Oral immunization using live Lactococcus lactis co-expressing LACK and IL-12 protects BALB/c mice against Leishmania major infection.

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. Current treatments are laborious, expensive, cause severe side effects, and emerging drug resistance has been reported. While vaccination is the most cost-effective means to control infectious diseases there is no human vaccine currently available against Leishmania infections. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used for the expression and delivery of biologically active molecules, such as antigens and cytokines, in mice and humans. In this study, we report the generation of L. lactis(alr-) strains solely expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall or co-expressing mouse IL-12. We show that oral immunization using live L. lactis, secreting both LACK and IL-12 was the only regimen that partially protected BALB/c mice against subsequent Leishmania major challenge. This highlights the importance of temporal and physical proximity of the delivered antigen and adjuvant for optimal immune priming by oral immunization since co-administration of L. lactis strains independently expressing secLACK and secIL-12 did not induce protective immunity. Protected animals displayed a delay in footpad swelling, which correlated with a significant reduction of parasite burden. Immunization with the L. lactis strain secreting both LACK and IL-12 induced an antigen-specific mucosal immune response and a LACK-specific T(H)1 immune response in splenocytes and mesenteric lymph node cells. Further, protection in immunized animals correlated with a strong Leishmania-specific T(H)1 immune response post-challenge, detectable in splenocytes and lymph node cells draining the site of infection. This report demonstrates the use of L. lactis as an oral live vaccine against L. major infection in susceptible BALB/c mice. The vaccine strains generated in this study provide the basis for the development of an inexpensive and safe oral live vaccine against the human parasite Leishmania.

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling.

BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.

Generation and evaluation of A2-expressing Lactococcus lactis live vaccines against Leishmania donovani in BALB/c mice.

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. As current treatments are insufficient, the development of an effective vaccine is a priority. This study generated and assessed the efficacy of Leishmania vaccines engineered from the non-colonizing, non-pathogenic Gram-positive bacterium Lactococcus lactis. A truncated, codon-optimized version of the A2 antigen from Leishmania donovani was engineered for expression in Lactococcus lactis in three different subcellular compartments: in the cytoplasm, secreted outside the cell or anchored to the cell wall. These three A2-expressing Lactococcus lactis strains were tested for their ability to generate A2-specific immune responses and as live vaccines against visceral Leishmania donovani infection in BALB/c mice. Subcutaneous immunization with live Lactococcus lactis expressing A2 anchored to the cell wall effectively induced high levels of antigen-specific serum antibodies. It was demonstrated that Lactococcus lactis-based vaccines are a feasible approach in the generation of live vaccines against leishmaniasis. The Lactococcus lactis strains generated in this study provide an excellent foundation for further studies on live bacterial vaccines against leishmaniasis and other pathogens.