JP2002537269A - Yeast cell wall peptide and antibody thereto - Google Patents

Abstract

  (57) [Summary] The present invention provides a number of yeast cell wall proteins that are hydrophobic and are involved in hydrophobically mediated pathogenic events. The invention also provides antibodies to these proteins, as well as methods for inhibiting hydrophobically mediated pathogenic events, including administering these antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION

(Related Applications) This application is related to US Provisional Application No. 60 / 120,764, US Provisional Application No. 60 / 120,7
No. 65 and U.S. Provisional Application No. 60 / 122,216, which are incorporated herein by reference.

(Government Rights) The present invention relates to United States Public Health Se
Grant R29 Al31048, R0, awarded by rice / NIH
Partially made with US government support under 1 Al3048 and F32 Al09428. The United States Government has certain rights in the invention.

[0003] The present invention relates to yeast cell wall proteins.
n), detecting monoclonal antibodies recognizing them, and pathogenic yeast cells,
And methods for treating diseases associated with such organisms.

BACKGROUND OF THE INVENTION [0004] Various yeasts are pathogens for humans. Cell surface hydrophobicity is a property of adhesion and many yeast strains, especially Candida albicans, the most commonly yeast pathogen.
da albican). Cell filaments, including hyphae and pseudohyphae, produced by these organisms during the disease are hydrophobic. Hydrophobic cells bind more readily to epithelial cells and plastic than hydrophilic cells and exhibit a more generalized binding to host tissues. In addition, hydrophobic cells are more resistant to phagocytic attack than hydrophilic cells and are more toxic in mice.

[0005] Cell wall proteins, if not determined, are thought to contribute to the cell surface hydrophobicity of yeast cells. Hydrophobic proteins have been shown to be present on the cell wall surfaces of many yeasts in vitro. Adhesion of these microorganisms to host tissues is an early step in the formation of new environmental settlements and is thought to play an important role in the virulence of yeast cells.

[0006] Hydrophobic characteristics of fungal cell walls Yeast and molds can expose cell surfaces that are hydrophobic despite living in an aqueous environment. The presence of the hydrophobic surface of the fungal cell is determined by the substratum (substr).
Provides a mechanism of adhesion to ata) and, in the case of molds, aerial hyphae formation. How yeast and mold create a hydrophobic surface in an aqueous environment appears to be different. Molds produce small hydrophobic proteins. The proteins are generally classified as hydrophobins (a group of proteins with three properties: size, eight cysteine residues in a conserved pattern, and hydrophobic domains arranged in a similar pattern). The surface hydrophobicity of the yeast-form Candida species also appears to be due to proteins. However, the protein is not compatible with hydrophobin. The protein is present in vegetative cells and has a higher mass than hydrophobin. They do not appear to align as a filamentous layer at the cell surface, but can associate with mannosylated radioactive fibrils. At least one hydrophobic protein is candida (can)
saccharomyces cerevisiae (sacc)
haromyces cerevisiae). Candida albicans also contains a unique hydrophobic protein. Recent partial protein sequence analysis of two hydrophobic proteins of Candida albicans suggests that the protein has several sequences.

Introduction Fungal cell surfaces are hydrophobic. How an organism creates a hydrophobic surface, and how the structural arrangement of the hydrophobic surface varies, depends on the morphology of the organism and the cellular structure involved. For example, it is not surprising that vegetative cells, ie cells undergoing continuous cell growth, find that their hydrophobic surface is different from the rest of the cells or spores. Molds and yeasts appear to differ in which molecules determine the hydrophobicity of the surface.

[0008] Hydrophobin molds produce proteins that impart surface hydrophobicity. Proteins named hydrophobins are typically found in conidia and fruiting body tissues (eg, Ascoroman)
(Wessels, JGH (1996) Trends)
in Plant Science 1, 9-15). They are also responsible for certain fungal toxins, such as serato-ulmintoxin (respons
(molecular). All hydrophobins share certain characteristics.

[0009] 1. They have eight cysteine residues, which are generally arranged in a similar pattern along the protein. 2. They have a long stretch of hydrophobic amino acids (10-20aa). 3. They have hydrophobic regions arranged in a similar pattern. 4. They are low in mass (<20 kDa). 5. They have conserved intron sites. 6. They are only produced when the mycelium has reached a certain maturity.

[0010] Hydrophobin is secreted into the medium and forms a polymer on the surface of the mycelium (Wes
sels, J.M. G. FIG. H. (1996) Trends in Plant Sci
ence, 1, 9-15; A. B. (1993) Plan
t Cell 5, 1567-1574; Woesten, H .; A. B. (19
94) EMBO J. 13, 5848-5854). These polymers can lead to aerial mycelium formation. Aerial hyphae that secrete hydrophobin will secrete hydrophobin as an insoluble complex.

Assuming that the hydrophobin gene is found in a complete subclass, the surface hydrophobic proteins of defective yeast appear to be associated with hydrophobin. However, two teleological arguments for why the proteins are different are as follows: 1) Yeast is a vegetative cell. In turn, secreting highly hydrophobic proteins that polymerize on the surface of the cells substantially locks stagnant cells, ie, does not allow for growth. 2) Yeast does not reach sites with high nutritional value, but yeast does this in an aqueous environment. The release of hydrophobic molecules into aerobuoyants or aquabuoyants results in poor distribution in aqueous environments. A mixed population of hydrophobic and hydrophilic cell surface molecules can increase the ability of an organism to attach to nutrient-rich sites.

C. yeast hydrophobic surface molecule Investigations into the hydrophobic molecules of Albicans (Candida albicans), and perhaps the best studied yeast, suggest that the hydrophobic molecules are different from the mold hydrophobins. For example, C.I. albicans hydrophobic molecules are: High mass (> 20 kDa). 2. Production left constructively. 3. It does not appear to have long stretches of hydrophobic amino acids. 4. Possibly has a short sequence of hydrophobic amino acids.

The production of hydrophobic wall proteins is described in It occurs during all growth phases of Albicans (Candida albicans) and during yeast and hyphal morphogenesis. The proteins appear to be identical regardless of the growth phase (Hazen BW and Hazen KC (19).
98) Infect Immun 56, 2521-2525; Hazen K
C and Hazen B.C. (1993) FEMS Microbiol Let
t 107, 83-88). The protein has been detected in early buds, buds below, and in mature yeast cells, suggesting that the protein is made during all cell phases.

We have named three hydrophobic proteins we have named CAgp40, CAgp38 and CAgp37 against which we have monoclonal antibodies (Masuka J. et al. (1993) FEMS Immunol).
. Med. Microbiol. 24: 421-429) These proteins have a higher mass than hydrophobin.

CAgp40 and proteins with the same mass as the cross-reactive epitope for CAgp40 were present in all Candida species (seven species) and Saccharomyces cerevisiae we tested. Although only the epitope may be shared between species, it is surprising to find that the epitope-bearing protein is also of the same mass in all Candida species.

[0016] The protein CAgp38 is derived from C. albicans (Candida albicans), but was found in C, tropicalic, S. albicans. cerevisiae and C.I. kef
yr had a protein with a cross-reactive epitope. However, the epitope-bearing proteins differed in mass (Table 1).

[0017]

[Table 1]

The protein carrying the CAgp37 epitope is a 37 kGa protein S.p. cer
evisiae was present in wall extracts from several Candida species and had a cross-reactive epitope-bearing protein. However, its mass was different from protein from Candida species.

When CAgp40 and CAgp38 were analyzed for amino acid sequence by mass spectrometry, we obtained at least nine peptides from each that were at least partially sequenceable. Although extended lengths of adjacent hydrophobic amino acids were not obtained, at least one peptide had a sequence that conformed to the rules for surface exposure of hydrophobic groups on proteins (3 to 3 surrounded by polar amino acids). 5 hydrophobic amino acids). In addition, the two proteins have both tripeptide sequences. The tripeptide can be a marker for such a protein.

The role of mannosylation in hydrophobic surface exposure by yeast albicans are not commonly contrasted with other Candida species in that when grown to the stationary phase, their surface hydrophobicity changes with growth temperature. In addition, the hydrophobic state of its surface is influenced by morphology, age of culture, growth phase and growth medium.

The surface state that appears “constitutive” is hydrophobic. When hydrophilic cells are treated with an appropriate reagent, the hydrophilic cells become hydrophobic (eg, dithiothreitol treatment). Most Candida species are hydrophobic when grown at 37 ° C. Thus, the question is not what makes the yeast cells hydrophobic, albicans cells are made hydrophilic.

The topography of hydrophilic and hydrophobic cell surfaces was evaluated by freeze-fracture analysis. Remarkable contrast was obtained (Hazen KC and Hazen BW)
. (1992) Infect Immun 60, 1499-1508). Hydrophilic cells had radial, relatively long (200 nm) surface fibrils. The hydrophobic cells had a fibrillary layer, but the fibrils were short (100 nm), aggregated, smooth, and randomly oriented. Other researchers have described C.E. The fibril layer of Albicans (Candida albicans) was proposed to be based on a high molecular mass mannoprotein (
Cassona A. (1989) Curr Top Med Mycol 3
Hazen KC and Glee P., 248-314; M. (1994) C
and J Microbiol 40, 266-272). Based on this, the difference between hydrophobic and hydrophilic cells was tested for possible amounts or types of protein mannosylation present on the cell surface. No significant total cell wall protein or protein population differences between hydrophobic and hydrophilic cells were observed (Masuka J
And Hazen K .; C. (1997) Microbiology 143:
3015-3021). However, differences in the mannose content of the mannoprotein were detected. Western blots with monoclonal antibodies raised against outer mannosyl chains and phosphomannosyl groups showed that hydrophobic cells had few phosphomannosyl groups (Masuka J. and Hazen KC).
. (1999) Glycobiology 9: 1281-1286). Thus, exposure of the hydrophobic surface proteins depends on whether the wall surface proteins are heavily glycosylated and which wall surface proteins are so glycosylated.

SUMMARY OF THE INVENTION The present inventors have, for the first time, identified a number of yeast-produced cell wall proteins that are hydrophobic and are involved in hydrophobic-mediated pathogenic events. Antibodies to these proteins have been identified as hydrophobically mediated pathogens.
ed pathogenesis event).

More specifically, the present invention relates to C.I. Provided are hydrophobic peptides from C. albicans, and antibodies to the peptides. The antibodies are directed against yeast and the hydrophobic proteins found in the filamentous form of their pathogenic yeast. The antibodies recognize hydrophobic proteins in many pathogenic species of Candida and in other pathogenic yeasts and effectively block various pathogenic events involving hydrophobic proteins. Thus, the antibody is directed against a hydrophobic wall protein,
It is unique in that it can prevent pathogenic events. The antibodies offer therapeutic advantages over other treatments for yeast disease. That is, it prevents a number of disease events, most particularly, attachment to the host extracellular matrix, thereby preventing transmission. The antibodies are also useful in diagnostic tests to detect the presence of Candida and other diseased yeast in biological samples.

The present invention is further directed to treating pathogenic yeast-related pathological conditions. Methods for treating such diseases include the use of hybridoma cell lines F6-6C5-H4, F6-5F8-E1 to interfere with the ability of yeast cells to bind to host tissues.
Includes the use of antibodies produced by 0 and 5D8-A12CA and the use of peptides similar in structure to the binding domain displayed on the surface of hydrophobic yeast cell wall proteins. More specifically, the peptide has the general formula G—X ₁ —X ₂ —R, where G is glutamate or glutamine, X ₁ is a bond or an amino acid, X ₂ is an amino acid, and R is And at least one amino acid of the tripeptide is valine, leucine, isoleucine, phenylalanine, tyrosine or tryptophan).

With the foregoing and other objects, the advantages and features of the present invention will become apparent hereinafter.
The nature of the invention can be more clearly understood by referring to the following detailed description of preferred embodiments of the invention, and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides three hydrophobic proteins derived from Candida albicans (Candida albicans): 6C5, 5D8 and 5F8. These cell wall surface proteins contribute to the cell surface hydrophobicity of many yeast species, especially those of Candida albicans. Cell surface hydrophobicity promotes adhesion of yeast cells to host tissues, an important initial step in the formation of host organisms of pathogenic yeast cell colonies. Prevention of this binding to host tissues provides an effective means of treating diseases associated with yeast infection and pathogenic yeast cell colonization.

The 6C5 protein antigen is C. albicans is the product of one of a family of four or five similar gene products in Candida albicans. The gene sequence for 6C5 (FIG. 5) encodes a 337 amino acid protein with a predicted molecular weight of 38.228 kD and an isoelectric point of 5.80, which is in good agreement with experimental measurements. The genetically controllable yeast Saccharomyces cerevisiae has a single homologous gene of unknown function whose protein sequences are very similar. The 6C5 monoclonal antibody epitope was mapped by phage display panning and revealed the following epitopes.

Clone 4: RVDVGSEDAPSR-- Clone 54: SDRLEVGTEDLR Clone 43: DVEVGAEDALFS Clone 13: IEARVEIDWSLD --- Clone 26: --- GRNLEVDRALDT ------ Clone 11: --RSMLELDVILEG ------ Clone 12: -EGIGVEVVLE ------ Clone 4-3-3: -EIDLKLEPGTRV --- Clone 44: --- RVELAHELGLEVW --- Clone 38: --- ELD. AID. RPGIVV clone 39: --- VRLEID. QIDRGP --- Clone 5-4-1: -SFEVD. PIDENSG--clone 1-8: GETQCVEIA. SID ----- Clone 29: -IGFVERD. DIDAE-----Clone 57:----EID. SFELLLRER ----

The protein sequence “EIDPID” can be found in the open reading frame at residue number 107. Protein sequences that match the monoclonal antibody epitope may include other C.A. albicans (Candida albicans) sequence and is not present in the S. albicans sequence. It is not present in the cerevisiae sequence.

The 5D8 antigen epitope has been mapped by phage display panning and peptides exhibiting 5D8 reactivity are aligned below. Putative for the epitope] The consensus sequence is "E (E / P / K) L (F or Y) (I or V) (
S / T) ". Approximately 40 kDa molecular weight C.D. with 5D8 immunoreactivity adjusted by glass bead cell disruption. albicans (Candida albicans) protein has been analyzed by mass spectrometry. albicans
(Candida albicans) has been attributed as actin. In a second mass spectral analysis using the material prepared by limited litase digestion, 5D8
Proteins immunoreactive with do not show the same properties as actin and are currently under further investigation.

Pand 5D8 antigen epitope: {clone 1} PLLPEPLFIELG {clone 12} --- EKLYISAWDHLN {clone 19} -RFVEPLYVTAAG {clone 23} --- EELFISRLSRAP 5D8 peptide derived by second mass spectral analysis: PFVDAYPFNR TDQTSNNNNVA TWSV (1 / L) PT

The 5F8 antigen epitope has not been mapped by phage display. 40k
C produced by limited litase digestion containing Da immunoreactive protein
. albicans (Candida albicans) protein extract has been analyzed by mass spectrometry. Peptides derived from the 40 kDa protein produced by trypsin digestion show sequence similarity to the higher molecular weight two bacterial glucanases, 46 kDa apparent mass, and 58 kDa mass. The mass of some peptides does not show similarity with these glucanases.

Mass Spectrum Identifying Peptides: IWIPR CLDVR WAYDAGSK YQPQYGR ALNPSHIDIDVGK LVQPAVQNDSDPNR ANSALDGQGNLVITAR DLQAPNDHVVGPPIAR GAAVQVWTCNGTGAQKQPDATGN

Antibodies raised against the hydrophobic proteins of pathogenic yeasts, including but not limited to Candida species, target treatment against many types of infections, and those same proteins and microorganisms Useful in diagnosis. Such infections include, but are not limited to, opportunistic and nosocomial. These antibodies can also be used as research and development tools (research tools). Monoclonal and polyclonal antibodies, both are included as useful biological molecules, the individual components, including the antigen binding region (F _ab) or crystallization region (F _c) are useful in the treatment diagnosis and research.

[0036] Specific peptides and derivatives isolated from antibodies can also be used to develop biotechnology products. In the development of novel therapeutic, diagnostic and research tools targeted to yeast species, biologically active molecules such as toxins, peptide drugs and enzymes can be attached to these antibodies. All of the above biological molecules can be targeted for use in humans or can be targeted for use in animals. Such biological molecules are useful in prophylactic treatment of wounds, contaminated surfaces and surgical tools.

Also, these peptides can be used in the development of peptidomimetics and / or small organic molecules for therapeutic, diagnostic and / or research use.

Relevant genes may be used in the development of delivery systems for genes and / or gene vector constructs for actors, gene therapy, diseases associated with yeast infection, or for such opportunistic and / or nosocomial infections. It can be used as prophylactic therapy in patients at risk.

According to one embodiment of the invention, the antibodies are raised against a hydrophobic yeast cell surface protein. The antibodies are raised using standard techniques, and specific antibodies are selected that bind to regions of the hydrophobic protein that interfere with the ability of the yeast cells to adhere to host tissue surfaces.

The antibodies of the present invention have been shown to recognize these binding regions of hydrophobic yeast cell wall proteins. The antibodies of the present invention include polyclonal and monoclonal antibodies and fragments of F6, including fragments of any of these antibodies that exhibit similar chemical activity.
-6C5-H4, F6-5F8, and F6-5D8-A12 hybridoma cell lines. The F6-6C5-H4, F6-5F8-E
10, and the F6-5D8-A12 hybridoma cell line have been deposited with the ATCC.

The protein recognized by the antibody of the present invention exhibits a hydrophobic binding domain on the surface of the yeast cell wall. These binding domains are peptide sequences displayed on the outer surface of cell wall proteins. Antibodies directed against these antigens can inhibit yeast cell adhesion to host tissues by directly occupying these binding domains. Alternatively, yeast cell colonization can be blocked by occupying sites on the host tissue surface targeted by the yeast protein. Introduction of a peptide similar in structure to the hydrophobic binding domain recognized by the antibody should effectively inhibit the binding of yeast cells to host tissues.

The antibodies according to the present invention can be raised against natural or recombinant proteins, or antigenic fragments thereof. The antibodies of the present invention can be prepared using known techniques. Monoclonal antibodies are described in Kohler et al., Nature 256.
: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 5
11 (1976); Kohler et al., Eur. J. Immunol. 6: 292
(1976); Hammerling et al., In: Monoclonal Ant.
ibodies and T-Cell Hybridomas, Elsavier
, NY, pages 563-681 (1981). Such antibodies produced by the method of the present invention can protect against yeast infection.

The term “antibody” refers to both polyclonal and monoclonal antibodies, as well as, for example, Fvs, Fabs and Fs capable of binding antigens or haptens.
(Ab) Includes such fragments as _two fragments. Such fragments are typically of Fa
Produced by proteolytic cleavage, such as papain to generate the b fragment, or pepsin to generate the F (ab) ₂ fragment. Alternatively, hapten-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

As mentioned above, both polyclonal and monoclonal antibodies can be used in the present invention. Of particular interest to the present invention are antibodies produced in humans or "humanized" by recombinant or other techniques (ie, non-immunogenic in humans). A humanized antibody can be produced, for example, by placing an immunogenic portion of the antibody together with, but not limited to, an immunogenic portion (a chimeric antibody).
. For example, Robinson et al., International Patent.
Publication PCT / US86 / 02269; Akira et al., Eu.
ropen Patent Application 184, 187; Ta
niguchi, M .; European Patent Applicatio
n 171.496; Morrison et al., European Patent.
Application 173, 494; Neuberger et al., PCTA.
application WO 86/01533; Cabilly et al., Europe.
Ean Patent Application 125, 023; Bette
r et al., Science 240: 1041-1043 (1988); Liu et al., P.
ANS 84: 3439-3443 (1987); Liu et al. Immunol
. 139: 3521-526 (1987); Sun et al., PNAS 84:21.
4-218 (1987); Nishimura et al., Cancer Research.
ch 47: 999-1005 (1987); Wood et al., Nature 314.
: 446-449 (1985); and Shaw et al. National C
canceler Inst. 80: 1553-1559 (1988). Gerera
l Reviews or "humanized" chimeric Ant
ibodies are provided by Morrison, S.D. L
. Science 229: 1202-1207 (1985) and by Oi
Et al., BioTechniques 4: 214 (1986).

Using the antibodies or antibody fragments of the present invention, yeast infections can be detected, diagnosed, serum typed and treated. In this way, the antibodies or antibody fragments are particularly suitable for use in immunoassays. Such immunoassays include diagnostic tests to detect the presence of Candida and other pathogenic yeast in a biological sample.

An antibody or fragment thereof can be labeled using any of a variety of labels and labeling methods. Examples of types of labels that can be used in the present invention include, but are not limited to, enzyme labels, radioisotope labels, non-radioisotope labels, fluorescent labels, toxin labels, and chemiluminescent labels.

Examples of suitable enzyme labels include malate hydrogenase, staphdicocarnuclease, delta-5-steroid isomerase, yeast-alcohol dehumangenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase , Asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase and the like.

Examples of suitable radioisotope labels are ³ H, ¹²⁵ I, ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ C
^{r, 57 To, 58 Co,} 59 Fe, 75 Se, 152 Eu, 90 Y, 67Cu, 217 Ci, 21 1 At, 212 Pb, 47 Sc, and an ¹⁰⁹ Pd.

Examples of suitable fluorescent labels include ¹⁵² Eu label, fluorescein label, isothiocyanate label, rhodamine label, phycoerythrin label, phycocyanin label, allophycocyanin label, o-phthalaldehyde label, fluorescamine label and the like.

[0050] Examples of suitable toxin labels include diphtheria toxin, lysine, and cholera toxin. Examples of chemiluminescent labels are luminal label, isoluminal label,
Includes directional group acridinium ester labels and imidazole labels, as well as acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, aequorin labels and the like.

Those skilled in the art will know of other suitable labels that can be used in the present invention. Attachment of these labels to the antibody or fragment thereof can be accomplished using standard techniques commonly known to those skilled in the art. Typical techniques are described, inter alia, in Kennedy J. et al. H. (Clin. Chim. Acta 70: 1 to 3)
1 (1976)) and Schurs, A .; H. W. M. Et al., (Clin Ch
im. Acta 81: 1 to 40 (1997)). The coupling techniques described in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated herein by reference. Part.

Detection of an antibody (or antibody fragment) of the present invention can be improved through the use of a carrier. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. One of skill in the art will recognize many other suitable carriers that bind to the monoclonal antibody, or can identify it using routine experimentation.

By raising antibodies against a hydrophobic yeast surface protein that mimics the antigenicity of yeast, antibodies raised against such recombinant proteins are neutralizing and protective antibodies. The antibody can prevent subsequent infection of the same type of yeast from which the hydrophobic surface protein was obtained. That is, if C.I. alb
If antibodies are raised using recombinant or natural hydrophobic surface proteins from Candida albicans, these antibodies will albicans will protect against the subsequent infection of Candida albicans. Thus, the methods of the present invention provide for the prevention, treatment or detection of infection by any yeast type.

The present invention also provides a pharmaceutical composition. Since the antibodies can also be used to treat yeast infections in patients in need of such treatment. In the context of the present invention, a patient in need of treatment will be understood to be any mammal, especially a human, infected with any yeast. The antibodies or monoclonal antibodies can be used in pharmaceutical compositions to target drug therapy to sites of yeast infection. In this manner, the drug or compound of interest is linked to the antibody, allowing targeting of the drug or compound. Methods for linking antibodies to drugs or compounds are available. For example, EP0 (European Patent) 146,050
No., EP0 (European Patent) 187,658, and US Pat. No. 4,673,57.
No. 3, U.S. Pat. No. 4,368,149; U.S. Pat. No. 4,671,958 and U.S. Pat. No. 4,545,988.

[0055] Such pharmacotherapy includes antibiotics, toxic agents, and coumarin, plaralene, phthalocyanine, methylene blue, eosin, tetracycline, chlorophyll,
Includes photoactivatable compounds such as porphyrins. Such groups can be attached to the antibody via a suitable linking group. An antibody conjugate containing a photoactivatable compound is administered, followed by irradiation of the target cells.

The antibodies or antibody conjugates of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, such as by mixing with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulations are described, for example, in Remington's
on's Pharmaceutical Sciences (16th Ed
. Osol, A .; Ed. , Mack Easton PA (1980)). To form a pharmaceutically acceptable composition suitable for effective administration,
Such compositions will contain an effective amount of the antibody, alone or with an appropriate amount of a carrier vehicle.

The therapeutic or diagnostic compositions of the present invention are administered to a patient in a therapeutically effective amount. That is, an amount sufficient to diagnose or treat a yeast infection. The effective amount will vary according to the patient's weight, sex, age and medical history. Other factors include the severity of the condition of the patient, the type of yeast, the mode of administration, and the like. Generally, the composition will comprise about 0.01 to 2 mg.
/ Ml, more usually in doses ranging from about 0.001 to about 20 mg / ml.

Pharmaceutically prepared compositions can be administered topically, orally, intranasally, subcutaneously, intramuscularly, intravenously,
It can be provided to the patient by any means known in the art, including intra-arterial, parenteral, etc., with local administration being particularly preferred.

[0059] Another aspect of the invention relates to the development of a yeast type-specific vaccine. The vaccines of the present invention contain the necessary antigenic determinants to induce the formation of neutralizing antibodies in the host, possess high immunogenic potential, are sufficiently safe without the risk of clinical infection, and are toxic. Suitable for oral, nasal, topical or parenteral administration, without side effects, suitable for oral, nasal, topical or parenteral administration, mimics the natural infection situation, is stable under long-term storage conditions, and is usually inert It is suitable for various vaccine carriers.

The vaccine of the present invention comprises a conformationally correct recombinant yeast hydrophobic protein or fragment thereof that provides a conformational epitope present in intact yeast. Such an amino acid sequence of the yeast hydrophobic surface protein contains the antigenic components of the vaccine. It may be necessary or desirable to covalently attach the antigen to an antigenic carrier, ie, bovine serum albumin or keyhole limpet hemocyanin. The vaccine of the present invention can be administered to any patient susceptible to a yeast infection. Humans and non-animal mammals can benefit as hosts.

As noted above, administration of the vaccine can be by any route, including parenteral, but is preferably oral or nasal depending on the natural route of infection. The dose to be administered can depend on the age, health, weight, type, if any, and the nature and type of yeast of the co-treatment. The vaccine may be used for oral administration in dosage forms such as capsules, liquid solutions, suspensions or elixirs, or for sterile liquid formulations such as solutions or suspensions for parenteral or nasal use. An inert, immunologically acceptable carrier such as saline or phosphate buffered saline is preferably used.

The vaccine is administered in a therapeutically effective amount. That is, an amount sufficient to produce a protective immunological response. Generally, a vaccine will contain from about 0.1 mg protein to about 20 mg protein.

More usually, doses will be administered in the range of about 0.01 mg to about 100 mg protein. A carrier or multiple doses can be administered.

As more than one type of yeast can be associated with a yeast infection, the vaccine can include a yeast hydrophobic surface protein from more than one type of yeast or an antigenic fragment thereof.

In one embodiment of the invention, peptides are provided that are similar in structure and activity to these hydrophobic binding domains. Introduction of these peptides provides for inhibition of yeast cell binding to host tissue.

The peptide of the present invention is represented by the general formula GX ₁ -X ₂ -R, wherein G is glutamate or glutamine, X ₁ is a peptide bond or an amino acid,
X ₂ is an amino acid, and R is a sequence of three amino acids at least one of which is selected from the group consisting of valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan).

According to the present invention, X ₁ and X ₂ can be any natural or synthetic amino acid or any molecule capable of forming a peptide bond.

In one embodiment of the invention, G is glutamate, X ₁ is a peptide bond, X ₂ is proline, R is leucine-tyrosine-isoleucine,
A peptide of the above formula is provided which is a tripeptide sequence selected from the group consisting of leucine-tyrosine-valine and leucine-phenylalanine-isoleucine.

In another embodiment, a peptide of the above formula wherein G is glutamate, X ₁ is a peptide bond, X ₂ is proline, and R is a tripeptide sequence, leucine-phenylalanine-valine. Is provided.

[0070] Recognition of yeast cell surface proteins by the antibodies of the present invention allows for a method of detecting the presence of pathogenic yeasts and other organisms that exhibit similar hydrophobic surface binding domains. The antibodies of the present invention can be labeled with a number of detectable marker molecules known to those of skill in the art. The labeled antibody can then be applied to contact the antibody with a labeling environment or components of a labeling substance that are thought to contain antigens exhibiting these binding domains. The amount of labeled antibody bound to the labeled molecule or the amount of labeled antibody left unbound can be used to determine the level of antigen presence. This method of detection can be performed in vivo or in vitro.

Furthermore, this method of antigen detection can be performed by binding the antibody to a solid support and contacting the target substance-bound antibody. Possible forms of the solid support include a column of beads, or a fixed surface of a container or vessel through or into which the target is contacted with the antibody. The antibody bound to the solid support does not need to be labeled. Since it remains fixed to the solid support during the process.

In one embodiment of the invention, the suspected infection is passed through a column containing one or more agarose beads with the antibody bound to the beads, and the column is washed to displace the bound target. Remove any material that does not bind to the antibody with a substance that has insufficient affinity for the antibody, and then allow the antibody to pass through a fixed volume of substance with sufficient affinity to displace the bound target molecule. An antigen detection method is provided that includes the step of determining the amount of bound target antigen.

[0073] In addition to detection, recognition of the hydrophobic binding domain by the antibodies of the present invention allows for methods of treating yeast infections and related diseases. The hydrophobic cell wall proteins recognized by the antibodies of the present invention facilitate binding of yeast cells to host tissues (the first step in yeast cell pathology). Thus, inhibition of this binding should prove to be an effective treatment for diseases associated with yeast infection.

This preliminary step of infection can be inhibited by occupying the hydrophobic binding domain of cell wall proteins or by occupying the host tissue to which these yeast cells bind. Such inhibition can be achieved through the introduction of an antibody or peptide of the invention.

In one embodiment of the invention, there is provided a method of treating yeast infection and related diseases, wherein the antibody of the invention is in an amount sufficient to inhibit binding of yeast cells to host tissue. Is applied topically to the area suspected of yeast infection. Furthermore, the antibodies can be applied in a pharmaceutically acceptable carrier that can prevent antibody degradation prior to and during the application. Many such carriers are well known in the art and include ointments, lotions, salves and emulsions.

In another embodiment, there is provided a method of treating yeast infection and related diseases, wherein an amount of a peptide of the invention is sufficient to inhibit binding of yeast cells to host tissue. Applied topically in quantity. Further, the peptides can be applied in a pharmaceutically acceptable carrier that can prevent degradation of the peptide prior to and during application.

In another embodiment, there is provided a method of treating yeast infection and related diseases, wherein the peptide of the invention is present in an amount sufficient to inhibit binding of yeast cells to host tissue. Is orally introduced in a pharmaceutically acceptable carrier capable of preventing gastrointestinal or other degradation. Such carriers are well known in the art and include solid, liquid and encapsulated forms.

Pharmaceutical Compositions and Vaccines In certain embodiments, polypeptides, polynucleotides, antibodies, T cells and / or binding agents described herein can be incorporated into pharmaceutical or immunogenic compositions (ie, vaccine). Pharmaceutical compositions comprise one or more such compounds and a physiologically acceptable carrier. A vaccine can include one or more such compounds and an immunostimulant. An immunostimulant can be any substance that enhances or enhances the immune response (antibody and / or cell mediated) to an exogenous antigen. Examples of immunostimulants are adjuvants, biodegradable, microspheres (eg, polylactide galactide) and liposomes (in which the compound is incorporated, eg, Full
lerton US Patent No. 4,235,877). Vaccine formulations are generally described, for example, in MF. Powell and M.W. J. Newman, eds., "Vac
cine Design (subunit and adjuvant approach) ", P
Lenum Press (NY, 1995). Also, the pharmaceutical compositions and vaccines within the scope of the present invention may contain other compounds which may be biologically active or inactive. For example, one or more immunogenic portions of another antigen can be present in a composition or vaccine, can be integrated into a fusion polypeptide, or can be present as separate compounds.

A pharmaceutical composition or vaccine can contain DNA encoding one or more of the polypeptides described above, such that the polypeptide occurs in situ. As noted above, the DNA can be in any of a variety of delivery systems known to those of skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Rol
land, Crit. Rev .. Therap. Drug Carrier Sy
Numerous gene delivery techniques are well known in the art, such as those described by Stems 15: 143-198, 1988 and the references cited therein. Suitable nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and termination signal). Bacterial delivery systems involve the administration of bacteria (such as Bacillus-Calmette-Guerrin) that express an immunogenic portion of the polypeptide on their cell surface or secrete such epitopes. In a preferred embodiment, the DNA comprises a viral expression system (eg, vaccinia or other poxvirus, retrovirus, or adenovirus) that can include the use of a non-pathogenic (defective), replication competent virus. Can be introduced. Suitable systems are, for example, Fisher-Ho
ch et al., Proc. Natl. Acad. Sci. USA 86: 317-32
1, 1989; Flexner et al., Ann. N. Y. Acad. Sci. 569
: 86-103, 1989; Flexner et al., Vaccine 8: 17-2.
No. 1,1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,1.
No. 27; GB2, 220, 651; EP0, 345, 242; WO91 / 028
No. 05; Berkner, Biotechniques 6: 616-627,
1988; Rosenfeld et al., Science 252; 431-434.
1991; Kolls et al., Proc. Natl. Acad. Sci USA 9
1: 215-219, 1994; Kass-Eisler et al., Proc. Nat
l. Acad. Sci. USA 90: 11498-11502, 1993; G
uzman et al., Circulation 88: 2838-2848, 1993.
And Guzman et al., Cir Res. 73: 1202-1207,199
3. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can be obtained, for example, according to Ulmer et al., Science 259:
1747-1549, 1993; Cohen, Science 25.
9: 1691-1692, as reviewed in 1993.
d) ". Naked DNA uptake can be increased by coating the DNA with biodegradable beads, which is effectively exported to cells. It will be apparent that vaccines can include both polynucleotide and polypeptide components. Such a vaccine can provide an enhanced immune response.

It will be apparent that vaccines can contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts include organic bases (eg, salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (eg, sodium, potassium, lithium, anonium, calcium and magnesium salts). It can be prepared from pharmaceutically acceptable non-toxic bases, including:

[0081] While any suitable carrier known to those skilled in the art can be used in the pharmaceutical compositions of the invention, the type of carrier will vary depending on the mode of administration. The compositions of the present invention can be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, cranial, intraperitoneal, subcutaneous or intramuscular. For parenteral administration, such as subcutaneous injection, the carrier preferably includes water, saline, alcohol, fat, wax or a buffer. For oral administration, any of the above carriers or a solid carrier may be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, and magnesium carbonate. Biodegradable microspheres (eg, polylactic acid, polyglycolate) can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described, for example, in US Pat. No. 4,897,268; US Pat. No. 5,075,109; US Pat. No. 5,928,647; US Pat. No. 5,811,128. US Patent No. 5,82;
No. 0,883; U.S. Pat. No. 5,853,763; U.S. Pat. No. 5,814,34.
No. 4 and U.S. Pat. No. 5,942,252.

Such compositions may also include buffers (eg, neutral buffered saline or phosphate buffered saline), carbohydrates (eg, glucose, mannose, sucrose or dextran), mannitol, proteins, Amino acids such as polypeptides or glycine; antioxidants; bacteriostats; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); It may also contain solutes, suspending agents, thickeners and / or preservatives. Alternatively, the compositions of the present invention can be formulated as a lyophilizate. Compounds can also be encapsulated in liposomes using well-known techniques.

[0083] Any of a variety of immunostimulants can be used in the vaccines of the present invention.
For example, an adjuvant can be included. Most adjuvants are substances designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and lipid A, Bortadella pertuss
Contains stimulants of the immune response, such as is or Mycobacterium tuberculosis inducing protein. Suitable adjuvants include, for example, Freund's incomplete and complete adjuvants (Difco Labora)
tories, Detoroit, MI); Merck Adjuvant 65
(Merck and Company, inc., Rahway, NJ); AS-
2 (Smithkine Beecham, Philadelphia, PA)
Aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; insoluble suspension of acylated tyrosine;
Acylated sugars; polysaccharides cationically or anionically derivatized; polyphosphazenes; biodegradable microspheres; commercially available as monophosphoryl lipid A and quill A. Cytokines such as GM-CSF or interleukin-2, -7 or -12 can also be used as adjuvants.

Preferred adjuvants used to induce a dominant antibody response include, for example, a combination of an aluminum salt and monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL). . MPL adjuvant is Co
Rixa Corporation (Seattle, WA; U.S. Pat. No. 4,4)
36,727; U.S. Pat. No. 4,877,611; U.S. Pat.
No. 34 and U.S. Pat. No. 4,912,094). Also, C
The pG-containing oligonucleotide (where the CpG dinucleotide is unmethylated) predominantly induces a Th1 response. Such oligonucleotides are described, for example, in WO 96/02555 and WO 99/33488. Immunostimulatory DNA sequences are also described, for example, in Sato et al., Science.
273: 352, 1996. Another preferred adjuvant is saponin, preferably QS21 (Aquila Biopharmaceutical).
medicals Inc. , Framingham, MA), which can be used alone or in combination with other adjuvants. For example, the augmented system is a QS21 or 3D, described in WO 94/00153.
-A combination of monophosphoryl lipid A and a saponin derivative, such as a combination of MPL, or a less reactive composition in which QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations include an oil-in-water emulsion and tocopherol. A particularly good adjuvant formulation comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Another preferred adjuvant is Montanide ISA 720 (Sep.
pic, France), SAF (Chiron, California, Un)
ited States), ISCOMS (CSL), MF-59 (Chiro
n), adjuvant SBAS series (eg, SBAS-2 or SBAS
-4, Smithkline Beecham, Rixensart, Belgium
ium), Detox (Corixa, Seattle, Wash)
inton), RC-529 (Corixa, Seattle, Washin).
gton) and aminoalkylglucosaminide 4-phosphate (AGP).

[0086] Any of the vaccines provided herein can be prepared using well known methods that result in combinations of antigens, immune response enhancers and appropriate carriers and excipients. The compositions described herein can be administered as part of a sustained release formulation, ie, a formulation such as a capsule, sponge, or gel that provides a slow release of compound following administration (eg, comprising a polysaccharide). Such prescriptions are generally
Well-known techniques (eg, Coombes et al., Vaccine 14: 1429-1).
438, 1996) and can be administered, for example, by oral, rectal or subcutaneous implantation, or by implantation into a desired target site. Sustained-release formulations can contain the polypeptide, polynucleotide or antibody dispersed in a carrier matrix and / or contained in a container surrounded by a rate controlling membrane.

The carriers used in such formulations are biocompatible and may be biodegradable. Preferably, the formulation provides a relatively constant level of active ingredient release. Such carriers include poly (lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose, and dextran microparticles. Other delayed-release carriers include supramolecular biovectors that include a non-lipid hydrophilic core (eg, a cross-linked polysaccharide or oligosaccharide) and, optionally, an outer layer that includes an amphiphilic compound such as a phospholipid (eg, US Patent No. 5,151,254 and PC
T application WO 94/20078, WO 94/23701 and WO 96/06
No. 638). The amount of active compound contained in the sustained release formulation depends on the site of implantation,
It depends on the rate of release and the expected duration and nature of the disease to be treated or prevented.

The vaccines and pharmaceutical compositions can be contained in unit-dose or multi-dose containers, such as sealed ampules or vials. Such containers are
Preferably, it is hermetically sealed to maintain the sterility of the formulation until use. In general, formulations can be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the vaccine or pharmaceutical composition can be stored in lyophilized conditions, which only require the addition of a sterile liquid carrier immediately before use.

In another embodiment, there is provided a method of treating yeast infection and related diseases,
Here, a sufficient amount of the peptide of the present invention to inhibit the binding of yeast cells to host tissues is introduced intravenously in a pharmaceutically acceptable carrier capable of preventing the degradation of the peptide. . Further, the peptide and carrier can be introduced intraparially or intramuscularly. In addition to a pharmaceutically acceptable carrier, any of the methods of treatment can be further enhanced by the addition of an adjuvant to initiate immune system activity. Many such adjuvants are well-known in the art.

The following examples further illustrate preferred embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention.

Example 1 Adhesion of microorganisms to host tissues is the first step in the formation of a new environmental colony. In certain organisms, such as the opportunistic fungal pathogen Candida albicans, adhesion to multiple host layers contributes to disease transmission. Cell surface hydrophobicity (CSH) is described in C.I. albic
and plays an important role in the adhesive properties of Candida albicans cells. Hydrophobic cells bind more readily to epithelial cells and plastic than hydrophilic cells and exhibit more generalized binding to host tissues. In addition, hydrophobic cells show greater resistance to phagocytosis killing than hydrophilic cells and are more virulent in mice. Thus, CSH is C.I. albicans are important factors in the development of the disease caused by Candida albicans.

[0092] The cell wall proteins can be determined by C.I. albicans cells are thought to contribute to the CSH status of the cells. A hydrophobic protein has been identified and the C. albicans has been extracted from the cell wall. Earlier studies in our laboratory have shown that these proteins have been used to transform C. albic
ans (Candida albicans) cells. Thus, evaluation of the structure and function of individual hydrophobic cell wall proteins will provide insight into the specific role of CSH in host-fungal interactions.

The evaluation was started by comparing ¹²⁵ I surface labeled wall proteins from hydrophilic and hydrophobic cells. This comparison identified a 38 kDa protein, which appeared to be unique to hydrophobic cells, and the molecular mass range of hydrophobic cell wall proteins (30- 40 kDa). Some preliminary characterization of hydrophobic proteins has shown that they can be glycosylated. Since they bound to the lectin Concanavalin A in Western blots.

It was not clear whether these proteins could perform functions related to virulence in addition to their contribution to the hydrophobic character of the cell. Several groups using affinity chromatography and western blotting techniques have recently identified cell wall proteins that bind to extracellular matrix (ECM) proteins. The ECM protein forms a layer in the host tissue that can provide a binding site for yeast attachment. C. The identified ECM-binding protein of C. albicans has an intermediate molecular mass (30-70 kDa), similar to the hydrophobic protein.

[0095] Recent studies in our laboratory have shown a relationship between CSH and adhesion of Candida cells to ECM proteins. In particular, our laboratory has shown that proteins in the 30-70 kDa size range bind to both fibronectin and laminin.
Thus, hydrophobic proteins may contribute to virulence by mediating attachment to host ECM proteins located throughout defect walls and interstitial sites.

We hypothesized that distinct exposed surfaces on hydrophobic proteins were responsible for the CSH state, and that these proteins were, at least in part, involved in the adhesion of Candida cells to host tissues. The role of these proteins in adhesion involves their interaction with ECM proteins, and this interaction between hydrophobic proteins and ECM proteins involves the hydrophobic region in both sets of proteins. As part of our ongoing investigation of this hypothesis,
We provide here an analysis of three monoclonal antibodies that recognize hydrophobic cell wall proteins (37, 38 and 40 kDa). These antibodies were selected for further experimentation due to the different distribution of their antigens among various pathogenic Candida species. Initial characterization of three monoclonal antibodies and their protein antigens and evaluation of antigen distribution in Candida cell walls is described.

Materials and Methods Protein reagents Human fibronectin (hFn) was obtained from Promega. Mouse laminin (mLn, ultrapure) is available from Collaborative Biomedical P
products (Becton Dickinson). Mouse I
gG2a mouse IgM and alkaline phosphatase (AP) -conjugated goat α-mouse IgG were purchased from Sigma Chemical Co. Purchased from. AP-conjugated goat α-mouse is Jackson Immu
obtained from noResearch Laboratories.

Strain and culture conditions albicans (Candida albicans) isolates were cultured as previously described. [Hazen, K .; C. And Hazen, B .; W. (1993)
FEMS. Microbiol. Left. 107, 83-88; Hazen
K. C. And Hazen, B .; W. (1987) J. Amer. Microbiol. M
methods. 6,289-299]. Briefly, phosphate-buffered (pH 7.2) yeast nitrogen base containing 2% (w / v) glucose (YNB2G) (
+ Amino acids, Difco Laboratories, Detroit, MI)
Cells were grown in stationary phase in. Cells grown to stationary phase at 23 ° C. encountered hydrophobic (cell surface hydrophobic (CSH ≧ 95%); those grown to stationary phase at 37 ° C. were hydrophilic (CSH ≦ 5%)) .

C. germinated albicans cells are required if required.
As previously described, biotin (250 mgl^-1), Glucose (9 gl) ^-1 ), Glycine (1 gl)^-1) And HEPES (6 gl^-1Eagle supplemented with)
Auto-P with salt, pH 6.8 (Flow Laboratories)
Incubation of stationary phase yeast cells from 23 ° C. culture in ow minimal essential medium (
2 hours, 37 ° C, 1.0 × 10⁶Cells ml^-1) (Hazen, KC and H
azen, B .; W. (1987) Microbiol. Immunol31,4
97-508) Only hydrophobic stationary phase yeast cells were induced and germinated. because,
The germination of hydrophilic yeast results in pseudohyphae rather than true embryo tubes.

Cell wall preparations from several species of Candida (see below) were compared for similarity in hydrophobic protein homologs. C. albicans (Candida albicans)
LGH1095 (strain used in protein characterization experiments) and LGH870 have been described previously (Antley, PP and Hazen, KC (198).
8) Infect Immun. 56, 2884-2890). Candida
Keffir, C.I. krusei, C.I. Parapsilosis and C.I. t
ropicalis isolates are from the University of Virginia.
Medical Center Clinical Microbiology
y It was a clinical specimen isolated in the laboratory. Candida g
labrata ZB5 was taken from our frozen stock collection. Other isolates are available from the American Type Culture Collection (ATC).
Obtained from C). Isolates were subcultured three times in YNB2G at 37 ° C.

Release of cell wall proteins and isolated proteins were released from the walls of stationary phase yeast or budding cells by the rapid crude digest (RCD) technique described previously (Glee PM et al., (1995). )
Infect Immun. 63: 1373-1379; Hazen, K. et al. C
. And Hazen, B .; W. (1992) Infect Immun. 60,
1499-1508). The concentration of released protein is ~ 300-500mg
Upon reaching ml ⁻¹ , digestion was stopped by centrifugation (2 × 10 min, 14000 g) followed by removal of supernatant fluid (Coomassie Plus assay, Coomassie Plus assay,
Pierce Chemical Co. ). Protease inhibitors were replenished to the final supernatant fluid and the final protein concentration was determined (Bicinchoninic Acid Protein Assay (Pi
erce) (Smith, PK, (1985) Anal. Biochem.
150, 76-85). This method of digestion minimizes cytoplasmic contamination based on the lack of ghost cells present at the end of the digestion period. The RCD material was not dialyzed. Because previous studies from our laboratory showed that most wall proteins were not lost during dialysis. (Glee, PM, et al., (1996).
J. Med. Vet. Mycol. 34, 57-61)

Polyclonal and monoclonal antibody production Separation of RCD proteins by hydrophobic interaction chromatography (HIC) -HPLC and polyclonal antiserum (α-HICF6) against hydrophobic HIC-HPLC fractions (6 and 7, HICF6 / 7) The production of pAb) has been described previously (Glee, PM, et al., (1995) Infect Immu).
n. 63, 1373-1379; Hazen, K .; C. And Hazen, B .;
W. (1992) Infect Immun 60, 1499-1508). C
. albicans Antibodies to hydrophobic proteins (mAb)
Is the University of Virginia Medical Cen
Produced in collaboration with ter Hybridoma Facility (
Chang, J.M. H. Et al. (1994) Methods, Enzymol. 25
4,430-435). (American Association for
the Accreditation of Laboratory Anni
A / J, or BALB / c mice (maintained at a mal Care approved facility) were immunized with the hydrophobic protein from HICF6 as previously described (
Glee, P .; M. Et al., (1995) Infect Immun. 63,137
3-1379). Antiserum reactivity was monitored by Western blot analysis of the HICF6 / 7 protein. Approximately 3 μg of gel-purified from HIC-HPLC fraction 6 (Glee, PM et al. (1996) J. Med. Vet. Mycol.
34, 57-61) A final intra-splenic boost of 32-40 kDa protein was administered (Spitz, M. et al. (1984) J. Immunol Methods. 70).
, 3943). Spleen cells were fused with Sp / O myeloma cells, plated in 96-well microtiter plates, and supernatant fluids were screened for antibody ELISA. Positive cells were screened by Western blot analysis of the HICF6 / 7 protein. Reactive hybridomas were subcloned and retested by Western blot. Monoclonal antibodies were evaluated for isotype (Pharmingen). The three hybridomas selected for further experiments were F6-6C5-H4 (from 6C5, A / J mice), F6-5D8-
A18 (5D8, derived from BALB / c mice) and F6-5F8-E10 (S
F8, derived from BALB / c mice) was produced in BALB / c mice.

Electrophoresis and Western Blotting Cell wall proteins from RCD were separated by SDS-PAGE using a 12.5% (w / v) acrylamide degradation gel. In addition, cell wall proteins were separated by preparative isoelectric focusing (Rotofor, Bio-Rad). Amphiphiles (pH 4-7, Bio-Lytes, Bio-Rad) were mixed with the RCD solution to a final concentration of 2% (w / v). The solution was introduced into the isoelectric chamber and electrophoresed according to the manufacturer's specifications. Fractions were collected after isoelectric focusing and loaded on SDS-PAGE Slabkel as described above. Following electrophoresis, the separated proteins were transferred to nitrocellulose (BA-85, Schleicher and Schuell) membranes as described (Glee, PM et al., (1995) Inf.
ect Immun. 63, 1373-1379).

The strips were cut from the membrane, rehydrated with water, and immersed in Dulbecco's phosphate buffered saline, pH 7.2 (DPBS). Incubation in DPBS containing 5% (w / v) dry nonfat milk and 0.2% (v / v) Tween (
(37 ° C., 1 hour). This solution was used for all antibody dilution and washing steps. The blocked strips were incubated in the primary antibody solution (37 ° C, 1-2 hours). 6C5 was used as a 1: 2000 dilution of ascites. 5
F8 and 5D8 were used without dilution as hybridoma culture supernatant.
Polyclonal antiserum α-HICF6 pAb was used at a 1: 1000 dilution. The strips were washed three times (10 minutes each) and incubated in the primary antibody solution (37 ° C. for 1 hour). The secondary antibody was an alkaline phosphatase (AP) conjugated goat α-mouse IgG (1: 500) that recognizes 6C5 or 5D8.
) And 5F8 with AP-conjugated goat α- or IgM (1: 5
00). Final washes and detection were as described (Gle
e, P. M. Et al., (1995) Infect Immun. 63,1373-1
379). In lanes treated with polyclonal antiserum, a mixture of the two secondary antibodies was used.

Cell Adhesion Assay Cell adhesion assays were performed in 48-well flat-bottom non-tissue culture treated polystyrene multiwell plates (Falcon # 1178). Fn and L
m was diluted to 66.7αg ml ^{−1 in} DPBS. The distilled water and all buffer solutions used in this assay were sterilized by autoclaving. Wells were incubated overnight at 4 ° C. with 150 μl of diluted ECM protein solution (10 μg protein). The wells were then washed three times with cold DPBS.

The wells were blocked with 250 μl DPBS containing immunoglobulin. The immunoglobulin used for blocking was purified from normal mice and used at the same concentration as the test antibody and in the same isotype as the test antibody. Wells were incubated for 2 hours in blocking solution at room temperature. During the block incubation, cells from a third transfer (23 ° C.) culture were harvested by centrifugation and washed twice with cold distilled water. Germination was induced at this point, after which the embryonic tube initials were harvested and washed as described above. Cell concentration CSH and percent germination were measured. From the washed cell suspension, 3 × 10 ⁶ cells were transferred to each of several glass tubes containing 2 ml of treatment solution.

For treatment with 6C5 and 5D8, dilutions were made in DPBS (1: 3 each).
00 and 1: 500) The cells were added to the ascites solution. Final antibody concentration is 12μ
g mll. In experiments using 5F8, cells were added to a solution of ascites 1: 300 diluted in DPBS (final concentration 23 μg / ml ⁻¹ ). At the same concentration, irrelevant mouse IgG was used as a control for 6C5 / 5D8 and 5F8, respectively.
2a and IgM were used. At room temperature, cells were incubated in the antibody solution for 15 minutes, centrifuged, and washed with DPBS (once). Following treatment, a final cell count was performed and, if necessary, the cell concentration was adjusted to 8 × 10 ² cells nl ⁻¹ .

Immediately before adding cells to the wells, the blocking solution was removed and the wells were
Washed gently once with BS. Following washing, 200 cells from each treatment were added to their respective wells. The plate was then incubated at 37 ° C. for 15 minutes. The wells were gently washed three times with DPBS and 300 μl previously aged at 45 ° C.
l of 1.7% (w / v) molten cornmeal agar (CMA, Difco). The agar was solidified and the plate was incubated overnight at 37 ° C. CMA plates were inoculated using replica aliquots of 200 cells from each treatment.
These plates were also incubated at 37 ° C. overnight. Percent adhesion was calculated by: (colony per well / colony per CMA plate) x 100. All samples were performed in duplicate.

The adhesion data was analyzed for percent relative binding. Binding in the presence of the test or control antibody was compared to binding in the absence of the antibody, defined as 100%. The final data is from a commercially available statistical package (Data Desk,
Data Description, Inc. ) Was used for comparison.

Two aspects of the cell adhesion experimental design are important. First, host / patient ECM
The ECM protein is used in a polymerized, immobilized state, as would be found in. Second, the yeast cells are induced to germinate. This is because the proteins recognized by the three mAbs are predominantly exposed and expressed in embryo tubes.

Results Characterization of the Monoclonal Antibody The results of the isotyping showed that 6C5 and 5D8 were IgG2a and 5F8 was
gM. All three antibodies are of the kappa haplotype. Western blots of RCD protein from hydrophobic cells were probed with three mAbs. 6
C5, 5F8, and 5D8 recognize proteins with approximate molecular masses of 30, 40, and 37 kDa, respectively (FIG. 1, lane 1). R based on isoelectric point
Separation of the CD protein (Rotofor, Bio-Rad) and subsequent Western blot analysis showed that the 37, 38 and 40 kDa proteins were respectively 6.
2, 7.3 and 6.4. Also, proteins of these molecular masses were among the RCD proteins recognized by polyclonal sera raised against hydrophobic wall proteins (FIG. 1). Using this mAb, HIC
-Blotted HPLC fractions. The results indicate that the antigen for the mAb is H
It was only present in IC-HPLC fractions 5-7, indicating that fraction 6 had the highest concentration. The latter result indicates that the proteins recognized by 6C5, 5D8 and 5F8 are in the set of hydrophobic wall proteins.

Distribution of Proteins Recognized by mAbs The RCD of hydrophilic yeast cells and embryo tubes was compared to that of hydrophobic cells (FIG. 1).
The three monoclonal antibodies recognized similar proteins from all three cell types. 5D8 recognized additional proteins in embryo tubes (Figure 1, lane 2) and hydrophilic cells (Figure 1, lane 3). It is unknown whether the 38 kDa protein recognized by 6C5, present in the cell wall from both hydrophobic and hydrophilic cells, is the same protein identified by ¹²⁵ I surface labeling detected only on hydrophobic cells. (See above). One possibility is that wider or altered glycosylation of cell wall proteins may render the iodinated site unavailable to hydrophobic cells.

[0113] Monoclonal antibodies were used to probe blots of wall proteins extracted from several species of Candida. C. as already seen by Western blot and IFA. The walls of albicans (Candida albicans) contain all three proteins. Candida tropicalis cell wall extract contains 6
It contained proteins that reacted with C5 and 5D8, while 6C5-reactive protein contained 38
It was 54 kDa rather than kDa. C. The kefyr cell wall preparation is 5F
It contained proteins bound to 8 and 6C5. In these extracts, 5F8
Recognized 59 kDa and 55 kDa proteins. The protein recognized by 6C5 was 36 kDa rather than 38 kDa. For all three antibodies, C
. glabrata, C.I. guilliermondii, C.I. parapsi
losis or C.I. It did not recognize proteins from ricase digests of cell walls from Krusei. Possibly, what is absent from other Candida species is that it is an epitope rather than the protein itself. Another possibility is that
Differences in cell wall architecture exist between different species. Preliminary results are:
The (38 or 37 kDa) protein, but not the 40 kDa protein, was present in all Candida species tested, indicating that further extraction methods were required for its release. Although potentially restricted to epitopes, this differential expression among various Candida species reinforces our interest in these three proteins and our efforts towards their characterization.

Antibody Inhibition of Cell Binding to ECM The ability of mAbs to block the binding of intact cells to fibronectin (Fn) and laminin (Ln) was examined. In some separate experiments, the database included outliers. Isolated points could not be attributed to any systematic or investigator errors. The median was chosen as a more appropriate comparison parameter because it was less affected by extreme values.

Pre-treatment of embryonic tube initials with the antibody, shown as a decrease in relative binding,
It significantly reduced the observed binding of cells to the immobilized ECM protein relative to untreated cells (FIG. 2). Also, as a result of pretreatment of cells with an irrelevant antibody or control medium,
Perhaps due to steric effects of proteins non-specifically adsorbed to the cell surface, resulting in reduced cell attachment.

While 5F8 significantly reduced cell adhesion to Ln relative to controls (FIG. 2D), this was not the case for cell adhesion to Fn (FIG. 2C). Pretreatment of cells with either 6C5 or 5D8 reduces cell binding to both Fn and Ln.
It was larger than the control (FIGS. 2A and 2B). From these results,
We have concluded that the protein recognized by the Mab is an ECM binding protein.
In addition, the results of cell adhesion to an irrelevant antibody control indicate that the interaction of the 38 and 40 kDa proteins with Fn and Ln has some specificity.

Discussion HPLC-fractionated cell wall proteins were used to generate three monoclonal antibodies, each recognizing a different hydrophobic protein. Using these monoclonal antibodies, we have three antigens on the cell and each monoclonal antibody is immobilized on ECM
It was determined that whole cell adhesion to cells could be inhibited. Thus, the result is that the hydrophobic cells
It suggests that it can adhere to host cells via the contribution of hydrophobic cell wall protein attachment to CM. If correct, C.I. albicans (Candida albicans)
It has multiple surface molecules that can be recognized by ECM.

The binding of microbial cell surface proteins to the extracellular matrix is thought to play an important role in adhesion (for a review, see Roberts, DD.
(1990) Am. J. Respir. Cell Mol. Biol. 3,18
1-186; Calderone, R .; A. . (1993) Trends. Mi
crobiol. 1, 55-58; Patti, J .; M. And Hoeek,
M. (1994) Curr. Op. Cell. Biol. 6,752-758;
Pendrak, M .; L. And Klotz, S .; A. (1995) FEMS.
Microbiol. Left 129, 103-114). C. albican
s (Candida albicans) cells were cultured in vitro and in vitro with fibronectin (Skerl, KG, et al. (1984) Can. J. Microbiol.
30, 221-227; Schelld, W .; M. (1985) Proc. So
c. Exp. Biol. Med. 180, 474-482; Jakab, E .; (1993) Apmis. 101, 187-193), laminin (Lopez-
Ribot, J .; L. (1994) Infect Immun. 62,742
-746; Bouchara, JP. (1990) Infect Immun
. 58, 45-54), fibrinogen (Bouali, A. et al. (1986)).
J. Med. Vet. Mycol. 24, 345-348; Bendel, C.I.
M. (1993) J. Am. Clin. Invest. 92, 1840-1849;
Robert, R.A. (1991) FEMS. Microbiol. Lett
78, 301-304), fibrin (Maisch, PA and Cald).
erone, R .; A. (1980) Infect. Immun. 27,650-
656), and entactin (Lopez-Ribot, JL and Ch.
affin, W.C. L. (1994) Infect. Immun. 62,6564
-6471) has been shown to bind to several ECM proteins.
(Gale et al. (1998) Science. 279, 1355-1358)
The gene INT1 encoding the integrin-protein was cloned and subsequently linked to hyphal growth, virulence and adhesion. The cited studies generally report the identification of individual ECM binding proteins. Comparison of these reports often reveals that several proteins of different molecular masses can each bind to various ECM proteins. For example, C.I. of 37, 60, 62, 67 and 68 kDa. a
Ibicans wall proteins have been observed to bind to laminin (Lopez-Ribot, JL et al. (1994) Infect.
Immun. 62, 742-746; Bouchara, J. M .; -P. (1990
) Infect Immun. 58, 48-54). These molecular masses are calculated from fibrinogen (Casanova, M. et al. (1992) Infect Immu).
n. 60, 4221-4229), fibronectin (Klotz, SA et al. (1994) Infect Immun. 62, 4679-4681), C3d
(Calderone, RA, et al. (1998) Infect Immuno.
56, 252-258); Saxena, A .; And Calderone. , R
. A. (1990) Infect Immun. 58, 309-314) and iC3b (Kanbe, T. et al. (1991) Infect Immun. 59,
1832-1838) is very similar to that of the protein reported to bind. There is some evidence that this multiplicity is due to glycosylation of the protein (Kanbe, T. et al. (1991) Infect Immun.
59, 1832-1838), and these proteins have not been shown to be structurally related.

The monoclonal antibodies described herein first recognize proteins that are characterized based on their hydrophobicity, rather than a direct functional assay, as is most often done. Common aspects of hydrophobicity can provide evidence of the structural relevance described above. We hypothesized that fungal cell wall proteins and ECM proteins interact through their hydrophobic regions.

[0120] The peptide sequence RGD has been identified as a cell binding site on the ECM protein (
Ayad, S.M. (1994) The Extracellular Matr.
ix FactsBook. Academic Press. , Inc. , Sa
n. Diego, CA, 1-163). However, some groups have R
GD alone, ECM and host cells C.I. albicans (Candida albicans)
Are not sufficient to significantly inhibit binding (Bendel, C
. M. (1993) J. Am. Clin. Invest. 92, 1840-1849
Nagre, E .; (1994) J. Am. Biol Chem. 269, 2203
9-22045; Ollert, M .; W. (1993) Infect. Imm
un. 61, 4560-4568; Klotz, S .; A. And Smith, R
. L. (1991) J. Am. Infect. Dis. 163, -604-610).
The results presented here suggest that hydrophobicity is also a shared property beyond the RGD sequence and can contribute to a common mechanism driving ECM-cell interactions.

The involvement of cell surface hydrophobicity in adhesion extends to other microorganisms as well. Va
zquez-Juarez. ((1997) Mol. Mar. Biol. Bi).
otechnical. 6, 64-71) reported the involvement of CSH in the attachment of yeast strains to trout intestine. Davies et al. ((1996) Parasitolo)
gy. 112, 553-559) is the bacterium Pasteuria penetra.
It has been shown that there is a hydrophobic interaction between ns and its host nematode. (Coun
tney et al. (1990) in: Microbial Cell Surface.
Hydrophobicity (Doyle, RJ and Rosenbe)
rg, M .; Eds. ), American Society for Micr
obiology, Washington, D.C. C. pp. 361-386) have shown that fibronectin interacts hydrophobically with the Streptococcus cell surface.

Although the invention has been described and described herein with reference to various specific materials, techniques, and examples, the invention is not limited to this specific material. It is understood that the combination of materials and the technique selected for that purpose are not limited. Numerous variants of such details are:
It will be shown and appreciated by those skilled in the art.

[Brief description of the drawings]

FIG. 1 shows a representative Western blot of Candida albicans cell wall digests.
Gluconase-extracted wall proteins from hydrophobic yeast cells (1), embryonic tube early (2) and hydrophilic yeast cells (3) are shown. The protein is polyclonal α-H
Arrow heads in the (5F8, 6C5 and 5D8) α-HICF6 sections visualized by their recognition by CIF6 antisera or monoclonal antibodies,
The positions of the 37, 38 and 40 kDa proteins are indicated.

FIG. 2 shows C.I. to immobilized Fn (a, c) or Ln (b, d). albican
Inhibition of s (Candida albicans) adhesion. The results of these experiments are shown in box plot passages, where the top and bottom of the box represent the 75th and 25th percentiles, respectively, and the internal horizontal line represents the median. Gray shaded areas indicate 95% confidence intervals around the median. These confidence intervals are calculated such that if the shaded areas do not overlap, the medians differ at the 5% significance level (α = 0.05). n = 4 except for mlgG2a (n = 6).

FIG. 3 is a schematic diagram of the effect of amino acid sequence on surface exposure. A short sequence of contiguous hydrophobic amino acids surrounded by polar amino acids allows it to be anchored to other molecules (
If not restricted to C), it will not undergo a conformational (inward) change (A), but will be able to undergo a longer sequence (B).

FIG. 4 shows the effect of hydrophobic peptide conformation on interaction with hydrophobic sites on target molecules. Two molecules can have hydrophobic regions on their molecular surface, but the interaction is structure-constrained.

FIG. 5 shows the DNA sequence and deduced amino acid sequence of the 6C5 antigen.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/14 G01N 33/50 T 4H045 C12N 5/10 33/53 D G01N 33/50 33/569 A 33 / 53 33/577 B 33/569 C07K 14/39 33/577 C12P 21/08 // C07K 14/39 C12N 5/00 B C12P 21/08 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , K G, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Singleton, David Earl 22968, Virginia, USA Luckersville, William Road 205 (72) Inventor Masouca, James Ame United States 22903, Virginia, Charlottesville, Ivy Drive 116, Apartment # 8 (72) Inventor Woo, Jean G. 77479, Texas, United States of America, Ashland Way Court, Sugar Land 5411 (72) Inventor Gree, Patti M 59715, Minnesota, USA Boseman, West Village 813, # 75 F-term (reference) 2G045 AA25 AA28 BB20 CB21 DA36 FB03 FB05 FB07 FB15 JA20 4B064 AG27 CA10 CA20 CC24 DA01 DA15 4B065 AA914 AB05 CA08 CA25 AA06 AA07 BA17 MA52 MA66 NA05 NA14 ZB311 ZC781 4C085 AA03 AA13 AA14 BB11 EE01 FF24 4H045 AA10 AA11 AA20 AA30 BA13 BA14 CA10 DA50 DA75 DA76 EA29 EA31 EA52 FA72 FA74

Claims (48)

[Claims] _1. A peptide represented by the general formula: G—X ₁ —X ₂ —R, wherein G is glutamate or glutamine, and X ₁ is a chemical bond or glycine, alanine, valine. , Leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine,
X is an amino acid selected from the group consisting of methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine and glutamine; X ₂ is glycine, alanine, valine, leucine, isoleucine, proline,
Phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine and glutamine are amino acids selected from the group consisting of: An isolated peptide, wherein at least one amino acid of said tripeptide is selected from the group consisting of valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. 2. The method according to claim 1, wherein X ₁ is a chemical bond, R is a tripeptide, and wherein only one amino acid of said tripeptide is selected from the group consisting of valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. Item 7. The peptide according to Item 1. 3. X ₁ is a chemical bond and R is a tripeptide, wherein the two amino acids of the tripeptide are independently selected from the group consisting of valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. The peptide according to claim 1. 4. X ₁ is a chemical bond and R is a tripeptide, wherein each of the amino acids of the tripeptide is independent of the group consisting of valine, leucine, isoleucine, phenylalanine, tyrosine, and tryptophan. The peptide according to claim 1, which is selectively selected. 5. The peptide according to claim 4, wherein X ₂ is proline. 6. The peptide according to claim 1, wherein the peptide is glutamate-proline-leucine-tyrosine-isoleucine, glutamate-proline-leucine-tyrosine-valine, glutamate-proline-leucine-phenylalanine-isoleucine, glutamate-proline-leucine-phenylalanine-. The peptide according to claim 1, which is selected from the group consisting of valine. 7. A composition comprising the peptide of claim 1 and a carrier for said peptide. 8. An isolated antibody against the peptide of claim 1. 9. The antibody according to claim 8, wherein said antibody is a monoclonal antibody. 10. The antibody according to claim 8, wherein said antibody is 6C5. 11. The antibody of claim 8, wherein said antibody is produced by cell line F6-6C5-H4. 12. The antibody according to claim 8, wherein said antibody is a polyclonal antibody. 13. The cell line F6-6C5-H4. 14. A method of treating a yeast infection in a patient in need thereof, comprising the step of administering to the patient a composition comprising an active agent that is the antibody of claim 8. . 15. Providing the antibody of claim 8 labeled with a detectable marker; contacting a sample with the antibody of claim 8; forming between the sample component and the labeled antibody. Isolating the resulting complex. A method for detecting a hydrophobic binding domain in a sample containing multiple components. 16. The method of claim 15, wherein said detecting is performed in vivo. 17. The method of claim 15, wherein said detecting is performed in vitro. 18. Providing the antibody of claim 8, labeled with a detectable marker and bound to a solid support, contacting a sample containing a plurality of components with said antibody; Washing the solid support for removal. A method for isolating a hydrophobic binding domain comprising: 19. A method comprising administering to the patient a composition comprising an active agent, wherein the active agent is a peptide represented by the general formula: GX ₁ -X ₂ -R, Wherein G is glutamate or glutamine; X ₁ is a chemical bond or glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, an amino acid selected from the group consisting of asparagine and glutamine, X ₂ is glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine, serine, threonine, lysine, Arginine An amino acid selected from the group consisting of histidine, aspartate, glutamate, asparagine and glutamine, wherein R is a tripeptide, wherein at least one amino acid of the tripeptide is valine, leucine, isoleucine, phenylalanine, tyrosine and A method of treating a yeast infection in a patient in need thereof, wherein the method is selected from the group consisting of tryptophan. 20. X ₁ is a chemical bond, R is a tripeptide, and one amino acid of said tripeptide is selected from the group consisting of valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. 20. The method according to 19. 21. The method of claim 19, wherein said active agent is introduced orally. 22. The method of claim 19, wherein said active agent is introduced intravenously. 23. The method of claim 19, wherein said active agent is applied topically. 24. An isolated peptide represented by the general formula: EX ₁ -LX ₂ -X ₃ -X ₄ , wherein X is glutamate, X ₁ is proline, X ₂ is an amino acid selected from the group consisting of phenylalanine and tyrosine; X ₃ is an amino acid selected from the group consisting of isoleucine and valine; X ₄ Is an amino acid selected from the group consisting of serine and threonine. 25. A composition comprising the peptide of claim 23 and a carrier for the peptide. 26. An isolated antibody against the peptide of claim 23. 27. The antibody according to claim 25, wherein said antibody is a monoclonal antibody. 28. The antibody according to claim 26, wherein said antibody is 5D8. 29. The antibody of claim 26, wherein said antibody is produced by cell line F6-5D8-A12. 30. The method according to claim 25, wherein said antibody is a polyclonal antibody. 31. Cell line F6-5D8-A12. 32. A method of treating a yeast infection in a patient in need thereof, comprising administering to the patient a composition comprising an active agent that is the antibody of claim 25. 33. Providing an antibody according to claim 25, labeled with a detectable marker; contacting said sample with an antibody according to claim 25; Isolating the resulting complex formed. A method for detecting a hydrophobic binding domain in a sample containing multiple components. 34. The method of claim 33, wherein said detecting is performed in vivo. 35. The method of claim 33, wherein said detecting is performed in vitro. 36. Providing the antibody of claim 25 labeled with a detectable marker and bound to a solid support; contacting a sample containing multiple components with the antibody; Washing the solid support in order to remove the protein. 2. A method for isolating a hydrophobic binding domain, comprising: 37. Administering a composition comprising an active agent to a patient, wherein the active agent is represented by the general formula: EX ₁ -LX ₂ -X ₃ -X ₄ A peptide, wherein E is glutamate; X ₁ is an amino acid selected from the group consisting of proline, lysine and glutamate; X ₂ is an amino acid selected from the group consisting of phenylalanine and tyrosine; X ₃ is an amino acid selected from the group consisting of isoleucine and valine; and X ₄ is an amino acid selected from the group consisting of serine and threonine. How to treat an infection. 38. The method of claim 37, wherein said active agent is introduced orally. 39. The method of claim 37, wherein said active agent is introduced intravenously. 40. The method of claim 37, wherein said active agent is applied topically. 41. An isolated 5F8 antibody. 42. The antibody of claim 41, wherein said antibody is produced by cell line F6-5F8-E10. 43. The cell line F6-5F8-E10. 44. A method of treating a yeast infection in a patient in need thereof, comprising administering to the patient a composition comprising an active agent that is the antibody of claim 41. 45. Providing the antibody of claim 41 labeled with a detectable marker; contacting the sample with the antibody of claim 41; Isolating any resulting complex formed. A method for detecting a hydrophobic binding domain in a sample containing multiple components. 46. The method of claim 45, wherein said detecting is performed in vivo. 47. The method of claim 46, wherein said detecting is performed in vitro. 48. Providing the antibody of claim 41 labeled with a detectable marker and bound to a solid support; contacting a sample containing multiple components with the antibody; Washing the solid support to remove the substance.