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Infection and Immunity, June 2005, p. 3810-3813, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3810-3813.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Ana Paula Mattos Arêas,1,2 Maria Leonor Sarno Oliveira,1 Eliane Namie Miyaji,1 Luciana Cezar Cerqueira Leite,1,2 and Paulo Lee Ho1,2,3*

Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil,1 Instituto de Química, USP, São Paulo, Brazil,2 Instituto de Biociências, USP, São Paulo, Brazil3

Received 7 December 2004/ Returned for modification 19 January 2005/ Accepted 3 February 2005


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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 administration 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. Pneumococcal 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 classified into three families and six clades (8) that also display immunological cross-reactivity among themselves (17). In several pneumoccocal infection challenge models, PspA has proved to be a good vaccine candidate when administered either in protein-adjuvant formulations or in DNA-based vaccines (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 antibodies 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 antigen A from Streptococcus pneumoniae by intranasal inoculation of mice (3). In this work, we have amplified the 5' terminus region (which encodes the {alpha}-helix region plus the proline-rich domain) of the pspA clade 3 gene from pTG-pspA'3 (11) (pspA'3 accession number, AY082389), which carried the gene isolated from S. pneumoniae St 259/98 strain (Instituto Adolpho Lutz, São 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 Escherichia coli. Using this expression system, the recombinant protein 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 · liter–1 imidazole.

Protein characterization. The recombinant CTB-PspA' purified from E. coli BL21-SI extracts was able to form pentamers, as evaluated by 6% sodium dodecyl sulfate-polyacrylamide 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 immunosorbent assay (ELISA) experiment, using 10 µg · ml–1 GM1 in phosphate-buffered saline for an overnight coating and increasing 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 µg · ml–1 bovine serum albumin was used as a coating (Fig. 1).



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  		FIG. 1. Analysis by GM1 ELISA of CTB-PspA' ability to bind GM1 receptor. The ELISA was performed by coating a 96-well plate with GM1 or bovine serum albumin. CTB and PspA' were used as controls.

 
Immunization of mice. To test the immunogenic potential of CTB-PspA', 6-week-old female BALB/c mice from Instituto Butantan, São Paulo, Brazil, received by the intradermal route either the CTB-PspA' protein (5.6 µg) or the controls—saline, CTB (1.6 µg), PspA' (4.0 µg), and PspA' (4.0 µg) coadministered with CTB (1.6 µg)—twice a week for three consecutive weeks. The total volume administered to each animal was 100 µl. One group received one dose of the commercial Pneumovax vaccine (Merck Sharp Dohme) intraperitoneally (i.p.) 2 weeks before the challenge as a positive control. Another group received 5.6 µg of CTB-PspA', diluted in 10 µl of saline, by the intranasal route. The mass of CTB and PspA' used in this experiment was determined according to the molar concentrations of the two proteins present in 5.6 µg of the fusion protein. Three weeks after the last immunization, the mice were bled through the retroorbital plexus. Anti-PspA' antibodies were measured in the sera by ELISA, using recombinant PspA' as a coating. The titer was defined as the dilution that gave an absorbance of 0.1 at 492 nm. The results were compared using Student's t test.

As observed in Fig. 2, the highest titers of anti-PspA' IgG were achieved in the animals that received the combination of CTB and PspA' or the fusion protein CTB-PspA'. The levels of anti-PspA IgG from these groups were significantly different from those of the groups that received saline (P < 0.001), CTB (P < 0.001), or PspA' alone (P < 0.05). The levels of anti-PspA IgG for the group that received CTB-PspA by the intranasal route were not significantly different from that of the saline group. As expected, Pneumovax, which is a polysaccharide-based vaccine, did not induce significant amounts of anti-PspA' IgG.



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  		FIG. 2. Induction of anti-PspA' IgG by CTB-PspA' after intradermal immunization i.p. Individual logs of reciprocal anti-PspA' IgG titers from serum are shown. The titer was defined as the last dilution in which an absorbance of 0.1 could be observed. The individual sera that displayed nondetectable anti-PspA' IgG titers were represented as <1. Pneumovax-23 vaccine and CTB-PspA' IN were inoculated intraperitoneally and intranasally, respectively, whereas the others were administered by the intradermal route. The results are representative of two independent experiments.

 
Intraperitoneal challenge. After analysis of anti-PspA' IgG in the sera, the animals underwent an intraperitoneal challenge with 102 CFU of Streptococcus pneumoniae strain St 679/99 (serotype 6B, clade 3, family 2) from Instituto Adolpho Lutz, São Paulo, SP, Brazil. Survival, observed up to 7 days after challenge, is shown in Table 1. Partial protection was observed in mice immunized intradermally with commercial Pneumovax vaccine (65%) or the CTB-PspA' fusion protein (56%). These values were significantly different from those for the groups that received either saline or CTB (P < 0.05 by the Fisher exact test). When the CTB-PspA' was administered by the intranasal route, no increase in survival was observed. Intradermal immunization with a mixture of PspA' and CTB elicited very high levels of IgG antibody to PspA (Fig. 2) but did not elicit protection (Table 1). In addition, no correlations between anti-PspA' IgG levels and survival or median survival time were observed when the animals were analyzed individually.


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  		TABLE 1. Survival of mice challenged with St 679/99a

 
In the search for protein candidates for the development of a vaccine against S. pneumoniae with good efficacy in children and elderly and immunocompromised individuals, PspA antigen has been extensively studied in recent years. Antibodies to PspA have proved to be protective in different pneumococcal challenge models developed in animals (19), even when used as therapy a few hours after infection (16). Moreover, PspA– pneumococcus strains are less pathogenic than their wild-type counterparts (5). The basis for the protection exerted by anti-PspA antibodies has been addressed in different works and appears to be related to a decrease in the inhibition of complement deposition caused by PspA (13) during infection and an enhancement in pneumococcal killing by apolactoferrin present in mucosal secretions (15). In this work, we investigated the potential of a CTB-PspA' fusion protein to induce protective anti-PspA antibodies upon intradermal immunization. The intradermal route was chosen based on our preliminary results showing that the intranasal route was less efficient for the induction of anti-PspA' IgG in the sera (data not shown). The lower antibody levels could be a problem, since protection in a sepsis model is attributed to the presence of IgG in the serum, which is corroborated by the efficiency of passive immunization at protecting mice from fatal infection with S. pneumoniae (6, 16). CTB has proved to be a good adjuvant for different antigens when administered through the mucosa (9, 10). More recently, some groups have published the adjuvant effect of CTB when inoculated through the skin, such as the transcutaneous or epidermal route (1, 7), by a Th1-predominant mechanism. Our results have shown that intradermal immunization with CTB-PspA' protein partially protected mice against an intraperitoneal challenge with an S. pneumoniae strain that expressed a homologous PspA. The protection was similar to that observed for the Pneumovax vaccine in our experiments but was significantly different from that observed for the group that received PspA' in combination with CTB. Interestingly, the levels of anti-PspA' IgG elicited by the group that received CTB-PspA' or a combination of the two proteins were about the same. Given the fact that the animals with the highest antibody titers were not necessarily protected, other mechanisms seem to be involved. So far, the only factor that we have been able to associate with this result is the lower IgG1/IgG2a ratio (r) displayed by immunization with CTB-PspA' (r = 4) compared to that induced by PspA' alone (r = 32) or by PspA' plus CTB (r = 16). It is thought that a balanced immune response, in terms of IgG1 and IgG2a production, is suitable for protection against a lethal challenge with a virulent strain of S. pneumoniae (11). In addition to the immunological efficacy of this fusion protein, the manufacturing process appears to be easier and less expensive, since a single fermentation step is required for the production of the adjuvant and the antigen desired. Also, the functionality of the final recombinant product can be easily assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in which the samples do not undergo heat treatment, and by a GM1 ELISA. The partial protection against S. pneumoniae intraperitoneal challenge indicates that CTB-PspA' may be considered a promising vaccine component.


   ACKNOWLEDGMENTS
 
We thank Vera R. F. Ferreira for the coordination of the animal facilities, and Amanda C. E. Xavier for technical assistance.

This work was supported by FAPESP, CNPq, and Fundação Butantan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Biotecnologia, Instituto Butantan, São Paulo, Brazil. Phone: 55 11 3726 7222, ext. 2244. Fax: 55 11 3726 1505. E-mail: hoplee{at}butantan.gov.br. Back

Editor: J. N. Weiser


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Infection and Immunity, June 2005, p. 3810-3813, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3810-3813.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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