Mucosal immunity of mannose-modified chitosan microspheres loaded with the nontyepable Haemophilus influenzae outer membrane protein P6 in BALB/c mice

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a common opportunistic pathogen that colonizes the nasopharynx. NTHi infections result in enormous global morbidity in two clinical settings: otitis media in children and acute exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Thus, there is an urgent need to design and develop effective vaccines to prevent morbidity and reduce antibiotic use. The NTHi outer membrane protein P6, a potential vaccine candidate, is highly conserved and effectively induces protective immunity. Here, to enhance mucosal immune responses, P6-loaded mannose-modified chitosan (MC) microspheres (P6-MCMs) were developed for mucosal delivery. MC (18.75%) was synthesized by the reductive amination reaction method using sodium cyanoborohydride (NaBH3CN), and P6-MCMs with an average size of 590.4±16.2 nm were successfully prepared via the tripolyphosphate (TPP) ionotropic gelation process. After intranasal immunization with P6-MCMs, evaluation of humoral immune responses indicated that P6-MCMs enhance both systemic and mucosal immune responses. Evaluation of cellular immune responses indicated that P6-MCMs enhance cellular immunity and trigger a mixed Th1/Th2-type immune response. Importantly, P6-MCMs also trigger a Th17-type immune response. They are effective in promoting lymphocyte proliferation and differentiation without toxicity in vitro. The results also demonstrate that P6-MCMs can effectively induce MHC class I- and II-restricted cross-presentation, promoting CD4+-mediated Th immune responses and CD8+-mediated cytotoxic T lymphocyte (CTL) immune responses. Evaluation of protective immunity indicated that immunization with P6-MCMs can reduce inflammation in the nasal mucosa and the lung and prevent NTHi infection. In conclusion, MCMs are a promising adjuvant-delivery system for vaccines against NTHi.

Fig 1. Synthetic route of mannose-modified chitosan derivatives.

Fig 2. Gene cloning and expression of the loaded antigen P6.

A Amplification product for the NTHi-P6 gene by PCR. Lane 1 P6 gene, Lane M DNA ladder DL2000. B SDS-PAGE gel analysis of tag-removed P6 protein expressed from the NTHi-P6 gene. Lane 1 P6 protein, Lane M prestained protein ladder. C Western blot analysis of tag-removed P6. Lane 1 polyclonal antibodies against P6, Lane M prestained protein ladder.

Fig 3. Characteristics and evaluation of P6-CMs and P6-MCMs.

A SEM images of P6-CMs and P6-MCMs (5000x); the scale bar represents 10 μm. B Particle size distribution by intensity (percent) of P6-CMs and P6-MCMs. C Standard curve for D-glucosamine hydrochloride (100% free amino, 0.1 mg ml^-1) at different volumes. The slope was 5.2233, R² = 0.9984. D Continuous release profiles of P6-CMs and P6-MCMs at different times in vitro. Mean ± SD, n = 3.

Fig 4. Analysis of P6-Specific antibody levels in different treatment groups of immunized mice.

A The levels of P6-specific systemic IgG antibody in serum. B The levels of the P6-specific mucosal IgA antibody in nasal and lung lavage fluid. The antibody levels were indirectly presented in the form of optical density (OD) values. Values are the mean ± SD, n = 3. Significant differences were expressed as *P<0.05, **P<0.01, ***P<0.001.

Fig 5. Analysis of cytokine levels in spleen lymphocyte culture supernatants and T lymphocyte proliferation assays.

The levels of IL-2 (A), IFN-γ (B), IL-4 (C), IL-5 (D), and IL-17 (E) in lymphocyte culture supernatants. (F) Stimulation index of spleen lymphocytes determined according to the absorbance at 450 nm of the stimulated group and the control group. Values are the mean ± SD, n = 3. Significant differences were expressed as *P<0.05, **P<0.01, ***P<0.001.

Fig 6. Flow cytometric analysis of spleen T lymphocyte subsets in different groups of immunized mice.

A “Three-Color, Dual Anchor” gating strategy to identify the lymphocyte subsets (CD3⁺, CD4⁺ and CD8⁺); the proportions are shown. Values are expressed as a percentage. Cells were stained with APC-Cy^™7 Rat Anti-Mouse CD3, FITC Rat Anti-Mouse CD4 and PE Rat Anti-Mouse CD8a. B Comparison of CD3⁺ T cell proportions in spleen lymphocytes. C Comparison of CD3⁺CD4⁺ T cell proportions in spleen lymphocytes. D Comparison of CD3⁺CD8⁺ T cell proportions in spleen lymphocytes. Mean ± SD, n = 3. *P<0.05, **P<0.01.

Fig 7. Hematoxylin-Eosin staining of the nasal mucosa and lung tissues to evaluate histopathologic alterations in mice.

Histological scores for lung, nasal mucosa tissues from mice (n = 3 per group). *P<0.05, **P<0.01 and *** P<0.001.