Microparticle RSV Vaccines Presenting the G Protein CX3C Chemokine Motif in the Context of TLR Signaling Induce Protective Th1 Immune Responses and Prevent Pulmonary Eosinophilia Post-Challenge

Abstract

Layer-by-layer microparticle (LbL-MP) fabrication was used to produce synthetic vaccines presenting a fusion peptide containing RSV G protein CX3C chemokine motif and a CD8 epitope of the RSV matrix protein 2 (GM2) with or without a covalently linked TLR2 agonist (Pam3.GM2). Immunization of BALB/c mice with either GM2 or Pam3.GM2 LbL-MP in the absence of adjuvant elicited G-specific antibody responses and M2-specific CD8+ T-cell responses. Following challenge with RSV, mice immunized with the GM2 LbL-MP vaccine developed a Th2-biased immune response in the lungs with elevated levels of IL-4, IL-5, IL-13, and eotaxin in the bronchoalveolar lavage (BAL) fluid and a pulmonary influx of eosinophils. By comparison, mice immunized with the Pam3.GM2 LbL-MP vaccine had considerably lower to non-detectable levels of the Th2 cytokines and chemokines and very low numbers of eosinophils in the BAL fluid post-RSV challenge. In addition, mice immunized with the Pam3.GM2 LbL-MP also had higher levels of RSV G-specific IgG2a and IgG2b in the post-challenge BAL fluid compared to those immunized with the GM2 LbL-MP vaccine. While both candidates protected mice from infection following challenge, as evidenced by the reduction or elimination of RSV plaques, the inclusion of the TLR2 agonist yielded a more potent antibody response, greater protection, and a clear shift away from Th2/eosinophil responses. Since the failure of formalin-inactivated RSV (FI-RSV) vaccines tested in the 1960s has been hypothesized to be partly due to the ablation of host TLR engagement by the vaccine and inappropriate Th2 responses upon subsequent viral infection, these findings stress the importance of appropriate engagement of the innate immune response during initial exposure to RSV G CX3C.

Keywords: CX3C; G protein; RSV; eosinophils; microparticle; vaccine.

Figure 1

Antibody responses in BAL fluid of mice immunized with FI-RSV or convalescent from RSV infection. BALB/c mice were immunized with FI-RSV on days 0 and 21, with RSV on day 0, or not treated (naïve), then challenged with RSV on day 35. BAL fluid was collected on day 28 (pre-challenge) and 4- or 8-days post-challenge and IgG antibody responses were measured by ELISA against G_169–198 peptide (G) or whole RSV. Mean ± SEM of 3 mice per group at 1:5 BAL fluid dilutions. * p < 0.05 compared to naïve BAL fluid tested against RSV at each time point; ** p < 0.05 compared to naïve BAL fluid tested against G_169–198 peptide at each time point by t-test.

Figure 2

Dose-dependent immunogenicity and efficacy of LbL-MP loaded with RSV GM2 epitopes. BALB/c mice were immunized on days 0 and 21 with the indicated doses of GM2 LbL-MP, challenged with RSV on day 35, and sacrificed for plaque assay on day 40. (A) Day 28 post-boost serum antibody titers measured by RSV G protein ELISA. Endpoint titers of individual mice (open circles) and group means (bars). * p < 0.05 compared to naïve. (B) Day 28 spleen cell ELISPOTs restimulated in vitro with a pool of M2_81–95 and G_169–198 peptides. Mean ± SEM spots/10⁶ cells of 3 mice/group. * p < 0.05 compared to naïve for both analytes by t-test. (C) Lung viral titers 5 days post-challenge. Number of plaques per gram of lung tissue for individual mice (circles) and group means (bars). Insets show number of mice with no detectable plaques (n = 12 mice per group), average % reduction in plaques per group, and * p < 0.05 compared to the naïve group.

Figure 3

Improvement of immunogenicity and efficacy of LbL-MP by addition of TLR2 ligand Pam3Cys. BALB/c mice were immunized on days 0 and 21 with the indicated doses of GM2 or Pam3.GM2 LbL-MP, challenged with RSV on day 35, and sacrificed for plaque assay on day 40. (A) Day 28 post-boost serum antibody titers measured by RSV G protein ELISA. Endpoint titers of individual mice (open circles) and group means (bars); * p < 0.05. (B) Day 28 spleen cell ELISPOTs restimulated in vitro with a pool of M2_81–95 and G_169–198 peptides. Mean ± SEM spots/10⁶ cells of 3 mice/group; * p < 0.05 by t-test compared to the corresponding dose level of GM2. (C) Lung viral titers 5 days post-challenge. Number of plaques per gram of lung tissue for individual mice (circles) and group means (bars). Insets show number of mice with no detectable plaques (n = 10 mice per group), average % reduction in plaques per group, and * p < 0.05 compared to the naïve group. (D) Day 28 serum G-specific antibody isotype levels measured by ELISA. Mean ± SEM of 10 mice per group at 1:50 serum dilution. * p < 0.05 compared to the same dose level of GM2.

Figure 4

Influence of TLR2 engagement on post-challenge Th2 and eosinophil response. BALB/c mice were immunized on days 0 and 21 as indicated (day 0 only for live RSV), challenged with live RSV on day 35, and sacrificed on day 41 for harvesting of BAL fluid and cells. All analyses were conducted on day 41 (day 6 post-challenge) BAL samples. (A) BAL RSV G-specific antibody isotype levels measured by ELISA. Mean ± SEM of 6 mice per group at 1:5 BAL dilution. (B) Th2 cytokine content of BAL fluids measured by Luminex. Mean ± SEM of 6 mice per group. * p < 0.05 vs. GM2 and FI-RSV groups for IL-5 and IL-13. (C) Th1/Th17 cytokine content of BAL fluids measured by Luminex. Mean ± SEM of 6 mice per group. (D) Eosinophil counts in BAL. Mean ± SEM of 6 mice per group. * p < 0.05 vs. GM2 and FI-RSV groups.

Figure 5

Impact of TLR2 engagement on post-challenge cytokine/chemokine and eosinophilia response in lungs. BALB/c mice were immunized on days 0 and 21 with GM2, Pam3.GM2, or FI-RSV, or infected on day 0 with RSV; control groups were not immunized (naïve and NNC). Mice were challenged with live RSV on day 35 (+) or not challenged (−). NNC = naïve, not challenged. Mice were sacrificed 5 days post-challenge and BAL were harvested for analysis. (A) G-specific antibody isotype levels measured by ELISA. Mean ± SEM of 3 mice per group at 1:5 BAL dilution. (B) Lung viral burdens. Plaques/gram of lung tissue for individual mice (open circles) and group means (red bars). Insets show number of mice with no detectable plaques (n = 6–7 mice per group), average % reduction in plaques per group, and * p < 0.05 compared to the naïve group. (C) Eosinophil counts in BAL collected on day 8 post-challenge. Mean ± SEM of 6 mice per group. * p < 0.05 compared to GM2 and FI-RSV groups. (D–I) Cytokine and chemokine content of BAL fluids. Mean ± SEM of 6 mice per group.