Evaluation of Lsa46 and Lsa77 Leptospiral Proteins for Their Immunoprotective Activities in Hamster Model of Leptospirosis

Abstract

Leptospirosis is a neglected tropical disease caused by pathogenic Leptospira spp. The lack of an effective vaccine favors the increase of the disease. Currently, surface-exposed proteins are the main targets for the search of vaccine candidates. In this study, we examined whether the surface Lsa46 and Lsa77 proteins, previously identified as laminin and plasminogen binding proteins, have the capacity of inducing protection and sterilizing immunity against challenge with virulent Leptospira in hamster model. Animals were subcutaneously immunized with Lsa46, Lsa77, or a combination of both in Alum adjuvant and challenged intraperitoneally with L. interrogans serovar Kennewicki strain Pomona Fromm. Hamster immunization with Lsa46 or Lsa77 or both promoted a strong IgG response. Th2- and Th1-biased immune responses were observed when Lsa46 and Lsa77 were individually administered, respectively, as detected by the IgG1/IgG2/3 ratio. Immunized hamsters with the combined proteins induced a Th1-biased immune response. Although the immunization with Lsa46 and Lsa77 stimulated protective immunity with reduction of bacterial burden, when compared to animals individually immunized with the proteins, the data was not statistically significant. Thus, although promising, more studies are needed before the role of these proteins in stimulating sterilizing immunity in mammals is conclusively determined.

Figure 1

Distribution and expression of LIC13479 and LIC10050 genes in Leptospira spp. (a) Genomic DNA from L. interrogans serovars: Copenhageni (1), Canicola (2), Icterohaemorrhagiae (3), Pomona (4), Hardjo (5), L. borgpetersenii serovar Whitticombi (6), L. kirshneri serovars Cynopteri (7) and Grippotyphosa (8), L. santarosai serovar Shermani (9), L. noguchii serovar Panama (10), and saprophytic species L. biflexa serovar Patoc (11) were subjected to PCR analysis with specific primers. The integrity of the DNA used was determined by amplification of 16S DNA. (b) Transcript levels of the LIC13479 and LIC10050 were determined by qRT-PCR in virulent L. interrogans serovar Copenhageni strain FIOCRUZ L1-130 and culture-attenuated L. interrogans strain M20. The relative gene expression was calculated by 2^−ΔΔCT method after normalization with 16S. Error bars show the mean ± SD from three independent experiments performed in triplicate.

Figure 2

Immune response induced by immunization with Lsa46. Hamsters were inoculated subcutaneously with Lsa46 combined with 12.5% Alum and booster was administered after two weeks. Immunized animals with PBS or bacterin were used as negative and positive control, respectively. Animals were bled two weeks after each immunization and sera were used to determine antigen-specific total IgG by ELISA (a) and IgG1 and IgG2/3 (b). The IgG1/IgG2/3 ratios are shown in (c). Error bars show the mean ± SD from two independent experiments performed in triplicate. Representative data are shown.

Figure 3

Antibodies induced by immunization with Lsa77. Hamsters were immunized subcutaneously with Lsa77 combined with 12.5% Alum and booster was administered after two weeks. Inoculated animals with PBS or bacterin were used as negative and positive control, respectively. Animals were bled two weeks after each immunization and sera were utilized to determine antigen-specific total IgG (a) and IgG1 and IgG2/3 (b) response by ELISA. The IgG1/IgG2/3 ratios are shown in (c). Error bars show the mean ± SD from two independent experiments performed in triplicate. Representative data are shown.

Figure 4

Humoral immune response induced by immunization with the combined recombinant proteins. Hamsters were immunized subcutaneously with Lsa46 and Lsa77 combined with 12.5% Alum; booster was administered after two weeks. PBS or bacterin immunized animals were used as negative and positive control, respectively. Animals were bled two weeks after each immunization and sera were utilized to determine Lsa46-specific total IgG, IgG1 and IgG2/3 response ((a) and (c)) or Lsa77-specific response ((b) and (d)). The IgG1/IgG2/3 ratios are shown in (e). Error bars show the mean ± SD from two independent experiments performed in triplicate. Representative data are shown.

Figure 5

Protective effect induced by immunization with Lsa46 and Lsa77. Animals were immunized subcutaneously with Lsa46 (a), Lsa77 (b), or combined Lsa46 and Lsa77 (c) with 12.5% Alum; booster was administered after two weeks. Inoculated animals with PBS or bacterin were used as negative and positive control, respectively. Two weeks after the second immunization, animals were challenged intraperitoneally with 1x10⁴ L. interrogans serovar Kennewicki strain Pomona Fromm. After challenge, hamsters were monitored for 21 days. Two independent experiments were performed and pictures represent the data of experiments 1 and 2. Rates of survival were compared using log-rank and Fisher's exact test. P-value < 0,05 was considered significant.