The F1F3 recombinant chimera induced higher vaccine efficacy than its independent F1 and F3 components against Leishmania (L.) infantum chagasi mice infection

Abstract

Introduction: Visceral leishmaniasis (VL) is a severe human vector-borne CD4-immunosuppressive disease that can be lethal if untreated soon after symptoms arise. No vaccine is available against human VL, and its chemotherapy is highly toxic and requires hospitalization. VL patients show substantially decreased CD4₊ total and Leishmania-specific CD4+ T cell counts. Leishmania (L.) donovani nucleoside hydrolase (NH36) is a DNA metabolism enzyme and a conserved marker of the Leishmania genus. It has been considered, among other Leishmania antigens, a vaccine candidate. In mice vaccinated with NH36, protection against VL is mediated by a CD4₊ T cell response to the NH36 C-terminal domain (F3), and against cutaneous leishmaniasis (CL), by a CD4+ response against F3 and a CD8+ response against the NH36 N-terminal (F1). Vaccination with a recombinant chimera containing the F1 and F3 domains expressed in tandem (F1F3) protected mice against the heterologous CL infection by L. (L.) amazonensis and L. (V.) braziliensis.

Methods: In this investigation, BALB/c mice were immunized with either F1, F3, a mixture of both, or with the F1F3 chimera, plus saponin and challenged with amastigotes of L. (L.) infantum chagasi, the agent of VL in America.

Results: Before and after infection, the F1F3 chimera and the F3 vaccines promoted the highest IgA, IgM, IgG, IgG1, IgG2a, IgG2b, and IgG3 antibody responses. The F1F3 chimera promoted the strongest intradermal response against the leishmanial antigen, the highest body weight gain, and the most potent reduction of the spleen and liver relative weights. In addition, the F1F3 chimera vaccine increased the secretion of IFN-γ, and, together with the F3 vaccine, the secretion of TNF-α by splenocytes. The F1F3 chimera and the F1 vaccine also promoted the strongest secretion of IL-10, which was very low in mice immunized with F3. Thus, the IFN-γ/IL-10 and TNF-α/IL-10 ratios, characteristic of a Th1 response, were increased in mice vaccinated with F3. The F1F3 chimera and the F3 vaccine reduced the parasite load in the liver.

Discussion: The F1F3 chimera, as described for the heterologous CL infections, also optimizes protection against the homologous visceral leishmaniasis infection by L. (L.) infantum chagasi, by a Th1 contribution from the F3 peptide and a regulatory response from the F1 peptide. Expression of the F1 and F3 domains in tandem induced higher efficacy than the simple mixture of the F1 and F3 domains.

Keywords: F1F3 recombinant chimera; Leishmania (L.) infantum chagasi; mixed or T-cell regulatory response; nucleoside hydrolase NH36; visceral leishmaniasis.

Figure 1

Predominance of NH36-antibody responses promoted by the F3 and chimeras after vaccination. The mice were vaccinated with the F1, F3, F1+F3 mixture, or F1F3 chimeras at 100 µg or 200 µg doses, all formulated with 100 µg saponin. Results represent the individual absorbance data of anti-IgA (a), IgG (b), IgG2a (c), IgG3 (d), IgM (e), IgG1 (f) and IgG2b (g) anti-NH36 antibodies in mice sera diluted 1/100, as measured by the ELISA assay, and the individual IgG2a/IgG1 antibody ratios (h). Statistical differences were evaluated using the Kruskal-Wallis method and ANOVA. Horizontal bars represent the means of two independent experiments with n = 26 mice per treatment. Asterisks and horizontal lines indicate significant differences between treatments (p < 0.001).

Figure 2

Predominance of NH36-antibody responses promoted by the F3 and chimeras after infection. Mice were vaccinated with the F1, F3, F1 + F3 mixture, or F1F3 chimeras at 100- or 200-µg doses, all formulated with 100 µg saponin. The results represent the individual absorbance data of anti-IgA (a), IgG (b), IgG2a (c), IgG3 (d), IgM (e), IgG1 (f), and IgG2b (g) anti-NH36 antibodies in mice sera diluted 1/100, as measured by the ELISA assay, and the individual IgG2a/IgG1 antibody ratios (h). Statistical differences were evaluated using the Kruskal–Wallis method and ANOVA. Horizontal bars represent the means of two independent experiments with n = 19 mice per treatment. Asterisks and horizontal lines indicate significant differences between treatments (p < 0.001).

Figure 3

The F1F3 vaccine induces the strongest intradermal response. The mice were vaccinated with the F1, F3, F1 + F3 mixture, or F1F3 chimeras at 100- or 200-µg doses, all formulated with 100 µg saponin. The results represent the individual intradermal size response to the leishmanial antigen in millimeter after vaccination (a, b) and after infection (c, d). Horizontal bars represent the means of two independent experiments with n = 14 mice per treatment after vaccination and n = 8 mice per treatment after infection. Asterisks and horizontal lines indicate significant differences between treatments as assessed using 95% confidence interval.

Figure 4

Th1-cytokine secretion promoted by the F3 vaccine and mixed inflammatory/regulatory cytokine response induced by the chimeras. The mice were vaccinated with the F1, F3, F1 + F3 mixture, or F1F3 chimeras at 100- or 200-µg doses, all formulated with 100 µg saponin. The results represent the means of IFN-γ (a, d), TNF-α (b, e), and IL-10 secretion (c, f) in response to NH36 after immunization and infection and the means of IFN-γ/IL10 (g, i) and TNF-α/IL10 ratios (h, j) secreted by splenocytes after immunization and after infection, as evaluated in the ELISA assay (expressed in pg/mL). Horizontal bars represent the means ± SE of two independent experiments with n = 6 mice per treatment after vaccination and n = 8 mice per treatment after infection. Asterisks and horizontal lines indicate differences between treatments as disclosed using 95% confidence interval.

Figure 5

Impact of vaccination on clinical variables. Mice were vaccinated with the F1, F3, F1 + F3 mixture, or F1F3 chimeras at 100- or 200-µg doses, all formulated with 100 µg saponin. The results represent the gain in corporal weight in grams (g) (a), the liver/body relative weight as percentages (%) (b), the spleen/body relative weight in percentages (%), (c) and the parasite load in the liver of mice infected with amastigotes of Leishmania (L.) infantum chagasi expressed as LDU values (d) after euthanasia. Horizontal bars represent means ± SE of two independent experiments with n = 8–10 mice per treatment. Asterisks and horizontal lines indicate differences between treatments as assessed using 95% confidence interval.