The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis

Abstract

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund's adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P<0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.

Fig. 1

Schematic representation of LigA and LigB proteins and recombinant fragments. Boxes represent bacterial immunoglobulin-like (Big) tandem repeat domains (∼90 amino acids). Amino acids 102 to 630 (Big domains 2-6 and part of 7) of LigA and LigB, the region with 100% amino acid sequence identity between these two proteins, are represented as grey boxes. The C-terminal Big domains of LigA (amino acid position 631-1,224) and LigB (amino acid position 631-1,119) have lower amino acid sequence identity (38%) and are represented as hatched boxes. Lines represent the three recombinant fragments, LigANI, LigBrep and LigBNI that were cloned and expressed.

Fig. 2

Recombinant Lig proteins and seroreactivity with sera from leptospirosis patients. (A) Coomassie blue-stained SDS-PAGE gel of purified LigANI, LigBNI and LigBrep recombinant protein fragments (1.5 μg/lane). (B) Immunoblot analyses of LigANI, LigBNI and LigBrep recombinant protein fragments. Membranes were probed with pooled convalescent sera from leptospirosis patients (+) and from healthy individuals (-). Positions of molecular mass markers (kDa) are shown on the left.

Fig. 3

Antibody responses in hamsters immunized with recombinant Lig proteins. ELISA reactions were performed to determine antibody levels against LigANI (A), LigBNI (B) and LigBrep (C) in sera from hamsters immunized with LigANI (A: pre-immune, □; immune, ■) LigBNI (B: pre-immune, □; immune, ▴) and LigBrep (C: pre-immune, □; immune, ▾). Mean absorbance (optical density, 450nm) +/-standard deviation (bars) are shown for a representative experiment among three that evaluated sera from groups of four immunized hamsters.

Fig. 4

Kinetics of IgG antibody response in hamsters immunized with recombinant LigANI protein fragment. ELISA was used to determine anti-LigANI IgG antibody levels in hamsters immunized on day 0 to 14 (arrows) with LigANI doses of 80 and 40 μg (■), 40 and 20 μg (●) and 20 and 10 μg (□), respectively. Normalized geometric mean endpoint titres (GMTs) and standard deviation (bars) for sera collected from groups of four hamsters at weekly intervals over a 63 day follow-up period are shown.

Fig. 5

Sera from hamsters immunized with recombinant Lig fragments recognize native LigA and LigB proteins. Immunoblots of whole-cell extracts of L. interrogans strain Fiocruz L1-130 (10⁸ organisms per lane) were probed with representative samples of pre-immune (PI) and immune sera from hamsters immunized with LigANI, LigBNI, LigBrep and PBS (Control) with Freund’s adjuvant. The mobility of molecular mass markers and expected positions of LigA (128 kDa) and LigB (201 kDa) are shown on the left and right side, respectively of the figure.

Fig. 6

Survival of hamsters immunized with recombinant Lig proteins after lethal challenge. A: Post-challenge survival is shown for a representative vaccine evaluation (Experiment 1, Table 2) in which hamsters were immunized on day -21 (80 μg) and day -7 (40 μg) with LigANI (■), LigBNI (▴), LigBrep (□) and PBS (▾) and challenged with L. interrogans strain Fiocruz L1-130 on day 0. B. Protective effect conferred by immunization with increasing LigANI doses (80/40 μg, ■; 60/30 μg, □; 40/20 μg, ▴; 20/10 μg, ●; and 0/0 μg, ▾) on post-challenge survival (Experiment 6, Table 2).