Performance of LBSap vaccine after intradermal challenge with L. infantum and saliva of Lu. longipalpis: immunogenicity and parasitological evaluation

Abstract

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21(+)) and both CD4(+) and CD8(+) T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4(+) T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.

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; white square); Sap (saponin; white triangle); LB (killed L. braziliensis vaccine; black square); LBSap (killed L. braziliensis vaccine plus saponin; black triangle).

IgG2/IgG1 ratio: C (control; white square) and LBSap (killed L. braziliensis vaccine plus saponin; black square). Figure 1A represents anti-L. infantum total IgG, (B) anti-L. infantum IgG1, (C) anti-L. infantum IgG2 and (D) IgG2/IgG1 ratio: 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 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 LBSap group and the control C, Sap and LB groups are indicated, respectively, by the letters a, b, and c.

Figure 2. Cellular profile of circulating lymphocytes and monocytes in dogs submitted to different vaccination protocols before and after challenge with L. infantum plus SGE.

C (control; white square); Sap (saponin; white triagle); LB (killed L. braziliensis vaccine; black square), LBSap (killed L. braziliensis vaccine plus saponin; black triagnle): 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 the (A) absolute counts of circulating lymphocytes and of (B) CD5⁺, (C) CD4⁺, (D) CD8⁺, (E) CD21⁺ cells and (F) CD14⁺ monocytes. 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.

Figure 3. Cell proliferation response of PBMCs after stimulation with (A) vaccine soluble antigen (VSA) and (B) soluble L. infantum antigen (SLcA).

The middle and lower panels show the immunophenotypic profile of in vitro peripheral blood mononuclear cells following stimulation with (C) VSA and (D) SLcA determined at 90, 435 and 885 dac (days after challenge with L. infantum plus SGE) for vaccinated groups: C (control; white); Sap (saponin; white with weak hatched); LB (killed L. braziliensis vaccine; white with strong hatched); LBSap (killed L. braziliensis vaccine plus saponin; black). The results are expressed as index (stimulated cultures over non-stimulated cultures (SC/CC)) of the mean frequencies of CD5⁺, CD21⁺, CD4⁺ and CD8⁺ cells and MHC II expression in lymphocytes (reported as Mean Fluorescence Channel - MFC values). 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 LBSap and the control C, Sap and LB groups are represented by the letters a, b and c, respectively.

Figure 4. Evaluation of cytokine mRNA levels in spleen samples at 885 dac (days after challenge with L. infantum plus SGE) for vaccinated groups: C (control; white); Sap (saponin; white with weak hatched); LB (killed L. braziliensis vaccine; white with strong hatched); LBSap (killed L. braziliensis vaccine plus saponin; black).

The Log number of messenger RNA molecules for (A) IFN-γ, (B) TNF-α (C) IL-10 and (D) TGF- β1 and the cytokines ratio (E) IFN-γ/IL-10 and (F) IFN-γ/TGF-β1 are shown. Results were plotted representing median values for each group. Significant differences (p<0.05) between the LBSap and the control C, Sap and LB groups are represented by the letters a, b and c, respectively.

Figure 5. Quantification of parasite burden in spleen samples at 885 dac (days after challenge with L. infantum plus SGE) for vaccinated groups: C (control; white); Sap (saponin; white with weak hatched); LB (killed L. braziliensis vaccine; white with strong hatched); LBSap (killed L. braziliensis vaccine plus saponin; black).

(A) The quantification of amastigote forms of Leishmania/mg of spleen using real time PCR with specific primers for a single-copy gene of DNA polymerase of Leishmania infantum. (B) Parasitism reduction (%) in Sap, LB and LBSap in comparison with the control C group. Results were plotted representing median values for each group. Significant differences (p<0.05) between the LBSap and the control C group are represented by the letter a.