Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses

Abstract

Background: Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA.

Results: In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220+ cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice.

Conclusion: These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

Figure 1

Characterization of SARS-CoV S DNA vaccine (pci-S)-PEI complexes. (A) Transmission electron micrographic image of PEI/pci-S complexes at N/P ratio 10. Scale bar represents 0.5 μm. (B) Size distribution of complexes prepared at N/P ratio 10. (C) The cell uptake of PEI/pci-S complexes was observed by confocal laser scanning microscope. Upper left, intracellular distribution of rhodamine-labeled PEI/pci-S (red). Upper right, cell nuclei by DAPI staining. Lower left, differential interference images of RAW 264.7 cells. Lower right, overlapping image of nuclei and rhodamine-labeled PEI/pci-S. (D) Expression of S mRNA in RAW 264.7 cells transfected with PEI/pci-S complexes was detected by RT-PCR. (E) S protein in RAW 264.7 cells with PEI/pci-S complexes was detected by Western blot.

Figure 2

SARS-CoV S protein-specific humoral and mucosal immune responses in BALB/c mice intranasally immunized with PEI/pci-S complexes. The samples were collected on day 7 after the last immunization. Induction of (A) S-specific IgG subclasses in serum and (B) S-specific IgA in various mucosal samples were determined by ELISA. The results were expressed as means ± SEM for the group (n = 5 to 8). Significant differences compared with pci-S group were expressed as *P < 0.05 and **P < 0.01, respectively. (C) Proliferation activity of B220⁺ cells from spleen of mice immunized with PEI/pci-S complexes. Bar and number in each panel present the percentage of proliferated cells.

Figure 3

Expression of cell surface molecules, CD80, CD83, CD86, CCR7 and MHC class II, on CD11c⁺ cells in cervical lymph nodes from mice immunized with PEI/pci-S complexes. BALB/c mice were immunized with PBS, pci-mock, pci-S, or PEI/pci-S complexes and then cell surface molecules on CD11c⁺ cells from cervical lymph nodes were analyzed on day 3 after the last immunization. (A) The expressions of major cell surface molecules, CD80, CD83, CD86, CCR7, and MHC class II (I-A^d) on CD11c⁺ DC were determined by flow cytometry. Data were expressed as the mean value of mean fluorescence intensity ± SD. Significant differences compared with pci-S group were expressed as *P < 0.05. (B) Expression of MHC class II molecules was represented by histogram.

Figure 4

Effector CD4⁺ and CD8⁺ T cell responses in BALB/c mice immunized with PEI/pci-S complexes. Multi-intracellular cytokine staining for IL-17, IFN-γ, TNF-α and IL-2 was performed on (A) CD4⁺ and (B) CD8⁺ T cells after in vitro re-stimulation with SARS peptide. SARS S-specific CD4⁺ T cells from lung were recovered on day 6 after the last immunization. ND indicates not detectable. Data were expressed as the mean value of mean fluorescence intensity ± SEM. Significant differences compared with pci-S group were expressed as *P < 0.01. (C) The results showed representative example of flow cytometry analysis.