Comparative study of using different yeast genera as vehicles for protein delivery to antigen-presenting cells

The use of yeasts as vehicle for protein antigens has been demonstrated to be a highly effective vaccination approach. In part, this can be attributed to the intrinsic adjuvant properties of yeast cell wall components. Moreover, the correct processing of recombinantly expressed proteins and the safety status of many yeast genera has encouraged the onset of preclinical and clinical trials using yeast vectors. However, the vast majority of such studies focused the attention on yeasts of the genus Saccharomyces as candidate T cell vaccine. In this work, different yeast genera were evaluated as potential antigen carrier in view of the development of novel yeast-based vaccines. For this purpose, yeasts were initially assessed for their ability to induce maturation and activation of human dendritic cells. Next, the internalization profile of selected yeasts by mammalian phagocytes was analyzed, as well as the involvement of pattern recognition receptors in the uptake process. Subsequently, yeasts engineered to express foreign proteins were assessed for their antigen delivery capacity. In vitro antigen presentation and ex vivo whole blood assays showed that recombinant yeast genera differently activate antigen-specific T cells. Furthermore, antigen localization played a decisive role in T cell activation. The data presented here strongly support the potential of recombinant yeast in the development of novel vaccine strategies in order to induce antigen-specific T cell responses.Der Einsatz von Hefen als Vehikel für Proteinantigene stellt eine vielversprechende Vakzinierungsstrategie dar, was u.a. auf adjuvante Eigenschaften der Hefe-Zellwandkomponenten zurückzuführen ist. Weiterhin haben der Nachweis der korrekten Prozessierung rekombinanter Proteine und der unbedenkliche Status vieler Hefegattungen ihren Einsatz in präklinischen und klinischen Studien gefördert. Bislang hat sich die Mehrzahl dieser Studien auf Hefen der Gattung Saccharomyces als Vakzinkandidaten für zellvermittelte Immunantworten konzentriert. Im Rahmen dieser Arbeit wurden verschiedene Hefegattungen als potentielle Antigenvehikel zur Etablierung neuartiger Hefe-basierter Vakzinen untersucht. Zunächst wurden Ausreifung und Aktivierung von Dendritischen Zellen durch diverse Hefegattungen analysiert. Danach wurden sowohl die Aufnahme bestimmter Hefegattungen durch Säuger-Phagozyten als auch die Beteiligung spezifischer Rezeptoren in diesem Prozess untersucht. Anschließend wurde die Fähigkeit rekombinanter Hefen zum Antigen "Delivery" evaluiert. Durch in vitro Antigenpräsentation und ex vivo Vollblut-Assays konnte gezeigt werden, dass verschiedene Hefegattungen Antigen-spezifische T-Zellen unterschiedlich aktivieren. Des Weiteren spielt die Antigenlokalisierung eine wichtige Rolle bei der T-Zellaktivierung. Die vorliegenden Ergebnisse unterstreichen das Potenzial rekombinanter Hefen bei der Entwicklung neuartiger Impfstrategien zur Induktion Antigen-spezifischer T-Zell-Immunantworten

In pulmonary paracoccidioidomycosis IL-10 deficiency leads to increased immunity and regressive infection without enhancing tissue pathology

BACKGROUND: \ud Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM.\ud METHODOLOGY/PRINCIPAL FINDINGS: \ud Wild type (WT) and IL-10(-/-) C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10(-/-) mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10(-/-) and WT mice were i.t. infected with 1×10(6) Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10(-/-) mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10(-/-) mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4(+) and CD8(+) T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10(-/-) mice.\ud CONCLUSIONS/SIGNIFICANCE: \ud Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 04/14518-2)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2011/51258-2)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2010/52275-5)CAPE

Vergleichende Untersuchungen zur Anwendung verschiedener Hefegattungen als Vehikel für das Protein-Delivery in Antigen-präsentierende Zellen

The use of yeasts as vehicle for protein antigens has been demonstrated to be a highly effective vaccination approach. In part, this can be attributed to the intrinsic adjuvant properties of yeast cell wall components. Moreover, the correct processing of recombinantly expressed proteins and the safety status of many yeast genera has encouraged the onset of preclinical and clinical trials using yeast vectors. However, the vast majority of such studies focused the attention on yeasts of the genus Saccharomyces as candidate T cell vaccine. In this work, different yeast genera were evaluated as potential antigen carrier in view of the development of novel yeast-based vaccines. For this purpose, yeasts were initially assessed for their ability to induce maturation and activation of human dendritic cells. Next, the internalization profile of selected yeasts by mammalian phagocytes was analyzed, as well as the involvement of pattern recognition receptors in the uptake process. Subsequently, yeasts engineered to express foreign proteins were assessed for their antigen delivery capacity. In vitro antigen presentation and ex vivo whole blood assays showed that recombinant yeast genera differently activate antigen-specific T cells. Furthermore, antigen localization played a decisive role in T cell activation. The data presented here strongly support the potential of recombinant yeast in the development of novel vaccine strategies in order to induce antigen-specific T cell responses.Der Einsatz von Hefen als Vehikel für Proteinantigene stellt eine vielversprechende Vakzinierungsstrategie dar, was u.a. auf adjuvante Eigenschaften der Hefe-Zellwandkomponenten zurückzuführen ist. Weiterhin haben der Nachweis der korrekten Prozessierung rekombinanter Proteine und der unbedenkliche Status vieler Hefegattungen ihren Einsatz in präklinischen und klinischen Studien gefördert. Bislang hat sich die Mehrzahl dieser Studien auf Hefen der Gattung Saccharomyces als Vakzinkandidaten für zellvermittelte Immunantworten konzentriert. Im Rahmen dieser Arbeit wurden verschiedene Hefegattungen als potentielle Antigenvehikel zur Etablierung neuartiger Hefe-basierter Vakzinen untersucht. Zunächst wurden Ausreifung und Aktivierung von Dendritischen Zellen durch diverse Hefegattungen analysiert. Danach wurden sowohl die Aufnahme bestimmter Hefegattungen durch Säuger-Phagozyten als auch die Beteiligung spezifischer Rezeptoren in diesem Prozess untersucht. Anschließend wurde die Fähigkeit rekombinanter Hefen zum Antigen "Delivery" evaluiert. Durch in vitro Antigenpräsentation und ex vivo Vollblut-Assays konnte gezeigt werden, dass verschiedene Hefegattungen Antigen-spezifische T-Zellen unterschiedlich aktivieren. Des Weiteren spielt die Antigenlokalisierung eine wichtige Rolle bei der T-Zellaktivierung. Die vorliegenden Ergebnisse unterstreichen das Potenzial rekombinanter Hefen bei der Entwicklung neuartiger Impfstrategien zur Induktion Antigen-spezifischer T-Zell-Immunantworten

Expression of the HPV-16 L1 capsid protein in methylotrophic yeasts

Papilomavírus humanos (HPVs) são vírus de DNA que infectam células epiteliais, podendo ser responsáveis pelo aparecimento de lesões benignas e malignas. Dentre os mais de 120 tipos identificados, o HPV -16 constitui o principal agente etiológico do câncer cervical, que é uma das maiores causas de morte por câncer em mulheres no mundo. Sendo assim, infecções associadas ao HPV devem ser prevenidas por vacinas indutoras de resposta imune vírus-específicas. A proteína L1 do capsídio viral é capaz de arranjar-se em partículas morfologicamente e antigenicamente semelhantes ao vírus, denominadas \"virus-like particles\" (VLPs), que induzem altos títulos de anticorpos neutralizantes. Neste trabalho, foram clonados os genes L1 selvagem e otimizado de HPV -16 em vetores de expressão de leveduras metilotróficas como Hansenula polymorpha e Pichia pastoris. Foi observada uma expressão consistente da proteína recombinante apenas em P. pastoris, com o gene L1 otimizado. Foram realizadas diversas tentativas de purificação da proteína heteróloga, empregando técnicas de cromatografia e ultracentrifugação em gradiente descontínuo de sacarose. A correta montagem das VLPs foi confirmada por microscopia eletrônica. Problemas de agregação, heterogeneidade e adsorção a superfícies apresentados pela proteína L1 foram resolvidos após utilização de surfactante não-iônico e de um procedimento de desmontagem e remontagem das partículas, gerando preparações mais homogêneas. Ensaios de hemaglutinação e inibição da hemaglutinação comprovaram a apresentação de epítopos conformacionais na superfície das VLPs. Este trabalho demonstrou pela primeira vez a expressão da proteína L1 de HPV -16 em P. pastoris, visando ao desenvolvimento de uma vacina profilática de baixo custo para o sistema público de saúde.Human papillomaviruses (HPVs) are DNA viruses that infect epithelial cells and can cause both benign and malignant lesions. From over 120 types catalogued so far, HPV-16 is the main etiologic agent of cervical cancer, which is the one of the most common causes of cancer deaths among women worldwide. Thus, HPV -associated infections might be prevented by vaccine inducing virus-specific immune responses. The L1 major capsid protein can self assemble into virus-like particles (VLPs), which are morphologically and antigenically indistinguishable from native viruses and induce high titers of neutralizing antibodies. In this work, we have cloned wild-type and codon-optimized L1 genes from HPV-16 in expression vectors of the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris. Consistent L1 expression was only observed in P. pastoris transformed with the construction containing the codon-optimized gene. Many attempts to purify the heterologous protein were made, including chromatography and ultracentrifugation in sucrose density gradients. The correct assembly of VLPs was confirmed by electron microscopy. Some problems presented by recombinant L1 like aggregation, surface adsorption and heterogeneity were solved by using non-ionic surfactants and a procedure of disassembly and reassembly of the particles. Hemagglutination and hemagglutination inhibition assays corroborated the display of surface conformational epitopes by VLPs. This work showed for the first time the expression of the HPV-16 L1 protein in P. pastoris, aiming the development of a prophylactic vaccine free of charge for the public health system in Brazil

TNF-alpha 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 1x10(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-beta impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-alpha 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-alpha 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-alpha and IFN-gamma 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-alpha 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilHosp Sirio Libanes São Paulo, Dept Patol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 04/14518-2FAPESP: 2011/51258-2Web of Scienc

CD8⁺ T cells control the early influx of inflammatory cells to the lungs of iNOS-deficient mice.

<p>Groups (n = 6–7) of WT and iNOS^−/− mice were given 200 µg of anti-CD8α mAbs (H-35) or normal rat IgG (controls) by the i.p. route, 48 and 24 h before infection, and 150 µg of the anti-CD8α mAbs or rat IgG at days 6 and 12 of infection with 1×10⁶<i>P. brasiliensis</i>. The severity of infection was assessed by organ CFU counts (A) at the 2^nd week of infection. The phenotypes of lung infiltrating lymphocytes (B), mononuclear phagocytes (C), and CD11c⁺ dendritic cells isolated by magnetic beads (D) were characterized by flow cytometry. The results are representative of two experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001).</p

iNOS activity controls the influx of activated mononuclear phagocytes and T cells to the lungs.

<p>Flow cytometry characterization of lung infiltrating leucocytes (LIL) from iNOS^−/− and WT mice after i.t. infection with 1×10⁶<i>P. brasiliensis</i> yeast cells. Lungs of iNOS^−/− and WT mice (n = 6–8) were excised, washed in PBS, minced, and digested enzymatically. At weeks 2 (A, C) and 10 (B, D) after infection, lung cell suspensions were obtained and stained as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002325#s2" target="_blank">Materials and Methods</a>. (A, B) Phenotypic characterization of CD11b⁺ mononuclear phagocytes expressing CD80, CD86 and CD40 and dendritic cells expressing high levels of CD11c and CD86 (CD11c^highCD86^high). An increased presence of mononuclear phagocytes was observed in deficient mice, while the number of dendritic cells was equivalent in the lungs of both mouse strains. (C, D) Characterization of T cell subsets by flow cytometry in LIL obtained at weeks 2 (C) and 10 (D) after infection. To characterize the expansion of regulatory T cells in LIL, surface staining of CD25⁺ and intracellular FoxP3 expression were back-gated on the CD4⁺ T cell population. The acquisition and analysis gates were restricted to macrophages (A, B) or lymphocytes (C, D). The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01) and *** (<i>P</i><0.001), compared with WT mice.</p

Neutralization of TNF-α is more detrimental to iNOS^−/− than WT mice.

<p>TNF-α neutralization leads to increased influx of activated cells to the lungs, increased mortality and non-organized lesions in iNOS-deficient mice. WT and iNOS^−/− mice were treated with anti-TNF-α mAb (MP6 XT 22) or normal rat IgG (control) and i.t. infected with 1×10⁶ fungal cells. Number and activation of lymphocytes (A) and mononuclear phagocytes (B) present in pulmonary lesions at the 2^nd week postinfection of anti-TNF-α treated and untreated mice were determined by flow cytometry. (C) Survival times of anti-TNF-α treated and untreated mice. TNF-α depleted iNOS^−/− mice showed a decreased survival time, which was significantly different (* <i>P</i><0.05) from all other studied groups. The results are representative of three independent experiments (n = 6–7). (D) Photomicrographs of pulmonary lesions developed by IgG-treated (D, superior panels) and TNF-α-depleted (B, lower panels) mice at the week 8 of infection. Anti-TNF-α treatment of iNOS-deficient mice resulted in less organized, fungi rich, confluent lesions, occupying the largest part of lung tissue. H&E stained lesions (100×).</p

Fungal loads and fungicidal activity of alveolar macrophages.

<p>Early in infection (week 2), bronchoalveolar lavage fluids (BALFs) of iNOS^−/− mice have a lower fungal load, but alveolar macrophages are refractory to IFN-γ activation. (A) Number of viable yeasts (CFU counts) recovered from BALFs of iNOS^−/− and wild type (WT) C57BL/6 mice, 2 weeks after <i>P. brasiliensis</i> infection with 1×10⁶ yeast cells. (B and D) Viable yeasts recovered from adherent alveolar macrophages obtained at weeks 2 and 10 after infection, respectively, stimulated or not by IFN-γ (100 U/ml) and cultivated <i>in vitro</i> for 48 h. (C and E) Levels of NO in the supernatants of alveolar macrophages. The bars depict means ± SEM (6–8 animals per group) and are representative of three experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with WT controls.</p

Neutralization of TNF-α does not alter fungal loads and has a minor effect on organ cytokines.

<p>WT and iNOS^−/− mice (n = 6–7) were treated with anti-TNF-α mAb (MP6 XT 22, i.p. injections of 0.25 mg/0.5 mL) 4 h before, and at days 6 and 12 of infection). Normal rat IgG was used as control. CFU counts were determined at weeks 1, 2 and 8 postinfection with 1×10⁶<i>P.brasiliensis</i> yeasts. (A, C, and D, respectively). Levels of pulmonary (B, E) and hepatic (F) cytokines were assessed in organ homogenates obtained at the 1^st (B) or 2^nd (E, F) weeks postinfection. The data represent the mean ± SEM of the results from 6–7 mice per group and are representative of two independent experiments. * (<i>P</i><0.05) and ** (<i>P</i><0.01).</p