In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

International audiencePSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in severalLeishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice.We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in aL. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L.braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals weresubdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantuminfection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high orlow levels of IFN-c in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detectedin sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-c, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-a in more. No significant cytokine response wasobserved in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increasein CD4+ T cells producing IFN-c after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between thepercentage of IFN-c-producing CD4+ T cells and the released IFN-c. We showed that the LaPSA-38S protein was able toinduce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infectionindicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacityof Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infectio

Design of multi-epitope peptides containing HLA class-I and class-II-restricted epitopes derived from immunogenic Leishmania proteins, and evaluation of CD4+ and CD8+ T cell responses induced in cured cutaneous leishmaniasis subjects

International audienceHuman leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis

SDS-PAGE and silver staining of purified recombinant <i>La</i>-PSA38S protein expressed in <i>L. tarentolae</i>.

<p>Purified <i>La</i>-PSA38S was separated by electrophoresis in a 12% SDS-PAGE and silver stained under reducing (R) and non reducing (NR) conditions. The arrow indicates the position of <i>La</i>-PSA-38S (45 kDa) and Molecular Weight markers were marked in kDa.</p

Total IgG prevalence against <i>La</i>PSA-38S in different endemic areas of leishmaniasis.

<p>The cut-off value of reactivity for <i>La</i>PSA-38S antigen was calculated as the mean plus 3 SD of the OD values observed in naive controls from each country. These cut-off values allowed us to distinguish between positive and negative sera and consequently to estimate performance parameters of each ELISA test.</p

Total Soluble <i>Leishmania</i> antigen (TSLA) specific IFN-γ, granzyme B, TNF-α responses.

<p>IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g002" target="_blank">Fig. 2a</a>), granzyme B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g002" target="_blank">Fig. 2b</a>), were detected and quantified from culture supernatants of PBMC exposed for 120h to local TSLA (10 µg/ml) and TSLA Ldd8 (10 µg/ml), by Cytokine Beads Array test (CBA) using Flow cytometry. Statistically significant differences between stimulated and non stimulated cultures and between groups (p≤0.03) are showed.</p

<i>La</i>PSA-38S specific IFN-γ, granzyme B, TNF-α and IL-10 responses.

<p>IFN-γ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3a</a>), granzyme B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3b</a>), TNF-α (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3c</a>) and IL-10 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092708#pone-0092708-g003" target="_blank">Fig. 3d</a>) were detected and quantified from culture supernatants of PBMC exposed for 120 h to SLA (10 µg/ml) or <i>La</i>PSA-38S (10 µg/ml) using Cytokine Beads Array test (CBA). Data were analyzed by Flow cytometry. PHA (10 µg/ml) was used for all cultures as positive control (data not shown). Statistically significant differences between stimulated and non stimulated cultures (p≤0.003) and between groups (p≤0.01) are showed.</p

Study population.

<p>The recruitment and sampling collection (186 donors) of different groups (patients, cured, immunes without clinical symptoms and naives) were performed in endemic and non-endemic areas in each country.</p><p>*CVLd: Cured Visceral Leishmaniasis due to <i>L. donovani</i> (India), aVLd: active Visceral Leishmaniasis due to <i>L. donovani</i> (India), HLR-I: Healthy Low Responders from India, CCLb: Cured Cutaneous Leishmaniasis due to <i>L. brasiliensis</i> (Peru), aCLb: active Cutaneous Leishmaniasis due to <i>L. brasiliensis</i> (Peru), HLR-P: Healthy Low Responders from Peru, CCLm: Cured Cutaneous Leishmaniasis due to <i>L. major</i> (Tunisia), HHR-Lm: Healthy High Responders living in an endemic area for <i>L. major</i> (Tunisia), HHR-LiT: Healthy High Responders living in an endemic area for <i>L. infantum</i> (Tunisia), aVLiT: active Visceral Leishmaniasis due to <i>L. infantum</i> (Tunisia), H-T: Healthy Low Responders from Tunisia, HHR-LiF: Healthy High Responders living in an endemic area for <i>L. infantum</i> (France), HLR-F: Healthy Low Responders from France, HHR-LiS: Healthy High Responders living in an endemic area for <i>L. infantum</i> (Spain), aVLiS: active Visceral Leishmaniasis due to <i>L. infantum</i> (Spain), HLR-S: Healthy Low Responders from Spain.</p