Intranasal immunization with multivalent group A streptococcal vaccines protects mice against intranasal challenge infections

Abstract

We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.

FIG. 1.

Hexavalent protein-specific antibody titers in sera from mice immunized i.n. with formulations of hexavalent protein containing liposomes and lipid A (H/L/MPL) or liposomes only (H/L) (A), CT-B with or without holotoxin (H/CT-B/CT or H/CT-B, respectively) (B), or proteosomes (H/Pr/LPS) (C). Specific antibody titers for control mice that received only liposomes (L), CT-B/CT, or proteosomes (Pr/LPS), respectively, are also indicated (titers < 100). Each bar represents the mean (± standard error of the mean) antibody titer in serum from 10 mice in each group 2 weeks after the final immunization. *, P < 0.05 for H/L/MPL versus H/L and versus L, for H/CT-B/CT versus CT-B/CT, and for H/Pr/LPS versus Pr/LPS.

FIG. 2.

Hexavalent protein-specific antibody levels in saliva from mice immunized i.n. with formulations of hexavalent protein containing liposomes and lipid A (H/L/MPL), CT-B with or without CT (H/CT-B/CT or H/CT-B, respectively), or proteosomes (H/Pr/LPS). Each bar represents the fold increase in absorbance at 405 nm between pre- and postimmunization samples for 10 mice in each group.

FIG. 3.

Survival of mice immunized i.n. with formulations of hexavalent group A streptococcal protein containing liposomes and lipid A (H/L/MPL) or liposomes only (H/L) (A), CT-B with or without CT (H/CT-B/CT or H/CT-B, respectively) (B), or proteosomes (H/Pr/LPS) (C) and challenged via the i.n. route with type 24 group A streptococci. Percent survival is also indicated for control mice that received only liposomes (L), CT-B/CT, or proteosomes (Pr/LPS), respectively. Each data set represents the percent survival for 10 mice challenged in each group (with the exception that only nine mice were challenged in each of the H/L and Pr/LPS groups, due to deaths prior to challenge). *, P < 0.05 for H/L/MPL versus L, for H/CT-B/CT and H/CT-B versus CT-B/CT, and for H/Pr/LPS versus Pr/LPS.