Antibody Isolation in C. neoformans.

The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.

Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

A hidden battle in the dirt: Soil amoebae interactions with Paracoccidioides spp.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.

Agglutination of Histoplasma capsulatum by IgG monoclonal antibodies against Hsp60 impacts macrophage effector functions.

Histoplasma capsulatum can efficiently survive within macrophages, facilitating H. capsulatum translocation from the lung into the lymphatics and bloodstream. We have recently generated monoclonal antibodies (MAbs) to an H. capsulatum surface-expressed heat shock protein of 60 kDa (Hsp60) that modify disease in a murine histoplasmosis model. Interestingly, the MAbs induced different degrees of yeast cell agglutination in vitro. In the present study, we characterized the agglutination effects of the antibodies to Hsp60 on H. capsulatum yeast cells by light microscopy, flow cytometry, dynamic light scattering, measuring zeta potential, and using optical tweezers. We found that immunoglobulin Gs (IgGs) to Hsp60 cause H. capsulatum aggregation dependent on the (i) concentration of MAbs, (ii) MAb binding constant, and (iii) IgG subclass. Furthermore, infection of macrophages using agglutinates of various sizes after incubation with different Hsp60-binding MAbs induced association to macrophages through distinct cellular receptors and differentially affected macrophage antifungal functions. Hence, the capacity of IgG MAbs to agglutinate H. capsulatum significantly impacted pathogenic mechanisms of H. capsulatum during macrophage infection, and the effect was dependent on the antibody subclass and antigen epitope.

Diagnosis of histoplasmosis

As micoses endêmicas podem ser um desafio com relação ao diagnóstico e a interpretação acurada dos dados laboratoriais é importante para garantir um tratamento mais apropriado para os pacientes. Embora o diagnóstico definitivo da histoplasmose (HP), uma das micoses endêmicas no mundo, seja determinado pelo exame direto com a observação de micro e macroconídeos do Histoplasma capsulatum (H. capsulatum), evidências sorológicas da infecção fúngica se torna importante uma vez que o isolamento dos agentes etiológicos é um procedimento demorado e com baixa sensibilidade. Uma variedade de imunoensaios tem sido usada para detectar anticorpos específicos contra o H. capsulatum. A técnica mais aplicada para a detecção de anticorpos é a imunodifusão, apresentando uma sensibilidade entre 70 a 100 por cento e especificidade de dependendo da forma clínica. A fixação de complemento (CF), uma metodologia que foi extensivamente usada no passado, apresenta uma menor especificidade (60 to 90 por cento). A detecção de antígenos fúngicos pelos imunoensaios é valiosa para indivíduos imunocomprometidos onde cada ensaio garante valores preditivos positivos variando de 96-98 por cento. A maioria dos testes atual ainda utiliza antígenos complexos não-purificados obtidos de parede celular fúngica ou filtrados de cultura. Contudo, importância tem sido dada a imunoensaios clínicos utilizando antígenos altamente purificados e caracterizados, incluindo antígenos recombinantes. Neste manuscrito, nós revisamos as ferramentas atuais utilizadas no diagnóstico, como fixação de complemento e imunodifusão, sumarizando o desenvolvimento de novas tecnologias, reagentes e métodos, e discutiremos aqui os seus méritos relativos e desvantagens no imunodiagnóstico desta micose.

Proteína M recombinante do histoplasma capsulatum: mapeamento de epítopos e aplicação no diagnóstico da histoplasmose / Protein M recombinant histoplasma capsulatum: epitope mapping and application in the diagnosis of histoplasmosis

A histoplasmose é causada pelo fungo dimórfico Histoplasma capsulatum. A infecção por este organismo é frequentemente diagnosticada pela determinação de anticorpos para a proteína M, imunodominante, mas o papel deste antígeno na patogênese da histoplasmose ainda permanece obscuro. A função do antígeno M não é conhecida, mas geneticamente é homóloga a catalases fúngicas. Em nosso estudo, nós procuramos determinar regiões imunodominantes do antígeno M, produzir um painel de anticorpos monoclonais contra esta proteína, caracterizá-los, usá-los para o mapeamento de epítopos, mostrar a localização do antígeno M na levedura do H. capsulatum, demonstrar a atividade de catalase desta proteína e desenvolver imunoensaios para a detecção de anticorpos contra esta proteína e antígeno M circulante nos fluidos corporais. Foi possível determinar peptídeos com maior atividade antigênica e suas localizações na molécula e caracterizá-los de acordo com as suas propriedades bioquímicas. Nós geramos três fragmentos que continham estes peptídeos e os utilizamos para avaliar a ligação dos anticorpos monoclonais e reconhecimento a cada fragmento separadamente. Um dos fragmentos, F3, mostrou maior ligação aos anticorpos monoclonais que os outros avaliados, entretanto estudos adicionais devem ser avaliados para um complete mapeamento. Para avaliar o papel do antígeno M na patogênese da histoplasmose, usando análise por imunoblot de um extrato de parede celular e membrana (CW/M) obtido de levedura, provamos que os mAbs gerados contra o antígeno M recombinante puderam reconhecer este antígeno no extrato utilizado...