Extracellular vesicles are conduits for E. coli heat-labile enterotoxin (LT) and the B-subunits of LT and cholera toxin in immune cell-to-cell communication.

Several pathogens excrete their toxins either directly into the host or through extracellular vesicles. Enterotoxigenic E. coli is capable of secreting heat-labile toxin LT in extracellular vesicles (EVs) which are delivered to mammalian cells. LT and its B-subunit, LTB, and their structurally and functionally related toxin from Vibrio cholerae, CT and CTB, are potent immunogens and adjuvants. However, despite their reported remarkable effects on immune cells, the mechanisms by which they mediate their immunological properties are still unclear. We show that B cells incubated with LT or LTB secreted EVs in the cell culture medium. However, compared to unstimulated cells, EVs and their internal protein content were significantly reduced in recipient B cells. Analysis of protein markers of the vesicles secreted by B cells were found to be enriched in exosomes of endosomal origin. B cells incubated with FITC-CTB secreted CTB in EVs which were taken up by recipient B and T cells. FITC-CTB transfected into exosomes from mouse dendritic cells were also taken up by recipient B cells. Moreover, B cells incubated with FITC-CTB secreted CTB in EVs which increased the number of recipient B cells expressing higher levels of CD25 and CD86. These results suggest that EVs from B cells are conduits for the enterotoxins, and play an important role in the enterotoxins immune cell-to-cell communication. This is the first report which looked at EVs as a mean to deliver these proteins from and to immune cells.

Ameliorative effect of dietary supplementation of copper oxide nanoparticles on inflammatory and immune reponses in commercial broiler under normal and heat-stress housing conditions.

Heat stress (HS) is one of the most serious adverse conditions that affect poultry causing immunosuppression and decreasing production. In a novel approach, we investigated effects of supplementing copper oxide nanoparticles (CuO-NPs) on the immune response in two commercial broiler strains (Ross 308 and Cobb 500). At one day old, birds were divided into 3 groups with 3 replicates for each. The first group received diet supplemented with 100% of their recommended copper requirements as CuO while, in the second and third groups, birds were given diets supplemented with 100% and 50% of the recommended Cu requirements in the form of CuO-NPs, respectively. At age of 21 day, each group was subdivided randomly into normal (24 ±â€¯2 °C) and heat stressed (33 ±â€¯2 °C for 5 h per day for two successive weeks) groups. Under normal housing temperature, CuO-NPs, significantly enhanced the immune response in these birds, compared to CuO shown by the increased levels of phagocytic activity (PA), lysozyme serum activity, and by upregulating immune-modulator genes including NF-κß, PGES, IL-1ß, TGF-1ß, IFN-γ, BAX and CASP8. The responses were different between the two studied strains especially at the level of gene expression. In HS birds, supplementation of CuO-NPs reduced HS induced inflammatory conditions, as shown by lower gene expression levels, lower degenerative changes in the spleen, and altered heterophils/lymphocytes (H/L) ratio. We suggest CuO-NPs supplementation, especially in those chickens that received diet supplemented with 50% of their recommended Cu requirements, could be used under normal housing temperature to enhance the birds' immune response, and during HS to lower heat stress-induced degenerative changes depending on the magnitude of the HS.

Cell clustering and delay/arrest in T-cell division implicate a novel mechanism of immune modulation by E. coli heat-labile enterotoxin B-subunits.

The B-subunits of heat-labile enterotoxins LT-I (LT-IB) and LT-IIa (LT-IIaB) are strong adjuvants that bind to cell-surface receptors, including gangliosides G(M1) and GD1b, respectively. LT-IIaB also binds TLR-2. We demonstrate for the first time that co-incubation with the B-subunits induces significant clustering of B cells after only 4h, and B and T cells in 24h. Clustering was dependent on intact B-subunits, but not on the TLR-2 binding activity of LT-IIaB, indicating it was ganglioside-mediated. Treatment of B cells with LT-IB, a mixture of LT-IB+LT-IIaB, but not LT-IIaB alone, caused a delay in T cell division following ovalbumin endocytosis. B cell receptor-mediated uptake in presence of each treatment caused an arrest, but with increased production of IL-2. Further, treatments differentially increased the proportion of macrophages expressing MHC class-II. These results highlight the outcomes of interplay between signals involving different receptors and implicate a novel mechanism of adjuvanticity.

Novel Therapeutic Approach for Inhibition of HIV-1 Using Cell-Penetrating Peptide and Bacterial Toxins.

Despite advancements in our understanding of HIV-1 pathogenesis, critical virus components for immunity, vaccines trials, and drugs development, challenges remain in the fight against HIV-1. Of great importance is the inhibitory function of microbicidal cell penetrating peptides and bacterial toxins that interfere with production and neutralize infection of HIV-1 particles. We demonstrate that the neutralizing activity of a cationic 18 amino acids peptide, is similar to a broadly neutralizing human antibody, and inhibits production of two HIV-1 strains in human cell lines. Pretreatment of cells with bacterial toxins or toxoids derived from enterotoxigenic E. coli, boost subsequent activity of the peptide against HIV-1, to inhibit simultaneously production and infection. The synthetic peptide crosses the cell membrane into the cytoplasm and nucleus. In vitro analysis of a possible target for this peptide revealed specific binding to recombinant HIV-1 gag p24. This is the first demonstration of a synergy between bacterial toxins and a cell-penetrating peptide against HIV-1.

Expression and Regulation of Cholecystokinin Receptor in the Chicken's Immune Organs and Cells.

Cholecystokinin (CCK) is a neuropeptide that affects growth rate in chickens by regulating appetite. CCK peptides exert their function by binding to two identified receptors, CCKAR and CCKBR in the GI tract and the brain, respectively, as well as in other organs. In mammals, CCK/CCKAR interactions affect a number of immunological parameters, including regulation of lymphocytes and functioning of monocytes. Thus, food intake and growth can potentially be altered by infection and the resulting inflammatory immune response. It is uncertain, however, whether chicken express CCKAR in immune organs and cells, and, if so, whether CCKAR expression is regulated by pathogen derived inflammatory stimuli. Herein, we identify expression of CCKAR protein in chicken peripheral blood mononuclear cells (PBMC) including monocytes, and expression of the CCKAR gene in PBMC, thymus, bursa, and spleen, in selected commercial and pure chicken breeds. Further, stimulation with various types of E. coli heat-labile enterotoxins or lipopolysaccharide significantly regulated expression of CCKAR on monocytes in the different breeds. Ligation of CCKAR with antibodies in PBMC induced mobilization of Ca2+, indicating that CCKAR is signal competent. Injection with polyinosinic: polycytidylic acid (poly I:C), a synthetic analogue of double stranded viral RNA that binds Toll-Like Receptor-3 (TLR3), also regulated gene expressions of CCKAR and proinflammatory cytokines, in the different breeds. Interestingly, variations in the expression levels of proinflammatory cytokines in the different breeds were highly correlated with CCKAR expression levels. Taken together, these findings indicate that the physiological function of CCKAR in the chicken is tightly regulated in immune organs and cells by external inflammatory stimuli, which in turn regulate growth. This is the first report CCKAR expression in immune organs and cells, in any species, and the initial observation that CCKAR is regulated by inflammatory stimuli associated with bacterial and viral infection.

The Quest for an HIV-1 Vaccine Adjuvant: Bacterial Toxins as New Potential Platforms.

While tremendous efforts are undergoing towards finding an effective HIV-1 vaccine, the search for an HIV-1 vaccine adjuvant lags behind and is understudied. More recently, however, efforts have focused on testing adjuvant formulations that can boost the immune response and generate broadly neutralizing antibodies to HIV-1 ENV (gp160). Despite this, there remain a number of challenges towards achieving this goal. These include safety of adjuvant formulations; stability of the incorporated antigens; maintenance of ENV immunogenicity; optimal inoculation sites; the effective combination of adjuvants; stability of ENV neutralizing epitopes in some adjuvant formulations; mucosal immunity; and long-term maintenance of the immune response. A new class of adjuvants for HIV-1 proteins is suggested to overcome many of the limitations of some other adjuvants. Type 1 (LT-I) and type 2 (LT-II) human E. coli enterotoxins (HLTs) and their non-toxic B-subunits derivatives are strong systemic and mucosal adjuvants and effective carriers for other proteins and epitopes. Their stable molecular structure in the presence of fused proteins and epitopes, and their ability to target surface receptors on antigen presenting cells make them ideal for the delivery of HIV-1 ENV or HIV other proteins. Importantly, unlike some other adjuvants, HLTs and derivatives have well-defined modes of immune system activation. The challenges in finding optimal HIV-1 vaccine adjuvant formulation and the important properties of HLTs are discussed.

E. coli Heat-labile Enterotoxin B Subunit as a Platform for the Delivery of HIV Gag p24 Antigen.

Multiple vaccination strategies have been devised against HIV-1 including delivery of HIV moieties in attenuated or replication defective recombinant microbial agents alone or in combination with priming agents in form of soluble proteins or naked DNA. For the priming agents to be effective, adjuvants might be essential in directing the immune response to a desired outcome. E. coli enterotoxin B subunit (LTB) is an effective adjuvant and carrier for other proteins and epitopes. Here we show that conjugation of HIV gag p24 to LTB enhances the T cell response to gag p24 by increasing rate of T cell division compared to other treatments. Because HIV vaccines are likely to be multivalent, we further investigated whether gag p24 inhibits antigen presentation of an unrelated antigen, OVA. Addition of gag p24 to OVA-responsive DO.11.10 cell culture did not have adverse effects on antigen presentation. Interestingly, the presence of LTB in these cultures significantly increased proliferation of DO.11.10 cells. In all, the results suggest the use of LTB to boost immune responses against HIV gag p24 in systemic priming regimens with oral recombinant HIV vaccines.

Oral immunization with a live coxsackievirus/HIV recombinant induces gag p24-specific T cell responses.

BACKGROUND: The development of an HIV/AIDS vaccine has proven to be elusive. Because human vaccine trials have not yet demonstrated efficacy, new vaccine strategies are needed for the HIV vaccine pipeline. We have been developing a new HIV vaccine platform using a live enterovirus, coxsackievirus B4 (CVB4) vector. Enteroviruses are ideal candidates for development as a vaccine vector for oral delivery, because these viruses normally enter the body via the oral route and survive the acidic environment of the stomach. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a live coxsackievirus B4 recombinant, CVB4/p24(73(3)), that expresses seventy-three amino acids of the gag p24 sequence (HXB2) and assessed T cell responses after immunization of mice. The CVB4 recombinant was physically stable, replication-competent, and genetically stable. Oral or intraperitoneal immunization with the recombinant resulted in strong systemic gag p24-specific T cell responses as determined by the IFN-gamma ELISPOT assay and by multiparameter flow cytometry. Oral immunization with CVB4/p24(73(3)) resulted in a short-lived, localized infection of the gut without systemic spread. Because coxsackieviruses are ubiquitous in the human population, we also evaluated whether the recombinant was able to induce gag p24-specific T cell responses in mice pre-immunized with the CVB4 vector. We showed that oral immunization with CVB4/p24(73(3)) induced gag p24-specific immune responses in vector-immune mice. CONCLUSIONS/SIGNIFICANCE: The CVB4/p24(73(3)) recombinant retained the physical and biological characteristics of the parental CVB4 vector. Oral immunization with the CVB4 recombinant was safe and resulted in the induction of systemic HIV-specific T cell responses. Furthermore, pre-existing vector immunity did not preclude the development of gag p24-specific T cell responses. As the search continues for new vaccine strategies, the present study suggests that live CVB4/HIV recombinants are potential new vaccine candidates for HIV.

Dynamics of MHC class II-activating signals in murine resting B cells.

MHC class II (MHC II) proteins are competent signaling molecules on APC. However, little is known about the mechanisms that control generation of their activating signals. Previous reports highlighted a number of factors that could affect the nature and outcome of MHC II signals, including the inability of MHC II ligation on resting vs activated murine B cells to induce mobilization of Ca2+. In the present study, we report that ligation of MHC II on resting murine B cells reproducibly induces mobilization of intracellular Ca2+ using both mAbs and cognate T cells as ligands. Mobilization of Ca2+ was independent of MHC II haplotype, isotype, or mouse genetic background. MHC II-mediated mobilization of Ca2+ is completely inhibited by inhibitors of src-like kinases and syk, and MHC II ligation increases overall tyrosine phosphorylation level. Moreover, MHC II ligation results in specific up-regulation of CD86. However, induction of these responses is dependent on the type of anti-MHC II Ab used, suggesting that epitope specificity and/or the nature of ligation is important. Moreover, we demonstrate that MHC II-derived signals are strictly regulated by the order and timing of BCR and CD40 signals, suggesting coordination of these signals preserves the integrity of early B cell priming events. Thus, the mode and the context of MHC II ligation influence generation of MHC II-derived activating signals in resting B cells. Based on these results, a new model that highlights the role of MHC II-activating signals in regulation of Ag presentation by B cells is proposed.

Functional and structural requirements for the internalization of distinct BCR-ligand complexes.

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCR-mediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk-mediated BCR signaling are not essential for BCR-mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin-coated pits, the internalization of Ab-BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag-BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR-ligand complexes.

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation.

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4(+) T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.

Antigen binding to GM1 ganglioside results in delayed presentation: minimal effects of GM1 on presentation of antigens internalized via other pathways.

Plasma membrane rafts are sphingolipid- and cholesterol-rich patches that function as membrane trafficking and surface signalling regions. Ganglioside GM1 is an integral component of these microdomains, and Escherichia coli enterotoxin B subunit (EtxB) is a pentamer that binds with high affinity to GM1 resulting in GM1 cross-linking. We previously demonstrated that antigen coupled directly to EtxB resulted in enhanced presentation relative to antigen taken up by fluid-phase endocytosis. Here we demonstrate a new role for GM1 in antigen presentation by examining the effects of cross-linking GM1 on the kinetics of presentation and processing of antigen by the B-cell receptor (BCR), fluid-phase endocytosis and GM1-targeted antigen. EtxB bound to B cells does not augment the subsequent kinetics or magnitude of presentation of either BCR-internalized antigen or soluble antigen. Moreover, presentation of GM1-bound antigen is significantly slower than antigen presentation following BCR-mediated uptake. In contrast to the rapid internalization of BCR-bound antigen (which has a half life of 60 min), the majority of EtxB-bound antigen forms a plasma membrane depot detectable for many hours after initial incubation (and with a half life of 12 hr). We conclude that cross-linking of GM1 by EtxB minimally affects the processing and presentation of antigens internalized via other pathways. Nevertheless, binding of antigens to GM1 results in delayed presentation that has important implications for in vivo immunization using GM1-targeted adjuvants.