J Med Microbiol 55 (2006), 375-378; DOI: 10.1099/jmm.0.46217-0
© 2006 Society for General Microbiology
ISSN 0022-2615
DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein
Daniela M. Ferreira,
Eliane N. Miyaji,
Maria Leonor S. Oliveira,
Michelle Darrieux,
Ana Paula M. Arêas,
Paulo L. Ho and
Luciana C. C. Leite
Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900, São Paulo, SP, Brazil
Correspondence
Eliane N. Miyaji
enmiyaji{at}butantan.gov.br
Received 29 June 2005
Accepted 16 November 2005
Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.
Abbreviations: BHK, baby hamster kidney; rPspA, recombinant PspA.
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INTRODUCTION
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Streptococcus pneumoniae is one of the most common aetiological agents of invasive diseases such as meningitis, septicaemia and pneumonia, especially in children under 2 years of age and in the elderly. Currently, two pneumococcal vaccines are available, a 23-valent polysaccharide and a 7-valent conjugate vaccine (Bogaert et al., 2004). Although the 23-valent is poorly immunogenic and fails to induce protection in young children, the 7-valent has shown high efficacy against invasive infections in infants and the coverage is estimated as over 85 % in the USA, 60–70 % in Europe (Pelton et al., 2003) and 58 % in Brazil (Brandileone et al., 2003). However, this latter vaccine elicits protection only against the seven included serotypes. To overcome these limitations, investigators have proposed the use of protective protein antigens conserved in all pneumococcal serotypes, such as pneumococcal surface protein A (PspA), pneumococcal surface antigen A and pneumolysin, as promising candidates for the development of cost-effective vaccines.
PspA acts in host–pathogen interactions, interfering with the activation and deposition of complement (Tu et al., 1999; Ren et al., 2004). Furthermore, it has been reported recently that PspA protects pneumococci from killing by apolactoferrin at mucosal sites (Shaper et al., 2004). Many groups have shown that recombinant PspA (rPspA) can elicit an antibody response that protects against lethal challenge in animal models, including passive protection (Briles et al., 2000a, b). Induction of protective immunity using PspA through genetic immunization has also been described (McDaniel et al., 1997; Borsage et al., 2001; Miyaji et al., 2002).
In the present study, DNA vaccine vectors expressing an N-terminal fragment of PspA were constructed with or without fusion to a secretory sequence. The immune response elicited by these vaccines was evaluated in terms of their protective efficacy compared with a recombinant protein administered with aluminium hydroxide as an adjuvant.
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METHODS
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Construction of DNA vaccine vectors.
The non-secretion (pTG-pspA3NS) and the secretion (pSec-pspA3NS) vectors used for DNA immunization were based on pTARGET (Promega) and pSecTag2A (Invitrogen), respectively. In both vectors, expression of the gene is controlled by the human cytomegalovirus immediate-early promoter/enhancer. The pSecTag vector carries the secretion signal from the V-J2-C region of the mouse Ig kappa-chain, promoting efficient secretion of the recombinant protein. The DNA fragments encoding the N-terminal region of PspA3, without a signal sequence, as far as the proline-rich region, were amplified by PCR from pTG-pspA3 (Miyaji et al., 2002), using the primers 5'-TAGCTCGAGACCATGATCTTAGGGGCTGGTTT-3' and 5'-TAGTTATCTAGATTTTGGTGCAGGAGCTGG-3' for pTG-pspA3NS and 5'-GGTACCGGTAAGAGCAGAAGAAGAAGCCC-3' and 5'-CTCGAGTTATTTGGTGCAGGAGCTGG-3' for pSec-pspA3NS. The original sequence was amplified from strain St 259/98 (PspA clade 3, serotype 14). Plasmid DNA was purified from Escherichia coli DH5
by anion-exchange chromatography with QIAFilter Maxi kit from Qiagen.
Expression and purification of recombinant protein in E. coli.
Expression of rPspA3NS in E. coli was performed using the pAE vector (Ramos et al., 2004), containing exactly the same insert as found in pTG-pspA3NS. The recombinant protein contains a 6x His tag at the N terminus to facilitate purification through Ni2+-charged column chromatography. Recombinant protein was purified from the soluble fraction of E. coli BL21 (SI) transformed with the vector, as described previously (Arêas et al., 2004).
Antigen expression in transfected mammalian cells.
To evaluate in vitro expression, baby hamster kidney (BHK) cells were transfected with the constructed vaccine vectors using Lipofectamine reagent (Invitrogen). Cells were harvested 24 h after transfection and transient expression was analysed by Western blotting, as previously described (Miyaji et al., 2002), using anti-rPspA3NS antiserum developed in mice. Specificity of the antiserum had been tested previously using whole-cell extracts from several S. pneumoniae strains.
Immunization of mice and detection of anti-PspA antibodies.
The DNA vaccine vectors constructed were used to immunize female BALB/c mice (5–7 weeks old, from Instituto Butantan, São Paulo, Brazil). Groups of at least six mice were inoculated intramuscularly with 50 µl of 10 µM cardiotoxin (Laxotan, Valence, France) into each tibialis anterior muscle, 5 days before immunization with 50 µg vaccine vector in PBS (100 µl). Mice received a booster after 3 weeks with the same dose of plasmid DNA. For immunization with recombinant protein, mice were injected subcutaneously with 5 µg rPspA3NS, using aluminium hydroxide (Production Division of Instituto Butantan) as the adjuvant, and also boosted 3 weeks after priming. Mice were bled from the retro-orbital plexus, 6 weeks after priming, for detection of serum anti-PspA antibodies by ELISA as described previously (Miyaji et al., 2002), using rPspA3NS as the coating antigen. Differences between groups were analysed by Student's t-test. Isotyping of pooled sera was also performed by ELISA using mouse monoclonal antibody isotyping reagents (Sigma). Reciprocal titres were considered as the last dilution of sera that registered an A492 of 0·1.
Intraperitoneal challenge.
Immunized mice were challenged by intraperitoneal injection of 200 c.f.u. S. pneumoniae strain St 679/99 (PspA clade 3, serotype 6B) in 0·5 ml saline 6 weeks after priming. Animals were then observed for 2 weeks and inactive sick animals were euthanized. Differences in the overall survival rate between groups were analysed by Fisher's exact test.
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RESULTS AND DISCUSSION
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In order to evaluate expression of PspA by the constructed DNA vaccine vectors, mammalian cells were transfected and antigen expression was analysed by Western blotting. As expected, PspA3 was detected in cell lysates of both non-secretion (pTG-pspA3NS; Fig. 1
, lane 2) and secretion (pSec-pspA3NS, lane 3) vectors, whereas secreted PspA3 was observed only in the supernatant of the cells transfected with pSec-pspA3NS (lane 6). A protein with a slightly higher molecular mass was detected in the cytosol of cells transfected with the secretion vector when compared with the non-secretion vector. This difference is due to the presence of the V-J2-C secretion signal in the antigen expressed by pSec-pspA3NS, which is cleaved when the protein is exported to the culture supernatant.
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Fig. 1. Transient expression of PspA3 by DNA vaccine vectors in BHK cells. Total extracts (lanes 1–3) and supernatants (lanes 4–6) of BHK cells transformed or not with the vectors were analysed by Western blotting using anti-rPspA3NS antiserum. Lanes 1 and 4 indicate non-transformed cells; 2 and 5, cells transformed with pTG-pspA3NS; 3 and 6, cells transformed with pSec-pspA3NS. Migration of standard molecular mass markers is indicated.
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BALB/c mice were then immunized with the constructed DNA vaccine vectors and the anti-PspA antibody response was analysed. As shown in Fig. 2(a), both vectors elicited a significant anti-PspA IgG response (P
0·0001 for both non-immunized compared with pTG-pspA3NS and non-immunized compared with pSec-pspA3NS). Furthermore, mice immunized with pSec-pspA3NS elicited significantly higher levels of anti-PspA IgG than those observed for mice immunized with pTG-pspA3NS (P=0·03), which indicates that expression of the antigen as a secreted protein is able to enhance the induced humoral response. Isotyping of antibodies demonstrated that mice immunized with both DNA vaccines showed a balanced IgG1 : IgG2a ratio (0·5 to 2·0), showing only a small difference due to the secretion of the antigen (Fig. 2b). Animals were then challenged with St 679/99 and survival was analysed. Both vectors elicited significant protection, at similar levels (Table 1), indicating that the lower IgG response elicited by the non-secretion vector is sufficient for the observed protection.
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Fig. 2. Evaluation of the antibody response elicited by immunization with DNA vaccine vectors. Groups of at least 12 BALB/c mice were immunized intramuscularly with two doses of 50 µg of the indicated vectors, sera were collected and total anti-PspA IgG of individual sera (a) or anti-PspA IgG1 (filled bars) and IgG2a (open bars) of pooled sera (b) was measured by ELISA in plates coated with rPspA3NS. Log of the reciprocal titre of anti-PspA IgG is shown. Numbers above columns are IgG1 : IgG2a titre ratios. *, Statistically significantly different from non-immunized controls; **, statistically significantly different from pTG-pspA3NS (P0·05).
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In order to compare genetic immunization and recombinant protein strategies, we next immunized mice twice with either pSec-pspA3NS intramuscularly or rPspA3NS subcutaneously (Fig. 3a). As expected, mice immunized with two doses of the recombinant protein showed significantly higher levels of anti-PspA IgG antibodies than mice immunized with pSec-pspA3NS (P=0·02). Several studies have demonstrated that combining two or more vaccine modalities can enhance the immune response both qualitatively and quantitatively (reviewed by Doria-Rose & Haigwood, 2003). We have thus tested a prime–boost strategy, using DNA vaccine as priming and recombinant protein as booster, or vice versa. The groups that were immunized with the prime–boost strategies showed an enhancement of the antibody response, when compared with the mice that were immunized with two doses of pSec-pspA3NS vaccine vector, but the difference was not statistically significant. Isotyping of antibodies demonstrated that mice immunized with DNA vaccines showed a more balanced IgG1 : IgG2a ratio (1·0), whereas animals immunized with recombinant protein induced a very high ratio (64) (Fig. 3b). Booster immunizations increased the anti-PspA levels but did not alter the isotypic nature of the response: DNA-vaccine-primed mice continued to show an antibody response with a more balanced ratio, whereas recombinant protein priming maintained a more Th2-biased humoral immune response, with higher IgG1 antibody production.
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Fig. 3. Evaluation of the antibody response elicited by DNA vaccine and recombinant protein. Groups of six BALB/c mice were immunized with DNA vaccine or rPspA3NS, according to the indicated schedule of priming and booster doses. Sera were collected and total anti-PspA IgG of individual sera (a) or anti-PspA IgG1 (filled bars) and IgG2a (open bars) of pooled sera (b) was measured by ELISA on plates coated with rPspA3NS. Non-immunized and empty vector (pSec) controls are also shown. Log of the reciprocal titre of anti-PspA IgG is shown. Numbers above columns are IgG1 : IgG2a titre ratios. *, Statistically significantly different from non-immunized controls; **, statistically significantly different from pSec-pspA3NS (P0·05). Results are representative of two independent experiments.
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Mice were then challenged with St 679/99 and survival was analysed. All the immunized groups showed significant protection when compared with control groups (Table 1
). Animals immunized with pSec-pspA3NS and those primed with DNA vaccine and boosted with recombinant protein showed a tendency towards increased survival when compared with animals primed with rPspA3NS, but the difference was not statistically significant. Thus, augmentation of the humoral response did not lead to higher survival. In our previous work we have used immunization with protein as a positive control for comparison with DNA vaccines expressing different non-secreted PspAs (Miyaji et al., 2002, 2003). The proteins used were purified directly from S. pneumoniae through a choline chloride wash, resulting in contamination with other choline-binding proteins. PspA purified from S. pneumoniae consistently elicited higher protection levels when compared with the DNA vaccines. With the elimination of the pneumococcal contaminants and the use of exactly the same fragment, protection elicited by DNA vaccination was slightly higher (though not statistically significant) than for recombinant protein.
Anti-PspA antibodies are believed to overcome the anticomplement effect of PspA and to have opsonic activity that appeared to be mainly contributed by IgG2a, a Th1-characteristic immunoglobulin (Arulanandam et al., 2001). Intramuscular immunization with DNA vaccines is known to induce immune responses dominated by Th1 cytokine production, induced in part by immunostimulatory CpG motifs within the bacteria-derived plasmid DNA (Chu et al., 1997; Klinman et al., 1997). Indeed, immunization with DNA vaccines expressing PspA elicited a humoral response with a much more balanced IgG1 : IgG2a ratio. Data on IgG isotyping (Fig. 3b) suggest that the higher antibody response elicited by recombinant protein is mostly due to IgG1, while IgG2a is induced at similar levels by both recombinant protein and DNA vaccination, with comparable protection. We thus conclude that DNA vaccines are able to elicit protection levels similar to those for recombinant protein, even though total IgG levels are considerably lower.
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ACKNOWLEDGEMENTS
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This work was supported by CAPES, CNPQ, FAPESP and Fundação Butantan.
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REFERENCES
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TOP
INTRODUCTION
METHODS
