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Cholera toxin B subunit is known for its adjuvant properties when associated with different antigens. In this work, we have fused the ctxB gene to the pneumococcal surface protein (pspA) gene from Streptococcus pneumoniae. Intradermal ad- ministration of the fusion protein in mice induced anti-PspA antibodies and protection against pneumococcus in a sepsis model. Streptococcus pneumoniae is the major cause of bacterial pneumonia, meningitis, and otitis media cases around the world, leading to up to 1 million deaths per year. Pneumococ- cal surface protein A (PspA) is a virulence factor partially conserved among the Streptococcus pneumoniae isolates that plays a role in complement inactivation during infection (18). Based on amino acid sequence diversity, PspAs can be classi- fied into three families and six clades (8) that also display immunological cross-reactivity among themselves (17). In sev- eral pneumoccocal infection challenge models, PspA has proved to be a good vaccine candidate when administered either in protein-adjuvant formulations or in DNA-based vac- cines (4, 11, 12). PspA was also shown to bind and to prevent the bactericidal activity of apolactoferrin (15), present in saliva and other mucosal secretions. In addition, anti-PspA antibod- ies were shown to enhance pneumococcal killing by lactoferrin, suggesting a mechanism for the reduction of pneumococcal carriage by these antibodies (15). In a recent work, we tested the immunogenic potential of a fusion protein composed of the cholera toxin B subunit (CTB) and pneumococcal surface an- tigen A from Streptococcus pneumoniae by intranasal inocula- tion of mice (3). In this work, we have amplified the 5 termi- nus region (which encodes the -helix region plus the proline- rich domain) of the pspA clade 3 gene from pTG-pspA3 (11) (pspA3 accession number, AY082389), which carried the gene isolated from S. pneumoniae St 259/98 strain (Instituto Adol- pho Lutz, Sao Paulo, SP, Brazil). The pspA gene was then cloned downstream of the ctxB gene in the pAE-CTB plasmid (2) in order to express a CTB-PspA fusion protein in Esche- richia coli. Using this expression system, the recombinant pro- tein contains an N-terminal six-His tag that allows purification through Ni2 charged columns. This system was used for the expression and purification of the fusion protein CTB-PspA and CTB, as previously described (2, 3), and PspA, with the observation that the last protein did not adsorb very well, and it was released from the column with 20 mmol · liter1 imida- zole. Protein characterization. The recombinant CTB-PspA pu- rified from E. coli BL21-SI extracts was able to form penta- mers, as evaluated by 6% sodium dodecyl sulfate-polyacrylam- ide gel electrophoresis of unboiled samples (data not shown). Since the adjuvant effect of CTB is dependent on the binding of the pentameric form to the GM1 gangliosides present on cellular surfaces (14), we performed an enzyme-linked immu- nosorbent assay (ELISA) experiment, using 10 g·m l1 GM1 in phosphate-buffered saline for an overnight coating and in- creasing amounts of CTB-PspA, as previously described (3). Briefly, after an incubation of 1 h with the proteins, anti-CTB antiserum was added at the proper dilution, followed by a 1-h incubation at 37°C, and the reaction was developed using anti- mouse immunoglobulin G (IgG)-peroxidase conjugate. The results show that CTB-PspA was able to bind to GM1 to the same extent as CTB alone and in a dose-dependent manner (Fig. 1). Recombinant PspA, also expressed and purified from E. coli extracts using the same expression vector and Ni2 affinity chromatography, was not able to bind to GM1 even in the highest concentration used. None of the proteins were able to bind when 10 g·m l1 bovine serum albumin was used as

infection and immunity

Intradermal Immunization of Mice with Cholera Toxin B-Pneumococcal Surface Protein A Fusion Protein Is Protective against Intraperitoneal Challenge with <i>Streptococcus pneumoniae</i>

Intradermal Immunization of Mice with Cholera Toxin B-Pneumococcal Surface Protein A Fusion Protein Is Protective against Intraperitoneal Challenge with Streptococcus pneumoniae