Genetic Engineering of Lactococcus lactis Co-producing Antigen and the Mucosal Adjuvant 3′ 5′- cyclic di Adenosine Monophosphate (c-di-AMP) as a Design Strategy to Develop a Mucosal Vaccine Prototype

Abstract

<i>Lactococcus lactis</i> 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 <i>L. lactis</i> strain. First, we demonstrate that homologous expression of <i>cdaA</i> gen in <i>L. lactis</i> is able to increase c-di-AMP levels. Thus, we hypothesized that <i>in vivo</i> 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 <i>Trypanosoma cruzi</i> 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 <i>L. lactis</i> strain expressing antigenic TScf combined with another <i>L. lactis</i> strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional <i>L. lactis</i> strain containing a single plasmid with both <i>cdaA</i> and <i>tscf</i> 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 <i>L. lactis</i> 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.