Carla Bromuro

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 β-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-β-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-β-glucan IgG fractions, as well as by administration of a β-glucan–directed IgG2b mAb. Passive protection was prevented by adsorption of antibodies on Candida cells or β-glucan particles before transfer. Anti-β-glucan antibodies bound to C. albicans hyphae and inhibited their growth in vitro in the absence of immune-effector cells. Remarkably, Lam-CRM–vaccinated mice also were protected from a lethal challenge with conidia of Aspergillus fumigatus, and their serum also bound to and markedly inhibited the growth of A. fumigatus hyphae. Thus, this novel conjugate vaccine can efficiently immunize and protect against two major fungal pathogens by mechanisms that may include direct antifungal properties of anti-β-glucan antibodies.

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-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 β-glucan epitope(s) and protection mechanisms, we studied two anti-β-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 β1,3-β1,6 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 β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-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-β-glucan antibodies may affect details of the β-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 β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.

An Anti-β-Glucan Monoclonal Antibody Inhibits Growth and Capsule Formation of Cryptococcus neoformans In Vitro and Exerts Therapeutic, Anticryptococcal Activity In Vivo

ABSTRACT In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-β-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of β-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 β-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.

Interplay between Protective and Inhibitory Antibodies Dictates the Outcome of Experimentally Disseminated Candidiasis in Recipients of a Candida albicans Vaccine

ABSTRACT 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. albicans cells. After adsorption, no antibodies specific to mannoprotein (MP)-rich extracts or secretions were detected by indirect enzyme-linked immunosorbent assay and no serum reaction with the fungal cell surface was seen in immunofluorescence assays. However, this serum had totally preserved the level of other antibodies, in particular those reacting with β-1,3 and β-1,6 glucan (GG). The hypothesis that anti-GG antibodies contributed to the passive protection was suggested by the following circumstantial evidence: (i) mice immunized with C. albicans cells treated with dithiothreitol and protease (YDP cells), which exposed GG on their surfaces and generated anti-GG but not anti-MP antibodies, were substantially protected against a lethal fungus challenge; (ii) the sera, and their immunoglobulin fractions, of mice immunized with YDP cells transferred protection to nonimmune animals; and (iii) this passive protection was substantially abolished by preadsorption on GG but not on intact cells. Overall, our findings demonstrate that some anti-Candida antibodies can block the protective potential of immune serum, a potential to which anti-GG antibodies appear to contribute. Our observations may also help explain why subjects with elevated anti-Candida antibody titers, inclusive of anti-MP and anti-GG antibodies, remain nonetheless susceptible to invasive candidiasis.

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.

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

ABSTRACT 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. albicans. Following stimulation by Y cells, monocytes produced appreciable levels of IL-12, which, upon addition of gamma interferon (IFN-γ), compared to those achievable by lipopolysaccharide (100 ng/ml) stimulation (140 ± 65 and 185 ± 80 pg/ml, respectively [mean ± standard deviation in four independent experiments]). In contrast, IL-12 production by GT cell-stimulated monocytes was much lower or absent (<5 pg/ml) and could not be brought to the level induced by Y cells by the addition of IFN-γ (30 ± 10 pg/ml in the four independent experiments above). Besides being observed as actual cytokine production, this lower response was also observed as specific IL-12 p40 mRNA transcript and was not associated with hyperproduction of the IL-12-competing cytokine IL-10. Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells of C. albicans were not phagocytized by the human monocytes. Importantly, however, Y and GT cells were equally killed by the monocytes. Thus, the virulence trait attributed to the Y-GT transition of C. albicans might also be related to the lack of induction by GT cells of a protective anticandidal immunity through defective IL-12 production.

Anti-β-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. However, the titer of anti-beta-glucan antibodies was overall about 1log lower than that of anti-mannan antibodies of the corresponding isotype. In particular, the level of anti-laminarin IgG was the lowest one, its geometrical mean titer (95% confidence interval, CI) being 1838 (1245-2714) as compared to 8157 (6067-10,931) and 3940 (2911-5332) for pustulan and Pool 1 fungal glucan, respectively. Analysis of IgG subclass composition showed that IgG2 was the prevalent subclass against any antigen, and again the concentration of anti-laminarin IgG2 was the lowest one, its geometrical mean concentration being 0.13 (0.07-0.24)microg/ml as compared to anti-pustulan and anti-Pool 1 glucan and mannan IgG2 levels, which were 0.33 (0.2-0.5), 1.35 (0.9-2.0), and 36.1 (25.2-51.3)microg/ml, respectively. These data show that anti-laminarin antibodies are present at low levels in humans as compared to other anti-beta-glucan and, mostly, anti-mannan antibodies, and suggest that a protective antifungal vaccination in humans should attempt to tip the balance of antifungal antibodies in favour of the anti-laminarin ones.

Endogenous PGE2 promotes the induction of human Th17 responses by fungal β-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. Inhibition of PGE2 synthesis or blockade of PGE2 receptors EP2 and EP4 drastically reduces IL‐23 production by β‐glucan‐activated DCs, suggesting that endogenous PGE2 amplifies IL‐23 synthesis in response to the C. albicans PAMP. Moreover β‐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 β‐glucan.

Candida albicans cell wall comprises a branched β-d-(1→6)-glucan with β-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.

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. When peripheral blood mononuclear cells from normal human subjects were stimulated in vitro with graded amounts of yeast or mycelial MP, the latter was about one order of magnitude more potent than the former in inducing lymphocyte proliferation. Following MP separation by gel permeation chromatography, an appreciable CMI response was stimulated only by the MP65-containing MP fractions, and to a degree apparently related to the amount of MP65 itself. Altogether, these data confirm our previous findings about the MP65 antigen as a major target of CMI response to C. albicans, and demonstrate that this antigen is released predominantly by the mycelial cells of the fungus in vitro.