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HOST RESPONSE |
Unité de Pathologie Infectieuse et Immunologie, INRA – Centre de Tours, 37380 Nouzilly, France
Correspondence Olivier Grépinet grepinet{at}tours.inra.fr
Received 5 June 2002 Accepted 21 August 2002
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Abstract |
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 TOP Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Introduction |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The predominant component and immunodominant antigen of the Chlamydiaceae surface membrane, the 40 kDa major outer-membrane protein (MOMP), presents four surface-exposed variable domains (VDI–VDIV) flanked by five conserved regions and exhibits an oligomeric form, probably trimeric, that functions as a porin (De Sa, 1996). In a murine model of C. abortus infection, protective immunity can be conferred by antibodies (Buzoni-Gatel et al., 1990) and, more particularly, by the passive transfer of specific antibodies to the 110 kDa oligomeric MOMP (De Sa et al., 1995), so MOMP has been considered as the most likely vaccine candidate against C. abortus infections. Native MOMP (Pal et al., 1997; Tan et al., 1990), recombinant MOMP (Tuffrey et al., 1992), synthetic MOMP peptides (Su et al., 1995) and live recombinant vectors (Murdin et al., 1995) expressing momp genes from members of the Chlamydiaceae have been evaluated in different animal models. Limited immunity and protection were generated in animals when the vaccinal approaches attempted to preserve the conformational structure of MOMP. Nevertheless, most trials were unsuccessful. A recent study showed that protective immunity against a Chlamydia trachomatis genital challenge was induced with a vaccine based on the MOMP associated with lipophilic immune response-stimulating complexes (Igietseme & Murdin, 2000).
DNA vaccination represents an exciting means to induce specific immunity to a single antigen and, consequently, fits in the marker vaccine family (van Oirschot et al., 1996). Indeed, the direct injection of a DNA plasmid encoding an immunogenic protein into animals has resulted in both humoral and cell-mediated specific immune responses (Donnelly et al., 1997). Since DNA vaccination preferentially elicits a Th1-type immune response, it appears to be particularly appropriate for preventing intracellular bacterial infection (Strugnell et al., 1997). This novel strategy of vaccination has been assessed in different animal models and seems suitable for veterinary medicine (Babiuk et al., 2000).
DNA vaccination with the momp gene has already been tested in C. trachomatis (Pal et al., 1999; Zhang et al., 1997), Chlamydophila pneumoniae (Penttilä et al., 2000) and Chlamydophila psittaci (Vanrompay et al., 1999). In contrast to that observed in the C. psittaci model, no or modest protection was observed in the C. trachomatis and C. pneumoniae models. A single attempt at DNA vaccination against C. abortus in an abortion model is described in the literature (Héchard et al., 2002). In this model, only a non-specific and partial protection, probably due to the CpG motifs of bacterial DNA, was observed in pregnant mice immunized with a plasmid encoding the heat-shock protein DnaK.
The purpose of the present study was to evaluate the protective effect of intramuscular injection of the C. abortus AB7 momp gene in pregnant and non-pregnant mouse models. Protection was evaluated by chlamydial clearance in spleens, placentas and fetuses and the humoral response was analysed by measuring isotype-specific anti-MOMP antibodies.
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METHODS |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of the momp gene.
To produce the vaccinal vector, the momp gene was amplified by PCR and cloned into the vector pcDNA3.1 (Invitrogen) carrying the human cytomegalovirus immediate early promoter and the bovine growth hormone polyadenylation signal. The PCR was performed using chlamydial genomic DNA (80 ng) as template with dNTPs (200 µM each), specific primers (1 µM each) and 1 U Pfu DNA polymerase (Promega) in a Perkin Elmer 9600 thermocycler with the following program: 94 °C for 5 min, 1 cycle; 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 containing an EcoRI site (underlined) and Kozak sequence (bold) in the forward primer (MOMP-FW; 5'-CTCGAATTCACCATGAAAAAACTCTTGAAATCG-3') and an XhoI site (underlined) in the reverse primer (MOMP-RV; 5'-CT CCTCGAGTTAGAATCTGAATTGAGCATTC-3'). The resulting fragment was inserted into the vector pcDNA3.1 after linearization by EcoRI/XhoI double digestion to generate pcDNA3.1 : : MOMP. The plasmid pcDNA3.1 : : MOMP and the pcDNA3.1 control plasmid were purified after an overnight Luria–Bertani culture of recombinant Escherichia coli DH5
using the EndoFree Plasmid Mega kit (Qiagen). Plasmid DNA was dissolved at 1 µg µl-1 in endotoxin-free PBS (Sigma). The sequence of the vaccinal vector pcDNA3.1 : : MOMP was verified by DNA sequencing.
Immunization and challenge.
All the studies were carried out with 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 by an intraperitoneal injection of ketamine and xylazine (respectively 80 and 8 mg kg-1 body weight) and immunized with pcDNA3.1 : : MOMP plasmid by intramuscular injections (50 µg in each tibialis anterior). Mice were boosted in the same way at days 21 and 42. 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 4 x 104 p.f.u. of the live attenuated 1B vaccine at day 1. Groups of 10 and 20 mice were similarly immunized for evaluation of the protective effect of the DNA vaccine and respectively constituted the non-pregnant and pregnant groups. 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 4 x 104 p.f.u. C. abortus AB7. One group of pregnant mice 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 for the detection of anti-MOMP-specific antibodies before each DNA injection (days 0, 19, 41), just before the challenge infection (day 56) and at day 63. 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-MOMP antibodies was checked by ELISA as described previously (Héchard et al., 2002). The recombinant MOMP used as antigen was kindly provided by Dr R. Ashley (University of Edinburgh, UK) (Wyllie et al., 1999). Recombinant MOMP (100 µl), denatured by heating (100 °C for 10 min) and diluted in Mg2+-free, Ca2+-free PBS (pH 7.4) at a concentration of 1 µg ml-1, was used for coating microtitre plates (Nunc-Immuno Plate Maxisorp Surface) for 16 h at 4 °C. Sera used for ELISA were serially diluted in Mg2+-free, Ca2+-free PBS (pH 7.4), 0.05 % Tween 20; 1 : 200 for the less reactive serum or starting from 1 : 200 to 1 : 800 for the more reactive serum.
Determination of antibody isotypes.
Determination of antibody isotypes was carried out using the ELISA as described above. Briefly, after incubation with dilutions (1 : 50) of the pooled sera of pcDNA3.1 : : MOMP-vaccinated mice collected at day 56 or 63, the isotypes were revealed by adding 100 µl of a 1 : 1000 dilution of peroxidase-conjugated anti-mouse IgG1 or IgG2a (Nordic Immunological Laboratories). The A414 was measured on a microplate reader (Multiskan RC, Thermo Labsystems) after 1 h of incubation with the substrate (ABTS) at room temperature.
In vitro neutralization assay in the presence of the complement.
The neutralization assay was done by the plaque-reduction assay using McCoy cells (Schachter et al., 1974). Pooled sera of PBS-, pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice were diluted in Mg2+-free, Ca2+-free PBS (pH 7.4) containing 10 % fetal bovine serum and 2.5 % guinea pig serum. Two hundred microlitres of these dilutions (10-1, 10-2 and 10-3) were added to an equal volume (200 µl) of an AB7 chlamydial suspension (104 p.f.u.), incubated for 2 h at 37 °C and then titrated on McCoy cells by the plaque assay method (Rodolakis & Chancerelle, 1977). The results are expressed for each serum as the percentage p.f.u. of the PBS control.
Evaluation of protection.
The protective effect of the DNA vaccine was assessed by the evaluation of the number of living offspring per litter at birth or 8 days after birth and the clearance of bacteria in spleens, placentas and fetuses. After challenge, the outcome of pregnant mice 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 for the virulence control as described previously (Rodolakis, 1983). All the non-pregnant mice and five mice of each group of pregnant mice were euthanized at day 63. The spleens from non-pregnant mice and the uteruses and spleens from pregnant mice were removed aseptically. All the 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 PBS-DEAE dextran (0.01 %) 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.
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 |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The isotypes of anti-MOMP antibodies of sera collected at days 56 and 63 from pcDNA3.1 : : MOMP-vaccinated mice were tested by ELISA. The predominant isotype was IgG2a (data not shown). No IgG1 was found.
The ability of the sera to neutralize the infectivity of the virulent AB7 strain of C. abortus was assessed by the plaque-reduction test. The sera of 1B-vaccinated mice had a substantial neutralizing effect (90 % at the 10-1 dilution; Fig. 2). while the sera of pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice respectively had a modest neutralizing effect (60 %) and a non-significant neutralizing effect (20 %).
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Efficacy of DNA immunization on pregnancy
As expected, the number of live newborn mice observed in the 1B group was not significantly different from the number of live newborns observed in the gestation control group (P > 0.05), but it was significantly higher (P < 0.01) than the number monitored in the virulence group (Fig. 3). The numbers of living offspring at birth and 8 days after birth for the gestation control and 1B groups were not significantly different. For the virulence group and the pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice, the number of living offspring at birth was more than at 8 days after birth, but this difference was not significant. The numbers of live newborn in the virulence, pcDNA3.1 and pcDNA3.1 : : MOMP groups were not significantly different (P > 0.05), but were significantly lower (P < 0.05) than in the gestation control and 1B groups. Therefore, pcDNA3.1 : : MOMP-vaccinated mice were not protected against abortion due to C. abortus AB7 challenge.
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Protection was also evaluated at a placental and fetal level by titration of C. abortus in these organs. Chlamydial titres in placentas were higher than in fetuses (P < 0.001). As expected, chlamydial titres in the placentas and fetuses of 1B-vaccinated mice were significantly lower (P < 0.01) than in the virulence group (Fig. 4). 1B-vaccinated mice were consequently protected against chlamydial infection. Chlamydial titres in the placentas of pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice were not significantly different from those of the virulence group (P > 0.05). This would indicate that DNA vaccination did not elicit sufficient protection against placental colonization. Nevertheless, the fetuses of pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice were significantly less infected than those of the virulence group (P < 0.05) (Fig. 4). Thus, partial protection was observed in fetuses of 1B-, pcDNA3.1- and pc DNA3.1 : : MOMP-vaccinated mice.
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Clearance of C. abortus AB7 from the spleen
The induced protection was evaluated by splenic titration of C. abortus on pregnant and non-pregnant mice. As expected, chlamydial titres in spleens from non-pregnant or pregnant 1B-vaccinated mice were significantly lower (P < 0.001 and P < 0.01, respectively) than in the virulence group. Thus, 1B-vaccinated mice were protected against chlamydial infection (Fig. 5). Chlamydial titres in the spleens from pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated, non-pregnant or pregnant mice were not significantly different from those from mice of the virulence group (P > 0.05). These results suggested that DNA vaccination did not induce splenic protection in pregnant or non-pregnant mice.
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DISCUSSION |
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Although DNA immunization with the momp gene elicited production of MOMP in vivo, since it induced specific anti-MOMP antibodies, it failed to reduce the abortive effect of C. abortus challenge. No protection was observed after MOMP DNA immunization in either the placental colonization or splenic colonization model. However, partial reduction of infection was observed in fetuses of pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice. As placentas were infected, fetuses were probably protected by a barrier effect, but this protective effect was not sufficient to allow the survival of living offspring. This non-specific and partial protection could be explained by the immunostimulating properties of the CpG sequences present in the vaccinal plasmid backbone (Krieg, 2000). These unmethylated CpG motifs are recognized as a danger signal by the vertebrate immune system and trigger a Th1-biased immune response with IgG2a antibody production. This hypothesis is therefore in agreement with the predominant IgG2a isotype of anti-MOMP antibodies found in our experiments. Analysis of cytokine profiles could have confirmed this Th1-like immune response. Buendía et al. (1999) reported high levels of interferon-
and interleukin-12 in the serum of mice infected with the C. abortus strains AB7 and 1B, suggesting strongly that effective resolution of the infection involves a Th1-like immune response.
The increased mortality of newborn mice observed in the pcDNA3.1 and pcDNA3.1 : : MOMP groups 8 days after birth could result from the loss of the nutritious properties of infected placentas, leading to frail living offspring, resulting in death even in the absence of fetal infection. Also, late contamination could be possible when the mice gave birth. Indeed, it has been reported that C. abortus was excreted when ewes gave birth and that they could therefore infect newborns as well as other ewes at this time (Rodolakis & Souriau, 1979). This hypothesis could be transposable in the mouse model, since the number of living offspring appeared to be higher at birth than 8 days after birth. We could also propose that the newborn of 1B-vaccinated mice would be protected by the transfer of maternal antibodies via the milk, since the sera of these mice showed neutralizing properties.
In contrast with the pooled sera of 1B-vaccinated mice, the sera of pcDNA3.1- and pcDNA3.1 : : MOMP-vaccinated mice failed to neutralize chlamydial penetration in cells. As protective anti-MOMP antibodies are known to be directed against conformational epitopes (De Sa et al., 1995), we could speculate that the MOMP DNA vaccine induced linear epitope-specific antibodies. Probably due to the weak humoral response induced by the DNA vaccination, we failed to detect either conformational or linear MOMP after immunoblotting on whole C. abortus extracts. Another observation came from the weak level of anti-MOMP antibodies induced by the MOMP DNA vaccine. Because induction of the immune response is known to be linked directly to protein expression following DNA immunization (Barry & Johnston, 1997), we supposed that MOMP expression in vivo was likely to be insufficient to induce a relevant level of anti-MOMP antibodies. This was consistent with the fact that the antibodies of pcDNA3.1 : : MOMP-vaccinated mice sera failed to detect MOMP by immunoblotting, a less sensitive technique compared with ELISA.
Different methods have been evaluated in an attempt to increase the immune response generated by DNA vaccines (Gurunathan et al., 2000). More particularly, the vaccination protocol could be improved, as has already been done in a C. trachomatis model (Dong-Ji et al., 2000), using a DNA prime and a protein boost strategy known to enhance both humoral immune response and protection (Song et al., 2000). In addition, it might be possible to amplify the protective immune response by co-administration of an immunoregulatory cytokine-encoding plasmid with the vaccinal plasmid (Gurunathan et al., 2000). A protein boost and the co-administration of cytokine-encoding plasmids can also be combined in order to achieve protection (Scheerlinck et al., 2001).
In summary, DNA vaccination with a plasmid vector encoding the momp gene of C. abortus elicited a weak humoral response, with the IgG2a isotype predominant. Despite the antigenic properties of MOMP, DNA vaccination failed to protect pregnant mice from abortion against C. abortus challenge. In contrast, fetuses from DNA-vaccinated mice were non-specifically and partially protected. Therefore, it would be of interest to optimize the vaccination protocol and to use genetic adjuvants including cytokines or co-stimulating molecule genes in order to enhance the immune response.
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Acknowledgments |
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Footnotes |
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Present address: Laboratoire d'Immuno-dermatologie, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Genève 4, Switzerland. 
Abbreviation: MOMP, major outer-membrane protein.
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TOPAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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) and tested individually by ELISA for titration of anti-MOMP-specific IgG antibodies. The mean A414 (+SEM) is shown for each time-point.
) vaccinated mice. Mouse sera collected at day 56 were diluted 10-1, 10-2 or 10-3 and then added to the chlamydial suspension. These solutions had been titrated and the results are expressed relative to the control.
