FEMS Immunology and Medical Microbiology 41 (2004) 187–196
www.fems-microbiology.org
MiniReview
Antibody response to Candida albicans cell wall antigens
Jose L. L
opez-Ribot a, Manuel Casanova b, Amelia Murgui c, Jose P. Martınez
b,*
a
b
Department of Medicine, Division of Infectious Diseases, The University of Texas Health Sciences Center, San Antonio, TX, USA
Departamento de Microbiologıa y Ecologıa, Facultad de Farmacia, Universitat de Valencia/Estudi General, Avda. Vicente Andres Estelles,
s/n. 46100 Burjasot, Valencia, Spain
c
Departmento de Bioquımica y Biologıa Molecular, Facultad de Farmacia, Universitat de Valencia/Estudi General, Valencia, Spain
Received 24 December 2003; received in revised form 25 March 2004; accepted 25 March 2004
First published online 3 May 2004
Available online
Abstract
The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant
source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and
glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune
responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of
interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to
profoundly influence the host–parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis
and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and
glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain
cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis
of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the
cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response
triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the
serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of
infections.
Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
Keywords: Candida albicans; Cell wall; Candidiasis; Serologic response; Protective antibodies; Vaccines; Immunodiagnostic
1. Introduction
Among members of the genus Candida, the dimorphic
species Candida albicans is the most common fungal
pathogen in humans. Although this microorganism
colonizes the gastrointestinal tract, vagina and some
cutaneous areas of normal individuals, it is able to cause
a variety of infections ranging from mucosal candidiasis
(oral and vaginal) to life threatening disseminated can-
*
Corresponding author. Tel.: +34-96-354-4770/386-4770; fax: +3496-354-4299/386-4770.
E-mail address: [email protected] (J.P. Martınez).
didiasis in patients, who are predisposed to severe underlying diseases (i.e. AIDS or leukemia), impaired
phagocytic function (i.e. granulocytopenia or neutropenia), changes in host stasis (i.e. age or treatment with
broad-spectrum antibiotics) and exogenous factors (i.e.
wide-spectrum antibiotic treatment, I.V. drug use,
transplantation medicine, trauma, abdominal surgery).
Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, since as
the outermost part of the cell it mediates the host–fungus interplay [1]. Moreover, the cell wall of C. albicans is
a significant source of antigens. Indeed several immunodominant antigens in candidiasis have been characterized as cell wall components [2].
0928-8244/$22.00 Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
doi:10.1016/j.femsim.2004.03.012
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The incidence of candidiasis has dramatically increased in the last decades and bloodstream infections
due to Candida spp. are becoming an important cause of
morbidity and mortality in different types of immunocompromised patients [3]. There are limited therapeutic
alternatives to combat these infections, mainly azole
derivatives and amphotericin B. However, the toxicity
and emergence of resistance to these antifungal agents
are potential problems and highlight the need for alternative treatment strategies. Thus, there is an increasing interest in novel, immune-based prophylactic
and therapeutic approaches in candidiasis treatment. In
this context, vaccine development has a priority [4].
In this review, we focus on the most recent findings
on C. albicans cell wall components (mainly proteins
and glyco[manno]proteins; see below), which may elicit
antibody response in humans and in animal models of
experimentally-induced candidiasis, making a special
emphasis on the repercussions of these findings in developing novel techniques used in the prevention, therapy and diagnosis of candidiasis.
component, also called phosphomannoprotein or
phosphopeptidemannan complex, contains homopolymers of D -mannose (as the main component), protein,
and phosphate. The mannose polymers are linked to
proteins by N -glycosidic bonds (through two GlcNAc
[di-N -acetylchitobiose] units) through asparagine residues and by O-glycosidic, alkali-labile linkages to threonine or serine residues.
Readers who wish to know more about the basic
aspects of cell wall biology are referred to several excellent classic reviews [1,2,5–7] that cover topics such as
composition and structure, physiological role and antigenic composition.
The major cell wall components that elicit a response
from the host immune system are proteins and mannoproteins [2]. Both carbohydrate and protein moieties are
able to trigger immune responses. Cell wall proteins and
mannoproteins are able to induce a strong humoral response from the host that may include production of
some protective antibodies [2,8,9]. Thus, the cell wall
protein components may be used in the design of innovative immune-based strategies to combat candidiasis
such as vaccine development.
2. The cell wall of C. albicans: composition, structure and
biological functions
3. Host immune responses in C. albicans infections
The cell wall of C. albicans is a dynamic and complex
multilayered structure located externally to the plasma
membrane. It is responsible for maintaining the shape
that characterizes each growth form (yeast and hyphae)
of the fungus. Moreover, in addition to mediating the
initial interaction between the microorganism and the
environment, the cell wall also plays nutritional roles
and acts as a permeability barrier that protects the
protoplast against physical and osmotic injuries. The
major components (80–90%) of the C. albicans cell wall
are carbohydrates: (i) mannan or polymers of mannose
covalently associated with proteins to form glycoproteins (also referred to as mannoproteins); (ii) b-glucans,
which are branched polymers of glucose containing b1,3 and b-1,6 linkages; and (iii) chitin, which is an unbranched homopolymer of N -acetyl-D -glucosamine
(GlcNAc) containing b-1,4 bonds. Proteins and lipids
are present as minor wall constituents. b-glucans and
chitin are the structural components of the wall that
form a rigid microfibrillar network, and proteins and
glyco(manno)proteins are bound to this skeleton as well
as being present in the outer surface. Mannose polymers
(mannan) represent 40% of the total cell wall carbohydrate and are the main material of the amorphous cell
wall matrix in which the structural polymers (b-glucans
and chitin) are embedded. Although mannan does not
exist as such in the cell wall but covalently associated
with proteins, the term mannan has been also used to
refer to the main soluble immunodominant component
present in the outer cell wall layer of C. albicans. This
During the host–fungus interaction, the cell wall
triggers and modulates the immune response, which in
case of C. albicans appears to rely on a complex interplay between natural and adaptive immunity, posing
interesting challenges to the host. Cell-mediated immunity (T cells) and innate immunity (macrophages, neutrophils and natural killer (NK) cells) are considered to
be the most important line of defense against candidiasis
[10]. Albeit the controversy about the role of antibody
immunity in protection against candidiasis, recent evidence demonstrates that antibodies with defined specificities show different degrees of protection against
systemic and mucosal candidiasis [11–14], favoring the
host during the course of infection [2,8]. In general, it is
accepted that a Th1 type immune response is protective
against candidiasis, whereas a Th2 response may be
deleterious [9,15–17]. However, it must be emphasized
that during candidiasis the different arms of the immune
system (innate, cell-mediated and antibody-mediated)
can function synergistically, co-operate and modulate
each other with the final goal of fighting infection [9].
The exact mechanisms by which these antibodies
protect against Candida infection are unknown but they
are likely to include adhesion inhibition or germ tube
formation, opsonization, neutralization of virulencerelated enzymes and direct candidacidal activity [8].
Inhibition of adhesion of C. albicans to host surfaces is
one of the well-documented activities mediated by antibodies and different degrees of inhibition have been
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described with saliva, polyclonal antisera and monoclonal antibodies (Mabs) [2,18–20]. However, it has been
reported that antibodies can also exert anti-Candida
activities, which may contribute to the ability of controlling Candida multiplication. A monoclonal antiidiotypic antibody mimicking the activity of a killer toxin
from the yeast Pichia anomala has been shown to be
candidacidal in vitro [21] and to confer significant immunoprotection against mucosal candidiasis [22]. Antibodies directed against secretory aspartyl proteinases
(Sap; see below) of C. albicans have been found to be
protective in a vaginal model of candidiasis, possibly
through neutralization of its enzymatic activity (for a
review see [23]). Han and collaborators [24,25] described
an agglutinating monoclonal antibody raised against
mannan, whose mechanism of action is due to a rapid
and efficient complement deposition on the fungal cell
surface, enhancing uptake and killing by phagocytic
cells [11,24–26]. Recently, Moragues et al. [27] described
a monoclonal antibody against a polypeptidic epitope of
a high molecular mass (>200 kDa) mannoprotein, which
is predominantly expressed on the cell wall surface of C.
albicans germ tubes. This epitope displays three different
biological anti-C. albicans activities, i.e. inhibition of
adherence of C. albicans to Hep-2 and buccal epithelial
cells, inhibition of C. albicans germination and direct
candidacidal activity [27].
4. Serologic response to C. albicans cell wall protein
antigens
Clinical observations indicate that antibodies play an
important role in host defense against disseminated
candidiasis because individuals with defects in cellmediated immunity mechanisms are particularly prone
to superficial but not disseminated candidiasis [28]. In
recent years, there has been increased evidence that
some Candida-specific antibodies can be immunoprotective during infection, suggesting the viability of an
immunotherapy and/or a vaccine approach in the
treatment of candidiasis [8,11–13,28–31].
Candida albicans antigens that appear to be potential
elicitors of these immunoprotective antibody responses
are all proteins and glycoproteins that play their structural/physiological role outside the plasma membrane
barrier (secreted species). They can be divided into two
main categories: proteins whose primary destination is
considered to be the cell wall (autochthonous structural
components) and molecules whose final destination is
the exocellular environment, though they may be transiently (or even permanently) associated to the cell wall
structure [1]. Members of both categories may elicit a
host humoral response. Accordingly, antibodies to several secreted candidal proteins have been detected in
sera from both human patients and animals with ex-
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perimentally-induced candidiasis. In addition, antigenic
components released from C. albicans cells may be of
potential use as specific markers in the serodiagnosis of
systemic candidiasis [2].
A complex variety of hydrolytic enzymes such as
proteinases (secreted aspartyl proteinase; Sap), phospholipases, acid phosphatases, chitinases, esterases, glucoamylases and others can be found in culture filtrates of
C. albicans cells [1]. Although some proteins present in
the culture filtrates may represent specifically secreted
products, others may be components discarded from the
cell wall or, alternatively, released from the cytoplasm as
a consequence of spontaneous cell lysis. Several enzymes
mentioned above are putative virulence factors of C. albicans [1,23,32] and, consequently, the development of
neutralizing antibodies could represent an effective host
defense barrier. Unfortunately, except for secreted aspartyl proteinase, the host humoral response to other
candidal moieties is essentially non-examined. Additional cell wall components of potential interest due to
their acting as elicitors of immunoprotective antibody
responses are the candidal surface recognition molecules
(adhesins). Adhesins are mainly mannoproteins and play
a key role in the attachment of the fungus to the host
tissues and cells or to inert surfaces or materials [1].
4.1. Secreted aspartyl proteinases
As mentioned above, hydrolytic enzyme production
is one of the putative virulence factors of C. albicans.
While little is known about the extracellular proteinases
of most dimorphic human pathogenic fungi, the proteolytic system of C. albicans is well described. The three
most significant extracellular hydrolytic enzymes produced by this fungal species are the secreted aspartyl
proteinases (Sap), phospholipase B enzymes and lipases.
The Sap proteins, which are encoded by a family of ten
SAP genes, have been the most comprehensively studied
as key virulence determinants of C. albicans. The significance of the different putative virulence factors in C.
albicans pathogenicity can be established by determining
whether similar homologous attributes exist in other
nonpathogenic or less pathogenic yeasts such as Saccharomyces cerevisiae. Interestingly, C. albicans SAP
genes appear to have no equivalent in S. cerevisiae. The
role of Sap, phospholipases and lipases in virulence and
pathogenicity of C. albicans has been recently reviewed
[23,33,34].
Several studies using indirect-immunofluorescence
microscopy and immunogold-labeling techniques have
revealed that Sap proteins are expressed in vivo and are
localized inside the cell wall of yeast and hyphal C. albicans cells [23,32]. However, given that specific antibodies to individual Sap proteins do not exist yet,
detection and identification of individual Sap proteinases that localise in the cell wall or that are detected in
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vivo during experimental infections has been unsuccessful. Development of such antibodies would be a
valuable addition to the existing molecular techniques in
determining the localization and expression patterns of
the individual Sap species during different stages and
types of C. albicans infections [23].
Candida albicans Sap proteinases are immunogenic
and elicit mucosal and systemic antibody responses.
Although a large number of studies have described the
presence of Sap proteins during C. albicans infections,
few studies describe an antibody response to the C. albicans proteinases in human patients [23].
The inhibitory and protective effects of Sap antibodies against systemic and mucosal Candida infections remain still unclear [23]. In this regard, De Bernardis and
collaborators [11] showed that immunization with Sap2
antigen or administration of an anti-Sap2 monoclonal
antibody or anti-Sap2 antibody-containing vaginal fluids, partially protected rats against candidal vaginitis in
an experimental model. Although the mechanism of
protection was not elucidated in this study, the results
strongly suggested that Sap2 proteinase could be the
main target of the host immune response providing
protection at the mucosal sites to C. albicans infection.
One study determined the B-cell epitopes of Candida
Sap proteins, particularly Sap2, which provided some
information regarding the Sap2 epitopes recognized by
serum IgG and IgM antibodies [35]. However, the
mapping of epitopes relevant to mucosal immune protection using saliva or vaginal secretions has not been
performed in animals or humans yet. This would allow
the identification of IgA epitopes, which would be particularly important for mucosal C. albicans infections.
There is now solid evidence indicating that other
members of the Sap family play distinct roles during
different candidal infections. Given the close relationship between C. albicans pathogenesis and the immune
status of the host and the fine balance between commensalism and infection, further studies in this area are
clearly required [23].
4.2. 58-kilodalton fibrinogen binding cell wall mannoprotein (mp58)
The mp58 species is present in the cell wall extracts
from both C. albicans yeast cells and germ tubes. The
mp58 is expressed by fungal cells in vivo and in infected
tissues, acting as an immunodominant antigen during
infection [1,2]. The DNA sequence of a mp58 clone
isolated by immunoscreening using a C. albicans expression library (with antibodies generated against the
purified molecule) [36] was almost identical to the sequence of a C. albicans pH-regulated antigen gene
(PRA1) [37]. This clone also showed homology to a
family of immunodominant antigens present in different
Aspergillus species, which suggests that antigenicity ra-
ther than binding properties may be the primary role of
this cell wall component [37]. Consequently, delineation
of the antibody responses to mp58 could be important in
the development of novel immunity-based prophylactic,
therapeutic and diagnostic techniques in candidiasis
management. In this context, continuous B-cell epitopes
on C. albicans mp58 protein species have been recently
identified using a complete set of overlapping dodecapeptides, i.e. synthesized based on the DNA sequence of
the mp58 encoding gene (FBP1/PRA1) [38]. This epitope-scanning study revealed the presence of multiple
immunoreactive continuous B-cell epitopes in the mp58
protein sequence. Regions of increased reactivity included both the amino and carboxy termini of the mature protein and four internal amino acid domains.
Besides, a synthetic peptide corresponding to the last ten
amino acids at the C-terminus was found to be immunogenic when injected into mice after being coupled to a
carrier protein [38]. The presence of antibodies against
the mp58 in sera from patients with different types of
candidiasis has been also investigated by immunoblotting. All sera from patients with confirmed systemic
candidiasis reacted to this antigen, whereas none of the
sera from control individuals and patients suffering from
superficial candidiasis contained detectable levels of
antibodies against the mp58 [39]. These results suggest
the potential usefulness of mp58 as a marker in the
serodiagnosis of systemic candidiasis [2].
4.3. Heat shock proteins
All living organisms, including C. albicans, have the
ability to respond to sudden changes of temperature by
increasing the production of a set of proteins, the
so-called heat shock proteins (hsps). Hsps are also immunodominant antigens and the major targets of host
immune response during different type of infections [40].
Their ubiquitous nature and their high degree of homology among species pose interesting challenges in the
host immune system. Firstly, the presence of epitopes
shared by several infectious agents may provide the
immune system with a universal signal for infection,
therefore antibodies against these conserved regions
could provide some natural resistance to infections,
bridging the gap between innate and acquired immunity.
Secondly, epitopes shared by the parasite and the host
may trigger deleterious autoimmune responses [40].
An interesting feature of some hsps of C. albicans is
that they are not confined to the intracellular compartment and are also present at the cell (wall) surface.
Family members of the Hsp70 of C. albicans and the 47kDa fragment of Hsp90 have been reported to be present in the cell wall (for a review see [1]). This superficial
location implies that the antigenic determinants are
naturally exposed and may be responsible for the immunodominant nature of C. albicans hsps.
J.L. Lopez-Ribot et al. / FEMS Immunology and Medical Microbiology 41 (2004) 187–196
4.3.1. Hsp90
Immunoblotting experiments with sera from patients
suffering from systemic candidiasis showed the existence
of a 47-kDa immunodominant antigen in whole cell
extracts of the fungus [41]. This 47-kDa antigen was
further identified as a heat-stable breakdown product of
Hsp90 whose cell wall location has recently been confirmed [42]. Antibodies to the 47-kDa antigen are present in serum samples of a high proportion of patients
with chronic mucocutaneous candidiasis (CMC) and
AIDS. Patients recovering from systemic candidiasis
produce a major antibody response to the 47-kDa
component, whereas fatal cases have little antibody or
falling titers [41,43]. An enzyme-linked immunodot assay (ELISA) using affinity-purified antibodies against
the 47-kDa moiety were capable to detect circulating
antigen in the serum of patients [41]. Using this assay,
systemic candidiasis was detected in 77% of neutropenic
patients and in 87% of non-neutropenic patients. The
sensitivity and specificity of detection was improved
compared to other commercially available products [41].
In a systemic candidiasis mouse model, pretreatment
with sera from two infected patients containing antibodies against Hsp90 resulted in a decreased mortality
rates [44]. Epitope mapping of C. albicans Hsp90 with
patients’ sera revealed that patients recovering from
systemic candidiasis produce antibodies against both
fungal-specific and conserved epitopes of Hsp90 [45]. In
particular, a highly conserved epitope NKILKVIRKNIVKK was recognized by sera from all patients with
antibodies against the 47-kDa antigen [45]. Homologous
epitopes have been identified in a range of Candida spp.
and Aspergillus fumigatus [46]. When given prophylactically, a murine monoclonal antibody raised against
this epitope reduced mortality in an invasive candidiasis
mouse model [44]. It was concluded that autoantibodies
against Hsp90 can protect against infection. Mycograb
(NeuTec Pharma plc.) is a human genetically recombinant antibody against fungal Hsp90. It consists of
the antigen-binding variable domains of the heavy and
light chains linked together to create a recombinant
protein, which is expressed in Escherichia coli. It does
not have a Fc component. Mycograb shows activity
against a wide range of yeast species and acts synergistically with amphotericin B both in vitro and in vivo
[47]. Based on these properties, Mycograb is now being
assessed in a multinational placebo-controlled trial in
patients with invasive candidiasis.
4.3.2. Hsp70
Several members of the Hsp70 family of proteins
have been reported in C. albicans [1,48–50]. Ssa1p and
Ssa2p, the main cell wall-located immunogens of C. albicans, are capable of inducing antibody and cell-mediated immune responses in mice and humans infected by
C. albicans [1,2,51]. The presence of Hsp70s, which ap-
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pears to be highly immunogenic, in the cell wall and
surface, implies that the antigens are readily exposed to
the host immune defenses. Accordingly, it has been reported that serum samples from both healthy volunteers
and patients suffering from candidiasis contained antibodies against the C-terminal portion of Hsp70 although vaccination with the recombinant gave no
protection against the disease but conferred some enhancement of Candida infection in a murine model of
candidiasis [2,51]. The major antibody response to systemic infection with C. albicans detected in CBA/H mice
was raised against 96-kDa antigen, which was not induced by heat shock and against a 75-kDa candidal hsp,
which appears to be a member of the Hsp70 family.
Given the high homology among the different members
of the Hsp70 family, the antibody responses reported by
different authors could result from a combination of
antibodies against the different fungal Hsp70s and the
Hsp70s from other organisms. These reactivity patterns
are also likely to be the result of a combination of antibodies against conserved epitopes and unique epitopes
to individual C. albicans Hsp70s [2].
4.4. Glycolytic enzymes
Glycolytic enzymes are abundant immunodominant
antigens in C. albicans infections. The presence of glycolytic enzymes in the cell wall of C. albicans has been
reported [1,2]. Thus, enolase is associated with glucan in
the inner layers of the cell wall and in culture supernatants; phosphoglycerate kinase (PGK) is located in the
cell wall and at the outermost cell surface; and a wallassociated form of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is located at the most external cell
surface layer [1,2]. Additional cell wall-associated glycolytic and fermentation enzymes including fructosebisphosphate aldolase, triose phosphate isomerase,
phosphoglycerate mutase, pyruvate kinase, pyruvate
decarboxylase and alcohol dehydrogenase have been
recently described [52].
Despite the complex antigenic composition and considerable heterogeneity of the antibody responses to
candidal antigens in humans, several immunodominant
antigens have been identified, being enolase the most
prominent of them [1,2]. Sera from patients with disseminated candidiasis contained circulating antibodies
against candidal enolase. Consequently, circulating antienolase antibodies may have potential value for the
diagnosis of candidiasis and different tests have been
devised to detect such antibodies in sera from patients
with proven candidiasis. Statistical analysis of the results
obtained using an ELISA-based method with purified C.
albicans enolase as target indicated that the assay was
able to discriminate between invasive infection and simple colonization, although the sensitivity of the test was
low (for a review see [2]). On the contrary, antigenemia
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with enolase from C. albicans was detected using an
ELISA assay in a murine model of disseminated candidiasis in the absence of fungemia and correlated with
deep tissue infection. In this context, a method was
commercialized (Directigen; Becton Dickinson) for antigen capture using anti-enolase monoclonal antibody
and subsequent detection with an anti-enolase polyclonal
antibody. This test, which is not any longer commercially
available, was very specific (96%) but its sensitivity was
low (only 54% in patients with proven deep tissue invasion). However, the sensitivity improved to 85% by testing multiple samples for antigen detection in patients
with proven deep tissue infection and to 64% in proven
cases of candidemia. Thus, C. albicans enolase antigenemia was a suitable marker for deep tissue invasion even
in the absence of fungemia [2,53].
5. Serologic response to C. albicans cell wall carbohydrate
antigens
As already mentioned, b-glucans, chitin and mannan
are the three major polysaccharide components in the
cell wall of C. albicans. Although b-glucans are present
in greater abundance compared to mannan in the wall of
this fungal species, they are immunologically less active
[2]. However, it has been recently described that antibodies reacting to b-1,3 and b-1,6 glucan (bG) contributed to the passive protection against Candida infection.
Moreover, mice immunized with C. albicans cells, which
were treated with dithiothreitol and protease, i.e. with
bG exposed on their surfaces, generated anti-bG antibodies but not anti-mannoprotein antibodies and were
substantially protected against a lethal fungus challenge.
Furthermore, the sera and immunoglobulin fractions of
C. albicans immunized mice transferred protection to
non-immune animals [54]. In contrast, b-glucan has recently been identified as a receptor of the killer toxin
produced by the yeast P. anomala [55,56]. The killer
toxin glycoprotein is by itself of no practical use due to
its instability in the physiological milieu of human tissues and its antigenicity and toxicity. However, peptides,
derived from the sequence of a recombinant, singlechain and antiidiotypic antibody representing the
internal image of the wide-spectrum yeast killer toxin
active domain, exhibited therapeutic activity in experimental mucosal and systemic candidiasis models [57].
Hence, this indirectly supports the initial contention that
killer toxin antiidiotypic antibodies could exert their
cytocidal action on a large variety of glucan-containing
pathogenic microorganisms [58].
Mannan is mostly found as large N-linked polymers
containing several hundred mannose residues associated
with high-molecular-weight mannoprotein (HMWM)
species, although smaller N-linked moieties and/or Olinked mannooligosaccharides are associated with gly-
coproteins of lesser size. Antibodies to these carbohydrate immunodeterminants are readily detectable in
serum samples [1,2]. Considerable effort has been made
to determine the chemical structure of mannan and to
define the epitopes present in this wall component that
may account for serospecificity [2]. In this context, the
antigenic specificity of serotypes A and B of C. albicans
appears to be determined by structural peculiarities of
the mannan carbohydrate moiety in strains belonging to
a particular serotype. Oligomannosyl-side chains containing both b-1,2 and a-1,2 linkages were characterized
as serotype A-specific epitopes (factor 6), while b-1,2linked oligomannosyl-side chains attached to phosphate
serve as the major common epitopes (factor 5) of both
C. albicans serotypes (see [2] for a review about the
different antigenic factors found in serotypes A and B of
C. albicans). In addition, sequences of b-1,2-linked
mannose residues located at the C. albicans cell surface
may act as adhesins [59], inducing the generation of
protective antibodies [11,60] and contributing to virulence (for a review see [1,2]). Thus, many Mabs generated against C. albicans have been shown to react to
b-1,2-oligomannosides [60–62]. In addition, Han and
collaborators [24,25] demonstrated that a mannanbased vaccine formulation elicited antibodies that
protected against disseminated systemic and vaginal
candidiasis and that a monoclonal agglutinating
IgM (MAb 6.1) specific for a C. albicans cell surface
b-1,2-mannotriose was also protective in both types of
infection [12,13,60,63].
Anti-mannan antibodies have been shown to be
ubiquitous in human sera, presumably because the immune system can be stimulated as a result of colonization by C. albicans in the absence of disease [2,7,64]. The
applications for a diagnosis of disseminated candidiasis
by detection of (i) circulating mannan upon infection or
(ii) anti-mannan antibodies will be discussed later in this
review. Although most of the recent serological tests for
invasive candidiasis are focused on the detection of
specific circulating cell wall-bound or cytoplasmic candidal antigens other than mannan, special emphasis has
been made on the detection of mannan by different
procedures. These included counterimmunoelectrophoresis, radioimmunoassay, ELISA and latex-agglutination (for a review see [1,64]). In general, all these studies
have evidenced variable sensitivity and specificity values
depending on the immunological method employed in
each case.
6. Immunological strategies to prevent candidiasis
Despite the contradictory results about the role of
humoral response in mucosal Candida infections, the
fact that protective antibodies against Candida exist and
can be effective in animal models supports its immuno-
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therapeutical use. According to Casadevall [8], ‘‘protective’’, ‘‘nonprotective’’ and ‘‘indifferent’’ antibodies
comprise any pool of antigen-specific antibodies and
humoral immunity in protecting against infection is
dictated by those antibodies present in the highest concentration. Even though antibodies are readily present
in human vaginal secretions, the concentration of protective antibodies may not be high enough to be effective, whereas in rat vaginal secretions such protective
antibodies appear to be abundant. Inasmuch as vast
variety of antibody production and/or protection is evident from the experimental models or clinical settings,
formulation of antibody-producing vaccines using an
immunogen capable to induce protective antibodies
would be extremely advantageous in the prevention of
candidiasis in immunocompetent and T-cell-immunocompromised individuals. However, accomplishment of
this might be difficult given the recently reported interplay between protective and inhibitory antibodies,
which indeed dictates the outcome of experimentally
disseminated candidiasis in recipients of a C. albicans
vaccine, thus explaining why subjects with elevated antibody titers against cell wall-bound Candida antigens
remained nonetheless susceptible to invasive candidiasis
[54].
Cell wall components may be used in the development of innovative immune-based strategies to combat
candidiasis. Vaccines containing live microorganisms
are associated with significant risk of infection even if
the microorganism is attenuated. Subunit vaccines
consisting of single immunogenic molecules or even
specific epitopes from the fungus should be safer. Indeed, the development of subunit vaccines is presently
the main strategy that is being evaluated in the prevention of infectious diseases. The next generation of vaccines will be based on rational design approaches and
better understanding of the microbial factors required
for virulence and the nature of the immune response to
infection [65,66]. In case of C. albicans, vaccine development should be provided by an increased knowledge
on the identity, expression, role in pathogenesis and
antigenic characteristics of individual cell wall components. Although different studies showed different levels
of protection by vaccination using purified C. albicans
proteins, C. albicans mannan, membrane fractions and
whole heat-killed cells [12,13,51,63,67–69] it could be
predicted that a successful vaccine formulation would
include multiple synthetic peptides corresponding solely
to protective epitopes able to induce both humoral and
cellular immune responses.
7. Serodiagnosis of invasive candidiasis
The experimental diagnosis of invasive Candida infections is another controversial aspect due to its diffi-
193
cult evaluation using clinical criteria. Consequently,
culture techniques and serodiagnostic tests for antigen
and antibody detection have been employed as a laboratory complement in diagnosis. However, blood cultures containing Candida species generally exhibited low
sensitivity and the tests to evaluate marker antigens need
further refinement of their sensitivity and specificity to
be a valuable tool in guiding clinical treatment decisions
[64,70].
Since mannan is the major circulating antigen in patients presenting an invasive candidiasis, several standard serological tests detecting antibodies against
Candida mannan have been devised. However, given that
anti-mannan antibodies are ubiquitous in human sera,
these tests have usually failed in discriminating between
colonization and invasive candidiasis, leading therefore
to poor specificity values (false positives). Furthermore,
its low sensitivity (false negatives) registered in severely
immunosuppressed patients could also be due to a delayed, reduced or an absent immunological response (see
[2] for further information). In spite of more than fifty
years experience in this field, to date the development of
reliable tests using antibody detection requires further
investigation. Early studies using immunological methods in the diagnosis used typically whole cells or crude
antigenic preparations that were difficult to standardize.
In addition, most of these antigenic preparations contained cell wall mannan and anti-mannan antibodies,
which are almost ubiquitous in human serum samples
[2,7,64]. These early immunological studies used techniques such as latex agglutination, complement fixation,
immunodiffusion, counterimmunoelectrophoresis and
indirect immunofluorescence. In general, these methods
lacked sensitivity and specificity and were of limited diagnostic value [71]. Recently, various groups have reevaluated the diagnostic value of anti-mannan antibody
detection. Several oligomannosidic epitopes were identified to react with antibodies in human sera [61]. Based
on this, the newer Platelia Candida antibody test (Bio
Rad, Redmon, Washington) uses a standardized mannan preparation coating ELISA plates to capture circulating anti-mannan antibodies in sera from patients,
which reported specificity and sensitivity values of 94%
and 53%, respectively [72]. When performed in combination with a mannan antigen detection test, the technique gave a specificity of 93% and a sensitivity of 80%
[72,73]. These combined tests were assayed to early diagnose systemic C. tropicalis infection in neutropenic
adults [74], but further evaluation and validation are
necessary to confirm its utility.
To overcome the lack of specificity in the detection of
anti-Candida antibodies, efforts were also focused on the
detection of circulating Candida antigens. Accordingly,
Gentry and collaborators [75] developed a reverse passive latex agglutination assay (commercialized as the
Cand–Tec test at Ramco Laboratories, Houston, Texas)
194
J.L. Lopez-Ribot et al. / FEMS Immunology and Medical Microbiology 41 (2004) 187–196
to detect a structurally uncharacterized 56 °C heat-labile
antigen of C. albicans [75]. It used latex particles that
were sensitized with polyclonal sera from rabbits immunized with heat-killed C. albicans blastoconidia. The
antigen seems to be a mixture of cell wall glycoproteins
and may represent a neo-antigen after processing by
human cells. The test did not require dissociation of the
immune complexes. Even though being easy to perform,
the test gave poor sensitivity and specificity and its use in
the diagnosis of invasive candidiasis was limited (for
further information see [2]).
Due to the ubiquitous nature of anti-mannan antibodies, mannan often circulates as immunocomplexes
and dissociation from the complexes is required for its
optimal detection [2,7,71,76]. Sensitivities of 23–100%
and specificities of 92–100% have been reported using
ELISA assays in mannan detection [76]. The majority
of these analyses considered mannan to be a single
molecule and did not take into account its chemical
and immunological complexity. A commercial system
to detect Candida mannan in serum, the Pastorex
Candida test (Sanofi Diagnostics Pasteur, Marnesla-Coquette, France) appeared promising [77] until
Gutierrez and collaborators [78] registered 0% sensitivity while conducting a prospective clinical trial. Lack
of sensitivity is due to the rapid clearance of the antigen from patients’ sera and the test properties (latex
agglutination). It is noteworthy to point out that the
sensitivity of the Pastorex test was improved when serial assays using multiple consecutive serum samples
were performed [79]. Lately, a double-sandwich ELISA
method using the same monoclonal antibody (EBCA1)
employed in the Pastorex Candida test has been developed. This assay improved the detection limit up to
0.1 ng of mannan/ml and resulted in increased sensitivity. This test constitutes now the basis for the Platelia Candida antigen test and has reported specificity
and sensitivity values of 98% and 40%, respectively
[72,73]. As mentioned before, the utility of this assay is
enhanced when used in combination with the antibody
detection test.
The data currently available shows that Candida antigen detection tests may serve as markers in invasive
disease but they need further refinement to improve their
sensitivity and specificity to be valuable in guiding
clinical treatment decisions. The development of reliable
detection tests is often difficult due to the use of crude
preparations of candidal antigens, which cannot be well
standardized and allow to a good experimental reproducibility among laboratories. Hence, identification,
characterization and detection of defined fungal antigens may provide a suitable procedure in the diagnosis
of invasive candidiasis and availability of a simple, reliable, and easy-to-perform serological assay in the diagnosis of systemic Candida infections is urgently
needed [2,46,64,71].
Acknowledgements
We are grateful to Dr. W.B. Van Leeuwen and to the
reviewers for their suggestions. J.P.M. was supported by
grants BMC2001-2975 from the Programa Nacional de
Promoci
on General del Conocimiento, Ministerio de
Ciencia y Tecnologıa, and grant CTIDIB/2002/3 from
the Subsecretaria de la Oficina de Ciencia y Tecnologıa
de la Generalitat Valenciana, Valencia, Spain. J.L.L.-R.
gratefully acknowledges grant support from the
National Institutes of Health. J.L.L.-R. is the recipient
of a New Investigator Award in Molecular Pathogenic
Mycology from the Burroughs Wellcome Fund.
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