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

Abstract

Background: 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.

Objectives: Determine how monoclonal antibodies against the RSV F and G proteins modify the type I and III IFN responses to RSV infection.

Design: 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.

Methods: 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.

Results: 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.

Conclusions: These findings show that anti-G protein antibodies improve the protective early antiviral response, which has important implications for vaccine and therapeutic design.

Plain language summary: 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.

Keywords: F protein; G protein; IFN; ISG; Line19 F; RSV; mAb; monoclonal antibody; palivizumab.

Figure 1.

Anti-G protein mAbs are non-neutralizing in vitro but neutralize in mice. (a) RSV Line19 F was pre-incubated with 10 μg/mL mAb for 1 h at 37°C then added onto MLE-15 cells for 24 h. (b) BALB/c mice were i.p. treated prophylactically with 1 mg/kg of indicated mAb 24 h before i.n. infection with 10⁶ PFU Line19 F. Twenty-four hours post infection, genome equivalent units (GEs) were determined in MLE-15 cells and homogenized mouse lung using a standard curve as described in materials and methods. (c) RSV Line19 F was pre-incubated with 10 μg/mL mAb for 1 h at 37°C then added onto MLE-15 cells for 24 h. After 24 h, plates were scanned using Cellomics Array Scan and fluorescent focus units (FFUs) were enumerated. (d) Viral titers in mouse lungs were determined by plaque assay. ND = not detected. * p < 0.05 as determined by one-way ANOVA with Dunnett’s post hoc test compared with isotype control. Experiments were individually repeated at least twice with technical replicates. Bars represent the mean ± SEM.

Figure 2.

RSV antibodies modify IFNβ responses. (a) RSV Line19 F was pre-incubated with 10 μg/mL mAb for 1 h at 37°C then added onto MLE-15 cells for 24 h and supernatant was assayed for IFNβ concentrations. (b) The BALB/c mice were i.p. treated prophylactically with 1 mg/kg of indicated mAb 24 h before i.n. infection with 10⁶ PFU Line19 F. At 24 hpi, BALF was collected and assayed for IFNβ concentrations. Concentration of IFNβ was determined by ELISA and quantified using a standard curve of IFNβ. Bars represent the mean ± SEM of IFNβ (pg/mL). * p < 0.05 as determined by one-way ANOVA with Dunnett’s post hoc test compared with isotype control. For in vitro, bars represent the mean of three independent studies with technical replicates. For in vivo, bars represent mean of three independent assays with technical replicates from one experiment of 5 mice/group.

Figure 3.

RSV antibodies modify type I IFN gene expression. RSV was pre-incubated with 10 μg/mL mAb for 1 h at 37°C then added onto MLE-15 cells for 24 h. Twenty-four hours post infection, expression of (a) IFNA1, (b) IFNB, and (c) ISG15 genes was determined in MLE-15 cells. Bars represent the mean ± SEM of gene expression fold change using ΔΔCt PCR method normalized to ACTB expression and mock-infected cells. *p < 0.05 as determined by one-way ANOVA with Dunnett’s post hoc test compared with isotype control. Experiments were individually repeated at least twice with technical replicates.

Figure 4.

RSV antibodies modify IFNλ2/3 responses. (a) RSV Line19 F was pre-incubated with 10 μg/mL mAb for 1 h at 37°C, then added onto MLE-15 cells for 24 h, and supernatant was assayed for IFNλ2/3. (b) The BALB/c mice were i.p. treated prophylactically with 1 mg/kg of indicated mAb 24 h before i.n. infection with 10⁶ PFU Line19 F. Twenty-four hours post infection, BALF was collected and assayed for IFNλ2/3. Concentration of IFNλ2/3 was determined by ELISA and quantified using a standard curve of IFNλ2/3. Bars represent the mean ± SEM of IFNλ2/3 (pg/mL). *p < 0.05 as determined by one-way ANOVA with Dunnett’s post hoc test compared with isotype control. For in vitro, bars represent the mean of three independent studies with technical replicates. For in vivo, bars represent mean of three independent assays with technical replicates from one experiment of 5 mice/group.

Figure 5.

RSV antibodies modify type III IFN gene expression. RSV Line19 F was pre-incubated with 10 μg/mL mAb for 1 h at 37°C then added onto MLE-15 cells for 24 h. At 24 hpi, the expression of IFNL3 genes was determined in MLE-15 cells. Bars represent the mean ± SEM of gene expression fold change using ΔΔCt PCR method normalized to ACTB expression and mock-infected cells. *p < 0.05 as determined by one-way ANOVA with Dunnett’s post hoc test compared with isotype control. Experiments were individually repeated at least twice with technical replicates.

Figure 6.

RSV antibodies modify the suppression of cytokine signaling (SOCS) gene expression. RSV Line19 F was pre-incubated with 10 μg/mL mAb for 1 h at 37°C, then added onto MLE-15 cells for 24 h. At 24 hpi, expression of (a) SOCS1, (b) SOCS2, and (c) SOCS3 genes was determined in MLE-15 cells. Bars represent the mean ± SEM of gene expression fold change using ΔΔCt PCR method normalized to ACTB expression and mock-infected cells. * p < 0.05 as determined by one-way ANOVA with Dunnett’s post hoc test compared with isotype control. Experiments were individually repeated at least twice with technical replicates.