Abstract
Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 3′ 5′- cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.
Highlights
Lactococcus lactis is one of the most frequently used microorganisms in the food industry across the world (de Vos, 2011; Smid and Kleerebezem, 2014)
CdaA was amplified and cloned in the pBV153 vector, resulting in plasmid pIQ101 (Figure 1A and Table 1). pBV153 was developed in our laboratory and it has the Pcit promoter upstream of the multiple cloning site, leaving the expression of the gene of interest under pH regulation (Marelli and Magni, 2010). pIQ101 plasmid was electroporated in L. lactis IL1403, originating L. lactis cdaA+ (LL1, Table 1)
L. lactis cdaA+ showed a saline hypersensitivity growth defect at 0.25 M NaCl or upon addition of antibiotic compounds (Ampicillin 0.25 μg/ml, Penicillin 0.10 μg/ml, Vancomicyn 0.50 μg/ml), or Lysozyme 0.10 μg/ml (Quintana, 2018). These results suggest that overproduction of CdaA mediates an increment of the intracellular synthesis of c-diAMP that was previously related to the observed phenotypes in L. lactis and other bacteria (Smith et al, 2012; Gundlach et al, 2015; Rismondo et al, 2016; Quintana, 2018)
Summary
Lactococcus lactis is one of the most frequently used microorganisms in the food industry across the world (de Vos, 2011; Smid and Kleerebezem, 2014). L. lactis has been successfully employed to produce specific viral and bacterial antigens to cope infections or non-antigenic immunomodulatory proteins like cytokines or proteases to control infections or more complex inflammatory diseases such as the inflammatory bowel disease (Miyoshi et al, 2002; Bermudez-Humaran et al, 2003; Foligne et al, 2007; Wells and Mercenier, 2008; Marelli et al, 2011; Cano-Garrido et al, 2015; Kim et al, 2015; Mancha-Agresti et al, 2017). It has been used for the expression and delivery of heterologous antigens to develop oral and mucosal vaccines (Wells and Mercenier, 2008; Cano-Garrido et al, 2015)
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