Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination

Abstract

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.

FIG. 1

(Top) A restriction map of the 2.5-kb fragment containing the hap1 gene from L. interrogans serovar lai and the 2.5-kb EcoRI fragment containing the ompL1 gene from L. kirshneri serovar grippotyphosa (15) is shown. (Bottom) Southern blot of L. interrogans serovar autumnalis genomic DNA digested by four restriction enzymes (see restriction maps) and probed with probes A and B, corresponding to peptides A and B. Lanes 1, BglII; lanes 2, DraI; lanes 3, EcoRI; lanes 4, HindIII. M, molecular mass markers.

FIG. 2

Coomassie brilliant blue-staining of SDS-PAGE gels showing purified rHap1 (Hap1). M, molecular mass markers.

FIG. 3

Anti-adenovirus ELISA antibody responses in intramuscularly immunized animals (IM) with Ad-hap1 (●), Ad-ompL1 (▪), Ad-hap1 plus Ad-ompL1 (♦), and Ad-null (▴), followed by intraperitoneal heterologous challenge (C). Each point corresponds to the pool of sera sampled at each time point during assay 2.

FIG. 4

Anti-rHap1 ELISA antibody responses in intramuscularly immunized animals (IM) expressed as optical densities with Ad-hap1 (●), Ad-ompL1 (▪), Ad-hap1 plus Ad-ompL1 (♦), and Ad-null (▴), followed by intraperitoneal heterologous challenge (C). Each point corresponds to the pool of sera sampled at each time point during assay 2.

FIG. 5

Survival curves of gerbils after intramuscular immunization with Ad-hap1 (●), Ad-ompL1 (▪), Ad-hap1 plus Ad-ompL1 (♦), or PBS for assay 1 or Ad-null for assay 2 (▴), followed by intraperitoneal heterologous challenge.