α-Gal-NGPs as biomarkers of active CL in patients with <i>L</i>. <i>major</i> infection.

<p>Assessment by chemiluminescent ELISA of α-Gal-containing NGPs and controls (Cysteine-BSA and Galβ-BSA) were immobilized on a microplate and reacted with pools of sera (at 1:100 dilution) from patients with active or cured CL, or heterologous skin (non-CL) infections (n = 5 per group, randomly selected) from an endemic region (Saudi Arabia), as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006039#pntd.0006039.ref026" target="_blank">26</a>]. RLU, Relative luminescence units. Error bars indicate S.E.M. of triplicate determinations. The fold difference in reactivity between active CL vs. cured CL, and active CL vs. heterologous infection are indicated. Two-way ANOVA with Tukey’s multiple comparisons: ns, non-significant; (*), <i>P</i><0.05; (**), <i>P</i><0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001.</p

Anti-NGP5B antibody response is specific against terminal α-Gal residues.

<p>(A-B) Chemiluminescent ELISA reactivity of mouse serum pools obtained at Boost 3 (n = 6) and endpoint (n = 3) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. Immunized groups are indicated in the legend and antigens on the microplate are shown in the Y-axis. (C) Chemiluminescent ELISA reactivity of mouse serum obtained at Boost 2. NGP5B (125 ng/well) was treated or not with green-coffee bean α-galactosidase. One-way ANOVA (compared with untreated sample): (**), <i>P<</i>0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001. (A-C) Error bars indicate S.E.M. of triplicate determinations.</p

Serum cytokine profile of NGP5B and NGP5B+CpG immunizations.

<p>(A-D) Th1 cytokines IL-12p40, IL-2, IFN-γ, and TNF-α. (E-G) Th2 cytokines IL-4, IL-10, and IL-5. (H) IFN-γ/IL4 ratio. (I) IFN-γ/IL10 ratio. Two-tailed unpaired Student’s <i>t</i>-test (compared with Naïve group): (*), <i>P</i><0.05; (***), <i>P</i><0.0001. (A-I) Error bars indicate S.E.M. of triplicate determinations.</p

α-Gal-NGPs as potential vaccine candidates against <i>L</i>. <i>major</i>.

<p>(A) Chemiluminescent ELISA to measure anti-α-Gal antibody levels in α1,3GalT-KO mice immunized with the α-Gal-containing NGP17B, NGP12B, or NGP5B. Immunizations groups are indicated in the legend, whereas NGPs and control (BSA) used as antigens in the chemiluminescent ELISA are shown in the Y-axis. Sera were used at 1:100 dilution. Two-way ANOVA with Tukey’s multiple comparisons: ns, non-significant; (***), <i>P<</i>0.001; (****), <i>P<</i>0.0001. (B) Lesion size (mm) in mice immunized with α-Gal-NGP (NGP12B, NGP17B, or NGP5B) or control (BSA), and then challenged with 1 x 10⁵ <i>L</i>. <i>major-luc</i> metacyclic promastigotes. One-way ANOVA (compared with BSA control): ns, non-significant; (**), <i>P<</i>0.01. (A and B) Error bars indicate S.E.M. of triplicate determinations.</p

Analysis of humoral immune response of NGP5B immunized mice.

<p>(A) Chemiluminescent ELISA reactivity against NGP5B of mouse sera obtained at prime (P), boost 1–3 (B1-B3), three weeks post-B3 (day 0) and at the endpoint (43 dpi) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. (B-C) Antibody isotyping (IgG1, IgG2a, IgG2b, IgG3, and IgE) of mouse sera obtained following boost 3 (B3) and at the experimental endpoint (43 dpi). Statistical analysis for A-C: Two-way ANOVA with Dunnett’s multiple comparison test: (*), <i>P</i><0.05; (****), <i>P</i><0.0001. (D) Percentage of lysis of <i>L</i>. <i>major-luc</i> metacyclic promastigotes incubated with sera from mice immunized with NGP5B, NGP5B+CpG, CpG or PBS. Control dead parasites: 10⁶ <i>L</i>. <i>major</i>-<i>luc</i> metacyclic promastigotes, heat-killed at 100^°C for 10 min, followed by 30-min incubation at RT with PI. Control live parasites: 10⁶ <i>L</i>. <i>major</i>-<i>luc</i> metacyclic promastigotes in DMEM (no FBS) without any treatment. C, mouse serum with active complement; iC, mouse serum with heat-inactivated complement; NMS, normal (non-infected) mouse serum. One-way ANOVA with Tukey’s multiple comparisons (compared with NMS control): ns, non-significant; (**), <i>P</i><0.01; (***), <i>P</i><0.001. (A-D) Error bars indicate S.E.M. of triplicate determinations.</p

Vaccination with NGP5B or NGP5B+CpG induces antigen-specific CD4⁺ and CD8⁺ memory T cell after <i>L</i>. <i>major</i> challenge.

<p>(A and B) Percentage of antigen specific CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, respectively, from splenocytes of mice 3 weeks post-last immunization and at the endpoint (immunized→challenged). Splenocytes were cultured and stimulated <i>in vitro</i> with 20 μg/ml of antigen. (C) Splenocytes stimulated and gated on CD4⁺CD44⁺, CD4⁺CD69⁺, and CD4⁺CD44⁺CD69⁺ T cell populations from immunized-challenged group, for the percentage of activated CD4⁺ T cells in mice vaccinated with NGP5B or NGP5B+CpG. (D) Splenocytes stimulated and gated on CD8⁺CD44⁺, CD8⁺CD69⁺, and CD8⁺CD44⁺CD69⁺ T cell populations from immunized-challenged group, for the percentage of activated CD8⁺ T cells in mice vaccinated with NGP5B or NGP5B+CpG. Statistical analysis for A-D: Two-tailed unpaired multiple Student’s <i>t</i>-test: (*), <i>P</i><0.05; (**), <i>P</i><0.01. (A-D) Error bars indicate S.E.M. of triplicate determinations.</p

Serodiagnosis and therapeutic monitoring of New-World tegumentary leishmaniasis using synthetic type-2 glycoinositolphospholipid-based neoglycoproteins

American tegumentary leishmaniasis (TL) caused by Leishmania braziliensis is characterized by a spectrum of clinical presentations, ranging from localized cutaneous ulcers (CL), mucosal (ML), or disseminated (DL) disease, to a subclinical (SC) asymptomatic form. Current diagnosis based on parasite culture and/or microscopy lacks sensitivity and specificity. Previous studies showed that patients with CL and ML have very high levels of Leishmania-specific anti-α-Gal antibodies. However, the native parasite α-Gal glycotope(s) is(are) still elusive, thus they have not yet been explored for a more accurate TL diagnosis. Using a chemiluminescent immunoassay, we evaluated the seroreactivity of TL patients across its clinical spectrum, and of endemic (EC) and nonendemic healthy controls (NEC) against three synthetic neoglycoproteins (NGP29b, NGP30b, and NGP28b), respectively comprising the L. major-derived type-2 glycoinositolphospholipid (GIPL)-1 (Galfβ1,3Manα), GIPL-2 (Galα1,3Galfβ1,3Manα), and GIPL-3 (Galα1,6Galα1,3Galfβ) glycotopes. Contrary to NGP29b and NGP30b, NGP28b exhibited high sensitivity and specificity to a CL serum pool. More importantly, NGP28b reacted strongly and specifically with individual sera from distinct clinical forms of TL, especially with SC sera, with 94% sensitivity and 97% specificity, by post-two-graph receiver-operating characteristic curve analysis. Contrary to NGP29b, NGP28b showed low cross-reactivity with Chagas disease and control (NEC/EC) sera. Additionally, seroreactivity of CL patients against NGP28b was significantly decreased after successful chemotherapy, indicating that L. braziliensis-specific anti-α-Gal antibodies may serve as an early biomarker of cure in CL. Our data also points towards the applicability of L. major type-2 GIPL-3-derived Galα1,6Galα1,3Galfβ glycotope for the serological diagnosis of American TL, particularly of the subclinical form.</p