Murine Systemic Candidiasis Research Articles

Toll-like receptor 4 (TLR4) is the major pattern recognition receptor triggering the protective effect of a Candida albicans extracellular vesicle-based vaccine prototype in murine systemic candidiasis.

Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of β-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

The F1Fo-ATP synthase α subunit of Candida albicans induces inflammatory responses by controlling amino acid catabolism

ABSTRACT Sepsis is a leading cause of fatality in invasive candidiasis. The magnitude of the inflammatory response is a determinant of sepsis outcomes, and inflammatory cytokine imbalances are central to the pathophysiological processes. We previously demonstrated that a Candida albicans F1Fo-ATP synthase α subunit deletion mutant was nonlethal to mice. Here, the potential effects of the F1Fo-ATP synthase α subunit on host inflammatory responses and the mechanism were studied. Compared with wild-type strain, the F1Fo-ATP synthase α subunit deletion mutant failed to induce inflammatory responses in Galleria mellonella and murine systemic candidiasis models and significantly decreased the mRNA levels of the proinflammatory cytokines IL-1β, IL-6 and increased those of the anti-inflammatory cytokine IL-4 in the kidney. During C. albicans-macrophage co-culture, the F1Fo-ATP synthase α subunit deletion mutant was trapped inside macrophages in yeast form, and its filamentation, a key factor in inducing inflammatory responses, was inhibited. In the macrophage-mimicking microenvironment, the F1Fo-ATP synthase α subunit deletion mutant blocked the cAMP/PKA pathway, the core filamentation-regulating pathway, because it failed to alkalinize environment by catabolizing amino acids, an important alternative carbon source inside macrophages. The mutant downregulated Put1 and Put2, two essential amino acid catabolic enzymes, possibly due to severely impaired oxidative phosphorylation. Our findings reveal that the C. albicans F1Fo-ATP synthase α subunit induces host inflammatory responses by controlling its own amino acid catabolism and it is significant to find drugs that inhibit F1Fo-ATP synthase α subunit activity to control the induction of host inflammatory responses.

P120 Vaccine induced protection by secreted aspartyl proteinase 2 from Candida parapsilosis in Candida tropicalis mediated murine systemic candidiasis: a role of B-cells and antibodies

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesNon-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. These challenges merit the need for developing novel immune therapies against Candida infections. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading Candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. Moreover, while the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. As such, we also investigated the role of humoral immunity in vaccine-mediated protection during murine systemic candidiasis.MethodsThe Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis strains prevalent in India. Groups of wild-type BALb/c mice were vaccinated with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with a lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Histopathological assessment was performed using H&E and PAS staining. Serum cytokine levels and antigen-specific antibody titers were measured by ELISA. Cellular responses were analyzed in detail using flow cytometry. Functional evaluation of antibody role was performed using in vitro (biofilm inhibition and neutrophil-mediated killing) and in vivo (passive transfer) studies. B-cell epitope analysis was carried out using immunoinformatics approaches.ResultsMice vaccinated with rSap2 cloned from C. parapsilosis (Sap2-parapsilosis) showed highest increase in survival time (P = .02) and maximum reduction in organ fungal burden (spleen, kidney, lungs, brain) (P <.05); compared with sham immunized controls. Vaccination with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ∼60% homology across species. Mice vaccinated with rSap2-parapsilosis also exhibited significantly higher levels of IFN gamma, IL-17, and IL-4 cytokine levels just before infection, which correlated with protection. In addition, rSap2-parapsilosis vaccination induced high titers of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (which were predominantly of IgM isotype). Notably, sera from rSap2-parapsilosis vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19 + B-cells, plasma cells, and Candida-binding B-cells in rSap2-parapsilosis vaccinated mice indicate a role of B-cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B-cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multi-epitope anti-Candida vaccine/s.ConclusionMice immunized with recombinant Sap2 cloned from C. parapsilosis exhibited increased survival during C. tropicalis mediated systemic candidiasis. Both Th1/Th17 mediated cellular immunity and humoral immunity contribute toward protection. Our results establish a role of B-cells and antibodies in generation of protective immune responses against Sap2 vaccine antigen during non-albicans Candida mediated murine systemic candidiasis.

Solidified Reverse Micellar Solution- (SRMS-) Based Microparticles for Enhanced Oral Bioavailability and Systemic Antifungal Efficacy of Miconazole Nitrate in Immunocompromised Mice.

Purpose To assess the improvement in oral bioavailability and efficacy in systemic candidiasis treatment of miconazole nitrate (MN) formulations in murine models of candidiasis. Methods Selected formulations containing 5% of Softisan + Phospholipon 90H lipid matrix with 3% of MN (A1), 5% of stearic acid + Phospholipon 90H lipid matrix with 3% of MN (B1), and 5% Softisan + stearic acid + Phospholipon 90H with 3% of MN (C1) from the in vitro investigation were used for the study. Their acute toxicity was assessed using Lorke's method (with slight modification) while bioavailability was determined using the bioassay method. The optimized batch (A1) was tested in murine systemic candidiasis induced in cyclophosphamide-immunosuppressed mice. The mice were treated with a single oral dose (100 mg/kg) of the formulations for five days. Serum fungal counts (cfu/mL) were determined on days 1, 3, and 5 of the treatment period. Haematological assessments were done. Results The lipid formulations were safer than MN powder with LD50 values of 3162.8 and 1118.3 mg/kg. Bioavailability determination revealed a higher area under the curve (AUC) value for formulations A1 (6.11 μg/hr/mL) and B1 (4.91 μg/hr/mL) while formulation C1 (1.80 μg/hr/mL) had a lower AUC than MN (4.46 μg/hr/mL). Fungi were completely cleared from the blood of animals treated with the optimized formulation by day 3 as opposed to the controls (MN and Tween® 20) which still had fungi on day 5. No significant increase (p > 0.05) in haematological parameters was observed in mice treated with A1. Conclusion Formulation A1 successfully cleared Candida albicans from the blood within a shorter period than miconazole powder. This research has shown the potential of orally administered MN-loaded SRMS-based microparticles in combating systemic candidaemia.

A role of B-cells and antibodies in generation of protective immune responses against a vaccinating antigen during non-albicans Candida mediated systemic candidiasis

Abstract While cellular immunity is acknowledged to be critical for vaccine-mediated protection during systemic candidiasis, the role of humoral immunity is not clear. We investigated the role of B cells and antibodies in conferring protective advantage during Candida tropicalis mediated murine systemic candidiasis, using a lead vaccine candidate, Sap2 (secreted aspartyl proteinase 2) obtained from C. parapsilosis. Compared to sham-immunized mice, Sap2-parapsilosis vaccinated mice exhibited significantly improved survival outcomes (p=0.02) and reduced organ fungal burdens (p&amp;lt;0.05). Sap2-parapsilosis vaccination induced high titres of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (of predominantly IgM isotype). Notably, sera from Sap2-parapsilosis-vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19+ B cells, plasma cells and Candida-binding B cells in Sap2-vaccinated mice indicate towards a role of B cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multiepitope anti-Candida vaccine/s. In summary, our results establishes a role of B cells and antibodies in contributing towards protective efficacy of Sap2 vaccine antigen during murine systemic candidiasis.

11g, a Potent Antifungal Candidate, Enhances Candida albicans Immunogenicity by Unmasking β-Glucan in Fungal Cell Wall

In the course of optimizing GPI biosynthesis inhibitors, we designed and synthetized a 2-aminonicotinamide derivative named 11g. After evaluating the antifungal activity of compound 11g in vitro, we investigated the influences of 11g on fungi immunogenicity. In addition, we also took advantage of murine systemic candidiasis model to investigate the protective effects of 11g in vivo. Results show that 11g exhibited potent antifungal activity both in vitro and in vivo. Further study shows that 11g caused the unmasking of fungi β-glucan layer, leading to stronger immune responses in macrophages through Dectin-1. These results suggest that 11g is a very promising antifungal candidate, which assists in eliciting stronger immune responses to help host immune system disposing pathogens. The discovery of 11g might expand the toolbox of fungal infection treatment.

Two New 1,3,4-Oxadiazoles With Effective Antifungal Activity Against Candida albicans.

Candida infections have become a serious public health problem with high mortality rates, especially in immunocompromised patients, since Candida albicans is the major opportunistic pathogen responsible for systemic or invasive candidiasis. Commercially available antifungal agents are restricted and fungal resistance to such drugs has increased; therefore, the development of a more specific antifungal agent is necessary. Using assays for antifungal activity, here we report that two new compounds of 1,3,4-oxadiazoles class (LMM5 and LMM11), which were discovered by in silico methodologies as possible thioredoxin reductase inhibitors, were effective against C. albicans. Both compounds had in vitro antifungal activity with MIC 32 μg/ml. Cytotoxicity in vitro demonstrated that LMM5 and LMM11 were non-toxic in the cell lines evaluated. The kinetic of the time-kill curve suggested a fungistatic profile and showed an inhibitory effect of LMM5 and LMM11 in 12 h that remained for 24 and 36 h, which is better than fluconazole. In the murine systemic candidiasis model by C. albicans, the two compounds significantly reduced the renal and spleen fungal burden. According to the SEM and TEM images, we hypothesize that the mechanism of action of LMM5 and LMM11 is directly related to the inhibition of the enzyme thioredoxin reductase and internally affect the fungal cell. In view of all in vitro and in vivo results, LMM5 and LMM11 are effective therapeutic candidates for the development of new antifungal drugs addressing the treatment of human infections caused by C. albicans.

Protective role of secreted aspartyl proteinase in murine systemic candidiasis caused by non-albicans Candida species

Abstract Non-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. The Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis. Groups of Balb/c mice were immunized with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Mice immunized with rSap2 cloned from C. parapsilosis showed highest increase in survival time (p=0.02) and maximum reduction in organ fungal burden (spleen, kidneys, lungs, brain) (p&amp;lt;0.05); compared to sham immunized controls. Immunization with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ~60% homology across species. Mice immunized with rSap2 cloned from C. parapsilosis also exhibited significantly higher levels of IFN gamma and IL-4 cytokine levels just before infection, which correlated with protection. The anti-Sap2 specific antibody titres were measured. No correlation was observed between level of Sap2-specific IgG titres and survival outcome. The IgG response was primarily Th2, belonging to IgG1 subtype. Further cellular and histological studies are in progress to characterize the murine rSap2-parapsilosis specific protective immune response in detail.

Bst1 is required for Candida albicans infecting host via facilitating cell wall anchorage of Glycosylphosphatidyl inositol anchored proteins.

Glycosylphosphatidyl inositol anchored proteins (GPI-APs) on fungal cell wall are essential for invasive infections. While the function of inositol deacylation of GPI-APs in mammalian cells has been previously characterized the impact of inositol deacylation in fungi and implications to host infection remains largely unexplored. Herein we describe our identification of BST1, an inositol deacylase of GPI-Aps in Candida albicans, was critical for GPI-APs cell wall attachment and host infection. BST1-deficient C. albicans (bst1Δ/Δ) was associated with severely impaired cell wall anchorage of GPI-APs and subsequen unmasked β-(1,3)-glucan. Consistent with the aberrant cell wall structures, bst1Δ/Δ strain did not display an invasive ability and could be recognized more efficiently by host immune systems. Moreover, BST1 null mutants or those expressing Bst1 variants did not display inositol deacylation activity and exhibited severely attenuated virulence and reduced organic colonization in a murine systemic candidiasis model. Thus, Bst1 can facilitate cell wall anchorage of GPI-APs in C. albicans by inositol deacylation, and is critical for host invasion and immune escape.

Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3)-glucan, a crucial pathogen-associated molecular pattern (PAMP) of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.

Vaccination with Recombinant Non-transmembrane Domain of Protein Mannosyltransferase 4 Improves Survival during Murine Disseminated Candidiasis.

Candida albicans is the most common cause of invasive fungal infections in humans. The C. albicans cell wall proteins play an important role in crucial host-fungus interactions and might be ideal vaccine targets to induce protective immune response in host. Meanwhile, protein that is specific to C. albicans is also an ideal target of vaccine. In this study, 11 proteins involving cell wall biosynthesis, yeast-to-hypha formation, or specific to C. albicans were chosen and were successfully cloned, purified and verified. The immune protection of vaccination with each recombinant protein respectively in preventing systemic candidiasis in BALB/c mice was assessed. The injection of rPmt4p vaccination significantly increased survival rate, decreased fungal burdens in the heart, liver, brain, and kidneys, and increased serum levels of both immunoglobulin G (IgG) and IgM against rPmt4p in the immunized mice. Histopathological assessment demonstrated that rPmt4p vaccination protected the tissue structure, and decreased the infiltration of inflammatory cells. Passive transfer of the rPmt4p immunized serum increased survival rate against murine systemic candidiasis and significantly reduced organ fungal burden. The immune serum enhanced mouse neutrophil killing activity by directly neutralizing rPmt4p effects in vitro. Levels of interleukin (IL)-4, IL-10, IL-12p70, IL-17A and tumor necrosis factor (TNF)-α in serum were higher in the immunized mice compared to those in the adjuvant control group. In conclusion, our results suggested that rPmt4p vaccination may be considered as a potential vaccine candidate against systemic candidiasis.

Novel oral amphotericin B formulation (iCo-010) remains highly effective against murine systemic candidiasis following exposure to tropical temperature

Purpose: To evaluate the antifungal activity of amphotericin B (AmB) in a mouse model of systemic candidiasis following administration of a novel oral AmB formulation (iCo-010) that has been pre-exposed to tropical temperatures.Methods: Amphotericin B (AmB) was prepared as a 5 mg/mL dispersion in a mixture of Peceol, Gelucire 44/14 and VitE-TPGS 2,3 (iCo-010). The formulation was protected from light and incubated in a sealed container at 43 °C for 60 days. Mice infected with Candida albicans were treated with either iCo-010 formulation pre-incubated at 43 °C for 60 days or freshly prepared iCo-010 formulation at doses of 5, 10 and 20 mg/kg once daily for five consecutive days. Single intravenous 5 mg/kg dose of AmBisome® was used as a positive control group. Seven days following the last dose, the kidney, liver, spleen, lung, heart and brain were removed and the number of colony forming units (CFUs) was determined as a measure of tissue fungal load. In addition, the concentration of AmB within each tissue was determined using high performance liquid chromatography (HPLC).Results: There were no significant differences in the reduction of CFUs and the concentration of AmB recovered in all organs at all iCo-010 doses tested between the freshly prepared iCo-010 formulation compared to the formulation that was incubated at 43 °C for 60 days.Conclusions: A novel oral AmB formulation, iCo-010, incubated at 43 °C for 60 days to simulate the exposure of the formulation to tropical temperatures remained highly effective against murine systemic candidiasis.

Inflammatory Monocytes Mediate Early and Organ-Specific Innate Defense During Systemic Candidiasis

Candida albicans is a commensal fungus that can cause systemic disease in patients with breaches in mucosal integrity, indwelling catheters, and defects in phagocyte function. Although circulating human and murine monocytes bind C. albicans and promote inflammation, it remains unclear whether C-C chemokine receptor 2 (CCR2)- and Ly6C-expressing inflammatory monocytes exert a protective or a deleterious function during systemic infection. During murine systemic candidiasis, interruption of CCR2-dependent inflammatory monocyte trafficking into infected kidneys impaired fungal clearance and decreased murine survival. Depletion of CCR2-expressing cells led to uncontrolled fungal growth in the kidneys and brain and demonstrated an essential antifungal role for inflammatory monocytes and their tissue-resident derivatives in the first 48 hours postinfection. Adoptive transfer of purified inflammatory monocytes in depleted hosts reversed the defect in fungal clearance to a substantial extent, indicating a compartmentally and temporally restricted protective function that can be transferred to enhance systemic innate antifungal immunity.

Hepcidin regulation by innate immune and infectious stimuli

AbstractHepcidin controls the levels and distribution of iron, an element whose availability can influence the outcome of infections. We investigated hepcidin regulation by infection-associated cytokines, pathogen-derived molecules, and whole pathogens in vitro and in vivo. We found that IL-22, an effector cytokine implicated in responses to extracellular infections, caused IL-6–independent hepcidin up-regulation in human hepatoma cells, suggesting it might represent an additional inflammatory hepcidin agonist. Like IL-6, IL-22 caused phosphorylation of STAT3 and synergized with BMP6 potentiating hepcidin induction. In human leukocytes, IL-6 caused potent, transient hepcidin up-regulation that was augmented by TGF-β1. Pathogen-derived TLR agonists also stimulated hepcidin, most notably the TLR5 agonist flagellin in an IL-6–dependent manner. In contrast, leukocyte hepcidin induction by heat-killed Candida albicans hyphae was IL-6–independent, but partially TGF-β–dependent. In a murine acute systemic candidiasis model, C albicans strongly stimulated hepcidin, accompanied by a major reduction in transferrin saturation. Similarly, hepcidin was up-regulated with concomitant lowering of serum iron during acute murine Influenza A/PR/8/34 virus (H1N1) infection. This intracellular pathogen also stimulated hepcidin expression in leukocytes and hepatoma cells. Together, these results indicate that hepcidin induction represents a component of the innate immune response to acute infection, with the potential to affect disease pathogenesis.

Activity of an Intralipid formulation of nystatin in murine systemic candidiasis

Since nystatin (NYT) is used only topically owing to its toxicity upon systemic administration, a study was initiated aiming to develop a formulation of NYT that could be used systemically against invasive mycoses. The present research is a continuation of previous in vitro investigation of the antifungal effect of nystatin–Intralipid (NYT-IL) against Candida, exploring its in vivo activity. NYT-IL was tested in murine systemic candidiasis induced in naïve as well as cyclophosphamide-immunosuppressed female ICR mice. The infection was assessed by survival rate (SR), mean survival time (MST) and qualitative and quantitative fungal organ colonisation. Mice were treated by intravenous administration of various doses of NYT-IL for 5 consecutive days starting either 24h or 48h after the initiation of infection. The experiments showed that NYT-IL is therapeutically effective in the murine candidiasis model. NYT-IL was found to be less toxic in vivo than NYT and therefore higher doses of NYT-IL could be used. The efficacy of NYT-IL was expressed in treated naïve and immunosuppressed mice by increased SR, prolonged MST and reduced fungal organ colonisation. Early initiation of treatment increased efficacy. In summary, the Intralipid formulation of NYT can be administered parenterally and is effective against systemic experimental Candida infection.

Dose Range Evaluation of Mycograb C28Y Variant, a Human Recombinant Antibody Fragment to Heat Shock Protein 90, in Combination with Amphotericin B-Desoxycholate for Treatment of Murine Systemic Candidiasis

Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t(1/2)(α) of ~0.198 h and a t(1/2)(β) of ~1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis.

Leukocyte Integrin αXβ2 Is the Principal Receptor for Candida Albicans on Macrophages and Is Essential for Antifungal Host Defense.

AbstractAbstract 1493The induction of cell-mediated immunity to pathogens is the prime task of cells of the innate immune system. Integrin αMβ2 (Mac-1, CR3, CD11b/CD18) is expressed on polymorphonuclear neutrophils (PMNs), monocytes, macrophages (MFC) and NK cells and has been implicated in diverse responses of these cells to infection, including phagocytosis, cell-mediated killing, chemotaxis and cellular activation. In PMNs integrin αMβ2 serves as the principal receptor involved in recognition of the common opportunistic fungal pathogen Candida albicans, and this integrin recognizes the fungal mannoprotein pH-regulated Antigen 1 (Pra1). Professional antigen-presenting cells of myeloid lineage MFC and dendritic cells express integrin αXβ2 (p150,95, CD11c/CD18) which is highly homologous to Integrin αMβ2, but the role of αXβ2 in the immune response to bacterial and fungal infections remains unclear. Employing αM - and αX-KO mice and mutated strains of C. albicans in a model of murine systemic candidiasis, we first demonstrate that mice deficient in αXβ2 exhibit significantly increased susceptibility to the invasive fungal infection. C. albicans induced lethality in αXβ2-KO mice more rapidly (2- to 3-fold faster) than the same inoculum in wild-type mice. MFC derived from mice deficient in α×β2 have a reduced ability to kill or phagocyte C. albicans. When the fungus was injected into mice, 6 hrs after peritoneal of thioglycollate, neutrophil recruitment to C. albicans showed a 20%-30% clearance of the fungus in the αMβ2-KO murine intraperitoneal lavages and a 50–60% rise in the αXβ2-KO lavages, which is consistent with our prior data showing αMβ2-dependency for PMNs-mediated antifungal defense activity. In contrast, when the fungus was injected into the peritoneal cavity 16 hrs after thioglycollate, when most recruited leukocytes are MFC, the C. albicans injected into αXβ2-KO mice showed a 23%-25% clearance of the fungus from the lavages while fungal clearance in αMβ2-KO lavages increased by only 30–40%. Thus, MFC antifungal activity is αXβ2-dependent. These results were further confirmed by in vitro fungicide assays employing isolated murine peritoneal PMNs and MFC. Fungal survival was greatest with PMN derived from αMβ2-KO mice and MFC derived from the αXβ2-KO mice-derived MFC. Disruption of the PRA1 gene of C. albicans, which is the primary ligand for αMβ2, had no effect on MFC fungicide activity, which suggests that αXβ2 recognizes a C. albicans ligand different from Pra1. Taken together, these results suggest that αXβ2 is a principal receptor for C. albicans on macrophages and plays critical role in host antifungal defense in a Pra1-independent mechanism. Disclosures:No relevant conflicts of interest to declare.

Alternative dosing regimens of liposomal amphotericin B (AmBisome) effective in treating murine systemic candidiasis

This study was done to determine whether high dose AmBisome (4-20 mg/kg), given intermittently, could reduce the frequency of dosing needed to treat murine systemic candidiasis when compared with conventional daily treatment. Mice were immunosuppressed with cyclophosphamide every 3 days, beginning day -3 before challenge with log(10) 5.0 cfu Candida albicans. Treatment was begun 48-72 h post-challenge with daily or intermittent dose regimens of AmBisome, followed by determination of kidney cfu for up to 1 month post-treatment. A single AmBisome dose of 4 mg/kg was as effective as four daily, 1 mg/kg treatments. A total of 8 mg/kg, given as 4 mg/kg on days 2 and 4, or as 5 mg/kg on day 2 followed by 1 mg/kg on days 3, 4, and 5, also produced comparable efficacy. While 20 mg/kg given day 2, 4 and 6 post-challenge as a 1 week loading dose, followed by one 10 mg/kg treatment on day 13, decreased the fungal burden by up to 5 logs compared with controls (log(10) 2.3 cfu/g and log(10) 7.5 cfu/g, respectively), 20 mg/kg given Monday, Wednesday and Friday for 5 weeks, reduced the fungal burden to undetectable levels (i.e. log(10) 1.0 cfu). Significant reduction or clearance of kidney cfu, following intermittent, high dose AmBisome treatment, indicated that non-daily dosing regimens could be successfully used instead of conventional daily dosing to treat established C. albicans infection in immunosuppressed mice.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 3

A new series of acid-stable antifungal agents having strong inhibitory activity against Candida albicans N-myristoyltransferase (CaNmt) has been developed starting from acid-unstable benzofuranylmethyl aryl ether 2. The inhibitor design is based on X-ray crystallographic analysis of a CaNmt complex with aryl ether 3. Among the new inhibitors, pyridine derivative 8b and benzimidazole derivative 8k showed clear antifungal activity in a murine systemic candidiasis model.

Reevaluation of the role of HWP1 in systemic candidiasis by use of Candida albicans strains with selectable marker URA3 targeted to the ENO1 locus.

Previous evaluation of HWP1 in systemic candidiasis in CBA/J mice was done with Candida albicans strains with differing genetic locations of URA3 as a result of Ura-blaster mutagenesis. In this study, the presence of HWP1 and the location of URA3 contributed to the severity of murine systemic candidiasis in BALB/c mice.