Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential

Abstract

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Keywords: Leptospira; Adjuvants; Chimeric protein; Recombinant bacterin; Recombinant inactivated E. coli vaccine; Recombinant subunit vaccine.

Fig. 1

Conservation of the OMPs LIC12287, LIC11711, and LIC13259 from L. interrogans in 16 other pathogenic Leptospira spp. The sequences were compared using the RBH method based on BLASTp searches. Abbreviations: kir., L. kirschneri; nog., L. noguchii; san., L. santarosai; may., L. mayottensis; bor., L. borgpetersenii; ale., L. alexanderi; wei., L. weilii; als., L. alstonii; yaz., L. yasudae; bar., L. barantonii; kme., L. kmetyi; tip., L. tipperaryensis; sti., L. stimsonii; adl., L. adleri; ell., L. ellisii; gom., L. gomenensis

Fig. 2

Schematic representation of the chimeric construction. Structural modeling of individual proteins LIC12287 (colored in blue), LIC11711 (colored in green), and LIC13259 (colored in pink) was performed using I-TASSER software. The 3D structure images were generated using UCSF Chimera software. Predicted B- and T-cell epitopes were mapped in each 3D model. Immunodominant regions as well as physicochemical parameters of each protein were considered to select the regions to compose the chimeric protein (indicated in parentheses). Each chimeric subunit protein is connected by a Gly4 × SerGly4 × flexible linker, presented in orange. The chimeric protein was fused to the 6 × histidine-tag of the pET28a vector at the N-terminal region

Fig. 3

Characterization of the processes of expression and purification of the subunit chimeric protein. a Analysis on SDS-PAGE. 1, molecular weight marker; 2, negative control (E. coli STAR); 3, pre-induction chimeric protein; 4, post-induction chimeric protein; 5, soluble fraction chimeric protein; 6, insoluble fraction chimeric protein; 7, purified chimeric protein. b Analysis by WB, with peroxidase-conjugated anti-polyhistidine antibody. 1, molecular weight marker; 2, negative control (E. coli STAR); 3, purified chimeric protein

Fig. 4

Characterization of the pET28a/chimera recombinant bacterin and pET28a bacterin (negative control) production processes. a Analysis on SDS-PAGE. 1, molecular weight marker; 2, negative control (E. coli STAR); 3, heat-inactivated bacterin pET28a; 4, heat-inactivated bacterin pET28a/chimera; 5, purified chimeric protein (positive control). b Analysis in WB, with peroxidase-conjugated anti-polyhistidine antibody. 1, molecular weight marker; 2, negative control (E. coli STAR); 3, heat-inactivated bacterin pET28a; 4, heat-inactivated bacterin pET28a/chimera; 5, purified chimeric protein (positive control)

Fig. 5

IgG antibody response in vaccinated hamsters with the chimeric protein evaluated by indirect ELISA, using serum samples collected on day 0 (pre-immune) and day 28 post-immunization, with the purified chimeric protein as antigen and a secondary anti-hamster IgG antibody. Results are presented as mean optical density (OD₄₉₂) with standard deviation bars. Significant differences were determined by two-way ANOVA. An asterisk (*) indicates a significance difference (P < 0.05) between pre-immune sera and day 28 post-immunization. IM, intramuscular via; SC, subcutaneous via

Fig. 6

Evaluation of the IgG isotype subclasses IgG1, IgG2/3, and IgG3 produced after immunization with the chimeric protein. The results are presented as the mean optical density (OD₄₉₂) with standard deviation bars. Significant differences were determined using a two-way ANOVA. An asterisk (*) indicates a significant difference (P < 0.05) compared to the control groups. IM, intramuscular via; SC, subcutaneous via

Fig. 7

Protection against lethal challenge of acute leptospirosis in the hamster model elicited by immunization with inactivated recombinant E. coli vaccine (recombinant bacterin) and recombinant subunit vaccines containing the chimeric protein. Hamsters received two doses of 100 µL of vaccines with a 14-day interval. The routes of administration are indicated in the figure. On day 28 after the first dose, hamsters were challenged with an ED₅₀ of 10.⁴ L. interrogans serovar Copenhageni strain L1–130 via intraperitoneal inoculation and monitored for an additional 28 days to assess clinical signs of leptospirosis. Endpoint criteria were observed in both the control and vaccinated groups between days 7 and 15 post-challenge. Only one animal from a vaccinated group and two animals from two of the control groups survived. No significant differences were observed (P > 0.05)

Fig. 8

Western blotting analysis in which aliquots of the purified chimeric protein were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Serum from human naturally infected by Leptospira were used. 1, molecular weight marker; 2, purified chimeric protein; 3, negative control (E. coli STAR); 4, LipL32 (positive control). Asterisks in the second lanes indicate a detected protein band with an apparent molecular weight of approximately 49.9 kDa, likely corresponding to our purified recombinant chimeric protein and, 30 kDa for rLipL32