scholarly journals Protection against Experimental Cryptococcosis following Vaccination with Glucan Particles Containing Cryptococcus Alkaline Extracts

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Charles A. Specht ◽  
Chrono K. Lee ◽  
Haibin Huang ◽  
Donald J. Tipper ◽  
Zu T. Shen ◽  
...  
Keyword(s):  
At Risk ◽  
T Cell ◽  
Cryptococcus Neoformans ◽  
Delivery System ◽  
Cell Walls ◽  
Vaccine Development ◽  
Cryptococcus Gattii ◽  
Content Type ◽  
Alkaline Extract ◽  
Virulent Strains

ABSTRACTA vaccine capable of protecting at-risk persons against infections due toCryptococcus neoformansandCryptococcus gattiicould reduce the substantial global burden of human cryptococcosis. Vaccine development has been hampered though, by lack of knowledge as to which antigens are immunoprotective and the need for an effective vaccine delivery system. We made alkaline extracts from mutant cryptococcal strains that lacked capsule or chitosan. The extracts were then packaged into glucan particles (GPs), which are purifiedSaccharomyces cerevisiaecell walls composed primarily of β-1,3-glucans. Subcutaneous vaccination with the GP-based vaccines provided significant protection against subsequent pulmonary infection with highly virulent strains ofC. neoformansandC. gattii. The alkaline extract derived from the acapsular strain was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the most abundant proteins were identified. Separation of the alkaline extract by size exclusion chromatography revealed fractions that conferred protection when loaded in GP-based vaccines. Robust Th1- and Th17-biased CD4+T cell recall responses were observed in the lungs of vaccinated and infected mice. Thus, our preclinical studies have indicated promising cryptococcal vaccine candidates in alkaline extracts delivered in GPs. Ongoing studies are directed at identifying the individual components of the extracts that confer protection and thus would be promising candidates for a human vaccine.IMPORTANCEThe encapsulated yeastCryptococcus neoformansand its closely related sister species,Cryptococcus gattii, are major causes of morbidity and mortality, particularly in immunocompromised persons. This study reports on the preclinical development of vaccines to protect at-risk populations from cryptococcosis. Antigens were extracted fromCryptococcusby treatment with an alkaline solution. The extracted antigens were then packaged into glucan particles, which are hollow yeast cell walls composed mainly of β-glucans. The glucan particle-based vaccines elicited robust T cell immune responses and protected mice from otherwise-lethal challenge with virulent strains ofC. neoformansandC. gattii. The technology used for antigen extraction and subsequent loading into the glucan particle delivery system is relatively simple and can be applied to vaccine development against other pathogens.

scholarly journals Pulmonary Iron Limitation Induced by Exogenous Type I IFN Protects Mice fromCryptococcus gattiiIndependently of T Cells

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Michael J. Davis ◽  
Shannon Moyer ◽  
Elizabeth S. Hoke ◽  
Edward Sionov ◽  
Katrin D. Mayer-Barber ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Cryptococcus Neoformans ◽  
Cryptococcus Gattii ◽  
Iron Limitation ◽  
Type I ◽  
Type I Ifn ◽  
Aids Patients ◽  
Content Type ◽  
Immune Pathways

ABSTRACTCryptococcus neoformanscauses deadly mycosis primarily in AIDS patients, whereasCryptococcus gattiiinfects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence ofC. gattii. As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome ofC. neoformansandC. gattiiinfections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. InC. neoformans-infected mice, pICLC activity was associated withC. neoformanscontainment and classical Th1 immunity. In contrast, pICLC activity againstC. gattiidid not require any immune factors previously associated withC. neoformansimmunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly,C. gattiipICLC activity depended on β-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggestingC. gattiipICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcusin vitro, suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection byC. gattiiandC. neoformansbut by distinct mechanisms; theC. gattiieffect was mediated by iron limitation, while the effect onC. neoformansinfection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for differentCryptococcusspecies suggests a possible mechanism by which HIV infection may select againstC. gattiibut notC. neoformans.IMPORTANCECryptococcus neoformansandCryptococcus gattiicause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species,C. gattiiinfects very few AIDS patients, whileC. neoformansinfection is an AIDS-defining illness, suggesting that the host response to HIV selectsC. neoformansoverC. gattii. We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action againstC. neoformanswas due to activation of well-defined immune pathways known to deterC. neoformans, whereas these immune pathways were dispensable for pICLC treatment ofC. gattii. Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to controlC. neoformansinfection but is protective againstC. gattii. Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibitedC. gattii, thus suggesting an entirely new mode of nutritional immunity activated by viral signals.

scholarly journals CD4 T Cell Antigens from Staphylococcus aureus Newman Strain Identified following Immunization with Heat-Killed Bacteria

2012 ◽  
Vol 19 (4) ◽  
pp. 477-489 ◽  
Author(s):  
Paulraj K. Lawrence ◽  
Bachra Rokbi ◽  
Nadège Arnaud-Barbe ◽  
Eric L. Sutten ◽  
Junzo Norimine ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
T Cell ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Protein A ◽  
Cd4 T Cell ◽  
Content Type ◽  
Intramammary Infections ◽  
T Cell Antigens ◽  
Ifn Γ

ABSTRACTStaphylococcus aureusis a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistantS. aureus(MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens fromS. aureushas made it difficult to design effective vaccines. The goal of this study was to identifyS. aureusproteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from theS. aureusNewman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.

scholarly journals Protection againstStaphylococcus aureusColonization and Infection by B- and T-Cell-Mediated Mechanisms

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Fan Zhang ◽  
Olivia Ledue ◽  
Maria Jun ◽  
Cibelly Goulart ◽  
Richard Malley ◽  
...  
Keyword(s):  
T Cell ◽  
Soft Tissue ◽  
Bacterial Infections ◽  
Vaccine Development ◽  
Pathological Condition ◽  
Treatment Options ◽  
Immune Defense ◽  
Invasive Infection ◽  
Content Type ◽  
Increased Risk

ABSTRACTStaphylococcus aureusis a major cause of morbidity and mortality worldwide.S. aureuscolonizes 20 to 80% of humans at any one time and causes a variety of illnesses. Strains that are resistant to common antibiotics further complicate management.S. aureusvaccine development has been unsuccessful so far, largely due to the incomplete understanding of the mechanisms of protection against this pathogen. Here, we studied the role of different aspects of adaptive immunity induced by anS. aureusvaccine in protection againstS. aureusbacteremia, dermonecrosis, skin abscess, and gastrointestinal (GI) colonization. We show that, depending on the challenge model, the contributions of vaccine-inducedS. aureus-specific antibody and Th1 and Th17 responses to protection are different: antibodies play a major role in reducing mortality duringS. aureusbacteremia, whereas Th1 or Th17 responses are essential for prevention ofS. aureusskin abscesses and the clearance of bacteria from the GI tract. Both antibody- and T-cell-mediated mechanisms contribute to prevention ofS. aureusdermonecrosis. Engagement of all three immune pathways results in the most robust protection under each pathological condition. Therefore, our results suggest that eliciting multipronged humoral and cellular responses toS. aureusantigens may be critical to achieve effective and comprehensive immune defense against this pathogen.IMPORTANCES. aureusis a leading cause of healthcare- and community-associated bacterial infections.S. aureuscauses various illnesses, including bacteremia, meningitis, endocarditis, pneumonia, osteomyelitis, sepsis, and skin and soft tissue infections.S. aureuscolonizes between 20 and 80% of humans; carriers are at increased risk for infection and transmission to others. The spread of multidrug-resistant strains limits antibiotic treatment options. Vaccine development againstS. aureushas been unsuccessful to date, likely due to an inadequate understanding about the mechanisms of immune defense against this pathogen. The significance of our work is in illustrating the necessity of generating multipronged B-cell, Th1-, and Th17-mediated responses toS. aureusantigens in conferring enhanced and broad protection againstS. aureusinvasive infection, skin and soft tissue infection, and mucosal colonization. Our work thus, provides important insights for future vaccine development against this pathogen.

scholarly journals Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

2017 ◽  
Vol 24 (11) ◽  
Author(s):  
Ahreum Kim ◽  
Yun-Gyoung Hur ◽  
Sunwha Gu ◽  
Sang-Nae Cho
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Vaccine Development ◽  
Protective Efficacy ◽  
Interleukin 2 ◽  
T Cell Epitopes ◽  
Content Type ◽  
Complete Form ◽  
Ifn Γ

ABSTRACT The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.

scholarly journals The Investigational Fungal Cyp51 Inhibitor VT-1129 Demonstrates PotentIn VitroActivity against Cryptococcus neoformans and Cryptococcus gattii

2016 ◽  
Vol 60 (4) ◽  
pp. 2528-2531 ◽  
Author(s):  
Shawn R. Lockhart ◽  
Annette W. Fothergill ◽  
Naureen Iqbal ◽  
Carol B. Bolden ◽  
Nina T. Grossman ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Geometric Mean ◽  
Cryptococcus Gattii ◽  
The Novel ◽  
Content Type ◽  
Large Panel

ABSTRACTThein vitroactivities of the novel fungal Cyp51 inhibitor VT-1129 were evaluated against a large panel ofCryptococcus neoformansandCryptococcus gattiiisolates. VT-1129 demonstrated potent activities against bothCryptococcusspecies as demonstrated by low MIC50and MIC90values. ForC. gattii, thein vitropotency was maintained against all genotypes. In addition, significantly lower geometric mean MICs were observed for VT-1129 than for fluconazole againstC. neoformans, including isolates with reduced fluconazole susceptibility.

scholarly journals Vaccination with Recombinant Cryptococcus Proteins in Glucan Particles Protects Mice against Cryptococcosis in a Manner Dependent upon Mouse Strain and Cryptococcal Species

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Charles A. Specht ◽  
Chrono K. Lee ◽  
Haibin Huang ◽  
Maureen M. Hester ◽  
Jianhua Liu ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mouse Strain ◽  
Subunit Vaccine ◽  
Vaccine Development ◽  
Inflammatory Responses ◽  
Cryptococcus Gattii ◽  
Candidate Vaccine ◽  
Effective Vaccine ◽  
Mouse Strains ◽  
Vaccine Antigens

ABSTRACT Development of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus -derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii . The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli , purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii . Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific. IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals. An effective vaccine could substantially reduce the burden of cryptococcosis. However, a major gap in cryptococcal vaccine development has been the discovery of protective antigens to use in vaccines. Here, six cryptococcal proteins with potential as vaccine antigens were expressed recombinantly and purified. Mice were then vaccinated with glucan particle preparations containing each antigen. Of the six candidate vaccines, four protected mice from a lethal cryptococcal challenge. However, the degree of protection varied as a function of mouse strain and cryptococcal species. These preclinical studies identify cryptococcal proteins that could serve as candidate vaccine antigens and provide a proof of principle regarding the feasibility of protein antigen-based vaccines to protect against cryptococcosis.

scholarly journals Altered Immune Response Differentially Enhances Susceptibility to Cryptococcus neoformans and Cryptococcus gattii Infection in Mice Expressing the HIV-1 Transgene

2013 ◽  
Vol 81 (4) ◽  
pp. 1100-1113 ◽  
Author(s):  
Kassandre Leongson ◽  
Vincent Cousineau-Côté ◽  
Mathieu Goupil ◽  
Francine Aumont ◽  
Serge Sénéchal ◽  
...  
Keyword(s):  
Immune Response ◽  
Cryptococcus Neoformans ◽  
Transgene Expression ◽  
Inflammatory Cell ◽  
Differential Susceptibility ◽  
Lung Parenchyma ◽  
Cryptococcus Gattii ◽  
Content Type ◽  
Altered Immune Response ◽  
Hiv 1

ABSTRACTCryptococcus neoformansvar.grubiiis the most frequent cause of AIDS-associated cryptococcosis worldwide, whileCryptococcus gattiiusually infects immunocompetent people. To understand the mechanisms which cause differential susceptibility to these cryptococcal species in HIV infection, we established and characterized a model of cryptococcosis in CD4C/HIVMutAtransgenic (Tg) mice expressing gene products of HIV-1 and developing an AIDS-like disease. Tg mice infected intranasally withC. neoformansvar.grubiistrain H99 or C23 consistently displayed reduced survival compared to non-Tg mice at three graded inocula, while shortened survival of Tg mice infected withC. gattiistrain R265 or R272 was restricted to a single high inoculum. HIV-1 transgene expression selectively augmented systemic dissemination to the liver and spleen for strains H99 and C23 but not strains R265 and R272. Histopathologic examination of lungs of Tg mice revealed large numbers of widely scattered H99 cells, with a minimal inflammatory cell response, while in the non-Tg mice H99 was almost completely embedded within extensive mixed inflammatory cell infiltrates. In contrast to H99, R265 was dispersed throughout the lung parenchyma and failed to induce a strong inflammatory response in both Tg and non-Tg mice. HIV-1 transgene expression reduced pulmonary production of CCL2 and CCL5 after infection with H99 or R265, and production of these two chemokines was lower after infection with R265. These results indicate that an altered immune response in these Tg mice markedly enhancesC. neoformansbut notC. gattiiinfection. This model therefore provides a powerful new tool to further investigate the immunopathogenesis of cryptococcosis.

scholarly journals Multicenter Study of Isavuconazole MIC Distributions and Epidemiological Cutoff Values for the Cryptococcus neoformans-Cryptococcus gattii Species Complex Using the CLSI M27-A3 Broth Microdilution Method

2014 ◽  
Vol 59 (1) ◽  
pp. 666-668 ◽  
Author(s):  
A. Espinel-Ingroff ◽  
A. Chowdhary ◽  
G. M. Gonzalez ◽  
J. Guinea ◽  
F. Hagen ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Multicenter Study ◽  
Species Complex ◽  
Cryptococcus Gattii ◽  
Broth Microdilution ◽  
Wild Type ◽  
Content Type ◽  
Microdilution Method ◽  
Cutoff Values ◽  
Broth Microdilution Method

ABSTRACTEpidemiological cutoff values (ECVs) of isavuconazole are not available forCryptococcusspp. The isavuconazole ECVs based on wild-type (WT) MIC distributions for 438Cryptococcus neoformansnongenotyped isolates, 870 isolates of genotype VNI, and 406Cryptococcus gattiiisolates from six laboratories and different geographical areas were 0.06, 0.12, and 0.25 μg/ml, respectively. These ECVs may aid in detecting non-WT isolates with reduced susceptibilities to isavuconazole.

scholarly journals Phagosomal F-Actin Retention by Cryptococcus gattii Induces Dendritic Cell Immunoparalysis

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Khusraw Jamil ◽  
Maria J. Polyak ◽  
David D. Feehan ◽  
Philip Surmanowicz ◽  
Danuta Stack ◽  
...  
Keyword(s):  
T Cell ◽  
Dendritic Cell ◽  
Immune Responses ◽  
Immune Evasion ◽  
Cryptococcus Gattii ◽  
Host Cells ◽  
Cell Mediated Immunity ◽  
Structured Illumination Microscopy ◽  
Content Type ◽  
Life Threatening

ABSTRACT Cryptococcus gattii is a major cause of life-threatening mycosis in immunocompetent individuals and responsible for the ongoing epidemic outbreak of cryptococcosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concomitant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolution structured illumination microscopy (SR-SIM) revealed that the persistent phagosomal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit phagosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also promoted DC costimulatory maturation and robust T cell activation and proliferation. Collectively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity. IMPORTANCE Cryptococcus yeast species typically display characteristics of opportunistic pathogens, with the exception of C. gattii, which can cause life-threatening respiratory and disseminated brain infections in otherwise healthy people. The pathogenesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.

scholarly journals Adaptation to Fluconazole via Aneuploidy Enables Cross-Adaptation to Amphotericin B and Flucytosine in Cryptococcus neoformans

2021 ◽  
Author(s):  
Feng Yang ◽  
Vladimir Gritsenko ◽  
Hui Lu ◽  
Cheng Zhen ◽  
Lu Gao ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Invasive Fungal Infection ◽  
Cryptococcus Gattii ◽  
Content Type ◽  
Therapeutic Drugs ◽  
Cross Adaptation ◽  
Pyrimidine Analogues ◽  
Globally Distributed

Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii . Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine).

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