Subcutaneous Immunization of Leishmania HSP70-II Null Mutant Line Reduces the Severity of the Experimental Visceral Leishmaniasis in BALB/c Mice

Abstract

Leishmania infantum parasites cause a severe form of visceral leishmaniasis in human and viscerocutaneous leishmaniasis in dogs. Recently, we reported that immunization with an attenuated L. infantum cell line, lacking the hsp70-II gene, protects against the development of murine cutaneous leishmaniasis. In this work, we analyzed the vaccine potential of this cell line towards the long-term protection against murine visceral leishmaniasis. This model shows an organ-dependent evolution of the disease. The infection can resolve in the liver but chronically affect spleen and bone marrow. Twelve weeks after subcutaneous administration of attenuated L. infantum, Bagg Albino (BALB/c) mice were challenged with infective L. infantum parasites expressing the luciferase-encoding gene. Combining in vivo bioimaging techniques with limiting dilution experiments, we report that, in the initial phase of the disease, vaccinated animals presented lower parasite loads than unvaccinated animals. A reduction of the severity of liver damage was also detected. Protection was associated with the induction of rapid parasite-specific IFN-γ production by CD4⁺ and CD8⁺ T cells. However, the vaccine was unable to control the chronic phase of the disease, since we did not find differences in the parasite burdens nor in the immune response at that time point.

Keywords: IFN-γ; T-cells; attenuated parasites; mice; vaccine; visceral leishmaniasis.

Figure 1

Evaluation of the L. infantum infection by in vivo bioluminescent imaging (BLI). BALB/c mice (n = 5 per group) were inoculated s.c. (left footpad) with PBS (control group) or vaccinated with 10⁷ LiΔHSP70-II attenuated parasites prepared in PBS. Twelve weeks after inoculation, animals were challenged i.v. with 10⁸ PpyRE9h⁺ L. infantum infective promastigotes. Radiance data are represented as box and whisker (min to max) plots. Statistics were obtained using a Mann–Whitney test. * shows the statistical differences between control and vaccinated mice (p < 0.05). Results are representative of two independent experiments.

Figure 2

Parasite load monitoring in the liver, spleen, and bone marrow. The presence of viable parasites of the PpyRE9h⁺ L. infantum line in control and vaccinated mice was analyzed by limiting dilution in the presence of puromycin at week 5 (initial phase; n = 8) and at week 11 (late phase; n = 8) after challenge with 10⁸ promastigotes. Samples from each mouse were processed and analyzed independently. The graph shows the mean (+SD) of parasites per g in the liver, spleen (total organ), or parasites per 1 × 10⁷ bone marrow cells. * (p < 0.05) shows the statistical differences between control and vaccinated mice. ⁺ (p < 0.05) shows the statistical differences between the initial and late phases of control mice. ^× (p < 0.05) shows the statistical differences between the initial and late phases of vaccinated mice. All samples were analyzed using a Student T test. Results are representative of at least two independent experiments.

Figure 3

Vaccinated animals develop an earlier parasite-specific antibody response than controls after infective challenge. The reciprocal end-point titer of IgG1 or IgG2a against leishmanial antigens was determined by ELISA from sera samples taken at the silent phase (a; 1 week post-challenge), at the initial phase (b; 5 weeks post-challenge), and at the late phase of infection (c; 11 weeks post-challenge). Sera samples from control (n = 8) and vaccinated (n = 8) animals were studied individually. Data are represented as box and whisker (min to max) plots and statistics were analyzed by a Mann–Whitney test. * (p < 0.05) shows the statistical differences between IgG1 and IgG2a titers in the control group. ⁺ (p < 0.05) or ^× (p < 0.05) shows the statistical differences between vaccinated and control groups of IgG1 or IgG2a titers, respectively. Results are representative of two independent experiments.

Figure 4

Analyses of parasite-specific IFN-γ production. BALB/c mice (n = 8 per group) were inoculated s.c. in the left footpad with PBS (control group) or vaccinated with 10⁷ LiΔHSP70-II attenuated parasites prepared in PBS. Twelve weeks after inoculation, animals were challenged i.v. with 10⁸ PpyRE9h⁺ L. infantum infective promastigotes. Spleen cell cultures from each mouse were independently established at the silent phase (1 week post-challenge), at the initial phase (5 weeks post-challenge), and at the late phase of infection (11 weeks post-challenge) and stimulated (SLA) or not (medium) with soluble leishmanial antigens for 72 h. IFN-γ levels were measured in culture supernatants by quantitative sandwich ELISA (a). Spleen cell cultures established at the indicated phases were independently stimulated for 24 h with BM-DCs pulsed or not with SLA. Afterwards, cells were processed for flow cytometry. The percentages of IFN-γ-secreting cells in CD4⁺ (b) or CD8⁺ (c) gates are shown. All data are represented as the mean (+ SD) and statistics were analyzed by a Student T test. * (p < 0.05) shows the statistical differences between control and vaccinated mice. ⁺ (p < 0.05) shows the statistical differences between SLA stimulation and non-stimulation in the control group. ^× (p < 0.05) shows the statistical differences between SLA stimulation and non-stimulation in the vaccinated group. Results are representative of two independent experiments.

Figure 5

Parasite-specific IL-10 and IL-4 production. BALB/c mice (n = 8 per group) were vaccinated and infected as in Figure 4. Spleen cell cultures from each mouse were independently established in the silent, initial, and late phases and stimulated for 72 h with soluble SLA or cultured without stimulus (medium). IL-10 (a) or IL-4 (b) levels were measured in culture supernatants by quantitative sandwich ELISA. Data are represented as the mean (+ SD) and statistics were analyzed by a Student T test. * (p < 0.05) shows the statistical differences between control and vaccinated mice. ⁺ (p < 0.05) shows the statistical differences between SLA-stimulated and unstimulated cytokine production in the control group. ^× (p < 0.05) shows the statistical differences between SLA-stimulated and unstimulated levels in the vaccinated group. Results are representative of two independent experiments.