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 SMT-expressing «Lactococcus lactis» live vaccines against visceral leishmaniasis

Lactococcus lactis is a Gram-positive lactic acid bacterium commonly used in the dairy industry. It has been deemed safe for human consumption by the United States Food and Drug Administration and holds Generally Recognised As Safe (GRAS) status. L. lactis is non-pathogenic, non-colonizing and non-invasive. The utility of this bacterium as a cell "factory" able to synthesis heterologous proteins is now well established. Previous work in our lab has also shown that L. lactis has the ability to stimulate the innate immune system. All these properties make L. lactis a suitable vector for biological delivery of antigens to stimulate an immune response.Visceral leishmaniasis is a disseminated protozoan infection, caused by the species of the Leishmania donovani complex, which affects the liver, spleen and bone marrow. There are 500,000 new cases of visceral leishmaniasis every year and 90% of clinical cases are fatal if left untreated. In addition to the disadvantages of high cost and side effects of chemotherapy, there is also emerging resistance of parasites to anti-leishmanial drugs. In light of these facts, the development of a vaccine against visceral leishmaniasis is a priority. We have engineered L. lactis (strains NZ9000 and PH3960) to heterologously express the protective Leishmania antigen, sterol 24-c-methyltransferase (SMT), in the cytoplasm. In separate studies, these strains of L. lactis were assessed for their protective capability as live vaccines against L. donovani in BALB/c mice, both individually and adjuvanted with IL-12-secreting L. lactis. Immune responses to subcutaneous immunizations were monitored by measuring serum antibody titers against SMT and cytokine production from splenocytes upon stimulation by soluble Leishmania antigen at the end point of infection. Mice immunised with SMT-expressing clones of L. lactis NZ9000 had high titers of both serum IgG1 and IgG2a as well as splenocyte cytokine response profiles which demonstrated a mixed Th1/Th2 response. The co-administration of SMT- and murine IL-12-producing L. lactis clones was able to reduce parasite load in the liver significantly in the case of L. lactis NZ9000. Overall, we demonstrated that L. lactis is a suitable vehicle for the delivery of vaccine antigens and generation of protective immune responses against visceral leishmaniasis.Lactococcus lactis est une bactérie lactique Gram-positive couramment utilisée dans l'industrie laitière. Elle a été jugée sans danger pour la consommation humaine par la "United States Food and Drug Administration" et elle détient le statut "GRAS" (Generally Recognised As Safe). L. lactis est non pathogène, non-colonisatrice et non-invasive. L'utilité de cette bactérie comme une cellule «usine» capable de synthétiser des protéines hétérologues est maintenant bien établie. Des travaux antérieurs dans notre laboratoire ont également montré que L. lactis a la capacité de stimuler le système immunitaire inné. Toutes ces propriétés font de L. lactis un vecteur approprié pour l'administration biologique d'antigènes afin de stimuler une réponse immunitaire. La leishmaniose viscérale est une infection protozoaire diffuse, causée par les espèces du complexe Leishmania donovani, qui affecte le foie, la rate et la moelle osseuse. Chaque année, 500,000 nouveaux cas de leishmaniose viscérale sont déclarés et 90% des cas cliniques sont mortels s'ils ne sont pas traités. En plus des inconvénients de coût élevé et les effets secondaires de la chimiothérapie, un autre problème majeur est l'émergence de parasites résistants aux médicaments anti-Leishmania. C'est pourquoi le développement d'un vaccin contre la leishmaniose viscérale est une priorité. Nous avons modifié L. lactis (souches NZ9000 et PH3960) afin qu'elle exprime l'antigène protecteur de Leishmania, stérol 24-C-méthyltransférase (SMT), de façon hétérologue dans le cytoplasme. Dans des études distinctes, ces souches de L. lactis ont été évaluées pour leur capacité de protection comme vaccins vivants contre L. donovani chez la souris BALB/c, individuellement, mais aussi en présence de L. lactis sécrétant de l'IL-12 en tant qu'adjuvant. Les réponses immunitaires à ces vaccinations sous-cutanées ont été suivies en mesurant les titres d'anticorps sériques dirigés contre SMT et la production de cytokines provenant de splénocytes stimulués par les antigènes solubles de Leishmania au point final de l'infection. Les souris immunisées avec les clones de L. lactis NZ9000 exprimant SMT montrent des taux sériques élevés d'IgG1 et d'IgG2a, ainsi qu'un profil de cytokines splénique démontrant une réponse mixte Th1/Th2. La co-administration de clones de L. lactis exprimant SMT et de l'IL-12 murin a permis de réduire la charge parasitaire dans le foie de manière significative dans le cas de L. lactis NZ9000. Dans l'ensemble, nous avons démontré que L. lactis est un vecteur approprié pour l'administration d'antigènes vaccins et la génération de réponses immunitaires protectrices contre la leishmaniose viscérale

Expression and localization of the <i>Leishmania</i> LACK antigen and single-chain mouse IL-12 in <i>L. lactis</i>.

<p>(A) Western blot of total protein extracts of LACK-expressing strains of <i>L. lactis</i>. T, total cell extract; S, culture supernatant. (B) Localization of expressed LACK was confirmed by whole cell ELISA using anti-LACK antibody. C, intact cells; S, culture supernatant. * <i>P</i><0.05 by unpaired T-test to <i>L. lactis</i>/vector. Data shown are representative of three independent experiments with similar results. (C) Total protein extracts of <i>L. lactis</i> were analyzed for IL-12 expression by Western blot using anti-IL-12p70 antibody. (D) Secretion of IL-12 by <i>L. lactis</i> was quantified using mouse IL-12p70 ELISA. * <i>P</i><0.05 by unpaired T-test to <i>L. lactis</i>/secIL-12 wt. Data shown are representative of at least three independent experiments with similar results. (E) IFN-γ secretion by BALB/c splenocytes stimulated with αCD3/αCD28 in combination with rIL-12 or concentrated supernatant from <i>L. lactis</i> secreting IL-12 was determined by ELISA. * <i>P</i><0.05 by unpaired T-test to αCD3/αCD28. Data shown are representative of three independent experiments with similar results.</p

Protection against <i>L. major</i> infection correlates with <i>Leishmania</i>-specific T_H1 response in immunized animals.

<p>Mice were immunized three times subcutaneously with PBS or different strains of <i>L. lactis</i> and challenged with <i>L. major</i>. Mice were sacrificed eight days (A, B), or ten weeks (C–G) after challenge (pm: <i>post mortem</i>). Lymph node cells (A–E), or splenocytes (F, G) were restimulated with SLA <i>ex vivo</i>. IFN-γ, IL-10, and IL-2 secretion by restimulated draining lymph node cells and splenocytes were determined by ELISA. Mean and SEM of four to five mice per group are shown. * <i>P</i><0.05; ** <i>P</i><0.01 by unpaired T-test to PBS and <i>L. lactis</i>/vector. ^▴<i>P</i><0.05 by unpaired T-test to PBS. Data shown are representative of two independent experiments with similar results.</p

Antigen-specific humoral immune response following immunization with LACK-expressing <i>L. lactis</i>.

<p>Mice were immunized three times subcutaneously with PBS or different strains of <i>L. lactis</i> and blood was collected one week after the last immunization (A), four weeks after challenge (B) or ten weeks after challenge (C). LACK-specific IgG₁ and IgG_2a antibody titers in sera were determined by ELISA. Mean and SEM of four to five mice per group are shown. * <i>P</i><0.05 by unpaired T-test to PBS and <i>L. lactis</i>/vector. Data shown are representative of two independent experiments with similar results.</p

Immunization with <i>L. lactis</i>/cwaLACK and <i>L. lactis</i>/secIL-12 induces systemic T_H1 immune responses and multifunctional T_H1 cells pre-challenge.

<p>Mice were immunized three times subcutaneously with PBS or different strains of <i>L. lactis</i>. Mice were sacrificed two weeks after the last immunization and splenocytes were restimulated with purified LACK antigen <i>ex vivo</i>. (A, B) IFN-γ, IL-10, and (C) IL-2 secretion after three days of restimulation was determined by ELISA. (D) Splenocytes were restimulated for 16 hours and cytokine expression was analyzed by flow cytometry. Frequencies of CD4⁺ and CD8⁺ T cells positive for IFN-γ, IL-2, or TNF-α or the combination of the different cytokines are shown. (E) Fraction of the total CD4⁺ or CD8⁺ response comprising cells expressing any two cytokines (2+), or any one cytokine (1+). Mean and SEM of four to five mice per group are shown. * <i>P</i><0.05; ** <i>P</i><0.01 by unpaired T-test to PBS and <i>L. lactis</i>/vector. ^▴<i>P</i><0.05 by unpaired T-test to PBS. Data shown are representative of two independent experiments with similar results.</p

Immunization with <i>L. lactis</i>/cwaLACK and <i>L. lactis</i>/secIL-12 delays footpad swelling and reduces parasite burden in <i>L. major</i> infected BALB/c mice.

<p>Mice were immunized three times subcutaneously with PBS or different strains of <i>L. lactis</i> and challenged with <i>L. major</i> promastigotes into the right hind footpad. (A) Change in footpad swelling post-infection was determined by measuring the thickness of the infected footpad and subtracting the thickness of the contralateral, uninfected footpad at weekly intervals. Animals were sacrificed ten weeks after parasite challenge and parasite burden in infected footpads was determined by (B) PCR-ELISA or (C) limiting dilution. Mean and SEM of four to five mice per group are shown. * <i>P</i><0.05; ** <i>P</i><0.01 by unpaired T-test to PBS and <i>L. lactis</i>/vector. ^▴<i>P</i><0.05 by unpaired T-test to PBS. Data shown are representative of three (A), one (B), or two (C) independent experiments with similar results.</p

Bacterial strains and primers used.

<p>Bacterial strains and primers used.</p