Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

Abstract

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.

FIG. 1.

(A) Expression of Hap1 in HEK293 cells. Western blotting was performed with a monoclonal antibody against rHap1, which showed the expression of Hap1 48 h after transfection of HEK293 cells in culture with a recombinant plasmid. Lane 1, pellet of cells transfected with pcDNA3.1 (15 μg); lane 2, control (cell-free supernatant of a Leptospira biflexa culture); lane 3, pellet of cells transfected with pcDNA-hap1(aut) (15 μg); lane 4, pellet of cells transfected with pcDNA-hap1(grip) (15 μg); lane 5, control (cell-free supernatant of serovar autumnalis leptospire culture; lanes M, protein molecular weight marker. (B) Expression of Hap1 in leptospire culture. Western blotting was performed with a monoclonal antibody against rHap1. Lane 1, rHap1 (100 ng); lane 2, pellet of serovar autumnalis leptospire culture; lane 3, pellet of saprophyte leptospire culture (L. biflexa sensu stricto serovar patoc strain Patoc); lane 4, cell-free supernatant of serovar autumnalis leptospire culture; lane 5, cell-free supernatant of a saprophyte leptospire culture.

FIG. 2.

Anti-rHap1 ELISA antibody responses in subcutaneously immunized gerbils (SC) expressed as optical density (OD) with rHap1 plus Freund adjuvant (FA) (group 1), Freund adjuvant (Te: group 2), rHap1 with aluminum hydroxide and QS2l (group 3), and aluminum hydroxide and QS2l (Te: group 4), followed by intraperitoneal leptospiral challenge (C).

FIG. 3.

Anti-rHap1 ELISA antibody responses in intramuscularly immunized gerbils (IM) expressed as optical density (OD) with pcDNA-hap1(aut) (group 5), pcDNA-hap1(grip) (group 6), and pcDNA-emp (plasmid devoid of transgene) (group 7), followed by intraperitoneal leptospiral challenge (C). Each point corresponds to the pool of sera sampled at one time during the assay.

FIG. 4.

Survival curves for gerbils after intramuscular immunization with pcDNA-hap1(aut) (group 5), pcDNA-hap1(grip) (group 6), and pcDNA-emp (plasmid devoid of transgene) (group 7), followed by intraperitoneal leptospiral challenge.