Immunisation with LRV1c alone did not induce protection.

<p>Mice were injected twice intramuscularly with 10μg of LRV1c, 3 weeks apart. After the second vaccination, mice were infected in the hind of the footpad with 3x10⁶ LRV1+ <i>Lg</i> parasites. Footpad thickness was measured weekly (A) and parasite load (B) was measured by bioluminescence at 4 weeks post-infection. (B) Statistical significance tested by a two-way ANOVA, using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005). Representative of 5 independent experiments.</p

LRV1 capsid is immunogenic.

<p>C57BL/6 mice infected with LRV1+ <i>Lg</i> or LRV1- <i>Lg</i> parasites. (A) Mice were sacrificed 4 weeks post infection and IFN-γ levels were analysed in the supernatant (SN) of restimulated cells with recombinant LRVc by ELISA to assess the cellular immune response against LRVc (white LRV1- <i>Lg</i> and black LRV1+ <i>Lg</i>) or control mice (grey). (B) Mice were bled 8 weeks post infection. Sera were collected and tested for the presence of IgG specific to the LRV1c protein by ELISA (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005240#sec002" target="_blank">Materials and Methods</a>). IgG were analyzed form sera of infected mice (white LRV1- <i>Lg</i> and black LRV1+ <i>Lg</i>) or control mice (grey). Results are means±SEM. Statistical significance tested by a two-way ANOVA, using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<.005, ***: P<0.0005). Representative of 2 independent experiments.</p

LRV1c formulated with CpG-ODN or ISA-720 induced a strong protection in C57BL/6 mice.

<p>LRV1c was formulated with various adjuvants like: squalene-in-water emulsion (SWE), squalene-in-water emulsion with MPL (SM), water-in-oil emulsion (Montanide ISA-720), liposome-saponin-based adjuvant (LQ) and CpG ODN-1826. Mice were vaccinated twice 21 days apart and then infected with LRV1+ L.g parasites 21 days after the second inoculation. (A-B) Footpad lesion size was measured weekly and parasite load was quantified by bioluminescence at the peak of infection (C). Results are means±SEM. (A-B) Statistical significance tested by a two-way ANOVA, using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<.005, ***: P<0.0005). Representative of 2 independent experiments. C) Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005 vs Vehicle Control). Representative of 2 independent experiments.</p

B cells played no role in the protection mechanism.

<p>Serum from LRV1c+CpG immunized or non-immunized mice was re-injected in naïve C57BL/6 mice. (A) Change in footpad swelling of mice receiving immunized serum (white square) versus naïve serum (black square). Sera were administrated two days before and after the infection with LRV+ <i>Lg</i>. (B) <i>In vivo</i> bioluminescence of mice challenged with LRV1c+CpG (white) or non-immunized (black) and infected with LRV1+ <i>Lg</i> at the peak of infection. Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005). Representative of 2 independent experiments.</p

Immunization with LRV1c+CpG was specific to the <i>Leishmania</i> RNA virus capsid.

<p>C57BL/6 mice were immunized 3 times IM, 15 days apart with LRV1c+CpG or PBS as a control. 21 days after the third vaccination, mice were infected with either LRV1+ <i>Lg</i> or LRV1- <i>Lg</i>. (A) Change in footpad swelling of mice immunized with LRV1c+CpG (white square) or vehicle control (black square) and infected with LRV1+ <i>Lg</i>. (B-C) <i>In vivo</i> bioluminescence of mice challenged with LRV1c+CpG (white) or vehicle control (black) and infected with LRV1+ <i>Lg</i> at the peak of infection. (D) Change in footpad swelling of mice immunized with LRV1c+CpG (white circle) or vehicle control (black circle) and infected with LRV1- <i>Lg</i>. (E-F) <i>In vivo</i> bioluminescence of mice immunised with LRV1c+CpG (white) or vehicle control (black) and infected with LRV1- <i>Lg</i> at the peak of infection. Results are means±SEM. (A and D) Statistical significance tested by a two-way ANOVA, using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<.005, ***: P<0.0005). (B-E) Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005). Representative of 8 independent experiments.</p

LRV1c formulated with CpG-ODN 1826 induced a strong Th1 immune response against the viral capsid.

<p>C57BL/6 mice vaccinated or not with the LRVc+ CpG were infected with LRV1+ <i>Lg</i> parasites and at 4 weeks post infection their dLN cells were restimulated ex vivo with either LRV1+ <i>Lg</i> (right side) or LRV1- <i>Lg</i> parasites (left side). By comparing these two re-stimulations, we discriminated between the immune response directed against the parasite (i.e. LRV1- <i>Lg</i> re-stimulation) and the response directed toward the LRV1 capsid (i.e. the difference between LRV1- <i>Lg</i> and LRV1+ <i>Lg</i> re-stimulation). IFN-γ, IL-4 and IL-10 level were detected by ELISA 72h after re-stimulation. Results are means±SEM. Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005). Representative of 3 independent experiments.</p

LRV1c Specific T cell transfer induced protection.

<p>C57BL/6 mice were vaccinated three times, 15 days apart, and sacrificed 7 days after the third vaccination. CD3+ T cells from vaccinated, or PBS injected mice (vehicle control), were purified (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005240#sec002" target="_blank">Materials and Methods</a>) and transferred into naïve C57BL/6 mice. These mice were infected 1 day after with LRV1+ <i>Lg</i> parasites. (A) Change in footpad swelling of mice infected with LRV1+ <i>Lg</i>. White squares represent the group carrying CD3+ cells from vaccinated mice, black squares indicate mice carrying vehicle control (B) Parasite load measured by <i>in vivo</i> luminescence at the peak of infection. Results are means±SEM. (A) Statistical significance tested by a two-way ANOVA, using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005). Representative of 3 independent experiments. (B) Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 5, *: P<0.05, **: P<0.005, ***: P<0.0005). Representative of 2 independent experiments.</p

LRV1c formulated with CpG-ODN 1826 induced LRV1c specific IFN-γ secreting T cells.

<p>Seven days after the third challenge, LRV1c+CpG, or vehicle control mice were sacrificed and splenocytes were restimulated with BMM infected with LRV1+ <i>Lg</i>, LRV1- <i>Lg</i> or stimulated with LRV1c (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005240#sec002" target="_blank">Material and Method</a>). (A) IFN-γ secretion from vehicle control mice, or LRV1c+CpG incubated with BMM stimulated with LRV1c. (B) IFN-γ secretion from vehicle control mice or LRV1c+CpG incubated with BMM infected with LRV1+ <i>Lg</i>, or LRV1- <i>Lg</i>. (C) Intracellular FACS on dLN cells stimulated with LRV1+ <i>Lg</i> or LRV1- <i>Lg</i> infected macrophage in presence of BrefeldinA. Cells were gated firstly on CD3+ T cells, then on CD8+ and CD4+, and finally on IFN-γ secretion. Results are means±SEM. (A-B) Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 3, *: P<0.05, **: P<0.005, ***: P<0.0005 vs Vehicle Control). Representative of 4 independent experiments. (C) Statistical significance tested by a 2-tailed Student’s t-test using Prism5 Graphpad software (n = 1, *: P<0.05, **: P<0.005, ***: P<0.0005 vs Vehicle Control). Representative of 3 independent experiments.</p