Protection against experimental cryptococcosis elicited by Cationic Adjuvant Formulation 01-adjuvanted subunit vaccines.

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.

Immunological correlates of protection mediated by a whole organism, Cryptococcus neoformans, vaccine deficient in chitosan.

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.

Chitosan-Deficient Cryptococcus as Whole-Cell Vaccines.

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.

Vaccine Strategies for Cryptococcus neoformans.

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.

Semisynthetic Glycoconjugate Vaccine Candidates against Cryptococcus neoformans.

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.

Design of a cryptococcus neoformans vaccine by subtractive proteomics combined with immunoinformatics.

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.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Introns in Cryptococcus

In Cryptococcus neoformans, nearly all genes are interrupted by small introns. In recent years, genome annotation and genetic analysis have illuminated the major roles these introns play in the biology of this pathogenic yeast. Introns are necessary for gene expression and alternative splicing can regulate gene expression in response to environmental cues. In addition, recent studies have revealed that C. neoformans introns help to prevent transposon dissemination and protect genome integrity. These characteristics of cryptococcal introns are probably not unique to Cryptococcus, and this yeast likely can be considered as a model for intron-related studies in fungi.

The status of cryptococcosis in Latin America

Cryptococcosis is a life-threatening fungal infection caused by the encapsulated yeasts Cryptococcus neoformans and C. gattii, acquired from the environment. In Latin America, as occurring worldwide, C. neoformans causes more than 90% of the cases of cryptococcosis, affecting predominantly patients with HIV, while C. gattii generally affects otherwise healthy individuals. In this region, cryptococcal meningitis is the most common presentation, with amphotericin B and fluconazole being the antifungal drugs of choice. Avian droppings are the predominant environmental reservoir of C. neoformans, while C. gattii is associated with several arboreal species. Importantly, C. gattii has a high prevalence in Latin America and has been proposed to be the likely origin of some C. gattii populations in North America. Thus, in the recent years, significant progress has been made with the study of the basic biology and laboratory identification of cryptococcal strains, in understanding their ecology, population genetics, host-pathogen interactions, and the clinical epidemiology of this important mycosis in Latin America.

Metodologia científica na formação do médico veterinário: experiênciateórico-prática na análise de fontes urbanas de Cryptococcus spp – Jaboticabal, SP – Brasil / Scientific methodology in veterinary medical training: a theoretical andpractical experience in the analysis of urban sources of Cryptococcus spp in Jaboticabal, SP – Brazil / Metodología científica en la formación médica veterinaria: la experienciateórica y práctica en el análisis de fuentes urbanas de Cryptococcus spp de Jaboticabal, SP – Brasil

O artigo apresenta resultados de um projeto de ensino de Metodologia Científica em Curso de Graduação deMedicina Veterinária. Pautou-se por uma reflexão crítica da necessidade de interação entre as dimensões teórica eprática na formação do profissional e realizou-se análise de fontes urbanas de Cryptococcus spp em fezes de pombos(Columbia livia), evidenciando a importância de orientação à população sobre os riscos de contágio, caracterizando a patogenicidade dos agentes identificados em dez ambientes públicos.

This article presents the results of an educational project for the Scientific Methodology degree course for veterinary medicine. The project was guided by the critical reflection on the necessity for the interaction between the theoretical and practical dimensions of professional training. An analysis was performed of urban sources of Cryptococcus spp in excrements of pigeons (Columbia livia), indicating the need for education of the population about the risks factors of transmission and characterizing the pathogenicity of the identified agents in ten public places.

El artículo presenta los resultados de un proyecto de enseñanza de Metodología Científica en el Curso de Graduaciónde Medicina Veterinaria. Se marcó por una reflexión crítica da la necesidad de la interacción entre las dimensionesteóricas y prácticas en la formación del profesional y se realizó un análisis de fuentes urbanas de Cryptococcus sppen excremento de palomas (Columbia livia), mostrando la importancia de la orientación a la población sobre losriesgos del contagio, caracterizando la patogenicidad de los agentes identificados en diez ambientes públicos.

Rapid advice: diagnosis, prevention and management of cryptococcal disease in HIV-infected adults, adolescents and children

Increasing access to antiretroviral therapy (ART) has transformed the prognosis of HIVinfected patients in resource-limited settings (RLS). However, treatment coverage remains relatively low, and HIV diagnosis occurs at a late stage. As a result, many patients continue to die of HIV-related opportunistic infections (OIs) in the weeks prior to, and months following initiation of ART. Cryptococcal disease is one of the most important OIs, and a major contributor to this early mortality, accounting for between 13% and 44% of deaths in HIV-infected cohorts in resource-limited countries. In subSaharan Africa alone, there are more than 500,000 deaths each year due to cryptococcal meningitis (CM), which may exceed those attributed to tuberculosis. This guideline will provide recommendations on the prevention, diagnosis and management of cryptococcal disease in HIV-infected adults, adolescents (10-19 years) and children (up to 10 years), with a focus on settings with limited health systems capacity and resources, and a high burden of cryptococcal disease, to outline standards for high quality care of persons living with HIV infection (PLHIV) and patients with cryptococcal disease, by providing evidence-based recommendations that consider the risks and benefits, acceptability, feasibility, cost and other resource implications and to identify gaps and prioritize areas where further clinical and operational research are required.

Interrupción exitosa de la profilaxis secundaria antifúngica en la criptococosis asociada al sida / Successful discontinuation of antifungal secondary prophylaxis in AIDS-related cryptococcosis

Se presentan los datos clínicos de 22 pacientes con criptococosis asociada al VIH que interrumpieron la profilaxis antifúngica secundaria, después de haber recibido la terapéutica antirretroviral de gran actividad (TARGA). Fueron 14 varones y 8 mujeres con edades comprendidas entre los 15 y los 50 años (X: 34 años). Todos presentaron un síndrome infeccioso general grave y 19 tuvieron meningoencefalitis. En el momento del diagnóstico 59% de los enfermos tenía recuentos de células CD4+ < 50/µL,la mediana de lascargas viralesfue de 134. 804 copias ARN/ml yla mediana de los títulosde antigenemiafue de 1/3.000. El tratamiento del episodio agudo se realizó con anfotericina B por vía venosa (0,7 mg/kg/día) o fluconazol(600 a 800 mg/día), hasta la negativización de los cultivos de LCR. La profilaxis secundaria consistió en la administración oral de 200 mg diarios de fluconazoló 2 dosis semanalesde 50 mg de anfotericina B. La profilaxis secundaria antifúngica fue interrumpida cuando los enfermos habían recibido la TARGA por un lapso medio 19 meses, la mediana de los recuentos de células CD4+ fue de 249/µl. Todos estaban asintomáticos y en buen esta-do general. El lapso medio de seguimiento posterior fue de 22 meses y ningún enfermo experimentó recidivas desu micosis.

The clinical and laboratory data of 22 patients with AIDS related cryptococcosis who were able to interrupt antifungal secondary prophylaxis afterHAART administration, are presented. They were 14 males and 8 females, between 15 and 50years old (X: 34 years old). All patients presented fever andsevere deterioration of their general health status, and 19 exhibited a meningeal syndrome. At the start of antifungal treatment, 59% of the cases presented < 50 CD4+ cells/µl, the median viral burden was 134,804 RNA copies/ml and the median titer ofserum cryptococcal antigen was 1/3,000. Amphotericin B by intravenous route, (0.7 mg/kg/day) or fluconazole (600 to 800 mg/day) were given as a treatment of the initial episode, up to CSF cultures negativization. Oral fluconazole (200 mg/day) or intravenous amphotericin B, 50 mg twice a week, were given as a secondary prophylaxis. The secondary prophylaxis was interrupted when the patients had received HAART for an average lapse of 19 months (6 to 36 months) and the medianCD4+ cells counts was 249/µl. The follow up after secondary prophylaxis discontinuation lasted for a median lapse of 22 months. These data seem to show that secondary prophylaxisis not necessary when the patient are clinically asymptomatic and the CD4+ cells counts are above 150/µl.

Diferenças clínicas, epidemiológicas e ecológicas entre as duas variedades de Cryptococcus Neoformans / Clinical, epidemiologic and ecologic differences of two different types of Cryptococcus Neoformans

Existem diferenças variedade-específicas na infecção por Cryptococcus neoformans. C. neoformans var. neoformans é cosmopolita e tem nicho ecológico urbano, habitando solo rico em fezes de aves e ocorre no paciente imunodeprimido (patógeno oportunista). C. neoformans var. gattii de nicho ecológico rural, é sazonal, relacionado à floração do eucalipto, com propensão a causar doença no hospedeiro hígido (patógeno primário). Ambos apresentam tropismo para o sistema nervoso central; a variedade neoformans frequentemente é isolada do sangue e da urina e a variedade gattii usualmente ocasiona consolidações pulmonares, sendo detectado no escarro. O tratamento antifúngico é mais prolongado na variedade gattii, frequentemente necessitando cirurgia (pulmonar ou cerebral) mas como regra evolui par a cura. A mortalidade entre os pacientes infectados com C. neoformans var. neoformans é alta