# Use of Recombinant Antigens for the Diagnosis of Invasive Candidiasis

Authors: Ana Laín; Natalia Elguezabal; Elena Amutio; Iñigo Fernández de Larrinoa; María Dolores Moragues; José Pontón
Journal: Clinical and Developmental Immunology
 (2008-02-19)

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## Abstract

Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against differentCandida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis.


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## Body

## 1. Introduction

Invasive candidiasis is one of the
leading infective complications found in immunocompromised and critically ill
patients, and is associated with high morbidity and mortality. The diagnosis of
disseminated candidiasis remains difficult, since signs and symptoms of the
disease are unspecific and many patients with invasiveCandida infections (up to 50%) show negative results by blood
culture [1, 2], which, in addition, may only become positive late in the
infection. Other standard techniques for the diagnosis of invasive candidiasis,
including microscopic visualization of the infecting fungus and histopathology,
usually lack specificity or sensitivity, or may require invasive procedures
that can not be accomplished due to the critical conditions of many of these
patients. Therefore, the diagnosis of invasive candidiasis should be based on
the combined interpretation of the patient’s risk factors to develop this
disease, clinical manifestations (usually the presence of fever that persists
despite the administration of wide spectrum antibiotics), and laboratory data
(blood cultures, antibody titers).Serological diagnosis of human
infections is based on two strategies: the detection of antigens from the
infecting agent in host’s samples, and the detection of the antibody response
elicited by these antigens in the host. This second approach is being widely
studied in the diagnosis of many infectious diseases caused by a large number
of microorganisms, including fungi [3, 4], bacteria [5, 6], or viruses [7].Serological tests have been subject
of much study but in many cases they can be difficult to interpret. The
investigations concerningCandida antigen detection in serum samples show that these methods are in general quite
specific but most antigens are often rapidly cleared from the circulation, so
that antigen detection tests may lack the desired level of sensitivity required
for a definitive diagnosis. Other serological tests sometimes require
hardworking procedures that make them practically impossible to establish as
routine techniques in the clinical laboratory [8, 9]. The specific antibody
response that is usually induced in patients with invasive Candida infections can help in the diagnosis. However, antibody
detection methods can also have limitations. Circulating antibodies may occur
in normal individuals as a result of commensal colonization of mucosal
surfaces, leading to the presence of false positive results. Thus a serological
test based on the detection of anti-Candida antibodies should be able to distinguish between the level of antibodies
detected in healthy individuals and in patients without invasive candidiasis
from those detected in patients with an invasive Candida infection. Another problem with the clinical usefulness of
antibody detection is the occurrence of false negative results in
immunocompromised patients who may produce low or undetectable levels of
antibodies. Anyhow, all these problems could be solved, at least in part, by
the use of suitable antigens and the development of more sensitive antigen or
antibody detection techniques.Traditionally, the techniques
developed to detect antibodies in patients with invasive fungal infections have
made use of crude extract mixtures, composed of a large number of fungal
antigens [10]. Although these extracts were easy to obtain, they did not allow
standardization and facilitated cross reactivity between antibodies of patients
with different invasive fungal and bacterial infections. However, over the last
decades, molecular biology techniques have allowed the production of
recombinant antigens. These antigens can be produced in a prokaryotic host in
large amounts making standardization processes easier, and eliminating the
cross-reactivity due to posttranslational modifications.The advances in molecular biology,
genomics, proteomics, and bioinformatics are helping in the design of new
strategies for the development of more sensitive and specific diagnostic tests.
The selection of new biomarkers for the diagnosis of systemic candidiasis has
been strongly supported by the combination of proteomics and bioinformatics
[11]. The recent completion of Candida albicans genome sequence has also
been achieved thanks to advances in molecular biology and genomics [12]. All
the above mentioned tools can be employed to select more specific fungal
antigens and to produce them as recombinant proteins of high quality to be used
for serological diagnosis.In the present article, we review
the recombinant antigens that have been investigated so far for their potential
use in the diagnosis of invasive candidiasis.


## 2. Recombinant Antigen Methodology

When using molecular biology
techniques to produce a recombinant antigen for future use in serodiagnosis,
the first step is to choose the expression system and design the DNA
constructs. An excellent source for the design of the DNA constructs in order
to achieve the isolation of the gene of interest is theCandida Genome
Database (CGD), where the entire sequence of the gene can be assessed by
inserting its name (http://www.candidagenome.org). After having access to the
sequence of the gene, knowledge of the protein to be expressed is important.
Features to be considered are glycosylation states and sites, occurrence of
disulphide bonds, posttranslational modifications, or overall stability of the
protein.The election of the vector and host
strain is also important. Most commercial plasmids work well containing
powerful promoters that show a minimal basal expression under certain
conditions and that suffer a fast and dramatic induction upon a change in growth
conditions of the host. Normally T7 bacteriophage promoter is used for the
expression of recombinant proteins inEscherichia
coli, being induced by IPTG. High levels of expression are normally
achieved with this option. There is a large selection of vectors commercially
available so  it should be easy to find
one with a suitable combination of markers, cloning sites, and epitopes or tags
that can be used as targets for affinity purification. For this reason, when
choosing the vector one should consider the size of the insert, the
purification strategy to be used once the protein is expressed, the restriction
sites included in the polylinker, or the antibiotic resistance for selection,
among other factors.E. coli is a
suitable host for proteins that are not glycosylated or when glycosylation is
not necessary. This is the case in some mannosylated antigens of Candida used in the diagnosis of
invasive candidiasis, where glycosylation is not desired since cross-reactivity
has been observed with antibodies that are directed to glycosylated regions of
proteins, rendering them less specific. E.
coli BL21 is a protease-deficient host strain yielding intact full-length
recombinant proteins [13], and it has been used in many recombinant antigen
production strategies [14–16]. Once the sequence of the gene
is known and the vector is chosen, primers can be designed in order to isolate
the entire gene or a specific domain of interest (e.g., the
amino-terminal fragment of the antigen). These primers can be used in a
polymerase chain reaction (PCR) to isolate the desired part of the gene from
genomic DNA of Candida if there are
no introns or from a cDNA library. The design of the primers should include
restriction sites, matching some of the sites in the polylinker region
contained in thevector. After
digestion of both insert and plasmid, subsequent ligation should lead to a
stable expression vector that can be cloned. Upon antibiotic selection,
positive colonies containing the vector should be selected. Screening of
potential PCR-amplified clones for proper insert orientation and sequence
analysis of positive clones must be performed, in order to confirm that a
proper reading frame has been obtained and that no errors introduced by PCR are
present. Once positive clones have been selected, induction of protein
expression can take place. At this point, it is important to select more than one colony since not all positive colonies are able to overexpress the
recombinant protein and yield can vary from clone to clone. Recombinant protein
yield and solubility are also highly dependent on the specific protein
sequence, as well as on the vector, host cell, and culture conditions used.
Different host growth conditions can be tested to set up the optimal conditions
for the induction of protein expression.The final step in recombinant
antigen technology involves purification of the protein. This process may be
simplified if affinity purification tags have been included. For
high-throughput processing, His-tag has been studied for purification in
soluble and insoluble conditions [17]. The incorporation of a His-tag allows for
generic single-step purification using nickel-nitrilotriacetate immobilized on
a resin. His-tag strategy has been employed for Hwp1 purification [15].
Hemagglutinin influenza virus (HA)-tag is also widely used for affinity
purification and easy detection by
immunoblotting using a anti-HA antibody. HA-tag
strategy was used in Ece1 purification [3, 18].After the recombinant, protein is
purified and its value in diagnosis can be assessed by different immunological
methods, such as ELISA, immunoblotting, or indirect immunofluorescence.


## 3. Candida Antigens and their Usefulness in the Diagnosis of Invasive Candidiasis

### 3.1. Enzymes

Antibodies against severalCandida enzymes have been detected in
sera from patients with systemic candidiasis. Some of them are available as
recombinant products. Among them, enolase is the most studied and has shown a
high diagnostic value (Table 1).Table 1
Candida recombinant antigens tested for serodiagnosis of invasive candidiasis.
AntigenMolecular mass (kDa)ReferenceEnzymesEnolase48[14, 16, 19]Sap—[20, 21]Hypha specific antigensEce135[18]Hyr190[18]N-Als350[18]Hwp161[15]


#### 3.1.1. Enolase

Enolase is an immunodominant
glycolytic enzyme, present in the cytoplasm and, in minor amounts, in the inner
layers of the cell wall ofC. albicans [22], either in the yeast form or
in the mycelial phase of the fungus. Previous work showed that this enzyme can
elicit an antibody response in the infected host [23–26]. Several authors have
studied the diagnostic utility of the detection of antibodies against enolase
with promising results. van Deventer et al. [25] studied 76 patients with
invasive candidiasis, 46 of which were immunocompromised. The detection of
anti-enolase antibodies by ELISA yielded sensitivity and specificity values of
50% and 86%, respectively, in the immunocompetent group, and 53% and 78% in the
immunocompromised group, suggesting that it is possible to detect anti-enolase
antibodies even in immunodepressed patients. Mitsutake et al. [24] detected the presence of antibodies against C. albicans enolase in 27 patients with
systemic candidiasis by immunoblotting. The specificity of the test was 95% and
the sensitivity 62.9%, but reached 92.5% when the number of samples tested was
increased. In addition, they observed anti-enolase antibodies in infections
caused by several species of the genus Candida,
including C. albicans, C. parapsilosis, C. tropicalis, C.
guilliermondii, and C. glabrata.
However, both studies were performed with a C.
albicans native enolase preparation, which is difficult to obtain and
standardize. A recombinant enolase could therefore constitute an advance in the
study of the immune response to this enzyme and its diagnostic usefulness.A number of studies have
demonstrated that recombinant enolase is useful to detect antibodies in
serological tests. Sundstrom and Aliaga [27] obtained a recombinant enolase
which was employed to study humoral and cellular responses in vitro [19]; and Sandini et al. [14] detected antibodies to a
recombinant enolase by immunoblotting in sera from seven patients with invasive
candidiasis. Laín et al. [16]
have recently described the performance of a new simple diagnostic ELISA test, Candida Enolasa ELISA IgG test, based on
the detection of antibodies against a recombinant enolase in 98 patients. Since
immunocompromised individuals have an increased risk for developing invasive
candidiasis, and they may produce lower antibody titers, the usefulness of this
test was assayed in two different populations, 47 immunocompromised, and 51
immunocompetent patients. The results were similar in both groups of patients,
with sensitivity values of 78.9% and 82.6%, and specificity values of 89.3% and
78.6%, respectively, confirming the utility of the detection of antibodies
against recombinant enolase for the diagnosis of invasive candidiasis, even in
immunocompromised patients. In agreement with previous studies [24], we were
able to detect anti-enolase antibodies in sera from patients infected with
different Candida species.


#### 3.1.2. Secreted Aspartyl Proteinases

Secreted aspartyl proteinases (Saps)
are proteins secreted byC.albicans and other members of the genus.
They have been described as immunodominant antigens and virulence factors
associated with adherence and tissue invasion and dissemination in animal
models of infection [28, 29] (Table 1). Na and Song [20] described an ELISA for
the detection of antibodies against Sap1 antigen in 33 patients with invasive
candidiasis, reaching a sensitivity and specificity of 69.7% and 76%,
respectively. More recently, Yang et
al. [21] produced a hybrid phage displaying the Sap epitope VKYTS and
studied its reactivity against sera from mice and patients with systemic C. albicans infection, by immunoblotting
and ELISA assay. The sensitivity and specificity were 77% and 88.3% in an
animal model of invasive candidiasis, respectively, and 60% and 85%,
respectively, in patients with invasive candidiasis.


### 3.2. Hyphal-Specific Candida Antigens

Candida albicans is a fungus that can grow
either as yeast or in a hyphal form. The reversible transition from yeast to
mycelium or morphogenesis is thought to be a key factor for the virulence of
this organism in vivo [30, 31].
Since the mycelial form is associated with the invasive phase of the fungus,
the detection of antibodies directed against antigens specifically expressed in
this phase may provide a way for differentiating a simple colonization from a
disseminated Candida infection. Our
group has previously reported that the detection of antibodies specifically
directed to antigens expressed on the C.
albicans germ tube surface by indirect immunofluorescence has a good
diagnostic value, with a sensitivity of 79–89% and a specificity of 91–100%,
on both competent and immunocompromised patients [32–36]. However, this method
requires the adsorption of the sera with heat-killed C. albicans yeast, in order to eliminate the reactivity with the
antibodies directed to the yeast form [37]. These antimannan antibodies are
commonly found in humans and are responsible for the majority of the false
positive results observed in many serological studies. The production of
hypha-specific recombinant antigens in a prokaryotic host would eliminate the
need to adsorbe the sera to remove the antimannan antibodies, since the
recombinant protein will be nonglycosylated. A number of antigens specifically
expressed on the C. albicans germ
tube have been recently identified. We hereby describe the potential of some of
them for Candida serodiagnosis.


#### 3.2.1. Hyr1 Protein

Hyphally regulated protein 1, Hyr1,
[38] is a C. albicans germ tube
specific cell wall glycoprotein of 937 aminoacids, with a
glycosylphosphatidylinositol (GPI) motive. Hyr1 is specifically expressed in
the mycelial form and its heterologous expression in Saccharomycescerevisiae did not show an obvious phenotype. Consequently, it was suggested to have a
structural role in the C. albicans cell wall architecture.Laín et al. [18, 39] studied the reactivity of a recombinant Hyr1
protein with sera from 36 patients with invasive candidiasis and 45 control
patients by immnunoblotting. The sensitivity and specificity values of the
assay were 39.1% and 100%, respectively, (Table 2). The detection of anti-Hyr1
antibodies by an ELISA assay increased the sensitivity to 58.3%, but reduced
the specificity to 82.2% in the diagnosis of invasive candidiasis (Table 2).Table 2
Diagnostic usefulness of the detection of antibodies against Hyr1, Ece1, Als3, and Hwp1 by immunoblotting and ELISA assay [18] (PPV: positive predictive
value, NPV: negative predictive value).
Sensitivity%Specificity%PPV%NPV%ImmunoblottingHyr139.110010076.3Ece134.893.372.773.7Als326.193.366.771.2Hwp127.897.583.362.3ELISA assayHyr158.382.272.471.2Ece155.680.069.062.5Als341.777.860.062.5Hwp188.982.680.090.2


#### 3.2.2. Ece1 Protein

Extent of cell ellongation 1, Ece1
[40], is another C. albicans germ
tube specific protein. An ECE1 null
mutant C. albicans strain did not
show morphological changes and the authors concluded that this protein is not
essential for cell elongation or hyphal formation, despite the strict
association of Ece1 expression with the mycelial form of C. albicans.By using a similar approach to that
followed to study the diagnostic potential of the Hyr1, theECE1 gene has been expressed in E. coli and used in both immunoblotting
and ELISA assays to detect antibodies in patients with invasive candidiasis
[18, 41]. As observed with the Hyr1 antigen, the results obtained by ELISA
showed higher sensitivity but lower specificity than those obtained by
immunoblotting in the diagnosis of invasive candidiasis (Table 2).


#### 3.2.3. Als3 Protein

TheALS (agglutinin-like sequence) gene family of Candidaalbicans encodes
cell surface glycoproteins implicated in adhesion of the organism to host
surfaces. ALS genes conform a basic
three-domain structure that includes a relatively well conserved domain of 1299
to 1308 nucleotides (433 to 436 aminoacids), a central domain of variable
length consisting entirely of a tandemly repeated 108-bp motif, and a
C-terminal domain of variable length and sequence that encodes a serine-threonine
rich protein, with a GPI motif. Presently, eight genes in the ALS family have been reported in the
literature [42]. ALS genes are
differentially regulated by physiological conditions such as changes in the
growth medium, morphogenesis, or the growing phase of the fungus. The
expression of these proteins is correlated with Candida infection, and there has also been found evidence of these
proteins in other Candida species,
such as C. dubliniensis and C. tropicalis [43]. Among the Als
family, Als3 is implicated in
endothelial and epithelial adhesion and it is specifically expressed in the C.albicans germ tube [44]. Several authors have established a functional model for the Als proteins, where the N-terminal domain is
exposed to the cell surface, and the central and C-terminal domain are
integrated in the cell wall layers [42, 45]. The N-terminal domain of the Als3 that has been described as a binding motif
[44] is the most exposed part of the protein and therefore to the immune system
of the host. However, the detection of antibodies against the N-terminal
recombinant fragment of the Als3 for the
diagnosis of invasive candidiasis has shown a very poor diagnostic value (Table 2) [18].


#### 3.2.4. Hwp1 Protein

Hyphal wall protein 1 (Hwp1),
described by Staab et al. [46], is a glycoprotein specifically
expressed in the cell wall surface of the hyphae of C. albicans, which has been studied as an important adhesin,
required as a virulence factor in invasive candidiasis [47]. Naglik et al. [48]
analysed by RT-PCR the presence of HWP1 mRNA in human subjects who were positive for C. albicans culture and had oral or vaginal symptoms, as well as in
asymptomatic patients, by RT-PCR. They also detected antibodies against a
recombinant fragment of the Hwp1, Hwp1N13, consisting of the transglutaminase
substrate domain, previously produced by Staab et al. [49]. HWP1 mRNA
was found to be correlated with the presence of C. albicans in both asymptomatic carriers and in cases of oral and
vaginal candidiasis, although the quantity of mRNA in candidiasis probably
exceeded that revealed in asymptomatic conditions. Antibody titers (IgG and
IgA) in oral-culture-negative carriers and candidiasis cases were found to be
equivalent, suggesting that Hwp1 is a common target of host responses to C. albicans that are recognized to
result from long-term colonization.Since this protein has a
surface-exposed N-terminal domain that binds antibodies, while the carboxyl
terminus is most probably covalently integrated in the cell wall [46], Laín et al. [15] selected the N-terminal
fragment of the Hwp1 (161 aminoacids) for serological studies in patients with
invasive candidiasis. While detection of antibodies by immunoblotting showed
very poor sensitivity and negative predictive values (27.8% and 62.3%,
resp.), detection of antibodies against the N-terminal fragment of Hwp1
by ELISA notably increased the diagnostic usefulness, with sensitivity,
specificity, positive and negative predictive values of 88.9%, 82.6%, 80.0%,
and 90.2%, respectively. Interestingly, these results were very similar to
those obtained by the detection of antibodies to the C. albicans germ tube by indirect immunofluorescence, a technique
that has been previously reported to be of diagnostic utility [35, 50].
However, the detection of antibodies against the Hwp1 recombinant fragment is
easier to perform, since it does not require the adsorption of the patient’s
serum and allows both an objective measurement and automatization of the test.
In addition to that, the ELISA test was able to detect invasive candidiasis in
patients infected by non-C. albicans species, including C. parapsilosis, C. tropicalis, C. utilis, C. glabrata, and C. dubliniensis, although this antigen was initially described as
specific of the germ tube of C. albicans.
The reasons for this reactivity are not known at present, but it has been
reported that HWP1 mRNA may also
arise from pseudohyphal and yeast growth forms in C. albicans [48], and an ORF of 1266 pb with homology to C. albicans Hwp1 has been found in C. dubliniensis [51]. We have also
observed that the region encoding the first 85 residues of the Hwp1 fragment
shows homology with the sequence of proteins of non albicans Candida species
[15].


## 3.1. Enzymes

Antibodies against severalCandida enzymes have been detected in
sera from patients with systemic candidiasis. Some of them are available as
recombinant products. Among them, enolase is the most studied and has shown a
high diagnostic value (Table 1).Table 1
Candida recombinant antigens tested for serodiagnosis of invasive candidiasis.
AntigenMolecular mass (kDa)ReferenceEnzymesEnolase48[14, 16, 19]Sap—[20, 21]Hypha specific antigensEce135[18]Hyr190[18]N-Als350[18]Hwp161[15]


### 3.1.1. Enolase

Enolase is an immunodominant
glycolytic enzyme, present in the cytoplasm and, in minor amounts, in the inner
layers of the cell wall ofC. albicans [22], either in the yeast form or
in the mycelial phase of the fungus. Previous work showed that this enzyme can
elicit an antibody response in the infected host [23–26]. Several authors have
studied the diagnostic utility of the detection of antibodies against enolase
with promising results. van Deventer et al. [25] studied 76 patients with
invasive candidiasis, 46 of which were immunocompromised. The detection of
anti-enolase antibodies by ELISA yielded sensitivity and specificity values of
50% and 86%, respectively, in the immunocompetent group, and 53% and 78% in the
immunocompromised group, suggesting that it is possible to detect anti-enolase
antibodies even in immunodepressed patients. Mitsutake et al. [24] detected the presence of antibodies against C. albicans enolase in 27 patients with
systemic candidiasis by immunoblotting. The specificity of the test was 95% and
the sensitivity 62.9%, but reached 92.5% when the number of samples tested was
increased. In addition, they observed anti-enolase antibodies in infections
caused by several species of the genus Candida,
including C. albicans, C. parapsilosis, C. tropicalis, C.
guilliermondii, and C. glabrata.
However, both studies were performed with a C.
albicans native enolase preparation, which is difficult to obtain and
standardize. A recombinant enolase could therefore constitute an advance in the
study of the immune response to this enzyme and its diagnostic usefulness.A number of studies have
demonstrated that recombinant enolase is useful to detect antibodies in
serological tests. Sundstrom and Aliaga [27] obtained a recombinant enolase
which was employed to study humoral and cellular responses in vitro [19]; and Sandini et al. [14] detected antibodies to a
recombinant enolase by immunoblotting in sera from seven patients with invasive
candidiasis. Laín et al. [16]
have recently described the performance of a new simple diagnostic ELISA test, Candida Enolasa ELISA IgG test, based on
the detection of antibodies against a recombinant enolase in 98 patients. Since
immunocompromised individuals have an increased risk for developing invasive
candidiasis, and they may produce lower antibody titers, the usefulness of this
test was assayed in two different populations, 47 immunocompromised, and 51
immunocompetent patients. The results were similar in both groups of patients,
with sensitivity values of 78.9% and 82.6%, and specificity values of 89.3% and
78.6%, respectively, confirming the utility of the detection of antibodies
against recombinant enolase for the diagnosis of invasive candidiasis, even in
immunocompromised patients. In agreement with previous studies [24], we were
able to detect anti-enolase antibodies in sera from patients infected with
different Candida species.


### 3.1.2. Secreted Aspartyl Proteinases

Secreted aspartyl proteinases (Saps)
are proteins secreted byC.albicans and other members of the genus.
They have been described as immunodominant antigens and virulence factors
associated with adherence and tissue invasion and dissemination in animal
models of infection [28, 29] (Table 1). Na and Song [20] described an ELISA for
the detection of antibodies against Sap1 antigen in 33 patients with invasive
candidiasis, reaching a sensitivity and specificity of 69.7% and 76%,
respectively. More recently, Yang et
al. [21] produced a hybrid phage displaying the Sap epitope VKYTS and
studied its reactivity against sera from mice and patients with systemic C. albicans infection, by immunoblotting
and ELISA assay. The sensitivity and specificity were 77% and 88.3% in an
animal model of invasive candidiasis, respectively, and 60% and 85%,
respectively, in patients with invasive candidiasis.


## 3.1.1. Enolase

Enolase is an immunodominant
glycolytic enzyme, present in the cytoplasm and, in minor amounts, in the inner
layers of the cell wall ofC. albicans [22], either in the yeast form or
in the mycelial phase of the fungus. Previous work showed that this enzyme can
elicit an antibody response in the infected host [23–26]. Several authors have
studied the diagnostic utility of the detection of antibodies against enolase
with promising results. van Deventer et al. [25] studied 76 patients with
invasive candidiasis, 46 of which were immunocompromised. The detection of
anti-enolase antibodies by ELISA yielded sensitivity and specificity values of
50% and 86%, respectively, in the immunocompetent group, and 53% and 78% in the
immunocompromised group, suggesting that it is possible to detect anti-enolase
antibodies even in immunodepressed patients. Mitsutake et al. [24] detected the presence of antibodies against C. albicans enolase in 27 patients with
systemic candidiasis by immunoblotting. The specificity of the test was 95% and
the sensitivity 62.9%, but reached 92.5% when the number of samples tested was
increased. In addition, they observed anti-enolase antibodies in infections
caused by several species of the genus Candida,
including C. albicans, C. parapsilosis, C. tropicalis, C.
guilliermondii, and C. glabrata.
However, both studies were performed with a C.
albicans native enolase preparation, which is difficult to obtain and
standardize. A recombinant enolase could therefore constitute an advance in the
study of the immune response to this enzyme and its diagnostic usefulness.A number of studies have
demonstrated that recombinant enolase is useful to detect antibodies in
serological tests. Sundstrom and Aliaga [27] obtained a recombinant enolase
which was employed to study humoral and cellular responses in vitro [19]; and Sandini et al. [14] detected antibodies to a
recombinant enolase by immunoblotting in sera from seven patients with invasive
candidiasis. Laín et al. [16]
have recently described the performance of a new simple diagnostic ELISA test, Candida Enolasa ELISA IgG test, based on
the detection of antibodies against a recombinant enolase in 98 patients. Since
immunocompromised individuals have an increased risk for developing invasive
candidiasis, and they may produce lower antibody titers, the usefulness of this
test was assayed in two different populations, 47 immunocompromised, and 51
immunocompetent patients. The results were similar in both groups of patients,
with sensitivity values of 78.9% and 82.6%, and specificity values of 89.3% and
78.6%, respectively, confirming the utility of the detection of antibodies
against recombinant enolase for the diagnosis of invasive candidiasis, even in
immunocompromised patients. In agreement with previous studies [24], we were
able to detect anti-enolase antibodies in sera from patients infected with
different Candida species.


## 3.1.2. Secreted Aspartyl Proteinases

Secreted aspartyl proteinases (Saps)
are proteins secreted byC.albicans and other members of the genus.
They have been described as immunodominant antigens and virulence factors
associated with adherence and tissue invasion and dissemination in animal
models of infection [28, 29] (Table 1). Na and Song [20] described an ELISA for
the detection of antibodies against Sap1 antigen in 33 patients with invasive
candidiasis, reaching a sensitivity and specificity of 69.7% and 76%,
respectively. More recently, Yang et
al. [21] produced a hybrid phage displaying the Sap epitope VKYTS and
studied its reactivity against sera from mice and patients with systemic C. albicans infection, by immunoblotting
and ELISA assay. The sensitivity and specificity were 77% and 88.3% in an
animal model of invasive candidiasis, respectively, and 60% and 85%,
respectively, in patients with invasive candidiasis.


## 3.2. Hyphal-Specific Candida Antigens

Candida albicans is a fungus that can grow
either as yeast or in a hyphal form. The reversible transition from yeast to
mycelium or morphogenesis is thought to be a key factor for the virulence of
this organism in vivo [30, 31].
Since the mycelial form is associated with the invasive phase of the fungus,
the detection of antibodies directed against antigens specifically expressed in
this phase may provide a way for differentiating a simple colonization from a
disseminated Candida infection. Our
group has previously reported that the detection of antibodies specifically
directed to antigens expressed on the C.
albicans germ tube surface by indirect immunofluorescence has a good
diagnostic value, with a sensitivity of 79–89% and a specificity of 91–100%,
on both competent and immunocompromised patients [32–36]. However, this method
requires the adsorption of the sera with heat-killed C. albicans yeast, in order to eliminate the reactivity with the
antibodies directed to the yeast form [37]. These antimannan antibodies are
commonly found in humans and are responsible for the majority of the false
positive results observed in many serological studies. The production of
hypha-specific recombinant antigens in a prokaryotic host would eliminate the
need to adsorbe the sera to remove the antimannan antibodies, since the
recombinant protein will be nonglycosylated. A number of antigens specifically
expressed on the C. albicans germ
tube have been recently identified. We hereby describe the potential of some of
them for Candida serodiagnosis.


### 3.2.1. Hyr1 Protein

Hyphally regulated protein 1, Hyr1,
[38] is a C. albicans germ tube
specific cell wall glycoprotein of 937 aminoacids, with a
glycosylphosphatidylinositol (GPI) motive. Hyr1 is specifically expressed in
the mycelial form and its heterologous expression in Saccharomycescerevisiae did not show an obvious phenotype. Consequently, it was suggested to have a
structural role in the C. albicans cell wall architecture.Laín et al. [18, 39] studied the reactivity of a recombinant Hyr1
protein with sera from 36 patients with invasive candidiasis and 45 control
patients by immnunoblotting. The sensitivity and specificity values of the
assay were 39.1% and 100%, respectively, (Table 2). The detection of anti-Hyr1
antibodies by an ELISA assay increased the sensitivity to 58.3%, but reduced
the specificity to 82.2% in the diagnosis of invasive candidiasis (Table 2).Table 2
Diagnostic usefulness of the detection of antibodies against Hyr1, Ece1, Als3, and Hwp1 by immunoblotting and ELISA assay [18] (PPV: positive predictive
value, NPV: negative predictive value).
Sensitivity%Specificity%PPV%NPV%ImmunoblottingHyr139.110010076.3Ece134.893.372.773.7Als326.193.366.771.2Hwp127.897.583.362.3ELISA assayHyr158.382.272.471.2Ece155.680.069.062.5Als341.777.860.062.5Hwp188.982.680.090.2


### 3.2.2. Ece1 Protein

Extent of cell ellongation 1, Ece1
[40], is another C. albicans germ
tube specific protein. An ECE1 null
mutant C. albicans strain did not
show morphological changes and the authors concluded that this protein is not
essential for cell elongation or hyphal formation, despite the strict
association of Ece1 expression with the mycelial form of C. albicans.By using a similar approach to that
followed to study the diagnostic potential of the Hyr1, theECE1 gene has been expressed in E. coli and used in both immunoblotting
and ELISA assays to detect antibodies in patients with invasive candidiasis
[18, 41]. As observed with the Hyr1 antigen, the results obtained by ELISA
showed higher sensitivity but lower specificity than those obtained by
immunoblotting in the diagnosis of invasive candidiasis (Table 2).


### 3.2.3. Als3 Protein

TheALS (agglutinin-like sequence) gene family of Candidaalbicans encodes
cell surface glycoproteins implicated in adhesion of the organism to host
surfaces. ALS genes conform a basic
three-domain structure that includes a relatively well conserved domain of 1299
to 1308 nucleotides (433 to 436 aminoacids), a central domain of variable
length consisting entirely of a tandemly repeated 108-bp motif, and a
C-terminal domain of variable length and sequence that encodes a serine-threonine
rich protein, with a GPI motif. Presently, eight genes in the ALS family have been reported in the
literature [42]. ALS genes are
differentially regulated by physiological conditions such as changes in the
growth medium, morphogenesis, or the growing phase of the fungus. The
expression of these proteins is correlated with Candida infection, and there has also been found evidence of these
proteins in other Candida species,
such as C. dubliniensis and C. tropicalis [43]. Among the Als
family, Als3 is implicated in
endothelial and epithelial adhesion and it is specifically expressed in the C.albicans germ tube [44]. Several authors have established a functional model for the Als proteins, where the N-terminal domain is
exposed to the cell surface, and the central and C-terminal domain are
integrated in the cell wall layers [42, 45]. The N-terminal domain of the Als3 that has been described as a binding motif
[44] is the most exposed part of the protein and therefore to the immune system
of the host. However, the detection of antibodies against the N-terminal
recombinant fragment of the Als3 for the
diagnosis of invasive candidiasis has shown a very poor diagnostic value (Table 2) [18].


### 3.2.4. Hwp1 Protein

Hyphal wall protein 1 (Hwp1),
described by Staab et al. [46], is a glycoprotein specifically
expressed in the cell wall surface of the hyphae of C. albicans, which has been studied as an important adhesin,
required as a virulence factor in invasive candidiasis [47]. Naglik et al. [48]
analysed by RT-PCR the presence of HWP1 mRNA in human subjects who were positive for C. albicans culture and had oral or vaginal symptoms, as well as in
asymptomatic patients, by RT-PCR. They also detected antibodies against a
recombinant fragment of the Hwp1, Hwp1N13, consisting of the transglutaminase
substrate domain, previously produced by Staab et al. [49]. HWP1 mRNA
was found to be correlated with the presence of C. albicans in both asymptomatic carriers and in cases of oral and
vaginal candidiasis, although the quantity of mRNA in candidiasis probably
exceeded that revealed in asymptomatic conditions. Antibody titers (IgG and
IgA) in oral-culture-negative carriers and candidiasis cases were found to be
equivalent, suggesting that Hwp1 is a common target of host responses to C. albicans that are recognized to
result from long-term colonization.Since this protein has a
surface-exposed N-terminal domain that binds antibodies, while the carboxyl
terminus is most probably covalently integrated in the cell wall [46], Laín et al. [15] selected the N-terminal
fragment of the Hwp1 (161 aminoacids) for serological studies in patients with
invasive candidiasis. While detection of antibodies by immunoblotting showed
very poor sensitivity and negative predictive values (27.8% and 62.3%,
resp.), detection of antibodies against the N-terminal fragment of Hwp1
by ELISA notably increased the diagnostic usefulness, with sensitivity,
specificity, positive and negative predictive values of 88.9%, 82.6%, 80.0%,
and 90.2%, respectively. Interestingly, these results were very similar to
those obtained by the detection of antibodies to the C. albicans germ tube by indirect immunofluorescence, a technique
that has been previously reported to be of diagnostic utility [35, 50].
However, the detection of antibodies against the Hwp1 recombinant fragment is
easier to perform, since it does not require the adsorption of the patient’s
serum and allows both an objective measurement and automatization of the test.
In addition to that, the ELISA test was able to detect invasive candidiasis in
patients infected by non-C. albicans species, including C. parapsilosis, C. tropicalis, C. utilis, C. glabrata, and C. dubliniensis, although this antigen was initially described as
specific of the germ tube of C. albicans.
The reasons for this reactivity are not known at present, but it has been
reported that HWP1 mRNA may also
arise from pseudohyphal and yeast growth forms in C. albicans [48], and an ORF of 1266 pb with homology to C. albicans Hwp1 has been found in C. dubliniensis [51]. We have also
observed that the region encoding the first 85 residues of the Hwp1 fragment
shows homology with the sequence of proteins of non albicans Candida species
[15].


## 3.2.1. Hyr1 Protein

Hyphally regulated protein 1, Hyr1,
[38] is a C. albicans germ tube
specific cell wall glycoprotein of 937 aminoacids, with a
glycosylphosphatidylinositol (GPI) motive. Hyr1 is specifically expressed in
the mycelial form and its heterologous expression in Saccharomycescerevisiae did not show an obvious phenotype. Consequently, it was suggested to have a
structural role in the C. albicans cell wall architecture.Laín et al. [18, 39] studied the reactivity of a recombinant Hyr1
protein with sera from 36 patients with invasive candidiasis and 45 control
patients by immnunoblotting. The sensitivity and specificity values of the
assay were 39.1% and 100%, respectively, (Table 2). The detection of anti-Hyr1
antibodies by an ELISA assay increased the sensitivity to 58.3%, but reduced
the specificity to 82.2% in the diagnosis of invasive candidiasis (Table 2).Table 2
Diagnostic usefulness of the detection of antibodies against Hyr1, Ece1, Als3, and Hwp1 by immunoblotting and ELISA assay [18] (PPV: positive predictive
value, NPV: negative predictive value).
Sensitivity%Specificity%PPV%NPV%ImmunoblottingHyr139.110010076.3Ece134.893.372.773.7Als326.193.366.771.2Hwp127.897.583.362.3ELISA assayHyr158.382.272.471.2Ece155.680.069.062.5Als341.777.860.062.5Hwp188.982.680.090.2


## 3.2.2. Ece1 Protein

Extent of cell ellongation 1, Ece1
[40], is another C. albicans germ
tube specific protein. An ECE1 null
mutant C. albicans strain did not
show morphological changes and the authors concluded that this protein is not
essential for cell elongation or hyphal formation, despite the strict
association of Ece1 expression with the mycelial form of C. albicans.By using a similar approach to that
followed to study the diagnostic potential of the Hyr1, theECE1 gene has been expressed in E. coli and used in both immunoblotting
and ELISA assays to detect antibodies in patients with invasive candidiasis
[18, 41]. As observed with the Hyr1 antigen, the results obtained by ELISA
showed higher sensitivity but lower specificity than those obtained by
immunoblotting in the diagnosis of invasive candidiasis (Table 2).


## 3.2.3. Als3 Protein

TheALS (agglutinin-like sequence) gene family of Candidaalbicans encodes
cell surface glycoproteins implicated in adhesion of the organism to host
surfaces. ALS genes conform a basic
three-domain structure that includes a relatively well conserved domain of 1299
to 1308 nucleotides (433 to 436 aminoacids), a central domain of variable
length consisting entirely of a tandemly repeated 108-bp motif, and a
C-terminal domain of variable length and sequence that encodes a serine-threonine
rich protein, with a GPI motif. Presently, eight genes in the ALS family have been reported in the
literature [42]. ALS genes are
differentially regulated by physiological conditions such as changes in the
growth medium, morphogenesis, or the growing phase of the fungus. The
expression of these proteins is correlated with Candida infection, and there has also been found evidence of these
proteins in other Candida species,
such as C. dubliniensis and C. tropicalis [43]. Among the Als
family, Als3 is implicated in
endothelial and epithelial adhesion and it is specifically expressed in the C.albicans germ tube [44]. Several authors have established a functional model for the Als proteins, where the N-terminal domain is
exposed to the cell surface, and the central and C-terminal domain are
integrated in the cell wall layers [42, 45]. The N-terminal domain of the Als3 that has been described as a binding motif
[44] is the most exposed part of the protein and therefore to the immune system
of the host. However, the detection of antibodies against the N-terminal
recombinant fragment of the Als3 for the
diagnosis of invasive candidiasis has shown a very poor diagnostic value (Table 2) [18].


## 3.2.4. Hwp1 Protein

Hyphal wall protein 1 (Hwp1),
described by Staab et al. [46], is a glycoprotein specifically
expressed in the cell wall surface of the hyphae of C. albicans, which has been studied as an important adhesin,
required as a virulence factor in invasive candidiasis [47]. Naglik et al. [48]
analysed by RT-PCR the presence of HWP1 mRNA in human subjects who were positive for C. albicans culture and had oral or vaginal symptoms, as well as in
asymptomatic patients, by RT-PCR. They also detected antibodies against a
recombinant fragment of the Hwp1, Hwp1N13, consisting of the transglutaminase
substrate domain, previously produced by Staab et al. [49]. HWP1 mRNA
was found to be correlated with the presence of C. albicans in both asymptomatic carriers and in cases of oral and
vaginal candidiasis, although the quantity of mRNA in candidiasis probably
exceeded that revealed in asymptomatic conditions. Antibody titers (IgG and
IgA) in oral-culture-negative carriers and candidiasis cases were found to be
equivalent, suggesting that Hwp1 is a common target of host responses to C. albicans that are recognized to
result from long-term colonization.Since this protein has a
surface-exposed N-terminal domain that binds antibodies, while the carboxyl
terminus is most probably covalently integrated in the cell wall [46], Laín et al. [15] selected the N-terminal
fragment of the Hwp1 (161 aminoacids) for serological studies in patients with
invasive candidiasis. While detection of antibodies by immunoblotting showed
very poor sensitivity and negative predictive values (27.8% and 62.3%,
resp.), detection of antibodies against the N-terminal fragment of Hwp1
by ELISA notably increased the diagnostic usefulness, with sensitivity,
specificity, positive and negative predictive values of 88.9%, 82.6%, 80.0%,
and 90.2%, respectively. Interestingly, these results were very similar to
those obtained by the detection of antibodies to the C. albicans germ tube by indirect immunofluorescence, a technique
that has been previously reported to be of diagnostic utility [35, 50].
However, the detection of antibodies against the Hwp1 recombinant fragment is
easier to perform, since it does not require the adsorption of the patient’s
serum and allows both an objective measurement and automatization of the test.
In addition to that, the ELISA test was able to detect invasive candidiasis in
patients infected by non-C. albicans species, including C. parapsilosis, C. tropicalis, C. utilis, C. glabrata, and C. dubliniensis, although this antigen was initially described as
specific of the germ tube of C. albicans.
The reasons for this reactivity are not known at present, but it has been
reported that HWP1 mRNA may also
arise from pseudohyphal and yeast growth forms in C. albicans [48], and an ORF of 1266 pb with homology to C. albicans Hwp1 has been found in C. dubliniensis [51]. We have also
observed that the region encoding the first 85 residues of the Hwp1 fragment
shows homology with the sequence of proteins of non albicans Candida species
[15].


## 4. Conclusions and Future Perspectives

There is an increasing interest in the
development of new, reliable, and simple diagnostic tests for the diagnosis of
invasive candidiasis. Molecular biology techniques have allowed the production
of recombinant antigens which may be useful for the detection of antibodies
against them. It has been demonstrated that the detection of antibodies against
purified and well-defined recombinant antigens allows the diagnosis of invasive
candidiasis. A mixture of different antigens in the same assay may optimize and
increase the diagnostic value of these tests, since the kinetics of antibodies
against each recombinant antigen in the same patient is different (Figure1).
Identification of the most immunogenic domains of each antigen can also improve
the results. A combined detection of antigen and antibody may also increase the
sensitivity. Finally, further search and production of new immunogenic
recombinant antigens could open new ways for an accurate diagnosis of invasive
candidiasis.Figure 1
Kinetics of antibody
levels against differentC. albicans recombinant proteins in a patient with invasive candidiasis. Ece 1 (O), Hyr1 (◆), Als3 (•), enolase (□), and Hwp1 (Δ). Arrows point to the days where
cultures of urine and blood yielded C. albicans. The bar shows the days the patient was treated with fluconazole.



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*Source: 721950-2008-03-11.xml*