Proteomic Analysis of the Promastigote Secretome of Seven Leishmania Species.

Leishmaniasis is one of the most impactful parasitic diseases worldwide, endangering the lives of 1 billion people every year. There are 20 different species of Leishmania able to infect humans, causing cutaneous (CL), visceral (VL), and/or mucocutaneous leishmaniasis (MCL). Leishmania parasites are known to secrete a plethora of proteins to establish infection and modulate the host's immune system. In this study, we analyzed using tandem mass spectrometry the total protein content of the secretomes produced by promastigote forms from seven Leishmania species grown in serum-free in vitro cultures. The core secretome shared by all seven Leishmania species corresponds to up to one-third of total secreted proteins, suggesting conserved mechanisms of adaptation to the vertebrate host. The relative abundance confirms the importance of known virulence factors and some proteins uniquely present in CL- or VL-causing species and may provide further insight regarding their pathogenesis. Bioinformatic analysis showed that most proteins were secreted via unconventional mechanisms, with an important role for vesicle-based secretion for all species. Gene Ontology annotation and enrichment analyses showed a high level of functional conservation among species. This study contributes to the current knowledge on the biological significance of differently secreted proteins and provides new information on the correlation of Leishmania secretome to clinical outcomes and species-specific pathogenesis.

A systematic review of peptide-based serological tests for the diagnosis of leishmaniasis.

Serological methods should meet the needs of leishmaniasis diagnosis due to their high sensitivity and specificity, economical and adaptable rapid diagnostic test format, and ease of use. Currently, the performances of serological diagnostic tests, despite improvements with recombinant proteins, vary greatly depending on the clinical form of leishmaniasis and the endemic area. Peptide-based serological tests are promising as they could compensate for antigenic variability and improve performance, independently of Leishmania species and subspecies circulating in the endemic areas. The objective of this systematic review was to inventory all studies published from 2002 to 2022 that evaluate synthetic peptides for serological diagnosis of human leishmaniases and also to highlight the performance (e.g., sensitivity and specificity) of each peptide reported in these studies. All clinical forms of leishmaniasis, visceral and tegumentary, and all Leishmania species responsible for these diseases were considered. Following PRISMA statement recommendations, 1,405 studies were identified but only 22 articles met the selection criteria and were included in this systematic review. These original research articles described 77 different peptides, of which several have promising performance for visceral or tegumentary leishmaniasis diagnosis. This review highlights the importance of and growing interest in synthetic peptides used for serological diagnosis of leishmaniases, and their performances compared to some widely used tests with recombinant proteins.

Title: Revue systématique des tests sérologiques à base de peptides pour le diagnostic de la leishmaniose. Abstract: D'une sensibilité et d'une spécificité élevées, faciles à réaliser, économiques et adaptables à un format de test de diagnostic rapide, les méthodes sérologiques devraient répondre aux besoins du diagnostic de la leishmaniose. Actuellement, les performances des tests de diagnostic sérologique, malgré des améliorations avec les protéines recombinantes, varient fortement selon la forme clinique de la leishmaniose et les zones d'endémie. Les tests sérologiques à base de peptides sont prometteurs car ils pourraient compenser la variabilité antigénique et améliorer les performances, indépendamment des espèces et sous-espèces de Leishmania circulant dans les zones endémiques. L'objectif de cette revue systématique était d'inventorier toutes les études publiées de 2002 à 2022 qui évaluent les peptides synthétiques pour le diagnostic sérologique des leishmanioses humaines et également de mettre en évidence les performances (dont la sensibilité et la spécificité) de chaque peptide rapporté dans ces études. Toutes les formes cliniques de leishmanioses, viscérales et tégumentaires, et toutes les espèces de Leishmania responsables de ces maladies ont été considérées. Suite aux recommandations de la déclaration PRISMA, 1405 études ont été identifiées mais seuls 22 articles répondaient aux critères de sélection et ont été inclus dans cette revue systématique. Ces articles de recherche originaux décrivent 77 peptides différents, dont plusieurs sont prometteurs pour le diagnostic de la leishmaniose viscérale ou tégumentaire. Cette revue met en évidence l'importance et l'intérêt croissant accordés aux peptides synthétiques utilisés pour le diagnostic sérologique des leishmanioses, et leurs performances par rapport à certains tests largement utilisés avec des protéines recombinantes.

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.

Human 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.

Peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis.

Dogs are the main reservoir of zoonotic visceral leishmaniasis. Vaccination is a promising approach to help control leishmaniasis and to interrupt transmission of the Leishmania parasite. The promastigote surface antigen (PSA) is a highly immunogenic component of Leishmania excretory/secretory products. A vaccine based on three peptides derived from the carboxy-terminal part of Leishmania amazonensis PSA and conserved among Leishmania species, formulated with QA-21 as adjuvant, was tested on naive Beagle dogs in a preclinical trial. Four months after the full course of vaccination, dogs were experimentally infected with Leishmania infantum promastigotes. Immunization of dogs with peptide-based vaccine conferred immunity against experimental infection with L. infantum. Evidence for macrophage nitric oxide production and anti-leishmanial activity associated with IFN-γ production by lymphocytes was only found in the vaccinated group. An increase in specific IgG2 antibodies was also measured in vaccinated dogs from 2 months after immunization. Additionally, after challenge with L. infantum, the parasite burden was significantly lower in vaccinated dogs than in the control group. These data strongly suggest that this peptide-based vaccine candidate generated cross-protection against zoonotic leishmaniasis by inducing a Th1-type immune response associated with production of specific IgG2 antibodies. This preclinical trial including a peptide-based vaccine against leishmaniasis clearly demonstrates effective protection in a natural host. This approach deserves further investigation to enhance the immunogenicity of the peptides and to consider the possible engineering of a vaccine targeting several Leishmania species.

Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein.

Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs.

Preventive 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 recombinant 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 recombinant 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.

Identification and characterization of new Leishmania promastigote surface antigens, LaPSA-38S and LiPSA-50S, as major immunodominant excreted/secreted components of L. amazonensis and L. infantum.

We have previously demonstrated that sera from dogs vaccinated with excreted/secreted antigens (ESA) of Leishmania infantum promastigotes (LiESAp) mainly recognized an immunodominant antigen of 54 kDa. An anti-LiESAp-specific IgG2 humoral response was observed and associated to Th1-type response in vaccinated dogs. This response was highly correlated with a long-lasting and strong LiESAp-vaccine protection toward L. infantum experimental infection. In addition, it was also shown that dogs from the vaccinated group developed a selective IgG2 response against an immunodominant antigen of 45 kDa of Leishmania amazonensis ESA promastigotes (LaESAp). In order to identify and characterize these immunodominant antigens, a mouse monoclonal antibody (mAb F5) was produced by immunization against LaESAp. It was found to recognize the major antigenic targets of both LaESAp and LiESAp. Analysis with mAb F5 of L. amazonensis amastigote and promastigote cDNA expression libraries enabled the identification of clones encoding proteins with significant structural homology to the promastigote surface antigens named PSA-2/gp-46. Among them, one clone presented a full-length cDNA and encoded a novel L. amazonensis protein of 38.6 kDa calculated molecular mass (LaPSA-38S) sharing an amino acid sequence consistent with that of the PSA polymorphic family and a N-terminal signal peptide, characteristic of a secreted protein. We then screened a L. infantum promastigote DNA cosmid library using a cDNA probe derived from the LaPSA-38S gene and identified a full-length clone of a novel excreted/secreted protein of L. infantum with a calculated molecular mass of 49.2 kDa and named LiPSA-50S. The fact that a significant immunological reactivity was observed against PSA, suggests that these newly identified proteins could have an important immunoregulatory influence on the immune response. This hypothesis is supported by the fact that (i) these proteins were naturally excreted/secreted by viable Leishmania promastigotes and amastigotes, and (ii) they are selectively recognized by vaccinated and protected dogs.

In vitro evaluation of a soluble Leishmania promastigote surface antigen as a potential vaccine candidate against human leishmaniasis.

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (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 a L. 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 were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection.