Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate

Abstract

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37's ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

Fig 1. Western blot of recombinant and native OmpL37 proteins.

A: rOmpL37 characterization with convalescent human sera. (1) Full-Range Rainbow Molecular Weight Marker (GE Healthcare); (2) Negative control (BSA); (3) Positive control (rLipL32); (4) rOmpL37. B: Anti-rOmpL37 serum characterization. (1) Full-Range Rainbow Molecular Weight Marker (GE Healthcare); (2) rOmpL37; (3) Negative control (BSA); (4) L. interrogans serovar Copenhageni Fiocruz L1-130 WCL.

Fig 2. Immunofluorescence (IFA) analysis of the expression of recombinant OmpL37 protein in CHO-K1 cells 24 h after transfection with pTargeT/ompL37 (A) or pTargeT alone (B).

The IFA was based on a mouse anti-rOmpL37 antibody and FITC conjugated anti-mouse IgG. Panels on the left shows Hoechst 33258 DNA staining and on the right, antibody reactions.

Fig 3. Specific IgG response in hamsters inoculated with different vaccine formulations.

Recombinant rOmpL37 expressed by E. coli was used as the antigen in ELISA. Mean values were calculated from serum samples assayed in triplicate. Results are expressed as the mean absorbance ± standard deviation. OD₄₉₂, optical density at 492 nm. Significant differences, at a P value of 0.001 in comparison to the control group, are shown by an asterisk.

Fig 4. Relative mRNA expression levels of IFN-γ, TNF-α, IL1-α and TGF-β in pooled hamster blood samples.

The relative C_T (ΔΔ C_T) method was used to quantify cytokine gene expression: C_Ts were normalized against the β-actin gene C_T (Δ C_T) and then compared to the same normalized gene in the pre-immune sera sample (calibrator). A 2-fold or greater change in mRNA levels was considered significant. The control groups were set to 1. The values represent grouped results of two independent experiments.

Fig 5. Survival of hamsters immunized with rOmpL37 vaccines after lethal challenge.

Survival curves were compared using log-rank analysis. The results are a summary of two independent experiments (Table 2).