Nanoparticle vaccines encompassing the respiratory syncytial virus (RSV) G protein CX3C chemokine motif induce robust immunity protecting from challenge and disease

Abstract

Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN-γ secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8(+) T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2-specific T cell responses, and a reduction in RSV disease pathogenesis.

Figure 1. Antibody responses elicited by vaccination with RSV G protein nanoparticles.

Groups of BALB/c mice (n=5) were vaccinated with nanoparticle vaccines diluted in PBS to yield 50 µg of designed peptide per dose, with 10 µg of purified G protein emulsified 1:1 with TiterMAX®, 10⁵ PFU of live RSV A2 or with 100 µL of PBS. Sera were obtained from blood taken 21 days after the secondary inoculation. RSV A2 (A, C, and E) and RSV B1 (B, D, and F)-specific IgG (A and B), IgG1 (C and D) and IgG2a (E and F) levels were determined by indirect ELISA. Bars represent the average titer of each group with error bars representing the SEM from n=5 mice per group. *, #, p<0.05, significant difference as determined by one-way ANOVA and Dunnett’s test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).

Figure 2. RSV A2 neutralizing antibodies in mice vaccinated with RSV G protein nanoparticles.

Sera from vaccinated mice were evaluated for neutralizing antibodies by plaque inhibition assay. Serum samples were collected 21 days after the secondary inoculation (grey bars) and 5 days post challenge (black bars) with 10⁶ PFU of RSV A2. Error bar represents the SEM from n=5 mice per group. *, #, p<0.05, significant difference as determined by one-way ANOVA and Dunnett’s test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).

Figure 3. Lung virus titers and body weight loss following RSV A2 challenge of vaccinated mice.

Groups of BALB/c mice were vaccinated with nanoparticle diluted in PBS to yield 50 µg of designed peptide per dose, with 10 µg of purified G protein emulsified 1:1 with TiterMAX®, 10⁵ PFU of live RSV A2 or with 100 µL of PBS on days 0 and 21, and challenged i.n. on day 42 with 10⁶ PFU of RSV A2. A) Lung virus titers were determined 5 days post-challenge by plaque assay (n=5). The data are presented as PFU/g of lung tissue. B) Animals were weighed daily and percentage of weight loss calculated based on day 0. C) Quantitation of lung inflammation at day 5 post challenge (n=3). Error bar represents the SEM from n=3-5 mice per group. *, #, p<0.05, significant difference as determined by one-way ANOVA and Dunnett’s test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).

Figure 4. Frequency of RSV-specific IFNγ and IL-4 secreting cells after virus challenge.

The number of G₁₈₃-specific (grey bars) and M2₈₂-specific (black bars) IL-4 (A) or IFNγ (B) producing splenocytes was determined by ELISPOT in cells harvested 5 days post-challenge. The data are presented as cytokine spots/10⁶ splenocytes. Error bars represent the SEM from n=5 mice per group. *, #, p<0.05, significant difference as determined by one-way ANOVA and Dunnett’s test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).

Figure 5. Enumeration of RSV M2-specific CD8⁺ T cells using MHC class I tetramers.

Spleen and BAL cell suspensions obtained from mice challenged with RSV A2 were stained with anti-mouse CD3e PE-Cy7-conjugated, anti-mouse CD8α PerCP-conjugated and APC-labeled M2-specific H-2Kd tetramer. FACS contour plots were gate on CD3/CD8 positive cells. Percentage of M2- H-2Kd tetramer positive CD8⁺ T cells in BAL (A and C) and spleen (B) are shown. Error bar represents the SEM from n=5 mice per group. *, #, p<0.05, significant difference as determined by one-way ANOVA and Dunnett’s test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).

Figure 6. Pulmonary cell recruitment and cytokine production upon RSV infection.

Mice were vaccinated with 50 µg of RSV G nanoparticles, 10 µg of purified G protein emulsified 1:1 with TiterMAX®, 10⁵ PFU of live RSV A2 or with 100 µL of PBS and challenged at 6 weeks post boost with 10⁶ PFU of RSV A2. BAL were collected at day 5 post challenge and cell suspensions were immunolabeled with anti-CD3 PE, anti-CD8 FITC and anti-CD4 PerCPCy5.5 to characterize T cells (A) or with a combination of anti-CD45 PerCP-Cy5.5, anti-siglec F PE and anti-CD11c FITC to detect eosinophils (B, grey bars) and macrophages (B, black bars). Based on cell surface markers eosinophils were defined as CD45⁺SiglecF ⁺ CD11c^low cells and alveolar macrophages as CD45⁺SiglecF ⁺ CD11c^high cells. The data are presented as the total number of cells. Values represent mean ± SEM of cells per BAL (n=5). The level of IFN-γ, IL-4, IL-5, IL-13, IL-17A and TNF-α (C) were measured in BAL supernatant by Luminex assay, and the data are presented as picograms of cytokine/ mL of BAL supernatant (n=5). *, #, p<0.05, significant difference as determined by one-way ANOVA and Dunnett’s test, compared with PBS vaccinated control mice (*) or compared to live RSV A2 vaccinated mice (#).