Evaluation of immunoprotective activity of six leptospiral proteins in the hamster model of leptospirosis

Abstract

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of L. interrogans serovar Copenhageni together with bioinformatics tools represent a great opportunity to search for novel antigen candidates that could be used as subunit vaccine against leptospirosis. We focused on six genes encoding for conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from Leptospira interrogans genomic DNA and were cloned and expressed in Escherichia coli. The recombinant proteins tagged with N-terminal hexahistidine were purified by metal-charged chromatography. The immunization of hamsters followed by challenge with lethal dose of virulent strain of Leptospira showed that the recombinant proteins Lsa21, Lsa66 and rLIC11030 elicited partial protection to animals. These proteins could be used combined or in a mixture with novel adjuvants in order to improve their effectiveness.

Keywords: Leptospira interrogans; leptospirosis; recombinant protein; vaccine..

Fig. (1)

Distribution of genes among saprophytic and pathogenic leptospires. Genomic DNA from L. biflexa Patoc and from ten serovars belonging to the pathogenic species of Leptospira were subjected to PCR analysis with specific primers designed according to L. interrogans serovar Copenhageni genome sequences. (A) Amplification of 16S DNA shows template integrity; (B) LIC10821 and (C) LIC10672. No DNA was added to the negative control reaction (-).

Fig. (2)

Analysis of purified recombinant proteins by metal chelating chromatography through SDS-12% PAGE. Purified protein eluted from Ni⁺² - charged chelating sepharose column with 1 M imidazole. Lane M, molecular mass protein marker; lane 1, rLIC10821 (37 kDa); lane 2, Lsa66 (65 kDa); lane 3, Lsa25 (24 kDa); lane 4, Lsa21 (21 kDa); lane 5, rLIC11030 (37 kDa); lane 6, rLIC10672 (23 kDa). Protein bands were visualized by Coomassie blue staining. Positions of molecular mass standards are indicated on the left (in kDa). (*) denotes each protein band.

Fig. (3)

Induction of humoral immune response in hamsters. The animals were immunized subcutaneously with PBS (negative control), Lsa21, Lsa66, rLIC11030, rLIC10821, Lsa25 or rLIC10672, 50 µg each and boostered after two weeks. Animals were bled on day 30 (prior challenge) and 51 (after the challenge) to determine antigen specific total IgG responses in sera of immunized groups by ELISA method. Microdilution plates were coated with recombinant proteins and incubated with a serial dilution of serum from the immunized hamsters for IgG measurements. The data represent a pooled serum from two hamsters per group (days 30) or survival animals after challenge (day 51). All antibody titers refer to challenge assay 1.

Fig. (4)

Survival curves of hamsters immunized with recombinant proteins and challenged with virulent leptospires. The animals were immunized subcutaneously with PBS (negative control), bacterin (killed whole-leptospires) (positive control), Lsa21, Lsa66, rLIC11030, rLIC10821, Lsa25 or rLIC10672. Two weeks after the second immunization (day 30) with the recombinant proteins, the animals were challenged intraperitoneally with 3 - 5 X 10⁷ of L. interrogans serovar Kennewicki strain Pomona Fromm (LPF) in PBS. The animals were monitored for morbidity until day 21 post-challenge. A and B depicted the data of two independent experiments.