2G should prevent disease and enhance the adaptive immune response with later RSV infection. The fact that 131-2G binds to the 13-amino-acid region conserved among all strains and that flanking sequences are conserved within group A or group B strains simplifies the task of developing a vaccine to induce 131-2G-like antibodies. If our findings in mice apply to humans, then including the 131-2G binding region of G in a vaccine should improve its safety and efficacy.

A respiratory syncytial virus (RSV) anti-G protein F(ab')2 monoclonal antibody suppresses mucous production and breathing effort in RSV rA2-line19F-infected BALB/c mice.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Increased airway resistance and increased airway mucin production are two manifestations of RSV infection in children. RSV rA2-line19F infection induces pulmonary mucous production and increased breathing effort in BALB/c mice and provides a way to assess these manifestations of RSV disease in an animal model. In the present study, we investigated the effect of prophylactic treatment with the F(ab')2 form of the anti-G protein monoclonal antibody (MAb) 131-2G on disease in RSV rA2-line19F-challenged mice. F(ab')2 131-2G does not affect virus replication. It and the intact form that does decrease virus replication prevented increased breathing effort and airway mucin production, as well as weight loss, pulmonary inflammatory-cell infiltration, and the pulmonary substance P and pulmonary Th2 cytokine levels that occur in mice challenged with this virus. These data suggest that the RSV G protein contributes to prominent manifestations of RSV disease and that MAb 131-2G can prevent these manifestations of RSV disease without inhibiting virus infection.

Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells.

RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection.

An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. To clarify the potential for an anti-G mAb, 131-2G which has both anti-viral and anti-inflammatory effects, to effectively treat RSV disease, we determined the kinetics of its effect compared to the effect of the anti-F mAb, 143-6C on disease in mice. Treatment administered three days after RSV rA2-line19F (r19F) infection showed 131-2G decreased breathing effort, pulmonary mucin levels, weight loss, and pulmonary inflammation earlier and more effectively than treatment with mAb 143-6C. Both mAbs stopped lung virus replication at day 5 post-infection. These data show that, in mice, anti-G protein mAb is superior to treating disease during RSV infection than an anti-F protein mAb similar to Palivizumab. This combination of anti-viral and anti-inflammatory activity makes 131-2G a promising candidate for treating for active human RSV infection.