Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection

Abstract

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His(6)-OmpL1 and His(6)-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.

FIG. 1

PCR mutagenesis of the lipoprotein signal peptidase cleavage site of LipL41. In step 1, the lipL41 gene of L. kirschneri was PCR amplified and inserted into pET15b. In step 2, a portion of pET15b-LipL41 plasmid was amplified by using primers that alter the sequence encoding the two amino acids preceding cysteine. The mutagenized FspI-ApaI fragment was then inserted into pET15b-LipL41. The location of the FspI site is underlined in the boxed sequences, which also show the amino acid sequences commencing with the leucine of the lipoprotein signal peptidase cleavage site before (LGNC) and after (LAGC) PCR mutagenesis.

FIG. 2

Immunoblot showing OmpL1 expression at different IPTG concentrations. A culture of E. coli XL1-Blue containing plasmid pMMB66-OmpL1 was grown to log phase, separated into four equal volumes, and treated with various concentrations of IPTG. Cells were incubated for an additional 3 h in a shaker incubator at 37°C, pelleted in a microcentrifuge, resuspended in final sample buffer, and analyzed by SDS-PAGE and immunoblotting. A companion SDS-polyacrylamide gel loaded with the same four samples was stained with Coomassie brilliant blue to verify that all four lanes contained comparable amounts of material (data not shown). The locations of molecular size standards are shown (in kilodaltons) on the left. Even though use of the tac promoter typically results in relatively high background expression levels, expression using plasmid pMMB66-OmpL1 was not detectable without IPTG induction.

FIG. 3

Coomassie brilliant blue-stained SDS-polyacrylamide gel showing material representative of E. coli membrane fractions used to immunize hamsters. Lanes: 1 and 2, E. coli XL1-Blue with plasmids pMMB66 and pMMB66-OmpL1, respectively; 3 and 4, E. coli JM109(DE3) with plasmids pET15b and pET15b-LipL41*, respectively. Locations of the OmpL1 doublet in lane 2 and of LipL41 in lane 4 are shown (arrowheads). Positions of molecular size markers (M) are given in kilodaltons.

FIG. 4

Immunoblot showing recombinant LipL41 expression before and after site-specific mutagenesis of the lipL41 gene. Expression studies were performed with plasmids pET15b-LipL41 (containing the native lipoprotein signal peptidase cleavage site, LGNC) and pET15b-LipL41* (containing the modified lipoprotein signal peptidase cleavage site, LAGC). E. coli JM109(DE3) was transformed with pET15b-LipL41 (left) or pET15b-LipL41* (right). In the left-hand lane, incomplete processing of LipL41 by E. coli lipoprotein signal peptidase results in formation of a doublet. The doublet's lower band represents processed LipL41, while the doublet's upper band represents unprocessed LipL41. As shown in the right-hand lane, expression and processing of LipL41 were much more efficient after modification of the lipoprotein signal peptidase cleavage site. The locations of molecular mass markers are shown on the right in kilodaltons.

FIG. 5

Antibody response of hamsters immunized with leptospiral OMPs. Hamsters were immunized with E. coli membrane fractions with (solid lines) and without (broken lines) OmpL1-M, LipL41-M, or a combination of both. Antibody levels were determined by ELISA against purified His₆-OmpL1 (A) and His₆-LipL41 (B) fusion proteins. Antibody levels were lower in hamsters immunized with both antigens than in those immunized with single antigens.

FIG. 6

Survival of hamsters challenged with L. kirschneri after immunization with leptospiral OMPs. Hamsters were immunized with E. coli membrane fractions with and without OmpL1-M, LipL41-M, or a combination of both. Bars denote percent survival at various time points after i.p. challenge with virulent L. kirschneri. Asterisks denote a significant difference in survival versus control (P < 0.05). Data represent the sum of three separate experiments.

FIG. 7

No evidence of infection in hamsters immunized with a combination of OmpL1-M and LipL41-M. Hamsters were immunized with E. coli membrane fractions with and without OmpL1-M, LipL41-M, or a combination of both. Bars denote percentages of animals surviving with or without evidence of infection 28 days after i.p. challenge with virulent L. kirschneri. Data represent the sum of three separate experiments. The largest number of infected survivors was seen in hamsters immunized with OmpL1-M alone. In no group were the differences between the total number of survivors and the number of uninfected survivors significant (P < 0.05).