New Insights into the Molecular Interplay between Human Herpesviruses and Alzheimer's Disease-A Narrative Review.

Human herpesviruses (HHVs) have been implicated as possible risk factors in Alzheimer's disease (AD) pathogenesis. Persistent lifelong HHVs infections may directly or indirectly contribute to the generation of AD hallmarks: amyloid beta (Aß) plaques, neurofibrillary tangles composed of hyperphosphorylated tau proteins, and synaptic loss. The present review focuses on summarizing current knowledge on the molecular mechanistic links between HHVs and AD that include processes involved in Aß accumulation, tau protein hyperphosphorylation, autophagy, oxidative stress, and neuroinflammation. A PubMed search was performed to collect all the available research data regarding the above mentioned mechanistic links between HHVs and AD pathology. The vast majority of research articles referred to the different pathways exploited by Herpes Simplex Virus 1 that could lead to AD pathology, while a few studies highlighted the emerging role of HHV 6, cytomegalovirus, and Epstein-Barr Virus. The elucidation of such potential links may guide the development of novel diagnostics and therapeutics to counter this devastating neurological disorder that until now remains incurable.

The Association of Human Herpesviruses with Malignant Brain Tumor Pathology and Therapy: Two Sides of a Coin.

The role of certain viruses in malignant brain tumor development remains controversial. Experimental data demonstrate that human herpesviruses (HHVs), particularly cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpes virus 6 (HHV-6), are implicated in brain tumor pathology, although their direct role has not yet been proven. CMV is present in most gliomas and medulloblastomas and is known to facilitate oncomodulation and/or immunomodulation, thus promoting cancer cell proliferation, invasion, apoptosis, angiogenesis, and immunosuppression. EBV and HHV-6 have also been detected in brain tumors and high-grade gliomas, showing high rates of expression and an inflammatory potential. On the other hand, due to the neurotropic nature of HHVs, novel studies have highlighted the engagement of such viruses in the development of new immunotherapeutic approaches in the context of oncolytic viral treatment and vaccine-based strategies against brain tumors. This review provides a comprehensive evaluation of recent scientific data concerning the emerging dual role of HHVs in malignant brain pathology, either as potential causative agents or as immunotherapeutic tools in the fight against these devastating diseases.

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.

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.

Designed positively charged cyclodextrin hosts with enhanced binding of penicillins as carriers for the delivery of antibiotics: The case of oxacillin.

In an effort to identify the optimal cyclodextrin (CD) host for delivery of penicillins to mammalian cells that will also offer protection against ß-lactamase-induced hydrolysis, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) have been employed to study the inclusion complexes formed in aqueous solution between designed CD derivatives and two aminopenicillins, ampicillin and amoxicillin, and two antistaphylococcal penicillins, methicillin and oxacillin. Anionic and cationic thioether-substituted-ß- and -γCD derivatives were thus synthesized and compared with the neutral, parent CDs for complexation with the penicillins. The synthesized derivatives were shown to present ∼20% elongated cavity space in solution. Moreover, the cationic ones are >98% protonated at physiological pH. The most efficient host was the positively charged octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) that formed the strongest complex with oxacillin (Kb ∼1700M-1) in an enthalpically and entropically favorable process (ΔHb=-10.5kJ/mol,TΔSb=8.0kJ/mol). In vitro biological tests demonstrated that γCys reduces 2.3-fold the rate of hydrolysis of oxacillin in the presence of oxa-1 ß-lactamase while displaying cell crossing capability and efficient internalization into macrophages as well as a sufficiently safe cytotoxicity profile. Overall, γCys could be considered as a promising vehicle for protection and delivery of oxacillin.

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.

Gold nanoparticle-based lateral flow biosensor for rapid visual detection of Leishmania-specific DNA amplification products.

Leishmaniasis is a disease, caused by Leishmania parasites, which infect humans and animals, posing a major social and economic burden worldwide. The need for accurate and sensitive disease diagnosis led to the widespread adoption of PCR amplification. Detection of the amplification products (i.e. gel electrophoresis) require time-consuming protocols performed by trained personnel, with high cost. Aim of the present study was the simplification of PCR product detection, using a nucleic acid lateral flow, combined with functionalized gold nanoparticles. Amplification reactions targeting kinetoplastid DNA of Leishmania spp were performed on canine blood samples and a positive signal was formed as a red test zone. The visual detection was completed in 20min. Extensive optimization enabled the detection of 100fmol of target DNA. Clinical samples of infected dog blood were analyzed with high specificity. Overall, the proposed lateral flow biosensor can be considered an appealing alternative platform for Leishmania-specific amplification products detection with low cost and attractive simplicity.

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.