New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31-to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

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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.

Scrambled hirudins consist of a collection of equilibrated isomers and serve as essential folding intermediates during the in vitro renaturation of hirudin (Chatrenet, B., and Chang, J.-Y. (1993) J. Biol. Chem. 268, 20988-20996). Ten fractions of scrambled hirudins have been isolated. Their disulfide structures were deduced from the analysis of thermolysin-digested peptides by amino acid sequencing and mass spectrometry. The results reveal 9 fractions of pure scrambled species, and, together, 11 species of scrambled structures have been identified. About all possible disulfide isomers of hirudin have been found to exist. The three native disulfides, Cys6-Cys14, Cys16-Cys28, and Cys22-Cys39, are detected in five different scrambled species and constitute 18% of the total disulfide bonds found in scrambled hirudins.

We have defined the molecular environment of a snake neurotoxin interacting with the high-and lowaffinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the highaffinity site, the probes on Lys-15 and Lys47 predominantly reacted with the high-affinity site of the AcChoR a subunit whereas the probe on Lys-51 reacted with the 8 subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the a chain and the I8 chain whereas those on Lys-15 and Lys-51 reacted with the y and 8 chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.Determination of protein-protein interactions is one of the most challenging issues in biology. Interacting domains can be identified by chemical modifications of selected residues. Such techniques have proven suitable for delineating functionally critical residues of curaremimetic toxins from snake venoms (1-11). The domain by which toxin-a, a neurotoxin present in venom of the spitting cobra Naja nigricollis (12) MATERIALS AND METHODS HPLC columns C18 p.Bondapak were purchased from Waters. Toxin-a from N. nigricollis was prepared from venom as described (12). Torpedo marmorata AcChoR-rich membranes were purified according to Saitoh et al. (15). The monoclonal antibody (Mal) was prepared and purified according to Boulain et al. (8). The concentration of toxin-a binding sites was determined using [3H]toxin-a as a radioactive tracer. All other chemicals were of the purest grade commercially available.The preparation and characterization of photoactivatable derivatives will be published in detail elsewhere. Briefly, 1 tmol of [3H]toxin-a (3 Ci/mmol; 1 Ci = 37 GBq) in 0.5 ml of 0.05 M sodium phosphate (pH 7) was incubated with 3 Amol of p-azidobenzoyl N-hydroxysuccinimide ester in 0.5 ml of acetonitrile for 3 hr at room temperature (10). The toxin derivatives were separated from excess reagent by filtration through Bio-Gel P-2 (1 cm x 18 cm) equilibrated in 10%6 (vol/vol) acetic acid and fractionated on a C18 reversed-phase HPLC column. Four major radioactive fractions were resolved. Their molar extinction coefficient determined ...