Immunoprotective properties of recombinant LigA and LigB in a hamster model of acute leptospirosis

Abstract

Leptospirosis is the most widespread zoonosis and is considered a major public health problem worldwide. Currently, there is no widely available vaccine against leptospirosis for use in humans. A purified, recombinant subunit vaccine that includes the last six immunoglobulin-like (Ig-like) domains of the leptospiral protein LigA (LigA7'-13) protects against lethal infection but not renal colonization after challenge by Leptospira interrogans. In this study, we examined whether the addition of the first seven Ig-like domains of LigB (LigB0-7) to LigA7'-13, can enhance immune protection and confer sterilizing immunity in the Golden Syrian hamster model of acute leptospirosis. Hamsters were subcutaneously immunized with soluble, recombinant LigA7'-13, LigB0-7, or a combination of LigA7'-13 and LigB0-7 in Freund's adjuvant. Immunization with Lig proteins generated a strong humoral immune response with high titers of IgG that recognized homologous protein, and cross-reacted with the heterologous protein as assessed by ELISA. LigA7'-13 alone, or in combination with LigB0-7, protected all hamsters from intraperitoneal challenge with a lethal dose of L. interrogans serovar Copenhageni strain Fiocruz L1-130. However, bacteria were recovered from the kidneys of all animals. Of eight animals immunized with LigB0-7, only three survived Leptospira challenge, one of which lacked renal colonization and had antibodies to native LigB by immunoblot. In addition, sera from two of the three LigB0-7 immunized survivors cross-reacted with LigA11-13, a region of LigA that is sufficient for protection. In summary, we confirmed that LigA7'-13 protects hamsters from death but not infection, and immunization with LigB0-7, either alone or in combination with LigA7'-13, did not confer sterilizing immunity.

Fig 1. Expression, purification, and antigenicity of recombinant His-tagged Lig proteins.

(A) E. coli BLR (DE3) pLysS transformed with pET-20b(+) containing ligA7’-13 or ligB0-7 were grown to an OD₆₀₀ = 0.5–0.6 prior induction with 0.5–1 mM IPTG for 2 h at 30^°C. Bacterial cells were harvested by centrifugation, followed by lysis in the presence of protease inhibitors. Supernatant was separated from pellet fraction by centrifugation at 16,000 x g at 4^°C. His-tagged Lig proteins were purified from the supernatant fraction by affinity chromatography using Ni-NTA resin. Fifteen microliters of whole cell lysates from uninduced (-) and induced cultures (WCL), supernatant (SUP) and pellet (PEL) fractions, and 2 μg purified protein (PUR) were separated by 4–12% SDS-PAGE followed by Coomassie staining. A replicate gel was also run loaded with 5 μl cell fractions and 200 ng purified proteins for immunoblot analysis. The proteins were transferred to a PVDF membrane and the blot was probed with rabbit α-Lig antiserum (dilution 1:10,000). The expressed His-tagged LigA7’-13 and LigB0-7 were found predominantly in the supernatant. Asterisk shows LigB0-7, produced at lower levels compared to LigA7’-13. (B) Two hundred nanograms of LigA7’-13 (lanes 1, 3, 5, 7, 9) or LigB0-7 (lanes 2, 4, 6, 8, 10) were run in 4–12% SDS-PAGE gel and transferred to PVDF. Membranes were probed with 1:10,000 control α-Lig antiserum (lanes 1, 2), or 1:250 of the following: pooled sera from hamsters before (lanes 3, 4) and after (lanes 5, 6) intraperitoneal challenge with L. interrogans, normal human serum (lanes 7, 8), and pooled sera from leptospirosis patients in convalescent stage (lanes 9, 10). Both Lig proteins were recognized by sera from Leptospira-infected hamsters and humans.

Fig 2. IgG response to immunization with purified recombinant Lig proteins.

Serum samples were collected from hamsters weekly during the immunization protocol. Anti-LigA7’-13 (A) or anti-LigB0-7 (B) antibody levels were measured in triplicate by ELISA. Each data line represents the IgG response of an individual animal over time; in black are animals that survived the challenge while in red are animals that met the endpoint criteria. Vertical dotted lines indicate immunization days (blue) or challenge with L. interrogans (red). Each data point represents the mean IgG level read at OD₆₅₅ minus pre-bleed read. Error bars indicate standard deviation. There was no significant difference in the IgG response among hamsters that received the same vaccine treatment at all time points (One-way ANOVA, P = 0.9).

Fig 3. Recognition of purified, recombinant LigA7’-13 and LigB0-7 proteins by pooled sera from immunized hamsters.

One to two hundred nanograms per well of purified His-tagged LigA7’-13 (A) or LigB0-7 (B) were run in 4–12% SDS-PAGE then transferred to PVDF membrane for western blot analysis. The membrane was cut to strips and probed with 1:5,000 pooled sera collected before immunization (pre-bleed, P) and at day 32 (last bleed, LB) from immunized and control groups. Another membrane strip was incubated with 1:5,000 rabbit α-Lig as positive control (Co). Sera collected from LigA7’-13-vaccinated animals cross reacted with recombinant LigB0-7 and vice versa.

Fig 4. Survival of hamsters immunized with purified recombinant Lig proteins after lethal challenge.

Groups of 5–8 female hamsters were immunized subcutaneously three times with 100 μg LigA7’-13 (●), LigB0-7 (■), LigA7’-13 + LigB0-7 (◆), or controls PBS (○) and PBS with adjuvant (□). Animals were challenged intraperitoneally with 1 x 10⁴ L. interrogans at day 0 and post-challenge survival was followed until day 28. Asterisks indicate significant difference between mortality rates compared (Fisher’s exact test, *P < 0.05, ***P < 0.001, ns not significant).

Fig 5. Bacterial load in kidney and liver of hamsters that survived the L. interrogans challenge.

Total genomic DNA was extracted from kidney (A) and liver (B), and analyzed by qPCR performed in duplicates with lipL32 primers and probes (Table 1) to quantify leptospiral tissue load. Bacterial burden was expressed as genomic equivalents (GEq) per gram of tissue. Black lines show mean bacterial load with error bars representing standard deviation, while dotted lines indicate limit of detection. There was no difference in the bacterial burden in kidneys among the different vaccine groups (Kruskal-Wallis test, P = 0.4050).

Fig 6. Recognition of native LigA and LigB in Leptospira lysate by sera from immunized hamsters.

A L. interrogans culture at an OD₄₂₀ = 0.29 was incubated in EMJH supplemented with 120 mM NaCl for 4 h at 30^°C. Bacteria was harvested by centrifugation, washed with PBS, resuspended in sample buffer, and boiled for 5 min. Fifteen microliters of bacterial lysate was separated in 4–12% SDS-PAGE, then transferred to a PVDF membrane for western blot analysis. (A) The membrane was cut to strips and probed with pooled sera (1:2,000 dilution) collected before immunization (pre-bleed, PB), and at day 32 (last bleed, LB) from immunized and control hamsters. (B) The membrane strips were probed with sera (1:2,000 dilution) from individual LigB0-7 vaccinated hamsters (1–8). The prominent band recognized by serum from hamster 7 migrates slower than LigB. The membranes were also probed with rabbit α-Lig (Co, 1:2,000 dilution) and rabbit α-LipL41 (1:10,000 dilution) as controls. Asterisks (*) represent hamsters that survived the challenge, while pound sign (#) designates animal that survived and was culture- and MAT-negative.

Fig 7. Recognition of purified, recombinant LigA11-13 proteins by sera from immunized hamsters.

One hundred nanograms per well of purified His-tagged LigA11-13 were run in 4–12% SDS-PAGE then transferred to a PVDF membrane for western blot analysis. The membrane was cut to strips and probed with 1:3,000 pooled sera collected before immunization (pre-bleed, P) and at day 32 (last bleed, LB), and sera from individual hamsters immunized with LigA7’-13 (A), LigB0-7 (B), and LigA7’-13 + LigB0-7 (C). Another membrane strip was incubated with 1:3,000 rabbit α-Lig as positive control (Co). In Panel B, asterisks (*) represent hamsters that survived the challenge, while pound sign (#) designates animal that survived and was culture- and MAT-negative. All animals immunized with LigA7’-13 alone, or in combination with LigB0-7, survived the challenge.