Immunization with native or recombinant Streptococcus pneumoniae neuraminidase affords protection in the chinchilla otitis media model

Abstract

Streptococcus pneumoniae neuraminidase has been implicated as a virulence factor in the pathogenesis of pneumococcal otitis media. In this study, native neuraminidase was partially purified from cultures of S. pneumoniae by serial chromatography with DEAE-Sepharose and Sephacryl S-200. Recombinant neuraminidase, a 3,038-bp fragment of the neuraminidase A (nanA) gene, was cloned into the pET-28b vector and then expressed at high levels in Escherichia coli. Chinchillas were immunized subcutaneously with either the gel-purified native or recombinant neuraminidase, and all responded with elevated titers of antineuraminidase antibody in serum. Immunization with neuraminidase resulted in a significant reduction in nasopharyngeal colonization as well as in the incidence of otitis media with effusion. These data demonstrate for the first time that neuraminidase affords protection against S. pneumoniae nasopharyngeal colonization and experimental otitis media.

FIG. 1.

Analysis of crude column-purified native S. pneumoniae neuraminidase (3 μg per lane) following SDS-PAGE. Lanes: A, column-purified native neuraminidase was subjected to SDS-PAGE and stained with Coomassie brilliant blue; B, the neuraminidase identified and visualized by a fluorimetric assay; C, Western blotting performed using sera from chinchillas immunized with gel-purified neuraminidase (1:10,000 dilution of serum). This figure represents a composite of lanes taken from separate gels.

FIG. 2.

Analysis of recombinant S. pneumoniae neuraminidase following SDS-PAGE. Lanes: A, lysate from the recombinant E. coli expression construct (prior to purification with Ni-NTA agarose) stained with Coomassie brilliant blue; B, the final purified NanA after elution from Ni-NTA agarose stained with Coomassie brilliant blue; C, the final product from E. coli without insertion of nanA stained with Coomassie brilliant blue; D, the final purified NanA identified and visualized by a fluorimetric assay; E, crude column-purified native neuraminidase (3 μg per lane) was probed with antisera from chinchillas immunized with gel-purified recombinant neuraminidase (1:10,000 dilution of serum). This figure represents a composite of lanes taken from separate gels.

FIG. 3.

Immunization with native neuraminidase (nNeu) or recombinant neuraminidase (r-nanA) reduces NP colonization (A) and middle ear invasion (B). Data shown represent the geometric mean number of CFU of S. pneumoniae ± the standard errors of the means per milliliter of nasal lavage fluid. *, P of <0.05 for the comparison.