Protection against Leptospira interrogans Sensu Lato Challenge by DNA Immunization with the Gene Encoding Hemolysin-Associated Protein 1
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感染与免疫杂志 2005年第7期
Leptospira Medical and Molecular Bacteriology Unit, Ecole Nationale Veterinaire de Nantes, BP 40706, 44307 Nantes cedex 03, France
Virbac Laboratories, BP 27, 06511 Carros Cedex, France
Immuno-endocrinology Unit, ENVN/INRA/University, BP 40706, 44307 Nantes cedex 03, France
ABSTRACT
The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.
INTRODUCTION
Leptospirosis is a widespread human and animal disease caused by pathogenic leptospires. Leptospire infection in mammals is transmitted by contact with infected animals or by exposure to water, moist soil, or vegetation contaminated by the urine of shedding animals (3, 37, 41, 48). Although leptospirosis has a worldwide distribution, it is most common in tropical and rural areas (8, 9, 16, 20). This acute febrile disease results in hepatic and renal dysfunction and hemorrhagic disorders that can cause death in 5 to 10% of human cases (32, 39) and fatality rates that are even higher in dogs. In livestock, Leptospira infection is associated with abortion, stillbirth, infertility, milk drop syndrome, and occasionally death (24, 34, 52).
The medical and economic losses caused by such forms of the disease justify the use of Leptospira vaccines in animal populations and humans at risk. However, currently available vaccines enhance a lipopolysaccharide-directed immune response, which is serogroup specific. These bacterins therefore provide no cross-protection against the different serogroups of pathogenic leptospires (53). However, these vaccines do generally provide protection against lethal onset of the disease, but they do not prevent persistent shedding from infected animals (1, 4).
New vaccine strategies are thus needed for the prevention of leptospirosis. The aim of our previous work was to identify a common immunogenic protein, hemolysis-associated protein 1 (Hap1) (22, 23, 53), whose gene is shared by pathogenic Leptospira genomospecies but is not present in the saprophytic genomospecies (12a). Hap1 is an immunogenic protein in both humans and animals. Immunization with Hap1 (also known as LipL32 or LP32) expressed by an adenovirus has been shown to protect gerbils against a virulent Leptospira challenge. To avoid the use of an adenovirus vector for safety reasons, we first evaluated the clinical protection induced by DNA vaccination against lethal challenge and subsequently tested the effect of recombinant Hap1 (rHap1) immunization with various adjuvants.
The observation that DNA immunization is able to elicit protective immunity has fostered the development of a new generation of vaccines. DNA vaccines provide prolonged antigen expression, leading to amplification of the immune response, and they appear to offer several advantages, such as easy construction, a low cost of mass production, a high level of temperature stability, and the ability to elicit both humoral and cell-mediated immune responses (2, 25, 43).
The purposes of the present study were to investigate the protective effect of Hap1 mediated by DNA vaccination and to compare the results of such an immunization to the results obtained with rHap1 immunization. Briefly, gerbils were immunized against leptospirosis with plasmid vectors coding for the hap1 gene from serovar autumnalis leptospires [hap1(aut)] or the hap1 gene from serovar grippotyphosa leptospires [hap1(grip)] under the control of a cytomegalovirus enhancer-promoter or with rHap1 associated with different adjuvants. We found that DNA vaccination with the hap1 gene induces protection against a lethal challenge by Leptospira interrogans serovar canicola.
MATERIALS AND METHODS
Leptospira strains. (i) Microagglutination test. Leptospires were grown in EMJH enriched medium at 29°C (19) to a concentration of 109 leptospires/ml, as estimated by turbidimetry with a Hach apparatus calibrated as previously described (53). Typically, 100 turbidimetric units was equivalent to 2 x 108 to 5 x 108 leptospires/ml. L. interrogans Autumnalis (serovar autumnalis strain 32) was isolated from the liver of a dead dog. This strain, which was selected based on the acute clinical effects observed in infected dogs, lost its virulence following numerous subcultures.
L. interrogans Icterohaemorrhagiae (serovar copenhageni strain M20 and serovar icterohaemorrhagiae strain RGA) and L. interrogans Canicola (serovar canicola strain Hond Utrecht) were kindly provided by the national reference center at the Pasteur Institute (Paris, France).
(ii) Challenge. A virulent strain, L. interrogans Canicola, was kindly provided by the Pasteur Institute (Paris, France). The virulence of this strain was maintained by regular passages (twice a year) in laboratory gerbils.
(iii) Western blotting. Leptospires were cultured in EMJH enriched medium at 29°C and harvested when the concentration reached 100 turbidimetric units by centrifugation for 30 min at 15,000 x g. The bacteria were resuspended in Laemmli sample buffer containing 2% 2-mercaptoethanol, and after addition of a protease inhibitor (Roche), the supernatants were stored at –80°C.
Construction of Hap1(aut) and Hap1(grip) mammalian expression vectors: cloning. Plasmid DNA preparation and restriction enzyme digestion were performed under standard conditions (49). The genomic DNA of L. interrogans Autumnalis strain 32 (serovar autumnalis) was used as a template for PCR isolation of the target sequences. The sequence of the forward primer for hap1 was 5'-GCTCTAGAATGAAAAAACTTTCGATTTTGGC-3'. This primer has an 8-bp sequence (underlined) at the 5' end to create an XbaI site. The sequence of the reverse primer for hap1 was 5'-CGGGGTACCTTACTTAGTCGCGTCAGAA-3'. This primer has a sequence (underlined) at the 5' end to create a KpnI site. The 836-bp PCR product amplified from the genomic DNA of serovar autumnalis strain 32 was cloned by TA cloning in the PCRII.1 vector (Invitrogen Corp.), which generated PCRII.1-hap1(aut). Both strands of the DNA insert were sequenced (Act gene, France). PCRII.1-hap1(aut) was digested, and the 836 bp was ligated into the XbaI and KpnI sites of a Puc19 vector in which the EcoRI site was previously removed, generating puc19-hap1(aut). The sequence of Hap1(grip) (GenBank accession number AF121192) from a serovar grippotyphosa strain differs from the sequence of Hap1(aut) (GenBank accession number AF366366) by only one amino acid, so the puc19-hap1(grip) plasmid was constructed by directed mutation of PCRII.1-hap1(aut). The sequence of the forward primer for ApoI-hap1(grip) was 5'-GGTCTTTACAGAATTTCTTTCCCTACCTACAAAC-3'. This primer has a 9-bp sequence (underlined) corresponding to an ApoI site. The 207-bp PCR product amplified from the genomic DNA of Autumnalis strain 32 was cloned by TA cloning in the PCRII.1 vector (Invitrogen Corp.), which generated PCRII.1-Apo-hap1(grip). Both strands of the DNA insert were sequenced (Act gene, France). The ApoI/KpnI fragment prepared from PCRII.1-Apo-hap1(grip) was ligated into the ApoI/KpnI site of pUC19-hap1(aut), generating puc19-hap1(grip).
The HindIII/KpnI fragments prepared from pUC19-hap1(aut) and pUC19-hap1(grip) were ligated into the HindIII/KpnI site of a pcDNA3.1 eukaryotic expression vector (Invitrogen Corp.), generating pcDNA-hap1(aut) and pcDNA-hap1(grip), respectively. The pcDNA3.1 plasmid contains the cytomegalovirus immediate-early promoter region and the BGH polyadenylation site. Both strands of the DNA insert were sequenced (Act gene, France).
Another vector without any foreign sequence was used as a control. Large-scale plasmid production was performed with an endotoxin-free QIAGEN kit as previously described (12). Plasmid stocks were stored at –20°C in phosphate-buffered saline (PBS) without calcium. These plasmids were used for in vitro transfection and vaccination studies in gerbils.
Transfection and visualization of expressed antigens. To confirm that the various DNA constructs were functional and to determine the efficiency of protein expression, HEK293 cells were transfected with each plasmid [pcDNA-hap1(aut) or pcDNA-hap1(grip)] using the transfecting reagent jetPEI (Polyplus transfection) according to the manufacturer's protocol. Briefly, HEK293 cells were maintained in flasks in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% (vol/vol) heat-inactivated fetal bovine serum. When 60 to 75% confluence was reached, the cells were transfected with 15 μg of plasmid DNA precondensed with jetPEI in serum-free Dulbecco's modified Eagle's medium. Transfection with an empty control vector (pcDNA3.1) was performed as a negative control. Forty-eight hours after transfection and after the protease inhibitor treatment (Roche), the transfected cells were removed from the plates by scraping, washed with PBS, resuspended in lysis buffer (PBS containing 4% [vol/vol] NP-40), and put on ice for 30 min.
SDS-polyacrylamide gel electrophoresis and Western blot analysis. Equal amounts of lysates or supernatants (transfected HEK293 cells or leptospires) were incubated in Laemmli sample buffer and boiled for 10 min. Twenty microliters of a sample was loaded onto a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, as previously described (33), and subjected to electrophoresis. One hundred nanograms of rHap1 (11) was used as a control. Following SDS-polyacrylamide gel electrophoresis, proteins were transferred onto nitrocellulose membranes (Bio-Rad) using a semidry system in Tris buffer (48 mM Tris, pH 9.2, 39 mM glycine, 1.3 mM SDS, 20% methanol). After overnight blocking at 4°C with 3% bovine serum albumin in 10 mM Tris (pH 8)-150 mM NaCl-0.05% Tween 20, proteins were selectively identified by Western blotting using a rabbit antileptospire serum prepared as previously described, followed by an alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG; Etablissement Franais du Sang) or a mouse monoclonal antibody against rHap1 (12) and then by alkaline phosphatase-conjugated goat anti-mouse IgG (Interchim).
Animals. Six- to twelve-week-old Mongolian gerbils (Meriones unguiculatus) bred in our laboratory were used in this study. The animals were managed by using welfare animal practices.
rHap1 immunization. For each immunization, gerbils were anesthetized by intraperitoneal injection of 0.2 mg/kg of ketamine and 0.08 mg/kg of xylazine.
(i) Freund adjuvant. Fifteen animals received a subcutaneous injection of 50 μg of rHap1 with complete Freund adjuvant in 500 μl. Two and four weeks later, the gerbils received a subcutaneous injection of 50 μg of rHap1 with incomplete Freund adjuvant in 500 μl. The controls (n = 15) received the same volume of the appropriate adjuvant mixed with PBS.
(ii) Aluminum hydroxide with QS21. rHap1 was incubated overnight at 4°C with aluminum hydroxide. QS21 (Superfoss biosector) was then added at a final concentration of 20 μg/ml. Fifteen animals received three subcutaneous injections (at 2-week intervals) consisting of 50 μg of rHap1 with QS2l and aluminum hydroxide. The controls (n = 15) received the same volume of the adjuvant mixed with PBS.
DNA immunization. For each immunization, gerbils were anesthetized by intraperitoneal injection of 0.2 mg/kg of ketamine and 0.08 mg/kg of xylazine. All animals received two intramuscular injections (with a 3-week interval between injections) of 100 μl of cardiotoxin (0.01pmol; Latoxan) in the left quadriceps. Five days after each cardiotoxin injection, the immunized animals received two intramuscular injections of 100 μg of vector containing the hap1(aut) or hap1(grip) gene in the left quadriceps. The controls (n = 20) received an empty DNA plasmid in the same conditions.
Challenge. (i) Recombinant protein immunization challenge. Two weeks after the second immunization, all gerbils were challenged by intraperitoneal injection. Animals in the groups immunized with complete Freund adjuvant and with QS21 received 107 and 108 leptospires, respectively, in 0.5 ml from a fresh culture of the virulent organism L. interrogans sensu stricto serovar canicola. The gerbils were observed daily, and the mortality rate was recorded for 30 days after the challenge. The surviving animals were sacrificed after 30 days for serum analysis by an enzyme-linked immunosorbent assay (ELISA).
(ii) Plasmid immunization assay. Three weeks after the second vaccination of plasmid DNA, all gerbils were challenged by intraperitoneal injection. Each animal received 107 leptospires in 0.5 ml from a fresh culture of the virulent organism L. interrogans sensu stricto serovar canicola. The gerbils were observed daily, and the mortality rate was recorded for 28 days after challenge. The surviving animals were sacrificed after 28 days for necropsy and to obtain blood samples used for serological analysis. Necropsies were performed with special attention to liver and kidney lesions.
Serum sampling. Blood sampling was done on days 0, 21, 42, and 70 with anesthetized animals.
Serological analysis. Sera of the animals were pooled because we collected the smallest blood sample so that we did not modify the health status of the small animals before challenge. Specific serum antibodies were measured by ELISA. Briefly, 96-well microtiter plates (Nunc) were coated overnight at 4°C with 4 μg of an rHap1fragment. The plates were then washed three times with TE-t (50 mM Tris, pH 7.6, 50 mM EDTA, 0.05% Tween 20) and blocked with TE-t containing 1% (wt/vol) gelatin at 37°C for 60 min. One hundred microliters of diluted serum (1:50 to 1:2,000) was incubated at 37°C for 60 min. After five washes, horseradish peroxidase-conjugated rabbit anti-gerbil IgG (1:1,000; Bioatlantic, France) was added and incubated for 30 min at 37°C. Finally, the plates were washed five times, and the chromogenic substrate 3,3'-5,5'-tetramethylbenzidine was added. After 10 min, the reaction was stopped by adding 2 M H2SO4. The optical density at 405 nm was measured with a Labsystems Multiskan MCC/340.
RESULTS
In vitro expression of Hap1 in mammalian cells. Significant expression of Hap1 was demonstrated for each of the plasmid constructs [pcDNA-hap1(aut) and pcDNA-hap1(grip)] by transient transfection of HEK293 cells, followed by Western blot analysis. The results of the Western blot analysis of Hap1 levels 48 h after transfection with the hap1 gene are shown in Fig. 1. As Fig. 1A shows, the extracts of cells transfected with pcDNA-hap1(aut) and pcDNA-hap1(grip) showed the presence of Hap1 when they were incubated either with the monoclonal antibody against rHap1 or the rabbit anti-leptospire serum (data not shown). Both constructs directed high-level expression of a protein with an apparent molecular mass of 31 kDa, which was equivalent to the molecular mass of the Hap1 expressed in the supernatant of a serovar autumnalis leptospire culture (Fig. 1B, lane 5). No band was detected for the pcDNA3.1 plasmid-transfected cells used as a negative control after incubation with both antibody preparations.
The following two positive controls were used: rHap1 corresponding to the recombinant Hap1 containing an N-terminal six-His tag produced in Escherichia coli (11) and the supernatant of a culture of pathogenic leptospires in which secreted proteins were expressed. Western blot analysis of the rHap1 showed three bands between 28 and 32 kDa, whereas a single band at 31 kDa was detected in the pathogenic leptospire culture supernatant (Fig. 1, lanes 1 and 4), as revealed with a monoclonal antibody against rHap1. Western blot analysis with rabbit antileptospire serum gave the same results for rHap1, whereas three bands at 25, 32, and 65 kDa were observed with a cell-free leptospire supernatant; these bands corresponded to several antigenic proteins secreted by leptospires into the medium (data not shown).
Antibody response to rHap1 immunization. The IgG humoral immune response of the gerbils immunized with rHap1 and either Freund adjuvant or aluminum hydroxide and QS21 was analyzed by an ELISA against the recombinant protein itself (Fig. 2). No rHap1 antibody was detected in the controls (groups 2 and 4) before challenge. After the first injection, the anti-rHap1 response increased rapidly in groups 1 and 3, reaching a plateau that was boosted neither by a second injection nor after the challenge. Moreover, there was no significant decrease in the humoral response throughout the experiment. No significant difference was observed between groups 1 and 3. The challenge induced a strong increase in the humoral response against rHap1 in the surviving gerbils in each control group (groups 2 and 4).
Antibody response to plasmid vector-mediated immunization. The humoral immune response of the gerbils immunized with either pcDNA-hap1(aut) or pcDNA-hap1(grip) was analyzed by IgG ELISA against the same rHap1 protein (Fig. 3). No antibody was detected in the control group (group 7) before challenge. In groups 5 [pcDNA-hap1(aut)] and 6 [pcDNA-hap1(grip)], a weak antibody response was observed after the first immunization, which was boosted by the second immunization. The challenge induced a strong immune response against rHap1 in the survivors in the control group (group 7) and an increase in the immune response in group 5. No significant difference was observed between groups 5 and 6.
Protective effect against challenge after rHap1 immunization. Gerbils were challenged with L. interrogans serovar canicola 2 weeks after the third protein injection, and mortality rates were recorded 30 days after the challenge (Table 1). No lesions were observed at necropsy (data not shown) in the surviving animals at the end of the experiment (30 days postchallenge). It is worth noting that in previous experiments (12, 53) the mortality rate for the same challenge dose was significantly lower in groups receiving Freund adjuvant than in the absolute control group that received PBS. Thus, a comparison was made for each immunized group and the control group that received the same adjuvant.
During the trial, the mortality rates of the vaccinated and control groups were not significantly different (Table 1). No protection was observed. Furthermore, the delays in the onset of death in the immunized groups and the corresponding control groups were identical.
Protective effect against challenge after DNA immunization. Gerbils were challenged with L. interrogans serovar canicola 2 weeks after the second plasmid injection, and the mortality rates were recorded 30 days after the challenge (Table 2 and Fig. 4). No lesions were observed at necropsy (data not shown) in the surviving animals at the end of the experiment (30 days postchallenge). The results of a statistical analysis of mortality incidence (log rank test) are shown in Table 3. During the trial, the mortality rates for animals vaccinated with recombinant plasmid pcDNA-hap1(aut) and animals vaccinated with pcDNA-hap1(grip) were not significantly different (9 of 15 animals versus 9 of 15 animals). In the trial, 9 of 15 pcDNA-hap1(aut)-vaccinated animals survived, compared with 7 of 20 controls (P < 0.02). The same results were observed for group 2 [for pcDNA-hap1(grip), 9 of 15 animals versus 7 of 20 animals (P < 0.04)]. The survival rate of gerbils vaccinated with pcDNA-hap1(aut) and pcDNA-hap1(grip) was significantly higher than that of the controls (P < 0.04). Combined analysis of the results for groups 1 and 2 showed significant protection with plasmid-expressed hap1 30 days after challenge (P < 0.003).
DISCUSSION
Our previous work showed that Hap1 induces significant protection against a leptospiral challenge in the gerbil model (11). This protection was observed only when the protein was expressed by adenoviral vectors. In human and veterinary medicine, vaccines need to overcome a large number of hurdles, including the cost of production, the ease of delivery, and safety (5). DNA vaccination has recently been tested for many pathogens and is a promising approach (25). The aim of this study was to investigate the protective effect of DNA vaccination with the Hap1-encoding gene from L. interrogans serovars autumnalis and grippotyphosa in a gerbil model challenged with serovar canicola and to evaluate the protection by the recombinant protein associated with different adjuvants.
Hemolysin-associated protein has been described as a secreted protein (35; M. J. Kim, K. A. Kim, S. J. Eom, S. C. Park, and Y. J. Lee, Meet. Int. Leptospirosis Soc. 1996, section F, 1996) with hemolytic activity for erythrocytes from humans, dogs (12), and sheep (35). The presence of Hap1 in the supernatant of leptospire cultures was demonstrated by Western blotting with a rabbit polyclonal serum against this protein. Furthermore, using a rabbit serum raised against live leptospires, we observed in the same supernatant other bands corresponding to sphingomyelinases A (26 kDa), B (63 kDa), and C (mature protein at 41 kDa) (20, 51), demonstrating that the signal observed by the monoclonal antibody against Hap1 was not due to degradation of leptospires in the culture. Protein secretion by L. interrogans has been analyzed by Zuerner et al. (58), who obtained similar results using radiolabeling. This protein is an extracellular protein, but its membrane expression is transient during secretion by living pathogenic leptospires; it is thought that it is not a structural outer membrane protein, as previously described (27). These data could explain (i) the results of a proteomics analysis of the Leptospira outer membrane in which 13 bands of Hap1 were found on a two-dimensional electrophoresis gel (the protein was identified by mass spectrometry), whereas 1 to 3 bands were identified for the other outer membrane proteins with a major band corresponding to the mature protein (15) and (ii) the atypical signal peptidase cleavage signal of Hap1, which differs from the signals of other leptospiral outer proteins (the putative signal peptide was defined by a predictive program because the protein possesses an N terminus blocked to Edman sequencing [26]). In another analysis, purified rHap1 which was blocked at the N terminus was subjected to mass spectrometry analysis in order to determine the signal peptide, and it had a different sequence (12) than the putative signal peptide described by Haake et al. (27). Purification of native Hap1 from a cell-free culture supernatant and analysis of this protein by mass spectrometry are essential steps for defining the signal peptide of this protein (10) and for explaining the results described below.
The direct protective effect of rHap1 obtained from serovar autumnalis and produced in E. coli against lethal disease was tested after a serovar canicola challenge. Different adjuvants that are known to elicit Th1 or Th2 immune responses (38) were used for these assays performed with the recombinant protein. Although immunizations with rHap1 elicited a strong antibody response, no clinical protection was observed after rHap1 immunization whatever adjuvant was used. Therefore, it seems likely that the mode of production or extraction of this recombinant protein and/or its presentation to the immune system was unsuitable for eliciting a protective immune response, while antibody production was elicited. To answer part of this question, the secreted Hap1 would need to be purified from the supernatant of a virulent leptospire culture and not extracted from the cell pellet. In fact, as determined by E. coli production, the recombinant protein (rHap1) was present both in bacteria and in the culture medium. Western blot analysis with antihistidine antibody of the bacterial lysate identified three bands between 28 and 32 kDa, whereas a single band at 31 kDa was detected in the culture medium (11). Furthermore, the protein expressed by the adenovirus, which induced a protective effect against the leptospire challenge, was present as a single band on the Western blot as the native Hap1 expressed in the supernatant of the culture (12). The protection potentially induced by this natural protein required characterization of this protein and comparison with the recombinant protein, and its role in the pathogenesis of leptospirosis has to be clarified.
Hap1 of L. interrogans serovar lai (GenBank accession number AAB68646) and Hap1 of serovar autumnalis (GenBank accession number AF366366) were found to be identical. Comparison of the Hap1 of L. interrogans serovar lai or autumnalis with the Hap1 of Leptospira kirschneri serovar grippotyphosa (GenBank accession number AF12192) revealed the same amino acid sequence, except for a proline residue (residue 215) which could play a role in the protein conformation (57) and consequently in its biological activities (44, 56). For example, the listeriolysin Hlys secreted by Listeria monocytogenes lost its hemolytic activity when only one amino acid in its sequence was changed (29).
We thus decided to evaluate the protection conferred by both plasmid-expressed Hap1(aut) and Hap1(grip). The gene expressed by the plasmid corresponded to the integral Hap1 with no His tag at the C terminus, which was the case for the adenovirus construct. Immunization with pcDNA-hap1(aut) and pcDNA-hap1(grip) induced significant protection against a serovar canicola challenge. These data show that the proline mutation in the amino sequence of the protein has no effect in terms of protection. This fact is essential for the development of a vaccine able to protect against each strain of pathogenic leptospires. The survival rate of gerbils vaccinated with pcDNA-hap1 was significantly higher than that of controls. Taking into account both experiments [pcDNA-hap1(aut) and pcDNA-hap1(grip)] improved the significance of the results from P < 0.04 and P < 0.01 to P < 0.003, indicating that the protective effect occurred throughout both experiments. These results are in accordance with those obtained previously using the adenovirus vector (11); we found the same significance (P < 0.01) for the protective effect observed against a Leptospira challenge after immunization with plasmid or adenovirus expressing Hap1(aut). The protective effect of immunization with Hap1 expressed by a plasmid vector was therefore as great as the protective effect of Hap1 expressed by the adenovirus vector.
As previously demonstrated for many genes (including those coding for proteins targeted to the cell nucleus), plasmid vectors can elicit not only T-cytotoxic responses but also a strong antibody response against the foreign gene product (18, 42), such as the adenovirus vector (30). It is noteworthy that the eukaryotic vectors can be used not only for intracellular bacteria but also for extracellular bacterial diseases (25, 47, 54). The presence of pathogenic leptospires in the intracellular compartment has already been demonstrated in vitro. Monocyte/macrophage-like and Vero cells were shown to be permissive host cells for virulent leptospire invasion (40). Moreover, Barocchi et al. (6) showed that Madin-Darby canine kidney cells were permissive for pathogenic leptospires but not for saprophytic leptospires. However, no data have confirmed the presence or persistence and multiplication of leptospires in cells in vivo. L. interrogans sensu lato is sensitive to currently used antibiotics, but persistence of leptospires in biological fluids has been observed after treatment with beta-lactams (40). It was guessed that leptospires could have an intracellular phase during which they are protected from antibiotics and the host immune system. Recent data showed that Th1 responses play an important role in the protective effect against disease. L. interrogans could activate and T-cell populations (31), and the roles of these populations in host defense and the pathology of leptospirosis have been investigated. Leptospire infection has been shown to induce a type 1 response comprising CD4 and T lymphocytes; however, although this response was too weak to prevent the establishment of chronic infection (46), a vaccine composed of dead Leptospira together with an aluminum hydroxide adjuvant induced a potent Th1-type cellular response in cattle which was associated with protective immunity to serovar hardjo infection (45, 13). The use of adjuvant-containing rabies-leptospirosis vaccines in dogs elicits a stronger immunogenic response than the use of leptospiral bacterins alone elicits (unpublished data). Follow-up studies are required to define the mechanisms of the protective effect resulting from pcDNA-hap1 immunization. Determination of the relative contributions of cellular immunity and humoral immunity observed in our study would provide insight into how immunity could be improved. Moreover, determination of the immune mechanism and the location of the immunoprotective epitopes would make it possible to use other types of immunization and to understand why immunization by the recombinant protein was unable to induce any significant protection against a Leptospira challenge.
It has been suggested that hemolysins (17, 50), particularly leptospiral hemolysins (7, 51, 55), play an important role in virulence and pathogenesis. Hap1 is produced only by pathogenic leptospires (21), which might reflect its role in this virulence. The protective effect induced by pcDNA-hap1(aut) and pcDNA-hap1(grip) immunization inhibits the virulent activity of the pathogenic strains, but the mechanism has to be explained. Moreover, the assay showed that this protein is a powerful immunogen, and the results are in agreement with the results for other bacteria for which hemolysins have been used as the main vaccine components (28, 29), particularly by plasmid vaccination (14).
In conclusion, our results show that a cross-protective effect with pathogenic strains of Leptospira was shared by Hap1 mediated by a plasmid vector. This could be helpful for designing and developing new generations of vaccines.
ACKNOWLEDGMENTS
This work was supported by the French Ministry of Agriculture and Virbac Laboratory (France).
We thank B. Blanchet for assistance with experimental work. M. Hebben is thanked for suggestions during the course of this work.
Present address: Department of Biology, Washington University, 1 Brookings Dr., Campus Box 1137, St. Louis, MO 63130.
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Virbac Laboratories, BP 27, 06511 Carros Cedex, France
Immuno-endocrinology Unit, ENVN/INRA/University, BP 40706, 44307 Nantes cedex 03, France
ABSTRACT
The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.
INTRODUCTION
Leptospirosis is a widespread human and animal disease caused by pathogenic leptospires. Leptospire infection in mammals is transmitted by contact with infected animals or by exposure to water, moist soil, or vegetation contaminated by the urine of shedding animals (3, 37, 41, 48). Although leptospirosis has a worldwide distribution, it is most common in tropical and rural areas (8, 9, 16, 20). This acute febrile disease results in hepatic and renal dysfunction and hemorrhagic disorders that can cause death in 5 to 10% of human cases (32, 39) and fatality rates that are even higher in dogs. In livestock, Leptospira infection is associated with abortion, stillbirth, infertility, milk drop syndrome, and occasionally death (24, 34, 52).
The medical and economic losses caused by such forms of the disease justify the use of Leptospira vaccines in animal populations and humans at risk. However, currently available vaccines enhance a lipopolysaccharide-directed immune response, which is serogroup specific. These bacterins therefore provide no cross-protection against the different serogroups of pathogenic leptospires (53). However, these vaccines do generally provide protection against lethal onset of the disease, but they do not prevent persistent shedding from infected animals (1, 4).
New vaccine strategies are thus needed for the prevention of leptospirosis. The aim of our previous work was to identify a common immunogenic protein, hemolysis-associated protein 1 (Hap1) (22, 23, 53), whose gene is shared by pathogenic Leptospira genomospecies but is not present in the saprophytic genomospecies (12a). Hap1 is an immunogenic protein in both humans and animals. Immunization with Hap1 (also known as LipL32 or LP32) expressed by an adenovirus has been shown to protect gerbils against a virulent Leptospira challenge. To avoid the use of an adenovirus vector for safety reasons, we first evaluated the clinical protection induced by DNA vaccination against lethal challenge and subsequently tested the effect of recombinant Hap1 (rHap1) immunization with various adjuvants.
The observation that DNA immunization is able to elicit protective immunity has fostered the development of a new generation of vaccines. DNA vaccines provide prolonged antigen expression, leading to amplification of the immune response, and they appear to offer several advantages, such as easy construction, a low cost of mass production, a high level of temperature stability, and the ability to elicit both humoral and cell-mediated immune responses (2, 25, 43).
The purposes of the present study were to investigate the protective effect of Hap1 mediated by DNA vaccination and to compare the results of such an immunization to the results obtained with rHap1 immunization. Briefly, gerbils were immunized against leptospirosis with plasmid vectors coding for the hap1 gene from serovar autumnalis leptospires [hap1(aut)] or the hap1 gene from serovar grippotyphosa leptospires [hap1(grip)] under the control of a cytomegalovirus enhancer-promoter or with rHap1 associated with different adjuvants. We found that DNA vaccination with the hap1 gene induces protection against a lethal challenge by Leptospira interrogans serovar canicola.
MATERIALS AND METHODS
Leptospira strains. (i) Microagglutination test. Leptospires were grown in EMJH enriched medium at 29°C (19) to a concentration of 109 leptospires/ml, as estimated by turbidimetry with a Hach apparatus calibrated as previously described (53). Typically, 100 turbidimetric units was equivalent to 2 x 108 to 5 x 108 leptospires/ml. L. interrogans Autumnalis (serovar autumnalis strain 32) was isolated from the liver of a dead dog. This strain, which was selected based on the acute clinical effects observed in infected dogs, lost its virulence following numerous subcultures.
L. interrogans Icterohaemorrhagiae (serovar copenhageni strain M20 and serovar icterohaemorrhagiae strain RGA) and L. interrogans Canicola (serovar canicola strain Hond Utrecht) were kindly provided by the national reference center at the Pasteur Institute (Paris, France).
(ii) Challenge. A virulent strain, L. interrogans Canicola, was kindly provided by the Pasteur Institute (Paris, France). The virulence of this strain was maintained by regular passages (twice a year) in laboratory gerbils.
(iii) Western blotting. Leptospires were cultured in EMJH enriched medium at 29°C and harvested when the concentration reached 100 turbidimetric units by centrifugation for 30 min at 15,000 x g. The bacteria were resuspended in Laemmli sample buffer containing 2% 2-mercaptoethanol, and after addition of a protease inhibitor (Roche), the supernatants were stored at –80°C.
Construction of Hap1(aut) and Hap1(grip) mammalian expression vectors: cloning. Plasmid DNA preparation and restriction enzyme digestion were performed under standard conditions (49). The genomic DNA of L. interrogans Autumnalis strain 32 (serovar autumnalis) was used as a template for PCR isolation of the target sequences. The sequence of the forward primer for hap1 was 5'-GCTCTAGAATGAAAAAACTTTCGATTTTGGC-3'. This primer has an 8-bp sequence (underlined) at the 5' end to create an XbaI site. The sequence of the reverse primer for hap1 was 5'-CGGGGTACCTTACTTAGTCGCGTCAGAA-3'. This primer has a sequence (underlined) at the 5' end to create a KpnI site. The 836-bp PCR product amplified from the genomic DNA of serovar autumnalis strain 32 was cloned by TA cloning in the PCRII.1 vector (Invitrogen Corp.), which generated PCRII.1-hap1(aut). Both strands of the DNA insert were sequenced (Act gene, France). PCRII.1-hap1(aut) was digested, and the 836 bp was ligated into the XbaI and KpnI sites of a Puc19 vector in which the EcoRI site was previously removed, generating puc19-hap1(aut). The sequence of Hap1(grip) (GenBank accession number AF121192) from a serovar grippotyphosa strain differs from the sequence of Hap1(aut) (GenBank accession number AF366366) by only one amino acid, so the puc19-hap1(grip) plasmid was constructed by directed mutation of PCRII.1-hap1(aut). The sequence of the forward primer for ApoI-hap1(grip) was 5'-GGTCTTTACAGAATTTCTTTCCCTACCTACAAAC-3'. This primer has a 9-bp sequence (underlined) corresponding to an ApoI site. The 207-bp PCR product amplified from the genomic DNA of Autumnalis strain 32 was cloned by TA cloning in the PCRII.1 vector (Invitrogen Corp.), which generated PCRII.1-Apo-hap1(grip). Both strands of the DNA insert were sequenced (Act gene, France). The ApoI/KpnI fragment prepared from PCRII.1-Apo-hap1(grip) was ligated into the ApoI/KpnI site of pUC19-hap1(aut), generating puc19-hap1(grip).
The HindIII/KpnI fragments prepared from pUC19-hap1(aut) and pUC19-hap1(grip) were ligated into the HindIII/KpnI site of a pcDNA3.1 eukaryotic expression vector (Invitrogen Corp.), generating pcDNA-hap1(aut) and pcDNA-hap1(grip), respectively. The pcDNA3.1 plasmid contains the cytomegalovirus immediate-early promoter region and the BGH polyadenylation site. Both strands of the DNA insert were sequenced (Act gene, France).
Another vector without any foreign sequence was used as a control. Large-scale plasmid production was performed with an endotoxin-free QIAGEN kit as previously described (12). Plasmid stocks were stored at –20°C in phosphate-buffered saline (PBS) without calcium. These plasmids were used for in vitro transfection and vaccination studies in gerbils.
Transfection and visualization of expressed antigens. To confirm that the various DNA constructs were functional and to determine the efficiency of protein expression, HEK293 cells were transfected with each plasmid [pcDNA-hap1(aut) or pcDNA-hap1(grip)] using the transfecting reagent jetPEI (Polyplus transfection) according to the manufacturer's protocol. Briefly, HEK293 cells were maintained in flasks in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% (vol/vol) heat-inactivated fetal bovine serum. When 60 to 75% confluence was reached, the cells were transfected with 15 μg of plasmid DNA precondensed with jetPEI in serum-free Dulbecco's modified Eagle's medium. Transfection with an empty control vector (pcDNA3.1) was performed as a negative control. Forty-eight hours after transfection and after the protease inhibitor treatment (Roche), the transfected cells were removed from the plates by scraping, washed with PBS, resuspended in lysis buffer (PBS containing 4% [vol/vol] NP-40), and put on ice for 30 min.
SDS-polyacrylamide gel electrophoresis and Western blot analysis. Equal amounts of lysates or supernatants (transfected HEK293 cells or leptospires) were incubated in Laemmli sample buffer and boiled for 10 min. Twenty microliters of a sample was loaded onto a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, as previously described (33), and subjected to electrophoresis. One hundred nanograms of rHap1 (11) was used as a control. Following SDS-polyacrylamide gel electrophoresis, proteins were transferred onto nitrocellulose membranes (Bio-Rad) using a semidry system in Tris buffer (48 mM Tris, pH 9.2, 39 mM glycine, 1.3 mM SDS, 20% methanol). After overnight blocking at 4°C with 3% bovine serum albumin in 10 mM Tris (pH 8)-150 mM NaCl-0.05% Tween 20, proteins were selectively identified by Western blotting using a rabbit antileptospire serum prepared as previously described, followed by an alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG; Etablissement Franais du Sang) or a mouse monoclonal antibody against rHap1 (12) and then by alkaline phosphatase-conjugated goat anti-mouse IgG (Interchim).
Animals. Six- to twelve-week-old Mongolian gerbils (Meriones unguiculatus) bred in our laboratory were used in this study. The animals were managed by using welfare animal practices.
rHap1 immunization. For each immunization, gerbils were anesthetized by intraperitoneal injection of 0.2 mg/kg of ketamine and 0.08 mg/kg of xylazine.
(i) Freund adjuvant. Fifteen animals received a subcutaneous injection of 50 μg of rHap1 with complete Freund adjuvant in 500 μl. Two and four weeks later, the gerbils received a subcutaneous injection of 50 μg of rHap1 with incomplete Freund adjuvant in 500 μl. The controls (n = 15) received the same volume of the appropriate adjuvant mixed with PBS.
(ii) Aluminum hydroxide with QS21. rHap1 was incubated overnight at 4°C with aluminum hydroxide. QS21 (Superfoss biosector) was then added at a final concentration of 20 μg/ml. Fifteen animals received three subcutaneous injections (at 2-week intervals) consisting of 50 μg of rHap1 with QS2l and aluminum hydroxide. The controls (n = 15) received the same volume of the adjuvant mixed with PBS.
DNA immunization. For each immunization, gerbils were anesthetized by intraperitoneal injection of 0.2 mg/kg of ketamine and 0.08 mg/kg of xylazine. All animals received two intramuscular injections (with a 3-week interval between injections) of 100 μl of cardiotoxin (0.01pmol; Latoxan) in the left quadriceps. Five days after each cardiotoxin injection, the immunized animals received two intramuscular injections of 100 μg of vector containing the hap1(aut) or hap1(grip) gene in the left quadriceps. The controls (n = 20) received an empty DNA plasmid in the same conditions.
Challenge. (i) Recombinant protein immunization challenge. Two weeks after the second immunization, all gerbils were challenged by intraperitoneal injection. Animals in the groups immunized with complete Freund adjuvant and with QS21 received 107 and 108 leptospires, respectively, in 0.5 ml from a fresh culture of the virulent organism L. interrogans sensu stricto serovar canicola. The gerbils were observed daily, and the mortality rate was recorded for 30 days after the challenge. The surviving animals were sacrificed after 30 days for serum analysis by an enzyme-linked immunosorbent assay (ELISA).
(ii) Plasmid immunization assay. Three weeks after the second vaccination of plasmid DNA, all gerbils were challenged by intraperitoneal injection. Each animal received 107 leptospires in 0.5 ml from a fresh culture of the virulent organism L. interrogans sensu stricto serovar canicola. The gerbils were observed daily, and the mortality rate was recorded for 28 days after challenge. The surviving animals were sacrificed after 28 days for necropsy and to obtain blood samples used for serological analysis. Necropsies were performed with special attention to liver and kidney lesions.
Serum sampling. Blood sampling was done on days 0, 21, 42, and 70 with anesthetized animals.
Serological analysis. Sera of the animals were pooled because we collected the smallest blood sample so that we did not modify the health status of the small animals before challenge. Specific serum antibodies were measured by ELISA. Briefly, 96-well microtiter plates (Nunc) were coated overnight at 4°C with 4 μg of an rHap1fragment. The plates were then washed three times with TE-t (50 mM Tris, pH 7.6, 50 mM EDTA, 0.05% Tween 20) and blocked with TE-t containing 1% (wt/vol) gelatin at 37°C for 60 min. One hundred microliters of diluted serum (1:50 to 1:2,000) was incubated at 37°C for 60 min. After five washes, horseradish peroxidase-conjugated rabbit anti-gerbil IgG (1:1,000; Bioatlantic, France) was added and incubated for 30 min at 37°C. Finally, the plates were washed five times, and the chromogenic substrate 3,3'-5,5'-tetramethylbenzidine was added. After 10 min, the reaction was stopped by adding 2 M H2SO4. The optical density at 405 nm was measured with a Labsystems Multiskan MCC/340.
RESULTS
In vitro expression of Hap1 in mammalian cells. Significant expression of Hap1 was demonstrated for each of the plasmid constructs [pcDNA-hap1(aut) and pcDNA-hap1(grip)] by transient transfection of HEK293 cells, followed by Western blot analysis. The results of the Western blot analysis of Hap1 levels 48 h after transfection with the hap1 gene are shown in Fig. 1. As Fig. 1A shows, the extracts of cells transfected with pcDNA-hap1(aut) and pcDNA-hap1(grip) showed the presence of Hap1 when they were incubated either with the monoclonal antibody against rHap1 or the rabbit anti-leptospire serum (data not shown). Both constructs directed high-level expression of a protein with an apparent molecular mass of 31 kDa, which was equivalent to the molecular mass of the Hap1 expressed in the supernatant of a serovar autumnalis leptospire culture (Fig. 1B, lane 5). No band was detected for the pcDNA3.1 plasmid-transfected cells used as a negative control after incubation with both antibody preparations.
The following two positive controls were used: rHap1 corresponding to the recombinant Hap1 containing an N-terminal six-His tag produced in Escherichia coli (11) and the supernatant of a culture of pathogenic leptospires in which secreted proteins were expressed. Western blot analysis of the rHap1 showed three bands between 28 and 32 kDa, whereas a single band at 31 kDa was detected in the pathogenic leptospire culture supernatant (Fig. 1, lanes 1 and 4), as revealed with a monoclonal antibody against rHap1. Western blot analysis with rabbit antileptospire serum gave the same results for rHap1, whereas three bands at 25, 32, and 65 kDa were observed with a cell-free leptospire supernatant; these bands corresponded to several antigenic proteins secreted by leptospires into the medium (data not shown).
Antibody response to rHap1 immunization. The IgG humoral immune response of the gerbils immunized with rHap1 and either Freund adjuvant or aluminum hydroxide and QS21 was analyzed by an ELISA against the recombinant protein itself (Fig. 2). No rHap1 antibody was detected in the controls (groups 2 and 4) before challenge. After the first injection, the anti-rHap1 response increased rapidly in groups 1 and 3, reaching a plateau that was boosted neither by a second injection nor after the challenge. Moreover, there was no significant decrease in the humoral response throughout the experiment. No significant difference was observed between groups 1 and 3. The challenge induced a strong increase in the humoral response against rHap1 in the surviving gerbils in each control group (groups 2 and 4).
Antibody response to plasmid vector-mediated immunization. The humoral immune response of the gerbils immunized with either pcDNA-hap1(aut) or pcDNA-hap1(grip) was analyzed by IgG ELISA against the same rHap1 protein (Fig. 3). No antibody was detected in the control group (group 7) before challenge. In groups 5 [pcDNA-hap1(aut)] and 6 [pcDNA-hap1(grip)], a weak antibody response was observed after the first immunization, which was boosted by the second immunization. The challenge induced a strong immune response against rHap1 in the survivors in the control group (group 7) and an increase in the immune response in group 5. No significant difference was observed between groups 5 and 6.
Protective effect against challenge after rHap1 immunization. Gerbils were challenged with L. interrogans serovar canicola 2 weeks after the third protein injection, and mortality rates were recorded 30 days after the challenge (Table 1). No lesions were observed at necropsy (data not shown) in the surviving animals at the end of the experiment (30 days postchallenge). It is worth noting that in previous experiments (12, 53) the mortality rate for the same challenge dose was significantly lower in groups receiving Freund adjuvant than in the absolute control group that received PBS. Thus, a comparison was made for each immunized group and the control group that received the same adjuvant.
During the trial, the mortality rates of the vaccinated and control groups were not significantly different (Table 1). No protection was observed. Furthermore, the delays in the onset of death in the immunized groups and the corresponding control groups were identical.
Protective effect against challenge after DNA immunization. Gerbils were challenged with L. interrogans serovar canicola 2 weeks after the second plasmid injection, and the mortality rates were recorded 30 days after the challenge (Table 2 and Fig. 4). No lesions were observed at necropsy (data not shown) in the surviving animals at the end of the experiment (30 days postchallenge). The results of a statistical analysis of mortality incidence (log rank test) are shown in Table 3. During the trial, the mortality rates for animals vaccinated with recombinant plasmid pcDNA-hap1(aut) and animals vaccinated with pcDNA-hap1(grip) were not significantly different (9 of 15 animals versus 9 of 15 animals). In the trial, 9 of 15 pcDNA-hap1(aut)-vaccinated animals survived, compared with 7 of 20 controls (P < 0.02). The same results were observed for group 2 [for pcDNA-hap1(grip), 9 of 15 animals versus 7 of 20 animals (P < 0.04)]. The survival rate of gerbils vaccinated with pcDNA-hap1(aut) and pcDNA-hap1(grip) was significantly higher than that of the controls (P < 0.04). Combined analysis of the results for groups 1 and 2 showed significant protection with plasmid-expressed hap1 30 days after challenge (P < 0.003).
DISCUSSION
Our previous work showed that Hap1 induces significant protection against a leptospiral challenge in the gerbil model (11). This protection was observed only when the protein was expressed by adenoviral vectors. In human and veterinary medicine, vaccines need to overcome a large number of hurdles, including the cost of production, the ease of delivery, and safety (5). DNA vaccination has recently been tested for many pathogens and is a promising approach (25). The aim of this study was to investigate the protective effect of DNA vaccination with the Hap1-encoding gene from L. interrogans serovars autumnalis and grippotyphosa in a gerbil model challenged with serovar canicola and to evaluate the protection by the recombinant protein associated with different adjuvants.
Hemolysin-associated protein has been described as a secreted protein (35; M. J. Kim, K. A. Kim, S. J. Eom, S. C. Park, and Y. J. Lee, Meet. Int. Leptospirosis Soc. 1996, section F, 1996) with hemolytic activity for erythrocytes from humans, dogs (12), and sheep (35). The presence of Hap1 in the supernatant of leptospire cultures was demonstrated by Western blotting with a rabbit polyclonal serum against this protein. Furthermore, using a rabbit serum raised against live leptospires, we observed in the same supernatant other bands corresponding to sphingomyelinases A (26 kDa), B (63 kDa), and C (mature protein at 41 kDa) (20, 51), demonstrating that the signal observed by the monoclonal antibody against Hap1 was not due to degradation of leptospires in the culture. Protein secretion by L. interrogans has been analyzed by Zuerner et al. (58), who obtained similar results using radiolabeling. This protein is an extracellular protein, but its membrane expression is transient during secretion by living pathogenic leptospires; it is thought that it is not a structural outer membrane protein, as previously described (27). These data could explain (i) the results of a proteomics analysis of the Leptospira outer membrane in which 13 bands of Hap1 were found on a two-dimensional electrophoresis gel (the protein was identified by mass spectrometry), whereas 1 to 3 bands were identified for the other outer membrane proteins with a major band corresponding to the mature protein (15) and (ii) the atypical signal peptidase cleavage signal of Hap1, which differs from the signals of other leptospiral outer proteins (the putative signal peptide was defined by a predictive program because the protein possesses an N terminus blocked to Edman sequencing [26]). In another analysis, purified rHap1 which was blocked at the N terminus was subjected to mass spectrometry analysis in order to determine the signal peptide, and it had a different sequence (12) than the putative signal peptide described by Haake et al. (27). Purification of native Hap1 from a cell-free culture supernatant and analysis of this protein by mass spectrometry are essential steps for defining the signal peptide of this protein (10) and for explaining the results described below.
The direct protective effect of rHap1 obtained from serovar autumnalis and produced in E. coli against lethal disease was tested after a serovar canicola challenge. Different adjuvants that are known to elicit Th1 or Th2 immune responses (38) were used for these assays performed with the recombinant protein. Although immunizations with rHap1 elicited a strong antibody response, no clinical protection was observed after rHap1 immunization whatever adjuvant was used. Therefore, it seems likely that the mode of production or extraction of this recombinant protein and/or its presentation to the immune system was unsuitable for eliciting a protective immune response, while antibody production was elicited. To answer part of this question, the secreted Hap1 would need to be purified from the supernatant of a virulent leptospire culture and not extracted from the cell pellet. In fact, as determined by E. coli production, the recombinant protein (rHap1) was present both in bacteria and in the culture medium. Western blot analysis with antihistidine antibody of the bacterial lysate identified three bands between 28 and 32 kDa, whereas a single band at 31 kDa was detected in the culture medium (11). Furthermore, the protein expressed by the adenovirus, which induced a protective effect against the leptospire challenge, was present as a single band on the Western blot as the native Hap1 expressed in the supernatant of the culture (12). The protection potentially induced by this natural protein required characterization of this protein and comparison with the recombinant protein, and its role in the pathogenesis of leptospirosis has to be clarified.
Hap1 of L. interrogans serovar lai (GenBank accession number AAB68646) and Hap1 of serovar autumnalis (GenBank accession number AF366366) were found to be identical. Comparison of the Hap1 of L. interrogans serovar lai or autumnalis with the Hap1 of Leptospira kirschneri serovar grippotyphosa (GenBank accession number AF12192) revealed the same amino acid sequence, except for a proline residue (residue 215) which could play a role in the protein conformation (57) and consequently in its biological activities (44, 56). For example, the listeriolysin Hlys secreted by Listeria monocytogenes lost its hemolytic activity when only one amino acid in its sequence was changed (29).
We thus decided to evaluate the protection conferred by both plasmid-expressed Hap1(aut) and Hap1(grip). The gene expressed by the plasmid corresponded to the integral Hap1 with no His tag at the C terminus, which was the case for the adenovirus construct. Immunization with pcDNA-hap1(aut) and pcDNA-hap1(grip) induced significant protection against a serovar canicola challenge. These data show that the proline mutation in the amino sequence of the protein has no effect in terms of protection. This fact is essential for the development of a vaccine able to protect against each strain of pathogenic leptospires. The survival rate of gerbils vaccinated with pcDNA-hap1 was significantly higher than that of controls. Taking into account both experiments [pcDNA-hap1(aut) and pcDNA-hap1(grip)] improved the significance of the results from P < 0.04 and P < 0.01 to P < 0.003, indicating that the protective effect occurred throughout both experiments. These results are in accordance with those obtained previously using the adenovirus vector (11); we found the same significance (P < 0.01) for the protective effect observed against a Leptospira challenge after immunization with plasmid or adenovirus expressing Hap1(aut). The protective effect of immunization with Hap1 expressed by a plasmid vector was therefore as great as the protective effect of Hap1 expressed by the adenovirus vector.
As previously demonstrated for many genes (including those coding for proteins targeted to the cell nucleus), plasmid vectors can elicit not only T-cytotoxic responses but also a strong antibody response against the foreign gene product (18, 42), such as the adenovirus vector (30). It is noteworthy that the eukaryotic vectors can be used not only for intracellular bacteria but also for extracellular bacterial diseases (25, 47, 54). The presence of pathogenic leptospires in the intracellular compartment has already been demonstrated in vitro. Monocyte/macrophage-like and Vero cells were shown to be permissive host cells for virulent leptospire invasion (40). Moreover, Barocchi et al. (6) showed that Madin-Darby canine kidney cells were permissive for pathogenic leptospires but not for saprophytic leptospires. However, no data have confirmed the presence or persistence and multiplication of leptospires in cells in vivo. L. interrogans sensu lato is sensitive to currently used antibiotics, but persistence of leptospires in biological fluids has been observed after treatment with beta-lactams (40). It was guessed that leptospires could have an intracellular phase during which they are protected from antibiotics and the host immune system. Recent data showed that Th1 responses play an important role in the protective effect against disease. L. interrogans could activate and T-cell populations (31), and the roles of these populations in host defense and the pathology of leptospirosis have been investigated. Leptospire infection has been shown to induce a type 1 response comprising CD4 and T lymphocytes; however, although this response was too weak to prevent the establishment of chronic infection (46), a vaccine composed of dead Leptospira together with an aluminum hydroxide adjuvant induced a potent Th1-type cellular response in cattle which was associated with protective immunity to serovar hardjo infection (45, 13). The use of adjuvant-containing rabies-leptospirosis vaccines in dogs elicits a stronger immunogenic response than the use of leptospiral bacterins alone elicits (unpublished data). Follow-up studies are required to define the mechanisms of the protective effect resulting from pcDNA-hap1 immunization. Determination of the relative contributions of cellular immunity and humoral immunity observed in our study would provide insight into how immunity could be improved. Moreover, determination of the immune mechanism and the location of the immunoprotective epitopes would make it possible to use other types of immunization and to understand why immunization by the recombinant protein was unable to induce any significant protection against a Leptospira challenge.
It has been suggested that hemolysins (17, 50), particularly leptospiral hemolysins (7, 51, 55), play an important role in virulence and pathogenesis. Hap1 is produced only by pathogenic leptospires (21), which might reflect its role in this virulence. The protective effect induced by pcDNA-hap1(aut) and pcDNA-hap1(grip) immunization inhibits the virulent activity of the pathogenic strains, but the mechanism has to be explained. Moreover, the assay showed that this protein is a powerful immunogen, and the results are in agreement with the results for other bacteria for which hemolysins have been used as the main vaccine components (28, 29), particularly by plasmid vaccination (14).
In conclusion, our results show that a cross-protective effect with pathogenic strains of Leptospira was shared by Hap1 mediated by a plasmid vector. This could be helpful for designing and developing new generations of vaccines.
ACKNOWLEDGMENTS
This work was supported by the French Ministry of Agriculture and Virbac Laboratory (France).
We thank B. Blanchet for assistance with experimental work. M. Hebben is thanked for suggestions during the course of this work.
Present address: Department of Biology, Washington University, 1 Brookings Dr., Campus Box 1137, St. Louis, MO 63130.
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