Immunogenicity of a killed Leishmania vaccine with saponin adjuvant in dogs

Abstract

Cellular and humoral immune responses of dogs to a candidate vaccine, composed of Leishmania braziliensis promastigote protein plus saponin as adjuvant, have been investigated as a pre-requisite to understanding the mechanisms of immunogenicity against canine visceral leishmaniasis (CVL). The candidate vaccine elicited strong antigenicity related to the increases of anti-Leishmania IgG isotypes, together with higher levels of lymphocytes, particularly of circulating CD8(+) T-lymphocytes and Leishmania chagasi antigen-specific CD8(+) T-lymphocytes. As indicated by the intense cell proliferation and increased nitric oxide production during in vitro stimulation by L. chagasi soluble antigens, the candidate vaccine elicited an immune activation status potentially compatible with effective control of the etiological agent of CVL.

Fig. 1

Comparative immunogenicity in the different treatment groups: C (control; □); Sap (saponin; ○); LB (killed L. braziliensis vaccine; ■); LBSap (killed L. braziliensis vaccine plus saponin; ●). Upper left and right panels and lower left panel show anti-L. chagasi total IgG, anti-L. chagasi IgG1, and anti-L. chagasi IgG2, respectively: the x-axis displays the times at which the assays were conducted (T0: prior to the first dose; T1: 15 days after the first dose; T2: 15 days after the second dose; and T3: 15 days after the third dose) and the y-axis represents the mean ELISA absorbance values determined at 492 nm in serum samples diluted 1:80. Significant differences (P < 0.05) between the LBSap group and the control C, Sap, and LB groups are indicated, respectively, by the letters a, b, and c. The lower right panel shows the correlation between IgG1 and IgG2 in the LBSap group and the Pearson's correlation indexes (r) at P < 0.05 are shown in figure.

Fig. 2

The cell profile of peripheral blood leucocytes in different treatment groups. Upper panel [C (control; □); Sap (saponin; ); LB (killed L. braziliensis vaccine; ), LBSap (killed L. braziliensis vaccine plus saponin; ■)] and middle left panels [C (control; □); Sap (saponin; ○); LB (killed L. braziliensis vaccine; ■); LBSap (killed L. braziliensis vaccine plus saponin; ●)]: the x-axis displays the times at which the assays were conducted (T0: prior to the first dose; T1: 15 days after the first dose; T2: 15 days after the second dose; and T3: 15 days after the third dose) and the y-axis represents the mean values (with standard deviations in the upper panel) of the absolute counts of circulating lymphocytes (upper panel), and of CD5⁺, CD21⁺, CD4⁺ and CD8⁺ cells (middle left panels). Significant differences (P < 0.05) between the LBSap group and the control C and Sap groups are indicated, respectively, by the letters a, b. The middle right panels and the lower panels show the correlations between circulating lymphocytes in the LBSap group and the Pearson's correlation indexes (r) at P < 0.05 are shown in figure.

Fig. 3

Cell proliferation response of peripheral blood mononuclear cells after stimulation with vaccine soluble antigen (VSA) (upper left panel) and soluble L. chagasi antigen (SLcA) (upper right panel). The middle and lower panels show the immunophenotypic profile of in vitro peripheral blood mononuclear cells following stimulation with vaccine soluble antigen (VSA) (left panels) and soluble L. chagasi antigen stimulation (right panel) determined at T3 for treatment groups: C (control; □); Sap (saponin; ); LB (killed L. braziliensis vaccine; ); LBSap (killed L. braziliensis vaccine plus saponin; ■). The results are expressed as the mean frequencies of CD5⁺, CD21⁺, CD4⁺ and CD8⁺ cells in the non-stimulated cultures (CC; controls) and in the stimulated cultures (SC). Significant differences (P < 0.05) between values measured at T0 (before the first dose) and T3 (15 days after the third dose) are indicated by connecting lines, and between the LBSap and the control C, Sap, and LB groups at T3 are represented by the letters a, b, and c, respectively.

Fig. 4

Antigen-presenting cells (APC) and the lymphocyte activation status in different vaccine groups: C (control; □); Sap (saponin; ○); LB (killed L. braziliensis vaccine; ■); LBSap (killed L. braziliensis vaccine plus saponin; ●). In the upper left panels, significant differences (P < 0.05) between the LBSap group and the control C, Sap, and LB groups with respect to the absolute counts of CD14⁺ monocytes and MHC-I expression in lymphocytes (reported as MFC values) are indicated, respectively, by the letters a, b, and c. The correlation between CD14⁺ cell counts and MHC-I in lymphocytes in the LBSap group at T0, T1, T2 and T3 is shown in the upper right panel and the Pearson's correlation indexes (r) at P < 0.05 are shown in figure. The middle panels show the correlations between CD14⁺ and CD21⁺ absolute cell counts and in vitro cell proliferation (counts per minutes – CPM) following stimulation by vaccine soluble antigen (VSA) or soluble L. chagasi antigen (SLcA) and the Pearson's correlation indexes (r) at P < 0.05 are shown in figure. The lower panels display NO production indices (antigen-stimulated culture/control culture) determined at T0 and T3 in culture supernatants from the different treatment groups: C (control; □); Sap (saponin; ); LB (killed L. braziliensis vaccine; ); LBSap (killed L. braziliensis vaccine plus saponin; ■). Significant differences (P < 0.05) between at T0 plus T3 are indicated by connecting lines.

Fig. 5

Correlations between cell proliferation (counts per minute) and frequencies of CD5⁺, CD4⁺ and CD8⁺ T-cells following in vitro peripheral blood mononuclear cell cultures derived from the LBSap group (upper panels), and C (control), Sap (saponin) and LB (killed L. braziliensis vaccine) groups (lower panel), stimulated by vaccine soluble antigen (VSA; upper panel) and soluble L. chagasi antigen (SLcA; middle panel) and determined at T0 (before the first dose) plus T3 (15 days after the third dose) and the Pearson's correlation indexes (r) at P < 0.05 are shown in figure.