Differentiation of and by Using Recombinant Human Antibody ... · dida antigens during...

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2003, p. 1152–1160 Vol. 41, No. 30095-1137/03/$08.00�0 DOI: 10.1128/JCM.41.3.1152–1160.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Differentiation of Candida albicans and Candida dubliniensis by UsingRecombinant Human Antibody Single-Chain Variable Fragments

Specific for HyphaeJoseph M. Bliss,1 Mark A. Sullivan,1 Jane Malone,2 and Constantine G. Haidaris2,3*

Department of Pediatrics,1 Department of Microbiology and Immunology,2 and Center for Oral Biology,3

University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received 23 August 2002/Returned for modification 10 October 2002/Accepted 5 November 2002

To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage displaylibrary expressing human immunoglobulin heavy and light chain variable regions was used to select phageclones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that boundspecifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibodyvariable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized indetail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsi-losis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filamentformation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase Ktreatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. Theantigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not withsodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weightbands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimensobtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratorymedia, confirming epitope expression in human infection. The availability of a monoclonal immunologicreagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C.albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely relatedspecies.

Candida albicans is a pathogenic fungus of humans thatcontinues to be of considerable medical importance. C. albi-cans may infect both skin and mucous membranes, and it isalso capable of causing life-threatening systemic infections,particularly in the immunocompromised host (7, 35). A num-ber of putative virulence factors have been proposed for C.albicans and have garnered experimental support (7, 13).Among these, the ability of the organism to convert morpho-genically from blastoconidia to filamentous forms has been thesubject of intensive study (7). C. albicans and C. dubliniensisare unique within the genus in that, in addition to thepseudohyphal form, they also form germ tubes and true hy-phae (7). Like blastoconidia, pseudohyphae form by budding;however, the new cell remains attached to the parent andelongates with constrictions where cells meet. In contrast,germ tubes are outgrowths of the blastoconidia that grow byapical extension and form septae behind the growing tip. Thegerm tube is the precursor to the true hypha, with parallel wallmorphology. The conversion to hyphae is accompanied by ex-pression of novel antigens on the filamentous form, identifiedby using both polyclonal sera and monoclonal antibodies (5,8–12, 14, 18, 25, 28, 31, 37, 39–41, 43, 49–53).

The expansion of medical technology and the increased sur-vival of immunocompromised patients has led to a generalincrease in mycoses over the past several decades (15, 21).Although C. albicans remains the most virulent Candida spe-cies, infections caused by other species, including C. tropicalis,C. glabrata, and C. parapsilosis, have increased in relative prev-alence (33). C. dubliniensis, a species closely related to C.albicans in both phenotype and genotype, was first described in1995, when it was associated with oral infection in patients withhuman immunodeficiency virus (48). This organism has sincebeen described across diverse geographic regions and has beenassociated with both superficial and systemic infections (33).Because of its similarities to C. albicans, particularly its abilityto form filaments, differentiating C. albicans from C. dublini-ensis has been challenging. A variety of phenotypic assays havebeen employed, but none have proven completely reliable (47).Growth of C. dubliniensis is inhibited at 45°C, but this methodrequires several days’ growth on synthetic media (38). Themost accurate methods currently available for differentiatingthese species are molecular techniques such as PCR, DNAfingerprinting, and pulsed-field gel electrophoresis, but theseare time-consuming and difficult to apply to large numbers ofisolates (47). A novel approach using fluorescent in situ hy-bridization with peptide nucleic acid probes (PNA-FISH) todetect differences in the rRNA sequences between these spe-cies has been reported and holds promise (36). Other recentefforts have been made to exploit serologic differences betweenthese two species, with some success (3, 28, 32), offering the

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Rochester School of Medicineand Dentistry, 601 Elmwood Ave., Box 672, Rochester, NY 14642.Phone: (585) 275-0678. Fax: (585) 473-9573. E-mail: [email protected].

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potential for immunologic reagents to provide more efficientand rapid identification.

We have been using phage display technology to study sur-face antigen expression on C. albicans (17). In the presentstudy, we have applied this technology to identify single-chainantibody fragments (scFv) that specifically recognize the fila-mentous form of C. albicans. This technique allows for therapid isolation of monoclonal, human immunologic reagentswith which to study the differential surface expression of Can-dida antigens during morphogenesis, and it has yielded re-agents that can distinguish C. albicans from C. dubliniensis, asshown in the present study.

MATERIALS AND METHODS

Organisms. Fungal strains used in this study are listed in Table 1.Identification and enrichment of C. albicans-binding scFv phage by successive

rounds of panning on intact cells. C. albicans strain 3153A was used as thestarting strain for panning the phage library (17). To isolate scFv against deter-minants expressed on the filament of C. albicans, a culture was grown overnighton a shaker platform at 37°C in liquid yeast extract-peptone-dextrose (YEPD)medium (Difco, Detroit, Mich.) with vigorous aeration (225 rpm) to stationaryphase (�2 � 108 cells/ml). Cells were diluted in Medium 199 (supplemented withEarle’s balanced salt solution, HEPES, and glutamine; BioWhittaker, Walkers-ville, Md.) to 105 cells/ml in 96-well flat-bottom tissue culture plates and weregrown at 37° for 4 h. Filament growth was confirmed by light microscopy. Themedium was removed, and wells were blocked with 0.5% casein in Tris-bufferedsaline (pH 7.5) (TBS) at room temperature for 1 h. We have previously describedthe construction of a large naïve human antibody library in a M13 phage displayformat (17). Approximately 5 � 1010 phage from the library were added to eachwell and incubated at room temperature for 90 min with gentle mixing on arotator platform. Wells were washed extensively with TBS containing 0.5%Tween 20, followed by two brief washes with water to remove detergent. Boundphage were eluted by incubation with 0.1 M glycine (pH 2.2) containing 1 mg ofbovine serum albumin (BSA)/ml for 15 min and were transduced into Escherichiacoli strain TG1. Ampicillin-resistant transductants were regrown and infectedwith helper phage for a subsequent round of enrichment.

Populations of phage that exhibited increased binding to the C. albicansfilaments after two rounds of enrichment were analyzed for sequence diversity byPCR and restriction enzyme digestion. The scFv coding sequence was amplifiedby using primers that flank the cloning sites followed by restriction digestion withBstNI. Clones that had distinct restriction patterns were chosen for furtheranalysis. The clones of interest were then manipulated to remove the M13 gene

III fragment, and mature scFv containing the FLAG epitope at the aminoterminus and the hexahistidine tag at the carboxy terminus were prepared asdescribed previously (17).

IFA detection of binding of phage or scFv protein to Candida strains. Blasto-conidia of Candida strains were induced to form filaments via inoculation at 5 �105 cells/ml either in Medium 199 in six-well culture dishes on the surfaces ofglass coverslips or in human serum in 24-well culture dishes on the surfaces ofplastic coverslips (35). Cells were incubated at 37°C for 3 to 4 h, and thefilamentous phenotype was determined by light microscopy (Table 1). Coverslipswere allowed to air dry, rinsed briefly with water, and blocked with 3% BSA(Sigma, St. Louis, Mo.) in phosphate-buffered saline (PBS) for 30 min. Theblocking solution was replaced with 3% BSA containing either 1012 CFU ofindividual M13 phage clones/ml or 10 �g of a scFv/ml. After a 2-h incubation atroom temperature (RT), the coverslips were washed extensively in PBS andincubated either in 10 �g of a biotinylated sheep polyclonal secondary antibodyspecific for M13 phage (5 Prime33 Prime, Inc., Boulder, Colo.)/ml for the phageindirect immunofluorescence assay (IFA) or in 10 �g of a monoclonal antibody(M2) specific for the FLAG epitope tag at the amino terminus of the scFv(catalog no. F3165; Sigma)/ml for the scFv IFA, followed by the appropriatetertiary streptavidin-fluorescein or antibody-fluorescein conjugate (Jackson Im-munoResearch, West Grove, Pa.). Specimens were allowed to air dry and wereviewed by using fluorescence microscopy.

Biochemical analysis of C. albicans antigens recognized by scFv. Organismswere grown on coverslips as described above for IFA. Coverslips were thentreated either with proteinase K or trypsin to degrade proteinaceous antigens orwith sodium periodate to degrade carbohydrate antigens on the cell surface. Forproteinase K, coverslips were first incubated in 50 mM Tris (pH 7.5) containing100 �g of proteinase K/ml for 2 h at 37°C and then washed with water (23). Fortrypsin, coverslips were first incubated in 0.05% trypsin–0.53 mM EDTA � 4Na inHanks balanced salt solution (Invitrogen, Carlsbad, Calif.) for 2 h at 37°C andthen washed with water. For sodium periodate treatment, coverslips were firstincubated in 50 mM sodium acetate (pH 4.5) containing 50 mM sodium perio-date for 30 min at 4°C in the dark and then washed with water. Mock-treatedsamples were incubated in the respective buffer alone. Adherent cells were thenanalyzed by IFA as described above. To confirm that loss of binding via scFv wasnot due to degradation of scFv via residual proteinase activity, blastoconidia ofC. albicans 3153A were used as a control in proteinase K experiments and wereanalyzed via IFA with scFv �2-18. This scFv has previously been shown torecognize a carbohydrate antigen (17).

IFA detection of binding of scFv protein to Candida in clinical specimens.Patients identified as having potential Candida infections in either the NeonatalIntensive Care Unit or the Pediatric Hematology/Oncology outpatient service atthe Golisano Children’s Hospital at Strong in Rochester, N.Y., were referred toone of the authors (J.M.B.). Referrals were obtained for three infants withoropharyngeal candidiasis (thrush) and one infant with urinary candidiasis. In-

TABLE 1. Fungal strains, their filamentation phenotypes in Medium 199 or in serum, and their reactivities in both by IFA with scFv5 andscFv12

Species Strain nameFilament phenotype in: Reactivity

M199 Serum scFv5 scFv12

Candida albicans Ca3153A,a Ca613p,b MRO2-O,c MRO4-O,MRO9-R, MRO17-O, SC5314, CAH7-1Ad

� �g � �

Candida dubliniensis CBS7987,e CBS7988, CBS8500 � � � �Candida glabrata MRO84-R � � � �Candida parapsilosis RO75-R1 � � � �Candida rugosa MRO63-V � � � �Candida tropicalis MRO84-O � � � �Saccharomyces cerevisiae T481f � NDh � �

a Ca3153A (27, 34, 42, 54) was used as the strain for panning of the recombinant human scFv phage library and was kindly provided by E. Rustchenko, Departmentof Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, N.Y.

b Ca613p is a clinical isolate (16, 30).c Strains designated “MRO” or “RO” were collected as part of a prospective study of Candida colonization in mothers and neonates at Strong Memorial Hospital,

Rochester, N.Y., and were kindly provided by W. Watson, formerly of the Department of Pediatrics, University of Rochester School of Medicine and Dentistry.d C. albicans strains CAH7-1A (hwp1/hwp1) and SC5314 (wild type) were kindly provided by P. Sundstrom, Ohio State University, Columbus (44).e C. dubliniensis strains were purchased from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and were kindly provided by S. Spinelli,

Department of Chemistry, University of Rochester, Rochester, N.Y.f T481 was kindly provided by J. S. Butler, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry.g C. albicans strains grown in serum had a more pleiomorphic appearance than when grown in Medium 199, but they retained their activity with the scFv.h ND, not determined.

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formed consent was obtained from the parents of each subject. Oropharyngealspecimens were collected with a moistened, sterile cotton swab. The urine spec-imen was collected from a spontaneous void and centrifuged to collect sediment.Immediately after collection, specimens were spread directly on microscopeslides and allowed to dry. IFAs with scFv were performed as described above.The specimen collection protocol was approved by the Institutional ReviewBoard at the University of Rochester, Rochester, N.Y.

Antigen extraction from C. albicans filaments. C. albicans 3153A filamentswere prepared by inoculation of 100 ml of Medium 199 with organisms at 106

cells/ml, followed by growth at 37°C for 3 to 4 h. Filamentous forms werecollected by centrifugation, washed with water, and stored at �20°C until use.Antigens were extracted by using either sodium dodecyl sulfate (SDS) or Zy-molyase. For SDS extraction, filaments were combined in a 1:1 ratio (wet weightto volume) with 2� SDS-polyacrylamide gel electrophoresis (PAGE) crackingbuffer (Novex, Carlsbad, Calif.) in the presence or absence of 5% 2-mercapto-ethanol and then boiled for 10 min. For Zymolyase treatment, a modification ofthe method described by Sundstrom and Kenny was utilized (50). Briefly, fila-ments were treated with 1 mg of Zymolyase/ml in 100 mM Tris (pH 8.0)–500 mMMgSO4 � 7H2O–1 mM phenylmethylsulfonyl fluoride at 30°C for 2 h. Extractswere centrifuged briefly to remove particulate matter, and supernatants weredialyzed overnight against 10 mM Tris (pH 8.0)–50 mM MgSO4 � 7H2O andconcentrated by lyophilization. Concentrated extracts were loaded onto an SDS–4-to-20% polyacrylamide gel (Novex) and electrophoresed for 2 h at 125 V.Extracts were electrotransferred to nitrocellulose filters and blocked for 30 min

at RT with 5% dried skim-milk in PBS (Blotto). Respective scFv were diluted to10 �g/ml in 5% Blotto, and the filters were probed overnight at RT. Anti-FLAGmonoclonal antibody M2 (Sigma) at 10 �g/ml and alkaline phosphatase-conju-gated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch) wereused as secondary and tertiary reagents, respectively.

Nucleotide sequence accession number. The GenBank accession numbers forscFv5 and scFv12 are AY143558 and AY143559, respectively.

RESULTS

Enrichment of C. albicans-binding scFv phage by successiverounds of panning on intact cells. A phage display libraryexpressing scFv of human immunoglobulin has been describedpreviously (17). The phage display library was panned againstwhole cells. After two rounds of panning, 100-fold enrichmentwas observed. From 16 clones examined by restriction diges-tion following the second round of panning, 9 distinct restric-tion patterns were observed. A phage IFA was performed witheach of these nine clones. Eight of the nine were positive forbinding to C. albicans by phage IFA (Fig. 1). Clone 1 did notbind and was included as a negative control. Five clones

FIG. 1. IFA detection of binding of phage clones to C. albicans. Representative fields are shown. Paired panels are immunofluorescent andbright-field photomicrographs of the same microscopic fields of C. albicans strain 3153A stained via IFA with representative M13 phage clonesdisplaying scFv. Numbers identify the clone from the original 16. Clone 1 did not bind and was included as a negative control. Arrows point toblastoconidia that were not stained by phage IFA. Bars, 10 �m.

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stained the filament specifically and uniformly (Fig. 1, clones 2,3, 5, 6, and 12), while three gave a speckled and nonuniformpattern of staining (clone 8 [Fig. 1] and clones 4 and 11 [datanot shown]). Two clones, scFv5 and scFv12, which showed thestrongest uniform staining of the filament via phage IFA, wereselected for further study. They were each converted to expressscFv protein as described previously (17) to facilitate furtheranalysis.

The nucleotide and deduced amino acid sequences of theVL and VH regions of scFv5 and scFv12 were determined.Deduced amino acid sequences are shown in Fig. 2. The VLregions of both scFv5 and scFv12 were of the lambda family.The VH region of scFv5 was of the VH3 family, and that ofscFv12 belonged to the VH4 family (22). Substantial differ-ences were observed in the complementarity-determining re-gions (CDRs) of scFv5 and scFv12 in both the VL and VHchains, suggesting that the two antibody fragments did notrecognize the same epitope on Candida filaments.

IFA of scFv5 and scFv12 against species of Candida. Thefilamentation phenotypes of the strains used in this study whengrown in Medium 199 (see Materials and Methods) are shownin Table 1, as are their reactivities with scFv5 and scFv12 byIFA. The photomicrographs in Fig. 3 show scFv binding byIFA to Candida species that formed filaments: C. albicansstrain 3153A (Fig. 3A through C), C. dubliniensis strainCBS7987 (Fig. 3D and E), and C. rugosa strain MRO63-V (Fig.3F). Figure 3A shows a previously described scFv, �2-18 (17),binding primarily to the parent blastoconidia of the fungus.Figure 3B and C show scFv5 and scFv12, respectively, bindingexclusively to the filamentous portion of the cell while sparingthe parent blastoconidia. This pattern is consistent with thatseen in the phage IFA. Figure 3D shows scFv5 binding tofilamentous portions of C. dubliniensis. In contrast to scFv5,

scFv12 showed no fluorescence in an IFA with C. dubliniensis(Fig. 3E). This differential pattern of fluorescence was consis-tent in all three C. dubliniensis strains examined. An indepen-dent experiment using phage from clones 5 and 12 in an IFAwith C. dubliniensis confirmed this difference in antigen recog-nition (data not shown). Neither scFv5 nor scFv12 bound toother species tested in these assays (Table 1). C. rugosa wasunique among the other species tested in that it formed fila-ments under the growth conditions used but showed no reac-tivity with either scFv clone (Fig. 3F; Table 1). To confirm thatthe same patterns of antigen expression would be obtainedwhen filament growth was induced by human serum, the IFAwas repeated with cells grown in serum rather than Medium199. The same pattern of antigen expression was observedunder these conditions (Table 1).

The patterns of binding by scFv5 and svFv12 depicted in Fig.3B and C, respectively, are representative of many indepen-dent experiments with multiple strains of C. albicans (Table 1).The scFv bound to the filament uniformly and to virtually allthe individual cells in a given IFA. The antigens could bedetected on very small, nascent filaments and were also de-tected in uniform distribution on mature filaments grown over24 h. These observations suggest that the cognate antigens areexpressed early in the process of filamentation and persistthroughout filament growth.

Biochemical analysis of antigens recognized by scFv5 andscFv12. To determine whether the cognate antigens recognizedby scFv5 and scFv12 were composed of carbohydrate or pro-tein, C. albicans cells were grown on coverslips in Medium 199to promote filament growth. Cells were dried and then treatedeither with proteinase K or trypsin, to degrade protein, or withsodium periodate, to degrade carbohydrate structures. Cellswere then analyzed by an IFA with scFv5 or scFv12. The

FIG. 2. Comparison of the deduced amino acid sequences of scFv5 and scFv12. The cloning strategy yields a VL-linker-VH orientation of thescFv. The framework (FR) and CDRs as defined by Kabat et al. (22) are indicated. The position and sequence of the linker between the VL andVH regions are also shown. A dot indicates amino acid identity at a given position in scFv12 to the corresponding residue in scFv5.



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proteolytic activity of proteinase K was confirmed by degrada-tion of BSA (data not shown). Cells treated with proteinase Klost their reactivity with the scFv (Fig. 4), while those treatedwith periodate maintained the same fluorescence as cellstreated with acetate buffer alone (data not shown). These re-sults suggest that the cognate antigen for both scFv5 andscFv12 was protein. An IFA with �2-18, previously reported torecognize a periodate-sensitive epitope (17), was included as acontrol. As expected, �2-18 scFv binding was maintained fol-lowing proteinase K treatment (Fig. 4) but was lost followingperiodate treatment (data not shown), demonstrating that theproteinase K treatment did not interfere with scFv binding tothe cell surface and that the periodate treatment was effectivein degrading carbohydrate surface antigens. Cells treated withtrypsin also lost fluorescence by IFA, providing additional sup-

port for the proteinaceous nature of the epitopes (data notshown).

IFA of scFv5 and scFv12 against C. albicans clinical isolates.To confirm that the antigens recognized by scFv5 and scFv12were expressed in actual human infection, Candida specimenswere obtained from infants with oropharyngeal candidiasis orwith urinary tract infections with Candida. These specimenswere obtained and processed as described in Materials andMethods. An IFA was performed on the primary patient spec-imens. Figure 5 shows representative fields from the IFA withboth scFv against specimens obtained from both sites. An IFAwith no added scFv was performed in parallel as a negativecontrol and showed no fluorescence (data not shown). Thesedata confirm that the cognate antigens recognized by scFv5and scFv12 are expressed in human infection.

FIG. 3. IFA detection of binding of scFv5 and scFv12 to Candida species. Paired panels are immunofluorescent and bright-field photomicro-graphs of the same microscopic fields. (A) scFv �2-18 binding to C. albicans, showing the highest intensity for binding to the blastoconidia aspreviously described (17). (B, D, and F) scFv5 binding to C. albicans (B), C. dubliniensis (D), and C. rugosa (F). C. albicans and C. dubliniensisstained intensely, while C. rugosa was negative. (C and E) scFv12 binding to C. albicans (C) and C. dubliniensis (E). scFv12 stained only C. albicans.Arrows indicate blastoconidia that did not stain. Bars, 10 �m.



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Western blotting of extracted antigens from C. albicans withscFv5 and scFv12. C. albicans was grown in Medium 199 toallow filamentation, and filaments were collected by centrifu-gation. They were then extracted by boiling in the presence ofSDS with or without 2-mercaptoethanol or, alternatively, bydigestion with Zymolyase. Extracts were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed withscFv5 or scFv12. No antigen was detected in the SDS extractsdespite several attempts (data not shown). Antigen was de-tected by both scFv as a disperse, high-molecular-weight bandin the Zymolyase-extracted samples (Fig. 6). Although notextractable by SDS and 2-mercaptoethanol, the antigen is ap-parently stable in their presence, as the immunoblots showedreactivity despite being separated by SDS-PAGE under reduc-ing conditions.

DISCUSSION

We have used a combinatorial phage display library express-ing single-chain, variable light (VL), and variable heavy (VH)human immunoglobulin fragments to identify phage clonescapable of binding to the filamentous forms of live C. albicans.After two rounds of enrichment, 16 clones were investigated;they yielded 9 unique scFv based on restriction length poly-morphisms. The advantage of using live cells in the panningprocess is that it increases the likelihood of obtaining scFvreactive with surface components exposed to the external en-

FIG. 4. IFA detection of scFv5 and scFv12 binding to C. albicansafter proteinase K treatment. Paired panels are immunofluorescentand bright-field photomicrographs of the same microscopic fields. C.albicans filaments were subjected to proteinase K digestion, and thepresence of antigen was detected by IFA. Mock-treated cells weretreated with buffer alone. scFv �2-18 has previously been shown torecognize a carbohydrate epitope (17) and was included as a control.Proteinase K eliminated binding by scFv5 and scFv12. Bars, 15 �m.

FIG. 5. IFA detection of scFv5 and scFv12 binding to C. albicans in clinical samples. Paired panels are immunofluorescent and bright-fieldphotomicrographs of the same microscopic fields. Clinical specimens were collected from infants with thrush (oral mucosa) or urinary candidiasis(urine) and spread immediately on microscope slides. Binding of scFv5 and scFv12 was detected by IFA. Bars, 10 �m.



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vironment. The high level of diversity among these 16 clonesmay reflect the antigenic complexity of the filament surface.Further, since human lymphocytes were the source of the vari-able-region genes used to construct the library (26), the “im-munologic history” of the individuals from whom the lympho-cytes were obtained will contribute to the specificities availablein the library. The diversity of clones obtained supports acomplex interplay between host and microbe and may reflect arole for these surface antigens in host immune interactions.

Two scFv clones that showed uniform and bright fluores-cence of filaments, both germ tubes and hyphae, by IFA wereselected for further study. These clones, scFv5 and scFv12,bound the entire population of cells in a given experiment andshowed binding to the entire filament length in true hyphaeafter 24 h of incubation (data not shown). Although the pat-terns of binding of these two scFv to C. albicans by IFA werevery similar, scFv5 recognizes an epitope present on the fila-ments of C. dubliniensis while scFv12 does not. Although thesespecies are morphologically and genetically similar, antigenicdifferences on their surfaces have been documented by adsorp-tion of a polyclonal C. dubliniensis antiserum with C. albicansblastoconidia (2). Interestingly, although there were clear dif-ferences between the antigenic profiles of extracts from thesetwo species, this antiserum recognized an epitope on C. albi-cans germ tubes, and adsorption of the serum with C. albicansgerm tubes eliminated binding to C. dubliniensis. Differences inantigen recognition by use of sera from experimentally inducedinfections with these two species have also been documented(32). To our knowledge, there is a single report of a monoclo-nal antibody that binds C. albicans but not C. dubliniensis, andthe usefulness of this antibody for diagnostic differentiationbetween these closely related species has been proposed (28).Our finding of a phage clone that recognizes an antigen sharedby the filamentous forms of these two species constitutes thefirst description of a monoclonal immunologic reagent that hasthis property. Further, its specificity for filaments and the ab-sence of binding to other Candida species make it a candidatefor use as a diagnostic reagent. By using the two scFv together,

one could envision a parallel test for identification of filamen-tous Candida in which one reagent would be positive for bothC. albicans and C. dubliniensis (scFv5), while the other wouldbe positive for C. albicans and negative for C. dubliniensis(scFv12). Such a test would provide an internal control for thevalidity of the result.

We have confirmed the expression of the scFv cognate an-tigens in actual human infection with specimens obtained frominfants with thrush or urinary candidiasis tested without priorgrowth on laboratory media. These reagents thus provide theflexibility to identify organisms in direct clinical samples byIFA, which we used successfully in this study, or by immuno-histochemistry. This obviates the need for incubation on lab-oratory media and leads to more-rapid identification. Further,an IFA used as a diagnostic tool to distinguish C. dubliniensisfrom C. albicans is preferable to many of the phenotype-basedor molecular methods currently available in that it affords easeof use, rapidity, reliability, affordability, and the ability to beapplied to a large number of samples. These properties areessential for epidemiologic studies, which are needed to fur-ther define the clinical importance of this recently identifiedspecies (47). The observation that C. dubliniensis rapidly de-velops fluconazole resistance in vitro may provide additionalclinical relevance to such epidemiologic studies and under-scores the importance of reagents that can identify this organ-ism rapidly and efficiently (47).

Although the scFv described in this study detect antigensexpressed only on filaments and do not detect other Candidaspecies aside from C. albicans and C. dubliniensis, we havepreviously described scFv that were raised against C. albicansblastoconidia (17). These scFv detected carbohydrate antigensexpressed on many Candida species. By using these scFv incombination, one could tailor the specificities for detection ofCandida as appropriate for a given application, be it distinctionbetween C. albicans and C. dubliniensis, detection of yeastforms versus hyphae in specimens, or detection of Candidaspecies versus other pathogenic fungi.

Many Candida filament antigens characterized to date areextractable under reducing conditions and with Zymolyase (29,40, 43, 50). The cognate antigens of scFv5 and scFv12 were notextractable with 2-mercaptoethanol and SDS despite multipleattempts. The antigens are stable under these conditions, how-ever, as they are detectable by Western blotting after separa-tion by SDS-PAGE under reducing conditions. Their sensitiv-ity to proteinase K, but not to periodate, confirms theproteinaceous nature of the antigens and is also typical ofantigens unique to the filamentous forms (49). The antigenswere extractable by Zymolyase treatment and ran as polydis-perse bands of high molecular weight. This pattern may reflectglycosylation of the antigens. Despite their similarities, thenotion that scFv5 and scFv12 recognize distinct antigens issupported by several observations: (i) they have unique aspectsto their coding sequences; (ii) the VH regions of scFv5 andscFv12 are of distinct families; (iii) scFv5 recognizes an epitopeexpressed on C. dubliniensis while scFv12 does not; and (iv) themobilities of the cognate antigens in Zymolyase extracts aredifferent. However, the possibility that they recognize distinctepitopes of the same protein remains a reasonable alternative.

To date, six hypha-specific genes have been identified andcharacterized in C. albicans (6). These include ALS3/ALS8 (19,

FIG. 6. Western blotting of C. albicans antigens by using scFv5 andscFv12. C. albicans filaments were extracted with Zymolyase, and ex-tracts were separated by SDS-PAGE (4 to 20% polyacrylamide), trans-ferred to nitrocellulose filters, and probed with scFv. A polydisperse,high-molecular-weight band was detected with each scFv clone (5,scFv5; 12, scFv12). The position of the 205-kDa molecular size markeris indicated.



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20), ECE1 (4), HWP1 (45, 46), HYR1 (1), and CSA1 (24). Allexcept ECE1 appear to encode cell surface proteins. None ofthese gene products are required for filamentous growth, how-ever, as evidenced by the fact that mutations in these loci donot result in a morphological defect in hyphal development.Hwp1 has been the most extensively characterized and is a cellsurface exposed mannoprotein that is developmentally regu-lated and expressed in germ tubes and true hyphae of C.albicans (44). This protein serves as a substrate for mammaliantransglutaminases and is important for adhesion to epithelialcells. We have confirmed that the cognate antigens of scFv5and scFv12 are distinct from Hwp1, as they both bind filamentsof the hwp1/hwp1 knockout strain CAH7-1A (44) (Table 1).The possibility that the antigens recognized by the scFv clonesrepresent one of the other cloned gene products remains andawaits further analysis. However, Csa1 is extractable with di-thiothreitol and is unlike the scFv-reactive antigens in thatrespect, as they were not extractable under reducing condi-tions.

The use of phage display technology to identify antigensunique to the filamentous forms of C. albicans is a simple andrapid method that can further our understanding of antigenexpression by this medically important and complex fungus.The antibody fragments identified are useful tools for studyingthe antigenic variation of C. albicans. This technology also hasthe potential to identify structural proteins of the germ tubethat are required for hyphal growth and that have not beendescribed to date. We have already demonstrated a potentialdiagnostic use for the reagents developed in these studies thathas both clinical and research applications. Further, since thescFv are entirely human, they may, if converted to matureantibody forms, also provide a therapeutic benefit as agents forpassive immunization. Studies are under way to identify thegenes encoding the antigens recognized by these scFv andshould allow a better understanding of the antigenic complex-ity of the Candida cell wall.

ACKNOWLEDGMENTS

We are grateful to Paula Sundstrom for providing the hwp1/hwp1strain CAH17-1A. We thank Carl D’Angio and Frank Gigliotti forcritical review of the manuscript.

J.M.B. was supported by National Institutes of Health grant T32AI07464.

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