A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis

Abstract

The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.

Fig 1

(A to F) Indirect immunofluorescence detection of LigAni (A), LigBni (B), LigBrep (C), LigBct₁ (D), and LigBct₂ (E) proteins expressed in Vero cells, using anti-Lig polyclonal sera; the negative control (F) corresponds to Vero cells that were transfected with the empty pTARGET plasmid and incubated with anti-Lig polyclonal sera. Visualization was performed with a 40× objective on an Olympus BX71 fluorescence microscope. (a to f) Corresponding bright-field microscopic views of the Vero cells.

Fig 2

Antibody responses of hamsters immunized with Lig DNA vaccines, as measured by ELISA. Recombinant LigAni, LigBni, LigBrep, LigBct₁, and LigBct₂ proteins were used as antigens in the ELISA. The results are expressed as the mean absorbances for all of the animals in each group. OD₄₉₂, optical density at 492 nm. *, P < 0.05 compared to the group that received the empty pTARGET plasmid.

Fig 3

Survival times of hamsters immunized with Lig DNA vaccines or with killed whole leptospires after heterologous challenge with 10¹ leptospires of the virulent L. interrogans serovar Copenhageni strain Spool. The Wilcoxon log rank test was used to determine significant survival differences between the immunized groups and the control (empty pTARGET plasmid) group. The groups immunized with killed whole leptospires and LigBrep show P values of <0.05.