The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects.

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.

Identification of two anti-Candida antibodies associated with the survival of patients with candidemia.

IMPORTANCE: Candidemia (bloodstream invasion by Candida species) is a major fungal disease in humans. Despite the recent progress in diagnosis and treatment, therapeutic options are limited and under threat of antimicrobial resistance. The disease mortality remains high (around 40%). In contrast with deep-seated invasive candidiasis, particularly that occurring in patients with hematologic malignancies and organ transplants, patients with candidemia are often not immunocompromised and therefore able to mount memory anticandidal immune responses, perhaps primed by Candida commensalism. We investigated antibody immunity in candidemia patients and report here on the ability of these patients to produce antibodies that react with Candida antigens. In particular, the patients with high titers of IgG reactive with two immunodominant, virulence-associated antigens (Als3 and MP65) had a higher 30-day survival. If confirmed by controlled, prospective clinical studies, our data could inform the development of antibody therapy to better treat a severe fungal infection such as candidiasis.

Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans.

BACKGROUND: Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions. RESULTS: In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results. CONCLUSIONS: Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans.

Comparative analysis of plant-produced, recombinant dimeric IgA against cell wall ß-glucan of pathogenic fungi.

Immunoglobulins A (IgA) are crucially involved in protection of human mucosal surfaces from microbial pathogens. In this work, we devised and expressed in plants recombinant chimeric antifungal antibodies (Abs) of isotype A (IgA1, IgA2, and scFvFcA1), derived from a murine mAb directed to the fungal cell wall polysaccharide ß-glucan which had proven able to confer protection against multiple pathogenic fungi. All recombinant IgA (rIgA) were expressed and correctly assembled in dimeric form in plants and evaluated for yield, antigen-binding efficiency and antifungal properties in vitro, in comparison with a chimeric IgG1 version. Production yields and binding efficiency to purified ß-glucans showed significant variations not only between Abs of different isotypes but also between the different IgA formats. Moreover, only the dimeric IgA1 was able to strongly bind cells of the fungal pathogen Candida albicans and to restrain its adhesion to human epithelial cells. Our data indicate that IgG to IgA switch and differences in molecular structure among different rIgA formats can impact expression in plant and biological activity of anti-ß-glucans Abs and provide new insights for the design of recombinant IgA as anti-infective immunotherapeutics, whose potential is still poorly investigated.

Antibodies against a ß-glucan-protein complex of Candida albicans and its potential as indicator of protective immunity in candidemic patients.

Sera from candidemic and non-candidemic subjects were examined for antibodies against the cell wall ß1,3- and ß1,6-glucans, as well as the ß-glucan-associated protein MP65 of Candida species. Although antibodies against each of the above components were detected in all subjects, candidemic patients had lower antibody titers against ß1,3-glucan, but higher antibody titers against ß1,6-glucan and MP65, than non-candidemic subjects. The elevated levels of anti-ß1,6-glucan and -MP65 antibodies found in candidemic patients were independent on the patient risk category, APACHE II score, presence of co-morbidities, ß1,3-glucanemia level, Candida isolate, and antifungal treatment. Interestingly, however, the anti-MP65, but not the anti-ß1,6-glucan antibodies, of candidemic patients had higher titers in survivors than in non-survivors, particularly in those subject categories with the highest mortality (>65-years old, diabetic, or septic shock patients). Thus, candidemic patients are capable of boosting anti-Candida immune responses upon infection, and some of these responses might be associated to the generation of protective immunity in patients with candidemia.

A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing ß-Glucan and MP65 Determinants in Candida Species.

There is a real medical need of new diagnostic tools for the early recognition of invasive Candida infections. We exploited a rather simple and rapid redox methodology to construct a bispecific monoclonal antibody (bsmAb) that combines a monoclonal antibody (mAb) directed against 1,3-ß-D-glucan, a well-known, pan-fungal diagnostic biomarker, with a mAb recognizing MP65, a major immunogenic mannoprotein secreted by C.albicans and other Candida species. The bsmAb (MP65/bglu mAb) was successfully produced and purified at high yields and proved to bind and reveal simultaneously, with high sensitivity, the ß-glucan and MP65 antigens in both purified and native forms. The MP65/bglu mAb is the first bispecific antibody generated against a fungal microorganism and may prove useful for the concurrent detection of different and clinically significant Candida biomarkers in patient sera.

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-ß-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated ß-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a "glucome" microarray, the first sequence-defined glycome-scale microarray, using a "designer" approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear "homo" and "hetero" and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or ß-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting ß-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Endogenous PGE2 promotes the induction of human Th17 responses by fungal ß-glucan.

The interaction of PAMPs with cells of the innate immune system shapes the adaptive host response. Here, we report that ß-glucan, a major fungal PAMP purified from Candida albicans, stimulates human DCs to secrete a pro-Th17 cytokine pattern. Notably, ß-glucan induces PGE2 production, which has been shown to play a pivotal role in Th17 cell expansion. Inhibition of PGE2 synthesis or blockade of PGE2 receptors EP2 and EP4 drastically reduces IL-23 production by ß-glucan-activated DCs, suggesting that endogenous PGE2 amplifies IL-23 synthesis in response to the C. albicans PAMP. Moreover ß-glucan promotes the expansion of Th17 cells, which is strongly decreased by EP2 and EP4 receptor blockade on DCs. Our results highlight a novel role for PGE2 in the regulation of innate and adaptive immune response triggered by recognition of a prominent, highly conserved fungal PAMP such as ß-glucan.

Beta-glucan-CRM197 conjugates as candidates antifungal vaccines.

A laminarin-diphtheria toxoid (CRM197) conjugate vaccine conferred protection against fungal infections in mice. We have now generated novel beta-glucan-CRM197 vaccines, with either natural (Curd-CRM197) or synthetic linear (15mer-CRM197), or beta-(1,6)-branched (17mer-CRM197) beta-(1,3)-oligosaccharides, formulated with the human-acceptable adjuvant MF59. Curd-CRM197 and 15mer-CRM197 conjugates, which induced high titers of anti-beta-(1,3)-glucan IgG, but no antibodies against beta-(1,6)-glucan, conferred protection to mice lethally challenged with C. albicans. In contrast, the 17mer-CRM197 conjugate, which induced anti-beta-(1,6)-glucan antibodies in addition to the anti-beta-(1,3)-glucan IgG, was non-protective. These data provide some insights on beta-glucan epitope(s) mediating antifungal protection and open the way to develop a synthetic oligosaccharide vaccine against fungal diseases.

A beta-glucan-conjugate vaccine and anti-beta-glucan antibodies are effective against murine vaginal candidiasis as assessed by a novel in vivo imaging technique.

The protective capacity of a parenterally administered beta-glucan-conjugate vaccine formulated with the human-compatible MF59 adjuvant was assessed in a murine model of vaginal candidiasis. To monitor infection, an in vivo imaging technique exploiting genetically engineered, luminescent Candida albicans was adopted, and compared with measurements of colony forming units. The vaccine conferred significant protection, and this was associated with production of serum and vaginal anti-beta-glucan IgG antibodies. Vaginal IgG molecules were the likely mediators of protection as inferred by the efficacy of passive transfer of immune vaginal fluid and passive protection by an anti-beta-1,3-glucan mAb. Overall, the in vivo imaging technique was more reliable than vaginal CFU counts in assessing the extent and duration of the vaginal infection, and the consequent protection level.

Protection by anti-beta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence.

Anti-beta-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective beta-glucan epitope(s) and protection mechanisms, we studied two anti-beta-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the beta1,3-beta1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to beta1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to beta1,6- and beta1,4-linked glucose sequences in addition to beta1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-beta-glucan antibodies may affect details of the beta-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize beta-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

Candida albicans cell wall comprises a branched beta-D-(1-->6)-glucan with beta-D-(1-->3)-side chains.

The structure of immunogenic and immunomodulatory cell wall glucans of Candida albicans is commonly interpreted in terms of a basic polysaccharide consisting of a beta-D-(1-->3)-linked glucopyranosyl backbone possessing beta-D-(1-->6)-linked side chains of varying distribution and length. This proposed molecular architecture has been re-evaluated by the present study on the products of selective enzymolysis of insoluble C. albicans glucan particles (GG). High resolution 1H (400 and 700 MHz) and 13C (100 and 175 MHz) NMR analyses were performed on a soluble beta-glucan preparation (GG-Zym) obtained by GG digestion with endo-beta-D-(1-->3)-glucanase and on its high- (Pool 1) and low-molecular weight (Pool 2) sub-fractions. The resonances typical of uniformly beta-D-(1-->6)- and beta-D-(1-->3)-linked linear glucans, together with additional multiplets assigned to short-chain oligoglucosides, were detected in GG-Zym. Pool 1 (46.3+/-6.4% of GG-Zym content) consisted of beta-D-(1-->6)-linked glucopyranosyl polymers, with short beta-D-(1-->3)-branched side chains of 2.20+/-0.02 units (branching degree (DB)=0.14+/-0.03). Pool 2 was a mixture of glucose and linear short-chain beta-D-(1-->3)-oligoglucosides. Further digestion of Pool 1 by beta-D-(1-->6)-glucanase yielded a mixture of glucose and short beta-D-(1-->6)-linked, either linear or beta-D-(1-->3,6) branched, oligomers. These endoglucanase digestion patterns were consistent with the presence in C. albicans cell wall glucans of beta-D-(1-->6)-linked glucopyranosyl backbones possessing beta-D-(1-->3)-linked side chains, a structure very close to that of beta-D-(1-->6)-glucan from Saccharomyces cerevisiae yeast. This finding may provide the grounds for further elucidation of the cell wall structure and a better understanding of the biological properties of C. albicans beta-glucans.

Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans.

There is concern about the rise of antifungal drug resistance, but little is known about comparative biological properties and pathogenicity of drug-resistant strains. We generated fluconazole (FLC; CO23 RFLC)- or micafungin (FK; CO23 RFK)-resistant strains of Candida albicans by treating a FLC- and FK-susceptible strain of this fungus (CO23 S) with stepwise-increasing concentrations of either drug. Molecular analyses showed that CO23 RFLC had acquired markedly increased expression of the drug-resistance efflux pump encoded by the MDR1 gene, whereas CO23 RFK had a homozygous mutation in the FSK1 gene. These genetic modifications did not alter to any extent the growth capacity of the drug-resistant strains in vitro, either at 28 degrees C or at 37 degrees C, but markedly increased their experimental pathogenicity in a systemic mouse infection model, as assessed by the overall mortality and target organ invasion. Interestingly, no apparent increase in the vaginopathic potential of the strains was observed with an estrogen-dependent rat vaginal infection. The increased pathogenicity of drug-resistant strains for systemic infection was associated with a number of biochemical and physiological changes, including (i) marked cellular alterations associated with a different expression and content of major cell wall polysaccharides, (ii) more rapid and extensive hypha formation in both liquid and solid media, and (iii) increased adherence to plastic and a propensity for biofilm formation. Overall, our data demonstrate that experimentally induced resistance to antifungal drugs, irrespective of drug family, can substantially divert C. albicans biology, affecting in particular biological properties of potential relevance for deep-seated candidiasis.

beta-Glucan of Candida albicans cell wall causes the subversion of human monocyte differentiation into dendritic cells.

The functional consequences of treating human monocytes with purified and chemically characterized Candida albicans beta-glucan -- a major microbial pathogen associated molecular pattern -- on their differentiation into dendritic cells (DC) were investigated. We show here that beta-glucan-treated monocytes differentiated into mature DC (Glu-MoDC) with altered phenotype and functional behavior, similarly to DC derived from C. albicans germ-tubes-infected monocytes (Gt-MoDC). They failed to express CD1a and to up-regulate CD80 and DR molecules. Moreover, they produced IL-10 but not IL-12 and primed naive T cells without inducing their functional polarization into effector cells. Since C. albicans beta-glucan is a mixture of both beta-(1,3) and beta-(1,6) glucan, we investigated their relative contribution by the use of non-Candida beta-glucan structural analogs. We found that high molecular weight (MW) glucans beta-(1,6) pustulan and beta-(1,3) curdlan totally mimicked the effect of C. albicans beta-glucan, while the low MW beta-(1,3) glucan laminarin did not have any effect. Because beta-glucan is a common constituent of all fungi and is abundantly released in vivo during systemic fungal infection, this novel effect of beta-glucan has potential implications for host-parasite relationship in candidiasis and other mycoses. In particular, our data suggest that beta-glucan could bias noninfected, bystander monocytes, thus aggravating the general immunodeficiency, predisposing to systemic fungal infection.

An anti-beta-glucan monoclonal antibody inhibits growth and capsule formation of Cryptococcus neoformans in vitro and exerts therapeutic, anticryptococcal activity in vivo.

In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-beta-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of beta-glucan-exposing Candida albicans cells. Here we show that MAb 2G8 binds to the cell wall of C. neoformans and inhibits its growth to an extent comparable to that observed for C. albicans. Binding and growth inhibition were detected almost equally for encapsulated and acapsular C. neoformans strains. In addition, at subinhibitory concentrations, MAb 2G8 reduced the capsule thickness without affecting protease or phospholipase production. Acapsular fungal cells, but not encapsulated fungal cells, were opsonized by the antibody and more efficiently phagocytosed and killed by human monocytes and by murine peritoneal macrophages. A single administration of MAb 2G8 resulted in a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain. This protective effect was also detected in neutropenic mice. Overall, these findings demonstrate that cell wall beta-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.

Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and Toll-like receptor 2.

Aspergillus fumigatus is a fungal pathogen causing severe infections in immunocompromised patients. For clearance of inhaled conidia, an efficient response of the innate immune system is required. Macrophages represent the first line of defence and ingest and kill conidia. C-type lectins represent a family of receptors, which recognize pathogen-specific carbohydrates. One of them is beta1-3 glucan, a major component of the fungal cell wall. Here we provide evidence that beta1-3 glucan plays an important role for the elimination of A. fumigatus conidia. Laminarin, a soluble beta1-3 glucan and antibodies to dectin-1, a well known beta1-3 glucan receptor, significantly inhibited conidial phagocytosis. On resting conidia low amounts of surface accessible beta1-3 glucan were detected, whereas high amounts were found on small spores that appear early during germination and infection as well as on resting conidia of a pksP mutant strain. Swollen conidia also display larger quantities of beta1-3 glucan, although in an irregular spotted pattern. Resting pksP mutant conidia and swollen wild-type conidia are phagocytosed with high efficiency thereby confirming the relevance of beta1-3 glucans for conidial phagocytosis. Additionally we found that TLR2 and the adaptor protein MyD88 are required for efficient conidial phagocytosis, suggesting a link between the TLR2-mediated recognition of A. fumigatus and the phagocytic response.

Opportunistic fungi and fungal infections: the challenge of a single, general antifungal vaccine.

A vaccine made up by an algal beta-glucan (laminarin), conjugated with a protein component, protects against infections by different fungi and induces antibodies capable of inhibiting fungal growth. Although taking a premium on a common molecular theme, this remains a sort of 'cross-kingdom' vaccine because the immunizing antigen and the vaccination target belong to organisms from two different kingdoms and this is certainly the first case in the field of human vaccines. Thus, it is possible to convey in a single immunological tool the potential to protect against multiple infections, in theory all those caused by beta-glucan-expressing fungi. The generation of antibodies with the potential to directly inhibit the growth of, or kill the fungal cells also opens an exciting perspective for both active and passive vaccination in immunocompromised subjects.