All proteins used in this study were identified in previous studies (14, 16, 23). The genomic DNA of L. interrogans was used as a template for amplification of the target sequences. PCR products were cloned into pAE (25), pQE30 (Qiagen), or pET100-D/TOPO (Invitrogen) plasmid vector. Those vectors contain a 6×His tag that is expressed fused to the recombinant protein. In general, primers were designed to include most of the target genes but not their highly hydrophobic signal sequences. They also included a restriction enzyme site to allow direct cloning of the PCR product. Full primer information is presented in Table 1. Recombinant plasmids were used to transform E. coli strains by electroporation, and E. coli organisms containing the constructs were then cultured at 37°C. Expression was induced by the use of IPTG (isopropyl-β-d-thiogalactopyranoside) at a 1 mM final concentration. Cells were harvested by centrifugation, resuspended in column buffer containing 8 M urea (no urea was used for soluble protein; see Table 1), and disrupted by sonication. His-tagged proteins were purified by affinity chromatography in a nickel (Ni⁺²)-charged Sepharose column. Columns containing bound protein were washed with 10 volumes of wash buffer containing 10 mM imidazole. His-tagged proteins were eluted from the column with elution buffer containing 250 mM imidazole. A dialysis procedure was used to remove urea and imidazole and to promote refolding of the recombinant proteins. Proteins in the final preparation were quantified by the Bradford (4) and bicinchoninic acid (BCA) (Pierce) methods (Table 1 contains further information regarding solubility, vectors, and protein size).

Female golden Syrian hamsters (4 to 5 weeks of age) were obtained from the animal house facility at the Federal University of Pelotas. The animals were immunized twice in the quadriceps muscle with the recombinant protein in an aluminum hydroxide solution (15%) on day 0 (zero) and day 14. All vaccine doses contained 60 μg of purified recombinant protein, with administration of a standard volume of 200 μl at a single injection site. Negative-control groups in all experiments were inoculated with a 200 μl of phosphate-buffered saline (PBS)–aluminum hydroxide (15%). Positive-control groups were immunized with 200 μl of a bacterin inoculum containing approximately 10⁸ leptospires per dose. All hamsters were challenged on day 28 (at age 8 to 9 weeks) with an intraperitoneal inoculum of 100 leptospires (~2 × the 50% lethal dose [LD₅₀]) of strain Fiocruz L1-130 (28) 14 days after the last immunization. The inoculum was produced from log-phase cultures and consisted of a 1-ml dose. Hamsters were monitored daily for clinical signs of leptospirosis and euthanized when clinical signs of terminal disease appeared.