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.

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.

Induction of innate immune responses by KPC-producing Klebsiella pneumoniae of the pandemic sequence type 258-clade I.

Klebsiella pneumoniae -carbapenemase-producing K. pneumoniae (KPC) sequence-type 258 (ST258) has emerged as an important human pathogen throughout the world. Although lacking known virulence factors, it is associated with significant morbidity and high mortality rates. The pathogenicity of KPC K. pneumoniae ST258 strains has not been fully elucidated yet. We sought to investigate the interactions of the KPC K. pneumoniae ST258-clade I with different components of innate immunity. Human serum was used to evaluate the serum bactericidal activity and the J774A.1 murine (BALB/c mice) macrophage cell-line was used to examine phagocytosis, mRNA expression and production of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6. L-78, a KPC-producing K. pneumoniae ST258-clade I strain was used as representative of the strains circulating in Greek hospitals. K. pneumoniae ATCC 43816, a virulent K2 strain, was used for comparison. Strain L-78 was susceptible to human serum and rapidly phagocytosed by J774A.1 cells, in contrast to the virulent K2 strain, which was serum-resistant and slowly phagocytosed. Stimulation of the J774A.1 cells with the L-78 strain induced production of IL-1ß at concentration levels significantly higher compared to K2, whereas production of TNF-α and IL-6 levels were comparable by the two strains. L-78 was able to induce IL-1ß mRNA and NLRP3 mRNA expression. Our findings indicate that K. pneumoniae ST258-clade I is serum sensitive, rapidly phagocytosed and is capable of eliciting adequate innate immune response in terms of production of pro-inflammatory cytokines.

Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass, Dicentrarchus labrax.

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues-eye, oesophagus, gills and skin-as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.

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.

Development of a Nanoparticle-based Lateral Flow Strip Biosensor for Visual Detection of Whole Nervous Necrosis Virus Particles.

Effective analysis of pathogens causing human and veterinary diseases demands rapid, specific and sensitive detection methods which can be applied in research laboratory setups and in field for routine diagnosis. Paper lateral flow biosensors (LFBs) have been established as attractive tools for such analytical applications. In the present study a prototype LFB was designed for whole particles (virions) detection of nodavirus or fish nervous necrosis virus. Nodavirus is an important threat in the aquaculture industry, causing severe economic losses and environmental problems. The LFB was based on polyclonal antibodies conjugated on gold nanoparticles for signal visualization. Brain and retinas from fish samples were homogenized, centrifuged and the supernatant was directly applied on the LFB. Formation of a red test line was indicative of nodavirus virions presence. Nodavirus visual detection was completed in short time (30 min). Key factors of the LFB development influencing the assays' detection limit were characterized and the optimum parameters were determined, enabling increased efficiency, excluding non-specific interactions. Therefore, the proposed LFB assay consists a robust, simple, low cost and accurate method for detection of nodavirus virions in fish samples. The proposed biosensor is ideal for development of a commercial kit to be used on aquaculture facilities by fish farmers. It is anticipated that disease monitoring and environmental safety will benefit from the simplification of time consuming and costly procedures.

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.

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.

Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes.

Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities.

Nanoparticle-based lateral flow biosensor for visual detection of fish nervous necrosis virus amplification products.

Lateral flow paper biosensors are an attractive analytical platform for detection of human and veterinary disease pathogens because they are optimal for accurate, rapid and sensitive analysis in research laboratory setups, as well as field analysis. Since diseases of viral etiology have been wreaking havoc in aquaculture industry, as well as the environment, the present study aims at the development of a gold nanoparticle-based biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification. The PCR products were mixed with a specific oligonucleotide probe and applied next to oligonucleotide conjugated Au NPs. A red test line was formed when nodavirus product was present. The visual detection of the RT-PCR product was completed within 20 min. Following optimization, the biosensor was able to visually detect 270 pg of nodavirus initial total RNA. The present study describes a simple, accurate, robust and low cost method for nodavirus detection in biological samples. Apart contribution on basic research, the proposed biosensor offers great potential for commercial kit development for use on the site of fish culture by fish farmers. This fact will have great impact on environmental safety and disease monitoring without time consuming and costly procedures.

Effect of dietary incorporation of a multi-strain probiotic on growth performance and health status in rainbow trout (Oncorhynchus mykiss).

Dietary supplementation with a multi-strain probiotic containing Bacillus subtilis, Enterococcus faecium, Pediococcus acidilactici and Lactobacillus reuteri has been examined for its effects on growth performance, intestinal microbiota, non-specific immune response and antioxidant status of rainbow trout. Three groups of sub-adult trout were stocked into experimental tanks. A commercial diet was used as control, while the other two groups received diets supplemented with the multi-strain probiotic at levels of 1 and 5 g kg(-1) diet. The fish were fed to apparent satiation three times daily for 8 weeks. Dietary probiotic at 1 g kg(-1) diet improved (P < 0.05) growth and feed efficiency compared to control diet. Lactic acid bacteria loads were higher in probiotic fed fish at both inclusion levels compared to control; however, Enterobacteriaceae, Coliforms and Aeromonas spp. were similar between groups. Dietary probiotics decreased (P < 0.05) malondialdehyde formation on day 0 compared to control fish but not on day 5 of storage. Probiotics also increased (P < 0.05) the activity of glutathione-based enzymes. Serum lysozyme levels were similar among dietary treatments. Probiotic supplementation at 1 g kg(-1) diet reduced serum nitric oxide levels compared to control. In conclusion, dietary probiotics at the level of 1 g kg(-1) of diet exerted both a growth promoting and antioxidant protective activity.

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.