Leishmania donovani nucleoside hydrolase (NH36) Domains induce T-cell cytokine responses in human Visceral leishmaniasis

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-gamma, IL-1 beta, and TNF-a and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3(+)CD4(+)IL-2(+)TNF-alpha-IFN-gamma(-), CD3(+)CD4(+)IL-2(+)TNF-alpha+IFN-gamma(-), CD3(+)CD4(+)IL-2(+)TNF-alpha-IFN-gamma(+), and CD3(+)CD4(+)IL-2(+)TNF-alpha+IFN-gamma(+) T cells in cured and asymptomatic subjects. Consistent with this, the IFN-gamma increase was correlated with decreased spleen (R = -0.428, P = 0.05) and liver sizes (R = -0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = -0.595, P = 0.005) and F2 (R = -0.462, P = 0.04). Conversely, F1 and F3 increased the CD3(+)CD8(+)IL-2(+)TNF-alpha-IFN-gamma(-), CD3(+)CD8(+)IL-2(+)TNF-alpha+IFN-gamma(-), and CD3(+)CD8(+)IL-2(+)TNF-alpha+IFN-gamma(+) T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4(+)-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8(+) T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4(+) and CD8(+) T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ)Fundacao Carlos Chagas de Amparo a Pesquisa do Estado de Rio de Janeiro (FAPERJ)Coordenacao de Aperfeicoamento de Pessoal de Nivel SuperiorCNPQ-Fundacao de Apoio a Pesquisa e a Inovacao Tecnologica do Estado de Sergipe-PRONEXFAPITEC CNPq (PRONEX)VII PN I+D+IFEDER FundsUniv Fed Sergipe HU UFS, Dept Med, Univ Hosp, Mol Biol Lab, Aracaju, Sergipe, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Paulo de Goes, Dept Microbiol Geral, Lab Biol Bioquim Leishmania, Rio De Janeiro, RJ, BrazilPontificia Univ Catolica Rio de Janeiro, Lab Biometrol, Programa Posgrad Metrol, Rio De Janeiro, RJ, BrazilInst Salud Carlos III, WHO Collaborating Ctr Leishmaniasis, Ctr Nacl Microbiol, Madrid, Comunidad De Ma, SpainInst Oswaldo Cruz, Lab Imunoparasitol, Rio De Janeiro, RJ, BrazilUniv Fed Rio de Janeiro, Inst Microbiologia Paulo de Goes, Dept Imunol, Lab Imunol Integrada, Rio De Janeiro, RJ, BrazilUniv Sao Paulo, Fac Med, Inst Invest Imunol, Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Microbiol Imunol & Parasitol, Lab Vacinas Expt, Sao Paulo, SP, BrazilUniv Fed Rio de Janeiro, Fac Med, Lab Imunohematol, Hosp Univ Clementino Fraga Filho, Rio De Janeiro, RJ, BrazilUniv Fed Sergipe HU UFS, Dept Morfol, Aracaju, Sergipe, BrazilUniv Fed Sao Paulo UNIFESP, Dept Microbiol Imunol & Parasitol, Lab Vacinas Expt, Sao Paulo, SP, BrazilCNPq: 300639/2003-1CNPq: 310977/2014-2CNPq: 310797/2015-2CNPq: 404400/2012-4FAPERJ: E-26-201.583/2014FAPERJ: E-26-102957/2011FAPERJ: E-26/111.682/2013FAPERJ: E-26/102415/2010FAPERJ: E-26/201747/2015CAPES: 23038.005304/2011-0CNPQ-PRONEX: 12/2009FAPITEC CNPq (PRONEX): 019.203.02712/2009-8FEDER Funds: RICET RD12/0018/0003Web of Scienc

Recombinant forms of Leishmania amazonensis excreted/secreted promastigote surface antigen (PSA) induce protective immune responses in dogs

International audiencePreventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombi-nant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21-and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recom-binant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates

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

Interleukin-2 as a marker for detecting asymptomatic individuals in areas where Leishmania infantum is endemic

AbstractNo field method exists for identifying asymptomatic individuals in areas where Leishmania infantum is endemic. This work reports that, 24 h after stimulating whole blood with soluble Leishmania antigen (SLA), plasma interferon-γ (IFN-γ) and interleukin-2 (IL-2) become significantly elevated in samples from asymptomatic individuals (n=47) compared with those from negative controls (n=50), all of them recruited from a blood bank. When compared with the reference test SLA-lymphoproliferative assay, IL-2 appears as a new, 100% sensitive and specific marker for asymptomatic individuals with a positive cellular response (compared with 100% and 84.78%, respectively, for IFN-γ). Further studies in other transmission areas and in other cohorts of exposed people need to be performed to confirm these results. Once validated, IFN-γ and IL-2 levels in SLA-stimulated whole blood could be reliably used in the field to estimate the prevalence of those asymptomatic individuals with Leishmania-specific cellular immune responses

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

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

<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

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