LBSapSal-vaccinated dogs exhibit increased circulating T-lymphocyte subsets (CD4⁺ and CD8⁺) as well as a reduction of parasitism after challenge with Leishmania infantum plus salivary gland of Lutzomyia longipalpis

Abstract

Background: The development of a protective vaccine against canine visceral leishmaniasis (CVL) is an alternative approach for interrupting the domestic cycle of Leishmania infantum. Given the importance of sand fly salivary proteins as potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in the last few decades. In this context, we previously immunized dogs with a vaccine composed of L. braziliensis antigens plus saponin as the adjuvant and sand fly salivary gland extract (LBSapSal vaccine). This vaccine elicited an increase in both anti-saliva and anti-Leishmania IgG isotypes, higher counts of specific circulating CD8⁺ T cells, and high NO production.

Methods: We investigated the immunogenicity and protective effect of LBSapSal vaccination after intradermal challenge with 1 × 10⁷ late-log-phase L. infantum promastigotes in the presence of sand fly saliva of Lutzomyia longipalpis. The dogs were followed for up to 885 days after challenge.

Results: The LBSapSal vaccine presents extensive antigenic diversity with persistent humoral and cellular immune responses, indicating resistance against CVL is triggered by high levels of total IgG and its subtypes (IgG1 and IgG2); expansion of circulating CD5⁺, CD4⁺, and CD8⁺ T lymphocytes and is Leishmania-specific; and reduction of splenic parasite load.

Conclusions: These results encourage further study of vaccine strategies addressing Leishmania antigens in combination with proteins present in the saliva of the vector.

Figure 1

Anti-Leishmania reactivity in serum from dogs submitted to different vaccination protocols before and after intradermal challenge with L. infantum plus SGE: C (control;); Sal (SGE;); LBSal (killed L. braziliensis vaccine plus SGE;); LBSapSal (killed L. braziliensis vaccine plus saponin plus SGE;). IgG2/IgG1 ratio: C (control;) and LBSapSal (killed L. braziliensis vaccine plus saponin plus SGE;). (A) anti–L. infantum total IgG; (B) anti–L. infantum IgG1; (C) anti–L. infantum IgG2; (D) IgG2/IgG1 ratio: C (control; white square) and LBSapSal (killed L. braziliensis vaccine plus saponin and SGE; black square), the x-axis displays the times at which the assays were conducted (Tbc: time before challenge with L. infantum; and 20, 90, 274, 435, 541, and 885 days after challenge with L. infantum), and the y-axis represents the mean ELISA absorbance values determined at 492 nm in serum samples diluted 1:80 for IgG total and subclasses. The cut-off is represented by the dotted line. Significant differences (p < 0.05) between the LBSapSal group and the C, Sal, and LBSal groups are indicated, respectively, by the letters a, b, and c. *Significant difference (p < 0.05) between the LBSapSal group and the control C group in IgG2/IgG1 ratio.

Figure 2

Cellular profile of circulating lymphocytes in dogs submitted to different vaccination protocols before and after challenge with L. infantum plus SGE. C (control; ); Sal (SGE; ); LBSal (killed L. braziliensis vaccine plus SGE; ); LBSapSal (killed L. braziliensis vaccine plus saponin plus SGE; ): the x-axis displays the times at which the assays were conducted (Tbc: time before challenge with L. infantum; and 20, 90, 274, 435, 541, and 885 days after challenge [dac] with L. infantum), and the y-axis represents the mean values of (A) CD5⁺, (B) CD4⁺, (C) CD8⁺ cells, and (D) CD4⁺/CD8⁺ ratio, the white and black bars represented the control and LBSapSal groups, respectively. Significant differences (p < 0.05) between the LBSapSal group and the C, Sal, and LBSal groups are indicated, respectively, by the letters a, b, and c. *Significant difference (p < 0.05) between the LBSapSal group and the control C group in CD4⁺/CD8⁺ ratio.

Figure 3

Cell proliferation response of PBMCs after stimulation with (A) vaccine soluble antigen (VSA) and (B) soluble L. infantum antigen (SLiA). The lower panels show the immunophenotypic profile of in vitro PBMCs following stimulation with (C) VSA and (D) SLiA determined at 90, 435, and 885 dac (days after challenge with L. infantum plus SGE) for vaccinated groups: C (control; ); Sal (SGE; ); LBSal (killed Leishmania braziliensis vaccine plus SGE; ); LBSapSal (killed L. braziliensis vaccine plus saponin plus SGE; ). The results are expressed as the ratio of mean frequencies of CD4⁺ and CD8⁺ cells in the stimulated cultures over non-stimulated cultures (SC/CC). Significant differences (p < 0.05) between values measured at 90, 435, and 885 dac under the same group are indicated by connecting lines, and between the LBSapSal and the control C, Sal and LBSal groups are represented by the letters a, b, and c, respectively.

Figure 4

Quantification of parasite burden in spleen samples at 885 dac (days after challenge with L. infantum plus SGE) for vaccinated groups: C (control;); Sal (SGE;); LBSal (killed L. braziliensis vaccine plus SGE;); LBSapSal (killed L. braziliensis vaccine plus saponin plus SGE; ). The y-axis displays the quantification of amastigote forms of Leishmania per milligram of spleen using real-time PCR with specific primers for a single-copy gene of DNA polymerase of Leishmania infantum. The y-axis displays inside the respective square of group the parasitism reduction (%) in Sal, LBSal, and LBSapSal in comparison with the C group. Results were plotted representing median values for each group. Significant differences (p < 0.05) between the LBSapSal and LBSal groups and the C group are represented by the letter a. C = mean 1657; Sal = mean 823, reduction = 50%; LBSal = mean 667, reduction = 60%; LBSapSal = mean 523, reduction = 69%.