LigB subunit vaccine confers sterile immunity against challenge in the hamster model of leptospirosis

Abstract

Neglected tropical diseases, including zoonoses such as leptospirosis, have a major impact on rural and poor urban communities, particularly in developing countries. This has led to major investment in antipoverty vaccines that focus on diseases that influence public health and thereby productivity. While the true, global, impact of leptospirosis is unknown due to the lack of adequate laboratory diagnosis, the WHO estimates that incidence has doubled over the last 15 years to over 1 million cases that require hospitalization every year. Leptospirosis is caused by pathogenic Leptospira spp. and is spread through direct contact with infected animals, their urine or contaminated water and soil. Inactivated leptospirosis vaccines, or bacterins, are approved in only a handful of countries due to the lack of heterologous protection (there are > 250 pathogenic Leptospira serovars) and the serious side-effects associated with vaccination. Currently, research has focused on recombinant vaccines, a possible solution to these problems. However, due to a lack of standardised animal models, rigorous statistical analysis and poor reproducibility, this approach has met with limited success. We evaluated a subunit vaccine preparation, based on a conserved region of the leptospiral immunoglobulin-like B protein (LigB(131-645)) and aluminium hydroxide (AH), in the hamster model of leptospirosis. The vaccine conferred significant protection (80.0-100%, P < 0.05) against mortality in vaccinated animals in seven independent experiments. The efficacy of the LigB(131-645)/AH vaccine ranged from 87.5-100% and we observed sterile immunity (87.5-100%) among the vaccinated survivors. Significant levels of IgM and IgG were induced among vaccinated animals, although they did not correlate with immunity. A mixed IgG1/IgG2 subclass profile was associated with the subunit vaccine, compared to the predominant IgG2 profile seen in bacterin vaccinated hamsters. These findings suggest that LigB(131-645) is a vaccine candidate against leptospirosis with potential ramifications to public and veterinary health.

Fig 1. Schematic of the Lig proteins, expression and purification of rLigB(131–645).

A) The full length amino acid sequences for LigA (1224 amino acids, 128.1 kDa) and LigB (1890 amino acids, 200.8 kDa) are indicated (black line), the square boxes indicate the BIDs and the LigB C-terminal domain is shown (rectangle). The recombinant proteins used as vaccine candidates are indicated: LigB(131–645) (green boxes) includes amino acids 131–645 (53.5 kDa) and is highly identical (97.9% pairwise identity) to the same region in LigA; the LigA(631–1224), also known as LigANI, (red boxes, amino acids 631–1224, 62.8 kDa) and LigB(625–1259), also known as LigBNI, (blues boxes, amino acids 625–1259, 66.2 kDa) fragments are not highly conserved (38.1% pairwise identity). B) Expression and purification of rLigB(131–645) analysed by 10% SDS-PAGE and Coomassie staining. Lanes 1: molecular mass marker (kDa); Expression of rLigB(131–645) in an E. coli(pLigB(131–645)) clone, lane 2: supernatant (soluble) fraction and lane 3: insoluble fraction; lane 4: IMAC purified rLigB(131–645), expected molecular mass of 57.2 kDa. C) Immunoblot analysis of rLigB(131–645), following transfer the nitrocellulose membrane was probed with an anti-His-HRP antibody, lane 1: molecular mass marker (kDa); lane 2: purified rLigB(131–645).

Fig 2. Protection against lethal challenge.

Representative experiment of survival among hamsters vaccinated with rLigB(131–645), bacterin or a PBS control, followed by the administration of a potentially lethal dose of L. interrogans serovar Copenhageni strain Fiocruz L1-130, see Table 1. Groups of hamsters were immunized (days -28 and -14) with two doses (80/40 μg) of either rLigB(131–645)/AH; PBS/AH control; bacterin; or a PBS only control, and challenged with 200 leptospires (day 0). The rLigB(131–645)/AH vaccine preparation significantly protected 90.0% (9/10) of hamsters against challenge (P < 0.001), compared to 100% (4/4) protection in hamsters vaccinated with the bacterin (P < 0.05).

Fig 3. Pathological findings in the hamster model.

Animals vaccinated with rLigB(131–645) (A, C, E and G) or the PBS control group (B, D, F and H) were euthanized 10 days PC and tissue samples were collected. Vaccinated animals showed no gross pulmonary lesions (A) or microscopic pulmonary lesions (C). Liver (E) and kidney samples (G) showed no evidence of microscopic abnormalities. Unvaccinated animals showed gross pulmonary haemorrhaging (B) and they were confirmed to be alveolar haemorrhages by microscopic analysis (D). Dystrabeculaton (loss of cohesion) of hepatocytes (F) and swelling of kidney tubular epithelial cells (H) were prominent features. (C-F, haematoxylin-eosin, 100× magnification and G-H, haematoxylin-eosin, 200× magnification).

Fig 4. IgM and IgG induced by rLigB(131–645).

ELISAs were performed to determine antibody levels in hamsters immunized with A) rLigB(131–645)/AH (80/40 μg) or B) bacterin vaccine (see Table 1). Pre-immune (PI), post-vaccination (PV) and post-challenge (PC) serum samples were collected and characterized at a single serum dilution (1:100) with anti-hamster IgM and IgG secondary antibodies. The mean optical density (OD_{450 nm}) ± standard deviation (bars) from three independent experiments are shown. Significance was determined by one-way ANOVA (Tukey multiple comparison) analysis, the presence of lower case letters, where different, indicates a significant difference (P < 0.05) between samples.

Fig 5. IgG subclasses induced by vaccination with LigB(131–645).

IgG subclasses were characterized using ELISAs to determine antibody levels in hamsters immunized with A) rLigB(131–645)/AH or B) bacterin vaccine, see Table 1. Pre-immune (PI), post-vaccination (PV) and post-challenge (PC) serum samples were collected and characterized with anti-hamster IgG1, IgG2/3 or IgG3 conjugates. The mean OD ± standard deviation (vertical bars) from three independent experiments are shown. Significance was determined by one-way ANOVA (Tukey multiple comparison) analysis and the presence of lower case letters, where different, indicates a significant difference (P < 0.05) between samples.