A Recombinant Chimeric Protein-Based Vaccine Containing T-Cell Epitopes from Amastigote Proteins and Combined with Distinct Adjuvants, Induces Immunogenicity and Protection against Leishmania infantum Infection

Abstract

Currently, there is no licensed vaccine to protect against human visceral leishmaniasis (VL), a potentially fatal disease caused by infection with Leishmania parasites. In the current study, a recombinant chimeric protein ChimT was developed based on T-cell epitopes identified from the immunogenic Leishmania amastigote proteins LiHyp1, LiHyV, LiHyC and LiHyG. ChimT was associated with the adjuvants saponin (Sap) or monophosphoryl lipid A (MPLA) and used to immunize mice, and their immunogenicity and protective efficacy were evaluated. Both ChimT/Sap and ChimT/MPLA induced the development of a specific Th1-type immune response, with significantly high levels of IFN-γ, IL-2, IL-12, TNF-α and GM-CSF cytokines produced by CD4+ and CD8+ T cell subtypes (p < 0.05), with correspondingly low production of anti-leishmanial IL-4 and IL-10 cytokines. Significantly increased (p < 0.05) levels of nitrite, a proxy for nitric oxide, and IFN-γ expression (p < 0.05) were detected in stimulated spleen cell cultures from immunized and infected mice, as was significant production of parasite-specific IgG2a isotype antibodies. Significant reductions in the parasite load in the internal organs of the immunized and infected mice (p < 0.05) were quantified with a limiting dilution technique and quantitative PCR and correlated with the immunological findings. ChimT/MPLA showed marginally superior immunogenicity than ChimT/Sap, and although this was not statistically significant (p > 0.05), ChimT/MPLA was preferred since ChimT/Sap induced transient edema in the inoculation site. ChimT also induced high IFN-γ and low IL-10 levels from human PBMCs isolated from healthy individuals and from VL-treated patients. In conclusion, the experimental T-cell multi-epitope amastigote stage Leishmania vaccine administered with adjuvants appears to be a promising vaccine candidate to protect against VL.

Keywords: T-cell epitopes; adjuvants; immune response; polypeptide-based protein; vaccine; visceral leishmaniasis.

Figure 1

A flowchart for the experimental protocol to examine the immunogenicity of ChimT in mice.

Figure 2

Cytokine production before L. infantum infection. Mice (n = 8 per group) received saline or were immunized with saponin, MPLA, ChimT, ChimT/Sap or ChimT/MPLA. Thirty days after the last vaccine dose, they were euthanized and their spleen cells (5 × 10⁶ cells per mL) were cultured in DMEM and non-stimulated (medium) or stimulated with ChimT or SLA (10 and 25 µg/mL, respectively) for 48 h at 37 °C in 5% (v/v) CO₂. The cell supernatant was collected and levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were measured by capture ELISA. Bars indicate the mean ± standard deviation of groups. (****) indicates significant difference in relation to the saline, saponin, MPLA and ChimT groups (p < 0.00001).

Figure 3

Cellular response developed after challenge. Mice (n = 8 per group) received saline or were immunized with saponin, MPLA, ChimT, ChimT/Sap or ChimT/MPLA. Thirty days after the last vaccine dose, they were infected with L. infantum promastigotes, and 45 days post-challenge, their spleen cells (5 × 10⁶ cells per mL) were cultured in DMEM and non-stimulated (medium) or stimulated with ChimT or SLA (10 and 25 µg/mL, respectively) for 48 h at 37 °C in 5% (v/v) CO₂. The cell supernatant was collected and levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were also measured by capture ELISA. Bars indicate the mean ± standard deviation of groups. (****) indicates significant difference in relation to the saline, saponin, MPLA and ChimT groups (p < 0.00001). (**) indicates significant difference in relation to the ChimT/Sap and ChimT/MPLA groups (p < 0.001).

Figure 4

Involvement of CD4⁺ and CD8⁺ T cell subtypes in the (A) nitrite and (B) IFN-γ pro-duction production after infection. Mice (n = 8 per group) were vaccinated and later challenged with L. infantum promastigotes. Forty-five days post-infection, their spleen cells (5 × 10⁶ cells per mL) were cultured in DMEM and non-stimulated (medium) or stimulated with ChimT or SLA (10.0 and 25.0 μg/mL, respectively) for 48 h at 37 °C in 5% (v/v) CO₂. In some wells, anti-CD4 or anti-CD8 monoclonal antibodies were added in the cultures (5 µg/mL each). In those, the cell supernatant was collected, and IFN-γ production was also measured by ELISA capture. (****) indicates significant difference in relation to the saline, saponin, MPLA and ChimT groups (p < 0.00001). In addition, in the wells without the addition of monoclonal antibodies, supernatants were collected, and nitrite secretion was evaluated by Griess reaction. Bars indicate the mean ± standard deviation of groups. (**) and (***) indicate statistically significant difference in relation to the control cell cultures (incubated without monoclonal antibody) (p < 0.001 and p < 0.0001, respectively).

Figure 5

IFN-γ mRNA expression after challenge infection. Mice (n = 8 per group) received saline or were immunized with saponin, MPLA, ChimT, ChimT/Sap or ChimT/MPLA. Then, they were challenged with L. infantum promastigotes, and 45 days post-infection, their spleen cells (5 × 10⁶ cells per well) were stimulated with SLA (25.0 μg/mL) for 48 h at 37 °C in 5% (v/v) CO₂. Cells were collected and mRNA was obtained and used to evaluate IFN-γ expression by qRT-qPCR. Data were normalized with control primers from housekeeping genes ACTB and GAPDH. Bars indicate the mean ± standard deviation of groups. Significant difference was observed for both ChimT/Sap and ChimT/MPLA over the saline, saponin, MPLA and ChimT groups with p < 0.00001. ChimT/MPLA showed significant difference over ChimT/Sap group with p < 0.05.

Figure 6

Intracytoplasmic cytokine-producing T-cell profile evaluated by flow cytometry. IFN-γ, TNF-α, IL-2 and IL-10-producing CD4⁺ and CD8⁺ T-cell subtypes were evaluated by the ratio between the frequency from stimulated versus non-stimulated cultures. The IFN-γ, TNF-α, IL-2 and IL-10-producing CD4⁺ and CD8⁺ T-cell percentages are shown. Bars indicate the mean plus standard deviation of groups. Lines between experimental groups indicate significant difference between them (p < 0.05).

Figure 7

Cellular proliferationevaluated after infection. Spleen cells (5 × 10⁶ cells per well) were obtained from infected and vaccinated mice (n = 8 per group), and they were cultured in complete RPMI 1640 medium and stimulated with SLA (25.0 μg/mL) for 24 h at 37 °C in 5% (v/v) CO₂. Cellular lymphoproliferation was evaluated by the MTT method. Bars indicate the mean ± standard deviation of groups. (****) indicates significant difference in relation to the indicated groups by lines (p < 0.00001).

Figure 8

Antibody response (A) before and (B) after challenge. Mice (n = 8 per group) were vaccinated and later challenged with L. infantum promastigotes. Forty-five days post-infection, they were euthanized, and their sera samples were collected to evaluate the anti-ChimT and anti-SLA IgG1 and IgG2a levels by ELISA technique. Bars indicate the mean ± standard deviation of groups. (****) indicates significant difference in relation to the saline, saponin, MPLA and ChimT groups (p < 0.00001). (&&&&) indicates significant difference in relation to the ChimT/Sap and ChimT/MPLA groups (p < 0.00001).

Figure 9

Parasite load estimated by a limiting dilution technique. Mice (n = 8 per group) received saline or were immunized with saponin, MPLA, ChimT, ChimT/Sap or ChimT/MPLA. Then, they were challenged with L. infantum promastigotes, and 45 days post-infection, their livers, spleens, bone marrow (BM) and draining lymph nodes (dLN) were collected to evaluate the parasite load through a limiting dilution technique. Data are expressed as the negative log of the titer adjusted per milligram of liver (A), spleen (B), BM (C) and dLN (D). Bars indicate the mean ± standard deviation of groups. (*), (**), (***) and (****) indicate significant difference in relation to the indicated groups by lines (p < 0.005, p < 0.001, p < 0.0001, and p < 0.00001, respectively).

Figure 10

Splenic parasitism evaluated by qPCR. Mice (n = 8 per group) were vaccinated and later challenged with L. infantum promastigotes. Forty-five days post-infection, their spleens were collected and the parasite load was also evaluated by qPCR technique. Data are expressed as the number of parasites per 1000 nucleated cells. Bars indicate the mean plus standard deviation of the groups. (****) indicates significant difference in relation to the saline, saponin and MPLA groups (p < 0.00001). (***) indicates significant difference in relation to the ChimT group (p < 0.05). (**) indicates significant difference in relation to the ChimT/Sap group (p < 0.05).

Figure 11

Immunogenicity in human cell cultures. PBMCs (1 × 10⁷ cells per mL) collected from healthy subjects (n = 6) and non-treated and treated visceral leishmaniasis (VL) patients (n = 6) were non-stimulated (medium) or stimulated with ChimT or SLA (10 and 25 µg/mL, respectively) for 5 days at 37 °C in 5% (v/v) CO₂. The cell culture supernatants from healthy subjects (A,B) and from non-treated (C,D) and treated (E,F) VL patients were used to evaluate the IFN-γ and IL-10 production specific to ChimT (A,C,E) or SLA (B,D,F), which was measured by capture ELISA. Bars indicate the mean ± standard deviation of groups. (*) and (****) indicate significant difference in relation to the indicated groups by lines (p < 0.005 and p < 0.00001, respectively).