Development of a novel squalene/α-tocopherol-based self-emulsified nanoemulsion incorporating Leishmania peptides for induction of antigen-specific immune responses.

Vaccination has emerged as the most effective strategy to confront infectious diseases, among which is leishmaniasis, that threat public health. Despite laborious efforts there is still no vaccine for humans to confront leishmaniasis. Multi-epitope protein/peptide vaccines present a number of advantages, however their use along with appropriate adjuvants that may also act as antigen carriers is considered essential to overcome subunit vaccines' low immunogenicity. In the present study, a stable self-emulsified nanoemulsion was developed and double-adjuvanted with squalene and α-tocopherol. The prepared nanoemulsion droplets exhibited low cytotoxicity in a certain range of concentrations, while they were efficiently taken up by macrophages and dendritic cells in vitro as well as in vivo in secondary lymphoid organs. To further characterize nanoformulation's potent antigen delivery capability, three multi-epitope Leishmania peptides were incorporated into the nanoemulsion. Peptide encapsulation resulted in dendritic cells' functional differentiation characterized by elevated levels of maturation markers and intracellular cytokine production. Intramuscular administration of the nanoemulsion incorporating Leishmania peptides induced antigen-specific spleen cell proliferation as well as elicitation of CD4+ central memory cells, supporting the potential of the developed nanoformulation to successfully act also as an antigen delivery vehicle and thus encouraging further preclinical studies on its vaccine candidate potency.

Intramuscular Immunization with a Liposomal Multi-Epitope Chimeric Protein Induces Strong Cellular Immune Responses against Visceral Leishmaniasis.

Control of the intracellular parasite Leishmania (L.) requires the activation of strong type 1 cellular immune responses. Towards this goal, in the present study, a multiepitope chimeric protein named LiChimera was encapsulated into cationic liposomes and its protective efficacy against experimental visceral leishmaniasis was investigated. Liposomal LiChimera conferred significant protection against L. infantum as evidenced by the significantly reduced parasite loads in the spleen and liver. Protection detected in Lipo:LiChimera-immunized mice was dependent on the differentiation of long-lasting cellular immune responses and particularly the induction of antigen-specific multifunctional memory CD4+ TH1 and CD8+ T cells that persisted during infection, as evidenced by the persistent high production of IFN-γ and IL-2 and proliferation activity. Notably, protected mice were also characterized by significantly low numbers of non-regulatory CD4+ T cells able to co-produce IFN-γ and IL-10, an important population for disease establishment, as compared to non-immunized control group. Collectively, these results demonstrate that cationic liposomes containing LiChimera can be considered an effective candidate vaccine against visceral leishmaniasis.

A liposomal vaccine promotes strong adaptive immune responses via dendritic cell activation in draining lymph nodes.

Subunit proteins provide a safe source of antigens for vaccine development especially for intracellular infections which require the induction of strong cellular immune responses. However, those antigens are often limited by their low immunogenicity. In order to achieve effective immune responses, they should be encapsulated into a stable antigen delivery system combined with an appropriate adjuvant. As such cationic liposomes provide an efficient platform for antigen delivery. In the present study, we describe a liposomal vaccine platform for co-delivery of antigens and adjuvants able to elicit strong antigen-specific adaptive immune responses. Liposomes are composed of the cationic lipid dimethyl dioctadecylammonium bromide (DDAB), cholesterol (CHOL) and oleic acid (OA). Physicochemical characterization of the formulations showed that their size was in the range of ∼250 nm with a positive zeta potential which was affected in some cases by the enviromental pH facilitating endosomal escape of potential vaccine cargo. In vitro, liposomes were effectively taken up by bone marrow dendritic cells (BMDCs) and when encapsulated IMQ they promoted BMDCs maturation and activation. Upon in vivo intramuscular administration, liposomes' active drainage to lymph nodes was mediated by DCs, B cells and macrophages. Thus, mice immunization with liposomes having encapsulated LiChimera, a previously characterized anti-leishmanial antigen, and IMQ elicited infiltration of CD11blow DCs populations in draining LNs followed by increased antigen-specific IgG, IgG2a and IgG1 levels production as well as indcution of antigen-specific CD4+ and CD8+ T cells. Collectively, the present work provides a proof-of-concept that cationic liposomes composed of DDAB, CHOL and OA adjuvanted with IMQ provide an efficient delivery platform for protein antigens able to induce strong adaptive immune responses via DCs targeting and induction of maturation.

Immunoinformatics Approach to Design a Multi-Epitope Nanovaccine against Leishmania Parasite: Elicitation of Cellular Immune Responses.

Leishmaniasis is a vector-borne disease caused by an intracellular parasite of the genus Leishmania with different clinical manifestations that affect millions of people worldwide, while the visceral form may be fatal if left untreated. Since the available chemotherapeutic agents are not satisfactory, vaccination emerges as the most promising strategy for confronting leishmaniasis. In the present study, a reverse vaccinology approach was adopted to design a pipeline starting from proteome analysis of three different Leishmania species and ending with the selection of a pool of MHCI- and MHCII-binding epitopes. Epitopes from five parasite proteins were retrieved and fused to construct a multi-epitope chimeric protein, named LeishChim. Immunoinformatics analyses indicated that LeishChim was a stable, non-allergenic and immunogenic protein that could bind strongly onto MHCI and MHCII molecules, suggesting it as a potentially safe and effective vaccine candidate. Preclinical evaluation validated the in silico prediction, since the LeishChim protein, encapsulated simultaneously with monophosphoryl lipid A (MPLA) into poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles, elicited specific cellular immune responses when administered to BALB/c mice. These were characterized by the development of memory CD4+ T cells, as well as IFNγ- and TNFα-producing CD4+ and CD8+ T cells, supporting the potential of LeishChim as a vaccine candidate.

Antibody and T-Cell Subsets Analysis Unveils an Immune Profile Heterogeneity Mediating Long-term Responses in Individuals Vaccinated Against SARS-CoV-2.

BACKGROUND: Based on the fact that coronavirus disease 2019 (COVID-19) is still spreading despite worldwide vaccine administration, there is an imperative need to understand the underlying mechanisms of vaccine-induced interindividual immune response variations. METHODS: We compared humoral and cellular immune responses in 127 individuals vaccinated with either BNT162b2, mRNA-1273, or ChAdOx1-nCoV-19 vaccine. RESULTS: Both mRNA vaccines induced faster and stronger humoral responses as assessed by high spike- and RBD-specific antibody titers and neutralizing efficacy in comparison to ChAdOx1-nCoV-19 vaccine. At 7 months postvaccination, a decreasing trend in humoral responses was observed, irrespective of the vaccine administered. Correlation analysis between anti-S1 IgG and interferon- (IFN-) production unveiled a heterogeneous immune profile among BNT162b2-vaccinated individuals. Specifically, vaccination in the high-responder group induced sizable populations of polyfunctional memory CD4 helper T cells (TH1), follicular helper T cells (TFH), and T cells with features of stemness (TSCM), along with high neutralizing antibody production that persisted up to 7 months. In contrast, low responders were characterized by significantly lower antibody titers and memory T cells and a considerably lower capacity for interleukin-2 and IFN- production. CONCLUSIONS: We identified that long-term humoral responses correlate with the individuals ability to produce antigen-specific persistent memory T-cell populations.

Transcriptional Profiling of Leishmania infantum Infected Dendritic Cells: Insights into the Role of Immunometabolism in Host-Parasite Interaction.

Leishmania parasites are capable of effectively invading dendritic cells (DCs), a cell population orchestrating immune responses against several diseases, including leishmaniasis, by bridging innate and adaptive immunity. Leishmania on the other hand has evolved various mechanisms to subvert DCs activation and establish infection. Thus, the transcriptional profile of DCs derived from bone marrow (BMDCs) that have been infected with Leishmania infantum parasite or of DCs exposed to chemically inactivated parasites was investigated via RNA sequencing, aiming to better understand the host-pathogen interplay. Flow cytometry analysis revealed that L. infantum actively inhibits maturation of not only infected but also bystander BMDCs. Analysis of double-sorted L. infantum infected BMDCs revealed significantly increased expression of genes mainly associated with metabolism and particularly glycolysis. Moreover, differentially expressed genes (DEGs) related to DC-T cell interactions were also found to be upregulated exclusively in infected BMDCs. On the contrary, transcriptome analysis of fixed parasites containing BMDCs indicated that energy production was mediated through TCA cycle and oxidative phosphorylation. In addition, DEGs related to differentiation of DCs leading to activation and differentiation of Th17 subpopulations were detected. These findings suggest an important role of metabolism on DCs-Leishmania interplay and eventually disease establishment.

A Canine-Directed Chimeric Multi-Epitope Vaccine Induced Protective Immune Responses in BALB/c Mice Infected with Leishmania infantum.

Leishmaniases are complex vector-borne diseases caused by intracellular parasites of the genus Leishmania. The visceral form of the disease affects both humans and canids in tropical, subtropical, and Mediterranean regions. One health approach has suggested that controlling zoonotic visceral leishmaniasis (ZVL) could have an impact on the reduction of the human incidence of visceral leishmaniasis (VL). Despite the fact that a preventive vaccination could help with leishmaniasis elimination, effective vaccines that are able to elicit protective immune responses are currently lacking. In the present study, we designed a chimeric multi-epitope protein composed of multiple CD8+ and CD4+ T cell epitopes which were obtained from six highly immunogenic proteins previously identified by an immunoproteomics approach, and the N-termini of the heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis served as an adjuvant. A preclinical evaluation of the candidate vaccine in BALB/c mice showed that when it was given along with the adjuvant Addavax it was able to induce strong immune responses. Cellular responses were dominated by the presence of central and effector multifunctional CD4+ and CD8+ T memory cells. Importantly, the vaccination reduced the parasite burden in both short-term and long-term vaccinated mice challenged with Leishmania infantum. Protection was characterized by the continuing presence of IFN-γ+TNFα+-producing CD8+ and CD4+ T cells and increased NO levels. The depletion of CD8+ T cells in short-term vaccinated mice conferred a significant loss of protection in both target organs of the parasite, indicating a significant involvement of this population in the protection against L. infantum challenge. Thus, the overall data could be considered to be a proof-of-concept that the design of efficacious T cell vaccines with the help of reverse vaccinology approaches is possible.

Transcriptome Analysis Identifies Immune Markers Related to Visceral Leishmaniasis Establishment in the Experimental Model of BALB/c Mice.

Visceral leishmaniasis (VL) caused by Leishmania donovani and L. infantum is a potentially fatal disease. To date there are no registered vaccines for disease prevention despite the fact that several vaccines are in preclinical development. Thus, new strategies are needed to improve vaccine efficacy based on a better understanding of the mechanisms mediating protective immunity and mechanisms of host immune responses subversion by immunopathogenic components of Leishmania. We found that mice vaccinated with CPA162-189-loaded p8-PLGA nanoparticles, an experimental nanovaccine, induced the differentiation of antigen-specific CD8+ T cells in spleen compared to control mice, characterized by increased dynamics of proliferation and high amounts of IFN-γ production after ex vivo re-stimulation with CPA162-189 antigen. Vaccination with CPA162-189-loaded p8-PLGA nanoparticles resulted in about 80% lower parasite load in spleen and liver at 4 weeks after challenge with L. infantum promastigotes as compared to control mice. However, 16 weeks after infection the parasite load in spleen was comparable in both mouse groups. Decreased protection levels in vaccinated mice were followed by up-regulation of the anti-inflammatory IL-10 production although at lower levels in comparison to control mice. Microarray analysis in spleen tissue at 4 weeks post challenge revealed different immune-related profiles among the two groups. Specifically, vaccinated mice were characterized by similar profile to naïve mice. On the other hand, the transcriptome of the non-vaccinated mice was dominated by increased expression of genes related to interferon type I, granulocyte chemotaxis, and immune cells suppression. This profile was significantly enriched at 16 weeks post challenge, a time-point which is relative to disease establishment, and was common for both groups, further suggesting that type I signaling and granulocyte influx has a significant role in disease establishment, pathogenesis and eventually in decreased vaccine efficacy for stimulating long-term protection. Overall, we put a spotlight on host immune networks during active VL as potential targets to improve and design more effective vaccines against disease.

Detection of Antigen-specific T cells in Spleens of Vaccinated Mice Applying 3[H]-Thymidine Incorporation Assay and Luminex Multiple Cytokine Analysis Technology.

For many infectious diseases T cells are an important part of naturally acquired protective immune responses, and inducing these by vaccination has been the aim of much research. Here, we describe a protocol for the analysis of vaccine-induced antigen-specific immune responses. For this purpose, cells of whole spleens obtained from vaccinated BALB/c mice were ex vivo stimulated with the antigen incorporated in the vaccine. Evaluation and characterization of vaccine-induced adaptive T cell responses was performed by assaying spleen cell proliferation through radioactive 3[H]-thymidine incorporation and multiplex cytokine analysis of IL-2, IFN-γ and TNFα in supernatants from spleen cell suspensions. This protocol can be very useful as a starting point for assessing vaccine-induced memory T cell populations in pre-clinical studies.

Induction of protective cellular immune responses against experimental visceral leishmaniasis mediated by dendritic cells pulsed with the N-terminal domain of Leishmania infantum elongation factor-2 and CpG oligodeoxynucleotides.

Leishmania elongation factor 2 (EF-2) has been previously identified as a TH1-stimulatory protein. In this study, we assayed the protective potential of the N-terminal domain of EF-2 (N-LiEF-2, 1-357 aa) that has been predicted to contain several overlapping MHC class I and II-restricted epitopes injected in the form of dendritic cell (DC)-based vaccine. Ex vivo pulsing of DCs with the recombinant N-LiEF-2 domain along with CpG oligodeoxynucleotides (ODNs) resulted in their functional differentiation. BALB/c vaccinated with CpG-triggered DCs pulsed with N-LiEF-2 were found to be the most immune-reactive in terms of induction of DTH responses, increased T cell proliferation and IL-2 production. Moreover, vaccination induced antigen-specific TH1 type immune response as evidenced by increased IFN-γ and TNFα levels followed by a significant increase of nitrite (NO) and reactive oxygen species (ROS) in splenocyte cultures. Vaccinated mice showed a pronounced decrease in parasite load in spleen and liver when challenged with L. infantum, increased expression of Stat1 and Tbx21 mRNA transcripts versus reduced expression of Foxp3 transcripts and were able to produce significantly elevated levels of IL-2, IFN-γ and TNFα but not IL-10 compared to non-vaccinated mice. Both antigen and parasite-specific CD4+ T and CD8+ T cells contributed to the IFN-γ production indicating that both subtypes contribute to the resistance to infection and correlated with robust nitrite generation, critical in controlling Leishmania infection. Together, these findings demonstrated the immunogenic as well as protective potential of the N-terminal domain of Leishmania EF-2 when given with CpG-triggered DCs representing a basis for the development of rationalized vaccine against leishmaniasis.

Vaccination with poly(D,L-lactide-co-glycolide) nanoparticles loaded with soluble Leishmania antigens and modified with a TNFα-mimicking peptide or monophosphoryl lipid A confers protection against experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) persists as a major public health problem, and since the existing chemotherapy is far from satisfactory, development of an effective vaccine emerges as the most appropriate strategy for confronting VL. The development of an effective vaccine relies on the selection of the appropriate antigen and also the right adjuvant and/or delivery vehicle. In the present study, the protective efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which were surface-modified with a TNFα-mimicking eight-amino-acid peptide (p8) and further functionalized by encapsulating soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand, was evaluated against challenge with L. infantum parasites in BALB/c mice. Vaccination with these multifunctionalized PLGA nanoformulations conferred significant protection against parasite infection in vaccinated mice. In particular, vaccination with PLGA-sLiAg-MPLA or p8-PLGA-sLiAg NPs resulted in almost complete elimination of the parasite in the spleen for up to 4 months post-challenge. Parasite burden reduction was accompanied by antigen-specific humoral and cellular immune responses. Specifically, injection with PLGA-sLiAg-MPLA raised exclusively anti-sLiAg IgG1 antibodies post-vaccination, while in p8-PLGA-sLiAg-vaccinated mice, no antibody production was detected. However, 4 months post-challenge, in mice vaccinated with all the multifunctionalized NPs, antibody class switching towards IgG2a subtype was observed. The study of cellular immune responses revealed the increased proliferation capacity of spleen cells against sLiAg, consisting of IFNγ-producing CD4+ and CD8+ T cells. Importantly, the activation of CD8+ T cells was exclusively attributed to vaccination with PLGA NPs surface-modified with the p8 peptide. Moreover, characterization of cytokine production in vaccinated-infected mice revealed that protection was accompanied by significant increase of IFNγ and lower levels of IL-4 and IL-10 in protected mice when compared to control infected group. Conclusively, the above nanoformulations hold promise for future vaccination strategies against VL.

A Poly(Lactic-co-Glycolic) Acid Nanovaccine Based on Chimeric Peptides from Different Leishmania infantum Proteins Induces Dendritic Cells Maturation and Promotes Peptide-Specific IFNγ-Producing CD8+ T Cells Essential for the Protection against Experimental Visceral Leishmaniasis.

Visceral leishmaniasis, caused by Leishmania (L.) donovani and L. infantum protozoan parasites, can provoke overwhelming and protracted epidemics, with high case-fatality rates. An effective vaccine against the disease must rely on the generation of a strong and long-lasting T cell immunity, mediated by CD4+ TH1 and CD8+ T cells. Multi-epitope peptide-based vaccine development is manifesting as the new era of vaccination strategies against Leishmania infection. In this study, we designed chimeric peptides containing HLA-restricted epitopes from three immunogenic L. infantum proteins (cysteine peptidase A, histone H1, and kinetoplastid membrane protein 11), in order to be encapsulated in poly(lactic-co-glycolic) acid nanoparticles with or without the adjuvant monophosphoryl lipid A (MPLA) or surface modification with an octapeptide targeting the tumor necrosis factor receptor II. We aimed to construct differentially functionalized peptide-based nanovaccine candidates and investigate their capacity to stimulate the immunomodulatory properties of dendritic cells (DCs), which are critical regulators of adaptive immunity generated upon vaccination. According to our results, DCs stimulation with the peptide-based nanovaccine candidates with MPLA incorporation or surface modification induced an enhanced maturation profile with prominent IL-12 production, promoting allogeneic T cell proliferation and intracellular production of IFNγ by CD4+ and CD8+ T cell subsets. In addition, DCs stimulated with the peptide-based nanovaccine candidate with MPLA incorporation exhibited a robust transcriptional activation, characterized by upregulated genes indicative of vaccine-driven DCs differentiation toward type 1 phenotype. Immunization of HLA A2.1 transgenic mice with this peptide-based nanovaccine candidate induced peptide-specific IFNγ-producing CD8+ T cells and conferred significant protection against L. infantum infection. Concluding, our findings supported that encapsulation of more than one chimeric multi-epitope peptides from different immunogenic L. infantum proteins in a proper biocompatible delivery system with the right adjuvant is considered as an improved promising approach for the development of a vaccine against VL.

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

BACKGROUND: 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 Leishmania Cysteine Protease A (CPA160-189) co-encapsulated with Monophosphoryl lipid A (MPLA) in PLGA NPs against L. infantum in BALB/c mice and identify immune markers correlated with protective responses. METHODOLOGY/PRINCIPAL FINDINGS: The DCs phenotypic and functional features exposed to soluble (CPA160-189, CPA160-189+MPLA) or encapsulated in PLGA NPs forms of peptide and adjuvant (PLGA-MPLA, PLGA-CPA160-189, PLGA-CPA160-189+MPLA) was firstly determined using BALB/c bone marrow-derived DCs. The most potent signatures of DCs maturation were obtained with the PLGA-CPA160-189+MPLA NPs. Subcutaneous administration of PLGA-CPA160-189+MPLA NPs in BALB/c mice induced specific anti-CPA160-189 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 L. infantum 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. CONCLUSIONS/SIGNIFICANCE: This study provide a basis for the development of peptide-based nanovaccines against leishmaniasis, since it reveals that vaccination with well-defined Leishmania 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.

PLGA nanoparticles modified with a TNFα mimicking peptide, soluble Leishmania antigens and MPLA induce T cell priming in vitro via dendritic cell functional differentiation.

Poly(lactide-co-glycolide) nanoparticles (PLGA NPs) represent a new approach for vaccine delivery due to their ability to be taken up by phagocytes and to activate immune responses. In the present study PLGA NPs were surface-modified with a TNFα mimicking peptide, and encapsulated soluble Leishmania antigens (sLiAg) and MPLA adjuvant. The synthesized PLGA NPs exhibited low cytotoxicity levels, while surface-modified NPs were more efficiently taken up by dendritic cells (DCs). The prepared nanoformulations induced maturation and functional differentiation of DCs by elevating co-stimulatory molecule levels and stimulating IL-12 and IL-10 production. Sensitized DCs promoted T cell priming, characterized by the development of mixed T cell subsets differentiation expressing Th lineage-specific transcriptional factors and cytokine genes. Moreover, PLGA NPs were biocompatible, while they were located in lymphoid organs and taken up by phagocytic cells. Our results suggest that surface-modified PLGA NPs encapsulating sLiAg and MPLA could be considered as an effective vaccine candidate against leishmaniasis.

Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis.

Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests.

Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins.

Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

Vaccination with Leishmania histone H1-pulsed dendritic cells confers protection in murine visceral leishmaniasis.

Visceral leishmaniasis is the most severe form of leishmaniases affecting millions of people worldwide often resulting in death despite optimal therapy. Thus, there is an urgent need for the development of effective anti-infective vaccine(s). In the present study, we evaluated the prophylactic value of bone marrow-derived dendritic cells (BM-DCs) pulsed with the Leishmania (L.) infantum histone H1. We developed fully mature BM-DCs characterized by enhanced capacity of IL-12 production after ex vivo pulsing with GST-LeishH1. Intravenous administration of these BM-DCs in naive BALB/c mice resulted in antigen-specific spleenocyte proliferation and IgG1 isotype antibody production and conferred protection against experimental challenge with L. infantum independently of CpG oligonucleotides (ODNs) co-administration. Protection was associated with a pronounced enhancement of parasite-specific IFNγ-producing cells and reduction of cells producing IL-10, whereas IL-4 production was comparable in protected and non-protected mice. The polarization of immune responses to Th1 type was further confirmed by the elevation of parasite-specific IgG2a/IgG1 ratio in protected mice. The above data indicate the immunostimulatory capacity of Leishmania histone H1 and further support its exploitation as a candidate protein for vaccine development against leishmaniasis.

Cellular vaccination with bone marrow-derived dendritic cells pulsed with a peptide of Leishmania infantum KMP-11 and CpG oligonucleotides induces protection in a murine model of visceral leishmaniasis.

The use of dendritic cells (DCs) pulsed with defined Leishmania antigens could be a potential immune intervention tool for the induction of protection against infection. In the present study, bone marrow-derived DCs (BM-DCs) pulsed ex vivo with the peptide 12-31aa portion of kinetoplastid membrane protein (KMP)-11 (KMP-11(12-31aa) peptide) acquired a semimature phenotype expressing IL-12 and IL-10, whereas pulsing with the combination of the peptide and CpG oligodeoxynucleotides (ODNs) resulted in their functional maturation expressing mainly IL-12. Vaccination of genetically susceptible to parasite BALB/c mice with both peptide-pulsed BM-DCs elicited a peptide-specific mixed Th1/Th2 immune response, characterized by the production of IFNγ, IL-10 and IgG1 and IgG2a isotype antibodies. However, only BM-DCs pulsed with the combination of KMP-11(12-31aa) peptide and CpG ODNs induced the differentiation of peptide-specific Th17 cells, indicating the adjuvanticity of CpG ODNs. When BALB/c mice were vaccinated with KMP-11(12-31aa) peptide-pulsed BM-DCs, they exhibited only partial protection against Leishmania infantum challenge, whereas (KMP-11(12-31aa) peptide+CpG ODNs)-pulsed BM-DCs reduced efficiently the parasite load in visceral organs. Protective immunity was correlated with restoration of lymphoproliferative responses and a modulation of parasite-specific cellular responses towards Th1 and Th17 profile, confirmed by the isotype switching towards IgG2a, the enhanced production of IFNγ against IL-10, the absence of TGF-ß and the overproduction of IL-17. Thus, ex vivo antigen-pulsed BM-DCs represent a powerful tool for the study of protective immune responses against leishmanial infection. Moreover, these findings suggest the use of BM-DCs as effective tools in antigen and adjuvant screening in the design of a protective vaccine against leishmaniasis and other pathogen-related infections.