Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs

Abstract

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly (p < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.

Keywords: chitosan nanoparticles; intranasal vaccination; mucosal immune response; pigs; swine influenza virus.

Figure 1

In vitro characteristics of chitosan nanoparticles (CNPs)-KAg. Diameter of (A) empty CNPs and (B) swine influenza A virus (SwIAV)-killed antigen (KAg)-loaded CNPs (CNPs-KAg) determined by dynamic light scattering. Scanning electron microscope (SEM) images of (C) empty CNPs and (D) CNPs-KAg. (E) Release of KAg from CNPs-KAg suspended in phosphate-buffered saline over a period of 15 days. (F) Uptake of soluble SwIAV KAg or CNPs-KAg formulation by monocytes/macrophages at indicated time points determined by fluorescent microscopy (Olympus, IX70, 20× magnifications). The frequency of monocytes/macrophages uptaken SwIAV KAg treated with soluble antigen or CNPs-KAg determined by flow cytometry: (G) SwIAV-infected Madin-Darby canine kidney (MDCK) cells as positive control; (H) a representative picture of SwIAV KAg or CNPs-KAg uptake by porcine monocytes/macrophages after 150 min treatment; and (I) percentage of cells with internalized SwIAV antigen at 10, 30, and 150 min treatment.

Figure 2

Production of innate, pro-inflammatory and T helper 1 cytokines by porcine MoDCs treated for 48 h with medium, KAg, chitosan nanoparticles (CNPs)-KAg or LPS control. Levels of cytokines (A) IFN-α, (B) TNF-α, (C) interleukin (IL)-1β, (D) IL-12, (E) IL-6, and (F) IL-10 were estimated in stimulated cell culture supernatant by ELISA. Data represent mean value of seven pig-derived DCs ± SEM. Statistical analysis between two groups was carried out using Mann–Whitney test. Asterisk refers to statistical significant difference between the indicated two pig groups. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

Antibody response after chitosan nanoparticles (CNPs)-KAg prime-boost vaccination at day post-vaccination 35/day post-challenge 0 (DPV 35/DPC 0) in pigs. Mucosal secretory IgA antibody response in nasal swab, systemic IgG antibody, and hemagglutination inhibition (HI) titers in serum samples against (A,D,G) H1N2-OH10, (B,E,H) H1N1-OH7, and (C,F,I) H3N2-OH4 influenza A virus (IAVs). Data represent the mean value of three to five pigs ± SEM. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test. Asterisk refers to statistical significant difference between the indicated two pig groups (*p < 0.05). In antibody dilution curves (A–F), A, B, and C refer to significant difference between unvaccinated vs KAg vaccinates, unvaccinated vs CNPs-KAg vaccinates, and KAg vs CNPs-KAg vaccinates, respectively, at the indicated dilution.

Figure 4

Expression of T helper 1 (Th1) and T helper 2 (Th2) response inducing specific transcription factors after prime-boost vaccination in pigs. The expression of (A) Th2 transcription factor GATA-3 and (B) Th1 transcription factor T-bet in peripheral blood mononuclear cells of pigs at day post-vaccination (DPV) 35/day post-challenge (DPC) 0 were determined by quantitative reverse transcription PCR (qRT-PCR). Data represent the mean value of three to five pigs ± SEM. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test. Asterisk refers to the statistical significant difference between the indicated two pig groups. *p < 0.05.

Figure 5

Mucosal IgA antibody response in the respiratory tract of pigs vaccinated with chitosan nanoparticles (CNPs)-KAg at day post-challenge 6. Specific IgA antibody response in nasal swab, bronchoalveolar lavage (BAL) fluid, and lung lysate samples against H1N2-OH10 (A,D,G), H1N1-OH7 (B,E,H), and H3N2-OH4 (C,F,I) influenza A virus (IAVs). Data represent the mean value of three to five pigs ± SEM at all indicated dilutions. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test where A, B, and C refer to significant difference (p < 0.05) between unvaccinated vs KAg vaccinates, unvaccinated vs CNPs-KAg vaccinates, and KAg vs CNPs-KAg vaccinates, respectively, at the indicated dilution.

Figure 6

Serum IgG response and bronchoalveolar lavage (BAL) fluid hemagglutination inhibition (HI) antibody titers in pigs vaccinated with chitosan nanoparticles (CNPs)-KAg at day post-challenge 6. Specific IgG antibody response in serum and BAL fluid HI titers against H1N2-OH10 (A,E), H1N1-OH7 (B,D), and H3N2-OH10 (C,F) influenza A virus (IAVs). Data represent the mean value of three to five pigs ± SEM. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test. Asterisk refers to the statistical significant difference between the indicated two pig groups (*p < 0.05). In antibody dilution curves (A–C), A, B, and C refers to significant difference between unvaccinated vs KAg vaccinates, unvaccinated vs CNPs-KAg vaccinates, and KAg vs CNPs-KAg vaccinates, respectively, at the indicated dilution.

Figure 7

Cell-mediated immune response after prime-boost vaccination was enhanced in chitosan nanoparticles (CNPs)-KAg-vaccinated pigs at pre-challenge days post-vaccination (DPV) 35/day post-challenge (DPC) 0. Peripheral blood mononuclear cells (PBMCs) isolated from blood were stimulated with different variant influenza A virus (IAVs). IFNγ secretion in the culture supernatant and antigen-specific lymphocyte proliferation was determined after 72 h of stimulation with (A,E) H1N2-OH10, (B,F) H1N1-OH7, and (C,G) H3N2-OH4 IAVs. (D) Flow cytometry analysis of PBMCs showed enhanced frequency of CTLs (CD3⁺CD4⁻CD8αβ⁺) in CNPs-KAg-vaccinated pigs. Data represent the mean value of three to five pigs ± SEM. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test. Asterisk refers to the statistical significant difference between the indicated two pig groups (*p < 0.05).

Figure 8

Clinical and pathological changes in pigs vaccinated with chitosan nanoparticles (CNPs)-KAg post-challenge. (A) Rectal temperature was recorded daily after challenge until the day of necropsy. (B) Gross pneumonic lesions in lungs determined at day post-challenge (DPC) 6. (C) Representative Hematoxylin and eosin (H&E)-stained lung pictures showing bronchial exudates (dotted black circle), perivascular inflammation (black arrow), peribronchial inflammation (dashed black arrow), and interstitial pneumonia (small black triangle). (D) Secretion of cytokine interleukin (IL-6) in bronchoalveolar lavage (BAL) fluid. Data represent the mean value of three to five pigs ± SEM.

Figure 9

Cell-mediated immune response in TBLN-MNCs of pigs vaccinated with chitosan nanoparticles (CNPs)-KAg at day post-challenge (DPC) 6. TBLN-MNCs isolated on the day of necropsy were stimulated with different variant swine influenza A viruses (SwIAVs), and secreted IFNγ into the culture supernatant was measured by cytokine ELISA against (A) H1N2-OH10, (B) H1N1-OH7, and (C) H3N2-OH4 influenza A virus (IAVs). (D) The frequency of T helper (Th)/memory cells (CD3⁺CD4⁻CD8α⁺) in TBLN-MNCs of CNPs-KAg-vaccinated pigs was analyzed by flow cytometry. The expression of Th1 (E) and Th2 (F) transcription factors were also determined in TBLN at DPC 6. Data represent the mean value of three to five pigs ± SEM. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test. Asterisk refers to the statistical significant difference between the indicated two pig groups (*p < 0.05).

Figure 10

Infectious challenge swine influenza A virus (SwIAV) H1N1 titer in the respiratory tract of chitosan nanoparticles (CNPs)-KAg-vaccinated and influenza A virus (IAV)-challenged pigs. Titers of challenge SwIAV shedding through nostrils at (A) day post-challenge (DPC) 4 and (B) DPC 6, and in bronchoalveolar lavage (BAL) fluid at DPC 6 (C) determined by using cell culture technique. Data represent the mean value of three to five pigs ± SEM. Statistical analysis was carried out using Kruskal–Wallis test followed by Dunn’s post hoc test. Asterisk refers to the statistical significant difference between the indicated two pig groups (*p < 0.05).