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Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.
Spirochetes from the genus Leptospira are the causative agents of leptospirosis, a zoonosis with a worldwide distribution. Leptospirosis is recognized as an emerging infectious disease and affects humans and wild and domestic animals (1). Leptospires colonize the proximal renal tubules of carrier animals (34) and are shed in the urine. The disease is associated with direct or indirect contact with contaminated urine (1, 25). Due to the impacts on animal production and public health and the severity of the disease, an efficient prophylactic measure is urgently needed. Current vaccines against leptospirosis are whole-cell preparations that produce only short-term immunity, with adverse reactions due to both leptospiral lipopolysaccharide (LPS) and residual medium components (1). Furthermore, the protection conveyed by these whole-cell preparations is serovar specific, with limited or no cross-protection (10) among the more than 260 serovars of Leptospira reported (1). Therefore, an effective multiserovar vaccine against leptospirosis with no collateral effects remains a challenge.
Efforts to develop recombinant vaccines against leptospirosis have focused on outer membrane proteins (OMPs) (10). The most abundant protein in the entire leptospiral proteome is an outer membrane lipoprotein of 32 kDa, LipL32 (27), accounting for 75% of the outer membrane proteome (7). This protein can be considered a promising antigen for the development of a multiserovar vaccine. LipL32 is expressed in all pathogenic Leptospira spp., and it is highly conserved (19) and not expressed in the saprophytic L. biflexa (29). This protein binds to extracellular matrix components, as indicated by in vitro assays (22, 23) and crystal structure analyses (36). Moreover, LipL32 is expressed during mammalian leptospiral infection (18). Different immunization strategies that have been tested with LipL32 have shown some immune protection when administered with naked-DNA (4), recombinant adenoviral (3), and Mycobacterium bovis BCG (30) delivery systems. However, LipL32 produced no protection by recombinant subunit protein vaccination with either a Freund or aluminum hydroxide adjuvant (4, 26). These findings suggest that the immune protection induced by LipL32 is correlated with a modulation of the immune system.
The Escherichia coli heat-labile enterotoxin (LT), and its closely related homologue Vibrio cholerae cholera toxin (CT), consists of one A subunit with ADP-ribosyltransferase activity linked to five B subunits (8). The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is highly immunogenic upon systemic (6, 9, 15) and mucosal (14, 37) immunizations. Its adjuvant activity has been demonstrated with unrelated antigens, both coadministered (14, 15) and linked by chemical conjugation or genetic fusion (6, 14, 37), exhibiting no toxic effect (8). LTB has a pentameric structure that binds to the ubiquitously expressed monosialotetrahexosylganglioside (GM1-ganglioside) receptor on the surface of mammalian cells, and this binding is essential for adjuvant properties (8). Therefore, the aim of the present study was to assess the immune protection induced by recombinant LipL32 coadministered or coupled with recombinant LTB. Our findings reveal the protective potential of LipL32 and suggest a new vaccine against leptospirosis using LTB and LipL32.
L. interrogans serovar Copenhageni strain Fiocruz L1-130 was cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco Laboratories) at 29°C. The procedures for the maintenance of the culture and challenge experiments were conducted as previously described (33).
Three recombinant vectors were used in this study. Two of them had been previously constructed, pAE/ltb (16) and pAE/lipL32 (31), and one was generated as follows: the lipL32 coding sequence from L. interrogans serovar Copenhageni strain Fiocruz L1-130 was amplified by PCR from pAE/lipL32. The following primers were used: LipL32-For (5′-GGGGTACCGGCGGCGGTGGTCTGCCAAGCCT) and LipL32-Rev (5′-GGAATTCTTACTTAGTCGCGTCAGAAGC). After amplification, the 771-bp fragment was cut with the KpnI and EcoRI (New England BioLabs) restriction enzymes and cloned into pAE/ltb cut with the same enzymes. The KpnI restriction site was modified to allow the insertion of lipL32 in the correct reading frame of the ltb coding sequence. The forward primer was constructed to allow a 4-amino-acid (aa) linker/spacer between ltb and lipL32 (Gly-Thr-Gly-Gly). The resulting plasmid, pAE/ltb::lipL32, was confirmed by PCR and restriction digestion. The recombinant vectors pAE/ltb, pAE/lipL32, and pAE/ltb::lipL32 were used to transform E. coli BL21 Star(DE3) cells (Invitrogen). The 6×His-tagged recombinant LTB (rLTB), recombinant LipL32 (rLipL32), and rLTB::LipL32 proteins were expressed and purified by affinity chromatography as previously described (32).
Western blot characterization was conducted as described elsewhere previously (32). The antibodies used were anti-LipL32 monoclonal antibody (MAb) 1D9 (13), diluted 1:5,000; rabbit anti-cholera toxin IgG (Sigma-Aldrich), diluted 1:6,000; sera from a human leptospirosis patient (21), diluted 1:500; a goat IgG–anti-mouse Ig–peroxidase conjugate (Sigma-Aldrich), diluted 1:6,000; a goat IgG–anti-rabbit IgG–peroxidase conjugate (Sigma-Aldrich), diluted 1:6,000; and a rabbit IgG–anti-human Ig–peroxidase conjugate (Abcam), diluted 1:2,000.
The abilities of rLTB and rLTB::LipL32 to bind to GM1-ganglioside were determined by an enzyme-linked immunosorbent assay (ELISA) as previously described (16), with minimal modifications. Briefly, plates were coated with 100 ng/well of bovine GM1-ganglioside (Sigma-Aldrich), and after blocking, the plates were incubated with 100 ng/well of rLTB, rLipL32, rLTB::LipL32, or choleric toxin (Sigma-Aldrich). The plates were then incubated with anti-LipL32 MAb 1D9 diluted 1:5,000 or rabbit IgG anti-cholera toxin antibody diluted 1:6,000, followed by a goat IgG anti-mouse- or anti-rabbit IgG-peroxidase conjugate diluted 1:6,000, respectively. The reactions were revealed with O-phenylenediamine dihydrochloride (Sigma-Aldrich) and hydrogen peroxide (Sigma-Aldrich) and read at 492 nm. Wells with GM1 but without proteins and wells without GM1 but with proteins were used as controls.
Four- to five-week-old female Golden Syrian hamsters were individually identified and distributed into three treatment groups. Each treatment group was composed of five animals, and three independent experiments were conducted, for a total of 45 animals. Hamsters were inoculated in the quadriceps muscle with 60 μg of rLTB::LipL32 (coupled) or 16.5 μg of rLTB and 43.5 μg of rLipL32 (coadministered), and the control group received 16.5 μg of rLTB (control). This dose design was used to administer equal amounts of adjuvant and antigen as coupled and coadministered proteins. Each animal received two doses, administered at days 0 and 14. The animals were inoculated with a maximum of 300 μl per injection site. Serum samples were collected from each animal by phlebotomy of the retro-orbital venous plexus on the day before the first immunization (preimmune; day zero) and on the day before challenge (postimmune; day 34). The animals were manipulated in accordance with the guidelines and approval of the Federal University of Pelotas Ethics Committee in Animal Experimentation.
For the determination of the humoral immune response induced by rLipL32 coupled or coadministered with rLTB, the serum from each animal was serially diluted and tested by a recombinant LipL32 ELISA (30). A preliminary checkerboard analysis was performed to determine ideal antigen concentrations and primary and secondary antibody dilutions. Polystyrene microtiter plates were coated with 100 ng/well of rLipL32 diluted in carbonate-bicarbonate buffer (pH 9.6) at 4°C overnight. After three washes with phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBS-T), the serum from each animal, diluted 1:800 to 1:25,600, was added in triplicate and incubated for 1 h at 37°C. Following three washes with PBS-T, the reaction mixtures were incubated for 1 h at 37°C with a 1:3,000 dilution of a goat polyclonal anti-hamster IgG–peroxidase conjugate (Abcam). After five washes with PBS-T, the reactions were revealed with O-phenylenediamine dihydrochloride (Sigma-Aldrich) and hydrogen peroxide (Sigma-Aldrich). The color reaction was allowed to develop for 15 min and stopped by the addition of 25 μl of 4 N H2SO4 to the mixture, and the optical densities were read at 492 nm.
To determine vaccine-induced protection, the same animals used for the serological analysis were challenged 21 days after the second dose. The animals received an intraperitoneal injection of 102 cells of L. interrogans serovar Copenhageni strain Fiocruz L1-130 (5× the 50% lethal dose [LD50]) (30). Two additional control groups of five animals were included in each of the three independent experiments. Similar to the treatment groups, on days 0 and 14, one group received 300 μl of PBS (negative control), and another received homologous bacterin (108 cells in 300 μl of PBS). The hamsters were observed daily for mortality. Survivors were euthanized at 21 days postchallenge.
Statistical analyses for ELISAs were carried out with Student's t test. The Fisher exact test and log-rank test were used to determine significant differences in mortality and survival rates, respectively, among the experimental groups. P values of <0.05 were considered to be indicative of statistical significance. All analyses were carried out with GraphPad Prism 4 software (GraphPad Software).
The construction of the recombinant vector carrying the fusion gene was successful. The lipL32 coding sequence without its signal sequence was ligated onto the 3′ end of ltb. rLTB, rLipL32, and rLTB::LipL32 were expressed in E. coli BL21 Star(DE3) cells. Purified recombinant proteins were analyzed by SDS-PAGE (Fig. 1A). The apparent molecular masses were as expected for each protein: 13 kDa, 30 kDa, and 41 kDa for rLTB, rLipL32, and rLTB::LipL32, respectively. rLTB and rLTB::LipL32 were expressed as inclusion bodies and required the addition of the denaturing agent N-lauroyl-sarcosine for purification, while rLipl32 was expressed and purified as a soluble protein. The yield of purified proteins varied from 3 to 10 mg per liter of culture. The pentameric form of rLTB was easily identified when the sample was not heated before SDS-PAGE was performed (data not shown). The pentamerization of rLTB::LipL32 (205 kDa) was not visualized.
The antigenic characterization of recombinant proteins was performed by Western blot analysis with antibodies specific for rLTB (Fig. 1B) and rLipL32 (Fig. 1C). Recombinant LTB was recognized by anti-CT antibodies. This serum did not react with rLipL32. MAb 1D9, as well as human convalescent-phase sera, reacted with rLipL32. As expected, the fusion protein was recognized by all tested antibodies. The negative-control E. coli extract did not react with any antibody, and the L. interrogans whole-cell extract reacted with human convalescent-phase sera (Fig. 1D). The GM1-ELISA with the recombinant proteins (Fig. 2) revealed the GM1-binding affinity of rLTB and rLTB::LipL32. These proteins showed a binding affinity as high as that of commercial choleric toxin, while rLipL32 did not bind to GM1-ganglioside. The binding activity obtained for rLTB::LipL32 was the same when anti-LipL32 or anti-CT antibody was used.
In order to assess whether rLipL32 coupled or coadministered with rLTB was able to promote IgG anti-LipL32 antibody responses in hamsters, preimmune and postimmune sera from each animal were evaluated by an indirect ELISA with rLipL32 as the immobilized antigen. The mean absorbances of the sera from each experimental group are shown in Fig. 3. The highest titer (over 1:25,600) was observed for animals receiving two doses of rLTB::LipL32, significantly higher than those of the other groups (P < 0.01 at a titer of 1:25,600). The coadministration of rLTB and rLipL32 also induced high anti-LipL32 titers.
Animals were monitored for 21 days postchallenge for the occurrence of death. Three independent experiments were accomplished, and statistical analyses of lethality rates are shown in Table 1, while survival rates (which also consider days to death) are shown in Fig. 4. In the first experiment, three and two animals died in the groups treated with rLTB::LipL32 and rLTB plus rLipL32, respectively. In subsequent experiments, no death occurred in these groups. All animals receiving rLTB and PBS in experiment 1 died, while just two deaths in each subsequent experiment with rLTB and two and four deaths with the PBS treatment in experiments 2 and 3, respectively, were registered. The survival analyses showed statistically significant results (log-rank test) when either of the experimental groups (rLTB::LipL32 or rLTB plus rLipL32) was compared to control groups (PBS or rLTB) in the first experiment and in the grouped results. Furthermore, in the third experiment, both experimental groups were statistically different from the PBS-treated negative-control group; however, no statistically significant survival was observed for the second experiment. Regarding lethality, no significance (Fisher exact test) was found by the statistical analysis of each experiment alone. rLipL32 coadministered with rLTB protected 87% of the animals, which is statistically different from the 40% of animals protected in the rLTB group (P = 0.02). The group receiving the fusion protein rLTB::LipL32 had a combined level of protection of 80%, which is statistically different from the level of protection in the PBS group (P < 0.01). When survival or lethality was considered, no difference was observed between the rLTB- and PBS-treated groups. Similarly, no difference could be attributed to the groups treated with rLTB plus rLipL32, rLTB::LipL32, and bacterin among themselves.
A truly effective and cross-protective leptospirosis vaccine is yet to be developed. At present, the highest level of protection afforded by a recombinant vaccine was observed with the recombinant leptospiral immunoglobulin-like protein LigA from L. interrogans (10, 32). However, even after six full genome sequences, molecular studies of multiple strains (1, 25), and several vaccine trials (5, 11, 12) have been reported, LipL32 remains the most promising recombinant vaccine candidate. In this work, we assessed the immunogenic properties of recombinant LipL32 in different subunit preparations using LTB as an adjuvant. To our knowledge, this is the first time that LTB has been used with a leptospiral antigen and the first study to report significant protection afforded by LipL32 administered as a subunit vaccine.
Challenge experiments are the most reliable assays to measure vaccine effectiveness (32). LipL32 has been extensively studied, with promising results when vaccine vectors or naked DNA was used (3, 4, 30). However, studies that used purified protein have thus far failed to produce significant protection with either Freund's adjuvant (4), aluminum hydroxide (26), or aluminum hydroxide and QS21 saponin (4). Our results show high survival rates for animals receiving recombinant LipL32 coadministered with or coupled to an LTB adjuvant, representing significant protection compared to that for any of the control groups (log-rank test). The level of significance of the results would be even higher if there were no surviving animals in the control groups; however, survival of a few animals from negative-control groups challenged with L. interrogans strain L1-130 is not uncommon (2, 10–12, 30).
Several studies have described LTB adjuvant efficiency when coupled (6, 37) or coadministered (15, 37) with different antigens; however, few studies have compared these two delivery systems (37). Our study shows that the rLTB::LipL32 protein was capable of stimulating significantly higher antibody titers than those elicited by the coadministration of the rLTB and rLipL32 proteins. Similar results were found for LipL32 coupled to the B subunit of CT (CTB), which induced higher antibody titers than those elicited by treatment with recombinant CTB (rCTB) plus rLipL32 (20). The animals vaccinated with rLTB::LipL32 had the highest antibody titers among all groups, but no statistical difference in survival rates was observed between the rLTB::LipL32 and rLTB-plus-rLipL32 treatment groups. Furthermore, within the same group, surviving animals did not necessarily have the highest antibody titers (data not shown). The demonstration that protection against leptospirosis is not necessarily associated with antibody titers is important, since most studies aimed at finding effective immunogens have been based solely on antibody responses (17, 31). Protection with LipL32 has been shown by use of recombinant BCG, adenovirus, and naked-DNA delivery systems (3, 4, 30), which are effective stimulants of cellular immunity. Therefore, not only humoral immunity but also cell-mediated immunity seems to play an important role in protection against leptospirosis (28, 35). Some studies have shown that cattle vaccines stimulating cellular immune responses are able to provide protection, while those stimulating high antibody titers are not (38). This phenomenon is not well understood for other species. In our study, protection may have occurred because LTB presents powerful immunostimulatory and immunomodulatory effects, such as enhancing antigen presentation via both major histocompatibility complexes, activating the selective differentiation of lymphocytes, increasing the expression levels of activation markers on B lymphocytes, and influencing the maturation and activation of dendritic cells (8). However, these effects must still be assessed.
The Western blot data (Fig. 1) suggest that the recombinant proteins retained antigenic epitopes present on native proteins. In addition, rLTB::LipL32 and rLipL32 were identified in serum from human leptospirosis patients, indicating that the immune response induced by these proteins is able to recognize native LipL32. The GM1-ELISA (Fig. 2) data show that the fusion did not impair the binding affinity of LTB. Variable numbers of amino acids in spacers/linkers between subunits in fusion proteins have been tested, and most fusions reported were successful (24, 37). In a recent study, Chen and coworkers (6) reported that 10 but not 6 amino acids in the flexible linker between LTB and the antigen were necessary to induce prolonged protection against BCL1 lymphomas. Therefore, we believe that a linker longer than 4 amino acids in LTB::LipL32 could allow the further folding and/or pentamerization of the coupled protein, thus affording a higher level of protection against leptospirosis.
In this study, we described a leptospirosis vaccine using a recombinant LipL32 antigen and rLTB as an adjuvant. We showed that rLipL32 coadministered or coupled with rLTB is highly immunogenic and protects hamsters from lethal leptospirosis. Studies are being carried out to assess the optimum dose, protection against other serovars, and vaccine dynamics. This approach may result in a formulation that could replace traditional vaccines against leptospirosis.
We are grateful to Michele dos Santos and Kátia R. Pimenta Cardoso for technical assistance.
This work was supported by the CNPq (grant number 475540/2008-5). A.A.G., S.R.F., A.C.P.S.N., and M.Q.F. were supported by scholarships from CAPES.
Published ahead of print 29 February 2012