RESUMO
The literature holds few descriptions on immune response findings for laryngeal cryptococcosis. Immunology has been more extensively described in cases involving the central nervous system and the lungs, although many of these studies were conducted in animal models. We aimed to analyze the clinical and immunological characteristics of three patients with laryngeal cryptococcosis. We observed a weak participation of the innate immune response, whereas adaptive immunity showed the predominance of a Th2-type response over a Th1-type response. Most cases occur in male older adults with immunosuppressive conditions, of which HIV infection was absent. Hoarseness configured the main symptom. We found a disease that was restricted to the larynx and possibly the lungs by contiguity. Patients with hoarseness and lesions in nasal endoscopy should be investigated for cryptococcosis by a biopsy of the larynx, including with negative serum cryptococcal antigen. The immunological aspects of our findings of laryngeal involvement resembled those in the most commonly affected systems.
Assuntos
Criptococose , Doenças da Laringe , Humanos , Criptococose/imunologia , Doenças da Laringe/imunologia , Doenças da Laringe/microbiologiaRESUMO
The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4+ T cells which produced interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8+ T cells were also found, albeit in lesser numbers. A significant, durable IFNγ response was observed in the lungs, spleen, and blood. Moreover, IFNγ secretion following ex vivo stimulation directly correlated with fungal control in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials.
Assuntos
Adjuvantes Imunológicos , Criptococose , Cryptococcus neoformans , Vacinas Fúngicas , Vacinas de Subunidades Antigênicas , Criptococose/imunologia , Criptococose/prevenção & controle , Animais , Camundongos , Vacinas Fúngicas/imunologia , Vacinas Fúngicas/administração & dosagem , Cryptococcus neoformans/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Feminino , Camundongos Endogâmicos C57BL , Adjuvantes de Vacinas/administração & dosagem , Antígenos de Fungos/imunologia , Modelos Animais de DoençasRESUMO
BACKGROUND: Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. METHODS: We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). RESULTS: We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. CONCLUSIONS: We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.
Assuntos
Anticorpos Neutralizantes , Autoanticorpos , Criptococose , Cryptococcus gattii , Cryptococcus neoformans , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Masculino , Colômbia , Feminino , Adulto , Cryptococcus gattii/imunologia , Pessoa de Meia-Idade , Cryptococcus neoformans/imunologia , Criptococose/imunologia , Criptococose/diagnóstico , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Estudos Retrospectivos , Soronegatividade para HIV/imunologia , Adulto Jovem , IdosoRESUMO
The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4+ T-cell counts. Previously, we deleted three chitin deacetylase genes from Cryptococcus neoformans to create a chitosan-deficient, avirulent strain, designated as cda1∆2∆3∆, which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8+ T cells. In contrast, protection was lost in mice lacking α/ß T cells or CD4+ T cells. Moreover, CD4+ T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4+ T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4+ T cells after vaccination but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in interferon-γ (IFNγ), tumor necrosis factor alpha (TNFα), or interleukin (IL)-23p19. A robust influx of leukocytes and IFNγ- and TNFα-expressing CD4+ T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFNγ production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8+ T cells are dispensable, IFNγ and CD4+ T cells have overlapping roles in generating protective immunity prior to cda1∆2∆3∆ vaccination. However, once vaccinated, protection becomes less dependent on CD4+ T cells, suggesting a strategy for vaccinating HIV+ persons prior to loss of CD4+ T cells. IMPORTANCE: The fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4+ T-cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans, designated as cda1∆2∆3∆. When used as a vaccine, cda1∆2∆3∆ protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8+ T cells were dispensible, protection was lost in mice genetically deficient in CD4+ T cells and the cytokines IFNγ, TNFα, or IL-23. A robust influx of cytokine-producing CD4+ T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4+ T cells following vaccination, suggesting a strategy to protect persons who are at risk of future CD4+ T-cell dysfunction.
Assuntos
Linfócitos T CD4-Positivos , Quitosana , Criptococose , Cryptococcus neoformans , Vacinas Fúngicas , Animais , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/genética , Criptococose/imunologia , Criptococose/prevenção & controle , Criptococose/microbiologia , Vacinas Fúngicas/imunologia , Vacinas Fúngicas/administração & dosagem , Vacinas Fúngicas/genética , Quitosana/imunologia , Camundongos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Camundongos Endogâmicos C57BL , Interferon gama/imunologia , Interferon gama/metabolismo , FemininoRESUMO
Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.
Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Glucosiltransferases , Pulmão , Fatores de Virulência , Animais , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/imunologia , Criptococose/microbiologia , Criptococose/imunologia , Camundongos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Interações Hospedeiro-Patógeno , Encéfalo/microbiologia , Baço/microbiologia , Feminino , Camundongos Endogâmicos C57BL , Imunidade Inata , Evasão da Resposta Imune , Deleção de GenesRESUMO
Cryptococcus deneoformans is a yeast-type fungus that causes fatal meningoencephalitis in immunocompromised patients and evades phagocytic cell elimination through an escape mechanism. Memory T (Tm) cells play a central role in preventing the reactivation of this fungal pathogen. Among these cells, tissue-resident memory T (TRM) cells quickly respond to locally invaded pathogens. This study analyzes the kinetics of effector T (Teff) cells and Tm cells in the lungs after cryptococcal infection. Emphasis is placed on the kinetics and cytokine expression of TRM cells in the early phase of infection. CD4+ Tm cells exhibited a rapid increase by day 3, peaked at day 7, and then either maintained their levels or exhibited a slight decrease until day 56. In contrast, CD8+ Tm cells reached their peak on day 3 and thereafter decreased up to day 56 post-infection. These Tm cells were predominantly composed of CD69+ TRM cells and CD69+ CD103+ TRM cells. Disruption of the CARD9 gene resulted in reduced accumulation of these TRM cells and diminished interferon (IFN) -γ expression in TRM cells. TRM cells were derived from T cells with T cell receptors non-specific to ovalbumin in OT-II mice during cryptococcal infection. In addition, TRM cells exhibited varied behavior in different tissues. These results underscore the importance of T cells, which produce IFN-γ in the lungs during the early stage of infection, in providing early protection against cryptococcal infection through CARD9 signaling.
Assuntos
Antígenos CD , Antígenos de Diferenciação de Linfócitos T , Criptococose , Cryptococcus , Interferon gama , Lectinas Tipo C , Pulmão , Animais , Criptococose/imunologia , Criptococose/microbiologia , Interferon gama/metabolismo , Interferon gama/imunologia , Camundongos , Antígenos de Diferenciação de Linfócitos T/metabolismo , Cryptococcus/imunologia , Antígenos CD/metabolismo , Antígenos CD/genética , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Pulmão/imunologia , Pulmão/microbiologia , Células T de Memória/imunologia , Células T de Memória/metabolismo , Camundongos Endogâmicos C57BL , Memória Imunológica , Imunidade Inata , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologiaAssuntos
Cryptococcus , Meningite Criptocócica , Humanos , Antifúngicos/uso terapêutico , Criptococose/diagnóstico , Criptococose/epidemiologia , Criptococose/imunologia , Criptococose/terapia , Cryptococcus/imunologia , Cryptococcus/patogenicidade , Hospedeiro Imunocomprometido , Meningite Criptocócica/diagnóstico , Meningite Criptocócica/epidemiologia , Meningite Criptocócica/imunologia , Meningite Criptocócica/terapia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fatores de Risco , Imunossupressores/efeitos adversos , Antígenos de Fungos/sangue , Antígenos de Fungos/líquido cefalorraquidiano , Hipertensão Intracraniana/etiologia , Hipertensão Intracraniana/terapia , Síndrome Inflamatória da Reconstituição Imune/etiologiaRESUMO
Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.
Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Animais , Criptococose/microbiologia , Criptococose/tratamento farmacológico , Criptococose/imunologia , Camundongos , Cryptococcus neoformans/patogenicidade , Cryptococcus gattii/patogenicidade , Antifúngicos/farmacologia , Antifúngicos/uso terapêuticoRESUMO
Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen found ubiquitously in the environment that causes pneumonia and life-threatening infections of the central nervous system. Following inhalation of yeasts or desiccated basidiospores into the lung alveoli, resident pulmonary phagocytic cells aid in the identification and eradication of Cryptococcus yeast through their arsenal of pattern recognition receptors (PRRs). PRRs recognize conserved pathogen-associated molecular patterns (PAMPs), such as branched mannans, ß-glucans, and chitins that are the major components of the fungal cell wall. However, the key receptors/ligand interactions required for cryptococcal recognition and eventual fungal clearance have yet to be elucidated. Here we present an imaging flow cytometer (IFC) method that offers a novel quantitative cellular imaging and population statistics tool to accurately measure phagocytosis of fungal cells. It has the capacity to measure two distinct steps of phagocytosis: association/attachment and internalization in a high-throughput and quantitative manner that is difficult to achieve with other technologies. Results from these IFC studies allow for the potential to identify PRRs required for recognition, uptake, and subsequent activation of cytokine production, as well as other effector cell responses required for fungal clearance.
Assuntos
Cryptococcus neoformans , Citometria de Fluxo , Fagocitose , Citometria de Fluxo/métodos , Cryptococcus neoformans/metabolismo , Animais , Camundongos , Fagócitos/metabolismo , Fagócitos/microbiologia , Criptococose/microbiologia , Criptococose/metabolismo , Criptococose/imunologia , Cryptococcus/metabolismo , Humanos , Citometria por Imagem/métodos , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
The ability of C. neoformans to survive and replicate within host phagocytes enables it to evade the immune system and allows for persistence of the infection. As such, measuring fungal burden of C. neoformans strains-and indeed how drug treatments can influence fungal burden-provides important information about C. neoformans pathogenesis. In this chapter, we describe two methods that may be used to appraise fungal burden: a standard end-point colony-formation assay for calculating the average number of yeast per host cell and a fluorescence microscopy-based method that may be used to measure changes in fungal burden in individual living macrophages in real time.
Assuntos
Criptococose , Cryptococcus neoformans , Macrófagos , Microscopia de Fluorescência , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Criptococose/microbiologia , Criptococose/imunologia , Microscopia de Fluorescência/métodos , Animais , Camundongos , Contagem de Colônia Microbiana/métodos , HumanosRESUMO
One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.
Assuntos
Criptococose , Cryptococcus neoformans , Ensaio de Imunoadsorção Enzimática , Polissacarídeos , Ensaio de Imunoadsorção Enzimática/métodos , Cryptococcus neoformans/imunologia , Criptococose/diagnóstico , Criptococose/microbiologia , Criptococose/imunologia , Polissacarídeos/análise , Polissacarídeos/imunologia , Humanos , Antígenos de Fungos/imunologia , Antígenos de Fungos/análise , Polissacarídeos Fúngicos/imunologia , Polissacarídeos Fúngicos/análise , Anticorpos Monoclonais/imunologia , Anticorpos Antifúngicos/imunologiaRESUMO
Monocyte/macrophage cells play a central role in innate immunity against C. neoformans and C. gattii, species known to cause human disease. Cryptococcus is the only fungal genus known to possess such a large extracellular polysaccharide capsule, which impacts interactions of innate cells with the yeast. This interaction results in different fates, such as phagocytosis and intracellular proliferation and, as the interaction progresses, vomocytosis, cell-to-cell transfer, lysis of macrophages, or yeast killing. Differentiating internalized versus external Cryptococcus cells is thus essential to evaluate monocyte-macrophage phagocytosis. We describe here a protocol that allows quantification of Cryptococcus spp. phagocytosis using quantitative flow cytometry in human monocytes and a murine macrophage cell line (J774).
Assuntos
Cryptococcus neoformans , Citometria de Fluxo , Macrófagos , Monócitos , Fagocitose , Cryptococcus neoformans/imunologia , Animais , Camundongos , Humanos , Monócitos/imunologia , Monócitos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Citometria de Fluxo/métodos , Linhagem Celular , Criptococose/imunologia , Criptococose/microbiologiaRESUMO
The interaction between macrophages and Cryptococcus neoformans is crucial in the pathogenesis of cryptococcosis. These phagocytes are important immune effectors, but also a niche in which facultative intracellular parasites, such as C. neoformans, thrive. Consequently, phagocytosis of cryptococcal cells and its outcomes are very frequently studied. One major issue with several of the tests used for this, however, is that macrophage-C. neoformans interaction does not always result in phagocytosis, as fungi may be attached to the external surface of the phagocyte. The most used methodologies to study phagocytosis of cryptococcal cells have varying degrees of precision in separating fungi that are truly internalized from those that are outside macrophages. Here we describe two assays to measure phagocytosis that can differentiate internal from external C. neoformans cells.
Assuntos
Criptococose , Cryptococcus neoformans , Macrófagos , Fagocitose , Cryptococcus neoformans/imunologia , Macrófagos/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Criptococose/microbiologia , Criptococose/imunologia , Animais , Camundongos , Humanos , Interações Hospedeiro-Patógeno/imunologiaRESUMO
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.
Assuntos
Anticorpos Antifúngicos , Anticorpos Monoclonais , Cryptococcus neoformans , Hibridomas , Cryptococcus neoformans/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Animais , Humanos , Hibridomas/imunologia , Anticorpos Antifúngicos/imunologia , Anticorpos Antifúngicos/isolamento & purificação , Camundongos , Linfócitos B/imunologia , Criptococose/imunologia , Criptococose/diagnóstico , Antígenos de Fungos/imunologia , ImunizaçãoRESUMO
Creating a safe and effective vaccine against infection by the fungal pathogen Cryptococcus neoformans is an appealing option that complements the discovery of new small molecule antifungals. Recent animal studies have yielded promising results for a variety of vaccines that include live-attenuated and heat-killed whole-cell vaccines, as well as subunit vaccines formulated around recombinant proteins. Some of the recombinantly engineered cryptococcal mutants in the chitosan biosynthesis pathway are avirulent and very effective at conferring protective immunity. Mice vaccinated with these avirulent chitosan-deficient strains are protected from a lethal pulmonary infection with C. neoformans strain KN99. Heat-killed derivatives of the vaccination strains are likewise effective in a murine model of infection. The efficacy of these whole-cell vaccines, however, is dependent on a number of factors, including the inoculation dose, route of vaccination, frequency of vaccination, and the specific mouse strain used in the study. Here, we present detailed methods for identifying and optimizing various factors influencing vaccine potency and efficacy in various inbred mouse strains using a chitosan-deficient cda1Δcda2Δcda3Δ strain as a whole-cell vaccine candidate. This chapter describes the protocols for immunizing three different laboratory mouse strains with vaccination regimens that use intranasal, orotracheal, and subcutaneous vaccination routes after the animals were sedated using two different types of anesthesia.
Assuntos
Quitosana , Criptococose , Cryptococcus neoformans , Vacinas Fúngicas , Animais , Quitosana/química , Camundongos , Vacinas Fúngicas/imunologia , Vacinas Fúngicas/genética , Vacinas Fúngicas/administração & dosagem , Criptococose/imunologia , Criptococose/prevenção & controle , Criptococose/microbiologia , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/genética , Modelos Animais de Doenças , Vacinação/métodos , Feminino , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genéticaRESUMO
Extracellular vesicles (EVs) are produced by all domains of life. In fungal pathogens, they participate in virulence mechanisms and/or induce protective immunity, depending on the pathogenic species. EVs produced by pathogenic members of the Cryptococcus genus mediate virulence, antifungal resistance, as well as humoral and cell-mediated immunity. The isolation of cryptococcal EVs has been laborious and time-consuming for years. In this chapter, we detail a fast protocol for the isolation and analysis of EVs produced by members of the Cryptococcus genus.
Assuntos
Cryptococcus , Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Criptococose/microbiologia , Criptococose/imunologia , HumanosRESUMO
Cryptococcus neoformans infections are a major worldwide concern as current treatment strategies are becoming less effective in alleviating the infection. The most extreme and fatal cases are those of immunocompromised individuals. Clinical treatments for cryptococcosis are limited to a few classes of approved drugs, and due to a rise in drug resistance, these drugs are becoming less effective. Therefore, it is essential to develop innovative ways to control this infection. Vaccinations have emerged as a safe, viable, and cost-effective solution to treat a number of diseases over the years. Currently, there are no clinically available vaccines to treat cryptococcal infections, but a number of studies have shown promising results in animal models. Here, we present step-by-step experimental protocols using live-attenuated or heat-killed C. neoformans cells as a vaccination strategy in a preventive or in a therapeutic murine model of cryptococcosis.
Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Vacinas Fúngicas , Cryptococcus neoformans/imunologia , Criptococose/imunologia , Criptococose/prevenção & controle , Animais , Vacinas Fúngicas/imunologia , Camundongos , Vacinação/métodos , Vacinas Atenuadas/imunologia , HumanosRESUMO
Objectives: This manuscript undertakes a systematic examination of the research landscape concerning global Cryptococcus species and their dynamism with the host immune system spanning the past decade. It furnishes a detailed survey of leading knowledge institutions and critical focal points in this area, utilizing bibliometric analysis. Methods: VOSviewer and CiteSpace software platforms were employed to systematically analyze and graphically depict the relevant literature indexed in the WoSCC database over the preceding ten years. Results: In the interval between October 1, 2013, and October 1, 2023, a corpus of 795 publications was amassed. The primary research institutions involved in this study include Duke University, the University of Minnesota, and the University of Sydney. The leading trio of nations, in terms of publication volume, comprises the United States, China, and Brazil. Among the most prolific authors are Casadevall, Arturo; Wormley, Floyd L., Jr.; and Olszewski, Michal A., with the most highly cited author being Perfect, Jr. The most esteemed journal is Mbio, while Infection and Immunity commands the highest citation frequency, and the Journal of Clinical Microbiology boasts the most significant impact factor. Present research foci encompass the intricate interactions between Cryptococcus pathogenesis and host immunity, alongside immune mechanisms, complications, and immunotherapies. Conclusion: This represents the first exhaustive scholarly review and bibliometric scrutiny of the evolving landscapes in Cryptococcus research and its interactions with the host immune system. The analyses delineated herein provide insights into prevailing research foci and trajectories, thus furnishing critical directions for subsequent inquiries in this domain.
Assuntos
Bibliometria , Criptococose , Cryptococcus , Animais , Humanos , Criptococose/imunologia , Cryptococcus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Sistema Imunitário/imunologiaRESUMO
Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, which poses a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semisynthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semisynthetic glycoconjugate vaccines contain an identical synthetic decasaccharide (M2 motif) antigen. This antigen is present in serotype A strains, which constitute 95% of the clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity toward M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced weakly opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). These findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. This antigen could serve as a component in a multivalent GXM motif vaccine.
Assuntos
Anticorpos Antifúngicos , Criptococose , Cryptococcus neoformans , Vacinas Fúngicas , Glicoconjugados , Vacinas Conjugadas , Cryptococcus neoformans/imunologia , Animais , Vacinas Fúngicas/imunologia , Camundongos , Criptococose/prevenção & controle , Criptococose/imunologia , Glicoconjugados/imunologia , Glicoconjugados/química , Vacinas Conjugadas/imunologia , Anticorpos Antifúngicos/imunologia , Feminino , Polissacarídeos/imunologia , Polissacarídeos/química , Camundongos Endogâmicos BALB C , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/química , Antígenos de Fungos/imunologiaRESUMO
The emergence of Cryptococcus neoformans has posed an undeniable burden to many regions worldwide, with its strains mainly entering the lungs through the respiratory tract and spreading throughout the body. Limitations of drug regimens, such as high costs and limited options, have directed our attention toward the promising field of vaccine development. In this study, the subtractive proteomics approach was employed to select target proteins from databases that can accurately cover serotypes A and D of the Cryptococcus neoformans. Further, two multi-epitope vaccines consisting of T and B cell epitopes were demonstrated that they have good structural stability and could bind with immune receptor to induce desired immune responses in silico. After further evaluation, these vaccines show the potential for large-scale production and applicability to the majority of the population of the world. In summary, these two vaccines have been theoretically proven to combat Cryptococcus neoformans infections, awaiting further experimental validation of their actual protective effects.