Maturation and cytokine pattern of human dendritic cells in response to different yeasts.

Activated dendritic cells (DC) induce and polarize T-cell responses by expression of distinct maturation markers and cytokines. This study systematically investigated the capacity of different biotechnically relevant yeast species and strains including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Candida glabrata to initiate maturation of human DC. As important prerequisite for T-cell activation, all yeasts were shown to effectively induce, though to a different extent, the expression of the activation marker CD83, the co-stimulatory molecules CD80, CD86, CD54, CD58, and CD40, as well as the antigen-presenting molecules MHCs I and II. Furthermore, yeast-activated DC secreted various cytokines including inflammatory TNF-α, IL-6, IL-8, and IL-1ß or T-cell polarizing IL-12, IL-10, IL-23, and IL-27. Variability was observed in the expression of TNF-α, IL-6, IL-8, IL-1ß, and IL-10 in response to the tested yeasts, whereas expression levels of IL-12, IL-23, and IL-27 were similar. Interestingly, maturation marker expression and cytokine secretion were not negatively affected after application of yeast mutants with altered cell wall mannoprotein structure (Δmnn11) or defective in protein N-glycosylation (Δost3), indicating that elongated cell wall mannoproteins at the outer yeast cell surface are not a prerequisite for the observed yeast-mediated DC maturation. Thus, our data provide a valuable basic knowledge for the future design of effective yeast-based delivery approaches.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris.

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.

Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera.

A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, ß-glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on ß-1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines.

TNF-α and CD8+ T cells mediate the beneficial effects of nitric oxide synthase-2 deficiency in pulmonary paracoccidioidomycosis.

BACKGROUND: Nitric oxide (NO), a key antimicrobial molecule, was previously shown to exert a dual role in paracoccidioidomycosis, an endemic fungal infection in Latin America. In the intravenous and peritoneal models of infection, NO production was associated with efficient fungal clearance but also with non-organized granulomatous lesions. Because paracoccidioidomycosis is a pulmonary infection, we aimed to characterize the role of NO in a pulmonary model of infection. METHODOLOGY/PRINCIPAL FINDINGS: C57Bl/6 wild type (WT) and iNOS(-/-) mice were i.t. infected with 1×10(6) Paracoccidioides brasiliensis yeasts and studied at several post-infection periods. Unexpectedly, at week 2 of infection, iNOS(-/-) mice showed decreased pulmonary fungal burdens associated with an M2-like macrophage profile, which expressed high levels of TGF-ß impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-α synthesis and intense migration of activated macrophages, CD4(+) and CD8(+) T cells into the lungs. By week 10, iNOS(-/-) mice showed increased fungal burdens circumscribed, however, by compact granulomas containing elevated numbers of activated CD4(+) T cells. Importantly, the enhanced immunological reactivity of iNOS(-/-) mice resulted in decreased mortality rates. In both mouse strains, depletion of TNF-α led to non-organized lesions and excessive influx of inflammatory cells into the lungs, but only the iNOS(-/-) mice showed increased mortality rates. In addition, depletion of CD8(+) cells abolished the increased migration of inflammatory cells and decreased the number of TNF-α and IFN-γ CD4(+) and CD8(+) T cells into the lungs of iNOS(-/-) mice. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that NO plays a deleterious role in pulmonary paracoccidioidomycosis due to its suppressive action on TNF-α production, T cell immunity and organization of lesions resulting in precocious mortality of mice. It was also revealed that uncontrolled fungal growth can be overcome by an efficient immune response.

Uptake of various yeast genera by antigen-presenting cells and influence of subcellular antigen localization on the activation of ovalbumin-specific CD8 T lymphocytes.

Yeasts of the genus Saccharomyces expressing recombinant antigens are currently evaluated as candidate T cell vaccines. Here, we compared the interaction kinetics between four biotechnologically relevant yeast genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Pichia pastoris) and human dendritic cells as well as the involvement of Dectin-1 and mannose receptor in phagocytosis. Further, we analyzed the activation capacity of recombinant yeasts expressing ovalbumin (OVA) either intracellular, extracellular or surface-displayed by OVA-specific CD8 T lymphocytes. We found that the kinetic patterns of yeast uptake by phagocytic cells varied between the tested yeast genera and that both genus and subcellular OVA antigen localization influenced the strength of T cell activation. In particular, in S. cerevisiae, a secreted antigen was less effectively delivered than its cytosolic variant, whereas most efficient antigen delivery with P. pastoris was obtained by cell surface bound antigen. Our data indicate that protein secretion might not be an effective delivery pathway in yeast.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.