Cytokine production before and after challenge of mice with <i>L. major</i> -infected sand flies.

<p>Mice were vaccinated subcutaneously with 10 µg KSAC+20 µg GLA-SE, 10 µg L110f+20 µg GLA-SE, or 20 µg GLA-SE alone. IFN-γ (A) or IL-10 (B) production was measured by ELISA after <i>in vitro</i> stimulation of spleen cells with 100 µg SLA, 10 µg recombinant KSAC, or 10 µg recombinant L110f. Results are from stimulations performed at two time points: three weeks after the last immunization and one week post challenge. (C) Ratio of IFN-γ∶ IL-10 in vaccinated mice. Mean and SEM of three mice per group. Data are representative of two independent experiments. nd = none detected.</p

IFN-γ-producing CD4+ T cells before and after challenge with <i>L. major</i> -infected sand flies.

<p>Mice were vaccinated subcutaneously with 10 µg KSAC+20 µg GLA-SE, 10 µg L110f+20 µg GLA-SE, or 20 µg GLA-SE alone. Splenocytes were analyzed three weeks after the last immunization (Pre-challenge) or one week Post challenge with <i>L. major</i>. Splenocytes gated on TCRβ⁺CD4⁺ T cells for the percentage of IFN-γ⁺ CD4⁺ T cells produced after antigen stimulation (A–C). (A) Splenocytes from KSAC+GLA-SE- or GLA-SE-vaccinated mice stimulated with 20 µg recombinant KSAC <i>in vitro</i>; (B) Splenocytes from L110f+GLA-SE- or GLA-SE-vaccinated mice stimulated with 20 µg recombinant L110f; or (C) Splenocytes from KSAC+GLA-SE-, L110f+GLA-SE-, or GLA-SE-vaccinated mice stimulated with 100 µg SLA <i>in vitro</i>. (D) Splenocytes gated on TCRβ⁺CD4⁺CD62L^lowCCR7^low T cell population for the percentage of effector memory CD4⁺ splenic T cells in mice vaccinated with GLA-SE, KSAC+GLA-SE or L110f+GLA-SE after stimulation with either KSAC, L110f or SLA. Three mice were used per group. Data are representative of two independent experiments.</p

Antibody responses induced by vaccination with KSAC+GLA-SE or L110f+GLA-SE.

<p>BALB/c mice were vaccinated by the subcutaneous route three times every three weeks with either 20 µg adjuvant alone (GLA-SE), 10 µg KSAC+20 µg GLA-SE, or 10 µg L110f+20 µg GLA-SE. Serum was obtained one day before each immunization and 20 days after the last immunization. (A) KSAC- and L110f-specific IgG1 and IgG2a antibody levels in mice vaccinated with the respective antigen. (B) Ratio of antigen-specific IgG2a∶IgG1 levels in mice vaccinated with KSAC+GLA-SE or L110f+GLA-SE. The OD values are presented after subtraction of the OD value of mice vaccinated with GLA-SE alone. Ten mice were used in each group. Data are representative of two independent experiments.</p

Vaccination of mice with KSAC+GLA-SE provides better protection from vector-transmitted <i>L. major</i> than does L110f+GLA-SE.

<p>BALB/c mice were challenged three weeks after the last vaccination in one ear with a needle injection of 2000 purified <i>L. major</i> metacyclics or exposed in one ear to bites of 10 <i>L. major</i>-infected sand flies (<i>P. duboscqi</i>). (A and C) Ear lesion thickness was measured in mice vaccinated with GLA-SE (•), KSAC+GLA-SE (▪), or L110f+GLA-SE (▴) and challenged with needle (A) or infected-sand fly bites (C). Panels show representative ears of vaccinated mice eight weeks after challenge. (B and D) Parasite load determined by Real-Time PCR eight weeks after challenge with needle (B) or infected-sand fly bites (D). Statistical significance was determined for KSAC+GLA-SE- or L110f+GLA-SE-vaccinated mice compared to the GLA-SE-vaccinated mice using a two-tailed, unpaired Student's t-test (*, p<0.05; **, p<0.01; ***, p<0.001). Five to seven mice were used in each group. Data are representative of three independent experiments.</p

Vaccination with KSAC+GLA-SE confers long-term protection against <i>L. major</i> -infected <i>P. duboscqi</i> sand flies.

<p>Mice were vaccinated subcutaneously with 10 µg KSAC+20 µg GLA-SE, 10 µg L110f+20 µg GLA-SE, or 20 µg GLA-SE alone. (A) The ratio of IgG2a∶IgG1 was determined 12 weeks after the last vaccination and prior to the delayed challenge. (B) Lesion thickness in mice vaccinated with KSAC+GLA-SE (▪), L110f+GLA-SE (▴), or GLA-SE alone (•) and challenged with <i>L. major</i>-infected sand flies in the right ear 12 weeks (delayed challenge) after the last immunization. Panels show representative ears of vaccinated mice five weeks after challenge. (C) Parasite load determined by Real-Time PCR five weeks after challenge with infected-sand fly bites. Statistical significance was determined for mice vaccinated with KSAC+GLA-SE or L110f+GLA-SE compared to the adjuvant group using a two-tailed unpaired Student's t-test (*, p<0.05; **, p<0.01; ***, p<0.001). Ten mice were used per group. Data are representative of two independent experiments.</p