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
). However, even after six full genome sequences, molecular studies of multiple strains (1
), and several vaccine trials (5
) 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
). 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
Several studies have described LTB adjuvant efficiency when coupled (6
) or coadministered (15
) 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
). Protection with LipL32 has been shown by use of recombinant BCG, adenovirus, and naked-DNA delivery systems (3
), 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
). 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 () 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 () 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
). 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.