Identification of Potentially Therapeutic Immunogenic Peptides From Paracoccidioides lutzii Species.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Serological tests using Sporothrix species antigens for the accurate diagnosis of sporotrichosis: a meta-analysis.

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I2 = 90.7%; 87% CI = (84-89), P < 0.001] and specificity [I2 = 89.2%; 93% CI = (92-95), P < 0.001] was observed. The performance of diagnostic tests was 0.93. Enzyme-linked immunosorbent assay was the main tool used, and the ConA-binding fraction antigen of the strain 1099-18 appears as a promising diagnostic biomarker candidate.

Prospective cohort of AIDS patients screened for cryptococcal antigenaemia, pre-emptively treated and followed in Brazil.

BACKGROUND: Cryptococcal meningitis has a high morbidity and mortality among AIDS population. Cryptococcal antigen (CrAg) detection is considered an independent predictor for meningitis and death. Since 2011, the World Health Organization recommends CrAg screening for people living with HIV/AIDS (PLHAs) with CD4 counts <100-200 cells/µl. Its implementation is still limited in low-middle-income countries. We aimed to estimate the prevalence and predictors of CrAg positivity in PLHAs. We also evaluated outcomes among those who were CrAg-positive. METHODS: Prospective cohort conducted at an infectious diseases hospital, in Brazil. Adults with CD4 <200 cells/µl, without previous cryptococcal disease and regardless of symptoms, were enrolled from 2015 to 2018. CrAg tests were performed by LFA. Lumbar puncture was done in CrAg+ individuals and pre-emptive therapy was offered for those without meningitis. RESULTS: Of 214 individuals recruited, 88% were antiretroviral experienced, of which only 11.6% with viral suppression. Overall, CrAg prevalence was 7.9% (95% CI, 4.7-12.4). In CD4 ≤100 cells/µl group it was 7.5% (95% CI, 4.1-12.6) and 9.1% (95% CI, 3.4-19.0) in the group with CD4 101 to 199 cells/µl (p = 0.17). Prevalence in asymptomatic subjects was 5.3% (95% CI, 1.4-13.1). One among 17 CrAg+ participants had documented meningoencephalitis and no subclinical meningitis was detected. Adherence to pre-emptive treatment was 68.7% (11/16). There were no statistically significant differences in sociodemographic, clinical or laboratory characteristics to predict CrAg positivity. No case of cryptococcal disease was diagnosed among CrAg + subjects, followed by a median of 12 months. CONCLUSIONS: CrAg screening for severely immunosuppressed PLHAs in Brazil yielded a prevalence of 7.9%. No difference was found in the prevalence of CrAg stratified by CD4 values (CD4 <100 versus CD4 101-199 cells/µl). No clinical nor laboratory factors predicted CrAg positivity, corroborating the need for the implementation of universal CrAg screening for PLHAs with CD4 <200 cells/µl in similar settings.

Cryptococcal antigen positivity combined with the percentage of HIV-seropositive samples with CD4 counts <100 cells/µl identifies districts in South Africa with advanced burden of disease.

INTRODUCTION: Cryptococcal meningitis (CM) is an opportunistic fungal disease with a high mortality among HIV-positive patients with severe immunosuppression (CD4 count <100 cells/µl). Reflexed screening for cryptococcal antigen (CrAg) in remnant blood samples was initially piloted at selected CD4 testing laboratories of the National Health Laboratory Service (NHLS) prior to the implementation of a national screening programme using a lateral flow assay (LFA) (IMMY, Norman, OK, USA). The aim of this study was to assess CrAg positivity nationally, per province and district in combination with the percentage of CD4 samples tested with a CD4 count <100 cells/µl to identify areas with advanced HIV/CrAg disease burden. METHODS: CrAg and CD4 laboratory result data were extracted from the NHLS corporate data warehouse. Monthly test volumes were used to assess CrAg test volumes and coverage, while bubble charts were used to display the relationship between CD4 <100 cells/µl, CrAg positivity and number of positive CrAg samples by district. ArcGIS software was used to spatially report CrAg positivity. RESULTS: CrAg screening coverage was stable at around 96% after November 2016. Samples with a CD4 <100 cell/µl and CrAg positivity were also stable over the study period at 10% and ~5% respectively. The highest CrAg positivity was reported for the Kwa-Zulu Natal province (7.3%), which also had the lowest percentage of samples with a CD4 <100 cells/µl (7.2%). Uthungulu and Umkhanyakude districts had the highest CrAg positivity (9.3% and 8.9% respectively). Ethekwini and Johannesburg Metro districts contributed to 22% of the total number of CrAg-positive samples tested across South Africa for the period reported. CONCLUSION: Existing CD4 testing services were used to rapidly scale up CrAg reflex testing in South Africa. Districts with advanced HIV and CrAg disease burden were identified that need further investigation of patient management interventions.

Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.

Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying ß-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater ß-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the ß-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and ß-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.

Mp1p homologues as virulence factors in Aspergillus fumigatus.

Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days after challenge with wild-type A. fumigatus QC5096, AFMP1 knockdown mutant, AFMP2 knockdown mutant and AFMP3 knockdown mutant and 28 days after challenge with AFMP4 knockdown mutant (P<.0001). Only 11% of mice died 30 days after challenge with AFMP1-4 knockdown mutant (P<.0001). For mice challenge with AFMP1-4 knockdown mutant, lower abundance of fungal elements was observed in brains, kidneys, and spleens compared to mice challenge with QC5096 at day 4 post-infection. Fungal counts in brains of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.01 and P<.05). Fungal counts in kidneys of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.001 and P<.001) and those of mice challenge with QC5096 were significantly higher than those challenge with AFMP4 knockdown mutant (P<.05). There is no difference among the survival rates of wild-type A. fumigatus, AFMP4 knockdown mutant and AFMP1-4 knockdown mutant, suggesting that Mp1p homologues in A. fumigatus do not mediate its virulence via improving its survival in macrophage as in the case in T. marneffei. Afmp1p, Afmp2p, Afmp3p, and Afmp4p in combination are important virulence factors of A. fumigatus.

Cryptococcal antigen negative meningoencephalitis in HIV/AIDS.

Diagnosis of central nervous system cryptococcosis relies on a spectrum of methods but has improved with lateral flow diagnostic assays that detect capsular polysaccharide antigens of Cryptococcus. Here, we present the case of an HIV-infected African-American man with cryptococcal meningoencephalitis caused by a strain producing little or no capsule.

Anti-apoptotic effects of decyl gallate on the induction of apoptosis in A549 pneumocytes by Paracoccidioides brasiliensis gp43.

Apoptosis is considered an escape mechanism from the host immune system for the fungus Paracoccidioides spp, and it serves as a vehicle for entry into macrophages without stimulating microbicidal activities. Recently, gp43 of P. brasiliensis was demonstrated to be involved in this process. Therefore, as a new therapeutic alternative, it is very important to study compounds that could reduce the modulation of the induction of apoptosis caused by this fungus. Decyl gallate (G14) is a known antifungal compound, and we decided to investigate its anti-apoptotic properties. Our results demonstrate that G14 was effective against apoptosis induced by gp43, as observed in epithelial cells, and led to a reduction in DNA damage, Bak down-regulation and Bcl-2 up-regulation. Together, these data show that G14 presents promising anti-apoptotic activity.

Improved Detection of Invasive Pulmonary Aspergillosis Arising during Leukemia Treatment Using a Panel of Host Response Proteins and Fungal Antigens.

Invasive pulmonary aspergillosis (IPA) is an opportunistic fungal infection in patients undergoing chemotherapy for hematological malignancy, hematopoietic stem cell transplant, or other forms of immunosuppression. In this group, Aspergillus infections account for the majority of deaths due to mold pathogens. Although early detection is associated with improved outcomes, current diagnostic regimens lack sensitivity and specificity. Patients undergoing chemotherapy, stem cell transplantation and lung transplantation were enrolled in a multi-site prospective observational trial. Proven and probable IPA cases and matched controls were subjected to discovery proteomics analyses using a biofluid analysis platform, fractionating plasma into reproducible protein and peptide pools. From 556 spots identified by 2D gel electrophoresis, 66 differentially expressed post-translationally modified plasma proteins were identified in the leukemic subgroup only. This protein group was rich in complement components, acute-phase reactants and coagulation factors. Low molecular weight peptides corresponding to abundant plasma proteins were identified. A candidate marker panel of host response (9 plasma proteins, 4 peptides), fungal polysaccharides (galactomannan), and cell wall components (ß-D glucan) were selected by statistical filtering for patients with leukemia as a primary underlying diagnosis. Quantitative measurements were developed to qualify the differential expression of the candidate host response proteins using selective reaction monitoring mass spectrometry assays, and then applied to a separate cohort of 57 patients with leukemia. In this verification cohort, a machine learning ensemble-based algorithm, generalized pathseeker (GPS) produced a greater case classification accuracy than galactomannan (GM) or host proteins alone. In conclusion, Integration of host response proteins with GM improves the diagnostic detection of probable IPA in patients undergoing treatment for hematologic malignancy. Upon further validation, early detection of probable IPA in leukemia treatment will provide opportunities for earlier interventions and interventional clinical trials.

Abolishing Cell Wall Glycosylphosphatidylinositol-Anchored Proteins in Candida albicans Enhances Recognition by Host Dectin-1.

Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans, can shield ß-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed ß-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo. These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition.

Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.

Immunoproteome of Aspergillus fumigatus using sera of patients with invasive aspergillosis.

Invasive aspergillosis is a life-threatening lung or systemic infection caused by the opportunistic mold Aspergillus fumigatus. The disease affects mainly immunocompromised hosts, and patients with hematological malignances or who have been submitted to stem cell transplantation are at high risk. Despite the current use of Platelia™ Aspergillus as a diagnostic test, the early diagnosis of invasive aspergillosis remains a major challenge in improving the prognosis of the disease. In this study, we used an immunoproteomic approach to identify proteins that could be putative candidates for the early diagnosis of invasive aspergillosis. Antigenic proteins expressed in the first steps of A. fumigatus germination occurring in a human host were revealed using 2-D Western immunoblots with the serum of patients who had previously been classified as probable and proven for invasive aspergillosis. Forty antigenic proteins were identified using mass spectrometry (MS/MS). A BLAST analysis revealed that two of these proteins showed low homology with proteins of either the human host or etiological agents of other invasive fungal infections. To our knowledge, this is the first report describing specific antigenic proteins of A. fumigatus germlings that are recognized by sera of patients with confirmed invasive aspergillosis who were from two separate hospital units.

Paracoccidioides lutzii Plp43 is an active glucanase with partial antigenic identity with P. brasiliensis gp43.

BACKGROUND: Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii ("Pb01-like") apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43. METHODOLOGY: We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients' sera from Southwestern and Midwestern Brazil. PRINCIPAL FINDINGS: We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species. CONCLUSION: Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.

Structure and function of a fungal adhesin that binds heparin and mimics thrombospondin-1 by blocking T cell activation and effector function.

Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.

Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis.

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.

Dectin-1-triggered recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens.

Dectin-1 is a pattern recognition receptor that is important for innate immune responses against fungi in humans and mice. Dectin-1 binds to ß-glucans in fungal cell walls and triggers phagocytosis, production of reactive oxygen by the NADPH oxidase, and inflammatory cytokine production which all contribute to host immune responses against fungi. Although the autophagy pathway was originally characterized for its role in the formation of double-membrane compartments engulfing cytosolic organelles and debris, recent studies have suggested that components of the autophagy pathway may also participate in traditional phagocytosis. In this study, we show that Dectin-1 signaling in macrophages and bone marrow-derived dendritic cells triggers formation of LC3II, a major component of the autophagy machinery. Further, Dectin-1 directs the recruitment of LC3II to phagosomes, and this requires Syk, activation of reactive oxygen production by the NADPH oxidase, and ATG5. Using LC3-deficient dendritic cells we show that whereas LC3 recruitment to phagosomes is not important for triggering phagocytosis, killing or Dectin-1-mediated inflammatory cytokine production, it facilitates recruitment of MHC class II molecules to phagosomes and promotes presentation of fungal-derived antigens to CD4 T cells.

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Biodegradable microspheres containing leukotriene B(4) and cell-free antigens from Histoplasma capsulatum activate murine bone marrow-derived macrophages.

Because of the potential protective role of leukotrienes (LTs) in histoplasmosis and the therapeutic and prophylactic effects of cell-free antigens from Histoplasmacapsulatum (CFAgs), the aim of this study was to develop and characterise biodegradable LTB(4)/CFAgs-loaded microspheres (MS) that could promote cellular activation for future immunisation purposes. LTB(4)/CFAgs-loaded MS that were developed through a double emulsion/extraction process were characterised according to their size, zeta potential, morphology, entrapment efficiency and in vitro release kinetics. We evaluated the uptake of LTB(4)/CFAgs-loaded MS by bone marrow derived-macrophages (BMDM). The TNF-α and chemokines, and nitrite production, in the supernatant of BMDM cultures were analysed by enzyme-linked immunosorbent assay (ELISA) and Griess reaction, respectively. We found an instantaneous release of CFAgs and a prolonged release of LTB(4) from the poly-(d,l-lactide-co-glycolide) (PLGA) MS. The microencapsulation process did not alter the zeta potential nor the spherical morphology of the MS. The appropriate size of the LTB(4)/CFAgs-loaded MS (smaller than 10µm) enabled the efficient uptake by BMDM and also induced TNF-α, CXCL1/KC, CCL2/MCP-1, CCL5/RANTES and nitrite oxide release by these cells. In conclusion, the biodegradable LTB(4)/CFAgs-loaded MS were able to efficiently activate murine BMDM and thereby have the potential to be used in an effective vaccine against H. capsulatum infection.

Characterizing the role of cell-wall ß-1,3-exoglucanase Xog1p in Candida albicans adhesion by the human antimicrobial peptide LL-37.

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37-cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall ß-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p(90-115), and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another ß-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion.

Interactions between TLR2, TLR4, and mannose receptors with gp43 from Paracoccidioides brasiliensis induce cytokine production by human monocytes.

The glycoprotein gp43 is an immunodominant antigen secreted by Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. The present study evaluated whether gp43 can interact with toll-like (TLR2, TLR4) and mannose (MR) receptors on the surface of human monocytes, and how that affects their expression and cytokine production. Monocytes were incubated with or without monoclonal antibodies anti-TLR2, anti-TLR4, or anti-MR, individually or in combination, prior to the addition of gp43. The gp43 binding to monocyte surface, as well as expression of TLR2, TLR4, and MRs were analyzed by flow cytometry, while production of TNF-α and IL-10 was monitored by ELISA. The results suggested that gp43 binds to TLR2, TLR4, and MR receptors, with TLR2 and MR having the strongest effect. All three receptors influenced the production of IL-10, while TNF-α production was associated with expression of TLR4 and MR. The modulatory effect of gp43 was demonstrated by high levels of TLR4 expression associated with increased production of TNF-α after 4 h of culture. Alternatively, high levels of TLR2 expression, and elevated production of IL-10, were detected after 18 h. We showed that interaction between gp43 and monocytes may affect the innate immune response by modulating the expression of the pattern recognition receptors TLR2, TLR4 and MR, as well as production of pro- and anti-inflammatory cytokines.