Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Interplays between inflammasomes and viruses, bacteria (pathogenic and probiotic), yeasts and parasites.

In recent years, scientists studying the molecular mechanisms of inflammation have discovered an amazing phenomenon - the inflammasome - a component of the innate immune system that can regulate the functional activity of effector cells during inflammation. At present, it is known that inflammasomes are multimolecular complexes (cytosolic multiprotein oligomers of the innate immune system) that contain many copies of receptors recognizing the molecular structures of cell-damaging factors and pathogenic agents. Inflammasomes are mainly formed in myeloid cells, and their main function is participation in the cleavage of the pro-IL-1ß and pro-IL-18 cytokines into their biologically active forms (IL-1ß, IL-18). Each type of microorganism influences particular inflammasome activation, and long-term exposure of the organism to viruses, bacteria, yeasts or parasites, among others, can induce uncontrolled inflammation and autoinflammatory diseases. Therefore, this review aims to present the most current scientific data on the molecular interplay between inflammasomes and particular microorganisms. Knowledge about the mechanisms responsible for the interaction between the host and certain types of microorganisms could contribute to the individuation of innovative strategies for the treatment of uncontrolled inflammation targeting a specific type of inflammasome activated by a specific type of pathogen.

Diagnosis and management of hypersensitivity reactions to vaccines.

INTRODUCTION: Many countries in Europe now recommend and enforce mandatory vaccinations to improve vaccination coverage. Thus, the number of adverse events following immunization (AEFI) may show an increase. Among these events, severe hypersensitivity reactions to vaccines are rare. However, it is important that they be identified and recognized so that they may be adequately managed. AREAS COVERED: The literature search was undertaken through PubMed and Embase to identify English-language papers focusing on hypersensitivity to vaccines. EXPERT OPINION: Hypersensitivity reactions following vaccinations are rare and are classified according to their chronology and extension: immediate when they occur within the first 4 hours following administration and non-immediate when they occur later. Local reactions are the most common adverse event following injection of vaccines and generally do not require any allergy workup. Immediate reactions, however, are potentially IgE-mediated and require an allergy workup. In general, a previously known food allergy (i.e., egg or milk) is not a contraindication to immunizations. Patients with a known allergy to gelatin, yeast, latex, antibiotics, or other specific components of vaccines require an allergy workup before administration of the vaccine.

CD4 Inhibits Helper T Cell Activation at Lower Affinity Threshold for Full-Length T Cell Receptors Than Single Chain Signaling Constructs.

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.

Edible Vaccines: Promises and Challenges.

Vaccines are biological preparations that improve immunity to particular diseases and form an important innovation of 19th century research. It contains a protein that resembles a disease-causing microorganism and is often made from weak or killed forms of the microbe. Vaccines are agents that stimulate the body's immune system to recognize the antigen. Now, a new form of vaccine was introduced which will have the power to mask the risk side of conventional vaccines. This type of vaccine was produced from plants which are genetically modified. In the production of edible vaccines, the gene-encoding bacterial or viral disease-causing agent can be incorporated in plants without losing its immunogenic property. The main mechanism of action of edible vaccines is to activate the systemic and mucosal immunity responses against a foreign disease-causing organism. Edible vaccines can be produced by incorporating transgene in to the selected plant cell. At present edible vaccine are developed for veterinary and human use. But the main challenge faced by edible vaccine is its acceptance by the population so that it is necessary to make aware the society about its use and benefits. When compared to other traditional vaccines, edible vaccines are cost effective, efficient and safe. It promises a better prevention option from diseases.

Subunit vaccines based on recombinant yeast protect against influenza A virus in a one-shot vaccination scheme.

Here we report on new subunit vaccines based on recombinant yeast of the type Kluyveromyces lactis (K. lactis), which protect mice from a lethal influenza A virus infection. Applying a genetic system that enables the rapid generation of transgenic yeast, we have developed K. lactis strains that express the influenza A virus hemagglutinin, HA, either individually or in combination with the viral M1 matrix protein. Subcutaneous application of the inactivated, but otherwise non-processed yeast material shows a complete protection of BALB/c mice in prime/boost and even one-shot/single dose vaccination schemes against a subsequent, lethal challenge with the cognate influenza virus. The yeast vaccines induce titers of neutralizing antibodies that are readily comparable to those induced by an inactivated virus vaccine. These data suggest that HA and M1 are produced with a high antigenicity in the yeast cells. Based on these findings, multivalent, DIVA-capable, yeast-based subunit vaccines may be developed as promising alternatives to conventional virus-based anti-flu vaccines for veterinary applications.

Microbial Colonization at Early Life Promotes the Development of Diet-Induced CD8αß Intraepithelial T Cells.

Intraepithelial lymphocytes (IELs) develop through the continuous interaction with intestinal antigens such as commensal microbiome and diet. However, their respective roles and mutual interactions in the development of IELs are largely unknown. Here, we showed that dietary antigens regulate the development of the majority of CD8αß IELs in the small intestine and the absence of commensal microbiota particularly during the weaning period, delay the development of IELs. When we tested specific dietary components, such as wheat or combined corn, soybean and yeast, they were dependent on commensal bacteria for the timely development of diet-induced CD8αß IELs. In addition, supplementation of intestinal antigens later in life was inefficient for the full induction of CD8αß IELs. Overall, our findings suggest that early exposure to commensal bacteria is important for the proper development of dietary antigen-dependent immune repertoire in the gut.

Immunomodulatory effects of whole yeast cells and capsicum in weanling pigs challenged with pathogenic Escherichia coli1.

An experiment was conducted to evaluate growth performance, fecal bacterial counts, frequency of diarrhea, and clinical blood parameters in weanling pigs inoculated with enterotoxigenic Escherichia coli (ETEC) who were fed a whole yeast cell (WYC) product and capsicum, a plant essential oil. Weanling pigs (34 barrows and 30 gilts, 21 d of age, 5.90 ± 1.03 kg BW) were allotted to experimental treatments in a randomized complete block design based on litter, sex, and initial BW. Four pigs were individually housed within each containment chamber and assigned to 1 of 4 dietary treatments, which included a control diet without or with 0.2% WYC (CitriStim; ADM, Decatur, IL) or 10 ppm of capsicum (XTract 6933; Pancosma, Geneva, Switzerland), provided either alone or in combination. After receiving diets for 13 d, pigs were orally inoculated with F18+ ETEC and maintained on their assigned diets for an additional 10 d; a separate cohort of 12 pigs receiving the control diet was sham-inoculated using PBS. Body and feeder weights were recorded, and fecal swabs collected, on 0, 5, and 10 d postinoculation (DPI), with blood sampled at 0, 2, 7, and 10 DPI for isolation of peripheral blood mononuclear cells. Pigs challenged with ETEC and fed diets containing WYC or capsicum alone had a higher frequency of diarrhea when compared with pigs receiving diets without those compounds (P < 0.05). Total fecal bacterial counts in pigs fed the combination of additives were highest when compared with either additive alone (interaction, P = 0.03) at 10 DPI. Blood leukocyte counts were increased in challenged pigs receiving the combination of additives compared with all other challenged treatment groups (interaction, P = 0.04). The addition of WYC increased (main effect, P = 0.01) lymphocyte counts at 7 DPI. Proportions of CD8+ and CD4+CD8+ cells were lower in pigs fed the combination of additives compared with pigs fed either additive alone at 0 and 7 DPI. In conclusion, these data indicate that the combination of the 2 additives elicited higher ETEC shedding and circulating leukocyte counts, and reduced the proportions of cytotoxic and memory T-cells than either additive alone.

Characterization of nuclear factor of activated T-cells-c3 (NFATc3) and gene expression of upstream-downstream signaling molecules in response to immunostimulants in Pacific red snapper cells.

The nuclear factor of activated T cells (NFAT) proteins have crucial roles in the development and function of the immune system since they not only regulate activation of T cells but are also involved in the control of thymocyte development and T-cell differentiation. In this study, NFATc3 was characterized from the Pacific red snapper, Lutjanus peru. LpNFAtc3, which contains an open reading of 3300 bp frame coding for a protein of 1100 aa with a predicted molecular weight of 118.52 kDa. The predicted protein showed a conserved NFAT family structure with signature motifs and domains, sharing high identity (up to 76%) compared to other fish sequences. NFATc3 gene expression was analyzed by real time-PCR in head-kidney cells (leukocytes and lymphocytes) following yeast, zymosan and Vibrio parahaemolyticus stimulation along with the expression of upstream (ILF2, ILF3 and CaN) and downstream (CD3, TCRß, IL-6 and IL-12) molecules. This study revealed a broad expression of NFATc3 with a relative strong expression in intestine and lymphocytes. The expression of NFATc3 was differentially up-regulated after stimulation with yeast in head-kidney leukocytes and after bacterial infection in lymphocytes at 24 h. Interestingly, the yeast and zymosan were able to activate ILF2, ILF3 and CaN mRNA gene expression in both kinds of cells. On the other hand, NFAT downstream genes such as CD3, TCRß, IL-6 and IL-12 were significantly up-regulated in leukocytes stimulated with yeast or zymosan at 12 h; however in lymphocytes, this up-regulation was detected when cells reacted to V. parahaemolyticus stimuli at 24 h. Stimulating Pacific red snapper leukocytes with immunostimulants as yeast significantly up-regulated the expression of NFATc3, and up- and down-stream molecular genes and NFATc3 lymphocytes expression were potentially involved in responses to invasion of bacterial pathogens in an early immune response.

Tongue Sole CD209: A Pattern-Recognition Receptor that Binds a Broad Range of Microbes and Promotes Phagocytosis.

CD209 is an immune receptor that plays an important role in the initiation of innate immunity and activation of adaptive immunity in mammals. However, much less is known about the immunological function of CD209 in lower vertebrates. In the present study, we examined the immune effect of a CD209 homologue (CsCD209) from the teleost fish tongue sole Cynoglossus semilaevis. CsCD209 possesses a lectin domain that shares high levels of similarity with the lectin domains of human and mouse CD209. CsCD209 expression was most abundant in kidney and blood and was significantly upregulated during bacterial infection. CsCD209 exhibited a subcellular localization mainly on the cell surface of myelomonocytes. Recombinant CsCD209 displayed apparent binding capacities to a broad range of bacteria and fungi, and significantly promoted the phagocytosis of the bound bacteria by C. semilaevis leukocytes. Collectively, the results indicate that teleost CD209 serves as a pattern recognition receptor that exerts an influence on the phagocytosis process during pathogen infections.

Heat-Killed Yeast as a Pan-Fungal Vaccine.

Fungal infections continue to rise worldwide. Antifungal therapy has long been a mainstay for the treatment of these infections, but often can fail for a number of reasons. These include acquired or innate drug resistance of the causative agent, poor drug penetration into the affected tissues, lack of cidal activity of the drug and drug toxicities that limit therapy. In some instances, such as coccidioidal meningitis, therapy is life-long. In addition, few new antifungal drugs are under development. In light of this information a preventative vaccine is highly desirable. Although numerous investigators have worked toward the development of fungal vaccines, none have become commercially available for use in humans. In the course of our studies, we have discovered that heat-killed yeast (HKY) of Saccharomyces cerevisiae can be used as a vaccine and have shown that it has efficacy in the prevention and reduction of five different fungal infections when used experimentally in mice, which raises the possibility of a pan-fungal vaccine preparation. In our studies we grow S. cerevisiae in broth and heat-kill the organism at 70 ° C for 3 h. The number of dead yeast cells is adjusted and mice are vaccinated subcutaneously beginning 3-7 weeks prior to infection. After infection, efficacy is assessed on the basis of survival and residual burden of the fungus in the target organs. Alternatively, efficacy can be assessed solely on fungal burden at a predetermined time postinfection. Although itself it is unlikely to be moved toward commercialization, HKY can be used a positive control vaccine for studies on specific molecular entities as vaccines, and as a guidepost for the key elements of potential, more purified, pan-fungal vaccine preparations.

Preparation of an Oral Vaccine by Proteome Analysis and Molecular Display Technology.

In recent years, genetic engineering and protein expression technologies have promoted the development of recombinant protein vaccines. To accelerate the development of efficient vaccines for mycosis, screening candidate antigens, and determining the optimal route of administration are indispensable steps. Two methods for identifying novel antigens and producing antigens specific to Candida albicans, as a model causative pathogen of mycosis, are discussed in this chapter. Specifically, the application of liquid chromatography/tandem mass spectrometry using a long monolithic column for proteome analysis to identify virulence factors of C. albicans, followed by molecular display technology to produce an oral vaccine using antigens found by the proteomic study, is described.

Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes ß-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

Yeast-containing feed additive alters gene expression profiles associated with innate immunity in whole blood of a rodent model.

Feeding a yeast-containing additive (YCA; OmniGen-AF) improves immune responses in ruminant livestock and reduces subsequent production losses. The objective was to identify molecular pathways by which dietary YCA may modify immune responses using a rodent model. Thirty-seven healthy, unchallenged CD rats received a diet containing 0 (control; n = 5, only 28 d), 0.5% (n = 15) or 1% (n = 17) YCA for 7 (n = 4/group), 14 (n = 3 or 4/group), 21 (n = 3 or 4/group) or 28 (n = 5/group) d. At the end of the feeding periods, whole blood was collected and the isolated RNA was analyzed for the expression of 84 genes involved in innate and cell-mediated adaptive immune responses. Three bacterial pattern recognition receptors TLR1 (0.5%: + 2.01; 1%: + 2.38), TLR6 (0.5%: + 2.11; 1%: + 2.34) and NOD2 (0.5%: + 2.32; 1%: + 2.23), two APC surface receptors CD1D1 (0.5%: + 1.75; 1%: + 2.33) and CD80 (0.5%: +2.45; 1%: +3.00), and the cell signaling molecule MAPK8 (0.5%: +1.87; 1%: +2.35) were significantly up-regulated by YCA at both inclusion rates. In conclusion, feeding YCA may potentially increase recognition and responses to bacterial pathogens and T-cell activation and differentiation and thereby maintain health and prevent production losses.

The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania.

BACKGROUND: Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5. METHODS: The expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania. RESULTS: These studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi. CONCLUSIONS: We demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.

Understanding ForteBio's Sensors for High-Throughput Kinetic and Epitope Screening for Purified Antibodies and Yeast Culture Supernatant.

Real-time and label-free antibody screening systems are becoming more popular because of the increasing output of purified antibodies and antibody supernatant from many antibody discovery platforms. However, the properties of the biosensor can greatly affect the kinetic and epitope binning results generated by these label-free screening systems. ForteBio human-specific ProA, anti-human IgG quantitation (AHQ), anti-human Fc capture (AHC) sensors, and custom biotinylated-anti-human Fc capture (b-AHFc) sensors were evaluated in terms of loading ability, regeneration, kinetic characterization, and epitope binning with both purified IgG and IgG supernatant. AHC sensors proved unreliable for kinetic or binning assays at times, whereas AHQ sensors showed poor loading and regeneration abilities. ProA sensors worked well with both purified IgG and IgG supernatant. However, the interaction between ProA sensors and the Fab region of the IgG with VH3 germline limited the application of ProA sensors, especially in the epitope binning experiment. In an attempt to generate a biosensor type that would be compatible with a variety of germlines and sample types, we found that the custom b-AHFc sensors appeared to be robust working with both purified IgG and IgG supernatant, with little evidence of sensor-related artifacts.

N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies.

The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662-667 in the MPER) but also several other residues (aa 652-655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

Characterization of a S-adenosyl-l-methionine (SAM)- accumulating strain of Scheffersomyces stipitis

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism (AU)

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Recognition of Candida albicans by Dectin-1 induces mast cell activation.

Mast cells are crucial elements of the innate immune response. They reside in tissues that are commonly exposed to the external environment, such as the skin and mucosae, where they can rapidly detect the presence of pathogens and mount a potent inflammatory response that recruits other cellular effectors of the immune response. The contribution of mast cells to the immune response to viruses, bacteria, protozoa and multicellular parasites is well established, but there is scarce information about the role of these cells in fungal infections. In this study, we analyzed if mast cells are activated by Candida albicans and if the C-type lectin receptor Dectin-1 is involved in its recognition. We found that both yeasts and hyphae of C. albicans-induced mast cell degranulation and production of TNF-α, IL-6, IL-10, CCL3 and CCL4, while only yeasts were able to induce IL-1ß. Mast cells also produced ROS after stimulation with both dimorphic phases of C. albicans. When mast cells were activated with yeasts and hyphae, they showed decreased expression of IκBα and increased presence of phosphorylated Syk. Blockade of the receptor Dectin-1, but not Toll-like receptor 2, decreased TNF-α production by mast cell in response to C. albicans. These results indicate that mast cells are capable of sensing the two phases of C. albicans, and suggest that mast cells participate as an early inductor of inflammation during the early innate immune response to this fungus.