Immunostimulatory Peptides Research Articles

Mass spectrometry-based quantification of immunostimulatory gliadin proteins and peptides in coloured wheat varieties: Implications for Celiac Disease

Pigmented wheat varieties (Triticum aestivum spp.) are getting increasingly popular in modern nutrition and thoroughly researched for their functional and nutraceutical value. The colour of these wheat grains is caused by the expression of natural pigments, including carotenoids and anthocyanins, that can be restricted to either the endosperm, pericarp and/or aleurone layers. While contrasts in phytochemical synthesis give rise to variations among purple, blue, dark and yellow grain’s antioxidant and radical scavenging capacities, little is known about their influence on gluten proteins expression, digestibility and immunogenic potential in a Celiac Disease (CD) framework. Herein, it has been found that the expression profile and immunogenic properties of gliadin proteins in pigmented wheat grains might be affected by anthocyanins and carotenoids upregulation, and that the spectra of peptide released upon simulated gastrointestinal digestion is also significantly different. Interestingly, anthocyanin accumulation, as opposed to carotenoids, correlated with a lower immunogenicity and toxicity of gliadins at both protein and peptide levels. Altogether, this study provides first-level evidence on the impact modern breeding practices, seeking higher expression levels of health promoting phytochemicals at the grain level, may have on wheat crops functionality and CD tolerability.

Nucleic acid and oligonucleotide delivery for activating innate immunity in cancer immunotherapy.

A group of nucleic acids and oligonucleotides play various roles in the innate immune system. They can stimulate pattern recognition receptors to activate innate immune cells, encode immunostimulatory proteins or peptides, or silence specific genes to block negative regulators of immune cells. Given the limitations of current cancer immunotherapy, there has been increasing interest in harnessing innate immune responses by nucleic acids and oligonucleotides. The poor biopharmaceutical properties of nucleic acids and oligonucleotides make it critical to use carriers that can protect them in circulation, retain them in the tumor microenvironment, and bring them to intracellular targets. Therefore, various gene carriers have been repurposed to deliver nucleic acids and oligonucleotides for cancer immunotherapy and improve their safety and activity. Here, we review recent studies that employed carriers to enhance the functions of nucleic acids and oligonucleotides and overall immune responses to cancer, and discuss remaining challenges and future opportunities in the development of nucleic acid-based immunotherapeutics.

Optimization of Expression and Purification of Recombinant Mouse plac1.

Background:Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated.Methods:A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA.Results:The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1 antibody titer was significantly higher in sera of mice immunized with main compared to control plac1.Conclusion:In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses.

Synthetic Antibody Mimics Based on Cancer-Targeting Immunostimulatory Peptides.

De novo cancer-targeting immunostimulatory peptides have been designed and developed as synthetic antibody mimics. A series of bifunctional peptides incorporating NKp30-binding and NK-cell-activating domains were synthesized as linear dimers and then extended into branching trimeric peptides by the incorporation of GRP78-targeting and tumor-cell-binding sequences. A selected trimeric peptide from this small set of peptides displayed binding capabilities on GRP78+ HepG2 and A549 target cells. Cell binding diminished in the presence of an anti-GRP78 peptide blocker, thus suggesting GRP78-binding dependence. Similarly, the selected trimeric peptide was also found to exhibit NK cell binding in an NKp30-dependent manner, which translated into NK cell activation as indicated by cytokine secretion. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Accordingly, A549 cells were found to be compromised, as evidenced by the loss of GFP signal and notable detection of early-/late-stage apoptosis. Investigation of the immunological markers related to toxicity revealed detectable secretion of pro-inflammatory cytokines and chemokines, including IFN-γ, TNF-α, and IL-8. Furthermore, administration of peptide-activated NK cells into A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. Taken together, the identification of a lead trimeric peptide capable of targeting and activating NK cells' immunotoxicity directly towards GRP78+ /B7H6- tumors provides a novel proof-of-concept for the development of cancer-targeting immunostimulatory peptide ligands that mimic antibody-targeting and -activating functions related to cancer immunotherapy applications.

Immuno-Stimulatory Peptides as a Potential Adjunct Therapy against Intra-Macrophagic Pathogens.

The treatment of infectious diseases is increasingly prone to failure due to the rapid spread of antibiotic-resistant pathogens. Antimicrobial peptides (AMPs) are natural components of the innate immune system of most living organisms. Their capacity to kill microbes through multiple mechanisms makes the development of bacterial resistance less likely. Additionally, AMPs have important immunomodulatory effects, which critically contribute to their role in host defense. In this paper, we review the most recent evidence for the importance of AMPs in host defense against intracellular pathogens, particularly intra-macrophagic pathogens, such as mycobacteria. Cathelicidins and defensins are reviewed in more detail, due to the abundance of studies on these molecules. The cell-intrinsic as well as the systemic immune-related effects of the different AMPs are discussed. In the face of the strong potential emerging from the reviewed studies, the prospects for future use of AMPs as part of the therapeutic armamentarium against infectious diseases are presented.

Immunological properties and protective efficacy of a single mycobacterial antigen displayed on polyhydroxybutyrate beads.

SummaryIn 2015, there were an estimated 10.4 million new tuberculosis (TB) cases and 1.4 million deaths worldwide. Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the vaccine available against TB, but it is insufficient for global TB control. This study evaluated the immunogenicity of the Mycobacterium tuberculosis antigen Rv1626 in mice while assessing the effect of co‐delivering either Cpe30 (immunostimulatory peptide), CS.T3378–395 (promiscuous T helper epitope) or flagellin (TLR5 agonist) or a combination of all three immunostimulatory agents. Rv1626 and the respective immunostimulatory proteins/peptides were co‐displayed on polyhydroxybutyrate beads assembled inside an engineered endotoxin‐free mutant of Escherichia coli. Mice vaccinated with these beads produced immune responses biased towards Th1‐/Th17‐type responses, but inclusion of Cpe30, CS.T3378–395 and flagellin did not enhance immunogenicity of the Rv1626 protein. This was confirmed in a M. bovis challenge experiment in mice, where Rv1626 beads reduced bacterial cell counts in the lungs by 0.48 log10 compared with the adjuvant alone control group. Co‐delivery of immunostimulatory peptides did not further enhance protective immunity.

A fragment of the alarmin prothymosin α as a novel biomarker in murine models of bacteria-induced sepsis.

Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.

Inhibition of Gliadin Digestion by Green Tea Polyphenols and the Potential Implications for Celiac Disease

Green tea has been suggested to have beneficial effects on human health through a variety of biological mechanisms. (−)‐Epigallocatechin‐3‐gallate (EGCG), the major phenolic constituent of green tea, has been reported to mitigate a number of inflammatory and hypersensitivity disorders in laboratory models, and has been shown to moderate pathways related to food allergies in vitro. Previous studies by our group have characterized interactions between EGCG and a2‐gliadin (amino acids 57–89), an immunodominant peptide associated with stimulation of the immune response in celiac disease, and have demonstrated that this polyphenol is able to form complexes with proline‐rich immunostimulatory epitopes on the peptide. Here, we sought to determine the impact of decaffeinated green tea extract (GTE) on the digestion of gliadin protein and production of smaller immunostimulatory peptides in vitro. Complexation of GTE and gliadin was confirmed via an increase in turbidity as a result of co‐precipitation upon titration of GTE into a gliadin solution. Co‐precipitation was observed again during in vitro digestion when gliadin was exposed to digestive proteases pepsin and trypsin. SDS‐PAGE of post‐digestion suspensions and supernatants revealed that GTE not only precipitated gliadin from solution, but also impeded the hydrolytic activity of pepsin and trypsin, resulting in decreased production of low molecular weight gliadin digestion products. An enzyme inhibition assay revealed the GTE dose‐dependently inhibits trypsin (IC50 = 113 mg/ml) in an uncompetitive manner with regard to substrate concentration. Ongoing studies are focused on determining the inhibitory effect of GTE against pepsin, and characterizing the effects of GTE on the immunogenic properties of gliadin using a cell‐culture based model of intestinal permeability and inflammation. We expect that the sequestration of gliadin via GTE complexation will result in decreased permeability and inflammation. Our results to date provide further support for the potential beneficial effects of GTE in the context of celiac disease.Support or Funding InformationThis project was supported by a National Institute of Food and Agriculture Predoctoral Fellowship awarded to C.V. under Grant no. 2016‐67011‐24702, USDA Agriculture and Food Research Initiative.

B7H6-derived peptides trigger TNF-α-dependent immunostimulatory activity of lymphocytic NK92-MI cells.

The rise of biologics that can stimulate immune responses towards the eradication of tumors has led to the evolution of cancer-based immunotherapy. Representatively, B7H6 has been recently identified as a protein ligand on tumor cells that binds specifically to the NKp30 receptor and triggers NK cell-derived cytokine production, which ultimately leads to tumor cell lysis and death. In an effort to develop effective immunotherapy approaches, the rational design of a novel class of immunostimulatory peptides (IPs) derived from the binding interface of B7H6:NKp30 is described in this study. The IPs comprised the B7H6 active site sequence for NKp30 binding and immunostimulatory activity. An aminohexanoic acid linker was also introduced at the N-terminus of the peptides for FITC-labeling by Fmoc-solid phase peptide synthesis. The peptides were characterized by LCMS to confirm identities and purities >95%. The secondary structures of the peptides were examined by CD spectroscopy in H2 O, PBS and a H2 O:TFE mixture which demonstrated versatile peptide structures which transitioned from random coil (H2 O) to α-helical (PBS) and turn-type (H2 O:TFE) conformations. Their biological properties were then evaluated by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and cell death assays. The occupancy of the synthetic peptides to a human NK cell line demonstrated comparable binding relative to the natural NKp30 ligand, B7H6, and the human anti-NKp30 monoclonal antibody (mAb), in a concentration dependent manner. A competitive binding assay between the human anti-NKp30 mAb or B7H6, and the synthetic peptides, demonstrated partial displacement of the ligands upon anti-NKp30 mAb treatment, suggesting NKp30 receptor specificities by the synthetic peptides. Moreover, the immunostimulatory activity of B7H6 was demonstrated by the secretion of the pro-inflammatory cytokines tumor necrosis factor-alfa (TNF-α) and interferon gamma (IFN-γ) by the human NK cell line. The immunostimulatory effects of IPs on the NK cells was assessed by the production of TNF-α alone as IFN-γ was undetectable. In a cell death assay, the IPs were found to be nontoxic, without any observable evidence of early or late stage apoptosis within the NK92-MI cells. Taking these findings together, this novel class of synthetic peptides may prove to be a promising lead in the development of a peptide-based immunotherapy approach, especially against B7H6 expressing tumors. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 658-672, 2016.

Prothymosin Alpha and Immune Responses: Are We Close to Potential Clinical Applications?

The thymus gland produces soluble molecules, which mediate significant immune functions. The first biologically active thymic extract was thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, including prothymosin alpha (proTα).ProTα displays a dual role, intracellularly as a survival and proliferation mediator and extracellularly as a biological response modifier. Accordingly, inside the cell, proTα is implicated in crucial intracellular circuits and may serve as a surrogate tumor biomarker, but when found outside the cell, it could be used as a therapeutic agent for treating immune system deficiencies. In fact, proTα possesses pleiotropic adjuvant activity and a series of immunomodulatory effects (eg, anticancer, antiviral, neuroprotective, cardioprotective). Moreover, several reports suggest that the variable activity of proTα might be exerted through different parts of the molecule. We first reported that the main immunoactive region of proTα is the carboxy-terminal decapeptide proTα(100–109). In conjunction with data from others, we also revealed that proTα and proTα(100–109) signal through Toll-like receptor 4. Although their precise molecular mechanism of action is yet not fully elucidated, proTα and proTα(100–109) are viewed as candidate adjuvants for cancer immunotherapy.Here, we present a historical overview on the discovery and isolation of thymosins with emphasis on proTα and data on some immune-related new activities of the polypeptide and smaller immunostimulatory peptides thereof. Finally, we propose a compiled scenario on proTα’s mode of action, which could eventually contribute to its clinical application.

Gliadin-dependent cytokine production in a bidimensional cellular model of celiac intestinal mucosa.

The downstream cascade of the inflammatory response to gliadin in celiac intestinal mucosa encompasses the early activation of the innate immunity that triggers the adaptive response. Therefore, the in vitro study of the pathogenic mechanism of celiac disease (CD) on enterocytes alone or mucosal T lymphocytes alone does not fully consider all the aspects of gliadin-dependent inflammation. Although the in vitro culture of specimens of intestinal mucosa obtained from celiac patients is the gold standard for the study of CD, this technique presents several technical challenges and the bioptic specimens are not easily available. So, in this paper, we described the gliadin-dependent cytokine production in a bidimensional cellular system, which is able to mimic both the innate and the adaptive steps of the mucosal immune response of CD. In the upper compartment, the intestinal epithelial cells are grown on a filter, and in the lower compartment, the mononuclear cells isolated from peripheral blood of celiac patients are cultured. Cells were apically exposed to the toxic gliadin peptide p31-43 for 3 h and then with the immunodominant gliadin fragment pα-9 for 21 h. The incubation with gliadin peptides resulted in increased levels of IL-15, INF-γ, IL-6, tumor necrosis factor (TNF)-α, IL-1β, and CCL 2, 3 and 4 in the basal supernatants, with respect to cells exposed to medium alone. The p31-43-driven epithelial priming of mucosal response consists of transglutaminase (TG2)-mediated deamidation of the immunostimulatory gliadin peptides, as demonstrated by the inhibition of pα-9 activity, when the system is exposed to blocking anti-TG2 antibody.

Immunomodulatory effects of protein hydrolysates from rohu (Labeo rohita) egg (roe) in BALB/c mice

Protein hydrolysates prepared from underutilized rohu (Labeo rohita) egg (roe), by enzymatic hydrolysis using pepsin, trypsin and Alcalase, were evaluated for their immunomodulatory effects in BALB/c mice. The female BALB/c mice (4–6weeks, 18–20g) were administered with the rohu egg protein hydrolysates (REPHs) daily for a period of forty five days with concentration of 0.25, 0.5 and 1g/kg body weight. Both innate and adaptive immune responses were studied. Pepsin hydrolysate significantly increased the splenic NK cell cytotoxicity, macrophage phagocytosis and level of serum immunoglobulin A (IgA). The mucosal immunity (S-IgA) in the gut was significantly enhanced by pepsin and Alcalase hydrolysates. Whereas trypsin hydrolysate induced significant increases in the percentages of CD4+ and CD8+ cells in the spleen. This study confirms that REPHs are able to modulate immune function and further reveals that different rohu egg protein hydrolysates may exert differential influences on the immune system. These results suggest that REPHs contain immunostimulatory peptides that could be useful in pharmaceutical, health food or nutraceutical industry for various applications.

Human Beta Casein Fragment (54–59) Modulates M. bovis BCG Survival and Basic Transcription Factor 3 (BTF3) Expression in THP-1 Cell Line

Immunostimulatory peptides potentiate the immune system of the host and are being used as a viable adjunct to established therapeutic modalities in treatment of cancer and microbial infections. Several peptides derived from milk protein have been reported to induce immunostimulatory activity. Human β -casein fragment (54–59), natural sequence peptide (NS) carrying the Val-Glu-Pro-Ile-Pro-Tyr amino acid residues, was reported to activate the macrophages and impart potent immunostimulatory activity. In present study, we found that this peptide increases the clearance of M. bovis BCG from THP-1 cell line in vitro. The key biomolecules, involved in the clearance of BCG from macrophage like, nitric oxide, pro-inflammatory cytokines and chemokines, were not found to be significantly altered after peptide treatment in comparison to the untreated control. Using proteomic approach we found that BTF3a, an isoform of the Basic Transcription Factor, BTF3, was down regulated in THP-1 cell line after peptide treatment. This was reconfirmed by real time RT-PCR and western blotting. We report the BTF3a as a novel target of this hexapeptide. Based on the earlier findings and the results from the present studies, we suggest that the down regulation of BTF3a following the peptide treatment may augment the M. bovis BCG mediated apoptosis resulting in enhanced clearance of M. bovis BCG from THP-1 cell line.

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements.

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.

Treatment of HER2-positive breast cancer: current status and future perspectives

The advent of HER2-directed therapies has significantly improved the outlook for patients with HER2-positive early stage breast cancer. However, a significant proportion of these patients still relapse and die of breast cancer. Trials to define, refine and optimize the use of the two approved HER2-targeted agents (trastuzumab and lapatinib) in patients with HER2-positive early stage breast cancer are ongoing. In addition, promising new approaches are being developed including monoclonal antibodies and small-molecule tyrosine kinase inhibitors targeting HER2 or other HER family members, antibodies linked to cytotoxic moieties or modified to improve their immunological function, immunostimulatory peptides, and targeting the PI3K and IGF-1R pathways. Improved understanding of the HER2 signaling pathway, its relationship with other signaling pathways and mechanisms of resistance has also led to the development of rational combination therapies and to a greater insight into treatment response in patients with HER2-positive breast cancer. Based on promising results with new agents in HER2-positive advanced-stage disease, a series of large trials in the adjuvant and neoadjuvant settings are planned or ongoing. This Review focuses on current treatment for patients with HER2-positive breast cancer and aims to update practicing clinicians on likely future developments in the treatment for this disease according to ongoing clinical trials and translational research.

Hemophilia B Mouse Lines Doubly Humanized for Human F9 and MHC Class II Genes Advance Specificity of Inhibitor Study

Abstract 200The development of neutralizing alloantibodies (inhibitors) against factor IX (FIX) in humans with hemophilia B is a potentially devastating complication; clinical investigation and evidence-based intervention for FIX inhibitors is complicated by their rarity. Current hemophilia B mouse models are not ideal for investigating the potential immunogenicity of hemophilia B therapies due to differences in mouse and human MHC class II structure and FIX epitope recognition. This recognition depends on CD4+ T cells and their direct interaction with surface-expressed MHC class II molecules complexed with antigen. We have developed a series of mouse lines that do not express mouse FIX or mouse MHC II, but instead express human FIX genes and humanized MHC II. The potential immunogenicity of human FIX and specific hFIX epitopes for CD4+ T cell recognition have been characterized in each of these “double humanized” (2H) mouse lines.Our derivation of the 2H mouse lines, all having first been bred through a complete knockout of mouse MHCII (mMHC II−/−), resulted in four lines of human HLA-DRB1*1501 mice. These four lines include 1) mouse FIX−/− (complete deletion; Cross-reacting material negative (CRM-)); 2) human R29X (expresses human FIX gene with early nonsense mutation; CRM-); 3) human R333Q (expresses human factor IX defective circulating protein with missense mutation; CRM+), and 4) human WT FIX (WT2 expresses hemostatic human FIX; CRM+). All lines were first shown to have similar numbers of MHCII and CD4+ expressing T cells in peripheral blood based on flow cytometry when compared with conventional (mouse MHC H2b) hemophilia B mice. To determine whether (hu-) mice have the same incidence and degree of immune response to hFIX compared to conventional hemophilia B mice, we immunized subcutaneously with hFIX emulsified in CFA. Importantly, control mMHC II−/− mice did not develop anti-FIX antibodies. Both strains of CRM- 2H hu-mice developed inhibitors and anti-hFIX IgG antibodies with similar intensity. CD4+-enriched splenocytes proliferated in vitro in response to hFIX challenge and secreted high amount of Th1-derived IFN-γ and moderate Th2 (IL-4 and IL-10) cytokines. However, when compared to the inhibitor-prone strains, conventional (mouse MHC H2b) and hu-DRB1*1501 CRM+ mice (both R333Q and WT2) tolerated hFIX, had larger total Treg populations, increased TGF-β1 cytokine, and demonstrated regulatory apoptosis of CD4+ Teffector cells. Additional lymphocyte response studies mapped four peptides derived from FIX catalytic domain as responsible for anti-FIX antibodies in the CRM- lines. (CD4+ T cells from CRM+ animals (hu-R333Q or hu-WT2 mice) did not proliferate to these four or any other hFIX peptides). The peptides identified in 2H hu- mice differed from immunostimulatory peptides identified in reported epitope mapping studies using FIX−/− mice having mouse MHC H2b, H2d, and H2kbackgrounds. These differences may reflect the fundamental structural and epitope recognition differences between murine and human MHC class II complexes.Finally, we have examined systemic and muscle-directed AAV-mediated gene therapy expressing either WT hFIX or a hFIX with a single amino acid gain of function variant (FIX Padua, R338LFIX). We have confirmed that expression of this single amino acid change does not increase inhibitor risk, as modeled in this double humanized hemophilia B model. This observation appears consistent with the extreme rarity of clinical inhibitor development in patients having underlying FIX missense mutations. In summary, the series of double humanized hemophilia B mouse models have hFIX recognition profiles that make them important tools for preclinical evaluation of novel hemophilia B therapeutics and to advance understanding of FIX inhibitors. Disclosures:No relevant conflicts of interest to declare.

DNA Vaccine Constructs Expressing Mycobacterium tuberculosis-Specific Genes Induce Immune Responses

RD1 PE35, PPE68, EsxA, EsxB and RD9 EsxV genes are present in Mycobacterium tuberculosis genome but deleted in Mycobacterium bovis BCG. The aim of this study was to clone these genes into DNA vaccine vectors capable of expressing them in eukaryotic cells as fusion proteins, fused with immunostimulatory signal peptides of human interleukin-2 (hIL-2) and tissue plasminogen activator (tPA), and evaluate the recombinant DNA vaccine constructs for induction of antigen-specific cellular immune responses in mice. DNA corresponding to the aforementioned RD1 and RD9 genes was cloned into DNA vaccine plasmid vectors pUMVC6 and pUMVC7 (with hIL-2 and tPA signal peptides, respectively), and a total of 10 recombinant DNA vaccine constructs were obtained. BALB/c mice were immunized with the parent and recombinant plasmids and their spleen cells were tested for antigen-induced proliferation with antigens of M.tuberculosis and pure proteins corresponding to the cloned genes. The results showed that antigen-specific proliferation responses were observed for a given antigen only with spleen cells of mice immunized with the homologous recombinant DNA vaccine construct. The mice immunized with the parent plasmids did not show positive immune responses to any of the antigens of the cloned genes. The ability of the DNA vaccine constructs to elicit cellular immune responses makes them an attractive weapon as a safer vaccine candidate for preventive and therapeutic applications against tuberculosis.

Toxic, Immunostimulatory and Antagonist Gluten Peptides in Celiac Disease

Celiac disease (CD) is an increasingly diagnosed, permanent autoimmune enteropathy, triggered, in susceptible individuals, by the ingestion of gluten, the alcohol - soluble protein fraction of some cereals, such as wheat, rye and barley. The main protein of wheat gluten is called gliadin, the similar proteins of rye and barley are secalin and hordein, respectively. Approximately 96% of CD patients express the HLA molecule DQ2, while the remainder mostly express the less common haplotype DQ8, reflecting the pivotal role of these molecules in the pathogenesis of CD. Because of their aminoacid sequence and tri-dimensional structure, gluten peptides selectively bind to these HLA alleles present on the surface of antigen presenting cells and then they are presented to the T lymphocytes in intestinal mucosa, thus starting the inflammatory immune response. CD is defined by the characteristic histological changes of small bowel mucosa: villous atrophy, crypts hyperplasia and T cells infiltration of the lamina propria, along with the increase of the number of intra-epithelial lymphocytes. The withdrawal of the gluten- containing food from the diet determines a complete recovery of the intestinal mucosa, whereas the reintroduction causes a relapse of the disease. This review focuses on the description of gluten peptides that elicit the mucosal immune response via the activation of innate and adaptive immunity in CD. It also describes the antagonist gluten peptides, obtained by artificial modification of gluten T epitopes or naturally occurring in the alcohol protein fraction of a cultivar of durum wheat, able to immuno-modulate the pathogenic immune response of CD.

Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac Disease

Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

Limited Efficiency of Prolyl-Endopeptidase in the Detoxification of Gliadin Peptides in Celiac Disease

The resistance of prolamines to digestive enzymes is thought to be a key contributor to the pathogenesis of celiac disease by promoting the intestinal entrance of peptides able to trigger inflammation in at-risk individuals. Oral administration of a bacterial prolyl-endopeptidase (PEP) therefore was proposed as a treatment for celiac disease. To delineate the feasibility of this treatment, the effect of PEP on gliadin peptides was assessed in vitro, and ex vivo during their transport across intestinal biopsy specimens of active celiac disease patients. In vitro degradation by PEP of 3H-labeled gliadin peptides 56-88 (33-mer) and 31-49, was analyzed by radio-reverse-phase high-performance liquid chromatography and mass spectrometry. For ex vivo studies, PEP and 3H-peptides were added together onto the mucosal side of duodenal biopsy specimens mounted in Ussing chambers, and peptide transport and digestion were assessed by radio-reverse-phase high-performance liquid chromatography. Gliadin peptides were degraded partly by 20 mU/mL PEP both in vitro and ex vivo. This concentration of PEP decreased the amount of intact peptides 31-49 and 56-88 crossing the intestinal biopsy specimens of celiac disease patients, but could not prevent the intestinal passage of toxic or immunostimulatory metabolites. PEP concentrations of at least 500 mU/mL for 3 hours were required to achieve full detoxification of peptides and to prevent intestinal transport of active fragments. After prolonged exposure to high concentrations of PEP, the amount of immunostimulatory gliadin peptides reaching the local immune system in celiac patients is decreased. These results provide a basis to establish whether such conditions are achievable in vivo.