EP1812063A2 - Anticorps monoclonaux du propeptide du candida albicans, et ses procédés d'utilisation - Google Patents

Anticorps monoclonaux du propeptide du candida albicans, et ses procédés d'utilisation

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Publication number
EP1812063A2
EP1812063A2 EP05825759A EP05825759A EP1812063A2 EP 1812063 A2 EP1812063 A2 EP 1812063A2 EP 05825759 A EP05825759 A EP 05825759A EP 05825759 A EP05825759 A EP 05825759A EP 1812063 A2 EP1812063 A2 EP 1812063A2
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European Patent Office
Prior art keywords
monoclonal antibody
protein
lntip
ser
lys
Prior art date
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EP05825759A
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German (de)
English (en)
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EP1812063A4 (fr
Inventor
Margaret K. Hostetter
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Yale University
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Yale University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
    • G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • 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.
  • the dimorphic yeast, Candida albicans is the leading fungal pathogen in normal hosts and in patients with damaged immune systems.
  • 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).
  • superficial disease e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water
  • chronic or recurrent infections e.g., candidal vaginitis
  • 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.
  • 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.
  • 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.
  • C. albicans adheres to epithelial and endothelial cells in the human host, often times by recognizing proteins of the extracellular matrix called ligands.
  • 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 ⁇ -subunit and a ⁇ - subunit. There are approximately 14 ⁇ and 8 ⁇ 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.
  • lntip of Candida albicans
  • 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.
  • 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- ⁇ ) which results in the colonization and progression of the yeast infection.
  • IFN- ⁇ interferon gamma
  • mortality rates from infections from organisms such as disseminated Candidas remain high despite aggressive antifungal therapy (Todischini, J. Intern Dis.
  • the present invention 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.
  • 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- ⁇ ).
  • 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
  • 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).
  • HBHA Mycobacterium tuberculosis heparin-binding hemagglutin adhesin
  • 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 V ⁇ 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 / ⁇ /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.
  • Lane 1 - S. cerevisiae lysate after expression of Pep 26 3; lane 2 - fraction 300-1 from nickel column; lane 3 - fraction 300-2; lane 4 - purification of Pep 263 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 Pep 263 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 V ⁇ expansion by the monoclonal antibodies generated in accordance with the present invention.
  • Fig. 22 shows the V ⁇ 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 V ⁇ Subsets 2 and 14 by soluble Pep 2 63; and the inhibition thereof by MAb 163.5 in accordance with the invention
  • Fig. 24 shows the induction of IFNy by soluble Pep 2 63; and the inhibition thereof by MAb 163.5 in accordance with the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • further information regarding lntip has been provided in pending U.S. patent application Ser. No. 09/264,604 and in U.S.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 V ⁇ subsets such as 2, 3, 14 or others, and the blocking of the secretion of IFN ⁇ .
  • 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.
  • 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.
  • 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 ⁇ M 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.
  • 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)
  • 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.
  • 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.
  • immunizations were as follows:
  • mice received 5 RIMMS immunizations of approximately 1-10 ⁇ g 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 ⁇ g 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 ⁇ l of the emulsion will be administered to each site.
  • the mice then received an injection of 0.5 to 5 ⁇ g of antigen emulsified in RIBI adjuvant at juxtaposed sites (lower and mid calf region, thigh, and axilla). 40-50 ⁇ l of the emulsion was administered to each site.
  • 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.
  • 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 V ⁇ subsets 2 and 14, and I FNy production stimulated by C. albicans, and the results are shown in the Table below:
  • 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 V ⁇ 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
  • the linear peptide is mixed with Freund's complete adjuvant and injected into Balb/c mice.
  • immunizations were as follows:
  • mice received 5 RIMMS immunizations of approximately 1-10 ⁇ g 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 ⁇ g 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 ⁇ l of the emulsion will be administered to each site.
  • the mice then received an injection of 0.5 to 5 ⁇ g of antigen emulsified in RIBI adjuvant at juxtaposed sites (lower and mid calf region, thigh, and axilla). 40-50 ⁇ l of the emulsion was administered to each site.
  • 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.).
  • 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.
  • mutations to these regions may also be utilized in order to disrupt the functioning of the lntip protein and to make the infectious microorganisms ineffective or less virulent.
  • 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 Pep 263 - As the present inventors have determined, the propeptide, Pep 263 constitutes a superantigen-like moiety which is released from lntip and which plays a major role in activating T lymphocytes in host cells.
  • 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.
  • 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 Pep 2 6 3 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 Pep 263 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, Pep 263 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.
  • agents and antibodies to Pep 2 63 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.
  • Fig. 4 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable 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.
  • 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.
  • the composition is formulated in the form of an ointment, cream, gel, lotion, drops or solution.
  • 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.
  • the isolated antibodies of the present invention 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.
  • these compositions may also be administered to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo.
  • the antibody may be modified as necessary so that, in certain instances, it is less immunogenic in the patient to whom it is administered.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 V ⁇ subsets, and/or the amount effective to block the eliciting of IFNy.
  • an effective amount will vary greatly depending on the nature of the infection and the condition of a patient.
  • 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).
  • 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.
  • 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.
  • kits 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.
  • 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.
  • 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.
  • 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.
  • a suitable detectable signal or label such as a biotin or enzyme that produces a detectable color, etc.
  • 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.
  • 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 Pep 263 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. Pep 263 eluted as a doublet.
  • 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.
  • 5x10 5 PBMCs were incubated with 5x10 5 C. albicans of the desired strain (wild type or int1- mutant) for 4 days at 37° C.
  • 0.2 ⁇ g/ml amphotericin B was added to all wells to prevent filamentation of C. albicans.
  • Antibody-treated wells received 25 ⁇ g/ml or 50 ⁇ g/ml MAb163.5 on day 1 through day 4.
  • an isotype control MAb (BD Biosciences) served as control.
  • PBMCs were pelleted and stained with a PE-conjugated monoclonal antibody against the lL-2 receptor (CD25).
  • CD25 PE-conjugated monoclonal antibody against the lL-2 receptor
  • 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.
  • SEB staphylococcal enterotoxin B
  • albicans were approximately 30-40% as active as SEB.
  • An isotype control MAb had no effect on / ⁇ /T7-induced T cell activation, but both 25 ⁇ g/ml and 50 ⁇ g/ml MAb 163.5 blocked T cell activation induced by //VT7-expressing C. albicans.
  • PBMCs Peripheral blood mononuclear cells were purified from the blood of adult humans by standard methods. In each well of a 96-well microtiter plate, 5x10 5 PBMCs were incubated with 5x10 5 C. albicans of the desired strain (wild type or int1- mutant) for 4 days at 37° C. 0.2 ⁇ g/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.
  • PE- conjugated monoclonal antibodies Beckman Coulter
  • 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/ ⁇ /cans-mediated expansion of Vbeta 2 and Vbeta14. This difference was statistically significant at p ⁇ 0.03.
  • Peripheral blood mononuclear cells were purified from the blood of adult humans by standard methods. All microtiter wells contained 5x10 5 PBMCs plus one of the following experimental mixtures: 200 pmoles of Pep 2 63 alone;
  • amphotericin B was added to all wells to prevent filamentation of C. albicans.
  • Control wells contained 200 pmoles SEB or SEB plus 25-50 ⁇ g/ml of MAb 163.5.
  • 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 Pep 263 was not effective in expanding Vbeta 2 or Vbeta 14.
  • Mab 163.5 does not block T lymphocyte activation induced by staphylococcal enterotoxin B. Thus, Mab 163.5 is specific for Intip.
  • 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.
  • PBMC Peripheral blood mononuclear cells
  • 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 ⁇ g/ml PHA, 4 ⁇ g/ml TSST-1 or 500,000 blastospores of either CAF2 or CAG3. Each culture condition was either left untreated or incubated with 10 ⁇ g/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 beta 2 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.
  • Vb ⁇ t a2 or Vbeta8 subsets were analyzed by flow cytometry for the expansion of either T cell Vb ⁇ t a2 or Vbeta8 subsets.
  • Significant expansion of the V b e ta 2 subset occurred in activations with the V beta 2 specific superantigen TSST-1 as well as with blastospores of CAF2 but not with CAG3.
  • the frequency of Vb eta 8 T cells was similar to the unactivated control for all conditions.
  • 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 beta 2 T cell subset of one of the donors evidences a role for lntip in activation-induced CD4 T cell loss.
  • T cells (2.5X10 5 ) were cultured with either 500,000 CAF2 blastospores or 4 ⁇ g/ml TSST-1 in the presence of APC (2.5X10 5 ) pretreated with or without 0.3% paraformaldehyde.
  • APC 2.5X10 5
  • T cell activation by CAF2 INT1/INT1 blastospores occurred despite the inability of MHC class Il expressing APCs to process antigen.
  • TSST-1 was not inhibited by paraformaldehyde fixation of APC.
  • 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 beta 2 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.
  • 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.
  • ICR mice given a tail vein injection of 10 5 wild type C. albicans expressing both INT1 alleles showed 100% mortality by day eleven, while 90% of mice given a homozygous double disruptant (genotype INT1/int1) survived.
  • PBMCs Peripheral blood mononuclear cells 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 ⁇ g/ml amphotericin B to prevent germ tube formation. See Figure 7. PBMCs were incubated at 37°C in 5% CO 2 with 10 5 blastospores of INT1/INT1 C. albicans or an equal number of int1/int1 C.
  • the superantigen TSST-1 800 ⁇ g/well
  • 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).
  • 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.
  • 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.
  • 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.
  • PFA paraformaldehyde
  • TSST-1 and INT1/INT1 C. albicans were used as stimuli.
  • 44% of T lymphocytes were activated with INT1/INT1 C. albicans as stimulus; in the presence of PFA, 43% were activated.
  • 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.
  • PFA treatment did not inhibit T lymphocyte activation in response to TSST-1 or to INT1/INT1 C. albicans.
  • lymphocyte activation in response to TSST-1 and INT1/INT1 C. albicans was not inhibited.
  • V ⁇ subsets were measured after stimulation of PBMCs with TSST-1 (800 ⁇ g), with INT1/INT1 C. albicans, with int1/int1 C. albicans, and with PHA. Unactivated PBMCs served as control ( Figure 9).
  • INT1/INT1 C. albicans induce a predominantly Th1 response in vitro with elevations in TNF ⁇ and IL-6 that are comparable to those induced by staphylococcal enterotoxin B (SEB), a well-characterized superantigen.
  • SEB staphylococcal enterotoxin B
  • 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.
  • V ⁇ 2 subset was preferentially expanded by S. cerevisiae expressing INT1; no expansion of V ⁇ 2 or V ⁇ 8 subsets was noted in response to S. cerevisiae transformed with vector alone.
  • X Activates T lymphocytes, up-regulates IL-2 receptor, and releases pro- inflammatory cytokines
  • APCs antigen-presenting cells
  • proprotein convertase 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.
  • 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.
  • a processing 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.
  • 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.
  • Figure 13 is a Western blot of supernatants from / ⁇ /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).
  • Pep 2 6 3 was responsible for the superantigen-like effects observed with INT1/INT1 C. albicans and INT1- expressing S. cerevisiae
  • Pep 263 was expressed as a recombinant, His-tagged protein in S. cerevisiae and assessed its effects on T lymphocyte activation and expansion of V ⁇ 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).
  • 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 V ⁇ 2 subset. Activation of T lymphocytes can be triggered by Pep 2 63, 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 Pep 263 are equivalent to INT1/INT1 C. albicans or / ⁇ /77-expressing S. cerevisiae in the ability to activate T lymphocytes. Like most microbial superantigens, Pep 263 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.
  • panel A the first 263 amino acids of lntip (Pep 263 ) are covert and cannot be detected by anti-INT600 antibodies ( Figure 12).
  • panel B Only in the presence of heparin (panel B) is the amino terminus of lntip exposed, at which point Pep 2 63 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 Pep 263 may also have superantigen-like effects, the activity of Pep 263 and the applicability of these interactions may be applicable to the problem of candidemia in the NICU infant.
  • IFNv secretion of IFNv is associated with MHC class Il haplotvpes.
  • MAM superantigen MAM from Mycoplasma arthritidis binds to antigen presenting cells expressing HLA DR1, DR4, DR7, and DR12.
  • release of IFN ⁇ is highest with cells expressing DR4, DR7, and DR12 (3).
  • MHC class Il binding site of MAM and an amino terminal sequence of Intip.
  • MHC class Il binding sites we also found that those people with "high" levels of IFN ⁇ in response to lntip expressed DR7, while "low” IFN ⁇ responders expressed DR1 , as has been reported for MAM.
  • the DQ locus appears to have no bearing on the IFN ⁇ response.
  • MAb 163.5 was able to inhibit T lymphocyte activation and expansion of V ⁇ subsets 2 and 14 in response to Int1 p + and inti p " C. albicans.
  • SEB as a stimulus
  • 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 "V ⁇ Expansion” column represent the percent of CD3 cells fluorescing with a monoclonal antibody to V ⁇ 2 or V ⁇ 14.
  • MAb 163.5 at 25 ⁇ g/ml or 50 ⁇ g/ml is able to block T lymphocyte activation and expansion of V ⁇ subsets 2 and 14 induced by Int1 p + C. albicans blastospores.
  • inti p C. albicans blastospores do not activate T lymphocytes or expand V ⁇ subsets.
  • MAb 163.5 fails to inhibit T cell activation or expansion of the V ⁇ 14 subset in response to SEB — an indication that MAb 163.5 is specific for Intip.
  • soluble Pep 263 for its ability to activate T lymphocytes, expand V ⁇ subsets 2 and 14, and trigger the release of IFND.
  • Pep 263 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.
  • C. albicans and S. cerevisiae are able to activate T lymphocytes by virtue of cell-surface polysaccharides that act as mitogens (2).
  • Pep 2 63 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 Pep 26 3-
  • the mutant may be contributing some post-translational modification of Pep 2 63 that augments its function: e.g. proteolytic processing or pH-dependent changes.
  • Pep 2 63 e.g. proteolytic processing or pH-dependent changes.
  • cleavage of malarial circumsporozoite protein by a malarial cysteine proteinase is essential for infectivity (5).
  • the cleavage site on malarial circumsporozoite protein is marked by the sequence KLKQP; the C-terminal sequence of Pep 2 63 is KLKH.
  • 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 His 263 -
  • Pro GIu lie His His Tyr Pro Asp Asn Arg VaI GIu GIu GIu Asp GIn 180 185 190
  • Lys Asn Lys Thr Asn GIu Asn lie His Asn Leu Ser Phe Ala Leu Lys 305 310 315 320
  • GIu GIn Ser lie Leu Asn Leu Leu Asn Ser Ala Ser GIn Ser GIn lie 450 455 460
  • 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
  • GIn Ala Thr lie GIy GIn Lys lie GIn GIu GIn Pro Ala Ser Lys 1130 1135 1140
  • Trp lie GIn Asn Leu GIn GIu lie He Tyr Arg Asn 1625 1630 1635
  • Lys Ser lie Met Lys Lys Ala Thr Pro Lys Ala Ser Pro Lys Lys 1 5 10 15

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Abstract

L'invention porte sur des anticorps monoclonaux pouvant se fixer au propeptide de la protéine Int1p de micro-organismes tels que la levure Candida albicans et s'avérant utiles pour traiter ou prévenir des infections dues à ces micro-organismes. L'invention porte en particulier sur des anticorps monoclonaux pouvant reconnaître l'épitope situé dans les acides aminés 252-260 de la protéine Intlp, et sur de tels anticorps monoclonaux pouvant reconnaître la région propeptide de l'Int1p ainsi que la protéine Int1p complète. De tels anticorps s'avèrent efficaces pour: bloquer l'activation des lymphocytes T; bloquer l'expansion des lymphocytes T porteurs de sous-ensembles V-béta tels que les 2, 3, 14 et autres; et bloquer la sécrétion de l'interféron gamma, ce qui les rend efficaces pour traiter et prévenir les infections dues à des levures telles que le Candida albicans.
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See also references of WO2006050343A2 *

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