Elsevier

Vaccine

Volume 29, Issue 18, 18 April 2011, Pages 3413-3418
Vaccine

Recombinant LipL32 and LigA from Leptospira are unable to stimulate protective immunity against leptospirosis in the hamster model

https://doi.org/10.1016/j.vaccine.2011.02.084Get rights and content

Abstract

The major antigenic component of pathogenic Leptospira spp. is lipopolysaccharide (LPS). However, due to the specificity of the immune response generated towards LPS and the diversity in leptospiral LPS carbohydrate structure, current commercial vaccines stimulate protection only against homologous or closely related serovars. Vaccines that confer heterologous protection would enhance protection in vaccinated animals and reduce transmission to humans. Several studies have investigated the potential of various leptospiral outer membrane proteins to stimulate protective immunity against pathogenic Leptospira species. These include the surface-exposed lipoproteins LipL32 and LigA. However, consistent protection from infection has proved difficult to reproduce. In this study we assessed the protective capacity of recombinant LipL32, the six carboxy-terminal unique Ig-like repeat domains of LigA (LigANI) and a LipL32–LigANI fusion protein in hamsters against infection with Leptospira interrogans serovar Manilae. Despite all of the proteins eliciting antibody responses, none of the hamsters was protected against infection.

Introduction

Leptospirosis is an emerging global zoonosis, resulting in a systemic infection with high mortality rates in tropical and developing countries. Leptospira is a member of the Spirochaetacae family, a group of helical, gram-negative organisms. Over 230 serovars of pathogenic Leptospira have currently been identified. The lipopolysaccharide (LPS) of Leptospira is the principle antigen to which agglutinating, opsonic antibodies bind [1]. Antigenically related serovars of Leptospira are grouped into serogroups depending on the similarity of the carbohydrate component of the LPS of each strain [2]. Due to the specificity of the immune response generated towards LPS and the diversity in leptospiral LPS carbohydrate structure, current commercial vaccines stimulate protection only against homologous or closely related serovars [3], [4]. Vaccines that confer heterologous protection would enhance protection in vaccinated animals and reduce transmission to humans. However, this goal has so far eluded researchers.

Several potential vaccine candidates have been identified. Many of these target antigens lie in the leptospiral outer membrane (OM). The OM of pathogenic Leptospira spp. contains a number of components including LPS, lipoproteins (including LipL32, LipL21 and LipL41 [5]), the leptospiral immunoglobulin-like proteins LigA and B, porins such as OmpL1 [6], and phospholipids. The OM proteins are highly conserved across the pathogenic species [7]. Therefore, these proteins have been the focus for development of novel vaccines for heterologous protection.

LipL32 is the major OM protein of pathogenic Leptospira spp. [8]. It is expressed in vivo and is highly immunogenic; over 95% of patients with leptospirosis produced antibodies against LipL32 [9]. Due to its antigenic properties, LipL32 has been a major candidate antigen in a number of vaccine trials and has been shown to confer partial protection against infection in some animal models [7], [10], while other trials have shown no protection (reviewed in [11]). LipL32 binds to extracellular matrix (ECM) proteins [12], [13], [14]. However, recent studies have shown that a LipL32 mutant retains ECM-binding capabilities in vitro and virulence in hamsters [15], suggesting redundancy in ECM-binding capability. The antibody dominant epitopes of LipL32 have been identified [13], [16] and have been shown to comprise two regions spanning amino acids 155–177 and 181–204 [16]. Hauk et al. [13] constructed three truncations of mature LipL32: N-terminal truncation (amino acids 21–92), an intermediate protein fragment (amino acids 93–184) and a C-terminal truncation (amino acids 185–272). The recombinant proteins were screened using serum samples from patients with confirmed leptospirosis. Both the intermediate and C-terminal truncations were recognised by the serum [13]. An additional study identified the specific amino acids in the immunodominant region through the screening of a synthetic peptide library against serum samples from leptospirosis patients [16]. Therefore, in the present study a truncated form of LipL32 (LipL32 Δ155–200) was included to determine the immunogenicity of LipL32 when the immunodominant epitopes are absent.

An alternative protein vaccine candidate is the leptospiral immunoglobulin-like protein A (LigA). LigA is a 130 kDa surface exposed lipoprotein expressed during infection and is highly immunogenic [17]. LigA is not expressed under normal in vitro growth conditions and requires the medium to be adjusted to physiological osmolarity to induce expression [17], [18]. LigA binds ECM proteins and fibrinogen [19], and therefore may be involved in cellular adhesion and colonisation. The six carboxy-terminal unique Ig-like repeat domains of the LigA (LigANI) may contribute to tissue specificity during infection [20]. Vaccination with LigANI was shown to protect hamsters from leptospirosis [21].

To assess the protective capacity of LigANI and LipL32 in the hamster model, we cloned and expressed variations of recombinant LigANI and LipL32 in Escherichia coli. These recombinant proteins included PBS-soluble mature LipL32 (minus the signal peptide), truncated LipL32 (LipL32 Δ155–200), the six C-terminal repeats of LigA (LigANI) based on the findings of Silva et al. [21] and a hybrid protein comprising truncated LipL32 (LipL32 Δ155–200) fused with LigANI. Immunity to leptospirosis is predominantly antibody mediated in a number of species, including humans and hamsters [11], therefore Alhydrogel was selected as the adjuvant for this study, as it stimulates a strong antibody response [22]. These proteins were then assessed for their capacity to protect hamsters from infection with Leptospira.

Section snippets

Bacterial strains and growth conditions

Leptospira interrogans serovar Manilae (L495), L. interrogans serovar Copenhageni (Fio Cruz L1-130), and Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (L550) were grown in EMJH medium [23] at 30 °C to a density of 4 × 108/mL. To induce LigA expression in serovar Copenhageni, NaCl was added to a final concentration of 0.15 M then all cultures were incubated at 30 °C for an additional 20 h before harvest. E. coli BL-21Codon Plus (Stratagene) was grown at 37 °C in Luria–Bertani medium

Expression, purification and immunogenicity of recombinant proteins

All recombinant proteins (Fig. 1) were highly expressed in E. coli and purified by metal affinity chromatography to >95% purity (Fig. 2).

Hamsters were immunised with formalin-killed L495 (control), PBS (control), 8 M urea (control), soluble LipL32, LipL32 Δ155–200, LigANI and LipL32 Δ155–200–LigANI, 30 μg in Alhydrogel on day 0 and day 14. Serum was collected from all hamsters on day 28 before they were challenged with 2 × 103 cells of serovar Manilae (L495). Sera from hamsters vaccinated with

Discussion

Each of the recombinant antigens elicited an antibody response against the homologous recombinant protein (Fig. 3) and against the corresponding protein in leptospiral WCL (Fig. 4). However, none of the immunised hamsters was protected when challenged with virulent L. interrogans, whereas the killed bactrin elicited 100% protection. Hamsters were challenged via the recommended intraperitoneal route of infection [26]. Serovar Manilae was selected for challenge as it causes systemic, lethal

Acknowledgements

We gratefully acknowledge the excellent technical assistance of Vicki Vallance, Kate Rainczuk and Chen Ai Khoo. This work was supported by grants from the National Health and Medical Research Council, and the Australian Research Council, Canberra, Australia.

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    These authors contributed equally to this work.

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