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.

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

Candidemia (bloodstream invasion by 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%).

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.

Comparative analysis of plant-produced, recombinant dimeric IgA against cell wall β-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 β-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.

Antibodies against a β-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 β1,3- and β1,6-glucans, as well as the β-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 β1,3-glucan, but higher antibody titers against β1,6-glucan and MP65, than non-candidemic subjects. The elevated levels of anti-β1,6-glucan and -MP65 antibodies found in candidemic patients were independent on the patient risk category, APACHE II score, presence of co-morbidities, β1,3-glucanemia level, Candida isolate, and antifungal treatment.

A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing β-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-β-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.

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-β-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 β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform.

Plant production of anti-β-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 β-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.

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 β-glucan, a major fungal PAMP purified from Candida albicans, stimulates human DCs to secrete a pro-Th17 cytokine pattern. Notably, β-glucan induces PGE2 production, which has been shown to play a pivotal role in Th17 cell expansion.

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.

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.

Anti-beta-glucan antibodies in healthy human subjects.

Previous data by our group demonstrated the antifungal efficacy of a vaccine consisting of laminarin (beta-(1,3)-glucan), conjugated with diphtheria toxoid, which generated protective anti-laminarin antibodies in mice. In this paper, we sought for the presence, isotype and subclass composition of natural anti-laminarin antibodies in an unselected population of human healthy subjects, in a comparison with antibodies directed against beta-(1,6)-glucan (pustulan) and branched beta-(1,3/1,6)-glucan (Pool 1) and mannan from Candida albicans. Almost all subjects showed detectable levels of anti-beta-glucan antibodies, with IgG largely prevailing on IgM, little, if any, IgA and no IgE.

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).

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.

A novel glyco-conjugate vaccine against fungal pathogens.

To generate a vaccine to protect against a variety of human pathogenic fungi, we conjugated laminarin (Lam), a well-characterized but poorly immunogenic beta-glucan preparation from the brown alga Laminaria digitata, with the diphtheria toxoid CRM197, a carrier protein used in some glyco-conjugate bacterial vaccines. This Lam-CRM conjugate proved to be immunogenic and protective as immunoprophylactic vaccine against both systemic and mucosal (vaginal) infections by Candida albicans. Protection probably was mediated by anti-beta-glucan antibodies as demonstrated by passive transfer of protection to naive mice by the whole immune serum, the immune vaginal fluid, and the affinity-purified anti-beta-glucan IgG fractions, as well as by administration of a beta-glucan-directed IgG2b mAb.

Gene expression of 70 kDa heat shock protein of Candida albicans: transcriptional activation and response to heat shock.

CaHSP70 (70 kDa heat shock protein) is a highly immunogenic protein of Candida albicans. We have studied heat shock-induced expression of the CaHSP70 gene under germ tube-inductive and non-inductive conditions. The CaHSP70 upstream regulatory region was cloned and sequenced.

Interplay between protective and inhibitory antibodies dictates the outcome of experimentally disseminated Candidiasis in recipients of a Candida albicans vaccine.

Mice immunized with heat-inactivated, whole yeast-form cells (Y cells) of Candida albicans developed intense, specific humoral and cell-mediated immune responses. However, they were modestly protected against a lethal challenge by the fungus, and their sera did not confer passive protection upon nonimmunized animals. Surprisingly, this immune serum conferred an elevated degree of passive protection to normal and SCID mice when preadsorbed on whole C.

Defective induction of interleukin-12 in human monocytes by germ-tube forms of Candida albicans.

Yeast (Y) to germ-tube (GT) transition of Candida albicans is considered a putative virulence trait. On the other hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen. We studied IL-12 production by human monocytes cocultured in vitro with Y or GT forms of C.

A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis.

The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects.

Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans.

By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa).

Differential release of an immunodominant 65 kDa mannoprotein antigen from yeast and mycelial forms of Candida albicans.

The release of mannoprotein (MP) antigen from Candida albicans grown at 28 degrees C (yeast form) or 37 degrees C (mycelial form), and the ability of each released material to stimulate a cell-mediated immune (CMI) response by human lymphocytes in vitro, were studied. Overall, the mycelial cells released more MP per unit of dry mass increase and the released material was relatively enriched with MP constituents of lower molecular mass with respect to the material released from yeast cells. Moreover, the mycelial MP contained a 65 kDa component (MP65) which was the largely predominant MP recognized by a rabbit anti-mycelium antiserum.

Identification of a 65-kDa mannoprotein as a main target of human cell-mediated immune response to Candida albicans.

To identify molecular targets of anticandidal cell-mediated immunity (CMI) in humans, a highly immunogenic mannoprotein fraction (MP-F2) of Candida albicans was studied. SDS-PAGE and gel-permeation chromatography separated MP-F2 into polydisperse mannoproteins of > 200-31.5 kDa.

Glucan synthesis and its inhibition by cilofungin in susceptible and resistant strains of Candida albicans.

The lipopeptide antimycotic agent, cilofungin, at a dose of 20 micrograms ml-1, inhibited beta 1-3 glucan synthesis in a drug-susceptible strain (3153; minimum inhibitory concentration (MIC) < 1 microgram ml-1) as well as in a drug-resistant strain of Candida albicans (CA-2, derived from 3153 by nitrosoguanidine mutagenesis; MIC > 50 micrograms ml-1). This was demonstrated for both whole cells under growing and non-growing conditions, and during protoplast regeneration. However, time-effect experiments, during growth of a CA-2 culture initially exposed to an inhibitory dose of cilofungin, showed that this strain was able to progressively regain both glucan synthesis and a growth rate comparable to that of cultures that had not been treated with the drug.

Biochemical and antigenic characterization of mannoprotein constituents released from yeast and mycelial forms of Candida albicans.

Yeast or mycelial cultures of Candida albicans released comparable amounts of Concanavalin A-reactive mannoprotein material after 24-h of growth, and in both cases this material showed a qualitatively similar SDS-PAGE pattern, with predominantly polydisperse constituents of high molecular mass. The two secretion mixtures also showed similar reactivity by ELISA with serum from a subject with high titre anti-Candida antibodies, as well as with an anti-Candida hyperimmune antiserum raised in rabbits. Both secreted extracts were separated by ion-exchange chromatography into two major fractions (designated F1 and F2), each containing mannoprotein antigens recognized by rabbit and human sera, although the immunoreactivity of the two fractions from the two growth forms was not uniform.