Vaccination with L. infantum chagasi nucleosomal histones confers protection against new world cutaneous leishmaniasis caused by Leishmania braziliensis

Abstract

Background: Nucleosomal histones are intracellular proteins that are highly conserved among Leishmania species. After parasite destruction or spontaneous lysis, exposure to these proteins elicits a strong host immune response. In the present study, we analyzed the protective capability of Leishmania infantum chagasi nucleosomal histones against L. braziliensis infection using different immunization strategies.

Methodology/principal findings: BALB/c mice were immunized with either a plasmid DNA cocktail (DNA) containing four Leishmania nucleosomal histones or with the DNA cocktail followed by the corresponding recombinant proteins plus CpG (DNA/Protein). Mice were later challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development, parasite load and the cellular immune response were analyzed five weeks after challenge. Immunization with either DNA alone or with DNA/Protein was able to inhibit lesion development. This finding was highlighted by the absence of infected macrophages in tissue sections. Further, parasite load at the infection site and in the draining lymph nodes was also significantly lower in vaccinated animals. This outcome was associated with increased expression of IFN-γ and down regulation of IL-4 at the infection site.

Conclusion: The data presented here demonstrate the potential use of L. infantum chagasi nucleosomal histones as targets for the development of vaccines against infection with L. braziliensis, as shown by the significant inhibition of disease development following a live challenge.

Figure 1. Antigen specific cytokine production following immunization with nucleosomal histones.

BALB/c mice (5 per group) were immunized with wild type DNA (WT DNA) or with recombinant DNA coding for nucleosomal histones (rDNA) (A–C). Alternatively, BALB/c mice (5 per group) were immunized with wild type DNA followed by CpG (WT DNA+CpG), or with recombinant DNA followed by recombinant nucleosomal histones+CpG (rDNA/rProtein+CpG) (D–F). Two weeks after the last immunization, dLNs were collected and cells were re-stimulated with the recombinant proteins or with Concanavalin A (Con A). Antigen specific cytokine production in culture supernatants was determined by flow cytometry, using a Th1–Th2 Cytometric Bead Array. Data are presented as the mean+SEM and are from two independent experiments.

Figure 2. Lesion development in mice immunized with nucleosomal histones following infection with L. braziliensis plus sand fly saliva.

BALB/c mice (5 per group) were immunized with WT DNA or with rDNA (A). Alternatively, BALB/c mice (5 per group) were immunized with WT DNA+CpG or with rDNA/rProtein+CpG (B). Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. The course of lesion development was monitored weekly and bars represent the means and standard errors from two independent experiments. The areas contained underneath the curves obtained in (A) and in (B) for each individual mouse from experimental and control groups were compared (C). Data are presented as the mean+SEM.

Figure 3. Histological aspects of ear lesions in mice immunized with nucleosomal histones and challenged with L. braziliensis plus sand fly saliva.

BALB/c mice (five mice per group) were immunized with WT DNA (A) or with rDNA (B). Alternatively, mice were immunized with WT DNA/CpG (C) or with rDNA/rProtein+CpG (D). Two weeks after the last inoculation, mice were challenged with L. braziliensis+sand fly saliva. Ears were removed at 5 weeks post infection and stained with hematoxylin and eosin. Panels represent 100×magnification. (C) Dark symbols indicate infected macrophages displaying a foamy aspect. (D) Red symbols indicate plasmocytes and dashed arrows indicate eosinophils.

Figure 4. Parasite load following immunization with nucleosomal histones and challenge with L. braziliensis plus sand fly saliva.

BALB/c mice (5 per group) were immunized with WT DNA or with rDNA. Alternatively, BALB/c mice (5 per group) were immunized with rDNA/rProtein+CpG. Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. Ear (A) and draining lymph node (B) parasite loads were determined five weeks post infection via a limiting dilution assay. Data are presented as the mean+SEM and are from two independent experiments.

Figure 5. Cytokine expression in the ear dermis following immunization with nucleosomal histones and challenge with L. braziliensis plus sand fly saliva.

BALB/c mice (5 per group) were immunized with rDNA (A) or with rDNA/rProtein+CpG (B). Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. Relative quantification of IFN-γ, IL-4 and IL-10 at the infection site was carried out five weeks after infection. Cytokine cycle threshold (C_t) values were normalized to GAPDH expression (housekeeping) as determined by ΔC_t = C_{t (cytokine)} – C_{t (GAPDH)}. Fold change was determined by real-time PCR, using the 2^–ΔΔCt method, where ΔΔC_t = ΔC_{t (experimental)} – ΔC_{t (control)} (see Materials and Methods). Data (mean+SEM) are presented as fold increase in gene expression of immunized mice over control mice and are from two independent experiments.

Figure 6. Intracellular cytokine production by CD4+ cells from mice immunized with nucleosomal histones and challenged with L. braziliensis plus sand fly saliva.

BALB/c mice (5 per group) were immunized as described. Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. Five weeks later, draining lymph nodes were pooled and cells were preincubated with Brefeldin A for four hours before staining. Data represent the frequency of CD4+ cells positive for IFN-γ (A, B), IL-4 (C, D) and IL-10 (D, E) with signals for the particular cytokine that were greater than the background signals established using isotype controls. Data are presented as the mean+SEM and are from two independent experiments.

Figure 7. Intracellular cytokine production by CD8+ cells from mice immunized with nucleosomal histones and challenged with L. braziliensis plus sand fly saliva.

BALB/c mice (5 per group) were immunized as described. Two weeks after the last immunization, mice were challenged with L. braziliensis+sand fly saliva. Five weeks later, draining lymph nodes were pooled and cells were preincubated with Brefeldin A for four hours before staining. Data represent the frequency of CD8+ cells positive for IFN-γ (A, B), IL-4 (C, D) and IL-10 (D, E) with signals for the particular cytokine that were greater than the background signals established using isotype controls. Data are presented as the mean+SEM and are from two independent experiments.