A β-mannan trisaccharide conjugate vaccine aids clearance of Candida albicans in immunocompromised rabbits
Highlights
► A synthetic β-(1 → 2)-linked mannose trisaccharide Candida albicans conjugate vaccine. ► Two injections with alum induce a robust secondary antibody response in rabbits. ► Antibodies recognized the cell wall β-mannan on hyphae and budding cells. ► C. albicans incubated with immune sera bound complement factor C3. ► The vaccine reduces C. albicans fungal burden in leukocytopenic rabbits.
Introduction
Candida species are the fourth most common cause of hospital-acquired bloodstream infections in the United States [1], [2]. These infections are associated with attributable mortality rates that have ranged from 38% between 1983 and 1986 [3] to 49% in the same institutions between 1997 and 2001 [4]. Mucocutaneous candidal infections are increasingly problematic in patients with acquired immunodeficiency syndrome (AIDS). Candidemia also occurred in 11–27% of patients with prolonged neutropenia from bone marrow transplantation or leukemia with associated mortality as high as 95% [5]. Invasive fungal infections had a 20% incidence in liver recipients within 100 days of transplantation and Candida accounted for 82% of all infections [6]. Vaccine strategies to increase host resistance are attracting attention [7], [8], [9], [10], [11] because mortality due to Candida infections remains high despite antifungal therapy [12], [13], [14], [15].
Experimental and clinical evidence shows the promise of acquired immunity in enhancing host defense mechanisms against the disease and the protective potential of antibody [16], [17], [18], [19], [20]. A successful vaccine in combination with antibiotic therapy could provide protection for individuals at high risk; such as patients scheduled to receive abdominal surgery; bone marrow, kidney or heart transplantations, immunosuppressive cancer therapy and those exposed to long-term hospitalization. Women may derive benefit from a safe vaccine that provides long-lasting immunity, since in North America, there are several million new cases annually of candidal vaginitis in otherwise healthy adult females [21]. Of those, 6–7% experience recurrent disease and some cases are recalcitrant to current antifungal therapy [22], [23].
Several lines of evidence support protection by antibody and the potential utility of vaccination [17], [24], [25], [26], [27]. Candida albicans cell wall polysaccharide antigens, the cell wall phosphomannan (Fig. 1) and the cell wall β-glucans are amongst the most promising candidate antigens for a vaccine. For example, monoclonal antibodies generated after immunization of mice with a liposome-encapsulated β-mannan conferred passive protection in both a vaginal Candida infection model [32] and against disseminated candidiasis [33]. Active immunity against C. albicans was achieved by immunization with a cell wall extract of the phosphomannan rich in β-mannan covalently coupled to bovine serum albumin (BSA) and antisera of immunized animals provided passive protection to naïve animals against disseminated disease [34]. When mouse dendritic cells were pulsed in vitro with β-mannotriose conjugated to a selection of six T-cell peptides and then administered to mice a range of activities from protection to enhancement of infection depending on the T-cell peptide of the glycoconjugate were observed [11]. If the glycopeptide with the most protective activity was conjugated to tetanus toxoid, dendritic cell stimulation could be avoid and a protective response elicited without the need for adjuvant [35]. Two distinct β-glucan glycoconjugate vaccines are also effective in affording antibody-mediated protection against C. albicans and Aspergillus fumigatus [10], [20].
The two protective monoclonal antibodies (IgM and IgG) described above that were produced in response to the liposomal β-mannan vaccine [32], [36] were optimally inhibited by a disaccharide and a trisaccharide [37]. Surprisingly tetra-, penta- and hexasaccharides were progressively worse inhibitors. These data are consistent with an antibody site that accepts a disaccharide or trisaccharide but binds larger homo-oligomers with sharply decreasing affinity, most likely due to the restricted conformational flexibility of the β-mannan, with the result that saccharide residues immediately adjacent to the binding site have steric clashes with the protein weakening or preventing binding of larger oligosaccharides [38].
The oligosaccharide component of a conjugate vaccine is often required to have a length in excess of ten residues [39], [40] but there are recent examples of protective conjugate vaccines composed of relatively short oligosaccharide epitopes [41], [42]. Based on our inhibition data we speculated that the β-(1 → 2) mannose trisaccharide might be a functional B-cell epitope. We report here a potentially viable conjugate vaccine based on a readily accessible, synthetic trisaccharide epitope conjugated to tetanus toxoid and its evaluation in a neutropenic rabbit model of C. albicans infection.
Section snippets
Glycoconjugates
Tetanus toxoid was obtained from the Statens Serum Institute (Copenhagen – Denmark). The synthesis and characterization of the trisaccharide and its conjugation to proteins was performed as previously reported [43]. The BSA conjugate was dialyzed against water and freeze-dried prior to use as an ELISA capture antigen. Tetanus toxoid conjugate was dialyzed against PBS, concentrated to 2 mg/mL, passed through a 0.22 μm sterile filter and stored at 4 °C. Hapten incorporation levels were determined by
Preparation of glycoconjugates
Conjugation of synthetic trisaccharide 1 to BSA and tetanus toxoid afforded a BSA glycoconjugate bearing from 9 to 13 residues of the trisaccharide hapten and a tetanus toxoid conjugate vaccine with 8–12 attached trisaccharide epitopes (Fig. 2) as determined by MALDI-TOF analysis [43].
Induction of anti-β-mannan antibodies
Two groups each of 16 rabbits were vaccinated with the glycoconjugate and with unconjugated tetanus toxoid each absorbed on alum. Each rabbit in the conjugate vaccine group received two injections 21 days apart of
Discussion
Two protective IgM and IgG β-mannan specific monoclonal antibodies described by Cutler's group [34], [36] exhibited closely similar β-mannan inhibition profiles [37]. This is consistent with binding sites that are complimentary to a β-mannan trisaccharide and points to the importance of this epitope as a protective antigen. These observations inspired us to consider the potential of a trisaccharide conjugate vaccine, since a fully synthetic vaccine of this type would be chemically defined and
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