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. 2017 Mar 7:8:217.
doi: 10.3389/fimmu.2017.00217. eCollection 2017.

A Vaccine Therapy for Canine Visceral Leishmaniasis Promoted Significant Improvement of Clinical and Immune Status with Reduction in Parasite Burden

Affiliations

A Vaccine Therapy for Canine Visceral Leishmaniasis Promoted Significant Improvement of Clinical and Immune Status with Reduction in Parasite Burden

Bruno Mendes Roatt et al. Front Immunol. .

Abstract

Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5-CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen-specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-γ+ and TCD8+IFN-γ+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-α and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment.

Keywords: L. infantum; canine visceral leishmaniasis; heterologous vaccine therapy; immune response; immunotherapy.

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Figures

Figure 1
Figure 1
Profile of peripheral blood leukocytes in dogs naturally infected by Leishmania infantum after immunotherapy with LBMPL vaccine or with MPL adjuvant alone. The immunophenotyping of peripheral blood was used to characterize the systemic cellular profile of circulating leukocytes in dogs submitted to immunotherapy with LBMPL vaccine (black rectangle) or MPL adjuvant alone (white rectangle), before (T0) and after 90 days (T90) of treatment. Data are reported as number of leukocytes in cubic meters of blood. Results are expressed as mean values ± SD of T cells (CD3+) (A) and subpopulations CD4+ (B) and CD8+ (C), B cells (CD21+) (D), NK cells (CD5−CD16+) (E), and monocytes (CD14+) (F). Significant differences (P < 0.05) are shown by connecting line—representing differences between the LBMPL and MPL groups—and by the "*"—representing differences between T0 and T90.
Figure 2
Figure 2
Profile of lymphocyte proliferation and cytokine production in peripheral blood mononuclear cells (PBMCs) of dogs naturally infected by Leishmania infantum after immunotherapy with LBMPL vaccine or with MPL adjuvant alone. The lymphocyte proliferation response and cytokine production were evaluated in PBMCs after in vitro stimulation with soluble Leishmania infantum antigen (SLiAg) in dogs submitted to immunotherapy with LBMPL vaccine (black rectangle) or MPL adjuvant alone (white rectangle); before (T0) and after 90 days (T90) of treatment. Lymphocyte proliferation (A) and cytokine production (B) indexes were calculated as the proportion of lymphocyte, T-CD4+ and T-CD8+ proliferation (A) and IL-10 and TNF-α cytokine production (B) observed in SLiAg-stimulated cultures divided by the control culture (SLiAg/CC ratio). The results of lymphocyte proliferation are expressed by median, and the results of cytokine production are expressed by mean. Significant differences (P < 0.05) are shown by connecting lines–representing differences between the LBMPL and MPL groups—and by the "*"—representing differences between T0 and T90.
Figure 3
Figure 3
Profile of intracytoplasmatic cytokines in peripheral blood mononuclear cells (PBMCs) of dogs naturally infected by Leishmania infantum after immunotherapy with LBMPL vaccine or with MPL adjuvant alone. Pseudocolor plots illustrating the analysis of intracellular cytokines+ (IL-4 and IFN-γ) (A) in CD4+ cells (B) and CD8+ cells (C) within gated lymphocytes in PBMCs after in vitro stimulation with soluble Leishmania infantum antigen (SLiAg) in dogs submitted to immunotherapy with LBMPL vaccine (black rectangle) or MPL adjuvant alone (white rectangle); before (T0) and after 90 days (T90) of treatment. The frequency of cytokines+ T-cell subsets were calculated by quadrant statistics approach and first reported as percentage of gated lymphocytes prior to the calculation of the SLiAg/control indexes. The CD4+ and CD8+ cytokine indexes (B,C) were calculated as the proportion of cytokine+ cells observed in SLiAg-stimulated cultures divided by the control culture (SLAg/CC ratio). Significant differences (P < 0.05) are shown by connecting lines—representing differences between the LBMPL and MPL groups—and by the "*"—representing differences between T0 and T90.
Figure 4
Figure 4
Quantification of parasite burden in bone marrow, skin, and sand flies of dogs naturally infected by Leishmania infantum after immunotherapy with LBMPL vaccine or with MPL adjuvant alone. The parasite burden was performed to evaluate the curative properties of LBMPL vaccine (black and white squares) or MPL adjuvant alone (white circle); before (T0) and after 90 days (T90) of treatment. The number of amastigotes was estimated in bone marrow (A), skin (B), and sand flies (C). Results were plotted representing individual values of each dog per group with connecting lines of T0 and T90. Significant differences (P < 0.05) are shown by "#"—representing difference between the LBMPL and MPL groups—and by the "*"—representing differences between T0 and T90.
Figure 5
Figure 5
Evaluation of clinical outcome [visceral leishmaniasis (VL) signs], clinical score, body weight, and splenomegaly of dogs naturally infected by Leishmania infantum after immunotherapy with LBMPL vaccine or with MPL adjuvant alone. (A) Percentage of dogs with suggested VL signs submitted to immunotherapy with LBMPL vaccine (right) or MPL adjuvant alone (left), before (T0 = black rectangle) and after 90 days (T90 = white rectangle) of treatment. (B) Representative graph of the percentage of reduction of clinical score in dogs submitted to immunotherapy with LBMPL vaccine (black square) or MPL adjuvant alone (white circle), before (T0) and after 90 days (T90) of treatment. (C) Percentage of gain weight in dogs submitted to immunotherapy with LBMPL vaccine (black rectangle) or MPL adjuvant alone (white rectangle), before (T0) and after 90 days (T90) of treatment. (D) Percentage of animals classified according to the absence of splenomegaly (normal = white rectangle), mild splenomegaly (light gray), moderate (medium gray), and severe (dark gray); in dogs submitted to immunotherapy with LBMPL vaccine (right) or MPL adjuvant alone (left), before (T0) and after 90 days (T90) of treatment. Significant differences (P < 0.05) are shown by connecting lines—representing differences between the LBMPL and MPL groups—and by the "*"—representing differences between T0 and T90.
Figure 6
Figure 6
Clinical efficacy of dogs naturally infected by Leishmania infantum after immunotherapy with LBMPL vaccine or with MPL adjuvant alone. Illustrative images of dogs submitted to immunotherapy with MPL adjuvant alone (A) or LBMPL vaccine (B), before (T0) and after 90 days (T90) of treatment.

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