Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice

Abstract

We have examined the efficacy of the administration in mice of a molecularly defined vaccine based on the Leishmania infantum acidic ribosomal protein P0 (rLiP0). Two different challenge models of murine cutaneous leishmaniasis were used: (i) subcutaneous inoculation of L. major parasites in susceptible BALB/c mice (a model widely used for vaccination analysis) and (ii) the intradermal inoculation of a low infective dose in resistant C57BL/6 mice (a model that more accurately reproduces the L. major infection in natural reservoirs and in human hosts). First, we demonstrated that C57BL/6 mice vaccinated with LiP0-DNA or rLiP0 protein plus CpG oligodeoxynucleotides (ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load. This protection was associated with production of gamma interferon (IFN-gamma) in the dermal site. Secondly, we showed that immunization with rLiP0 plus CpG ODN is able to induce only partial protection in BALB/c, since these mice finally developed a progressive disease. Further, we demonstrated that LiP0 vaccination induces a Th1 immunological response in both strains of mice. In both cases, the antibodies against LiP0 were predominantly of the immunoglobulin G2a isotype, which was correlated with an rLiP0-stimulated production of IFN-gamma in draining lymph nodes. Finally, we demonstrated that LiP0 vaccination does not prevent the Th2 response induced by L. major infection in BALB/c mice. Taken together, these data indicate that the BALB/c model of cutaneous leishmaniasis may undervalue the potential efficacy of some vaccines based on defined proteins, making C57BL/6 a suitable alternative model to test vaccine candidates.

FIG. 1.

Course of L. major infection in C57BL/6 vaccinated mice. Mice (eight per group) were vaccinated s.c. with three doses of either 100 μg of pcDNA3 or 100 μg of pcDNA3-LiP0, and twice with 10 μg of rLiP0 plus 50 μg of CpG ODN, 50 μg of ALM plus 50 μg of CpG ODN, 50 μg of CpG ODN alone or PBS. 4 weeks after the last inoculation, mice were infected by i.d. inoculation into the ear with 1,000 metacyclic promastigotes of L. major. Values represent the mean lesion diameter + standard deviation (SD). *, P < 0.001 significant decrease for ALM or rLiP0 plus CpG ODN compared with CpG ODN alone, and pcDNA3-LiP0 compared with pcDNA3. The experiment was repeated with similar results.

FIG. 2.

Parasite burden in the ear dermis (A) and in the local draining lymph nodes (B) in C57BL/6 mice quantitated at 4 weeks postinfection (white bars) and 16 weeks postinfection (grey bars). Results are expressed as the mean ± SD of eight ears and draining lymph nodes. *, P < 0.05, significant decrease for rLiP0 plus CpG ODN or ALM plus CpG ODN compared with CpG ODN alone, and pcDNA3-LiP0 compared with pcDNA3. Results represent the mean and standard deviation. The experiment was repeated with virtually the same results.

FIG. 3.

Leishmania-specific production of IFN-γ after infection with L. major. C57BL/6 mice were vaccinated as described above and then challenged with 1,000 metacyclic promastigotes of L. major. Dermal cell suspensions from the pooled infected ears (A) and DLN cells (B) were obtained 4 weeks after infection from C57BL/6 mice (four mice per group, eight ears and eight DLN). The cultures were stimulated with either uninfected or L. major-infected BMDDC. IFN-γ levels were measured in culture supernatants 48 h later by ELISA. Data are the mean concentrations of triplicate assays + SD. (C) The intracellular production of IFN-γ by CD4⁺ and CD8⁺ T cells in C57BL/6 mice was assessed in the dermal cells of mice vaccinated with pcDNA3-LiP0 and rLiP0 + CpG and their corresponding controls (empty DNA vector and CpG, respectively). After 24 h of in vitro restimulation with uninfected BMDDC or infected BMDDC, cells were processed for flow cytometry. Analyses are gated on TCRβ⁺ cells. Numbers represent the percentage of CD4⁺ or CD8⁺ T cells positive for IFN-γ.

FIG. 4.

Course of L. major infection in BALB/c vaccinated mice. (A) BALB/c mice (six per group) were immunized s.c. twice with either PBS, 50 μg CpG ODN or rLiP0 (0.5, 2, or 10 μg) plus 50 μg of CpG ODN. One month after the last immunization, the animals were infected in the left hind footpad with 5 × 10⁴ L. major promastigotes. Footpad swelling is given as the difference of thickness between the infected and the uninfected contralateral footpad. Four weeks after challenge, differences between controls (CpG ODN) and vaccinated mice (10 μg of rLiP0 plus CpG ODN) were found to be significant (*, P < 0.001). At weeks 4 (white bars) and 8 after infection (grey bars), the number of viable parasites present in the footpad per mg (B) and total parasites in the popliteal lymph node of the infected leg (C) were determined by limiting dilution. Results represent the mean and standard deviation representative of three independent experiments (*, P < 0.05).

FIG. 5.

Analysis of the specific humoral response induced in BALB/c and C57BL/6 mice immunized with 10 μg of rLiP0 plus CpG ODN. One month after the second inoculation, mice (n = 6) were bled and sera were tested by ELISA for specific anti-LiP0 antibody responses of both IgG1 and IgG2a isotypes (A). Each sample was assayed separately, and the titer was determined as indicated in the Materials and Methods section. The reactivity of these sera against the synthetic peptides covering the LiP0 sequence was analyzed by FAST-ELISA (B). The optical density at 450 nm (OD₄₅₀) values represent the reactivities obtained with the pooled sera diluted 1:500 using as second antibody a horseradish peroxidase-conjugated anti-IgG at 1:2,000 dilution. None of the preimmune, the PBS or the CpG ODN control sera showed reactivity against either the recombinant protein or the synthetic peptides (not shown).

FIG. 6.

Cytokine production by draining LNCs of LiP0-vaccinated mice. Mice (three per group) were immunized twice s.c. with either PBS, 50 μg CpG ODN or 10 μg of rLiP0 plus 50 μg CpG ODN. One month after the last immunization, the animals were euthanized, and their LNCs were obtained and cultured in vitro for 3 days in the presence of rLiP0 or medium alone (panel A). In parallel, DLN cultures were stimulated with rLiP0 in the presence of either anti-IL-12, anti-CD4 or anti-CD8 monoclonal antibodies (panel B). In both cases, the supernatants were harvested and assayed for cytokine production. The levels of IFN-γ were assessed by ELISA after 72 h of incubation. Data correspond to one representative experiment of two independent experiments with similar results. Differences in IFN-γ production between treatment with anti-CD8 monoclonal antibodies and treatment with control antibodies were statistically significant (*, P < 0.05).

FIG. 7.

Spleen cell IFN-γ response to LiP0 and its synthetic peptides. Splenocytes from BALB/c and C57BL/6 mice vaccinated with 10 μg of rLiP0 plus 50 μg CpG ODN were stimulated with BMDDC pulsed with whole rLiP0 or the synthetic peptides covering the LiP0 sequence. The levels of IFN-γ were assessed by ELISA after 72 h of incubation. Data correspond to one representative experiment of two independent experiments with similar results.

FIG. 8.

Analysis of the Th2 response after L. major infection. (A) Eight weeks after parasite challenge vaccinated BALB/c and C57BL/6 mice (n = 5) were bled and sera were tested by ELISA for specific anti-LiP0 antibody responses of both IgG1 and IgG2a isotypes. The titers of the anti-rLiP0 antibodies in the BALB/c CpG control mice at week 8 after challenge were 1/2,700 and 1/900 for IgG1 and IgG2a, respectively (not shown). (B) At weeks 4 and 8 after the L. major challenge, the animals were euthanized, and their splenocytes were obtained and cultured in vitro for 3 days in the presence of rLiP0 or SLA. The supernatants were harvested and assayed for IL-4 by ELISA.