Heat-killed Saccharomyces Cerevisiae Research Articles

Enhanced anti-cancer effect of curcumin loaded-niosomal nanoparticles in combination with heat-killed Saccharomyces cerevisiae against human colon cancer cells

Enhanced anti-cancer effect of curcumin loaded-niosomal nanoparticles in combination with heat-killed Saccharomyces cerevisiae against human colon cancer cells

Adhesion of Stenotrophomonas maltophilia, Delftia acidovorans, and Achromobacter xylosoxidans to Contact Lenses.

Contact lens cases become contaminated with microbes during use. We wished to compare the adhesion of uncommon bacterial contaminants isolated from lens cases to contact lenses with and without organic soil. Strains of Delftia acidovorans (001), Stenotrophomonas maltophilia (002 and 006), and Achromobacter xylosoxidans (001) isolated from contact lens cases (test strains) and Pseudomonas aeruginosa (Paer1) isolated from eyes at the time of infiltrative response (control strain) were used. Bacteria were grown and resuspended in phosphate-buffered saline (PBS) or 10% organic soil (heat-killed Saccharomyces cerevisiae resuspended in complement inactivated bovine serum). Two silicone hydrogel (senofilcon A and comfilcon A) and one hydrogel lens (etafilcon A) lens materials were used. Bacteria (1.0×10 and 1.0×10 colony-forming units/mL; CFU/mL) adhered to lenses for 24 hr and the numbers of bacteria adherent to each lens type (with and without organic soil) were estimated by culture. All the four test strains adhered in significantly greater numbers to contact lenses after incubation in inoculum prepared with organic soil compared with PBS-D. acidovorans 001 (0.7 log10 CFU; P<0.05), S. maltophilia 002 (1.7 log10 CFU; P<0.05), S. maltophilia 006 (0.9 log10 CFU; P<0.05), and A. xylosoxidans 001 (0.4 log10 CFU; P<0.05). However, the presence of organic soil did not increase adhesion of P. aeruginosa Paer1 (-0.1 log10 CFU; P>0.05). Achromobacter xylosoxidans 001 (P<0.01), D. acidovorans 001 (P<0.01), and S. maltophilia 002 (P<0.01) significantly differed in their adhesion to the three contact lens materials. Bacteria that are commonly found in contact lens cases adhered to contact lenses in relatively high numbers in the presence of organic soil. This might indicate that a similar phenomenon occurs in the presence of tears. This may facilitate their transfer from the lens to the cornea and the production of corneal infiltrates.

Heat-Killed Saccharomyces cerevisiae, A Dectin-1 Agonist, Selectively Induces IgG4 Production by Human B Cells.

Dectin-1 is a major receptor that recognizes fungal cell wall β-glucan. We previously reported that heat-killed Saccharomyces cerevisiae (HKSC), a Dectin-1 agonist, selectively induces IgG1 class switching in mouse B cells. Dectin-1 is also expressed on human B cells; however, Dectin-1 function in human B cells remains unknown. This study aimed to investigate the direct effect of in vitro stimulation using HKSC on Ig class switching in human B cells. HKSC selectively induced the expression of germline γ4 transcripts (GLTγ4) by human B cell line 2E2, and HKSC significantly augmented GLTγ4 promoter activity. Moreover, HKSC selectively enhanced GLTγ4 expression and IgG4 production by anti-CD40-activated human tonsillar resting B cells. Thus, these results suggest that Dectin-1 maybe involved in selective IgG4 class switching by human B cells.

SACCHAROMYCES CEREVISIAE-INDUCED APOPTOSIS OF MONOLAYER CERVICAL CANCER CELLS

Objective: The present study was undertaken to examine the effect of phagocytosis of killed yeast on the induction of apoptosis in monolayer of HeLa cells.Methods: HeLa cell line was incubated with different doses (1000-7.8 μg/ml) of heat-killed Saccharomyces cerevisiae for 24, 48, and 72 hrs. The cytotoxicity against HeLa cell line during different exposure hours was screened by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide assay. Induction of apoptosis was further confirmed by morphological and biochemical examination. Antiproliferative effect of yeast was examined under inverted microscope. Cell morphological changes were analyzed by fluorescent staining with propidium iodide.Results: The results showed that yeast induces cytotoxicity against HeLa cells in concentrations and during prolonged exposure periods. The viability of HeLa cells decreased from 85% to 45% during 72 hrs of treatment with 1000 μg/ml of yeast cells. The inhibitory concentration 50% of heat-killed yeast required to induce 50% inhibition of HeLa cells was 62.5 μg/ml. Apoptotic cells showed signs such as cell enlargement, membrane blebbing, and chromatin condensation. Furthermore, cell cycle analysis showed that S. cerevisiae treated HeLa cells and showed a typical apoptosis pattern of DNA content that reflected sub-G0 phase (corresponding to apoptotic cells).Conclusion: Results from the present work show that the heat-killed yeast has anticancer activity and it includes apoptosis of HeLa cells in vitro.

Different biosorption mechanisms of Uranium(VI) by live and heat-killed Saccharomyces cerevisiae under environmentally relevant conditions

Uranium adsorption mechanisms of live and heat-killed Saccharomyces cerevisiae in different pH values and biomass concentrations were studied under environmentally relevant conditions. Compared with live cells, the adsorption capacity of heat-killed cells is almost one order of magnitude higher in low biomass concentration and highly acidic pH conditions. To explore the mesoscopic surface interactions between uranium and cells, the characteristic of uranium deposition was investigated by SEM-EDX, XPS and FTIR. Biosorption process of live cells was considered to be metabolism-dependent. Under stimulation by uranyl ions, live cells could gradually release phosphorus and reduce uranium from U(VI) to U(IV) to alleviate uranium toxicity. The uranyl-phosphate complexes were formed in scale-like shapes on cell surface. The metabolic detoxification mechanisms such as reduction and “self-protection” are of significance to the migration of radionuclides. In the metabolism-independent biosorption process of heat-killed cells: the cells cytomembrane was damaged by autoclaving which led to the free diffusion of phosphorous from intracellular, and the rough surface and nano-holes indicated that the dead cells provided larger contact area to precipitate U(VI) as spherical nano-particles. The high biosorption capacity of heat-killed cells makes it become a suitable biological adsorbent for uranium removal.

Dectin-1 agonist selectively induces IgG1 class switching by LPS-activated mouse B cells

Heat-killed Saccharomyces cerevisiae (HKSC) is an agonist for Dectin-1, a major fungal cell wall β-glucan receptor. We previously reported that HKSC selectively enhances IgG1 production by LPS-activated mouse B cells. To determine if this IgG1 selectivity is caused by selective IgG1 class switching, we performed RT-PCRs for measuring germline transcripts (GLTs), flow cytometric analyses for detecting Ig-expressing cells, and ELISPOT assays for measuring the number of Ig-secreting cells in HKSC/LPS-stimulated mouse B cell cultures. HKSC selectively enhanced expression of GLTγ1, the number of IgG1-expressing cells, and the number of IgG1-secreting B cells in the presence of LPS stimulation. In addition, HKSC induced the expression of CD69, an activation marker for B lymphocytes, and the expression of surface Dectin-1. Two Dectin-1 antagonists, laminarin and a neutralizing Dectin-1 antibody, selectively diminished HKSC-reinforced IgG1 production by LPS-stimulated B cells. Furthermore, depleted zymosan (dzn), a Dectin-1 agonist with increased selectivity, also selectively enhanced GLTγ1 transcription. The Dectin-1 antagonists blocked dzn-induced IgG1 production by LPS-activated B cells. Collectively, these results suggest that Dectin-1 agonists selectively induce IgG1 class switching by direct stimulation of Dectin-1 on LPS-activated B cells resulting in selective production of IgG1.

17 Increased Expression of LRRK2, a Susceptibility Gene of IBD Results in Enhanced Pro-Inflammatory Response and Severe Experimental Colitis

Background Many genetic risk loci of inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have been identified by genome wide association studies in recent years. LRRK2/MUC19 locus has the third strongest association in IBD. LRRK2 gene has been intensively studied as a causative and susceptibility gene of Parkinson's disease. However the immunological function and the mechanisms about how this locus is involved in the pathogenesis of IBD remain largely unknown. The aim of this study is to investigate the immunological role of LRRK2 in the pathogenesis of IBD. Methods We used Lrrk2BAC transgenic mice (Lrrk2 Tg) that contains entire LRRK2 gene region including endogenous promoter and enhancer. Dextran sulfate sodium (DSS)-induced colitis was performed and bone marrow derived dendritic cells (BMDC) from Lrrk2 Tg and littermate control (CTL) were used to examine the immunological function. Immunoprecipitation and western blotting were performed to find the binding partners of LRRK2. Results Lrrk2 Tg exhibited more severe DSS induced colitis than CTL. BMDC from Lrrk2 Tg and CTL were stimulated with various TLR ligands including zymosan, a yeast wall extract. We found that TLR2 and TLR4 ligands did not show an increased response in Lrrk2 Tg compared to CTL. However zymosan induced significant increase in TNF-a synthesis in Lrrk2 Tg. Zymosan can stimulate Dectin1 as well. So we next stimulated BMDC with Dectin-1 agonists such as zymosan depleted (ZymD), a more specific ligand for Dectin-1, heat-killed Saccharomyces cerevisiae (HK-SC) and heat-killed Candida albicans (HK-CA). These stimulations significantly increased TNFa production in Lrrk2 Tg compared to CTL. To investigate the role of LRRK2 in Dectin-1 signaling, BMDC were stimulated with ZymD and the phosphorylation of NF-kB and MAP kinase-related molecules were examined by western blotting. BMDC from Lrrk2 Tg showed increased NF-kB signaling and slight increase in MAP kinase signaling compared to CTL. To identify the binding partners of LRRK2, HEK293T cells were transfected with TAK1, NEMO, TRAF6 and TAB2 expressing plasmids and the immunoprecipitants were subjected to western blotting. We found that these signaling molecules interacted with LRRK2. Conclusions The result of DSS colitis indicates that increased expression of LRRK2 leads to an increased inflammatory response in experimental colitis. Our data suggest that LRRK2 is involved in Dectin-1 signaling and activates NF-kB signaling through TAK1 complex. It has been shown that patients with Crohn's disease had increased LRRK2 expression in colonic mucosa and LRRK2 was phosphorylated during the course of DSS colitis. The activation and increased expression of LRRK2 cause more proinflammatory cytokines production in response to gut microbiota-derived innate immune stimuli and may lead to intestinal inflammation.

18 NOD2-Dependent IL-33 Expression Is an Important Mechanism in the Pathogenesis of Inflammatory Bowel Disease (IBD)

Background Many genetic risk loci of inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis have been identified by genome wide association studies in recent years. LRRK2/MUC19 locus has the third strongest association in IBD. LRRK2 gene has been intensively studied as a causative and susceptibility gene of Parkinson's disease. However the immunological function and the mechanisms about how this locus is involved in the pathogenesis of IBD remain largely unknown. The aim of this study is to investigate the immunological role of LRRK2 in the pathogenesis of IBD. Methods We used Lrrk2BAC transgenic mice (Lrrk2 Tg) that contains entire LRRK2 gene region including endogenous promoter and enhancer. Dextran sulfate sodium (DSS)-induced colitis was performed and bone marrow derived dendritic cells (BMDC) from Lrrk2 Tg and littermate control (CTL) were used to examine the immunological function. Immunoprecipitation and western blotting were performed to find the binding partners of LRRK2. Results Lrrk2 Tg exhibited more severe DSS induced colitis than CTL. BMDC from Lrrk2 Tg and CTL were stimulated with various TLR ligands including zymosan, a yeast wall extract. We found that TLR2 and TLR4 ligands did not show an increased response in Lrrk2 Tg compared to CTL. However zymosan induced significant increase in TNF-a synthesis in Lrrk2 Tg. Zymosan can stimulate Dectin1 as well. So we next stimulated BMDC with Dectin-1 agonists such as zymosan depleted (ZymD), a more specific ligand for Dectin-1, heat-killed Saccharomyces cerevisiae (HK-SC) and heat-killed Candida albicans (HK-CA). These stimulations significantly increased TNFa production in Lrrk2 Tg compared to CTL. To investigate the role of LRRK2 in Dectin-1 signaling, BMDC were stimulated with ZymD and the phosphorylation of NF-kB and MAP kinase-related molecules were examined by western blotting. BMDC from Lrrk2 Tg showed increased NF-kB signaling and slight increase in MAP kinase signaling compared to CTL. To identify the binding partners of LRRK2, HEK293T cells were transfected with TAK1, NEMO, TRAF6 and TAB2 expressing plasmids and the immunoprecipitants were subjected to western blotting. We found that these signaling molecules interacted with LRRK2. Conclusions The result of DSS colitis indicates that increased expression of LRRK2 leads to an increased inflammatory response in experimental colitis. Our data suggest that LRRK2 is involved in Dectin-1 signaling and activates NF-kB signaling through TAK1 complex. It has been shown that patients with Crohn's disease had increased LRRK2 expression in colonic mucosa and LRRK2 was phosphorylated during the course of DSS colitis. The activation and increased expression of LRRK2 cause more proinflammatory cytokines production in response to gut microbiota-derived innate immune stimuli and may lead to intestinal inflammation.

Abstract A28: Immune responses to mutated Ras - development of a yeast-based immunotherapeutic

Abstract Background: While mutations in the ras gene and corresponding product are known to be etiologic in a number of human cancers, efforts to block the mutated protein have been largely unsuccessful leading to its characterization as “undruggable.” We have taken an alternative approach involving generation of T cell responses to the mutated protein. GI-4000 is a series of proprietary immunotherapeutics designed to target cells with activating ras mutations using heat-killed Saccharomyces cerevisiae yeast (named Tarmogens: Targeted Molecular Immunogens) genetically engineered to express Ras G12 and Q61 mutations. Tarmogens activate antigen-specific T cell-mediated immune responses that kill target cells expressing a number of cancer antigens including mutated Ras. Activating mutations in ras occur in &amp;gt; 90% of pancreas cancer cases and &amp;gt;20% cases of NSCLC. GI-4000 has been evaluated in phase 2 trials in both these indications. GI-4000 has demonstrated: i) protection in a murine model of lung cancer (1), ii) in the pancreas trial, improvements in median RFS and OS vs placebo in subjects with a favorable proteomic signature (2), iii) also in the pancreas trial, 3 month improvement in median OS (p=NS) in R1 subjects, with 5 month improvement for immune responders (2), iv) improved OS in subjects with NSCLC treated with GI-4000 compared to case matched controls: HR=0.577, p=NS. In addition, Tarmogens specific for other oncologic targets decreased human regulatory T cells (Tregs) in vitro with a reciprocal increase in effector T cells (3). Here we discuss immunologic outcomes in R0 subjects enrolled in the pancreas cancer trial. Methods: In the pancreas cancer study 176 subjects with Ras mutant+ adenocarcinoma of the pancreas post resection were randomized 1:1 to GI-4000/gemcitabine or placebo/gemcitabine (stratified by resection status: R0/R1). Three weekly injections of GI-4000 or placebo were followed by 6 cycles of gemcitabine 1000 mg/m2 iv (day 1, 8, 15, then every 28 days). Monthly GI-4000 or placebo were administered on gemcitabine off-weeks and continued monthly until disease recurrence, intolerable toxicity or death. R0 subjects (n=102) with adequate blood samples available from timepoints throughout the study were assayed for immune response by interferon-γ (IFNγ) ELISpot assay using Ras peptide pools containing the G12 mutation present in the subject's tumor and a mismatched peptide set identical to the mutation-specific set except at G12. Frequencies of Tregs at baseline and pre-gemcitabine were measured by flow cytometry (n=76). Results: In contrast to the R1 group, there was no increase in Ras mutation-specific IFNγ responses in the R0 GI-4000 group compared to placebo: 16/52 (30.8%) vs 22/50 (44.0%) based on pre-specified criteria. However, naive Tregs (CD4+/CD45RA+/Foxp3low) in the GI-4000 treated group were significantly decreased compared to placebo: 11/42 (26.2%) vs 3/34 (8.8%) subjects had a &amp;gt;2-fold decrease in this fraction (p=0.048 Fisher's exact test). IFNγ responses and OS were strikingly influenced by G12 mutation and associations of specific Ras mutations with outcome will be discussed. Conclusions: GI-4000 decreases frequencies of Tregs, which could be an important attribute for an immunotherapeutic in the treatment of cancer. GI-4000 also generates mutation-specific immune responses and appears to have clinical activity in pancreas cancer and NSCLC. Immune targeting of the activating mutation may be a promising approach to Ras mutated cancers.

Whole glucan particles as a vaccine against murine aspergillosis.

Vaccination with heat-killed Saccharomyces cerevisiae (HKY) protects against experimental infection by pathogenic fungi of five genera. Here we tested whether purified Saccharomyces cell wall β-glucan could induce protection against systemic aspergillosis. CD-1 mice were given three weekly vaccine doses subcutaneously prior to intravenous infection with Aspergillus fumigatus. Mice received PBS, 2.5 mg HKY, whole glucan particles (WGP), WGP conjugated to BSA (0.06 to 12 mg per dose), a soluble medium molecular mass (MMW) β-glucan alone or MMW-BSA (≤24 mg per dose). Survival and c.f.u. were determined, and cytokine induction and anti-β-glucan antibodies were assessed in vaccinated mice. Neither soluble MMW glucan, nor MMW-BSA was effective. HKY protected in two studies (survival and c.f.u. were reduced in brain and kidney organs, P<0.004). Six or 12 mg WGP or WGP-BSA prolonged survival (P≤0.004) and reduced c.f.u. in each organ (P≤0.015) in both experiments; 0.6 mg WGP or WGP-BSA prolonged survival (P≤0.015) and reduced c.f.u. (P≤0.015) in one experiment. Cytokine profiles in serum and bronchoalveolar lavage from uninfected vaccinated mice showed an innate and adaptive immune profile (i.e. upregulation of colony stimulating factors, interferons, TNF-α, chemokines such as MCP-1, MIP-1α, RANTES and KC, and Th17-activating cytokines such as IL-6, IL-1β, IL-17). No anti-β-glucan antibodies were in the sera, suggesting an adaptive T cell-mediated, not a B cell-mediated, protective response. Vaccination with WGP or WGP-BSA proved protective against systemic aspergillosis, equivalent to that of HKY, supporting the potential of particulate β-glucans, alone or conjugated, as vaccines against aspergillosis.

Abstract 2561: Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses

Abstract The tumor-associated antigen MUC1 is overexpressed on various human hematological and epithelial malignancies. The MUC1 molecule, which has an N-terminal (MUC1-N) and a C-terminal (MUC1-C, which has been shown to act as an oncogene), is an attractive target for cancer immunotherapy. We have identified and reported 10 agonist epitopes (7 in the C-terminus, 2 in the N-terminus VNTR region, and 1 in the non-VNTR region) that enhance production of CD8 cytotoxic T lymphocytes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 MHC class I alleles, thus encompassing the majority of the North American population. Compared to native epitopes, these agonist epitopes more efficiently generate MUC1-specific T cells, induce production of IFN-gamma by MUC1-specific T cells, and lyse human tumor cells expressing MUC1 native epitopes in an MHC-restricted manner. We have previously shown that heat-killed recombinant Saccharomyces cerevisiae yeast genetically modified to express carcinoembryonic antigen can efficiently activate human dendritic cells (DCs) and stimulate CEA-specific CD8+ T cells. Here, we investigated the ability of a Saccharomyces cerevisiae vector containing the MUC1 transgene (yeast-MUC1) and encoding MUC1 with 8 agonist epitopes to activate human DCs, stimulate MUC1-N- and MUC1-C-specific T cells, and generate MUC1-specific T cells. We show here for the first time that human DCs treated with yeast-MUC1 vectors can activate MUC1-N and MUC1-C agonist-specific T-cell lines and can act as antigen-presenting cells to generate MUC1-N- and MUC1-C-specific T cells specific to each agonist epitopes, and that these T cells are capable of lysing MUC1-expressing tumor cells. Together, these findings provide a rationale for further clinical evaluation of yeast-MUC1 constructs encoding MUC1 agonist epitopes in cancer vaccine immunotherapy. Citation Format: Kwong Y. Tsang, Benjamin Boyerinas, Caroline Jochems, Tim Rodell, Thomas King, Jeffey Schlom. Yeast vector-encoding multiple MUC1 agonist epitopes (yeast-MUC1) can induce MUC1-specific T-cell immune responses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2561. doi:10.1158/1538-7445.AM2014-2561

Killed Saccharomyces cerevisiae protects against lethal challenge of Cryptococcus grubii.

Heat-killed Saccharomyces cerevisiae (HKY) vaccination protects mice against aspergillosis, coccidioidomycosis, mucormycosis, or candidiasis. We studied HKY protection against murine cryptococcosis. Once weekly subcutaneous HKY doses (S, 6 × 10(7); 2S, 1.2 × 10(8); 3S, 2.4 × 10(8)) began 28 (×3), 35 (×4), or 42 (×6) days prior to intravenous Cryptococcus grubii infection. Survival through 28 days, and CFU in the organs of survivors, were compared to saline-vaccinated controls. In the initial experiment, S, S×4, or 2S reduced brain CFU; liver or spleen CFU was reduced by S×4 or 2S. In a more lethal second experiment, 2S×6, 2S, or 3S×4 improved survival, and HKY regimens reduced CFU in the brain, liver, or spleen, with 2S×6, 2S, or 3S×4 most efficacious. Dose size appears more important than the number of doses: Regimens >S were superior, and 2S and 2S×6 were equivalent. 2S and 3S were equivalent, suggesting doses >2S do not provide additional protection. HKY protects against Cryptococcus, supporting components of HKY as a basis for the development of a panfungal vaccine.

Mechanism of uranium(VI) uptake by Saccharomyces cerevisiae under environmentally relevant conditions: Batch, HRTEM, and FTIR studies

Biosorption is of significance for the safety evaluation of high-level nuclear wastes repositories and remediation of radioactive contamination places. Quantitive study and structural characterization of uranium uptake by both live and heat-killed Saccharomyces cerevisiae at environmentally relevant uranium concentration and with different ionic strengths were carried out. Kinetic investigation showed the equilibrium reached within 15min. In equilibrium studies, pH shift towards neutral indicated release of hydroxyl ions. pH was the most important factor, which partly affected electrostatic interaction between uranyl ions and S. cerevisiae surface. The high ionic strength inhibited biosorption capacity, which can be explained by a competitive reaction between sodium ions and uranyl ions. Heat killing process significantly enhanced biosorption capacity, showing an order of magnitude higher than that of live cells. High resolution transmission electron microscopy (HRTEM) coupled with energy dispersive X-ray (EDX) showed needle-like uranium-phosphate precipitation formed on the cell walls for both live and heat-killed cells. Besides, dark-field micrographs displayed considerable similar uranium-phosphate precipitation presented outside the heat-killed cells. The phosphate released during heat-killing process. FTIR illustrated function groups hydroxyl, carboxyl, phosphate, and amino groups played important role in complexation with uranium.

Dectin-1 Stimulation Selectively Reinforces LPS-driven IgG1 Production by Mouse B Cells

Dectin-1, which specifically recognizes β-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, β-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.

O-0002 A Phase 2 Adjuvant Trial of GI-4000 Plus Gemcitabine vs. Gemcitabine Alone in Ras+ Patients with Resected Pancreas Cancer: R1 Subgroup Analysis

ABSTRACT Introduction Patients with resected pancreas cancer treated with standard of care gemcitabine have a median overall survival of 22 months (vs 20 months with observation). Activating mutations in ras occur in > 90% of pancreas cancer cases. GI-4000 is a proprietary immunotherapy designed to target cells with activating ras mutations using whole, heat-killed recombinant Saccharomyces cerevisiae yeast (called Tarmogens = Tar geted Mo lecular Immuno gens ). Tarmogens have demonstrated selective killing of target cells expressing a number of cancer antigens including mutated Ras in vivo by activating an antigen-specific T cell mediated response. This trial is designed to evaluate the efficacy, immunogenicity, and safety of GI-4000 plus gemcitabine in patients with Ras mutant positive resected pancreas cancer. Methods The study enrolled 176 subjects with Ras mutant positive adenocarcinoma of the pancreas post resection randomized 1:1 to GI-4000 plus gemcitabine or placebo plus gemcitabine (stratified by resection status; R0 or R1). Three weekly injections of GI-4000 or placebo were followed by 6 cycles of gemcitabine 1000 mg/m2 iv (day 1, 8, 15 every 28 days). Monthly GI-4000 or placebo were administered on the gemcitabine off-weeks and continued monthly until disease recurrence, intolerable toxicity, or death. The primary endpoint is recurrence-free survival. Data for the 39 R1 subjects (GI-4000 N = 19, Placebo N = 20) have been unblinded and analyzed. Results The GI-4000 group had an 11.4 week advantage in median overall survival (524 days vs 444 days), 16% advantage in 1 year survival (72% vs 56%), and a 4.6 week advantage in median RFS (287 days vs 255 days). The GI-4000 group showed a significantly higher rate of mutation specific T cell response to Ras by ELISpot assay; 7/15 (47%) vs 1/12 (8%), p = 0.032,with a more pronounced survival benefit in GI-4000 treated immune responders; 21.7 week advantage in median survival (596 Days vs 444 Days) compared to placebo. No significant novel toxicities have been observed to date. Conclusion GI-4000 in combination with adjuvant gemcitabine showed a clinically meaningful point estimate for the treatment effect on survival in R1 subjects with Ras mutant positive adenocarcinoma of the pancreas. GI-4000 was immunogenic and well tolerated. Ras specific immune response was associated with a more pronounced benefit in median survival. These data warrant further study in a definitively powered clinical trial for GI-4000 in the adjuvant setting in R1 subjects. The results for the R0 cohort will be reported when the data are mature.

A pilot trial of a combination of therapeutic vaccines (GI-4000 and GI-6207) as adjunctive therapy with first-line therapy with bevacizumab plus either FOLFOX or FOLFIRI in stage IV patients with newly diagnosed Ras-mutant positive or negative metastatic colorectal cancer.

TPS3638 Background: A promising approach for the treatment of cancer is the development of vaccines that target specific tumor antigens. In the metastatic CRC patient population, targeted and active immunotherapy may inhibit cancer progression and improve survival. This trial is designed to evaluate the efficacy, immunogenicity, and safety of GI-4000 plus standard therapy in patients with metastatic colorectal cancer. GI-4000 is a proprietary immunotherapy that uses whole, heat-killed recombinant Saccharomyces cerevisiae yeast (called Tarmogens = Targeted Molecular Immunogens). GI-4000 is designed to activate a cellular immune response to target cells with activating ras mutations. Tarmogens have been shown to elicit selective killing of target cells that express a number of cancer antigens, including mutated Ras, by activation of antigenspecific T cell mediated responses. Methods: The study population consists of subjects with metastatic colorectal cancer with an activating mutation in ras. Newly diagnosed subjects receive FOLFOX (or FOLFIRI) + bevacizumab (Bev) + GI- 4000; 3 weekly injections of GI-4000 are followed by 8 cycles of Bev + FOLFOX (or FOLFIRI); day 1 and 2 every 14 days. Doses of GI-4000 are administered on day 8 of each cycle. Upon completion of chemotherapy, GI-4000 continues along with Bev maintenance every 2 weeks for up to 5 years or until subjects experience intolerance, disease recurrence, or death. If Bev is stopped, GI-4000 may continue on the same maintenance schedule alone. Subjects that have already completed standard chemotherapy (FOLFOX or FOLFIRI) may enter the study and receive Bev + GI-4000 every 2 weeks for up to 5 years. Enrollment is ongoing and will continue up to 52 subjects.

Immune responses induced by heat killed Saccharomyces cerevisiae: A vaccine against fungal infection

Heat-killed Saccharomyces cerevisiae (HKY) used as a vaccine protects mice against systemic aspergillosis and coccidioidomycosis. Little is known about the immune response induced by HKY vaccination, consequently our goal was to do an analysis of HKY-induced immune responses involved in protection. BALB/c mice were vaccinated subcutaneously 3 times with HKY, a protective reagent, and bronchoalveolar lavage fluid, spleen, lymph nodes, and serum collected 2–5 weeks later. Cultured spleen or lymph node cells were stimulated with HKY. Proliferation of HKY-stimulated spleen or lymph node cells was tested by Alamar Blue reduction and flow cytometry. Cytokines from lymphocyte supernatants and antibody to glycans in serum collected from HKY-vaccinated mice were measured by ELISA. The results show that HKY promoted spleen cell and lymph node cell proliferation from HKY-vaccinated mice but not from PBS-vaccinated control mice (all P < 0.05). Cytokine measurement showed HKY significantly promoted IFNγ, IL-6 and IL-17A production by spleen cells and lymph node cells (all P < 0.05 and P < 0.01, respectively). Cytokine production by HKY-stimulated cells from PBS-vaccinated mice was lower than those from HKY-vaccinated ( P < 0.05). Cytokines in BAL from HKY-vaccinated were higher, 1.7-fold for IFNγ and 2.1-fold for TNFα, than in BAL from PBS-vaccinated. Flow cytometry of lymphocytes from HKY-vaccinated showed 52% of CD3 + or 56% of CD8 + cells exhibited cell division after stimulation with HKY, compared to non-stimulated controls (26 or 23%, respectively) or HKY-stimulated cells from PBS-vaccinated (31 or 34%). HKY also induced antibody against Saccharomyces glucan and mannan with titers 4- or 2-fold, respectively, above that in unvaccinated. Taken together, the results suggested that HKY vaccination induces significant and specific Th1 type cellular immune responses and antibodies to glucan and mannan.

Fungal Zymosan and Mannan Activate the Cryopyrin Inflammasome

Some fungal species are opportunistic pathogens that can cause infection in people with compromised immune systems. Activation of caspase-1 and the subsequent secretion of mature interleukin (IL)-1beta is a major signaling pathway of the innate immune system, but how yeasts induce caspase-1 activation is unknown. We show here that stimulation of macrophages and dendritic cells with heat-killed Saccharomyces cerevisiae or the purified cell wall components zymosan and mannan induced caspase-1 activation and IL-1beta secretion when combined with ATP. Macrophages deficient for the inflammasome adaptor ASC were defective in caspase-1 activation and IL-1beta secretion, suggesting involvement of an ASC-dependent inflammasome. Indeed, caspase-1 activation was abrogated in macrophages lacking the NOD-like (NLR) protein Cryopyrin/Nalp3 and in wild type macrophages pretreated with the pannexin-1 inhibitor probenecid. IL-1beta secretion further required the Toll-like receptor (TLR) adaptors MyD88 and TRIF, and partially relied on TLR2. We previously showed that bacterial molecules such as lipopolysaccharide (LPS) and peptidoglycan induce activation of caspase-7 through the Cryopyrin inflammasome. Similarly, Cryopyrin and ASC were required for activation of caspase-7 in macrophages stimulated with zymosan or mannan and ATP. These results demonstrate that the conserved fungal components zymosan and mannan require ASC and Cryopyrin for caspase-1 activation and IL-1beta secretion and suggest an important role for the Cryopyrin inflammasome during fungal infections.

Saccharomyces cerevisiae as a vaccine against coccidioidomycosis

Disseminated coccidioidomycosis is a life-threatening infection. In these studies, we examined protection against systemic murine coccidioidomycosis by vaccination with heat-killed Saccharomyces cerevisiae (HKY). CD-1 mice received HKY subcutaneously or by oral gavage with or without adjuvants once weekly beginning 3 or 4 weeks prior to infection; oral live Saccharomyces was also studied. All HKY sc regimens were equivalent, prolonging survival ( P ≤ 0.005) and reducing fungal burden versus controls. Oral live Saccharomyces, but not HKY, prolonged survival ( P = 0.03), but did not reduce fungal burden. Survival of mice given HKY was equivalent to vaccination with formalin-killed spherules, but inferior in reduction of fungal burden. HKY was superior to a successful recombinant vaccine, PRA plus adjuvant. This novel heterologous protection afforded by HKY vaccination offers a new approach to a vaccine against coccidioidomycosis.

Cellular-mediated reactions to foreign organisms inoculated into the hemocoel of Anopheles albimanus (Diptera: Culicidae).

The immune response against different organisms and particles inoculated in the hemocoel of female Anopheles albimanus Wiedemann was investigated. Histological and ultrastructural observations indicated that melanization and hemocyte type participation varied according to the particles inoculated. The initial responses against heat-killed Microccocus lysodeikticus and Escherichia coli included hemocyte lysis and melanization whereas the response to heat-killed Saccharomyces cerevisiae was only cellular, and an initial melanization of Sephadex G-25 (neutral charged) beads was followed by the formation of cellular aggregates. After 24 h, hemocytes were involved in all terminal encapsulation events. Plasmodium vivax Grassi and Feletti formalin-fixed sporozoites induced a weak response. Cellular aggregates were observed 1 h postinoculation, but participating hemocytes could not be identified because of the extensive cellular damage and lysis. Sporozoites were also observed in the core of these aggregates, mixed with cell debris and free in the hemolymph. The effect on the inoculated particles was also different-S. cerevisiae was encapsulated only by hemocytes, whereas M. lysodeikticus was lysed and E. coli was phagocytosed by plasmatocytes. These results indicate that hemocytes are important components in the immune response in An. albimanus.