Vaccination with a Leishmania infantum HSP70-II null mutant confers long-term protective immunity against Leishmania major infection in two mice models

Abstract

Background: The immunization with genetically attenuated Leishmania cell lines has been associated to the induction of memory and effector T cell responses against Leishmania able to control subsequent challenges. A Leishmania infantum null mutant for the HSP70-II genes has been described, possessing a non-virulent phenotype.

Methodology/principal findings: The L. infantum attenuated parasites (LiΔHSP70-II) were inoculated in BALB/c (intravenously and subcutaneously) and C57BL/6 (subcutaneously) mice. An asymptomatic infection was generated and parasites diminished progressively to become undetectable in most of the analyzed organs. However, inoculation resulted in the long-term induction of parasite specific IFN-γ responses able to control the disease caused by a challenge of L. major infective promastigotes. BALB/c susceptible mice showed very low lesion development and a drastic decrease in parasite burdens in the lymph nodes draining the site of infection and internal organs. C57BL/6 mice did not show clinical manifestation of disease, correlated to the rapid migration of Leishmania specific IFN-γ producing T cells to the site of infection.

Conclusion/significance: Inoculation of the LiΔHSP70-II attenuated line activates mammalian immune system for inducing moderate pro-inflammatory responses. These responses are able to confer long-term protection in mice against the infection of L. major virulent parasites.

Fig 1. Evolution of parasite burdens and immune response induced after LiΔHSP70-II infection in BALB/c mice.

Mice (n = 8 per group) were inoculated with PBS (Saline) or with 1 × 10⁷ LiΔHSP70-II promastigotes in the vein tail (i.v.) (A, B) or in the right footpad (s.c.) (C, D, E). In A and C, scatter plots of LiΔHSP70-II parasite burdens are shown including the mean ± standard deviation (SD). Parasite loads were analyzed by limiting dilution at week 12 (12 wk) for i.v. inoculated animals (A) and at week 4 (4 wk) and 12 wk for s.c. inoculated ones (C) in the spleen (parasites per total organ), liver (parasites per g) or bone marrow (parasite per 1 × 10⁷ cells) (A). In (C) parasite burdens in the right popliteal lymph node (RP, parasites per total lymph node) and in the right footpad (RFP, parasites per total footpad) are shown. * (P < 0.05) indicates the statistical differences in 4 wk and 12 wk parasite burdens determined by a Mann-Whitney test. Spleen cell cultures for i.v. (B) or s.c. (D), or right popliteal lymph node cell cultures for s.c. (E) vaccinated animals were established at the indicated times after the inoculation of the LiΔHSP70-II line. The presence of cytokines in supernatants was measured after growing cells in the absence (Med) or in the presence of L. infantum SLA. In B and D, data show the mean ± SD of at least 8 mice per group. In E, data show the mean ± standard error of mean (SEM) of two independent assays performed with pooled cells from 8 mice. * P < 0 .05 shows statistical differences between SLA-stimulated and non-stimulated cells (unpaired Student t-test). The IgG1 and IgG2a reciprocal end-point titers against L. infantum SLA were analyzed by ELISA at the indicated times for i.v and s.c. inoculated mice, and represented as whisker (min to max) plots (F). * (P < 0.05) indicates the statistical differences between IgG1 and IgG2a anti-SLA titers (Kruskal-Wallis test and Dunn's Multiple Comparison post-test). No parasite loads or SLA-specific antibodies or cytokines were detected in mice receiving saline. Results are representative of at least two independent experiments.

Fig 2. Analysis of splenic T cell populations in vaccinated mice.

Mice (n = 4 per group) were inoculated with PBS (Saline; s.c.) or with 1 × 10⁷ LiΔHSP70-II promastigotes in the vein tail (i.v.) or in the right footpad (s.c.). After 4 weeks (vaccinated) and 12 weeks (saline and vaccinated), T cells from the spleen were studied in pool by flow cytometry. Animals were age matched at the moment of the analysis. In (A) analyzed CD4⁺ T cells were: antigen-experienced cells CD44^high, central memory T cells (Tcm) CD44^highCD62L^high and effector T cells or effector memory T cells (Teff/Tem) CD44^highCD62L^low. Spleen cells (B) or right popliteal lymph node cells (C) of the indicated groups were ex vivo stimulated by anti-CD3/anti-CD28 and treated with brefeldin A to block cytokine secretion. Cells were characterized by using anti-CD4 and anti-CD8 antibodies as well as by intracellular staining for IFN-γ. The ratio between the percentages of CD4⁺ or CD8⁺ producing IFN-γ cells in vaccinated animals versus saline ones, derived from two independent experiments is shown (mean ± standard error of mean).

Fig 3. Mice vaccinated with the attenuated LiΔHSP70-II line showed protection against an infective challenge with L. major.

BALB/c mice (n = 8 per group) inoculated with PBS (Saline) or with 1 × 10⁷ LiΔHSP70-II promastigotes in the vein tail (i.v.) (A, B) or in the right footpad (s.c.) (C, D) were challenged with 5 × 10⁴ stationary-phase L. major promastigotes in the left footpad at week 4 (s.c.) or at week 12 after vaccination (i.v. and s.c.). Footpad swelling was monitored weekly. Mean ± standard deviation (SD) is shown (A, C). L. major parasite burdens were determined by limiting dilution in the spleen, liver and in the draining lymph node (left popliteal). Scatter plots with the individual number of parasite per total organ (spleen or lymph nodes) or per g of liver are shown including the mean ± SD (B, D). * (P < 0 .05) shows the statistical differences determined by the one-way ANOVA test followed by the Tukey post-test. Results are representative of two independent experiments.

Fig 4. Humoral and cellular parasite induced responses after L. major challenge.

BALB/c mice (n = 8 per group) inoculated with PBS (Saline) or with 1 × 10⁷ LiΔHSP70-II promastigotes in the vein tail (i.v.) (A, C, D, E) or in the right footpad (s.c.) (B, F, G, H) were infected with 5 × 10⁴ stationary-phase L. major promastigotes in the left footpad at week 4 or at week 12 after vaccination. Animals were euthanized 8 weeks after L. major challenge. IgG1 and IgG2a reciprocal end-point titers against L. major SLA were determined by ELISA (A, B). Results are shown as whisker (min to max) plots of at least 8 mice per group. * (P < 0.05) indicates the statistical differences between IgG1 and IgG2a anti-SLA titers within each group or differences in IgG2a between short- and long-term protected mice (Mann-Whitney test). For cytokine determinations spleen cells were cultured in the absence (Med) or in the presence of L. major SLA. Levels of IFN-γ (C, F), IL-10 (D, G) and IL-4 (E, H) were assessed by ELISA in culture supernatants and shown as whisker (min to max) plots of at least 8 mice per group. * (P < 0.01) shows the statistical differences of saline and short-term or long-term vaccinated mice groups, or the statistical differences between short-term and long-term vaccinated mice groups (Mann-Whitney test). Results are representative of two independent experiments.

Fig 5. C57BL/6 mice chronically infected with the LiΔHSP70-II showed a Th1 like response against Leishmania.

C57BL/6 mice (n = 8) were infected with 1 × 10⁷ LiΔHSP70-II promastigotes in the right footpad (s.c.). LiΔHSP70-II parasite burdens were analyzed at week 4 or at week 12 after vaccination by limiting dilution in the spleen (Sp, parasites per total organ), liver (Liv; parasites per g), bone marrow (BM; parasites per 1 × 10⁷ cells), in the right popliteal lymph node (RP; parasites per total lymph node) or right footpad (RFP; parasites per total footpad) (A). Scatter plots are shown including the mean ± standard deviation (SD). No statistical differences were found in the parasite burdens in the RP from week 4 to week 12 whereas a significant decrease was shown in the RFP (unpaired Student t-test). At week 4 or at week 12 after vaccination, animals were euthanized and spleen cell cultures and sera were prepared. The IgG1 and IgG2c reciprocal end-point titer against L. infantum SLA was determined by ELISA and represented as whisker (min to max) plots (B). * (P < 0.05) indicates the statistical differences between IgG1 and IgG2c anti-SLA titers within each group (Mann-Whitney test). For cytokine determinations spleen cells (C) or RP cells (D) were cultured in the absence (Med) or in the presence L. infantum SLA. Levels of IFN-γ, IL-10 and IL-4 were assessed by ELISA in culture supernatants. Mean ± standard deviations (SD) are shown in C and mean ± standard error of mean (SEM) are shown in D. * (P < 0.01) shows the statistical differences between SLA-stimulated and non-stimulated cells (unpaired Student t-test). No parasite loads or SLA-dependent cytokines were detected in saline immunized mice. Results are representative of two independent experiments.

Fig 6. Evolution of L. major infection in C57BL/6 vaccinated mice.

Animals (n = 8 per group) inoculated with PBS (Saline) or with 1 × 10⁷ LiΔHSP70-II promastigotes in the right footpad (s.c.) were challenged with 1 × 10³ metacyclic L. major promastigotes in the dermis of both ears at week 4 or at week 12 after vaccination. (A) Ear lesion diameter was monitored weekly (16 ears from week 1 to 5 and 8 ears from week 6 to 13). Mean ± standard deviation (SD) is shown. L. major parasite burdens were determined at week 5 after L. major challenge by limiting dilution in the ears (n = 8 per group) (B) or in the retromandibular draining lymph node (n = 8 per group) (C). Scatter plots with the individual number of parasite per total organ are shown including the mean ± standard deviation (SD), * (P < 0.05) shows the statistical differences determined by the one-way ANOVA test followed by the Tukey post-test. For cytokine determinations the DLNs (D) or the spleen (E) from mice sacrificed at week 5 after L. major challenge (n = 4 mice) cells were cultured in the absence (Med) or in the presence L. major SLA. Levels of IFN-γ and IL-10 were assessed by ELISA in culture supernatants. The means ± SD are shown. * (P < 0.01) shows the statistical differences among saline and short-term or long-term vaccinated mice groups (unpaired Student t-test). The IgG1 and IgG2c reciprocal end-point titer against L. major SLA was determined by ELISA at week 5 after L. major challenge (n = 8 mice per group) and represented as whisker (min to max) plots (F). * (P < 0.05) indicates the statistical differences between IgG1 and IgG2c anti-SLA titers within each group (Mann-Whitney test). Results are representative of at least two independent experiments.

Fig 7. Vaccinated mice mounted an early immune response against Leishmania.

Animals inoculated with PBS (Saline) or with 1 × 10⁷ LiΔHSP70-II promastigotes in the right footpad (s.c.) were challenged with 1 × 10³ metacyclic L. major promastigotes in the dermis of both ears at week 4 and at week 12 after vaccination. Mice (n = 5) per group were weekly sacrificed to analyze parasite loads in both ears (A and C), or in the corresponding retromandibular draining lymph nodes (B and D). Scatter plots with the individual number of parasites per total organ are shown including the mean ± standard deviation (SD). * (P < 0 .05) shows the statistical differences determined by the unpaired Student t-test. The IgG2c reciprocal end-point titer against L. major SLA was analyzed by ELISA weekly after L. major challenge and represented as whisker (min to max) plots (E). * (P < 0.05) indicates the statistical differences in IgG2c anti-SLA titers among the three groups (Kruskal-Wallis test and Dunn's Multiple Comparison post-test). Level of L. major dependent IFN-γ produced in retromandibular cell cultures (F). Mean ± SD are shown. * (P < 0.01) shows the statistical differences between saline vaccinated mice groups (unpaired Student t-test).

Fig 8. Analysis of the early response after L. major challenge in the site of infection.

C57BL/6 mice were inoculated with PBS (12 week saline group; n = 6) or with 1 × 10⁷ LiΔHSP70-II promastigotes (12 week vaccinated group; n = 10) in the right footpad (s.c.). After 12 week, vaccinated animals were inoculated with PBS (PBS challenge; n = 5) or infected with 1 × 10⁵ L. major metacyclic promastigotes (L major challenge; n = 5) in the dermis of both ears. Mice inoculated in the right footpad (s.c.) with PBS (12 week saline group) were also challenged with L. major in the ear dermis. Three days after infection T cells from the ears and retromandibular lymph nodes were analyzed in pool by flow cytometry after anti-CD3/anti-CD28 ex vivo stimulation. Contour plots of IFN-γ intracellular staining for CD4⁺ (A) or CD8⁺ (B) cells (gated on CD3⁺) are shown. Numbers represent the percentage of each population.