Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection

Abstract

Human respiratory syncytial virus (HRSV) is a leading cause of death in young children and there are no FDA approved vaccines. Bovine RSV (BRSV) is antigenically similar to HRSV, and the neonatal calf model is useful for evaluation of HRSV vaccines. Here, we determined the efficacy of a polyanhydride-based nanovaccine encapsulating the BRSV post-fusion F and G glycoproteins and CpG, delivered prime-boost via heterologous (intranasal/subcutaneous) or homologous (intranasal/intranasal) immunization in the calf model. We compared the performance of the nanovaccine regimens to a modified-live BRSV vaccine, and to non-vaccinated calves. Calves receiving nanovaccine via either prime-boost regimen exhibited clinical and virological protection compared to non-vaccinated calves. The heterologous nanovaccine regimen induced both virus-specific cellular immunity and mucosal IgA, and induced similar clinical, virological and pathological protection as the commercial modified-live vaccine. Principal component analysis identified BRSV-specific humoral and cellular responses as important correlates of protection. The BRSV-F/G CpG nanovaccine is a promising candidate vaccine to reduce RSV disease burden in humans and animals.

Keywords: bovine respiratory disease; bovine respiratory syncytial virus; human respiratory syncytial virus; immunology; nanoparticles; nanovaccines; neonatal calf model.

Figure 1

In Vitro Characterization of NPs. (A) NP size and morphology were determined by scanning electron microscopy. SEM image of 3% F/G CpG 20:80 CPTEG : CPH. NPs showed appropriate morphology and size (188.5 ± 55.8nm). (B) Antigen release kinetics were evaluated by suspending ~3 mg of particles in PBS and showed sustained release of protein for 30 days. (C) SDS-PAGE analysis was performed on the released proteins to confirm appropriate molecular weight for BRSV antigen released from the particles. (D) ELISA plates were coated with 1 µg/mL of released protein. Sera from BRSV-immune cows were diluted 1:1000 and added to the plates. The binding of bovine IgG to the virus or recombinant proteins was measured by absorbance.

Figure 2

Reduced gross and microscopic pathology in lungs of BRSV-F/G CpG nanovaccine-administered calves. (A) Schematic diagram of in vivo experiments. (B) Calves in all four groups from both studies were monitored daily post infection for fever, respiratory rate, appetite, and nasal discharge and assigned a clinical score. Data represents means ± SEM. Statistical significance determined by 2-way ANOVA with repeated measures, followed by Tukey’s multiple comparisons test(C) Representative images from an unvaccinated, control calf and a calf which received an In + Sc BRSV-F/G CpG nanovaccine (D) Cumulative gross pathology results from all groups in both studies. Results represent controls (n = 18), In + In BRSV-F/G CpG (n = 18), In + Sc BRSV-F/G CpG (n = 17), and commercial vaccine (n = 11). The graph depicts means ± SEM of each group. *p <0.05, **p<0.01 and ***p<0.001 compared to unvaccinated, control calves as determined by one-way ANOVA with repeated measures, followed by Sidak’s multiple comparisons test.

Figure 3

Reduced neutrophil infiltration in BAL of BRSV-F/G CpG nanovaccine-administered calves following BRSV challenge. Differential cell counts in BAL fluid of neonatal calves challenged with BRSV was performed by a blinded clinical pathologist on 7 d.p.i. (A) Proportion of granulocytes in BAL evaluated by cytology. Differences in the relative proportions of neutrophil (B) and macrophages (C). The results are expressed as a percentage composition. The graphs represent controls (n = 18), In + in BRSV-F/G CpG (n = 18), In + Sc BRSV-F/G CpG (n = 17), MLV (n = 11) treatment groups. The graph depicts means ± SEM of each group. Statistical significance was determined by two-way ANOVA with Tukey’s post test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.001 compared to unvaccinated control calves.

Figure 4

Nanovaccine induced viral protection in the lower and upper respiratory tract of neonatal calves. (A) Nasal swabs were collected on 0, 3, 5, and 7 d.p.i. to evaluate virus shedding. BRSV RNA was detected by RT-qPCR, and viral NS2 copy numbers were calculated using standard curves. The graphs include Controls (n = 18), In + In BRSV-F/G CpG (n = 18), In + Sc BRSV-F/G CpG (n = 17), MLV (n =11) and depict means ± SEM of each group. Statistical significance was determined by two-way ANOVA with a Tukey post hoc test (B) After euthanasia on 7 d.p.i. lung tissue samples were collected from 2-3 representative lesion and non-lesion sites of the lungs. Lung tissues were normalized to the housekeeping gene, RPS9, to correct for differences in input material. Statistical significance was determined by one-way ANOVA with a Holm-Šídák post hoc test. AUC, Area under curve.

Figure 5

BRSV-specific T cell responses in peripheral blood and TBLN of BRSV-F/G nanovaccine-administered calves. Calves were experimentally infected as described in Figure 2A . TBLN and PBMCs was collected at 7 d.p.i. and stimulated either with culture medium, 5 μg/mL ConA, 5 μg/mL post-F protein, 5 μg/mL G protein or BRSV for 5 days. After incubation, PBMCs and TBLN were surface stained for T cells markers and proliferation of T cells detected by flow cytometry. (A, B) Frequency of CD4 and CD8 dividing cells specific to BRSV, post-F protein, or G protein in TBLN collected on 7 d.p.i. (C, D) Frequency of CD4 and CD8 dividing cells specific to BRSV, post-F protein, or G protein in PBMCs collected on 7 d.p.i. Data is presented as percentages of cell proliferation expressed over mock treated cells. Data represent means ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post test. Controls (n = 18), In + in BRSV-F/G CpG (n = 18), In + Sc BRSV-F/G CpG (n = 17), commercial vaccine (n = 11).

Figure 6

Cytokine responses in the peripheral blood and tracheobronchial lymph node (TBLN) of BRSV-F/G nanovaccine-administered calves. TBLN cells (A, B) and PBMCs (C, D) were collected at necropsy and stimulated for 5 days either with culture medium as a negative control, 5 μg/mL Concanavalin A (ConA) as a positive control, 5 μg/mL post-F protein, 5 μg/mL G protein or BRSV. Supernatants from stimulated PBMCs and TBLN cells were collected and analyzed by ELISA for IFNγ (A, C) and IL-17 (B, D) secretion. Controls (n = 18), In + in BRSV-F/G CpG (n = 18), In + Sc BRSV-F/G CpG (n = 17), commercial vaccine (n = 11). Data represent means ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post hoc test. *p<0.10 compared to matched mock controls.

Figure 7

The results from the blinded PC analysis. (A) and (B) Scores plot from the analysis, with (B) showing the average treatment values. (C–E) show the impact of the treatment relative to control. The bars represent the disease protection descriptions and viral protection: total clinical score (dark blue), gross lung pathology (orange), lung histopathology score (gray), BAL cytology (yellow), nasal virus shedding (light blue) and lung viral burden (green). The numbered regions are the immune responses: (1) Serum neutralizing Ab titers, (2) nasal fluid IgA responses, (3) F specific IgG ELISA, (4) G specific IgG ELISA, (5) BRSV specific IgG ELISA, (6) cytokine responses by TBLN, (7) cytokine responses by PBMC, (8) TBLN proliferative responses, and (9) proliferative PBMC responses.