Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis

Abstract

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.

FIG. 1.

Serological responses to Leish-111f in vaccinated mice. Groups of eight BALB/c mice were immunized three times at 3-week intervals by s.c. injection of 10 μg of Leish-111f with or without 5 (A), 10 (B), or 20 (C) μg of either MPL-SE or Ribi 529-SE adjuvant, with adjuvant alone, or with saline. Serum samples were collected 2 weeks after the last injection and analyzed by ELISA for the presence of anti-Leish-111f IgG1 and IgG2a. Each point represents the mean and standard error of the mean of data from eight individual mice. This experiment was repeated with similar results. OD (450-570), optical density at 450 to 570 nm.

FIG. 2.

T-cell responses of BALB/c mice immunized with Leish-111f formulated with adjuvants suitable for human use. BALB/c mice were immunized s.c. in the right rear footpad and at the base of the tail with 10 μg of Leish-111f formulated with 5, 10, or 20 μg of either MPL-SE or Ribi 529-SE (Corixa Corporation). Mice were boosted twice with a 3-week interval, and 10 days later, their spleens were removed and the splenocytes were stimulated in vitro with Leish-111f, TSA, LmSTI1, LeIF, and SLA (all at 10 μg/ml), concanavalin A (Con A; 1 μg/ml), or medium alone. Elicitation of IFN-γ (A) and IL-4 (B) was assessed by sandwich ELISA with supernatants removed after 72 h of in vitro incubation. Cells from three mice per group were pooled. Each bar represents the mean and standard error of triplicate wells. The data shown are the means of duplicate wells. This experiment was repeated with similar results.

FIG. 3.

APCs infected with metacyclic L. major promastigotes recognize Leish-111f. Leish-111f-specific T-cell lines or SLA-specific T-cell lines generated from mice immunized with Leish-111f formulated with MPL-SE or SLA formulated with murine rIL-12, respectively, were stimulated for 96 h with bone marrow-derived macrophages that were uninfected or had been previously infected at a multiplicity of infection of two to four parasites per cell. Proliferation (A) was measured by incorporation of [³H]thymidine. After 72 h of culture, supernatants were taken and assessed by ELISA for production of IFN-γ (B). The data shown are the means of triplicate wells. These experiments were repeated with similar results. Con A, concanavalin A.

FIG. 4.

Selection of adjuvant and adjuvant dose for the Leish-111f vaccine formulation. Groups of five mice were immunized three times at 3-week intervals by s.c. injection in the right hind footpad and at the base of the tail with 10 μg of Leish-111f plus adjuvant (either 5, 10, or 20 μg of MPL-SE or Ribi 529-SE). Control animals received either adjuvant alone or saline. Three weeks after the third immunization, mice were challenged s.c. in the left hind footpad with 2 × 10⁵ metacyclic L. major promastigotes and footpad enlargement was measured weekly thereafter.

FIG. 5.

Efficacy of Leish-111f plus MPL-SE against L. amazonensis. Groups of five C57BL/6 mice were immunized three times at 3-week intervals by s.c. injection in the right footpad and at the base of the tail with 10 μg of Leish-111f plus 20 μg of MPL-SE. Control animals received either adjuvant alone or saline. Three weeks after the third immunization, mice were challenged s.c. in the left hind footpad with 10⁶ L. amazonensis promastigotes. Footpad enlargement was measured every second week thereafter.

FIG. 6.

Effective memory immunity against L. major challenge by immunization with Leish-111f vaccine. Groups of five BALB/c mice were immunized three times at 3-week intervals by s.c. injection in the right footpad and at the base of the tail with 10 μg of Leish-111f plus 20 μg of MPL-SE. Control animals received either adjuvant alone or saline. At 3 weeks (A) or 3 months (B) after the third immunization, mice were challenged s.c. in the left footpad with 4 × 10⁵ L. major promastigotes and footpad enlargement was measured weekly thereafter and is expressed as mean swelling ± the standard error of the mean.