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Comparative Study
. 2005 Sep 5;202(5):597-606.
doi: 10.1084/jem.20050749.

A novel glyco-conjugate vaccine against fungal pathogens

Antonella Torosantucci  1 , Carla Bromuro, Paola Chiani, Flavia De Bernardis, Francesco Berti, Chiara Galli, Francesco Norelli, Cinzia Bellucci, Luciano Polonelli, Paolo Costantino, Rino Rappuoli, Antonio Cassone
Affiliations
Comparative Study

A novel glyco-conjugate vaccine against fungal pathogens

Antonella Torosantucci et al. J Exp Med. .

Abstract

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. Passive protection was prevented by adsorption of antibodies on Candida cells or beta-glucan particles before transfer. Anti-beta-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-beta-glucan antibodies.

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Figures

Figure 1.
Figure 1.
Partial 600 MHz NMR proton spectra of (a) laminarin polysaccharide and (b) laminarin-CRM197 conjugate recorded at 25°C. Molecular structure of laminarin and labels are shown for the NMR peak assignment.
Figure 2.
Figure 2.
Vaccination with the Lam-CRM conjugate induces antibody-mediated anti-Candida protection in a murine experimental model of disseminated infection. (a) Anti-β-glucan IgG and IgM titers in Lam-CRM–vaccinated mice. The graph shows the ranges of ELISA titers against the indicated antigens measured in five groups of 6–12 mice (for a total of 46 animals) independently immunized with Lam-CRM. MP, mannoproteins; Pust, pustulan. (b) Survival rates of mice immunized with Lam or CRM or with the Lam-CRM conjugate, as compared with nonimmunized mice (Adj), after a lethal systemic challenge with C. albicans (cumulative data from three independent experiments and 28 mice per group). (c) Fungal burden in the kidneys from four Lam-CRM–vaccinated or four control CRM-vaccinated mice on day 2 after i.v. infection with C. albicans. (d) Number of fungal CFU in kidneys from naive mice given a single administration of anti-Lam-CRM, anti-CRM, or nonimmune (Adj) serum 2 h before an i.v. challenge with C. albicans. Data are from three independent experiments with a total of nine mice per group. (e) Reversal of the passive protection after serum adsorption with Candida cells. The experiment was performed with three mice per group. (f) Effect of the passive vaccination with Protein A affinity–separated fractions of the Lam-CRM serum on Candida kidney load. Data are from three mice per group. Some details on isotype and subclass of anti-β-glucan immunoglobulin in pool A, B and C are given in Fig. S2.
Figure 3.
Figure 3.
Antibodies raised by the vaccination with the Lam-CRM conjugate significantly accelerate the resolution of a rat vaginal infection with C. albicans. (a) Vaginal fungal clearance in rats vaccinated intravaginally with the Lam-CRM conjugate, with unconjugated CRM or Lam, or left immunized (CT) were intravaginally infected with C. albicans. Figure shows mean values plus SD of CFU counts from individual animals, measured in two independent experiments with a total of 10 animal per group (b) Anti-β-glucan antibodies in vaginal fluids from Lam-CRM–vaccinated rats. Results are expressed as mean OD 405 nm plus SEM readings of ELISA assays against the indicated solid-phase β-glucan antigens by two pooled vaginal fluids, obtained in two independent experiments from a total of 10 rats. OD values measured from CRM-vaccinated control rats (usually <0.15) have been subtracted. Pust, pustulan. (c) Course of the experimental vaginal infection in rats administered whole or β-glucan particle–adsorbed vaginal fluids from Lam-CRM–vaccinated animals, as compared with rats given fluconazole therapy. Results are expressed as mean CFU plus SD measured from five animals per group.
Figure 4.
Figure 4.
Specificity and anti-Candida protective activity by mAb 2G8, a murine anti-β-glucan, mAb. (a) ELISA reactivity of mAb 2G8 with various β-glucan or non–β-glucan polysaccharides. (b) Candida load in the kidney of mice given as a single i.p. administration of the mAb 2G8 or of the irrelevant anti-CRM mAb (Control) and subjected to a systemic challenge with C. albicans. Results are from three independent experiments with a total of 10 mice/group. (c) Vaginal clearance of C. albicans in rats (five per group) administered the anti-β-glucan mAb 2G8, the irrelevant anti-CRM mAb, or none and intravaginally infected with the fungus. Data are mean values of CFU counts from individual vaginal fluids. SD was always <15%.
Figure 5.
Figure 5.
Expression of anti-Lam-CRM serum– and mAb 2G8– reactive epitopes on the cell surface of C. albicans and A. fumigatus. (a and d) Indirect immunofluorescence staining of isolated β-glucan cell wall ghosts of C. albicans with the anti-Lam-CRM serum (a) or the mAb 2G8 (d). (b and e) Pattern of immunofluorescence reactivity in C. albicans germ tubes stained with the anti-Lam-CRM serum (b) or the mAb 2G8 (e). The b1 inset shows the preferential staining of the hyphal cell. (c and f) Hyphal filaments of C. albicans reacted with anti-Lam-CRM serum (c) or mAb 2G8 (f). (g) Immunofluorescence staining by the anti-Lam-CRM serum on a germinated conidium of A. fumigatus. (h and i): Reactivity of the anti-Lam-CRM serum (h) and of the mAb 2G8 (i) with A. fumigatus hyphae. Negative control staining of fungal cells is shown in Fig. S2.
Figure 6.
Figure 6.
In vitro growth of C. albicans and A. fumigatus is significantly restricted by the anti-β-glucan antibodies, and Lam-CRM vaccination significantly prolongs the survival of mice subjected to a systemic challenge with A. fumigatus. (a) CFU number in C. albicans cultures grown overnight in the presence of whole or 1:10 diluted anti-Lam-CRM or control sera. Data are from one representative experiment out of three performed with similar results and represent mean values from triplicate independent determinations. (b) Dose–response C. albicans CFU reduction by the anti-β-glucan, 2G8 and the irrelevant anti-CRM, mAbs. The figure shows a representative experiment as for Fig. 6 a. (c) Effect of anti-Lam-CRM and control anti-CRM serum on the in vitro growth of A. fumigatus, as evaluated by 3H-glucose incorporation assays. Mean count per minute values from four independent experiments, each performed in triplicate, are shown. For additional experimental details, see Fig. S3 and Table S1. (d) Survival of mice vaccinated with the Lam-CRM conjugate or with CRM only and infected i.v. with A. fumigatus. Data are from a single experiment with eight mice per group.
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