Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease

Abstract

Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis.

FIG. 1

T-cell responses of BALB/c mice immunized with the recombinant leishmanial antigens TSA and LmSTI1 formulated with IL-12 as an adjuvant. Mice were immunized subcutaneously in the rear footpad with either 10 μg of TSA, 10 μg of LmSTI1, or a mixture of 10 μg of each of these antigens. Before immunization, the antigens were mixed with IL-12 to achieve 1 μg of this cytokine per injection. Mice were boosted 3 weeks later with the same antigenic formulation used in the primary immunization. Ten days after the boost, animals were sacrificed and lymph node (popliteal) cells were obtained and cultured for 3 days in the presence of various concentrations of either TSA or LmSTI1 or with medium alone. Proliferative responses were assayed by incorporation of [³H]thymidine. Cytokine production (IFN-γ and IL-4) was assayed in culture supernatants by sandwich ELISA. (A) Proliferative responses and IFN-γ production of mice immunized with TSA (plus IL-12) or with a mixture of TSA plus LmSTI1 (plus IL-12) in response to stimulation with TSA. (B) Proliferative responses and IFN-γ production of mice immunized with LmSTI1 (plus IL-12) or with a mixture of TSA plus LmSTI1 (plus IL-12) in response to stimulation with LmSTI1. No IL-4 was detected in any culture supernatants (data not shown).

FIG. 2

Vaccination of BALB/c mice against L. major infection with the recombinant leishmanial antigens TSA and LmSTI1. Mice (five per group) were immunized subcutaneously in the left footpad twice (3 weeks apart) with 10 μg of either TSA or LmSTI1 or with a mixture of 10 μg of each of these antigens. Before immunization, the antigens were mixed with IL-12 (1 μg of this cytokine per injection). Three weeks after the last immunization, the mice were infected in the right footpad with 10⁴ amastigote forms of L. major; footpad swelling was measured weekly thereafter.

FIG. 3

Specific antibody response to the individual recombinant leishmanial proteins TSA and LmSTI1 in rhesus monkeys vaccinated with a mixture of these antigens formulated with recombinant human IL-12 and alum as adjuvants. Anti-LmSTI1 and anti-TSA antibody responses (IgG isotype) were tested by ELISA using a specific horseradish peroxidase-labeled goat anti-rhesus monkey IgG antiserum. Monkeys were immunized twice (approximately 1 month apart) with a vaccine containing, per dose, 25 μg of LmSTI1, 25 μg of TSA, 2 μg of IL-12, and 250 μg of alum in a final volume of 250 μl. The vaccine was administered subcutaneously in the deltoid area. One month after the second injection, the monkeys were boosted with a mixture containing 25 μg of each recombinant antigen suspended in 250 μl of saline-alum. All monkeys were anesthetized with ketamine before the injections. Results are from serum samples collected before immunization, at 3 weeks (w) after the first dose of vaccine, and at 1 week after each boost. All sera were diluted 1:20, and results are expressed as the optical density (OD) at 490 nm.

FIG. 4

Protection of rhesus monkeys against cutaneous leishmaniasis by vaccination with the recombinant leishmanial antigens LmSTI1 and TSA formulated with recombinant human IL-12 and alum as adjuvants. Monkeys were immunized as described in the legend to Fig. 2. Control monkeys were injected with saline only. Forty days after the last immunization, control and vaccinated monkeys (six animals per group) were infected intradermally in the left upper eyelid with 10⁷ metacyclic promastigotes of L. major suspended in 100 μl of RPMI medium. All monkeys were anesthetized with ketamine before the injections. Lesion development was inspected every 2 or 3 days, and lesion sizes, in square millimeters, were measured at 3-week intervals (bars show standard deviations).

FIG. 5

Morphological characteristics of lesions in control and vaccinated rhesus monkeys following infection with L. major. The typical macroscopic appearance of cutaneous leishmaniasis is illustrated at 6 and 8 weeks in control monkey O19 and at 8 weeks in control monkeys N15 and O25 (A). Vaccinated monkeys did not develop any lesions, as illustrated in four monkeys at 8 weeks after challenge (B). Also shown are microscopic characteristics of the inflammatory reactions in a control animal (A) and in a vaccinated animal (B). Note the large infiltration of granulocytes associated with nonspecific inflammation in the nonvaccinated monkey as opposed to the predominant mononuclear infiltration, a hallmark of the specific repair process, associated with fibrosis in the vaccinated animal.