EP1812063A2 - Monoclonal antibodies to the propeptide of candida albicans and methods of use - Google Patents

Abstract

Monoclonal antibodies which can bind to the propeptide of the Int1p protein of yeast microorganisms such as Candida albicans are provided which can be useful in methods for treating or preventing infections arising from such microorganisms. In particular, monoclonal antibodies are provided which can recognize the epitope located at amino acids 252-260 on the Intl p protein, and such monoclonal antibodies can recognize the propeptide region of Int1 p as well as the complete Int1 p protein. Such antibodies are effective in blocking of the activation of T-lymphocytes, blocking the expansion of T-cells bearing V-beta subsets, such as 2, 3, 14 or others, and blocking the secretion of interferon­gamma, so as to be effective in treating or preventing infection from yeast such as Candida albicans.

Description

MONOCLONAL ANTIBODIES TO THE PROPEPTIDE OF CANDIDA ALBICANS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application Serial No. 60/622, 845, filed October 29, 2004, incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates in general to antibodies which can bind to the propeptide sequence of the lntip protein of Candida albicans and methods of utilizing such antibodies to prevent and treat infections from microorganisms such as C. albicans, and in particular to monoclonal antibodies which can recognize the propeptide region and be useful in blocking the secretion of interferon gamma, blocking the production of T-lymphocytes and blocking the expansion of V-beta subsets so as to be useful in the treatment and prevention of infection from yeasts such as Candida albicans and other microorganisms expressing the Int1 p protein.

BACKGROUND OF THE INVENTION

The dimorphic yeast, Candida albicans, is the leading fungal pathogen in normal hosts and in patients with damaged immune systems. In normal hosts, disease caused by C. albicans ranges from mild, easily treated, superficial disease (e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water) to more severe, chronic or recurrent infections (e.g., candidal vaginitis). It is estimated that 5% of women of child-bearing age will suffer from recurrent candidal vaginitis (Hurley, Proc. R. Soc. Med. 70 (Suppl., 4), 1-8 (1970), and that virtually every woman will experience at least one episode during her reproductive years. Vaginitis is particularly frequent in otherwise normal females with diabetes or a history of prolonged antibiotic or oral contraceptive use. While short-term topical therapy is effective in treating individual episodes of vaginitis, such agents do not prevent recurrences. Thus, even in the normal host, infection with C. albicans can occur at epithelial surfaces, and recurrences are not prevented by presently available therapies.

In immunocompromised hosts such as cancer patients, transplant patients, post-operative surgical patients, premature newborns, or HIV-infected people, C. albicans ranks as the leading fungal pathogen. Invasion leading to systematic infection may also develop in neutropenic patients whose t-cell function is comprised. (Hostetter MK, Clinical Microbiology Reviews, Jan 1994, pp. 29-42.) In this population, disease ranges from aggressive local infections such as periodontitis, oral ulceration, or esophagitis in HIV-infected patients, to complex and potentially lethal infections of the bloodstream with subsequent dissemination to brain, eye, heart, liver, spleen, kidneys, or bone. Such grave prognoses require more toxic therapy, with attendant consequences from both the underlying infection and the treatment. Here again, the infection typically begins at an epithelial site, evades local defenses, and invades the bloodstream in the face of immunosuppression. Strategies to interrupt candidal adhesion therefore have broad applicability to the prevention of mild but recurrent disease in the normal host and to the reduction of substantial morbidity and mortality in the immunocompromised.

It is well recognized that C. albicans adheres to epithelial and endothelial cells in the human host, often times by recognizing proteins of the extracellular matrix called ligands. These ligands include proteins such as fibronectin, vitronectin, fibrinogen, the C3 degradation fragment iC3b, or the shorter C3 degradation fragment C3d. Because recognition of all of these proteins except C3d appears to be dependent upon the amino acid sequence ARGININE- GLYCINE-ASPARTIC ACID (or R-G-D), these candidal adhesions are thought to operate like the vertebrate integrins and are called "integrin-like proteins" or "integrin analogs."

Vertebrate integrins are composed of two subunits: an α-subunit and a β- subunit. There are approximately 14 α and 8 β subunits described to date in vertebrate cells. Using monoclonal or polyclonal antibodies to vertebrate integrins, several investigators have obtained evidence for integrin-like proteins in C. albicans.

One such protein is the protein lntip of Candida albicans, and this protein has been observed to function as an adhesin, to participate in morphologic switching of blastospores to hyphae, and has been linked to virulence in mice. Rapid mortality ascribable to INT1/INT1 strains suggested that lntip may have an immunomodulatory role. Pathogenesis studies using a mouse fungemia model have linked lntip mediated adhesion and filamentation to Candida albicans virulence (Gale et al., Science 279:1355-1358, 1998), and intravenous inoculation of an int1/int1 double disruption mutant (CAG3) is associated with reduced mortality and renal inflammation compared to the wild type INT1/INT1 strain (CAF2) (see Bendel et al., MoI. Genetics and Metabolism 67:343-351, 1999).

In particular, the mode of operation of the Candida albicans blastospores is to activate T-lymphocytes, expand T-cells bearing V-beta subsets such as 2, 3, 14 or others, and to elicit interferon gamma (IFN-γ) which results in the colonization and progression of the yeast infection. In this regard, mortality rates from infections from organisms such as disseminated Candidas remain high despite aggressive antifungal therapy (Todischini, J. Intern Dis. 1 :S37-S41 , 1997), and a highly effective method of treating or preventing diseases caused by Candida albicans and other similar microorganisms expressing lntip has yet to be obtained because all three of these of these modes, namely the activation of T-lymphocytes, the secretion of interferon gamma, and the expansion of V- beta subsets 2 and 14, have not been blocked by a single method of treatment.

SUMMARY OF THE INVENTION

It is thus an object of the present invention to provide a method of effectively treating infection caused by Candida albicans and other similar microorganisms which express the lntip protein.

It is further an object of the present invention to provide a method of blocking the activation of T-lymphocytes, blocking the expansion of T-cells bearing V-beta subsets such as 2, 3, 14 or others, and blocking the secretion of interferon gamma so as to effectively treat or prevent infection caused by Candida albicans.

It is still further an object of the present invention to provide a method of isolating a propeptide and treating or preventing infection caused by Candida albicans through generation of monoclonal antibodies against the propeptide.

It is even further an object of the present invention to isolate specific regions of the lntip protein from C. albicans and other similar microorganisms such as S. cerevisiae which express the lntip protein, and provide agents and antibodies capable of binding said regions.

It is still further an object of the invention to locate specific regions in the propeptide region and generate monoclonal antibodies to said regions that will be effective in binding the propeptide and the complete lnti p protein, and that will be effective in blocking the three modes of progression of the yeast infection, namely blocking of the activation of T-lymphocytes, blocking the expansion of T- cells bearing V-beta subsets, and blocking the secretion of interferon-gamma.

It is even further an object of the present invention to isolate specific regions of the lntip protein from C. albicans and other similar microorganisms such as S. cerevisiae which express the lnti p protein, and provide peptides or antibodies which can either disrupt the cleaving of the propeptide, or which can act to block the potential binding sites for the propeptide, namely the antigen- presenting cell or the T-lymphocyte binding region.

It is also an object of the present invention to provide a method of inhibiting the activity of the lntip protein of Candida albicans so as to prevent or treat infections caused by microorganisms expressing the lntip protein.

These and other objects are achieved by the present invention which comprises isolating a peptide from specific regions from the lntip protein of C. albicans including the propeptide region and generating monoclonal antibodies thereto, and treating or preventing an infection from C. albicans or other microorganism expressing the lntip protein by administering to a human or animal patient an effective amount of the monoclonal antibody which can bind to those specific regions and thus disrupt the activity of the lntip protein. In particular, the invention relates to the isolation of the propeptide of the lntip protein and the development of monoclonal antibodies which can bind to the propeptide and which have been unexpectedly also been able to disrupt the activity of the lnti p protein, such as by blocking the activation of T-lymphocytes, blocking the expansion of V-beta subsets, and by suppressing the secretion of interferon-gamma (IFN-γ). The invention thus relates to the generation of monoclonal antibodies which can suppress the superantigen of the host so as to be useful in methods of preventing or treating infections from C. albicans or other microorganisms expressing the lntip protein

BRIEF DESCRIPTION OF THE DRAWING FIGURES

Fig. 1 is a depiction of the amino acid sequence (SEQ ID NO:1) of the lntip protein from C. albicans.

Figs. 2A and 2B show the nucleic acid sequence (SEQ ID NO:2) coding for the lntip protein from C. albicans.

Fig. 3 is a schematic representation of the activation of a general proprotein convertase which shows the presence of a signal peptide, the propeptide, an inactive subtilisin and P-domain, and the manner of activation.

Fig. 4 is a schematic representation of the int1 p protein as compared to a generic proprotein convertase which illustrates the clipping of the lntip propeptide which is cleaved to become a superantigen at the same time the subtilisin regions are activated as well.

Fig. 5 shows the P Domain subtilisin motifs from a variety of proteins.

Fig. 6 shows a comparison of the high-affinity heparin binding site of Mycobacterium tuberculosis heparin-binding hemagglutin adhesin (HBHA) (SEQ ID NO:3) with the heparin-binding site of the lntip protein of Candida albicans (SEQ ID NO:4).

Fig. 7 depicts the activation of T lymphocytes after incubation with INT1/INT1 blastospores (squares) or int1/int1 blastospores (diamonds). Data from five normal adult donors are shown. *p<0.05. Fig. 8 depicts the effects of antibodies against the MHC Class Il determinant HLA-DR (black columns) on lymphocyte activation in response to PHA, TSST-1 , INT1/INT1 C. albicans, or int1/int1 C. albicans. An irrelevant murine IgG (hatched bars) served as isotype control. *p<0.04.

Fig. 9 shows the effects of TSST-1 , INT1/INT1 C. albicans, int1/int1 C. albicans, and phytohemagglutinin on stimulation of Vβ subsets. Unactivated T lymphocytes served as control. *p<0.05.

Fig. 10 is a schematic view showing the regions of a generic proprotein convertase.

Fig. 11 is a schematic representation of the lntip peptide regions in accordance with the present invention including an identification of regions recognized by certain anti-peptide polyclonal antibodies.

Fig. 12 illustrates the flow cytometry of surface-exposed domains of lntip when C. albicans blastospores are grown to exponential phase in the absence (left panel) or presence (right panel) of 2 units of heparin. X axis represents log- scale fluorescence; Y axis represents percent yeasts fluorescing. Hatched area - fluorescence with anti-INT600. Gray area-fluorescence with anti-CBS2. Fluorescence of C. albicans cells incubated with rabbit IgG serves as control - dotted line.

Fig. 13 is a Western blot of supematants from /Λ/T7-expressing S. cerevisiae grown in the absence or presence of heparin and probed with rabbit polyclonal antibodies to the lntip amino terminus (anti-INT600), to the second divalent cation binding site (anti-CBS2), or to the RGD domain (anti-RGD).

Fig. 14 are immunoblots showing the purification of Pep263. Silver stain lanes 1-4. Western blot lanes 5 and 6. Lane 1 - S. cerevisiae lysate after expression of Pep₂₆3; lane 2 - fraction 300-1 from nickel column; lane 3 - fraction 300-2; lane 4 - purification of Pep₂₆₃ to homogeneity; Lane 5 shows that a single band of 44 kDa on silver strain (lane 4) reacted with anti-His antibody on Western blot.

Fig. 15 is a graphic representation of the percent of T lymphocytes up- regulating the IL-2 receptor (Y axis) in response to Pep₂₆₃ presented as soluble antigen (leftmost group of three bars), as antigen bound to the plate (middle group), or as antigen bound to an anti-His antibody attached to protein A beads (right group).

Fig. 16 is a schematic representation of a model for the participation of lntip in Candidemia.

Fig. 17 shows the MHC-II Binding Sites in the lntip protein (SEQ ID NO:6), and in Mycoplasma arthritidis (SEQ ID NO:5), as disclosed in J. Exp. Med. 183:1105-1110 (1996), incorporated herein by reference.

Fig. 18 shows the linkage of the T lymphocyte to the antigen-presenting cell through the superantigen which is produced after the propeptide is cleaved.

Fig. 19 shows the detection of Pep263 (left panel) and full-length lntip (right panel) by the monoclonal antibodies generated in accordance with the present invention including 364.5 (lane 1), 253.4 (lane 2), 163.5 (lane 3) and 44.5 (lane 4). Purified Pep263 is shown in the lane marked with an arrow in the left panel. Full-length lntip is denoted by the arrow in the right panel.

Fig. 20 shows the inhibition of T cell activation by the monoclonal antibodies generated in accordance with the present invention.

Fig. 21 shows the inhibition of Vβ expansion by the monoclonal antibodies generated in accordance with the present invention.

Fig. 22 shows the Vβ expansion by the propeptide Pro263 and the blockade by the monoclonal antibodies generated in accordance with the present invention.

Fig. 23 shows the expansion of Vβ Subsets 2 and 14 by soluble Pep₂63; and the inhibition thereof by MAb 163.5 in accordance with the invention

Fig. 24 shows the induction of IFNy by soluble Pep₂63; and the inhibition thereof by MAb 163.5 in accordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, the present inventors have now discovered and isolated several distinct regions of the lntip protein, and the present invention is directed to treating or preventing infections from microorganisms which express the lntip protein, including yeast of the Candida species such as Candida albicans, and other microorganisms such as S. cerevisiae, by disrupting the regions, including the propeptide region, which are involved with the pathways by which the lntip protein is activated in a host. In particular the present invention is directed to the generation and use of monoclonal antibodies which can bind to the specific regions of the Intip protein and which thus can be useful in treating or preventing C. albicans infections. In general, it is desired to develop antibodies which can prevent the propeptide from cleaving, and/or antibodies that will bind to the propeptide and thus disrupt the activation of the lntip protein. The lntip protein is described in U.S. Pat. Nos. 6,774,219; 6,346,411 ; 5,886,151 ; WO 02/26257; and pending US application 09/964,858, filed September 28, 2001 , and all of these patents and applications are incorporated herein by reference.

In one of the embodiments, the invention relates to peptides, either linear or cyclic, which have the same sequence as that of the sites on the superantigen propeptide which will bind to two sites, namely the antigen-presenting cell (such as the MHC-II locus) and the T lymphocytes on the host cell. In the Intip protein, the MHC-II binding peptide appears to be in the region of from amino acid 239 through 254 (in the propeptide region of 1-263) of the sequence of the protein shown in Fig. 1 , and this sequence is shown in Fig. 17. Accordingly, the use of this peptide, or other blocking peptides, is contemplated in accordance with the invention in any suitable form, e.g., pharmaceutically acceptable compositions, as would be used for administration to a human or animal patient. These types of blocking peptides can thus be administered to the host as a method of blocking the sites that would become bound to the superantigen propeptide, and thus can be used to prevent or treat infections caused by the lntip protein.

In a further aspect of the present invention, it is contemplated that treatment or prevention of infections caused by microorganisms such as C. albicans may be achieved by causing mutations in the specific regions as set forth herein which can cause conformational or other changes to the peptides coded by these regions and thus disrupt the immunomodulatory ability of the lntip protein. The gene sequence and the peptide sequence for the lntip protein has previously been disclosed, e.g., in Proc. Natl. Acad. Sci. U.S.A. 93 (1), 357-361 (1996), incorporated herein by reference. In addition, further information regarding lntip has been provided in pending U.S. patent application Ser. No. 09/264,604 and in U.S. Pat. No. 5, 886,151, both incorporated herein by reference. Finally, the amino acid sequence of the lntip protein is shown in Figure 1 , and the DNA sequence is shown in Figures 2a-2b. In the preferred embodiments, those mutations in accordance with the invention will be those which can prevent the cleaving of the propeptide, or which can disrupt the binding of the propeptide superantigen to the antigen-presenting cells or to the T lymphocytes of the host. Accordingly, in accordance with the invention, mutations in either or both of these propeptide binding regions are preferred.

As will be shown further below, the present invention thus relates to antibodies which can bind to the specific regions from the C. albicans lntip protein as set forth below and the use of those antibodies in disrupting the C. albicans activity in human or animal hosts so as to prevent or treat infections caused by this or other similar microorganisms expressing the lntip organism. Accordingly, in one embodiment, the present invention relates to isolated and/or purified antibodies, such as polyclonal or monoclonal antibodies, which have been generated against specific regions of the C. albicans lntip protein which can be useful in methods of preventing and treating candidal and other yeast infections caused at least in part by the lntip protein and its immunomodulatory ability. The term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, including the products of an Fab immunoglobulin expression library, and generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. As indicated above, it is desirable to provide antibodies which can disrupt the activation of the lntip protein in any of a number of ways, including preventing the cleaving of the propeptide, or disrupting the binding of the cleaved superantigen to host cells at its binding sites, namely the antigen-presenting cell (such as the MHC-II locus) or the superantigen-binding site on T lymphocytes. As described further below, these antibodies are preferably used in amounts effective to prevent or treat infections from C. albicans and other similar microorganisms, and these antibodies may be produced in any of a number of suitable ways well known in the field to produce polyclonal or monoclonal antibodies. For example, in addition to generating polyclonal antibodies by injecting the isolated peptides of the invention into a suitable animal model and isolating the antibodies therefrom, monoclonal antibodies directed to the lntip regions described below may also be generated using the method of Kohler and Milstein (see, e.g., Nature 256:495-7, 1975), or other suitable ways known in the field. Antisera prepared using monoclonal or polyclonal antibodies in accordance with the invention are also contemplated and may be prepared in a number of suitable ways as would be recognized by one skilled in the art.

In a specific aspect of the present invention, monoclonal antibodies may be generated which can recognize the propeptide region or the complete lntip protein and thus be useful in methods of treating or preventing Candida albicans infections. In particular, the monoclonal antibodies of the present invention have unexpectedly been able to block the three key elements of Candida infection caused by the superantigen propeptide, namely the blocking of the activation of T-lymphocytes, the blocking of the expansion of T cells bearing Vβ subsets such as 2, 3, 14 or others, and the blocking of the secretion of IFNγ. Accordingly, as set forth further below, the monoclonal antibodies of the present invention are more effective than previous treatment regimens in that they can prevent all three modes of operation of Candida albicans.

In general, monoclonal antibodies of the invention may be generated in any manner conventionally used in the art, e.g., methods arising from the well known method taught by Kohler and Milstein. In the present case, monoclonal antibodies may be generated as follows: Antibody Scale-up and Purification

Hybridoma cells were grown in RPMI/DMEM, 1X Nutridoma-SP media containing 2mM sodium pyruvate, 4mM L-glutamine and 2X penicillin- streptomycin to 2-3 liter culture volumes. Hybridoma supernatants were then harvested by centrifugation. The supernatants were filtered through 0.45 μM filters and the IgG was affinity purified using protein G chromatography. The monoclonal antibodies were eluted using 0.1 M glycine, pH 2.7 and immediately neutralized with one tenth volume of 2M Tris, pH 8.0. The purified IgG was then dialyzed against 1X D-phosphate buffered saline, pH 7.4. Testing regarding these monoclonal antibodies is described further below in the Examples.

Generation of monoclonal antibodies to the INT-1A peptide.

Monoclonal antibodies may also be generated against the INT1A peptide, a synthetic peptide corresponding to amino acids of 248-277 of INT-1. The peptide was synthesized with an N-terminal cysteine. The peptide was coupled to either ovalbumin or KLH. The C at the beginning was added to couple to an ovalbumin as a carrier

INT-1 A = C-VNSEPEALTDMKLKRENFSNLSLDEKVNLY (SEQ ID NO:9)

Alternatively, one may start with other particular peptide sequences which may generate monoclonal antibodies that can recognize the propeptide at amino acids 1-263 of the Intip protein including the following sequence wherein histidine replaces the arginine at the 263 position The role of the histidine at position 263 is discussed further below:

C-VNSEPEALTDMKLKHENFSNLSLDEKVNLY (SEQ ID NO:11)

In both of these case, the sequences start with a Cysteine as a coupling agent, but antibodies may be generated from these sequences without the cysteine coupler if desired. The INT-1A-Ova coupled peptide as set forth above was mixed with Freund's complete adjuvant and injected into (2) Balb/c mice. In accordance with the invention, polyclonal or monoclonal antibodies may be generated against this peptide which will be useful in detecting the propeptide region and the complete Inti p protein. In one specific procedure for generating antibodies from this peptide, immunizations were as follows:

The mice received 5 RIMMS immunizations of approximately 1-10 μg of antigen emulsified in Freund's complete adjuvant and RIBI's adjuvant via subcutaneous (s.c.) injections. These immunizations were administered over the course of 7-11 days. For each immunization time point, the antigen was injected into twelve different subcutaneous sites that are proximal to the draining lymph nodes of the mice.

An emulsion of 0.5 to 5 μg of soluble antigen mixed with an equal volume of Freund's Complete adjuvant was delivered to 2 sites in the nape of the neck and bilaterally to the calf and groin. 40-50 μl of the emulsion will be administered to each site. The mice then received an injection of 0.5 to 5 μg of antigen emulsified in RIBI adjuvant at juxtaposed sites (lower and mid calf region, thigh, and axilla). 40-50 μl of the emulsion was administered to each site.

Three days after the final boost, the lymph nodes were removed, teased into a single cell suspension and the lymphocytes harvested. The lymphocytes were then fused to a P3X64Ag.653 myeloma cell line (ATCC #1580). Cell fusion, subsequent plating and feeding were performed according to the Production of Monoclonal Antibodies protocol from Current Protocols in Immunology (Chapter 2, Unit 2.).

Any clones that were generated from the fusion were then screened for specific anti-INT-1A antibody production using a standard ELISA assay with the INT-1A-KLH coupled peptide as the target protein. Positive clones were expanded and tested further. Numerous positive clones were originally identified and characterization by dot-blot analysis on the 1-263 INT-1 propeptide. Any clones that were generated from the fusion were then screened for specific antibody to the linear peptide using a standard ELlSA assay with the INT-1A-KLH coupled peptide as the target protein. Positive clones were expanded and tested further. Numerous positive clones were originally identified and characterization by dot-blot analysis on the 1-263 INT-1 propeptide indicated four that should be pursued for further characterization. These four were single cell cloned by limiting dilution. Single cell clones were tested for activity and all four produced single cell clones that generated specific anti-INT-1A antibodies, and these were identified as 44.10, 163.5, 253.4, and 364.5. These four were scaled up and purified material was analyzed for its ability to bind the 6X-HiS- tagged INT-1 (AA 1-263) on western blots.

Of the four monoclonal antibodies tested on Western blot, one (Mab 163.5) recognized both lntip and Pep263 (see Figure 19). Additional experiments indicated that the epitope recognized by Mab 163.5 was the region of from amino acids 252-260 or {252E P E A L T D M K280} (SEQ ID NO:8). This epitope includes a portion of the putative MHC class Il binding site in Pep263. The monoclonal antibody Mab 163.5 was then tested to determine its effectiveness in inhibiting T lymphocyte activation, expansion of Vβ subsets 2 and 14, and I FNy production stimulated by C. albicans, and the results are shown in the Table below:

TABLE 1

Accordingly, the monoclonal 163.5 was unexpectedly successful in achieving the necessary inhibition of the three main effects of the propeptide, namely it was able to block the activation of T-lymphocytes, it blocked the expansion of T cells bearing Vβ subsets 2 and 14, and it blocked the eliciting of I FNy. The monoclonal antibody of the present invention will thus be useful in treating or preventing candidal and other yeast infections caused at least in part by the lntip protein and its immunomodulatory ability

Generation and Testing of Additional Pep263 monoclonal antibodies:

An amino terminal sequence of lnti p shares 56% identity with the MHC Class Il binding site of the superantigen MAM from Mycoplasma arthritidis (see Figure 17), and this sequence is included in the propeptide region Pep263. As shown herein, the evidence shows that the first 263 amino acids of the lntip (Pep263) are a source of superantigen activity.

In the method of generating monoclonal antibodies, DNA encoding Pep263 was tagged at the 5' end with 6xHis, expressed in S. cerevisiae, and affinity purified on a nickel column. The resultant protein exhibited a mass of 44 kDa — much closer to that observed in the typical superantigen. Monoclonal antibodies were developed to a linear peptide encompassing both the putative MHC class Il binding site of lntip based on homology with the superantigen MAM from Mycoplasma arthritidis and the C-terminal histidine (H263):

239 F A Q L L N K N N E V N S {E P E A L T D M K} L K H₂₈₃ E N F S N (SEQ ID NO:7)

In the preferred method, the linear peptide is mixed with Freund's complete adjuvant and injected into Balb/c mice. In one exemplary procedure for generating antibodies from this peptide, immunizations were as follows:

The mice received 5 RIMMS immunizations of approximately 1-10 μg of antigen emulsified in Freund's complete adjuvant and RIBI's adjuvant via subcutaneous (s.c.) injections. These immunizations were administered over the course of 7-11 days. For each immunization time point, the antigen was injected into twelve different subcutaneous sites that are proximal to the draining lymph nodes of the mice.

An emulsion of 0.5 to 5 μg of soluble antigen mixed with an equal volume of Freund's Complete adjuvant was delivered to 2 sites in the nape of the neck and bilaterally to the calf and groin. 40-50 μl of the emulsion will be administered to each site. The mice then received an injection of 0.5 to 5 μg of antigen emulsified in RIBI adjuvant at juxtaposed sites (lower and mid calf region, thigh, and axilla). 40-50 μl of the emulsion was administered to each site.

Three days after the final boost, the lymph nodes were removed, teased into a single cell suspension and the lymphocytes harvested. The lymphocytes were then fused to a P3X64Ag.653 myeloma cell line (ATCC #1580). Cell fusion, subsequent plating and feeding were performed according to the Production of Monoclonal Antibodies protocol from Current Protocols in Immunology (Chapter 2, Unit 2.).

These antibodies recognized the epitope at amino acids 252-260 and thus could be useful in the present invention.

In addition to the above aspects, the invention relates to the use of agents which can bind to the specific regions below so as to disrupt these peptides and again inactivate the infectious and immunomodulatory pathways by which microorganisms expressing the lntip protein by become virulent. Finally, it is also contemplated that mutations to these regions, whether to the amino acid sequences or to the nucleic acid sequences coding these peptides, may also be utilized in order to disrupt the functioning of the lntip protein and to make the infectious microorganisms ineffective or less virulent.

In one embodiment of the present invention, the invention relates to the isolation of the propeptide of the lntip protein and the use of this propeptide in generating antibodies and other agents which will be useful in the treatment or prevention of C. albicans infection. This propeptide constitutes amino acids 1- 263 of the lntip protein, such as shown in Fig. 1 , and has been identified as peptide Pep₂₆₃- As the present inventors have determined, the propeptide, Pep₂₆₃ constitutes a superantigen-like moiety which is released from lntip and which plays a major role in activating T lymphocytes in host cells. Accordingly, an antibody or other agent capable of binding to this propeptide can be utilized in a method of disrupting the activation of T lymphocytes caused by microorganisms such as C. albicans and S. cerevisiae, and thus can be utilized in methods of preventing, treating, or reducing the virulence of infections from such microorganisms which express Inti p. In particular, the antibody to the propeptide in accordance with the present invention will be able to disrupt the functioning of the lntip protein, e.g., such as by binding the propeptide and/or preventing the cleaving of the propeptide and thus stopping the release of the propeptide in its superantigen form.

The propeptide Pep₂6₃ also contains a heparin binding site at amino acids 155-169, as shown, e.g., in Figure 6, and it appears that activation of T lymphocytes is triggered by Pep₂₆₃ when this peptide is cleaved from the amino terminus of lnti p in a reaction accelerated by physiologic doses of heparin. In the absence of heparin, Pep₂₆₃ appears to be covert and is generally not detectable by antibodies such as anti-INT600, an antibody to the first 600 amino acids of the lntip protein. However, in the presence of heparin, the amino terminus of lntip is exposed, at which point Pep₂₆₃ is cleaved and released into the fluid phase where it exhibits superantigen-like effects culminating in the release of pro-inflammatory cytokines that influence the clinical outcome and cause or enhance infection of microorganisms such as C. albicans and S. cerevisiae which express Intip. Accordingly, agents and antibodies to Pep₂63 in accordance with the present invention can be useful in methods to prevent or treat infections in microorganisms expressing the lntip protein and to eliminate or reduce the activation of T lymphocytes caused therefrom.

As shown in the schematic drawing Figs. 3 and 4, activation of "subtilisin- like" proprotein convertases occurs in the lnti p protein which ultimately leads to the cleaving of the propeptide and the activation of the virulent form of the microorganism. In Fig. 3, the schematic analysis of the lntip protein shows the presence of a signal peptide, the propeptide, an inactive subtilisin and the P- domain. The processing or "P-domain" is employed to clip the propeptide at the carboxy terminal side of dibasic residues, thereby releasing the propeptide. Exposed D-H-N-S (SEQ ID NO: 10) active site residues assume the subtilisin serine protease conformation. This amino terminal processing is shown further in Fig. 4 wherein the original form of lntip is transformed by the clipping of the propeptide, which includes heparin binding region 155-169, and which is cleaved to become a superantigen at the same time the subtilisin regions are activated as well. P Domain subtilisin motifs from a variety of proteins are compared as shown in Fig. 5. Fig. 6 shows a comparison of the high-affinity heparin binding site of Mycobacterium tuberculosis heparin-binding hemagglutin adhesin (HBHA) with the heparin-binding site of the Int1 p protein of Candida albicans.

As thus has been shown by the present inventors, the specific regions of the lntip protein which are involved in the activation of T lymphocytes by this protein all present target sites for disruption of infectivity and virulence of microorganisms that express this protein such as C. albicans and S. cerevisiae. As indicated above, the propeptide region at amino acids 1-263 which includes a heparin binding site is critical to the activation process in that this propeptide is cleaved from the protein in order to become a superantigen which has been shown to be able to immunomodulate host cells. In accordance with the invention, antibodies or other agents which can bind this region can thus be useful to prevent T-ceil activation and can thus be employed in methods of preventing or treating outbreaks of infections from microorganisms expressing Intip.

Still other specific regions utilized in the activation process have been identified, and these peptides can be isolated and/or purified so as to be used in generating antibodies and other agents which will bind to these proteins or otherwise be able to disrupt the lntip activation process. Included in these regions in addition to the propeptide region at amino acids 1-263 are the Catalytic domain 1 at amino acids 435-639, the Catalytic domain 2 at amino acids 738-949, and the Processing domain (or "P-domain") motif at amino acids 1022-1236, as best shown in Fig. 11. In accordance with the present invention, isolated and/or purified antibodies, produced for example in the manner described above, may be generated against the specific regions recited above, and effective amounts of said antibodies may be employed in methods of preventing or treating infections from C. albicans or other microorganisms that express the lntip protein. Similarly, other methods of treatment or prevention in accordance with the present invention would include agents which bind to or otherwise disrupt these specific regions so as to reduce or eliminate lntip activity, or mutations in these specific regions of wild-type sequences which also are effective in reducing or eliminating lntip activity. As indicated above, in the most desirable embodiment, these antibodies will function so as to disrupt Intip activity, such as by binding the peptide regions and/or preventing the cleaving of the propeptide and thus stopping the release of the propeptide in its superantigen form.

In addition, other peptides or antibodies which can disrupt the binding of the superantigen to the host cells are also provided in accordance with the invention. The superantigen enables the activation of T lymphocytes through a two-fold binding system wherein the superantigen binds to both the T cell and to the antigen-presenting cell, such as at the MHC Class Il locus, such as shown in Fig. 18.

It is also contemplated that isolated nucleic acids coding for the regions set forth above, namely the propeptide region at amino acids 1-263, the catalytic domains 1 and 2, and the processing domain as shown in Fig. 11, will be contemplated in accordance with the present invention. As would be recognized by one skilled in the art, nucleic acid sequences in accordance with the invention will include not only the specific regions of the nucleic acid sequence as shown in Figs. 2A-2B which correspond to the peptide regions as set forth above, but to any alternative nucleic acid sequences coding for those amino acid sequences. The isolated nucleic acids of the invention will be useful in many appropriate ways, including generating the peptide regions in accordance with the invention through recombinant means so that these recombinant peptides may be used to generate appropriate antibodies. In addition, it is contemplated that mutations to the peptide and nucleic acid sequences in these regions will also be useful in providing alternative methods by which to disrupt the lntip activation pathways.

In addition to the use of monoclonal antibodies which bind to the propeptide and/or other specific regions of the lntip protein as set forth above in methods of treating or preventing infection, the present invention also contemplates the use of these antibodies in a variety of ways, including the detection of the presence of microorganisms such as C. albicans or S. cerevisiae and thus using antibodies to diagnose infections caused by microorganisms expressing Intip, whether in a patient or in medical materials which may also become infected, is contemplated in accordance with the invention. For example, one such method of detecting the presence of infections by microorganisms expressing lntip involves the steps of obtaining a sample suspected of being infected, and lysing the cells so that the DNA can be extracted, precipitated and amplified. Following isolation of the sample, diagnostic assays utilizing the antibodies of the present invention may be carried out to detect the present of lntip microorganisms such as C. albicans or S. cerevisiae, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoasssay, Western blot analysis and ELISA assays.

Accordingly, antibodies in accordance with the invention may be used for the specific detection of Intip-producing microorganisms, for the prevention or treatment of infection from said microorganisms, or for use as research tools. As indicated above, the term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, including the products of an Fab immunoglobulin expression library. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art.

As would also be recognized by one skilled in the art, the monoclonal antibodies of the present invention may also be formed into suitable pharmaceutical compositions for administration to a human or animal patient in order to treat or prevent an infection caused by yeast such as C. albicans or S. cerevisiae. Pharmaceutical compositions containing the antibodies of the present invention, or effective fragments thereof, may be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such as saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the patient and the patient's condition, and a variety of modes of administration would be suitable for the compositions of the invention, as would be recognized by one of ordinary skill in this art. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.

For topical administration, the composition is formulated in the form of an ointment, cream, gel, lotion, drops or solution. Alternatively, when so desired, wound or surgical dressings, sutures and aerosols may be impregnated with the composition to further prevent infection. The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol.

Still further, the isolated antibodies of the present invention, or active fragments or portions as set forth above, may also be utilized in the development of vaccines for passive or active immunization against candidal-type infections or other infections associated with Intip-producing microorganisms. Further, these compositions may also be administered to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo. In addition, the antibody may be modified as necessary so that, in certain instances, it is less immunogenic in the patient to whom it is administered. For example, if the patient is a human, the antibody may be "humanized" by transplanting the complimentary determining regions of the hybridoma-derived antibody into a human monoclonal antibody as described, e.g., by Jones et al., Nature 321 :522- 525 (1986) or Tempest et al. Biotechnology 9:266-273 (1991).

In one embodiment, the isolated peptides in accordance with the invention may be used in the preparation of a vaccine which comprises one or more of the lntip peptides as described above in an amount sufficient to generate an immunological response. In addition, antibodies in accordance with the invention may be used as a passive vaccine which will be useful in providing suitable antibodies to treat or prevent candidal or other similar infections. As would be recognized by one skilled in this art, a vaccine may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. Although many methods of administering the vaccine will be suitable, the particular mode of administration will depend on the nature of the infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration, and the carrier may be include common materials such as water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The preferred dose for administration of an antibody composition in accordance with the present invention is that amount will be effective in preventing of treating a yeast infection or infection from other microorganisms that express the lntip protein. In particular, the preferred dose will be one that is suitable for effecting the inhibition of the modes of operation of the C. albicans blastophores, namely an amount effective to inhibit the activation of T- lymphocytes, the amount effective to block the expansion of T cells bearing Vβ subsets, and/or the amount effective to block the eliciting of IFNy. As one skilled in the art would recognize, such an effective amount will vary greatly depending on the nature of the infection and the condition of a patient. As indicated above, an "effective amount" of antibody or pharmaceutical agent to be used in accordance with the invention is intended to mean a nontoxic but sufficient amount of the agent, such that the desired prophylactic or therapeutic effect is produced. The exact amount of the antibody or a particular agent that is required will thus vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular carrier or adjuvant being used and its mode of administration, and the like. Accordingly, the "effective amount" of any particular antibody composition will vary based on the particular circumstances. However, an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The compositions may additionally contain stabilizers or pharmaceutically acceptable preservatives, such as thimerosal (ethyl(2-rnercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, MO).

Accordingly, the present invention contemplates a method of treating or preventing an infection from Candida albicans comprising administering to a patient in need thereof an effective amount of the monoclonal antibodies as discussed above. In addition, a method is contemplated for blocking of the activation of T-lymphocytes, blocking the expansion of T-cells bearing V-beta subsets (such as subsets 2, 3 14, or others), and/or blocking the secretion of interferon-gamma caused by the lntip protein comprising administering to a patient in need an effective amount of the monoclonal antibody of Claim 1.

In another embodiment of the present invention, a kit which may be useful in isolating and identifying infections caused by microorganisms expressing lntip which comprises the monoclonal antibodies of the present invention in a suitable form, such as lyophilized in a single vessel which then becomes active by addition of an aqueous sample suspected of being infected with C. albicans or other similar microorganism. Such a kit will typically include a suitable container for housing the antibodies in a suitable form along with a suitable immunodetection reagent which will allow identification of complexes binding to the specific regions of the lntip protein as set forth above. For example, the immunodetection reagent may comprise a suitable detectable signal or label, such as a biotin or enzyme that produces a detectable color, etc., which normally may be linked to the antibody or which can be utilized in other suitable ways so as to provide a detectable result when the antibody binds to the antigen. Additionally, a method of identifying or diagnosing an infection of C. albicans or other microorganism expressing the Intip protein is also provided wherein one or more antibodies to the peptide regions set forth above from the lntip protein are introduced into a sample thought to be infected with a microorganism expressing Intip, and the identification or diagnosis of the infection can be confirmed if binding to the sample is observed. Such binding can be observed in any of a number of suitable ways commonly used in the art, including, e.g., detectable labels, as described above.

In summary, the present invention thus provides isolated and/or purified regions of the lntip protein which have been shown to be involved in pathways of activation which results in the virulent spread of microorganisms expressing Intip, and also provides monoclonal antibodies which can bind to these specific regions, and/or which can disrupt the process of lntip activation in other ways. Such antibodies and agents can therefore be utilized in effective methods of treating or preventing infections from microorganisms such as C. albicans or S. cerevisiae which express the intip protein.

EXAMPLES

The following examples are provided which exemplify aspects of the preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 : MAb 163.5 Recognized In ti p and Pep₂β3

METHODS: The Monoclonal Antibody identified above as MAb 163.5 was tested for recognition of the propeptide region (amino acids 1-263) and the complete lnti p protein. The sequence of lntip was expressed in S. cerevisiae as a fusion protein with a 6X-His tag at the amino terminus. The sequence of Pep₂₆₃ was expressed in S. cerevisiae as a fusion protein with a 6X-His tag at the carboxy terminus. Lysates of transformed S. cerevisiae were applied to a nickel column and eluted with 300 mM imidazole, lnti p eluted as a single protein. Pep₂₆₃ eluted as a doublet. Each protein was electrophoresed on SDS-PAGE and transferred to nitrocellulose. Western blots were incubated with monoclonal antibodies 364.5 (lane 1), 253.5 (lane 2), 163.5 (lane 3), or 44.5 (lane 4). Blots were incubated with sheep anti-mouse IgG conjugated to HRP (Amersham) and developed with Supersignal West Pico chemiluminescent substrate (Pierce). As shown in Figure 19, of the four monoclonal antibodies tested, only MAb 163.5 in lane 3 recognized both full length lnti p (left panel) and purified Pep₂β₃ (right panel).

EXAMPLE 2: MAb 163.5 Inhibits T Cell Activation by Candida albicans

METHODS: The MAb 163.5 monoclonal antibody as described above was tested to determine its ability to inhibit T-cell activation by Candida albicans. Peripheral blood mononuclear cells were purified from the blood of adult humans by standard methods. In each well of a 96-well microtiter plate, 5x10⁵ PBMCs were incubated with 5x10⁵ C. albicans of the desired strain (wild type or int1- mutant) for 4 days at 37° C. 0.2 μg/ml amphotericin B was added to all wells to prevent filamentation of C. albicans. Antibody-treated wells received 25 μg/ml or 50 μg/ml MAb163.5 on day 1 through day 4. 50 μg/ml of an isotype control MAb (BD Biosciences) served as control. At the end of the incubation period, PBMCs were pelleted and stained with a PE-conjugated monoclonal antibody against the lL-2 receptor (CD25). The percent of PBMCs expressing CD25 was determined by fluorescence activated cell sorting. As depicted in Figure 20, The first bar shows that approximately 70% of T lymphocytes were activated after incubation with SEB (staphylococcal enterotoxin B). As expected, the MAb derived from the C. albicans sequence had no effect on SEB stimulation. In the third bar, the inti- mutant again failed to activate T lymphocytes, whereas the INT1+ C. albicans were approximately 30-40% as active as SEB. An isotype control MAb had no effect on /Λ/T7-induced T cell activation, but both 25 μg/ml and 50 μg/ml MAb 163.5 blocked T cell activation induced by //VT7-expressing C. albicans.

Other tests regarding the MAb 163.5 are described below.

EXAMPLE 3: MAb 163.5 Inhibits Vβ Expansion by Candida albicans

METHODS: Peripheral blood mononuclear cells were purified from the blood of adult humans by standard methods. In each well of a 96-well microtiter plate, 5x10⁵ PBMCs were incubated with 5x10⁵ C. albicans of the desired strain (wild type or int1- mutant) for 4 days at 37° C. 0.2 μg/ml amphotericin B was added to all wells to prevent filamentation of C. albicans. At the end of the incubation period, PBMCs were pelleted and stained with commercially available PE- conjugated monoclonal antibodies (Beckman Coulter) against Vbeta2 or Vbeta14. The percent of PBMCs expressing Vbeta2 or Vbeta14 was determined by fluorescence activated cell sorting. SEB is known to expand Vbeta14, and MAb 163.5 did not affect this response. However, as shown in Figure 21 , MAb 163.5 inhibited C. a/ύ/cans-mediated expansion of Vbeta 2 and Vbeta14. This difference was statistically significant at p<0.03.

EXAMPLE 4: Vβ Expansion by Pep₂6₃ and Blockade by MAb 163.5

Peripheral blood mononuclear cells were purified from the blood of adult humans by standard methods. All microtiter wells contained 5x10⁵ PBMCs plus one of the following experimental mixtures: 200 pmoles of Pep₂63 alone;

200 pmoles of Pep₂63 plus 5x10⁵ C. albicans (int1- mutant); or

200 pmoles of Pep₂63, C. albicans (int1 -mutant), and 25-50 μg/ml of MAb 163.5.

0.2 μg/ml amphotericin B was added to all wells to prevent filamentation of C. albicans. Control wells contained 200 pmoles SEB or SEB plus 25-50 μg/ml of MAb 163.5. At the end of the incubation period, PBMCs were pelleted and stained with commercially available PE-conjugated monoclonal antibodies (Beckman Coulter) against Vbeta2 or Vbeta14. The percent of PBMCs expressing Vbeta2 or Vbeta14 was determined by fluorescence activated cell sorting. As shown in Figure 22, 200 pmoles of Pep₂₆₃ was not effective in expanding Vbeta 2 or Vbeta 14. Co-incubation of Pep₂₆₃ with the int1- mutant led to a significant increase in Vbeta expansion. This effect was inhibited with MAb 163.5 MAb (p<0.03). MAb 163.5 had no effect on expansion of Vbeta14 induced by SEB. MAb 163.5 inhibits expansion of Vbeta2 and Vbeta 14 induced by Pep₂₆₃. The requirement for the int1- mutant presumably reflects an additional processing event that improves the ability of Pep₂₆3 to bind to the T lymphocyte.

EXAMPLE 5: OTHER INVESTIGATIONS USING MONOCLONAL

ANTIBODIES

Other studies regarding lntip monoclonal antibodies showed the following results:

A. Dose-response

1) 80 micrograms/ml of 163.5 inhibits T lymphocyte activation by 75% 70 mcg/ml inhibits by 75%

50 mcg/ml inhibits by 75% 25 mcg/ml inhibits by 50%

2) 70 micrograms of 253 inhibits T lymphocyte activation by 85% 50 mcg/ml inhibits by 85%

25 mcg/ml inhibits by 50%

Thus, 50-85% inhibition was obtained at doses of 25-50 mcg/ml.

B. Specificity of action

Mab 163.5 does not block T lymphocyte activation induced by staphylococcal enterotoxin B. Thus, Mab 163.5 is specific for Intip.

C. Urine Sample Testing

Three urine samples were obtained from unidentified infants suspected of C. albicans infection. Infant A had a proven C. albicans UTI with 10,000 organisms/ml of blood. Infants B and C had suspected fungemia. In the concentrated urine of infants A and C we found lntip fragments with Mab 163.5. These results suggest a) that lntip and Pep263 fragments are generated in vivo b) that Mab 163.5 and other monoclonal antibodies raised against the 248-277 lntip peptide (e.g. Mab 253 and the other MAb's described above) could serve as the basis for a rapid diagnostic test for C. albicans UTI and fungemia. For example, it is contemplated that the Mabs could be attached to protein A-coated latex beads and then used in a latex agglutination test.

EXAMPLE 6: INVESTIGATIONS OF THE INT1P PROTEIN.

The protein lntip of Candida albicans functions as an adhesin, participates in morphologic switching of blastospores to hyphae, and is linked to virulence in mice. Rapid mortality ascribable to INT1/INT1 strains suggested that lntip may have an immunomodulatory role. Therefore, we investigated whether expression of lnti p on the surface of C. albicans influenced T cell activation.

C. albicans strains used in the Investigations: CAF2 - INT1/INT1 - URA3/ura3 (supplied by WA Fonzi, Georgetown University, Washington D. C.)

CAG1 - INT1/int1 - URA3/ura3 (see Gale et al, Science 279:1355 -1358, 1998)

CAG3 - int1/int1 - URA3/ura3 (see Gale et al., Science 279:1355-1358, 1998)

HLC-54 - INT1/INT1 - URA3/ura3 cph1/cph1 efg1/efg1 (supplied by JR

Kohler, Whitehead Institute, MIT, Cambridge, MA).

Culture Conditions:

Blastospores grown to mid-exponential phase in YPD medium at 30⁰C shaking, were washed in PBS containing the subinhibitory dose of 0.2 μg/ml amphotericin B (Al-Bassam et al., J. Antimicrob Chemother, 15:263-269, 1985). Yeast were adhered to the bottom of 96 well culture plates by incubating 500,000 cells/well for 45 minutes at 37°C.

Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll- hypaque centrifugation of heparinized blood. Washed PBMCs were suspended in RPMI1640 supplemented with 10% human AB serum, L-glutamine, sodium pyruvate, nonessential amino acids and 0.2 μg/ml amphotericin B to maintain Candida albicans in the blastospores stage.

Cocultures were initiated by adding 200λ PBMCs at 2.5X10⁶ cells/ml to the adherent blastospores upon removal of PBS/amphotericin B. Cultures were incubated at 37°C 5% CO₂ for 1-7 days. Control wells containing PBMC + superantigen TSST-1 and the mitogen PHA were also established. The effect of MHC class Il inhibition was assessed by adding anti-HLA-DR antibody to PBMCs prior to coculture. Where appropriate, CD3 cells were isolated to >97% purity using cell separation columns. Antigen presenting cells (APC) expressing MHC class Il were isolated by plastic adherence. The antigen processing ability of APCs was abolished by pretreatment with 0.3% paraformaldehyde. Flow Cytometry:

Cultures harvested at appropriate time points were stained for 10 minutes at RT with PE and FITC conjugated monoclonal antibodies to IL2R and CD3, respectively, to assess T cell activation. Similarly, T cell subsets CD4 and CD8 as well as Vbeta2 and Vt,_eta8 were analyzed by using Cychrome and PE labelled Mabs, respectively. Cells were quantitated by flow cytometry using FACS Vantage. (BD Biosciences, San Jose, CA.) Data analysis was performed using WinMDI version 2.8 software. (Kindly supplied by Dr. Joseph Trotter, Scripps Research Institute, LaJoIIa, CA.)

Methods:

PBMCs from five normal healthy volunteers were cocultured with either CAF2 INT1/INT1 or CAG3 int1/int1 blastospores for days 1 through 7. IL2 receptor positive cells among the CD3 positive population indicate the frequency of activated T cells at each time point. Tests on five individuals showed that by day 4, the frequency of activated T cells was significantly greater for CAF2 cocultures. Clusters of activated T cells were also predominant by day 4 of PBMCs cocultured with INT1/INT1 blastospores. Under similar experimental conditions, int1/int1 blastospores do not induce these T cell activation clusters.

PBMCs from a single donor were cultured alone or with C. albicans strains CAF2, CAG1 , CAG3 or HLC-54 for five days. Only strain CAG3, the int1/int1 null mutant failed to activate T cells above the level of unstimulated control.

PBMCs were cultured with either 10 μg/ml PHA, 4 μg/ml TSST-1 or 500,000 blastospores of either CAF2 or CAG3. Each culture condition was either left untreated or incubated with 10 μg/ml of either anti-HLA-DR antibody or an isotype control. CD3 positive cells were analyzed for IL2 receptor upregulation by flow cytometry. T cell activation induced by PHA was unaffected by anti-HLA- DR antibody as anticipated since mitogen activation is independent of MHC class II. However, the response to superantigen TSST-1 was significantly inhibited since binding to V_beta2 of the TCR and the beta chain of class Il is required for activation. Anti-HLA-DR antibody significantly depressed the upregulation of IL2R on T cells cocultured with CAF2 INT1/INT1 but not with CAG3 int1/int1 suggesting a role for MHC class Il molecules in Intip-mediated T cell activation.

Cultures as described above were expanded during the last 24 hours of culture with human IL2. Cultures were analyzed by flow cytometry for the expansion of either T cell Vb_Θta2 or Vbeta8 subsets. Significant expansion of the V_be_ta2 subset occurred in activations with the V_beta2 specific superantigen TSST-1 as well as with blastospores of CAF2 but not with CAG3. The frequency of Vb_eta8 T cells was similar to the unactivated control for all conditions. Thus, there is preferential expansion of at least the V_beta2 subset by C. albicans expressing Intip.

PBMCs that were cocultured with either CAF2 INT1/INT1 or CAG3 int1/int1 blastospores were tested to determine frequencies of CD4 and CD8 T cells by flow cytometry. The ratio of CD4:CD8 cells was <1:1 for T cells expanded by CAF2 INT1/INT1, whereas all other activation conditions had ratios >1:1. CAF2 modulation of the CD4:CD8 ratio which was evident in the V_beta2 T cell subset of one of the donors evidences a role for lntip in activation-induced CD4 T cell loss.

T cells (2.5X10⁵) were cultured with either 500,000 CAF2 blastospores or 4 μg/ml TSST-1 in the presence of APC (2.5X10⁵) pretreated with or without 0.3% paraformaldehyde. In this case, T cell activation by CAF2 INT1/INT1 blastospores occurred despite the inability of MHC class Il expressing APCs to process antigen. Similarly, activation by TSST-1 was not inhibited by paraformaldehyde fixation of APC.

In summary, equal numbers of C. albicans blastospores from strains CAF2 INT1/INT1 and CAG3 int1/int1 in mid-exponential phase were incubated with PBMCs isolated from 5 normal healthy volunteers. IL2 receptor upregulation on CD3 cells was monitored by flow cytometry. The percentage of CD3 cells expressing IL2 receptor was significantly greater for activations using CAF2 blastospores (42.0 +/- 6.3) than for CAG3 (3.8 +/- 1.0), p=0.005. Antibodies to HLA-DR inhibited IL2 receptor expression on T cells activated by CAF2, but had no effect on T cells activated by CAG3. Preliminary data indicated a preference for V_be_ta2 T cell activation by CAF2. Analysis of CD4:CD8 subsets revealed that T cells activated by CAG3 blastospores had a normal CD4:CD8 ratio (2:1) similar to control T cells expanded with human IL2. However, after incubation with Intip-expressing CAF2 blastospores, the CD4:CD8 ratio was depressed (<1 :1) due to a reduction in the number of CD4 T cells. Thus, activation of a specific Vbeta subset of T lymphocytes and depletion of CD4 cells are two mechanisms by which Intip-bearing C. albicans modulate the cell-mediated immune response.

As a result of the above experiments, the following conclusions can be drawn:

Candida albicans blastospores expressing the protein lntip activate human T lymphocytes whereas blastospores lacking lntip surface expression do not.

Inhibition of IL2 receptor upregulation by anti-H LA-DR antibody indicates a dependence for MHC class Il in lntip induced T cell activation.

T cell activation by lntip expressing blastospores is independent of antigen processing as indicated by resistance to APC paraformaldehyde fixation.

Candida albicans expressing lntip preferentially activate the V_beta2 T cell subset.

Activation induced deletion of CD4 T cells by lntip is a mechanism by which Candida albicans can modify the host immune response.

EXAMPLE 7: ADDITIONAL INVESTIGATIONS OF THE INT1P PROTEIN

AND THE ISOLATION OF THE PROPEPTIDE FROM CANDIDA ALBICANS

INT1 Gene in Candida albicans and its Importance in Pathogenesis

Some years ago, we identified the Candida albicans gene INT1, which encodes a protein of Mr 188 kDa that mediates adhesion, medium-dependent filamentation, and virulence. See, e.g., Gale et al. PNAS 93:357-61 (1996); Gale et al. Science 279:1355-58 (1998), incorporated herein by reference. In particular, ICR mice given a tail vein injection of 10⁵ wild type C. albicans expressing both INT1 alleles (genotype INT1/INT1) showed 100% mortality by day eleven, while 90% of mice given a homozygous double disruptant (genotype INT1/int1) survived. Animals given a heterozygous mutant (genotype INT1/int1) or a re-integrant (genotype int1/int1/INT1) had intermediate mortality (40% survival). All strains replicated equally well and underwent filamentous growth in serum, and no differences in CFU in blood, kidney, or liver were found (36). Thus, defects in replication, filamentation, or organ dissemination did not explain /Λ/π-dependent mortality.

/Λ/Η-Expressing C. albicans Activate T Lymphocytes, but the int1 Knockout Strain Does Not

The question of why the presence of INT1 is associated with death in mice was also examined. Unlike other fungi, C. albicans does not produce mycotoxins, but based on the available evidence, we considered the possibility that the encoded protein Intip, or some part of it, might be a superantigen.

Peripheral blood mononuclear cells (PBMCs) from five normal donors were obtained by Ficoll-Hypaque centrifugation, suspended in RPMI 1640 supplemented with 10% human AB serum, L-glutamine, sodium pyruvate, non¬ essential amino acids, and 0.2 μg/ml amphotericin B to prevent germ tube formation. See Figure 7. PBMCs were incubated at 37°C in 5% CO₂ with 10⁵ blastospores of INT1/INT1 C. albicans or an equal number of int1/int1 C. albicans (homozygous double disruptant) for one to seven days (n=7 expts.) The superantigen TSST-1 (800 μg/well) or the mitrogen PHA served as controls. Two color flow cytometry was used to plot the percentage of CD3 positive cells that expressed the IL-2 receptor (CD25).

Results: Although no significant activation of T lymphocytes was observed for the first three days of culture, on days four through seven, PBMCs incubated with INT1/INT1 C. albicans showed a significant increase in expression of the IL- 2 receptor (CD25) on CD3+ cells, a marker for lymphocyte activation. PBMCs form the same donors did not increase expression of the IL-2 receptor when incubated with the int1/int1 double disruptant. These results indicate that lntip is required for activation of T lymphocytes by C. albicans.

Activated T lymphocytes were predominantly of the CD4 subset and were eliminated within 7-10 days after co-culture. In all donors, the CD4/CD8 ratio, which ranged from 1.8:1 to 2.2:1 on day 3, was reversed by day 7.

/Λ/T7 -Associated Activation of T Lymphocytes Can Be Blocked by Antibodies to MHC Class Il

PBMCs were pre-incubated with a monoclonal antibody to HLA-DR prior to stimulation with the mitogen PHA, the superantigen TSST-1 , INT1/INT1 C. albicans, or int1/int1 C. albicans. T cell activation (up-regulation of the IL-2 receptor CD25) was measured by two-color flow cytometry and plotted on the Y- axis. See Figure 8.

Results: Antibodies against MHC Class Il HLA-DR significantly inhibited T lymphocyte activation induced by toxic shock toxin TSST-1 and INT1/INT1 C. albicans. Anti-HLA-DR antibodies did not block T lymphocyte activation induced by the T cell mitogen PHA (which does not require antigen-presenting cells for its effects) or by int1/int1 C. albicans. These results confirm the participation of MHC Class Il in the activation response of T lymphocytes stimulated by TSST-1 or INT1/INT1 C. albicans.

T Lymphocyte Activation in Response to lntip Does Not Require Antigen Processing or Presentation by Antigen-Presenting Cells (APCs)

APCs were separated from PBMCs by a glass wool column and pre- treated with 0.3% paraformaldehyde (PFA) before being returned to co-culture with lymphocytes. TSST-1 and INT1/INT1 C. albicans were used as stimuli. In the absence of PFA, 44% of T lymphocytes were activated with INT1/INT1 C. albicans as stimulus; in the presence of PFA, 43% were activated. With TSST-1 as stimulus, 60% of T lymphocytes were activated in the absence of PFA treatment of APCs; in the presence of PFA, 56% were activated. Thus, PFA treatment did not inhibit T lymphocyte activation in response to TSST-1 or to INT1/INT1 C. albicans. When leupeptin and pepstatin were used to inhibit antigen processing and presentation, respectively, lymphocyte activation in response to TSST-1 and INT1/INT1 C. albicans was not inhibited. These results show that lymphocyte activation by lntip is independent of antigen processing and presentation by APCs.

Expansion of Identical Vβ Subsets by the Soluble Superantigen TSST-1 and Int1 p

The expansion of Vβ subsets was measured after stimulation of PBMCs with TSST-1 (800 μg), with INT1/INT1 C. albicans, with int1/int1 C. albicans, and with PHA. Unactivated PBMCs served as control (Figure 9).

Results: Incubation of PBMCs with wild type C. albicans (INT1/INT1) or TSST-1 preferentially expanded the Vβ2 subset of T-lymphocytes (black bars) but not the Vβ8 subset (open bars). There was no significant expansion of the Vβ2 subset when lymphocytes were incubated with int1/int1 C. albicans (double disruptant) or with phytohemagglutin. These preliminary experiments show Vβ subset specificity in response to TSST-1 and Intip; comparing INTI⁺ strains and intT strains confirms that expansion of the Vβ2 subset was /WT/ -dependent.

Release of Pro-Inflammatory Cytokines

Peripheral blood mononuclear cells were stimulated with 5x10⁵ INT1/INT1 C. albicans blastospores, and production of TNFα, IL-6 and IL-4 was measured in supernatants on days 2, 4 and 6. As can be seen from the table below, INT1/INT1 C. albicans induce a predominantly Th1 response in vitro with elevations in TNFα and IL-6 that are comparable to those induced by staphylococcal enterotoxin B (SEB), a well-characterized superantigen. There is virtually no production of IL-4 in response to C. albicans or SEB. Interestingly, the TN Fa response to C. albicans showed a near 40-fold variance (high responder = 7014 pg/ml on day 2; low responder = 165 pg/ml on day 2), while the response to SEB did not differ significantly in these two donors. Although one reason for the more consistent response to SEB could be its use as a soluble protein, another possible interpretation is that the response to C. albicans involves different MHC Class Il alleles, different Vβ subsets, or differing kinetics of T cell activation and apoptosis. Hypothesis Two will address this possibility.

Identical Effects with Saccharomvces cerevisiae Expressing INT1

In order to assess these effects apart from other candidal antigens, we expressed INT1 in S. cerevisiae YPH500 under the control of a galactose- inducible promoter. INT1 was ligated into plasmid pBM272 for transformation of S. cerevisiae YPH 500; the resultant plasmid was named pCGOl Expression of lntip was induced with 2% galactose. S. cerevisiae transformed with pBM272 served as control. Approximately 25% of donor PBMCs were activated after co- culture with S. cerevisiae expressing INT1, as measured by up-regulation of the IL-2 receptor on flow cytometry; no up-regulation occurred after co-culture with S. cerevisiae transformed with vector alone. These effects could be blocked by antibodies to HLA-DR. The Vβ2 subset was preferentially expanded by S. cerevisiae expressing INT1; no expansion of Vβ2 or Vβ8 subsets was noted in response to S. cerevisiae transformed with vector alone. EFFECTS of lntip PROTEIN in C. ALBICANS or S. CEREVISIAE

X Activates T lymphocytes, up-regulates IL-2 receptor, and releases pro- inflammatory cytokines

X Requires antigen-presenting cells (APCs) for co-stimulatory molecules but not for antigen presentation or processing

X Expands particular Vβ subsets

These experiments indicated that the presence of the protein lntip was associated with superantigen-like effects, both in wild type C. albicans and in transformed S. cerevisiae. These are imperfect experiments because they compare a soluble superantigen (TSST-1) with either INTI⁺ or intT strains of C. albicans; nevertheless, the results provide some indication that superantigen-like effects were associated with INTI⁺ C. albicans. However, because most superantigens are secreted proteins of 22-29 kDa, and lntip has a predicted mass of 188 kDa in its unglycosylated form, we hypothesized that limited proteolysis of lntip might generate a soluble polypeptide that could serve as a superantigen.

As a potential mechanism for proteolysis, we considered the possibility that lntip, like MMTV, might be cleaved by a proprotein convertase. A subset of serine endopeptidases, proprotein convertases cleave proproteins, or zymogens, to their active fragments by limited proteolysis at one or at most two specific cleave sites. In eukaryotes, these enzymes are called "subtilisin-like proprotein convertases" or SPCs. Most SPCs are autocatalytic and must be activated by cleavage of their propeptide before they can cleave their specific substrates. A model of a proprotein convertase is provided in Figure 10, and the canonical cleavage site is indicated with an arrow.

Most proprotein convertases exhibit several highly conserved features including a propeptide domain, distinguished by a canonical cleavage site just C- terminal to a pair of dibasic amino acids, most frequently KR or KK. A catalytic domain spans approximately 330 amino acids with an active site sequent of D-H- N-S [Asp-His-Asn-Ser], in which the initiating D is followed by a DX. This DDX motif has been shown in other systems (e.g., integrins) to be a recognition site for the binding of the RGD tripeptide; however, this interaction has never been explored with proprotein convertases. Catalytic domains may occur singly or in tandem. Lastly, a processing domain (or P-domain) also contains a D-H-N-S motif, but in six of the seven know SPCs, an RGD tripeptide is intercalated between the N and the S. The RGD motif is essential for cleavage of the propeptide; site-directed mutagenesis of the RGD tripeptide inhibits zymogen processing and mis-directs cellular trafficking of the unprocessed protein.

In Figure 11 , a comparison of the lntip sequence in C. albicans with the motifs essential for the proprotein convertases is shown, and this analysis disclosed several sites of interest, including a dibasic cleavage site at residue 263, two putative catalytic domains, and an RGD sequence correctly situation in a possible P domain. Regions recognized by specific rabbit anti-peptide polyclonal antibodies developed in our laboratory as shown in brackets.

Identification of an Amino-Terminal Peptide from lntip in Activating Supernatants

Subsequent experiments showed that the supernatants from exponentially replicating INT1/INT1 C. albicans or /Λ/77-expressing S. cerevisiae were just as active as yeast cells in activating T lymphocytes, and all activity was contained in a pool of proteins weighing less than 50 kDa. Indeed, as little as 500 pmoles of supernatant proteins served to activate T lymphocytes.

Exposure of a Covert Amino-Terminal Domain in lntip in the Presence of Heparin

Because colonization with C. albicans in the gastrointestinal tract does not induce superantigen-like effects, we hypothesized that some environmental factor relevant to fungemia might accelerate release of these <50 kDa amino- terminal polypeptides from Intip. Heparin is known to enhance autocatalysis and to accelerate cleavage of a propeptide from a zymogen. These effects suggested that heparin might accelerate release of Intip-derived fragments that might serve as superantigens. This possibility was all the more meaningful because heparin is an ever-present infusate in patients with intravascular catheters.

For these experiments, INT1/INT1 C. albicans blastospores were incubated in the absence or presence of heparin; flow cytometry with polyclonal antibodies to the second divalent cation binding site (anti-CBS2) or to the first 600 amino acids of the amino terminus (anti-INT600) was used to detect the appearance of these domains (see Figure 11 for domains recognized by these antibodies).

Results: In the absence of heparin (Figure 12 - left panel), C. albicans blastospores displayed intensity fluorescence with anti-CBS2 (gray area) but did not fluoresce with anti-lNT600. However with the addition of heparin, substantial fluorescence with anti-INT600 was now detectable (right panel). These results suggested that heparin exposed a covert amino-terminal domain and made it accessible for cleavage. If we were correct then a cleaved amino-terminal fragment of lntip should be found in the supernatants of organisms grown in the presence of heparin but not in its absence.

Absence of lnflp Amino-Terminal Fragments in Culture Supernatants is Accelerated by Heparin

Figure 13 is a Western blot of supernatants from /Λ/77-expressing S. cerevisiae grown in the absence or presence of heparin and probed with rabbit polyclonal antibodies to the lntip amino terminus (anti-INT600), to the second divalent cation binding site (anti-CBS2), or to the RGD domain (anti-RGD).

Results: Supernatants probed with anti-CBS2 (panel 2) or anti-RGD (panel 3) showed identical banding patterns in the absence or presence of heparin. However, probing the supernatants with anti-INT600 disclosed two novel fragments of 27 kDa and 44 kDa only in the supernatants of organisms grown in the presence of heparin for three hours. In the absence of heparin, these fragments appeared at much reduced levels after 18 hours or more. Thus, heparin accelerated the appearance of two Intip amino-terminal polypeptides in the supernatant. Supernatant containing the 27 and 44 kDa fragments activated T lymphocytes, while the other supernatants did not.

Localization of the Intip Superantiqen-like Fragment

From the foregoing experiments we had circumstantial evidence that amino-terminal fragments of lntip (Mr 27 or 44 kDa) could be exposed in the presence of heparin, cleaved, and detected in the supernatant of INT1- expressing S. cerevisiae. If heparin accelerated the cleavage of lnti p by a proprotein convertase as occurs with the superantigen vSAG7 from MMTV, then an amino-terminal fragment encompassing 263 amino acids should be released from lntip (Figure 11). A preliminary estimate of the mass of the first 263 amino acids of lntip was 35 kDa, rising to 42 kDa is glycosylated. We therefore tested the possibility that the first 263 amino acids of Intip, hereinafter called Pep263, constituted the superantigen-like moiety.

In order to obtain direct evidence that Pep₂6₃ was responsible for the superantigen-like effects observed with INT1/INT1 C. albicans and INT1- expressing S. cerevisiae, we expressed Pep₂₆₃ as a recombinant, His-tagged protein in S. cerevisiae and assessed its effects on T lymphocyte activation and expansion of Vβ subsets. S. cerevisiae was preferable to E. coli for expression in order to avoid the activating effects of lipopylsaccharide. C. albicans genomic DNA encoding amino acids 1 to 263 of lntip was amplified by PCR and ligated in-frame to a 6X-His tage at the 3' end. This construct was inserted as a BamH\/Sal\ fragment into pBM272 and expressed from a galactose-inducible promoter in S. cerevisiae BJ3501 , a protease-deficient strain. The His-tagged fusion protein appeared in the lysate (Figure 14, lane 1). S. cerevisiae iysate was chromatographed on a nickel column, and an anti-His Mab was used in a dotblot to detect the His-tagged protein as it was eluted from a nickel column by an imidazole gradient (0-500 mM imidazole). The His-tagged Pep₂₆₃ eluted at a concentration of 300 mM imidazole in fractions 300-1 and 300-2 (Figure 14, lanes 2 and 3). An additional filtration step yielded a single band of 44 kDa on silver strain (Figure 14, lane 4), which reacted with anti-His antibody on Western (Figure 14, lane 5). The use of these techniques has produced microgram quantities of the putative superantigen.

Activation of T Lymphocytes by Recombinant Pep?_Ra

100 picograms of Pep₂₆3was then incubated with 5x10⁶ PBMCs in each of three reactions: (a) purified Pep₂63 as a soluble peptide; (b) purified Pep₂6₃ bound to the bottom of the tissue-culture well; (c) purified Pep₂63 immobilized by anti-His antibodies covalently linked to Protein A Sepharose beads. Fractions which also eluted at 300 mM imidazole but contained no His-tagged protein were used as control.

Results: Unactivated T lymphocytes (left-most bar in each group of three) or T cells incubated with eluted peptides that did not contain the His tag (middle bar in each group of three) did not upregulate the IL-2 receptor. In contrast, as little as 100 picograms of soluble Pep₂₆₃ activated T lymphocytes (denoted with asterisk); up-regulation of the IL-2 receptor was two-fold higher than with control fractions. Substantial activation by Pep₂₆₃ occurred on day 3, one day sooner than had been observed with whole organisms (see Figure 7). Pep₂63 bound to the microtiter plate or immobilized by linkage to protein A beads failed to activate T lymphocytes. These results show that Pep₂₆3 is capable of activating T lymphocytes to an extent that surpasses what we observed with whole C. albicans cells. Experiments to confirm that Pep₂₆3 expands the Vβ2 subset are included in this proposal.

Model for the Participation of lntip in Candidemia

We have presented preliminary evidence that the C. albicans protein lntip exerts superantigen-like effects on human T lymphocytes. Activation of T lymphocytes as measured by up-regulation of the IL-2 receptor (CD25) is not dependent upon antigen processing and presentation, can be blocked by antibodies to MHC Class II, and results in the expansion of the Vβ2 subset. Activation of T lymphocytes can be triggered by Pep₂63, a 263 amino acid peptide that is cleaved from the amino terminus of lntip in a reaction accelerated by physiologic doses of heparin. Picogram inputs of Pep₂₆₃ are equivalent to INT1/INT1 C. albicans or /Λ/77-expressing S. cerevisiae in the ability to activate T lymphocytes. Like most microbial superantigens, Pep₂₆₃ is active when soluble, not when bound to a microtiter plate or to antibody-coated beads.

Figure 16 schematizes the apparent role of lnti p in C. albicans fungemia. In the absence of heparin (panel A), the first 263 amino acids of lntip (Pep₂₆₃) are covert and cannot be detected by anti-INT600 antibodies (Figure 12). Only in the presence of heparin (panel B) is the amino terminus of lntip exposed, at which point Pep₂63 is cleaved and released into the fluid phase (panel C), where it exerts superantigen-like effects culminating in the release of pro-inflammatory cytokines that influence the clinical outcome. While it is possible that there are other superantigens liberated by C. albicans, or that even smaller fragments of Pep₂₆₃ may also have superantigen-like effects, the activity of Pep₂₆₃ and the applicability of these interactions may be applicable to the problem of candidemia in the NICU infant.

EXAMPLE 8: BLOCKING OF INTERFERON-GAMMA BY MONOCLONALS TO THE PROPEPTIDE

Secretion of IFNv but not TNFα is Inti p-cfepencfenf. The ability to trigger secretion of pro-inflammatory cytokines from expanded Vβsubsets of T lymphocytes is a hallmark of most superantigens. We evaluated the ability of Int1p⁺ C. albicans and the isogenic intip^" mutant to elicit Th1/Th2 cytokines from T lymphocytes after 4 days of incubation.

TABLE 2

Results: We were unable to detect IL-4 or IL-10 in response to either C. albicans strain (Int1p⁺ or intip^" mutant). The absence of a Th2 response against C. albicans has been previously noted in humans (1). Levels of TNFα induced by Int1p⁺ C. albicans blastospores versus intip^" blastospores did not differ significantly. C. albicans and S. cerevisiae mannans have been shown to stimulate human monocyte secretion of TNFα by both LBP/CD14 and TLR4 dependent mechanisms, similar to LPS (2). Therefore, it is likely that the range of TNFD levels induced by wild type (Int1p⁺) and mutant (intip^") C. albicans in our assay reflects monocyte TNF production induced by cell wall polysaccharides.

In contrast, we found statistically significant differences in the amount of IFNγ induced by Int1p⁺ C. albicans blastospores versus intip^" blastospores. Thus, the I FNY response to C. albicans blastospores is Intip-dependent, while the TNFα response is not. The timeframe of IFNγ production correlates with the onset of T cell activation and Vβ expansion.

Secretion of IFNv is associated with MHC class Il haplotvpes. The well- characterized superantigen MAM from Mycoplasma arthritidis binds to antigen presenting cells expressing HLA DR1, DR4, DR7, and DR12. However, release of IFNγ is highest with cells expressing DR4, DR7, and DR12 (3). We found a 56% identity between the MHC class Il binding site of MAM and an amino terminal sequence of Intip. In keeping with the similarity in MHC class Il binding sites, we also found that those people with "high" levels of IFNγ in response to lntip expressed DR7, while "low" IFNγ responders expressed DR1 , as has been reported for MAM. The DQ locus appears to have no bearing on the IFNγ response.

TABLE 3

Effects of MAb 163.5 on the superantigen-like properties of Int1p⁺ C. albicans blastospores.

We next tested whether MAb 163.5 was able to inhibit T lymphocyte activation and expansion of Vβ subsets 2 and 14 in response to Int1 p⁺ and inti p^" C. albicans. As controls for specificity, we used SEB as a stimulus and an IgGI isotype control for MAb 163.5. Results in the "Activation" column are expressed as the percent of CD3 cells fluorescing with a monoclonal antibody to the IL-2 receptor. Results in the "Vβ Expansion" column represent the percent of CD3 cells fluorescing with a monoclonal antibody to Vβ2 or Vβ14.

TABLE 4

^* p < 0.002; t P < 0.02; tt p<0.03

Results: MAb 163.5 at 25 μg/ml or 50 μg/ml is able to block T lymphocyte activation and expansion of Vβ subsets 2 and 14 induced by Int1 p⁺ C. albicans blastospores. As in the past, inti p^" C. albicans blastospores do not activate T lymphocytes or expand Vβ subsets. Note that MAb 163.5 fails to inhibit T cell activation or expansion of the Vβ14 subset in response to SEB — an indication that MAb 163.5 is specific for Intip. These results evidence that MAb 163.5 is of therapeutic value in fungemia due to C. albicans. Superantiaen-like properties of soluble Pep^a/icf their inhibition by MAb163.5 We next tested soluble Pep₂₆₃ for its ability to activate T lymphocytes, expand Vβ subsets 2 and 14, and trigger the release of IFND. Pep₂₆₃ at a concentration of 200 pM was incubated with PBMCs either alone or in the presence of inti p^" C. albicans blastospores, S. cerevisiae yeast cells, or heat-killed intip^' C. albicans blastospores.

As has been reported by others, C. albicans and S. cerevisiae are able to activate T lymphocytes by virtue of cell-surface polysaccharides that act as mitogens (2). We found that Pep₂63 was also able to activate T lymphocytes, and that its effects were augmented in the presence of the inti p^" C. albicans mutant. Heat-killed mutant or heat-killed S. cerevisiae did not augment T-cell activation either alone or in conjunction with Pep₂₆3-

In order to show that the effects of Pep₂₆₃were ascribable to its activity as a superantigen, we examined whether Pep₂63_> S. cerevisiae, and heat-killed intip^" mutant could expand Vβ subsets 2 and 14, as we had previously shown with Int1p⁺ C. albicans (Figures 23, 24). Results are expressed as the percent of CD3 cells fluorescing with a monoclonal antibody to Vβ2 or Vβ14.

Results: As seen above, neither Pep₂₆₃ nor the int1 p- mutant (mt) expanded Vβ subsets 2 and 14 any better than medium alone. However, the combination of Pep₂₆₃ plus mutant (mt) was 50% more active (p<0.001). Heat-killed mutant (HK mt) and S. cerevisiae (Sc) were ineffective even in the presence of Pep₂₆3. Thus, activity of Pep₂₆₃ in the presence of replicating inti p^" blastospores is consistent with a superantigen-like effect. In addition, MAb 163.5 at a dose of 50 μg/ml inhibited the expansion of Vβ2 and 14 by 50% and 60%, respectively (data not shown).

The fact that the inti p^" mutant appears to augment the effects of Pep₂₆₃ has two important implications:

(1) Because preliminary data show that the int1 p^" mutant is significantly impaired in its ability to activate T lymphocytes, expand Vβ subsets, or elicit I FNy, the mutant may be contributing some post-translational modification of Pep₂63 that augments its function: e.g. proteolytic processing or pH-dependent changes. For example, cleavage of malarial circumsporozoite protein by a malarial cysteine proteinase is essential for infectivity (5). Interestingly, the cleavage site on malarial circumsporozoite protein is marked by the sequence KLKQP; the C-terminal sequence of Pep₂63 is KLKH. The potential contributions of the inti p^" mutant and the possible involvement of candidal proteinases are the subject of another grant proposal and will not be further addressed here. (2) Augmentation of Pep₂₆₃'s superantigen effects by the intip^" mutant may have physiologic correlates in vivo. A substantial proportion of C, albicans blastospores (~50%) do not express detectable Intip. In the setting of catheter-associated candidemia, inti p^" C. albicans blastospores may process or modify Pep₂₆3 liberated from Int1 p⁺ blastospores.

Next we examined the ability of Pep₂₆₃ to elicit the production of IFND from PBMCs.

Results: The intip- mutant elicited a small amount of IFNγ; this is not unexpected because C. albicans cell wall mannoproteins are potent stimuli for IFND production (8). Pep₂₆₃ was somewhat more active, but the combination of Pep₂₆₃ plus the inti p^" mutant was significantly more active than the mutant or Pep₂₆₃ alone (p=0.003, p=0.001 , respectively). MAb 163.5 inhibited the production of I FNY in response to Pep₂₆₃ plus mt (p=0.007). The isotype control MAb had no effect. The augmented activity of Pep₂63 in the presence of the mutant (mt) suggests the possibility that the mutant is modifying Pep₂₆3, perhaps by proteolytic processing. Structural requirements for activity of Pep?^.

Histidines are known to be essential for the activity of other superantigens (4-7). We made the following deletion constructs to test the requirement for His₂₆₃-

TABLE 5

Construct T Cell Activation* Vβ Expansion*

of mutant

Results: The C-terminal histidine is required for the potent effects of Pep₂₆₃ in activating T lymphocytes and expanding Vβ subsets.

BIBLIOGRAPHY:

The following references are incorporated as if set forth in their entirety herein:

1. Levitz SM, North EA. Gamma Interferon gene expression and release in human lymphocytes directly activated by Cryptococcus neoformans and Candida albicans. Infect lmmun 1996;64: 1595-9

2. Tada H, Nemoto E, Shimauchi H, et al. Saccharomyces cerevisiae- and Candida albicans-denved mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner. Microbiol Immunol 2002;46:503-12

3. Alvarez-Ossorio L, Johannsen M, Alvarez-Ossorio R, Nicklas W, Kirchner H and Rink L. Cytokine induction by Mycoplasma arthritidis-deήved superantigen (MAS), but not by TSST-1 or SEC-3, is correlated to certain HLA-DR types. Scand J Immunol 1998;47:43-7

4. Al-Daccak R, Mehindate K, Damdoumi F, et al. Staphylococcal enterotoxin D is a promiscuous superantigen offering multiple modes of interactions with the MHC class Il receptors. J Immunol 1998; 160:225-32

5. Earhart CA, Mitchell DT, Murray DL, et al. Structures of five mutants of toxic shock syndrome toxin-1 with reduced biological activity. Biochemistry 1998;37:7194-202

6. Gubba S, Cipriano V and Musser JM. Replacement of histidine 340 with alanine inactivates the group A Streptococcus extracellular cysteine protease virulence factor. Infect lmmun 2000;68:3716-9

7. Hoffman M, Tremaine M, Mansfield J and Betley M. Biochemical and mutational analysis of the histidine residues of staphylococcal enterotoxin A. Infect lmmun 1996;64:885-90

8. Coppi A, Pinzon-Ortiz C, Hutter C and Sinnis P. The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion. J Exp Med 2005;201 :27-33 SEQUENCE LISTING <110> HOSTETTER, Margaret K.

<120> MONOCLONAL ANTIBODIES TO THE PROPEPTIDE OF CANDIDA ALBICANS AND METHODS OF USE

<130> P08437WO00/BAS

<150> US 60/622,845 <151> 2004-10-29

<160> 11

<170> Patentln version 3.1

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<211> 1664

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<213> Candida albicans

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Met Asn Ser Thr Pro Ser Lys Leu Leu Pro lie Asp Lys His Ser His 1 5 10 15

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Pro Asn Ser Ser Ser Asp Thr Tyr Thr Ser GIu GIn Asp GIn GIu Lys 50 55 60

GIy Lys GIu GIu Lys Lys Asp Thr Ala Phe GIn Thr Ser Phe Asp Arg 65 70 75 80

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GIn GIn GIn GIn Pro GIn GIn GIn GIn GIn Leu Ser GIn Thr Asp Asn 100 105 110

Asn Leu lie Asp GIu Phe Ser Phe GIn Thr Pro Met Thr Ser Thr Leu 115 120 125

Asp Leu Thr Lys GIn Asn Pro Thr VaI Asp Lys VaI Asn GIu Asn His 130 135 140

Ala Pro Thr Tyr lie Asn Thr Ser Pro Asn Lys Ser lie Met Lys Lys 145 150 155 160 Ala Thr Pro Lys Ala Ser Pro Lys Lys VaI Ala Phe Thr VaI Thr Asn 165 170 175

Pro GIu lie His His Tyr Pro Asp Asn Arg VaI GIu GIu GIu Asp GIn 180 185 190

Ser GIn GIn Lys GIu Asp Ser VaI GIu Pro Pro Leu lie GIn His GIn 195 200 205

Trp Lys Asp Pro Ser GIn Phe Asn Tyr Ser Asp GIu Asp Thr Asn Ala 210 215 220

Ser VaI Pro Pro Thr Pro Pro Leu His Thr Thr Lys Pro Thr Phe Ala 225 230 235 240

GIn Leu Leu Asn Lys Asn Asn GIu VaI Asn Ser GIu Pro GIu Ala Leu 245 250 255

Thr Asp Met Lys Leu Lys His GIu Asn Phe Ser Asn Leu Ser Leu Asp 260 265 270

GIu Lys VaI Asn Leu Tyr Leu Ser Pro Thr Asn Asn Asn Asn Ser Lys 275 280 285

Asn VaI Ser Asp Met Asp Ser His Leu GIn Asn Leu GIn Asp Ala Ser 290 295 300

Lys Asn Lys Thr Asn GIu Asn lie His Asn Leu Ser Phe Ala Leu Lys 305 310 315 320

Ala Pro Lys Asn Asp lie GIu Asn Pro Leu Asn Ser Leu Thr Asn Ala 325 330 335

Asp lie Ser Leu Arg Ser Ser GIy Ser Ser GIn Ser Ser Leu GIn Ser 340 345 350

Leu Arg Asn Asp Asn Arg VaI Leu GIu Ser VaI Pro GIy Ser Pro Lys 355 360 365

Lys VaI Asn Pro GIy Leu Ser Leu Asn Asp GIy lie Lys GIy Phe Ser 370 375 380

Asp GIu VaI VaI GIu Ser Leu Leu Pro Arg Asp Leu Ser Arg Asp Lys 385 390 395 400 Leu GIu Thr Thr Lys GIu His Asp Ala Pro GIu His Asn Asn GIu Asn 405 410 415

Phe lie Asp Ala Lys Ser Thr Asn Thr Asn Lys GIy GIn Leu Leu VaI 420 425 430

Ser Ser Asp Asp His Leu Asp Ser Phe Asp Arg Ser Tyr Asn His Thr 435 440 445

GIu GIn Ser lie Leu Asn Leu Leu Asn Ser Ala Ser GIn Ser GIn lie 450 455 460

Ser Leu Asn Ala Leu GIu Lys GIn Arg GIn Thr GIn GIu GIn GIu GIn 465 470 475 480

Thr GIn Ala Ala GIu Pro GIu GIu GIu Thr Ser Phe Ser Asp Asn lie 485 490 495

Lys VaI Lys GIn GIu Pro Lys Ser Asn Leu GIu Phe VaI Lys VaI Thr 500 505 510

lie Lys Lys GIu Pro VaI Ser Ala Thr GIu He Lys Ala Pro Lys Arg 515 520 525

GIu Phe Ser Ser Arg He Leu Arg He Lys Asn GIu Asp GIu He Ala 530 535 540

GIu Pro Ala Asp He His Pro Lys Lys GIu Asn GIu Ala Asn Ser His 545 550 555 560

VaI GIu Asp Thr Asp Ala Leu Leu Lys Lys Ala Leu Asn Asp Asp GIu 565 570 575

GIu Ser Asp Thr Thr GIn Asn Ser Thr Lys Met Ser He Arg Phe His 580 585 590

He Asp Ser Asp Trp Lys Leu GIu Asp Ser Asn Asp GIy Asp Arg GIu 595 600 605

Asp Asn Asp Asp He Ser Arg Phe GIu Lys Ser Asp He Leu Asn Asp 610 615 620

VaI Ser GIn Thr Ser Asp He He GIy Asp Lys Tyr GIy Asn Ser Ser 625 630 635 640 Ser GIu lie Thr Thr Lys Thr Leu Ala Pro Pro Arg Ser Asp Asn Asn 645 650 655

Asp Lys GIu Asn Ser Lys Ser Leu GIu Asp Pro Ala Asn Asn GIu Ser 660 665 670

Leu GIn GIn GIn Leu GIu VaI Pro His Thr Lys GIu Asp Asp Ser lie 675 680 685

Leu Ala Asn Ser Ser Asn lie Ala Pro Pro GIu GIu Leu Thr Leu Pro 690 695 700

VaI VaI GIu Ala Asn Asp Tyr Ser Ser Phe Asn Asp VaI Thr Lys Thr 705 710 715 720

Phe Asp Ala Tyr Ser Ser Phe GIu GIu Ser Leu Ser Arg GIu His GIu 725 730 735

Thr Asp Ser Lys Pro He Asn Phe He Ser He Trp His Lys GIn GIu 740 745 750

Lys GIn Lys Lys His GIn He His Lys VaI Pro Thr Lys GIn He He 755 760 765

Ala Ser Tyr GIn GIn Tyr Lys Asn GIu GIn GIu Ser Arg VaI Thr Ser 770 775 780

Asp Lys VaI Lys He Pro Asn Ala He GIn Phe Lys Lys Phe Lys GIu 785 790 795 800

VaI Asn VaI Met Ser Arg Arg VaI VaI Ser Pro Asp Met Asp Asp Leu 805 810 815

Asn VaI Ser GIn Phe Leu Pro GIu Leu Ser GIu Asp Ser GIy Phe Lys 820 825 830

Asp Leu Asn Phe Ala Asn Tyr Ser Asn Asn Thr Asn Arg Pro Arg Ser 835 840 845

Phe Thr Pro Leu Ser Thr Lys Asn VaI Leu Ser Asn He Asp Asn Asp 850 855 860

Pro Asn VaI VaI GIu Pro Pro GIu Pro Lys Ser Tyr Ala GIu He Arg 865 870 875 880 Asn Ala Arg Arg Leu Ser Ala Asn Lys Ala Ala Pro Asn GIn Ala Pro 885 890 895

Pro Leu Pro Pro GIn Arg GIn Pro Ser Ser Thr Arg Ser Asn Ser Asn 900 905 910

Lys Arg VaI Ser Arg Phe Arg VaI Pro Thr Phe GIu lie Arg Arg Thr 915 920 925

Ser Ser Ala Leu Ala Pro Cys Asp Met Tyr Asn Asp lie Phe Asp Asp 930 935 940

Phe GIy Ala GIy Ser Lys Pro Thr lie Lys Ala GIu GIy Met Lys Thr 945 950 955 960

Leu Pro Ser Met Asp Lys Asp Asp VaI Lys Arg lie Leu Asn Ala Lys 965 970 975

Lys GIy VaI Thr GIn Asp GIu Tyr lie Asn Ala Lys Leu VaI Asp GIn 980 985 990

Lys Pro Lys Lys Asn Ser lie VaI Thr Asp Pro GIu Asp Arg Tyr GIu 995 1000 1005

GIu Leu GIn GIn Thr Ala Ser lie His Asn Ala Thr lie Asp Ser 1010 1015 1020

Ser lie Tyr GIy Arg Pro Asp Ser lie Ser Thr Asp Met Leu Pro 1025 1030 1035

Tyr Leu Ser Asp GIu Leu Lys Lys Pro Pro Thr Ala Leu Leu Ser 1040 1045 1050

Ala Asp Arg Leu Phe Met GIu GIn GIu VaI His Pro Leu Arg Ser 1055 1060 1065

Asn Ser VaI Leu VaI His Pro GIy Ala GIy Ala Ala Thr Asn Ser 1070 1075 1080

Ser Met Leu Pro GIu Pro Asp Phe GIu Leu lie Asn Ser Pro Ala 1085 1090 1095

Arg Asn VaI Ser Asn Asn Ser Asp Asn VaI Ala lie Ser GIy Asn 1100 1105 1110 Ala Ser Thr lie Ser Phe Asn GIn Leu Asp Met Asn Phe Asp Asp 1115 1120 1125

GIn Ala Thr lie GIy GIn Lys lie GIn GIu GIn Pro Ala Ser Lys 1130 1135 1140

Ser Ala Asn Thr VaI Arg GIy Asp Asp Asp GIy Leu Ala Ser Ala 1145 1150 1155

Pro GIu Thr Pro Arg Thr Pro Thr Lys Lys GIu Ser lie Ser Ser 1160 1165 1170

Lys Pro Ala Lys Leu Ser Ser Ala Ser Pro Arg Lys Ser Pro He 1175 1180 1185

Lys He GIy Ser Pro VaI Arg VaI He Lys Lys Asn GIy Ser He 1190 1195 1200

Ala GIy He GIu Pro He Pro Lys Ala Thr His Lys Pro Lys Lys 1205 1210 1215

Ser Phe GIn GIy Asn GIu He Ser Asn His Lys VaI Arg Asp GIy 1220 1225 1230

GIy He Ser Pro Ser Ser GIy Ser GIu His GIn GIn His Asn Pro 1235 1240 1245

Ser Met VaI Ser VaI Pro Ser GIn Tyr Thr Asp Ala Thr Ser Thr 1250 1255 1260

VaI Pro Asp GIu Asn Lys Asp VaI GIn His Lys Pro Arg GIu Lys 1265 1270 1275

GIn Lys GIn Lys His His His Arg His His His His His His Lys 1280 1285 1290

GIn Lys Thr Asp He Pro GIy VaI VaI Asp Asp GIu He Pro Asp 1295 1300 -1305

VaI GIy Leu GIn GIu Arg GIy Lys Leu Phe Phe Arg VaI Leu GIy 1310 1315 1320

He Lys Asn He Asn Leu Pro Asp He Asn Thr His Lys GIy Arg 1325 1330 1335 Phe Thr Leu Thr Leu Asp Asn GIy VaI His Cys VaI Thr Thr Pro 1340 1345 1350

GIu Tyr Asn Met Asp Asp His Asn VaI Ala lie GIy Lys GIu Phe 1355 1360 1365

GIu Leu Thr VaI Ala Asp Ser Leu GIu Phe He Leu Thr Leu Lys 1370 1375 1380

Ala Ser Tyr GIu Lys Pro Arg GIy Thr Leu VaI GIu VaI Thr GIu 1385 1390 1395

Lys Lys VaI VaI Lys Ser Arg Asn Arg Leu Ser Arg Leu Phe GIy 1400 1405 1410

Ser Lys Asp He He Thr Thr Thr Lys Phe VaI Pro Thr GIu VaI 1415 1420 1425

Lys Asp Thr Trp Ala Asn Lys Phe Ala Pro Asp GIy Ser Phe Ala 1430 1435 1440

Arg Cys Tyr He Asp Leu GIn GIn Phe GIu Asp Gin He Thr GIy 1445 1450 1455

Lys Ala Ser GIn Phe Asp Leu Asn Cys Phe Asn GIu Trp GIu Thr 1460 1465 1470

Met Ser Asn GIy Asn GIn Pro Met Lys Arg GIy Lys Pro Tyr Lys 1475 1480 1485

He Ala GIn Leu GIu VaI Lys Met Leu Tyr VaI Pro Arg Ser Asp 1490 1495 1500

Pro Arg GIu He Leu Pro Thr Ser He Arg Ser Ala Tyr GIu Ser 1505 1510 1515

He Asn GIu Leu Asn Asn GIu GIn Asn Asn Tyr Phe GIu GIy Tyr 1520 1525 1530

Leu His GIn GIu GIy GIy Asp Cys Pro He Phe Lys Lys Arg Phe 1535 1540 1545

Phe Lys Leu Met GIy Thr Ser Leu Leu Ala His Ser GIu He Ser 1550 1555 1560 His Lys Thr Arg Ala Lys lie Asn Leu Ser Lys VaI VaI Asp Leu 1565 1570 1575

lie Tyr VaI Asp Lys GIu Asn lie Asp Arg Ser Asn His Arg Asn 1580 1585 1590

Phe Ser Asp VaI Leu Leu Leu Asp His Ala Phe Lys lie Lys Phe 1595 1600 1605

Ala Asn GIy GIu Leu lie Asp Phe Cys Ala Pro Asn Lys His GIu 1610 1615 1620

Met Lys lie Trp lie GIn Asn Leu GIn GIu lie He Tyr Arg Asn 1625 1630 1635

Arg Phe Arg Arg GIn Pro Trp VaI Asn Leu Met Leu GIn GIn GIn 1640 1645 1650

GIn GIn GIn GIn GIn GIn GIn Ser Ser GIn GIn 1655 1660

<210> 2

<211>* 5194

<212> DNA

<213> Candida albicans

<400> 2 cccaaaaaag ataaaataaa aacaaaacaa aacaaaagta ctaacaaatt attgaaactt 60 ttaattttta ataaagaatc agtagatcta ttgttaaaag aaatgaactc aactccaagt 120 aaattattac cgatagataa acattctcat ttacaattac agcctcaatc gtcctcggca 180 tcaatattta attccccaac aaaaccattg aatttcccca gaacaaattc caagccgagt 240 ttagatccaa attcaagctc tgatacctac actagcgaac aagatcaaga gaaagggaaa 300 gaagagaaaa aggacacagc ctttcaaaca tcttttgata gaaattttga tcttgataat 360

tcaatcgata tacaacaaac aattcaacat cagcaacaac agccacaaca acaacaacaa 420 ctctcacaaa ccgacaataa tttaattgat gaattttctt ttcaaacacc gatgacttcg 480 actttagacc taaccaagca aaatccaact gtggacaaag tgaatgaaaa tcatgcacca 540 acttatataa atacctcccc caacaaatca ataatgaaaa aggcaactcc taaagcgtca 600 cctaaaaaag ttgcatttac tgtaactaat cccgaaattc atcattatcc agataataga 660 gtcgaggaag aagatcaaag tcaacaaaaa gaagattcag ttgagccacc cttaatacaa 720 catcaatgga aagatccttc tcaattcaat tattctgatg aagatacaaa tgcttcagtt 780 ccaccaacac caccacttca tacgacgaaa cctacttttg cgcaattatt gaacaaaaac 840 aacgaagtca atctggaacc agaggcattg acagatatga aattaaagca cgaaaatttc 900 agcaatttat cattagatga aaaagtcaat ttatatctta gtcccactaa taataacaat 960 agtaagaatg tgtcagatat ggatctgcat ttacaaaact tgcaagacgc ttcgaaaaac 1020 aaaactaatg aaaatattca caatttgtca tttgctttaa aagcaccaaa gaatgatatt 1080 gaaaacccat taaactcatt gactaacgca gatattctgt taagatcatc tggatcatca 1140 caatcgtcat tacaatcttt gaggaatgac aatcgtgtct tggaatcagt gcctgggtca 1200

cctaagaagg ttaatcctgg attgtctttg aatgacggca taaaggggtt ctctgatgag 1260 gttgttgaat cattacttcc tcgtgactta tctcgagaca aattagagac tacaaaagaa 1320 catgatgcac cagaacacaa caatgagaat tttattgatg ctaaatcgac taataccaat 1380 aagggacaac tcttagtatc atctgatgat catttggact cttttgatag atcctataac 1440 cacactgaac aatcaatttt gaatcttttg aatagtgcat cacaatctca aatttcgtta 1500 aatgcattgg aaaaacaaag gcaaacacag gaacaagaac aaacacaagc ggcagagcct 1560 gaagaagaaa cttcgtttag tgataatatc aaagttaaac aagagccaaa gagcaatttg 1620 gagtttgtca aggttaccat caagaaagaa ccagttctgg ccacggaaat aaaagctcca 1680 aaaagagaat tttcaagtcg aatattaaga ataaaaaatg aagatgaaat tgccgaacca 1740 gctgatattc atcctaaaaa agaaaatgaa gcaaacagtc atgtcgaaga tactgatgca 1800 ttgttgaaga aagcacttaa tgatgatgag gaatctgaca cgacccaaaa ctcaacgaaa 1860 atgtcaattc gttttcatat tgatagtgat tggaaattgg aagacagtaa tgatggcgat 1920 agagaagata atgatgatat ttctcgtttt gagaaatcag atattttgaa cgacgtatca 1980 cagacttctg atattattgg tgacaaatat ggaaactcat caagtgaaat aaccaccaaa 2040 acattagcac ccccaagatc ggacaacaat gacaaggaga attctaaatc tttggaagat 2100 ccagctaata atgaatcatt gcaacaacaa ttggaggtac cgcatacaaa agaagatgat 2160 agcattttag ccaactcgtc caatattgct ccacctgaag aattgacttt gcccgtagtg 2220 gaagcaaatg attattcatc ttttaatgac gtgaccaaaa cttttgatgc atactcaagc 2280 tttgaagagt cattatctag agagcacgaa actgattcaa aaccaattaa tttcatatca 2340 atttggcata aacaagaaaa gcagaagaaa catcaaattc ataaagttcc aactaaacag 2400 atcattgcta gttatcaaca atacaaaaac gaacaagaat ctcgtgttac tagtgataaa 2460 gtgaaaatcc caaatgccat acaattcaag aaattcaaag aggtaaatgt catgtcaaga 2520 agagttgtta gtccagacat ggatgatttg aatgtatctc aatttttacc agaattatct 2580 gaagactctg gatttaaaga tt±gaatttt gccaactact ccaataacac caacagacca 2640 agaagtttta ctccattgag cactaaaaat gtcttgtcga atattgataa cgatcctaat 2700 gttgttgaac ctcctgaacc gaaatcatat gctgaaatta gaaatgctag acggttatca 2760 gctaataagg cagcgccaaa tcaggcacca ccattgccac cacaacgaca accatcttca 2820 actcgttcca attcaaataa acgagtgtcc agatttagag tgcccacatt tgaaattaga 2880 agaacttctt cagcattagc accttgtgac atgtataatg atatttttga tgatttcggt 2940 gcgggttcta aaccaactat aaaggcagaa ggaatgaaaa cattgccaag tatggataaa 3000 gatgatgtca agaggatttt gaatgcaaag aaaggtgtga ctcaagatga atatataaat 3060 gccaaacttg ttgatcaaaa acctaaaaag aattcaattg tcaccgatcc cgaagaccga 3120 tatgaagaat tacaacaaac tgcctctata cacaatgcca ccattgattc aagtatttat 3180 ggccgaccag actccatttc taccgacatg ttgccttatc ttagtgatga attgaaaaaa 3240 ccacctacgg ctttattatc tgctgatcgt ttgtttatgg aacaagaagt acatccgtta 3300 agatcaaact ctgttttggt tcacccaggg gcaggagcag caactaattc ttcaatgtta 3360 ccagagccag attttgaatt aatcaattca cctgctagaa atgtgctgaa caacagtgat 3420 aatgtcgcca tcagtggtaa tgctagtact attagtttta accaattgga tatgaatttt 3480 gatgaccaag ctacaattgg tcaaaaaatc caagagcaac ctgcttcaaa atccgccaat 3540 actgttcgtg gtgatgatga tggattggcc agtgcacctg aaacaccaag aactcctacc 3600 aaaaaggagt ccatatcaag caagcctgcc aagctttctt ctgcctcccc tagaaaatca 3660 ccaattaaga ttggttcacc agttcgagtt attaagaaaa atggatcaat tgctggcatt 3720 gaaccaatcc caaaagccac tcacaaaccg aagaaatcat tccaaggaaa cgagatttca 3780 aaccataaag tacgagatgg tggaatttca ccaagctccg gatcagagca tcaacagcat 3840 aatcctagta tggtttctgt tccttcacag tatactgatg ctacttcaac ggttccagat 3900 gaaaacaaag atgttcaaca caagcctcgt gaaaagcaaa agcaaaagca tcaccatcgc 3960 catcatcatc atcatcataa acaaaaaact gatattccgg gtgttgttga tgatgaaatt 4020 cctgatgtag gattacaaga acgaggcaaa ttattcttta gagttttagg aattaagaat 4080 atcaatttac ccgatattaa tactcacaaa ggaagattca ctttaacgtt ggataatgga 4140 gtgcattgtg ttactacacc agaatacaac atggacgacc ataatgttgc cataggtaaa 4200

gaatttgagt tgacagttgc tgattcatta gagtttattt taactttgaa ggcatcatat 4260 gaaaaacctc gtggtacatt agtagaagtg actgaaaaga aagttgtcaa atcaagaaat 4320 agattgagtc gattatttgg atcgaaagat attatcacca cgacaaagtt tgtgcccact 4380 gaagtcaaag atacctgggc taataagttt gctcctgatg gttcatttgc tagatgttac 4440 attgatttac aacaatttga agaccaaatc accggtaaag catcacagtt tgatctcaat 4500 tgttttaatg aatgggaaac tatgagtaat ggcaatcaac caatgaaaag aggcaaacct 4560 tataagattg ctcaattgga agttaaaatg ttgtatgttc cacgatcaga tccaagagaa 4620 atattaccaa ccagcattag atccgcatat gaaagcatca atgaattaaa caatgaacag 4680 aataattact ttgaaggtta tttacatcaa gaaggaggtg attgtccaat ttttaagaaa 4740 cgttttttca aattaatggg cacttcttta ttggctcata gtgaaatatc tcataaaact 4800 agagccaaaa ttaatttatc aaaagttgtt gatttgattt atgttgataa agaaaacatt 4860 gatcgttcca atcatcgaaa tttcagtgat gtgttattgt tggatcatgc attcaaaatc 4920 aaatttgcta atggtgagtt gattgatttt tgtgctccta ataaacatga aatgaaaata 4980 tggattcaaa atttacaaga aattatctat agaaatcggt tcagacgtca accatgggta 5040

aatttgatgc ttcaacaaca acaacaacaa caacaacaac aaagctccca acagtaattg 5100 aaaggtctac ttttgatttt tttaatttta attggcaaat atatgcccat tttgtattat 5160 cttttagtct aatagcgttt tctttttttc cagt 5194

<210> 3

<211> 16

<212> PRT

<213> Mycobacterium tuberculosis

<400> 3

Lys Ala Ala Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala Ala Lys Lys 1 5 10 15

<210> 4

<211> 15

<212> PRT

<213> Candida albicans

<400> 4

Lys Ser lie Met Lys Lys Ala Thr Pro Lys Ala Ser Pro Lys Lys 1 5 10 15

<210> 5

<211> 16

<212> PRT

<213> Mycoplasma arthritidis

<400> 5 Phe VaI GIn Asn Leu Asn Asn VaI VaI Phe Thr Asn Lys GIu Leu GIu 1 5 10 15

<210> 6

<211> 16

<212> PRT

<213> Candida albicans

<400> 6

Phe Ala GIn Leu Leu Asn Lys Asn Asn GIu VaI Asn Ser GIu Pro GIu 1 5 10 15

<210> 7

<211> 30

<212> PRT

<213> Candida albicans

<400> 7

Phe Ala GIn Leu Leu Asn Lys Asn Asn GIu VaI Asn Ser GIu Pro GIu 1 5 10 15

Ala Leu Thr Asp Met Lys Leu Lys His GIu Asn Phe Ser Asn 20 25 30

<210> 8

<211> 9

<212> PRT

<213> Candida albicans

<400> 8

GIu Pro GIu Ala Leu Thr Asp Met Lys 1 5

<210> 9

<211> 31

<212> PRT

<213> Candida albicans

<400> 9

Cys VaI Asn Ser GIu Pro GIu Ala Leu Thr Asp Met Lys Leu Lys Arg 1 5 10 15

GIu Asn Phe Ser Asn Leu Ser Leu Asp GIu Lys VaI Asn Leu Tyr 20 25 30 <210> 10

<211> 4

<212> PRT

<213> Candida albicans

<400> 10

Asp His Asn Ser 1

<210> 11

<211> 31

<212> PRT

<213> Candida albicans

<400> 11

Cys VaI Asn Ser GIu Pro GIu Ala Leu Thr Asp Met Lys Leu Lys His 1 5 10 15

GIu Asn Phe Ser Asn Leu Ser Leu Asp GIu Lys VaI Asn Leu Tyr 20 25 30

Claims

What is claimed is:

1. A monoclonal antibody that recognizes the propeptide of the Intip protein from Candida albicans.

2. The monoclonal antibody of claim 1 wherein the antibody is generated against amino acids 239-268 of the Candida albicans lntip protein.

3. The monoclonal antibody of claim 1 wherein the antibody is generated against amino acids 248-277 of the Candida albicans lntip protein.

4. The monoclonal antibody of claim 1 wherein the antibody is generated against a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

5. The monoclonal antibody of claim 1 wherein the antibody recognizes the epitope at amino acids 252-260 of the Candida albicans lntip protein.

6. The monoclonal antibody of claim 1 wherein the antibody recognizes the full lntip protein in addition to recognizing the propeptide.

7. The monoclonal antibody of claim 1 wherein the propeptide has the sequence of the first 263 amino acids of the lntip protein of Candida albicans.

8. The monoclonal antibody according to claim 1 wherein said antibody is capable of preventing the cleaving of the propeptide.

9. The monoclonal antibody according to claim 1 wherein binding of said antibody to the propeptide limits T lymphocyte activity, prevents the secretion of interferon-gamma, or prevents the expansion of T-cells bearing V- beta subsets.

10. The monoclonal antibody according to claim 9 wherein the V-beta subsets are selected from the group consisting of subsets 2, 3 and 14.

11. The monoclonal antibody according to claim 1 wherein the host cell affecting the antibody is selected from the group consisting of peripheral blood mononuclear cells.

12. A pharmaceutical composition comprising the monoclonal antibody according to claim 1 and a physiologically acceptable carrier, vehicle or diluent.

13. A diagnostic kit comprising the monoclonal antibody according to claim 1 and means for detecting binding by that antibody.

14. A monoclonal antibody that recognizes the epitopes recognized by the monoclonal antibody Mab 163.5.

15. The monoclonal antibody of claim 14 wherein the antibody recognizes the epitope at amino acids 252-260 of the Candida albicans lntip protein..

16. The monoclonal antibody of claim 14 wherein the antibody recognizes the full lntlp protein in addition to recognizing the propeptide.

17. The monoclonal antibody according to claim 14 wherein binding of said antibody to the propeptide limits T lymphocyte activity, prevents the secretion of interferon-gamma, or prevents the expansion of T-cells bearing V- beta subsets.

18. The monoclonal antibody according to claim 17 wherein the V-beta subsets are selected from the group consisting of subsets 2, 3 and 14.

19. The monoclonal antibody according to claim 14 wherein the host cell affected by the antibody is selected from the group consisting of peripheral blood mononuclear cells.

20. A pharmaceutical composition comprising the monoclonal antibody according to claim 13 and a physiologically acceptable carrier, vehicle or diluent.

21. A diagnostic kit comprising the monoclonal antibody according to claim 13 and means for detecting binding by that antibody.

22. A method of treating or preventing an infection from Candida albicans comprising administering to a patient in need thereof an effective amount of the monoclonal antibody of Claim 1.

23. A method of preventing secretion of interferon-gamma caused by the lntlp protein comprising administering to a patient in need an effective amount of the monoclonal antibody of Claim 1.

24 A method of blocking of the activation of T-lymphocytes, blocking the expansion of T-cells bearing V-beta subsets, and blocking the secretion of interferon-gamma caused by the Intip protein comprising administering to a patient in need an effective amount of the monoclonal antibody of Claim 1.

25. The method according to claim 24 wherein the V-beta subsets are selected from the group consisting of subsets 2, 3 and 14.

26. A method of treating or preventing an infection from Candida albicans comprising administering to a patient in need thereof an effective amount of the monoclonal antibody of Claim 14.

27. A method of preventing secretion of interferon-gamma caused by the lntip protein comprising administering to a patient in need an effective amount of the monoclonal antibody of Claim 14.

28 A method of blocking of the activation of T-lymphocytes, blocking the expansion of T-cells bearing V-beta subsets, and blocking the secretion of interferon-gamma caused by the lntip protein comprising administering to a patient in need an effective amount of the monoclonal antibody of Claim 14.

29. The method according to claim 28 wherein the V-beta subsets are selected from the group consisting of subsets 2, 3 and 14.