Intranasal vaccination with chitosan-DNA nanoparticles expressing pneumococcal surface antigen a protects mice against nasopharyngeal colonization by Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a respiratory pathogen, and mucosal immune response plays a significant role in the defense against pneumococcal infections. Thus, intranasal vaccination may be an alternative approach to current immunization strategies, and effective delivery systems to mucosal organism are necessary. In this study, BALB/c mice were immunized intranasally with chitosan-DNA nanoparticles expressing pneumococcal surface antigen A (PsaA). Compared to levels in mice immunized with naked DNA or chitosan-pVAX1, anti-PsaA IgG antibody in serum and anti-IgA antibody in mucosal lavages were elevated significantly in mice immunized with chitosan-psaA. The balanced IgG1/IgG2a antibody ratio in serum, enhanced gamma interferon (IFN-γ) and IL-17A levels in spleen lymphocytes, and mucosal washes of mice immunized with chitosan-psaA suggested that cellular immune responses were induced. Furthermore, significantly fewer pneumococci were recovered from the nasopharynx of mice immunized with chitosan-psaA than for the control group following intranasal challenge with ATCC 6303 (serotype 3). These results demonstrated that mucosal immunization with chitosan-psaA may successfully generate mucosal and systemic immune responses and prevent pneumococcal nasopharyngeal colonization. Hence, a chitosan-DNA nanoparticle vaccine expressing pneumococcal major immunodominant antigens after intranasal administration could be developed to prevent pneumococcal infections.

FIG. 1.

Analysis of the expression of the pVAX1-psaA construct in 293T cells by RT-PCR (A) and Western blotting (B). Total RNA and proteins were extracted from 293T cells transfected with the pVAX1-psaA construct or pVAX1 vector. Lane 1, DNA marker; lane 2, PCR products from cells transfected with the pVAX1-psaA construct, showing the 867-bp psaA gene and 217-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene; lane 3, PCR products from cells transfected with the pVAX1 vector; lane 4, distilled water as a template serving as a negative control. Lanes 5 and 6, cells transfected with the pVAX1-psaA construct, showing the expression of a 37-kDa PsaA protein; lane 7, cells transfected with the pVAX1 vector only. The migration of standard molecular mass markers is indicated at the left.

FIG. 2.

Agarose gel electrophoresis of chitosan-psaA nanoparticles following DNase I digestion. Lane 1, pVAX1-psaA without DNase I; lane 2, pVAX1-psaA with DNase I; lane 3, chitosan-psaA without DNase I; lane 4, chitosan-psaA with DNase I.

FIG. 3.

ELISA analysis of total anti-PsaA IgG in serum (A), anti-PsaA IgG isotypes in serum (B), and IgA antibody in nasal washes, BALF, and MEL (C). Mice were intranasally immunized with four doses of chitosan-psaA, naked psaA, or chitosan-pVAX1 at 2-week intervals, and one group was immunized with recombinant PsaA proteins with CT adjuvants as a control. Numbers above columns are mean IgG1/IgG2a reciprocal titer ratios. Statistical analysis was performed using one-way ANOVA, and each column represents means ± standard deviations. Statistical difference between the group immunized with chitosan-psaA and that immunized with naked psaA (P < 0.05) is marked with an asterisk. All samples were obtained from individual mice. These results are representative of three experiments.

FIG. 4.

ELISA detection of cytokine levels in spleen cells induced by chitosan-psaA. Splenocytes were isolated from immunized BALB/c 2 weeks after the last immunization and then were incubated for 72 h with rPsaA (5 μg/ml). IL-17A and IFN-γ in the supernatants were detected through sandwich ELISA. Each column represents mean concentrations ± standard deviations, and statistical analysis was performed using one-way ANOVA. Statistical difference between the group immunized with chitosan-psaA and that immunized with naked psaA (P < 0.05) is marked with an asterisk. No significant difference was found between naked psaA and chitosan-pVAX1 groups in IL-17A (P = 0.851) and IFN-γ (P = 0.076). All data were the measurements of individual mouse samples. These results are representative of three experiments.

FIG. 5.

IL-17A responses in bronchoalveolar fluids (BALF) during pneumococcal challenge. The BALF were collected before and after pneumococcal challenge, and IL-17A levels were measured through sandwich ELISA. Each column represents mean concentrations ± standard deviations. Before challenge, the secretion of IL-17A was low in each group. Statistical difference after challenge between the group immunized with chitosan-psaA and that immunized with naked psaA (P < 0.05) is marked with an asterisk. These results are representative of three experiments.

FIG. 6.

Bacterial recovery from mice immunized with chitosan-psaA, naked psaA, or chitosan-pVAX1 after intranasal challenge with S. pneumoniae. Log₁₀ values of total CFU recovered from nasal washes after intranasal challenge with pneumococcal strain ATCC 6303 are shown. The median for each group is displayed as a line, and the absence of colonies in individual nasal washes is represented as 1.0. Statistical analysis was performed using the Mann-Whitney U test. Statistical difference between the groups immunized with chitosan-psaA and naked psaA (P < 0.05) is marked with an asterisk.