IgG reaction pattern associated with CVL status.

<p>Total <i>Leishmania infantum</i> promastigote cell extracts were separated with 12% SDS-PAGE and electrotransferred to nitrocellulose membranes. The membranes were probed with serum obtained from individual asymptomatic dogs (1:500) or symptomatic dogs (1:1,000) and secondary anti-dog (A) IgG, (B) IgG1 or (C) IgG2 (1:10,000). Representative western blots from each group of dogs are depicted (asymptomatic: n = 5; symptomatic: n = 3). Numbers represent the code numbers of the dogs enrolled in the study. Molecular weight markers are shown on the left-hand side.</p

<i>In silico</i> evaluation of the identified proteins antigenicity.

<p><i>In silico</i> evaluation of the identified proteins antigenicity.</p

Percentage (%) of amino acid sequence coverage of identified <i>L</i>. <i>infantum</i> proteins based on their content in high binding MHC class I and/or II-restricted peptides.

<p>Percentage (%) of amino acid sequence coverage of identified <i>L</i>. <i>infantum</i> proteins based on their content in high binding MHC class I and/or II-restricted peptides.</p

Proteins selected on the basis of predicted antigenicity and their content on highly scored MHC class I- and MHC class II-restricted epitopes.

<p>Proteins selected on the basis of predicted antigenicity and their content on highly scored MHC class I- and MHC class II-restricted epitopes.</p

Comparison of serospecificities against total promastigote cell extracts of symptomatic (SD) and asymptomatic (AD) dogs suffering from CVL.

<p>(A) Total promastigote cell extracts were separated in the first dimension with a pH gradient of 5–8 (7-cm strips) followed by 12% SDS-PAGE. The separated proteins were electroblotted onto a nitrocellulose membrane and probed with pools of sera from (B) asymptomatic (n = 9; 1:500) and (C) symptomatic dogs (n = 5; 1:1,000). Protein spots recognized by AD sera only are numbered.</p

2DE and western blot analysis of serospecificity for samples from different dogs suffering from CVL.

<p>(A) Total promastigote cell extracts were separated in the first dimension with a pH gradient of 3–10 (7 cm strips) followed by 12% SDS-PAGE. The separated proteins were electroblotted onto nitrocellulose membranes and probed with different pools of sera from (B) healthy dogs (n = 5; 1:500), (C, E) asymptomatic dogs (n = 5 and n = 4, respectively; 1:500), and (D, F) symptomatic dogs (n = 3 and n = 2, respectively; 1:1,000).</p

Protein antigens recognized by IgG antibodies in asymptomatic dog (AD) sera.

<p>Protein antigens recognized by IgG antibodies in asymptomatic dog (AD) sera.</p

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

Vaccination and challenge schedule in BALB/c mice.

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