Vaccine 30 (2011) 95–102 Contents lists available at SciVerse ScienceDirect Vaccine jou rn al h om epa ge: www.elsevier.com/locate/vaccine Mucosal co-immunization of mice with recombinant lactococci secreting VapA antigen and leptin elicits a protective immune response against Rhodococcus equi infection S. Cauchard a , L.G. Bermúdez-Humarán b , S. Blugeon b , C. Laugier a , P. Langella b , J. Cauchard a,∗ a Anses, Dozulé Laboratory for Equine Diseases, Bacteriology and Parasitology Unit, Goustranville, 14430 Dozulé, France b Laboratory of Commensal and probiotics-host interactions, UMR INRA-AgroParisTech 1319 MICALIS-Microbiologie de l’Alimentation au Service de la Santé humaine, F-78350, Jouy-en-Josas, France a r t i c l e i n f o Article history: Received 16 May 2011 Received in revised form 29 September 2011 Accepted 10 October 2011 Available online 20 October 2011 Keywords: Rhodococcus Immunization Mucosal VapA Lactococcus Leptin a b s t r a c t Rhodococcus equi causes severe pneumonia in foals and has recently gained attention as a significant opportunistic pathogen in immunocompromised humans. However, no effective vaccine to prevent rhodococcosis is currently available. In this study, we have engineered the food-grade bacterium Lac- tococcus lactis to secrete the virulence-associated protein A from R. equi (LL-VapA). The immunogenic potential of LL-VapA strain was then evaluated after either intragastric or intranasal immunization in mice either alone or in combination with LL-Lep, a recombinant strain of L. lactis secreting biologically active leptin, a pleiotropic hormone with significant immunomodulatory properties. Intragastric admin- istration of LL-VapA led to the highest VapA-specific mucosal response whereas intranasal administration led to the highest systemic immune responses. Cytokines released from in vitro-stimulated spleen cells show both a strong IFN- response and an increase of IL-4 level in all immunized groups, except for the group intranasally co-administered with both LL-VapA and LL-Lep. Strikingly, a significant reduction in R. equi viable counts in liver and spleen was observed four days after intravenous challenge with a viru- lent strain of R. equi in all immunized groups except for the group vaccinated by intragastric route with LL-VapA. Altogether, our results demonstrate that LL-VapA can evoke a T H 1-based protective immune response in intranasally immunized mice. This response is enhanced when co-administered with LL-Lep strain, whereas only co-administration of LL-VapA and LL-Lep can induce a protective immune response in intragastric vaccinated mice, associated with a T H 1/T H 2 cytokine response. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Rhodococcus equi, a facultative intracellular Gram-positive pathogen, is a leading cause of pneumonia in foals less than 6 months of age and is of great concern to horse-breeding farms [1]. This soil bacterium has also emerged as a significant opportunistic pathogen in immunocompromised humans, especially those with acquired immunodeficiency syndrome (AIDS), or patients under- going an immunosuppression treatment [2]. In addition, some rare cases of infection in immunocompetent patients have also been reported [3]. R. equi intracellular survival in macrophages is consid- ered to be necessary for the development of the disease [4], which is characterized by severe and sometimes fatal pyogranulomatous pneumonia in both humans and foals, although numerous other clinical manifestations may also occur [1]. There is now evidence ∗ Corresponding author. Tel.: +33 0 2 31 79 22 76; fax: +33 0 2 31 39 21 37. E-mail address: julien.cauchard@anses.fr (J. Cauchard). that the disease is regulated by the virulence of the pathogen and the peculiar susceptibility of the host. R. equi virulence is associated with the presence of a 85–90- kb plasmid carrying a pathogenicity island which encodes, among other genes, nine genes of the unique and R. equi-specific Vap (virulence-associated proteins) proteins family [5,6]. One mem- ber of this family, VapA, has been widely investigated since large amounts of antibodies directed against a highly immunogenic pro- tein of approximately 15–17 kDa were invariably found in the serum of foals infected with R. equi [7]. This lipoprotein, located at the bacterial surface, has been proven to be essential for virulence [8] and suggested to be important in inducing immunity against R. equi in mice and foals [9,10]. Because of the facultative intracellular nature of R. equi, protec- tion of foals against R. equi relies on both humoral and cell-mediated immune responses but its exact mechanism remains to be eluci- dated. Evidence from immune adult horses, supported by studies in murine models, indicate that production of opsonizing antibod- ies enhanced by CD4+ T cells together with macrophages activation 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.10.026