Synthesis and immunological studies of linear oligosaccharides of β-glucan as antigens for antifungal vaccine development

Abstract

Antifungal vaccines have recently engendered considerable excitement for counteracting the resurgence of fungal infections. In this context, β-glucan, which is abundantly expressed on all fungal cell surfaces, functionally necessary for fungi, and immunologically active, is an attractive target antigen. Aiming at the development of effective antifungal vaccines based on β-glucan, a series of its oligosaccharide derivatives was designed, synthesized, and coupled with a carrier protein, keyhole limpet hemocyanin (KLH), to form new semisynthetic glycoconjugate vaccines. In this article, a convergent and effective synthetic strategy using preactivation-based iterative glycosylation was developed for the designed oligosaccharides. The strategy can be widely useful for rapid construction of large oligo-β-glucans with shorter oligosaccharides as building blocks. The KLH conjugates of the synthesized β-glucan hexa-, octa-, deca-, and dodecasaccharides were demonstrated to elicit high titers of antigen-specific total and IgG antibodies in mice, suggesting the induction of functional T cell-mediated immunity. Moreover, it was revealed that octa-, deca-, and dodeca-β-glucans were much more immunogenic than the hexamer and that the octamer was the best among these. The results suggested that the optimal oligosaccharide sequence of β-glucan required for exceptional immunogenicity was a hepta- or octamer and that longer glucans are not necessarily better antigens, a finding that may be of general importance. Most importantly, the octa-β-glucan-KLH conjugate provoked protective immunity against Candida albicans infection in a systemic challenge model in mice, suggesting the great potential of this glycoconjugate as a clinically useful immunoprophylactic antifungal vaccine.

Figure 1

The structure of designed β-glucan oligosaccharides and their protein conjugates 1–8

Figure 2

ELISA results of the day 48 antisera obtained with conjugates 1 (A), 2 (B), 3 (C) and 4 (D) combined with Titermax Gold adjuvant, respectively. The titers of corresponding antigen-specific antibodies are displayed. Each dot represents the antibody titer of an individual mouse, and the black bar shows the average titer.

Figure 3

Comparison of the average antibody titers of corresponding antigen-specific (A) total (anti-kappa) antibodies and (B) IgG1 antibodies in the day 48 pooled antisera of mice immunized with conjugates 1–4, respectively. Each error bar is the standard deviations for three parallel experiments. * P << 0.01 as compared to 1; # P < 0.05 as compared to 2.

Figure 4

Survival time of mice immunized with 2 compared with mice immunized with PBS after i.v. injection of C. albicans (7.5 × 10⁵ cells per mouse and 11 mice per group).

Scheme 1

Synthesis of β-glucan oligosaccharides 22–25 Reagents and conditions: a) Bu₂SnO, toluene, reflux, 6 h; then 2-naphthylmethyl bromide, CsF, DMF, 70 °C, 12 h, 72%; b) BzCl, Et₃N, CH₂Cl₂, rt, 12 h, 96%; c) DDQ, CH₂Cl₂/H₂O (18:1), rt, 8 h, 92% for 12, 95% for 14; d) AgOTf, TTBP, p-TolSCl, CH₂Cl₂, −78 °C to rt, 4 h, 90% for 13, 86% for 15 ; e) AgOTf, TTBP, p-TolSCl, CH₂Cl₂, −78 °C, rt, 4 h; then DDQ, CH₂Cl₂/H₂O (18:1), rt, 8 h, 91% for 16, 90% for 17, 87% for 18, 81% for 19, 80% for 20, 85% for 21; f) Zn, AcOH, CH₂Cl₂, 24 h, rt; then AcOH/H₂O (5:1), 60 °C, 24 h; finally NaOH, t-BuOH:H₂O, 40 °C, 24 h, 80% for 22, 88% for 23, 85% for 24, 88% for 25.

Scheme 2

Preparation of oligo-β-glucan-protein conjugates 1–8 Reagents and conditions: a) DSG, DMF and PBS buffer (4:1), rt, 4 h; b) KLH or HSA, PBS buffer, rt, 2.5 days.