Identification of BALB/c Immune Markers Correlated with a Partial Protection to <i>Leishmania infantum</i> after Vaccination with a Rationally Designed Multi-epitope Cysteine Protease A Peptide-Based Nanovaccine

<div><p>Background</p><p>Through their increased potential to be engaged and processed by dendritic cells (DCs), nanovaccines consisting of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with both antigenic moieties and adjuvants are attractive candidates for triggering specific defense mechanisms against intracellular pathogens. The aim of the present study was to evaluate the immunogenicity and prophylactic potential of a rationally designed multi-epitope peptide of <i>Leishmania</i> Cysteine Protease A (CPA_160-189) co-encapsulated with Monophosphoryl lipid A (MPLA) in PLGA NPs against <i>L</i>. <i>infantum</i> in BALB/c mice and identify immune markers correlated with protective responses.</p><p>Methodology/Principal Findings</p><p>The DCs phenotypic and functional features exposed to soluble (CPA_160-189, CPA_160-189+MPLA) or encapsulated in PLGA NPs forms of peptide and adjuvant (PLGA-MPLA, PLGA-CPA_160-189, PLGA-CPA_160-189+MPLA) was firstly determined using BALB/c bone marrow-derived DCs. The most potent signatures of DCs maturation were obtained with the PLGA-CPA_160-189+MPLA NPs. Subcutaneous administration of PLGA-CPA_160-189+MPLA NPs in BALB/c mice induced specific anti-CPA_160-189 cellular and humoral immune responses characterized by T cells producing high amounts of IL-2, IFN-γ and TNFα and IgG1/IgG2a antibodies. When these mice were challenged with 2x10⁷ stationary phase <i>L</i>. <i>infantum</i> promastigotes, they displayed significant reduced hepatic (48%) and splenic (90%) parasite load at 1 month post-challenge. This protective phenotype was accompanied by a strong spleen lymphoproliferative response and high levels of IL-2, IFN-γ and TNFα versus low IL-4 and IL-10 secretion. Although, at 4 months post-challenge, the reduced parasite load was preserved in the liver (61%), an increase was detected in the spleen (30%), indicating a partial vaccine-induced protection.</p><p>Conclusions/Significance</p><p>This study provide a basis for the development of peptide-based nanovaccines against leishmaniasis, since it reveals that vaccination with well-defined <i>Leishmania</i> MHC-restricted epitopes extracted from various immunogenic proteins co-encapsulated with the proper adjuvant or/and phlebotomine fly saliva multi-epitope peptides into clinically compatible PLGA NPs could be a promising approach for the induction of a strong and sustainable protective immunity.</p></div

Properties of synthesized PLGA NPs.^a.

<p>Properties of synthesized PLGA NPs.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005311#t002fn001" target="_blank">^a</a>.</p

Co-encapsulation of CPA_160-189 and MPLA in PLGA NPs induced activation of DCs.

<p>(A-F) Diagrams showing expression (MFI values) of CD40, CD80, CD83, CD86, MHCI and MHCII molecules on DCs and (G) (%) IL-12-producing DCs after pulsing with PLGA NPs. Results are expressed as mean±SD from three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 were assessed by one-way ANOVA and Tukey’s multiple comparison tests.</p

Evaluation of CPA_160-189 immunogenicity in naturally infected dogs.

<p>Detection of (A) CPA_160-189- and (B) SLA-specific IgG antibodies in sera samples from asymptomatic (n = 12) and symptomatic (n = 14) <i>L</i>. <i>infantum</i>-infected and non-infected healthy (n = 6) dogs. Lines indicate the mean±SD.</p

Characterization of the synthesized PLGA NPs.

<p>(A) PLGA-CPA_160-189 NPs and (B) PLGA-CPA_160-189+MPLA NPs were analyzed for their morphology by scanning electron microscopy analysis (SEM). Indicative SEM photomicrographs with scale bar at 1 μm. Size distribution of (C) PLGA-CPA_160-189 NPs and (D) PLGA-CPA_160-189+MPLA NPs. (E) CPA_160-189 peptide and (F) MPLA <i>in vitro</i> release profile of PLGA-CPA_160-189 NPs and PLGA-CPA_160-189+MPLA NPs in PBS at 37°C. Data represent the mean±SD from three independent experiments.</p

Evaluation of vaccine-mediated protection against <i>L</i>. <i>infantum</i> infection in BALB/c mice 1 month post intravenous challenge.

<p>Determination of parasite load in (A) the liver and (B) the spleen by limiting dilution assay in vaccinated and non-vaccinated mice 1 month post challenge with <i>L</i>. <i>infantum</i>. Results are presented as means±SD of five individual mice per group, representative of two independent experiments with similar results. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 were assessed by one-way ANOVA and Tukey’s multiple comparison tests.</p

Determination of CPA_160-189-specific cellular responses in PLGA NPs-vaccinated mice.

<p>(A) CPA_160-189-specific proliferation of spleen cells obtained from mice vaccinated at different time points. Control group was vaccinated with PBS. Results are expressed as mean stimulation index (S.I.)±SD from three independent experiments. (B) CPA_160-189-specific IL-2, IFN-γ, IL-4, IL-10 and TNFα production from spleen cells isolated two weeks after the end of vaccinations from vaccinated and non-vaccinated mice. (C) Diagram showing detection of CPA_160-189-specific IFN-γ production by CD4⁺ and CD8⁺ T cells by flow cytometry analysis with representative dot plots, two weeks after the end of vaccinations. Results are presented as means±SD of five individual mice per group, representative of two independent experiments with similar results. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 for proliferation and cytokine assays were assessed by two-way ANOVA and Bonferroni’s multiple comparison tests, whereas flow cytometry results were assessed by one-way ANOVA and Tukey’s multiple comparison tests.</p

Vaccination and challenge schedule in BALB/c mice.

<p>Vaccination and challenge schedule in BALB/c mice.</p

Evaluation of vaccine-mediated protection against <i>L</i>. <i>infantum</i> infection in BALB/c mice 4 months post intravenous challenge.

<p>Determination of (A-B) parasite load in the spleen and the liver by limiting dilution assay and (C-D) hepatosplenomegaly in vaccinated and non-vaccinated mice 4 months post challenge with <i>L</i>. <i>infantum</i>. Results are presented as means±SD of five individual mice per group, representative of two independent experiments with similar results. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 were assessed by one-way ANOVA and Tukey’s multiple comparison tests.</p

Antigen-presentation efficacy of PLGA NPs-pulsed DCs.

<p>(A) Antigen-presentation efficacy of PLGA NPs-pulsed DCs to naive splenocytes. Splenocytes treated with medium alone served as negative control. The results are the mean stimulation index (S.I.)±SD from three independent experiments. (B) IFN-γ, IL-4 and IL-10 mRNA expression in DCs-primed splenocytes by qRT-PCR. Results are presented as means±SD of three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 were assessed by one-way ANOVA and Tukey’s multiple comparison tests.</p