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Unité de Pathologie Infectieuse et Immunologie, INRA-Centre de Tours, 37380 Nouzilly, France
Correspondence Olivier Grépinet grepinet{at}tours.inra.fr
Received August 21, 2003
Accepted May 17, 2004
The immunogenicity and protective effect of a DNA vaccine encoding the heat-shock protein (Hsp) GroEL of Chlamydophila abortus AB7, an obligate intracellular bacterium that causes abortion in sheep, was evaluated in pregnant and non-pregnant mouse models. The C. abortus groEL gene was cloned by screening a genomic library constructed in 
FIX II arms with a nucleic acid probe corresponding to the central portion of the groEL gene from C. abortus. Sequence analysis of a positive clone revealed an open reading frame of 1632 bp encoding a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da and highly similar to GroEL of Chlamydia trachomatis (93 %) and Chlamydophila pneumoniae (94 %). As observed in other sequenced chlamydial genomes, the groEL gene belongs to an operon comprising another gene encoding the Hsp GroES. OF1 outbred mice were immunized intramuscularly with plasmid DNA carrying the groEL gene three times at 3 week intervals and challenged 2 weeks after the last DNA injection. In pregnant mice, no reduction in abortion was observed and the DNA vaccination failed to reduce the bacterial infection in the placenta and spleen of mice. Nevertheless, partial protection of fetuses was obtained. Immunization of non-pregnant mice with the groEL gene resulted in a specific humoral response with the predominant IgG2a isotype, suggesting a Th1-type immune response. The anti-GroEL antibodies showed no neutralizing effect in vitro on C. abortus infectivity. Although the DNA vaccine induced a delayed-type hypersensitivity response, it failed to elicit an efficient cellular immune response since the mice were not protected against bacterial challenge.
The GenBank accession number for the groES–groEL operon of Chlamydophila abortus is AY052785.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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DNA vaccination has emerged as a promising strategy for the induction of protective immune responses (Donnelly et al., 1997). Plasmid DNA encoding antigenic proteins is introduced directly into animals by intramuscular or intradermal injection. After the direct transfection of host cells, the antigen is expressed in situ and thus constitutes the target of the host immune response. DNA vaccination induces a Th1-type immune response, including both humoral and cellular immune responses (Donnelly et al., 1997), and has already been tested for preventing viral, parasitic and bacterial diseases (Hasan et al., 1999). More particularly, DNA vaccines appear to be especially successful at preventing intracellular bacterial diseases (Strugnell et al., 1997). In previous studies, the genes encoding the heat-shock protein (Hsp) DnaK and the major outer-membrane protein (MOMP) were used to immunize mice against C. abortus (Héchard et al., 2002, 2003). Despite a specific humoral response directed against the proteins encoded by the injected vaccine genes, no significant protection was found. Indeed, DNA vaccination with these two genes failed to reduce the number of abortion cases significantly, as well as the quantities of C. abortus in the spleen, placenta and fetus of mice after challenge.
Among the several potential immunogens of chlamydiae, the protein GroEL has been considered as one of the most likely candidates for a vaccine (Brunham & Peeling, 1994). GroEL (Hsp60) belongs to the Hsp family, which is widely distributed in nature. Hsps are commonly involved in the folding/unfolding or translocation of proteins, as well as the assembly/disassembly of protein complexes, and are known to be highly immunogenic (Srivastava et al., 1998; Zugel & Kaufmann, 1999). GroEL is synthesized in the early stage of the developmental cycle of chlamydiae and is one of the major targets of the humoral response in infected animals. Moreover, DNA vaccination with the groEL gene has been tested successfully against extracellular (Noll et al., 1999) and intracellular bacteria (Tascon et al., 1996) and more particularly against Chlamydophila pneumoniae infection in a murine experimental model (Penttilä et al., 2000; Svanholm et al., 2000).
In the present study, humoral and cellular immune responses were characterized after intramuscular injection of the groEL gene in a murine model of systemic C. abortus infection. The protection induced by the DNA vaccine was also assessed in pregnant and non-pregnant mouse models after chlamydial challenge.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Construction and screening of the C. abortus AB7 genomic library.
Chromosomal DNA from C. abortus AB7 was extracted and purified as described previously (Boumedine & Rodolakis, 1998). After partial digestion with Sau3AI (Promega), genomic DNA fragments were partially filled in with dGTP and dATP using Klenow DNA polymerase (Promega) and ligated with T4 DNA ligase to FIX II arms prepared by a partial fill-in reaction with dTTP and dCTP after digestion with XhoI (Stratagene). Recombinant phage DNA was packaged in vitro using Gigapack III XL packaging extracts (Stratagene) and transduced into Escherichia coli XL-1 Blue MRA(P2).
Plaque replicas were screened with a DIG-labelled probe corresponding to the central portion of the groEL gene from C. abortus AB7. Briefly, gene-specific primers GroE-FW (5'-GATAAAGCTGGTGATGGAAC-3') and GroE-RV (5'-ATACCTTCTTCAACAGCAGC-3') were designed according to a partial sequence encoding GroEL from C. abortus B577 (GenBank accession no. AF109790) and used to amplify a 986 bp fragment from C. abortus AB7 genomic DNA by PCR. This fragment was labelled with DIG-11-dUTP using the High Prime DNA Labelling kit (Roche Diagnostics). Hybridization of the probe and washes were performed according to standard protocols (Sambrook & Russell, 2001). Binding of the probe was detected with an anti-DIG–alkaline phosphatase conjugate (Roche Diagnostics) followed by incubation with BCIP/NBT. Positive plaques were removed from the plates and the phages were eluted in SM buffer (0.01 % gelatin, 100 mM NaCl, 8 mM MgSO4, 50 mM Tris/HCl, pH 7.5). The procedure was repeated until all plaques were positive.
Southern hybridization and subcloning into pBluescript II KS(+).
DNA from recombinant phages bearing the groEL gene was purified with the Qiagen Lambda Mini kit (Qiagen). Phage DNA was then digested with NotI, SacI, SalI and XbaI. Restriction fragments were separated by electrophoresis on a 0.7 % agarose gel and transferred to a nylon membrane. The groEL probe used for hybridization was previously described in the screening of the genomic library. A 7.0 kb XbaI restriction fragment hybridizing with the groEL probe was subcloned into pBluescript II KS(+). Additional subcloning steps generated pCH603, which contained a 4.1 kb insert bearing the entire groEL gene.
DNA sequencing and analysis.
Recombinant plasmid pCH603 was sequenced on both strands (Génome Express) using a primer-walking strategy. DNA and protein sequence analysis were performed with the DNA Strider 1.3 program (Marck, 1988). The BLAST2 program (Infobiogen) was used to search for similarities in the nucleic acid and protein sequence databases. Alignment of sequence was performed using CLUSTAL W (Infobiogen).
Plasmid constructions.
To produce recombinant GroEL and the vaccine vector, the groEL gene was amplified by PCR and cloned into appropriate vectors. PCR was performed using chlamydial genomic DNA (40 ng) as template, dNTPs (200 µM each), specific primers (1 µM each) and 1 U Pfu DNA polymerase (Promega) in an Applied Biosystems 9600 thermocycler with the following program: one cycle of 94 °C for 5 min; 30 cycles of 50 °C for 45 s, 72 °C for 2 min and 94 °C for 45 s; and 1 cycle of 50 °C for 45 s and 72 °C for 10 min. Specific primers were determined according to the DNA sequence of C. abortus AB7 as follows. (i) Primers contained a BamHI site (underlined) in the forward primer (GroEL-His1; 5'-CTCGGATCCGCAGCAAAAAAT ATTAAATAT-3') and an XhoI site (underlined) in the reverse primer (GroEL-His2; 5'-CTCCTCGAGTTAATAATCCATTCCTGCGCC-3'). The resulting fragment was inserted in a 5'-end histidine tag (His6) prokaryotic expression vector, pTrcHisA (Invitrogen), linearized by BamHI/XhoI double digestion to generate pTrcHis : : GroEL. (ii) Primers contained a BamHI site (underlined) and Kozak sequence (bold) in the forward primer (GroEL-B; 5'-CTCGGATCCACCATGG CAGCAAAAAATATTAAA-3') and an XbaI site (underlined) in the reverse primer (GroEL-X; 5'-CTCTCTAGATTAATAATCCATT CCTGCGCC-3'). The resulting fragment was inserted into the pcDNA3.1 (Invitrogen) eukaryotic vaccine vector, carrying the human cytomegalovirus immediate-early promoter and the bovine growth hormone polyadenylation signal, after linearization by BamHI/XbaI double digestion to generate pcDNA3.1 : : GroEL. The plasmid pcDNA3.1 : : GroEL and the pcDNA3.1 control plasmid were amplified in E. coli DH5
and purified using the EndoFree Plasmid Mega kit (Qiagen). Plasmid DNA was dissolved at a concentration of 1 µg µl–1 in endotoxin-free PBS (Sigma). The sequences of pTrcHis : : GroEL and of the vaccine vector pcDNA3.1 : : GroEL were verified by DNA sequencing.
Purification of recombinant GroEL.
E. coli TG1 was transformed with the plasmid pTrcHis : : GroEL and grown overnight in 3 ml Luria–Bertani (LB) medium supplemented with ampicillin (100 µg ml–1). One millilitre of this culture was transferred into 100 ml LB/ampicillin medium. After 3 h growth at 37 °C, IPTG (1 mM final concentration) was added and the culture was left overnight. Bacteria were pelleted by centrifugation for 15 min at 7000 g and recombinant GroEL was extracted and purified under native conditions according to the protocol of Xpress System Protein Purification (Invitrogen). After desalting and concentration of the protein using an Ultrafree-4 centrifugal filter (Millipore), the quantity of recombinant GroEL was measured using the BCA protein assay reagent (Pierce) and its concentration was adjusted to 1 mg ml–1 in PBS, pH 7.4.
Cell transfection.
COS-7 cells were cultivated in Dulbecco's modified Eagle's medium containing 10 % fetal calf serum and transfected with 10 µl Lipofectamine (Invitrogen) and 1 µg pcDNA3.1 : : GroEL plasmid per 3–4 x 105 cells. Expression of recombinant GroEL was tested in cell extracts after SDS-PAGE and immunoblotting (Héchard et al., 2002). Blots were probed with a 1 : 1000 dilution of the anti-GroEL-specific mAb A57-B9 (IgG1 isotype), kindly provided by Dr R. Morrison (Montana State University, Bozeman, USA) (Yuan et al., 1992).
Immunization and challenge.
All studies were carried out using 6-week-old female outbred OF1 Swiss mice (IFFA Credo). Prior to DNA immunization, each mouse was injected with cardiotoxin (Latoxan) into the tibialis anterior muscles of both hind legs to enhance the uptake of plasmid DNA (Davis et al., 1993). Five days later, mice were anaesthetized with an intraperitoneal injection of ketamine and xylazine [80 and 8 mg (kg body weight)–1, respectively] and immunized with pcDNA3.1 : : GroEL plasmid by intramuscular injection (50 µg in each tibialis anterior). Mice were boosted in the same way at days 21 and 41. The negative-control mice were immunized intramuscularly with endotoxin-free PBS (virulence control) or pcDNA3.1 plasmid. Positive-control mice were immunized with one subcutaneous injection of 105 p.f.u. live-attenuated 1B vaccine (Rodolakis, 1983) at day 1 (1B group). Groups of 22 and 20 mice were similarly immunized for evaluation of the protective effect of the DNA vaccine and constituted the non-pregnant and pregnant groups, respectively. The five groups of pregnant mice were mated at day 44. Non-pregnant and pregnant mice were challenged at day 58 by an intraperitoneal injection of 1.5x105 p.f.u. C. abortus AB7. Three mice from each non-pregnant group were not challenged for evaluation of the cellular immune response. One group of pregnant mice that was neither immunized nor challenged was kept as a control for the pregnancy (gestation control).
Antibody response.
Blood samples were collected from the retro-orbital sinus of non-pregnant mice immunized with PBS, pcDNA3.1 or pcDNA3.1 : : GroEL for the detection of anti-GroEL-specific antibodies before each DNA injection (days 0, 19 and 41), just before the challenge infection (day 56) and at days 63 and 69. Blood samples were stored overnight at room temperature and then centrifuged (600 g, 15 min, 4 °C) and sera were collected and stored at –20 °C. The specificity of anti-GroEL antibodies was checked by ELISA and immunoblotting, as described previously (Héchard et al., 2002). For ELISA, microculture plates were coated with 100 µl recombinant GroEL diluted in magnesium-/calcium-free PBS (pH 7.4) at a concentration of 1 µg ml–1. The anti-GroEL mAb A57-B9 was used as the reference in order to express the results in arbitrary units. For immunoblotting, 0.1 µg recombinant GroEL or crude C. abortus extracts (2.4 x 106 p.f.u.) were transferred from polyacrylamide gel to a nitrocellulose membrane. Blots were probed with a 1 : 200 serum dilution from mice of pcDNA3.1 and pcDNA3.1 : : GroEL groups collected at day 56. Determination of antibody isotypes was performed using sera from pcDNA3.1 : : GroEL-vaccinated mice collected at day 56 or 63 (Héchard et al., 2002). Due to the limited quantity of mice sera available, ELISA tests were performed on individual animals for total IgG and on pooled sera for the determination of isotypes. The in vitro neutralization assay was carried out using the plaque-reduction assay (Héchard et al., 2003).
Evaluation of protection.
The protective effect of the DNA vaccine was assessed by evaluation of the number of living offspring per litter 8 days after birth (Rodolakis et al., 1981) and the clearance of bacteria in the spleen, placenta and fetus. After challenge, the outcome of pregnant mice (15 mice per group) was monitored daily. Mice were considered as protected when the number of living offspring per litter 8 days after birth was significantly different (P < 0.05) from the number in the virulence group. Five mice from each pregnant group were euthanized at day 63. Ten mice of each non-pregnant group were euthanized at day 63 and nine mice were euthanized at day 69. The spleens from non-pregnant mice and the uterus and the spleens from pregnant mice were aseptically removed. All placentas from the same uterine horn were dissected from the fetuses. Placentas from the same uterine horn were pooled as well as the fetuses. Pooled placentas, pooled fetuses and spleens were weighed and frozen at –80 °C. Organs were homogenized in PBS with a glass grinder, diluted in DEAE dextran (0.01 %) in PBS and titrated by plaque assay on McCoy cells (Rodolakis & Chancerelle, 1977). The course of infection was evaluated by counting the number of p.f.u. and expressed as log10 p.f.u. per organ for spleens and log10 p.f.u. per uterine horn for placentas and fetuses.
Measurement of GroEL-specific delayed-type hypersensitivity (DTH) response.
The DTH response was evaluated 3 weeks after the third DNA injection in three non-pregnant immunized mice of each group. Recombinant GroEL (10 µg in 30 µl) was injected into the right hind footpads and the same volume of PBS was injected into the left hind footpad as a control. Footpad swelling was measured at 48 and 72 h after the injection using a dial-gauge calliper (Mettallex). The difference in thickness between the right and the left footpads was used as a measure of the DTH response.
Statistical tests.
Analysis of the results was performed using the software InStat 2.03 for Macintosh. For all protective results, the mean was calculated using a one-way analysis of variance and a comparison of the means was then carried out through a Student–Newman–Keuls multiple comparison test. The minimal statistical significance was judged at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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FIX II arms and screened with a DIG-labelled probe complementary to the central portion of the C. abortus AB7 groEL gene. DNA from a recombinant positive phage was subcloned into pBluescript II KS(+) to yield plasmid pCH603, which contained the entire groEL gene. DNA sequencing of the insert carried by plasmid pCH603 revealed the presence of two open reading frames. The larger one (1632 bp) encoded a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da. The encoded polypeptide was highly similar to GroEL of Chlamydia trachomatis (93 %) and C. pneumoniae (94 %), which confirms the extreme interspecies conservation of Hsps throughout evolution. The smaller one (306 bp), located 50 bp upstream of the groEL start codon, encoded a 102 amino acid polypeptide with a calculated molecular mass of 11 267 Da. The encoded polypeptide exhibited 83 % identity with GroES of C. trachomatis and C. pneumoniae. Therefore, as in the case of other members of the Chlamydiaceae, the groES and groEL genes belong to the same operon.
The groEL gene sequence was then inserted into the eukaryotic expression vector pcDNA3.1. Production of C. abortus GroEL from the pcDNA3.1 : : GroEL construct was confirmed by transfecting the plasmid into COS-7 cells. Using the anti-GroEL mAb A57-B9, GroEL was readily detected by immunoblotting using whole-cell lysate of pcDNA3.1 : : GroEL-transfected COS-7 cells but not in control pcDNA3.1-transfected COS-7 cells (Fig. 1). The smaller size of the GroEL protein expressed by the COS-7 cells compared with the recombinant GroEL produced in E. coli and the chlamydial GroEL (60–62 kDa) could be explained by a slight proteolysis of the protein synthesized in COS cells. Thus, eukaryotic cells correctly expressed C. abortus GroEL and the plasmid pcDNA3.1 : : GroEL could consequently be used for DNA immunization.
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Anti-GroEL antibody response to DNA vaccination
An anti-GroEL IgG antibody response was found in mice immunized with pcDNA3.1 : : GroEL (Fig. 2). Anti-GroEL IgG were readily detectable after the second DNA injection and increased after the third injection to reach a maximum just before challenge (day 56). A significant increase in IgG was observed 11 days after challenge (day 69), probably due to the booster effect of the challenge (data not shown). No GroEL-specific IgG antibodies were identified in sera from non-immunized mice (data not shown) or from mice immunized with pcDNA3.1 (Fig. 2).
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The specificity of anti-GroEL antibodies was also tested by immunoblotting with chlamydial extracts and purified recombinant GroEL on the immunized mice sera collected before challenge (day 56). The sera collected at day 56 from pcDNA3.1 : : GroEL-vaccinated mice and the anti-GroEL mAb A57-B9 detected a protein with a similar molecular mass (62 kDa) (Fig. 3). Thus, the antibodies of the pcDNA3.1 : : GroEL-vaccinated mice sera were specifically directed against GroEL.
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The isotypes of anti-GroEL antibodies of sera from pcDNA3.1 : : GroEL-vaccinated mice were determined by ELISA. IgG2a was the predominant isotype at days 56 and 63 (65 and 60 % of total IgG, respectively). No IgG1, -2b or -3 was found at day 56, although IgG3 was found at day 63 (data not shown). These data indicated that plasmid vaccination using the groEL gene generated predominantly IgG2a antibodies, suggesting a Th1-like immune response. This hypothesis could have been confirmed by analysis of cytokine profiles. Buendía et al. (1999) showed the presence of high levels of IFN-
and IL12 in the serum of mice infected with the C. abortus AB7 and 1B strains, which strongly suggests that resolution of the infection effectively involves a Th1-like immune response.
The neutralizing effect of the anti-GroEL antibodies was assessed. The 10–1 and 10–2 dilutions of 1B-vaccinated mice sera reduced the infectivity of C. abortus (90 and 68 %, respectively; Fig. 4). There was no difference between the effect of a 10–2 dilution of pcDNA3.1- and pcDNA3.1 : : GroEL-vaccinated mice sera as neither significantly reduced the infectivity of the bacteria. Nevertheless, the 10–1 dilution of pcDNA3.1-vaccinated mice sera had a modest neutralizing effect (41 %) while the 10–1 dilution of pcDNA3.1 : : GroEL-vaccinated mice sera had a non-significant effect (20 %). Therefore, the pcDNA3.1 : : GroEL-vaccinated mice sera failed to reduce the infectivity in vitro of C. abortus on McCoy cells. This negative result could be explained by the inaccessibility of the antigen on the cell surface. Different studies have suggested that GroEL is not a surface-displayed antigen on elementary bodies (Bavoil et al., 1990; Morrison et al., 1989). Neutralizing sera against chlamydiae have been obtained with recombinant antigens such as the MOMP of C. trachomatis, which is a surface-exposed ligand (Murdin et al., 1995).
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Efficacy of DNA immunization in pregnant mice
The number of live newborn mice was evaluated at birth and 8 days after birth. The numbers of newborn mice from 1B-vaccinated and gestation control groups were not significantly different but were significantly greater (P < 0.05) than in the virulence group at birth or 8 days after birth (Fig. 5). The numbers of live newborn mice in pcDNA3.1 and pcDNA3.1 : : GroEL groups 8 days after birth were not significantly different but were significantly less (P < 0.05) than in the 1B-vaccinated group and not significantly greater than in the virulence group. Thus, according to the mean number of living offspring 8 days after birth, mice in the pcDNA3.1 and pcDNA3.1 : : GroEL groups were not protected from C. abortus AB7 challenge. Nevertheless, at birth, a protective effect was observed in the pcDNA3.1 : : GroEL group. This effect was not statistically significant, probably because of the high variability of numbers or living offspring in each group.
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The protective effect was also evaluated in placental and fetal murine models of infection, which allow a graded differentiation between virulent and non-virulent C. abortus strains (Rodolakis et al., 1989). As already observed, the level of infection was higher in the placenta than in the fetus (Fig. 6). 1B-vaccinated mice had significantly reduced levels of placental and fetal infection in comparison with the other groups. No effect of DNA vaccination was observed on the placental level of infection, but fetuses from virulence and pcDNA3.1 groups were similarly infected, while fetuses from the pcDNA3.1 : : GroEL group presented a reduced level of infection. This partial protection was not statistically significant, probably due to the high variability of the fetal level of infection. As placentas were infected, fetuses were most likely protected by a barrier effect, but this protective effect was not sufficient to allow the survival of the living offspring. The mortality of the newborn mice could be caused by the infected placentas, which may have lost their nutritious properties and led to a frailty of living offspring, resulting in death, even in the absence of fetal infection. Also, late contamination during the birth process is possible. Indeed, it has been reported that C. abortus was excreted when ewes gave birth and that they could therefore infect newborn fetuses, as well as other ewes, at this time (Rodolakis & Souriau, 1979). This hypothesis could be transposable to the mouse model, since the number of living offspring appeared to be higher at birth than 8 days after birth.
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Clearance of C. abortus AB7 challenge in the spleen of pregnant and non-pregnant mice
Pregnant and non-pregnant mice were euthanized and spleens were collected 5 days after challenge, since it has been shown that chlamydial titres in spleen are maximal on this day (Rodolakis, 1983). Spleens from other non-pregnant mice were also taken 11 days after challenge. The level of infection 11 days after challenge was reduced in comparison with that observed 5 days after challenge, due to the natural clearance of C. abortus (Rodolakis, 1983). Chlamydial titres in 1B-vaccinated mice spleens 5 or 11 days after challenge were significantly lower (P < 0.05) than in the virulence group, so 1B-vaccinated mice were protected against chlamydial infection (Fig. 7). Splenic chlamydial titres of the virulence, pcDNA3.1 and pcDNA3.1 : : GroEL groups did not show significant differences at 5 or 11 days after the challenge. These results suggested that DNA vaccination did not induce splenic protection in pregnant or non-pregnant mice.
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Cellular response
A positive but not statistically significant DTH response to recombinant GroEL was detected at 48 h among 1B- and pcDNA3.1 : : GroEL-immunized mice (0.23 ± 0.12 mm and 0.33 ± 0.03 mm, respectively). No difference was found between the DTH responses elicited in PBS- or pcDNA3.1-vaccinated mice (0.10 ± 0.10 mm). The protection against C. abortus infection is mediated mainly by the induced cellular immune response, especially cytotoxic CD8+ cells, while the humoral immune response provides only a moderate contribution (Buzoni-Gatel et al., 1987). The non-protective effect of the groEL DNA vaccine was not surprising as it induced a weak DTH. In this study, we used a mouse model of abortion, since it was currently employed at our laboratory and was successfully utilized for the evaluation of C. abortus strain virulence (Rodolakis et al., 1989), the protective effect of vaccines (Rodolakis, 1983) and immune responses induced by C. abortus infection (Buzoni-Gatel et al., 1987). Nevertheless, this model involves outbred mice and therefore presents two major disadvantages. Firstly, it is not suitable for an extensive analysis of the cellular immune response. Secondly, the higher variability of the results obtained with outbred mice could explain the fact that the different effects of pcDNA3.1 : : GroEL immunization observed in this study were not statistically significant. Thus, it would be interesting to evaluate in further experiments the cellular immune response induced by groEL DNA vaccines in an inbred model of mice and, in the same way, their protective effect in the abortive outbred mouse model, since this vaccine is ultimately destined for outbred goats and sheep.
In a C. pneumoniae murine experimental model of infection, DNA vaccination with a secreted or non-secreted form of GroEL induced partial protection against challenge (Penttilä et al., 2000; Svanholm et al., 2000). In contrast with the present study, DNA immunization elicited both cellular and humoral immune responses in this model. Nevertheless, DNA vaccination with the groEL gene induced only a 1.5 log reduction in the mean lung bacterial counts in comparison with a plasmid control. Another recent report evaluated the protective efficacy of a DNA vaccine encoding the groEL gene from Brucella abortus in a murine model (Leclerq et al., 2002). In spite of a strong humoral response and a Th1-predominant immune response, DNA immunization conferred no protection against a virulent challenge, as observed in our experiments.
DNA immunization is known preferentially to induce both cellular and humoral immune responses (Donnelly et al., 1997). Nevertheless, the generation of these immune responses is partly dependent on the expression of the target protein, i.e. GroEL. Expression of the encoded antigen is generally low (Wolff et al., 1990), and thus, before it can be concluded that the GroEL protein is not suitable for inducing protective immunity after DNA vaccination, it would be necessary to enhance expression of GroEL. A number of methods can be used to enhance the efficiency of DNA vaccines, including in vivo electroporation (Widera et al., 2000), a DNA priming–protein boosting protocol (Dong-Ji et al., 2000) or the use of replicon-based DNA vaccines (Mena et al., 2000). Despite these moderate results obtained with the groEL gene, DNA vaccination could offer the opportunity for development of an acellular vaccine against ovine chlamydiosis, which would allow the detection of infected animals in vaccinated flocks.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) and tested individually by ELISA for titration of anti-GroEL-specific IgG antibodies. The mean titre (+SEM) was calculated for each time point.
) or pcDNA3.1 : : GroEL (
