Recombinant LipL32 stimulates interferon-gamma production in cattle vaccinated with a monovalent Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis vaccine

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Abstract

Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) is the main causative agent of bovine leptospirosis in Australia, New Zealand, North America and elsewhere. Bovine leptospirosis can result in spontaneous abortion, stillbirth and reduced milk output. The organism is shed in the urine of infected animals and contact with contaminated materials can result in zoonotic infections in humans. Protective immunity in cattle against Hardjobovis involves stimulation of a Th1 cell mediated immune response, which can be characterized by the production of IFN-γ when blood from vaccinated animals is exposed to Hardjobovis antigens. However, the leptospiral components involved in stimulating this response have yet to be identified. In this study, 238 recombinant leptospiral proteins were evaluated for their ability to stimulate IFN-γ production in blood of cattle vaccinated with a commercial monovalent Hardjobovis vaccine. The conserved lipoprotein LipL32 is the major outer membrane protein of pathogenic Leptospira spp. A pool of soluble recombinant proteins which included LipL32, as well as LipL32 alone, stimulated significant IFN-γ production in blood of vaccinated cattle. A number of recombinant LipL32 fragments was generated, which identified the amino acids between 20 and 200 as containing the bovine T-cell reactive regions of LipL32. However, whether LipL32 plays a role in stimulating protective immunity in mammals has yet to be conclusively determined.

Introduction

Bovine leptospirosis is a disease of medical and agricultural economic significance worldwide. It is a systemic disease, with infection resulting in abortion, fetal death, and decreased milk production (Hanson, 1975). Historically, bovine leptospirosis has been associated with several serovars, including Grippotyphosa, Pomona, Icterohaemorrhagiae, and Hardjo (subtypes Hardjoprajitno and Hardjobovis) (Tripathy et al., 1976).

Cattle are the maintenance hosts for Hardjobovis; endemically infected animals are responsible for spreading the organism via excretion in the urine (Faine et al., 1999). Humans become infected through contact with contaminated materials (Adler and de la Peña Moctezuma, 2010). Hardjobovis is also a common serovar identified in cases of human leptospirosis in Northern Australia (Smythe et al., 1997) and a significant percentage of cases in the USA (Miller et al., 1991). Several vaccines containing Hardjobovis have been tested for their ability to stimulate a protective immune response, but despite stimulating strong antibody responses (Bolin et al., 1989a, Bolin et al., 1989b, Bolin et al., 1991), none of the vaccines protected cattle from infection upon challenge. In contrast, cattle vaccinated with a monovalent Hardjobovis vaccine, Spirovac™, were protected against infection by Hardjobovis (Bolin and Alt, 2001). Protection was correlated with stimulation of a cell-mediated immune response (Naiman et al., 2001).

Brown et al. (2003) evaluated the cellular immune response in cattle vaccinated with the protective monovalent Spirovac™ vaccine, the monovalent Leptavoid vaccine and the pentavalent vaccine, Leptoshield 5. Lymphocytes from the vaccinated animals were cultured with Hardjobovis whole cell lysate (WCL). The lymphocytes from animals vaccinated with the monovalent vaccines produced high levels of IFN-γ in comparison to lymphocytes from unvaccinated animals, or animals vaccinated with Leptoshield 5, neither of which were protected against Hardjobovis infection. The production of IFN-γ correlated with a strong type 1 cell-mediated immune response (Brown et al., 2003). Characterization of these cells indicated that vaccination appears to confer protection against Hardjobovis by activation and clonal expansion of IFN-γ positive γδ and CD4+ T cells (Baldwin et al., 2002). Further characterization of the γδ T-cells identified the WC1.1 isoform as predominant in the subset (Rogers et al., 2005) and the γδ T-cell response appears to develop before the CD4+ T cell response (Blumerman et al., 2007). The γδ T cells also constitute the major population of T-cells in the ruminant uterus (Hansen and Liu, 1996), an organ commonly affected during bovine leptospiral infections. Although γδ T-cells can be activated in response to non-protein peptides such as mycobacterial isopental phosphate (Smyth et al., 2001), lymphocytes from cattle vaccinated with the protective monovalent vaccine stimulated with protease-digested Hardjobovis WCL produced far lower levels of IFN-γ (similar levels to unvaccinated animals), suggesting that the cell-mediated response was due to protein components of the bacterium and not due to mitogenic components such as LPS (Naiman et al., 2002).

The purpose of this study was to identify the Hardjobovis proteins involved in stimulating an IFN-γ response in cattle vaccinated with Spirovac™. The work presented in this study describes the quantitation of the immune response (both cell mediated and humoral) to the Spirovac™ preparation. Bioinformatic analysis of the Hardjobovis strain L550 genome was used to predict 260 potential surface-exposed, candidate vaccine antigens, as per Murray et al. (2013), of which 238 proteins or protein fragments were successfully expressed. These proteins were screened for their ability to stimulate IFN-γ in blood from cattle vaccinated with Spirovac™. Identification of the proteins involved in stimulating an IFN-γ response may shed light on essential leptospiral protein components required in effective vaccines, leading to optimization of vaccine formulation and/or development of a recombinant vaccine.

Section snippets

Bacterial strains and culture conditions

Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis strain L550 was isolated in Australia from a human patient with leptospirosis contracted from exposure to infected cattle, while strain L683 was provided by Pfizer Animal Health, Melbourne, Australia. Strain L692, maintained for National Association of Testing Authorities, Australia (NATA)-accredited serology testing was used for microscopic agglutination tests (MATs). Bacteria were cultured at 30 °C in EMJH medium (Difco).

Cloning and expression of recombinant proteins

Genome

Cattle vaccinated with Spirovac™ produced agglutinating antibodies against serovar Hardjobovis

None of the animals selected for this study had antibodies against serovar Hardjobovis before vaccination. All of the non-vaccinated animals remained sero-negative for the duration of the trial, with MAT titers of <20, log2 4.25 (Fig. 1). All of the vaccinated animals sero-converted after the first vaccination with an average titer of log2 6.92, increasing to 8.52 following the first boost vaccination and peaking at day 189 to an average titer of log2 10.22, following the second boost vaccination

Discussion

The Spirovac™ vaccine generates protective immunity against Hardjo infection correlated with IFN–γ release. However, the identity of protein antigens involved is unknown. In this study, we have investigated 238 purified recombinant proteins for this capacity. Sera from the cattle used in the study were screened by MAT prior to vaccination to confirm that there were no antibodies against Hardjobovis. As expected, all of the vaccinated animals seroconverted after the initial vaccination, while

Acknowledgements

This study was funded by an Australian Research Council Linkage Grant in conjunction with Pfizer Animal Health. The authors acknowledge the excellent technical assistance of Vicki Vallance, Kate Rainczuk, Chenai Khoo, Nik Sotirellis, Dr. Lisa Walter, and Lauren Jory.

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