Elsevier

Vaccine

Volume 30, Issue 44, 28 September 2012, Pages 6263-6269
Vaccine

A β-mannan trisaccharide conjugate vaccine aids clearance of Candida albicans in immunocompromised rabbits

https://doi.org/10.1016/j.vaccine.2012.08.010Get rights and content

Abstract

A β-(1  2)-linked mannose trisaccharide epitope that is the optimal inhibitor of two protective monoclonal antibodies specific for the Candida albicans cell wall phosphomannan was used to create a synthetic conjugate vaccine. Two injections of the trisaccharide-tetanus toxoid conjugate administered with alum induced a robust secondary antibody response in rabbits with trisaccharide specific IgG ELISA titers in excess of 1:100,000. Fluorescent labeling studies demonstrated these antibodies (i) recognized the cell wall β-mannan of C. albicans on hyphae and budding cells and (ii) C. albicans incubated with immune sera bound complement factor C3. The synthetic conjugate vaccine but not the carrier protein, tetanus toxoid reduced Candida load in vaccinated rabbits subsequently rendered leukocytopenic by injection of cyclophosphamide and then challenged with live C. albicans. These data support the contention that antibody mediated immunity plays a role in combating C. albicans infections and suggests that a surprisingly simple, readily accessible synthetic conjugate vaccine may have therapeutic potential.

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

References (49)

  • M.B. Edmond et al.

    Nosocomial bloodstream infections in United States hospitals: a three-year analysis

    Clin Infect Dis

    (1999)
  • S.B. Wey et al.

    Risk factors for hospital-acquired candidemia. A matched case–control study

    Arch Intern Med

    (1989)
  • O. Gudlaugsson et al.

    Attributable mortality of nosocomial candidemia, revisited

    Clin Infect Dis

    (2003)
  • A.W. Maksymiuk et al.

    Systemic candidiasis in cancer-patients

    Am J Med

    (1984)
  • S.W. Crawford

    Bone-marrow transplantation and related infections

    Semin Respir Infect

    (1993)
  • A. Espinel-Ingroff et al.

    Antifungal agents and susceptibility testing

  • J.E. Cutler et al.

    Advances in combating fungal diseases: vaccines on the threshold

    Nat Rev Microbiol

    (2007)
  • A. Torosantucci et al.

    A novel glyco-conjugate vaccine against fungal pathogens

    J Exp Med

    (2005)
  • H. Xin et al.

    Synthetic glycopeptide vaccines combining beta-mannan and peptide epitopes induce protection against candidiasis

    Proc Natl Acad Sci U S A

    (2008)
  • G.D. Taylor et al.

    Trends and sources of nosocomial fungemia

    Mycoses

    (1994)
  • M. Thaler et al.

    Hepatic candidiasis in cancer-patients – the evolving picture of the syndrome

    Ann Intern Med

    (1988)
  • D.L.R. Yamamura et al.

    Candidemia at selected Canadian sites: results from the fungal disease registry, 1992–1994

    Can Med Assoc J

    (1999)
  • A. Casadevall

    Antibody immunity and invasive fungal-infections

    Infect Immun

    (1995)
  • A. Casadevall et al.

    Antibody and/or cell-mediated immunity, protective mechanisms in fungal disease: an ongoing dilemma or an unnecessary dispute?

    Med Mycol

    (1998)
  • Cited by (33)

    • Prospects for anti-Candida therapy through targeting the cell wall: A mini-review

      2021, The Cell Surface
      Citation Excerpt :

      It was previously thought that the oligosaccharide component of conjugate vaccines required ≥ 10 residues, but recent studies prove that conjugate vaccines composed of shorter oligosaccharide epitopes are also protective. A vaccine candidate derived from a synthetic β-(1,2)-linked mannose oligosaccharide hapten conjugated to tetanus toxoid could induce high titer antibody, capable of opsonizing the surface of C. albicans cells after two injections in an immunocompromised rabbits model (Lipinski et al., 2012). Significant reduction of fungal burden in animals’ vital organs exhibited after vaccination.

    • Role of carbohydrate antigens in antifungal glycoconjugate vaccines and immunotherapy

      2020, Drug Discovery Today: Technologies
      Citation Excerpt :

      Synthesis of oligosaccharide ligands and the design of synthetic fungal vaccines are described in details by Krylov and Nifantiev in the review “Synthetic carbohydrate based vaccines against fungal pathogens” [29]. Bundle et al. synthesized various β-mannan oligosaccharides to study the β-mannan protective epitope and the synthetic β-(1,2) linked mannose trisaccharide was then conjugated to the tetanus toxoid (TT), showing a robust secondary antibody response in rabbits, but poor immunogenicity in mice [30,31]. Subsequently, the β-mannan trisaccharide was attached to Fba, a T-cell peptide present in a Candida albicans cell wall protein, offering protection from both saccharide and peptide side when delivered in an ex-vivo antigen-pulsed dendritic cell-based vaccine strategy [32].

    • Antifungal glycoconjugate vaccines

      2020, Recent Trends in Carbohydrate Chemistry: Synthesis and Biomedical Applications of Glycans and Glycoconjugates
    • Synthesis and immunological studies of β-1,2-mannan-peptide conjugates as antifungal vaccines

      2019, European Journal of Medicinal Chemistry
      Citation Excerpt :

      For example, β-1,2-mannan oligosaccharides, which are relatively short oligosaccharides attached as side chains to the main α-mannan backbone of C. albicans cell wall phosphomannan complex (Fig. 1) [10], were proven to be highly immunogenic. It was demonstrated that β-1,2-mannan oligosaccharides covalently linked to immunogenic carrier proteins, such as tetanus toxoid (TT) and bovine serum albumin (BSA), and to other immunologic stimulants could induce effective and specific humoral immune responses and the raised antibodies were protective [11–14]. The C. albicans cell wall proteins (CWPs), many of which are involved in fungal adhesion, aggregation and invasion to host cells [15–19], are considered as another group of promising antigens useful for antifungal vaccine design.

    View all citing articles on Scopus
    View full text