CA2528851A1

CA2528851A1 - Extracellular aspergillus polypeptides

Info

Publication number: CA2528851A1
Application number: CA002528851A
Authority: CA
Inventors: Andrew Roche; Martin Chr. Hansen; Inge D. Villsen; Petra Schrotz-King; Jeanette Henningsen; Trine Louise Lund Joergensen
Original assignee: Individual
Current assignee: Zymenex Holding AS
Priority date: 2003-06-10
Filing date: 2004-06-10
Publication date: 2004-12-16
Also published as: WO2004108765A2; EP1636269A2; AU2004245174A1; JP2007535897A; WO2004108765A3

Abstract

The present invention relates to extracellular polypeptides ofAspergillus fumigatus, to fragments of these polypeptides, to compositions comprising such polypeptides and fragments and to exposed domains and epitopes of these polypeptides. The invention also relates to the use of these polypeptides and fragments for immunisation and for production of antibodies, and to antibodies that recognize and bind the polypeptides. Furthermore, the invention relates to methods of identifying binding partners and inhibitors, and to methods of preventing, treating and diagnosingAspergillus infections.

Description

Extracellular Aspergillus polypeptides This application is a non-provisional of U.S. provisional applications Serial No.
60/477,355, filed June 11, 2003 and Serial No. 60/482,451, filed June 26, 2003, which are hereby incorporated by reference in their entirety.
All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety.
Field of the invention The present invention relates to extracellular polypeptides of Aspergillus fumigatus, to fragments of these polypeptides, to compositions comprising such polypeptides and fragments and to exposed domains and epitopes of these polypeptides. The invention also relates to the use of these polypeptides and fragments for immunisation and for production of antibodies, and to antibodies that recognise and bind the polypeptides. Furthermore, the invention relates to methods of identifying binding partners and inhibitors, and to methods of diagnosing Aspergillus infections.
Background of the invention The rise of diseases that attack the immune system, such as AIDS, and medical treatments that depress the immune system, such as cancer chemotherapy or organ transplantation, have resulted in an increase in the death rate caused by fungal infections. Since the mid-1980's, fungal pathogens have begun to rival their bacterial counterparts in many different medical settings. Species of the Aspergillus family account for a substantial number of these fungal infections and in particular Aspergillus fumigatus has emerged world-wide as a frequent cause of nosocomial infection in virtually every major medical centre. For almost 30 years, amphotericin B
was the only drug approved for treating serious fungal infections despite significant kidney toxicity. Azoles were introduced in the 1980s for treating the most common fungal pathogen, Candida albicans, which is responsible for approximately 50%
of fungal infections. Widespread use of azoles encouraged the development of resistant strains to this drug. Unfortunately, most currently marketed azoles are largely ineffective against the more severe forms of fungal disease, such as CONFIRMATION COPY

infections caused by Aspergillus. The increase in drug-resistant strains of fungal pathogens further underscores the need for new antimicrobial treatments.
Aspergillus fumigates is a saprophytic fungus found ubiquitously in the environment, particularly in soil and in water and may be readily found in very large numbers in hay, grain and decaying organic matter. Aspergillus fumigates plays an essential role in recycling environmental carbon and nitrogen. Reservoirs in hospitals and other institutions include unfiltered air, ventilation systems, contaminated dust during construction work, carpeting, food, ornamental plants and water and water supply systems. It is generally believed that aspergillosis occurs as a consequence of the exogenous acquisition of spores; they are small enough (2.5-3.0 pm) to reach the alveoli upon inhalation and hardy enough to survive for prolonged periods in fomites.
It remains unclear what the size of the infectious inoculum needs to be, although this probably depends upon the immunological status of the host. There are around recognised species, but only a small number have been identified as pathogenic.
Among these A. fumigates which causes over 80% of human infections caused by Aspergillus species. A. fumigates is an opportunistic pathogen and normal individuals are not susceptible to disease except after inhalation of large quantities of spores. Aspergillus can cause illness in at least three ways: an allergic reaction in asthmatics (allergic aspergillosis); a colonisation in scarred lung tissue (aspergilloma); and an invasive infection with pneumonia which can affect the heart, lungs, brain and kidneys (invasive aspergillosis).
Allergic aspergillosis In the first type of aspergillosis illness, people with allergic asthma or genetic predisposition may develop this form of asthma upon becoming sensitised to Aspergillus species. Asthmatics may find their asthmatic condition aggravated upon exposure to A. fumigates. Some people develop allergic bronchopulmonary aspergillosis (ABPA), a condition in which Aspergillus spores germinate and the resultant mycelial growth can potentially block the bronchi. Patients may cough up small, brown plugs of mycelia. There is no invasion of tissue. However, the patient may suffer lung fibrosis and may, over time, become more susceptible to other lung diseases. ABPA is currently the most severe allergic pulmonary complication caused by Aspergillus species. It occurs in patients suffering from atopic asthma or cystic fibrosis. Another disease entity, related to ABPA only because it is immune-WO 2004/108765 PCT/DK2004/000407 _ _ ..._ mediated, hypersensitivity pneumonitis (also called extrinsic allergic alveolitis) is often associated with repeated exposure to an identified -- often occupational --source of high levels of antigen.
Asperailloma Aspergilloma, commonly referred to as "fungus ball," occurs in pre-existing pulmonary cavities that were caused by tuberculosis, sarcoidosis, or other bullous lung disorders and in chronically obstructed paranasal sinuses.
Invasive aspergillosis~A) Invasive aspergillosis (iA) is seen in people whose normal immune systems are compromised by other serious diseases such as leukaemia, lymphoma, carcinoma, tuberculosis, emphysema, diabetes, HIV/AIDS or by use of immunosuppressive drugs (often used in connection with organ or bone marrow transplant operations);
or by large doses of corticosteroids. In IA, there is an actual invasion of lung tissue or skin. Infection can also occur in many organs or tissues, e.g. heart, liver, eye, nose, ear and skeletal muscle. Pathologically invasive infections show clear invasion of the underlying tissue, eventually leading to bloodstream dissemination or contiguous spread to adjacent structures. The prognosis for IA is serious illness and death.
A fourfold increase in IA has been observed in the last 12 years. In 1992, IA
was responsible for approximately 30% of fungal infections in patients dying of cancer, and it is estimated that IA occurs in 10 to 25% of all leukaemia patients, in whom the mortality rate is 80 to 90%, even when treated. The average incidence of IA is estimated to be 5 to 25°to in patients with acute leukaemia, 5 to 10%
after alfogenic bone marrow transplantation (BMT), and 0.5 to 5% after cytotoxic treatment of blood diseases or autologous BMT and solid-organ transplantation. IA which follows solid-organ transplantation is most common in heart-lung transplant patients (19 to 26%) and is found, in decreasing order, in liver, heart, lung, and kidney recipients (1 to 10%) (Patel and Paya, 1997, Clin. Microbiol. Rev. 10: 86-124). IA also occurs in patients with nonhematogenous underlying conditions; it is increasingly reported in AIDS patients 1 to 12%) (Denning et al., 1991, N. Engl. J. Med. 324: 654-662) and is also a common infectious complication of chronic granulomatous disease (25 to 40%) Four types of IA have been described (Denning, 1998, Clin, Infect. Dis.
26:

781-805) : (i) acute or chronic pulmonary aspergillosis, the most common form of IA;
(ii) tracheobronchitis and obstructive bronchial disease with various degrees of invasion of the mucosa and cartilage as well as pseudomembrane formation, seen predominantly in AIDS patients; (iii) acute invasive rhinosinusitis; and (iv) disseminated disease commonly involving the brain (10 to 40% in BMT patients) and other organs (for example, the skin, kidneys, heart, and eyes).
Diagnosis of Aspergillus infections Unlike bacterial infections, cultures from blood or cerebrospinal fluid and other sterile body fluids - are rarely positive for Aspergillus species, even in patients with endocarditis and disseminated disease. Given the ubiquitous nature of the spores, recovering Aspergillus from cultures of the respiratory tract does not discriminate between genuine infection, colonization or contamination. A number of clinical findings may trigger a diagnosis of invasive aspergillosis, such as neutropenic fever not responding to broad-spectrum antibiotics, the development of new pulmonary infiltrates on chest X-ray and the presence of clinical signs suggestive of invasive mycosis (e.g. pleuritic chest pain, hemoptysis, etc.). Unfortunately, most of these triggers have low predictive value. Therefore, the only way to reach a precise and early diagnosis is to make intense efforts to collect specimens for culture and histopathological examination (by biopsy or needle aspiration). However, this gold standard approach involves aggressive procedures (open lung biopsy, brain biopsy, etc.) that are often precluded by cytopenia or by the critical condition of the patient.
Hence, definitive diagnosis is infrequently made before fungal proliferation becomes overwhelming and therapy may no longer be successful.

The detection of anti-Aspergillus antibodies has no place in the diagnosis of aspergillosis in neutropenic patients and hematopoietic stem cell transplant recipients because these populations are not capable of mounting an adequate antibody response. Diagnostic tools used at the moment are galactomannan detection (a major cell wall constituent released during growth), high-resolution pulmonary CT-scanning and detection of aspergillar DNA. Obtaining both high sensitivity and high selectivity remains a problem, and there is a need for novel reliable diagnostic'markers.

Currently available anti-Aspergillus agents The antifungal armamentarium that is currently available for the treatment of invasive aspergillosis is limited in number. It includes:
1. The polyene macrolide, amphotericin-B and its lipid-based formulations;
5 2. the triazole, itraconazole;
3. the fluorinated pyrimidine, 5-fluorocytosine; and 4. the allylamine, terbinafine.
The lack of a highly selective fungal target, not present in other eukaryotic cells, has for a long time precluded the development of new agents. With the exception of fluorocytosine, all available agents act by interfering with the structural or functional integrity of the fungal plasma membrane, either by physical disruption or by blocking the biosynthesis of membrane sterols. This strategy remains far from ideal since the non-selective nature of the therapeutic target results in concomitant cross-inhibition (or toxicity) in mammalian cells.
Treatment with antifungal drugs such as amphotericin-B and/or itraconazole involves many difficulties. Amphotericin-B, flucytosine and itraconazole are associated with low success rates and are hampered by serious infusion- or drug-related toxicity, by hazardous drug-drug interactions, by pharmacokinetic problems and by the development of resistance. Amphotericin-B has to be given by vein in large doses. In some patients the treatment can damage kidney and other organs.
The overall success rate of Amphotericin-B therapy for IA is 34%. In addition, most IA cases occur in spite of empirical administration of Amphotericin-B in response to a fever unresponsive to antibacterial agents. Itraconazole is generally given orally (also in large doses, e.g. at least 400 mg daily) and has been used for many years as a treatment, but even so, mortality is still as high as 35%.
Vaccination Vaccination may be another approach for combating Aspergillus infections. As explained above, IA is a severe problem for immunocompromised patients and especially in neutropenic patients, who have lost all their acquired immune response and are virtually without memory, Aspergillus infection is lethal in most cases. It seems that vaccination of these patients prior to immune suppression would not be a viable strategy. However, vaccination of a bone marrow donor could assist in the clearance of infection post donation. Also passive immunisation with immunoglobulins may be an option. Until now there have been no extensive preclinical and/or clinical data available concerning the efficacy of specific immunoglobulins. However, there are reports from invasive Aspergillosis studies in mice that show that active vaccination has influence on their mortality rate (Ito and Lyons (2002) J~ Infect. Dis. 186, 869-871 ).
Targets As A. fumigatus is becoming a major fungal pathogen of humans there is an urgent need for identification of suitable biochemical targets in A. fumigatus and for the discovery and development of new effective antifungal agents active against such biochemical targets. Recently, the A. fumigatus genome was analysed by random shotgun DNA sequencing. By sequence comparison with Candida albicans genes known to be essential for survival, a large number of potentially essential A.
fumigatus genes was identified (WO 02/086090). Such genes may potentially be interesting drug targets, but information on structure, function or cellular localisation of most of the encoded gene products is not yet available.
Summary of the invention In a main aspect, the present application relates to extracellular polypeptides of A.
fumigatus. In the context of this application, an 'extracellular polypeptide' is defined as a polypeptide which is entirely or partially (i.e. part of the polypeptide chain or part of the population of polypeptide molecules) localised outside the plasma membrane of a fungal cell. Thus, extracellular polypeptides include plasma-membrane polypeptides which have extracellular parts, cell-wall polypeptides, periplasmic polypeptides, secreted polypeptides and all other polypeptides that are fully or partially exposed to or released into the space outside the plasma membrane. Extracellular polypeptides furthermore include all polypeptides or polypeptide fragments that can be found in cell-wall, cell-surface-exposed and diffusate fractions isolated as described herein.
Extracellular polypeptides are attractive targets for antifungal therapy and/or diagnosis since the exposure of such polypeptides to the extracellular space means that compounds that interact with these peptides (e.g. compounds used to prevent, treat or diagnose fungal infections) often do not need to pass through the plasma membrane to be effective. This is a considerable advantage as the plasma membrane constitutes a major barrier for most types of compounds.
Extracellular localisation of a fungal protein can usually not be predicted from its amino-acid sequence. The presence of a signal sequence mediating entrance of protein into the secretory pathway can be predicted with a high degree of certainty, but many proteins carrying such sequences remain intracellular, in compartments such as the endoplasmic reticulum, the Golgi complex, endosomes and lysosomes.
Very little is known about sorting signals in A. fumigatus.
In principle, localisation of A. fumigatus proteins could be inferred from a known localisation of homologous proteins in other fungi, such as Saecharomyces cerevisiae or the pathogenic yeast Candida albicans, which are much better characterised than A. fumigatus. However, in practice, such predictions are highly uncertain. A recently performed genetic screening for putative exported C.
albicans proteins identified a number of such proteins whose closest homologue was an intracellular protein (Monteoliva et al. (2002) Eukaryotic Cell 1, 514-525).
Thus, even with the genome sequence of A, fumigatus available, it is not easy to predict which polypeptides can be found extracellularly.
The inventors have isolated and analysed cell-wall-, cell-surface-exposed- and diffusate fractions of A. fumigatus and thus determined extracellular localisation of the following polypeptides:
1. The polypeptide set forth in SEQ ID N0:1. This polypeptide has not previously been detected in A, fumigatus, as it was only previously proposed as a putative gene product on the basis of a nucleotide sequence. It is herein proposed to name this polypeptide Cssl, for Conidial Surface and Secreted protein I.
2. Hydrophobin (SEQ ID N0:2). Previously described in Parta et al. (1994) Infect.
Immun. 62, 4389-4395.
3. GAPDH-B, glyceraldehyde 3-phosphate dehydrogenase (SEQ ID NO:3). A 172 amino-acid fragment of this sequence has been described in the NCBI database under accession number AAL25819 (SEQ ID N0:35). However, the full-length polypeptide has not been described previously.

4. enolase (SEQ ID NO: 4). Described in the NCBI database under accession number AAK49451.
5. catalase B (SEQ ID N0:5). Described in the NCBI database under accession number AAB71223 and in Calera et al. (1997) Infect. Immun. 65, 4718-4724.
6. catalase A (SEQ ID N0:6). Described in the NCBI database under accession number 087630.
7. isopropylmalate dehydrogenase B (IMDH B) (SEQ ID NO: 36). This A. fumigatus polypeptide has not been described previously.
For several of these polypeptides, no localisation was known previously. For all polypeptides, novel polypeptide fragments that are useful in prevention, therapy or diagnosis of Aspergillus infections were identified by the inventors. Several of these fragments are relatively accessible from the extracellular space or released into it.
In a first main aspect, the invention relates to the polypeptide set forth in SEQ ID
N0:3 and variants and fragments thereof, with the proviso that the fragment does not consist of the sequence set forth in SEQ ID N0:35.
In another main aspect, the invention relates to the polypeptide set forth in SEQ ID
NO: 36 and variants and fragments thereof.
In a further main aspect, the invention relates to polypeptide fragments which are derived from the polypeptides set forth in SEQ ID NOs: 1-6 and 36, and comprise one or more amino-acid residues from the sequences set forth in SEQ ID NOs:7-and 37. The invention also relates to variants of these polypeptide fragments.
The invention also relates to exposed domains and epitopes which are comprised within or comprise part of the polypeptides or polypeptide fragments of the invention.
Furthermore, the invention relates to compositions comprising one or more extracellular Aspergillus polypeptides or polypeptide fragments of the invention The techniques that were used by the inventors in the identification of polypeptides in the different cell-wall-, cell-surface-exposed and/or diffusate fractions favour 35. identification of highly expressed proteiris. Thus, the polypeptides that were identified are relatively abundant. This, added to the determination that they are exposed to the extracellular environment of the cell, make them highly suitable as biochemical targets or diagnostic markers. Thus, the identification of these polypeptides in these fractions by the inventors formed the basis for the development of methods aimed at prevention, treatment and/or diagnosis of Aspergillus infections.
Accordingly, in a main aspect, the invention relates to use of polypeptides or fragments of the invention for generating a medicament. Preferably, a medicament that can be used for the immunisation or vaccination of a mammal, preferably a human being, preferably to generate a protective immune response.
Furthermore, in another main aspect, the invention relates to methods of raising antibodies against these polypeptides or fragments thereof in non-human mammals.
The invention also, in a further main aspect, relates to antibodies capable of binding an extracellular Aspergillus fumigatus polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID N0:36), Cssl (SEQ ID
N0:1 ), hydrophobin (SEQ ID N0:2), GAPDH-B (SEQ ID . N0:3), and catalase A
(SEQ ID N0:6). Use of such antibodies for the manufacture of a medicament for treatment or prevention of infection with Aspergillus is also an aspect of the invention. Thus, the invention also relates to pharmaceutical compositions comprising antibodies of the invention and a pharmaceutically-acceptable carrier.
Moreover, the invention relates to methods of treating or preventing Aspergillus fumigatus infections and/or other fungal infections comprising the step of administering antibodies of the invention to an individual in need thereof.
Furthermore, the invention relates to methods for screening for binding partners andlor inhibitors of these extracellular polypeptides, to methods for screening for antifungal agents and to methods aimed at diagnosing Aspergillus infections.

Detailed description of the invention Definitions A 'fragment' or 'polypeptide fragment' is defined as a non-full-length part of a 5 polypeptide. The length of fragments may vary from 2 amino-acid residues to the full-length polypeptide minus one amino-acid residue. Preferably, fragments are less than 100 amino acids, such as less than 50 amino acids, e.g. less than 40 amino acids, such as less than 30 amino acids, e.g. less than 25 amino acids, such as less than 20 amino acids in length. Thus, for example fragments can be 10 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 amino acids in length.
In further embodiments, fragments comprise more than 5 amino acids, such as more than 9 amino acids, e.g. more than 10 amino acids, such as more than 15 amino acids, e.g.
more than 20 amino acids, such as more than 30 amino acids. Expressed in another way, a fragment consists of a part of an amino-acid sequence which is less than 100% in length as compared to the full-length polypeptide. The length of the fragment can be less than 50%, such as less than 25%, such as less than 10% of the length of the full-length polypeptide. In other embodiments, the length of the fragment can be more than 5%, such as more than 10%, such as more than 25% of the length of the full-length polypeptide.
'Variants' of a given polypeptide or fragment are polypeptides or peptides that display a certain degree of sequence identity to said polypeptide or fragment.
Variants preferably have at least 75% sequence identity, for example at least 80%
sequence identity, such as at least 85% sequence identity, for example at least 90%
sequence identity, such as at least 91 % sequence identity, such as at least 92%
sequence identity, for example at least 93% sequence identity, such as. at least 94%
sequence identity, for example at least 95% sequence identity, such as at least 96%
sequence identity, for example at least 97% sequence identity, such as at least 98%
sequence identity, for example 99% sequence identity with the given polypeptide or fragment. Sequence identity is determined with any of the algorithms GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
Preferred variants of a given polypeptide or fragment are variants in which all amino-acid substitutions between the variant and the given polypeptide or fragment are conservative substitutions: Conservative amino-acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino-acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
Variants of a polypeptide or of a fragment thereof also include fom~s of the polypeptide or fragment wherein one or more amino acids have been deleted or inserted. Preferably, less than 5, such as less than 4, e.g. less than 3, such as less than 2, e.g. only one amino acid has been inserted or deleted. 'Variants' of a polypeptide or of a fragment thereof also include forms of these polypeptides or fragments modified by post-translational modifications of the amino-acid sequence.
Also included are fusion proteins wherein the given polypeptide or fragment thereof has been fused (on the gene level or post-translationally) to another peptide or polypeptide.
An 'exposed domain' is defined as a part of a polypeptide that is exposed to the external environment. Secreted or released parts of polypeptides, which are not cell-associated, are examples of exposed domains. Exposed domains can also be found in polypeptides that are cell-associated. This can e.g. be determined by protease treatment as described herein in the Examples. 1.e. an exposed domain of a polypeptide is a part of the polypeptide which is more accessible for proteases, such as trypsin or chymotrypsin, than other parts of the same polypeptide, and can be released from cellular association by protease treatment without disrupting the integrity of the cell. Surface exposure of a domain can also be determined using indirect immunofluorescence analysis, e.g. as described by Sanjuan et al.
(1996) Microbiology 142, 2255-2262. Exposed domains of plasma-membrane-associated polypeptides are parts of such polypeptides that are located immediately adjacent to membrane-spanning regions and are located on the extracellular side of the plasma membrane. An exposed domain can be flanked on both or on only one side by a membrane-spanning region. Membrane-spanning regions can be predicted by a variety of methods, reviewed in Moller et al. (2001 ) Bioinformatics 17, 646-653. In a preferred embodiment, an exposed domain of a plasma-membrane-associated polypeptide is a part of a polypeptide located on the extracellular side of the plasma membrane, immediately adjacent to a membrane-spanning region (transmembrane helix) as predicted by the TMHMM program 2.0 (Krogh et al. (2001 ) J. Mol.
Biol.
305, 567-580.
'Epitope' in this context covers any part of a polypeptide capable of being recognised by an antibody or functional equivalent thereof. Epitopes may consist of a stretch of consecutive amino-acid residues or of non-consecutive parts of a polypeptide. Typically, an epitope consists of 2-20 amino acids, such as 3-10 amino acids, preferably 3-8 amino acids, such as 3,4,5,6,7 or 8 amino acids.
'Expression vector' refers to a plasmid or phage or virus, for producing a polypeptide from a polynucleotide sequence. An expression vector comprises an expression construct, comprising an assembly of (1 ) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and which is operably linked to the elements of (1); and (3) appropriate transcription initiation and termination sequences.
'Vaccine' is used to indicate a composition capable of inducing a protective immune response against a microorganism in a human being or animal.
'Protective immune response' is used to indicate an immune response (humoral/antibody and/or cellular) inducing memory in an organism, resulting in the infectious agent, being met by a secondary rather than a primary response, thus reducing its impact on the host organism.
A 'binding partner' of a polypeptide refers to a molecule that can bind to said polypeptide. Such binding can be indirect, through another molecule, but is preferably direct. A binding partner can be any type of molecule, such as e.g.
small hydrophobic molecules or e.g. a cellular or extracellular macromolecule, such as a protein, a carbohydrate or a nucleic acid. Preferred types of binding partners include antibodies, ligands or inhibitors.
The term 'plurality' indicates more than one, preferably more than 10.
'Secreted' in the present context refers to soluble polypeptides or fragments thereof that are not cell-associated and thus in principle diffuse freely in the surrounding medium. This includes fragments of polypeptides that are released from cellular association, for instance through proteolysis.
The term 'indicator moiety' covers a molecule or a complex of molecules that can be detected or generates a detectable signal. Preferably, the indicator moiety is an antibody or includes an antibody molecule_ Thus, a preferred indicator moiety is an antibody coupled to a detectable substance. The detectable substance can in some embodiments comprise a second antibody.
'Host-derived molecule' or 'host molecule' refers to a molecule which is normally found in a host organism that can be infected with A. fumigates. A host-derived molecule is preferably a host polypeptide, preferably a human polypeptide.
Examples of host-derived molecules that interact with pathogenic fungi are serum albumin and transferrin, fibrinogen, complement fragment C3d, complement fragment iC3b, laminin, fibronectin, entactin, vitronectin, mannan adhesins, epithelial binding lectin-like protein, and agglutinin-like proteins.
The term 'antibodies' when used herein is intended to cover antibodies as well as functional equivalents thereof. Thus, this includes polyclonal antibodies, monoclonal antibodies (mAbs) ) derived from any species including but not limited to mouse, rat, hamster, rabbit and lama, human, humanised or chimeric antibodies, single-chain antibodies, and also Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies, hybrids comprising antibody fragments, and epitope-binding fragments of any of the these. The term also includes mixtures of monoclonal antibodies.
'Isolated' used in connection with polypeptides and polynucleotides disclosed herein refers to these having been identified and separated andlor recovered from a component of . its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
Polypeptides of the invention Fragments of extracellular Aspergillus polypeptides The analysis of three different Aspergillus fractions (diffusate, cell-surface-exposed and cell wall) that was performed by the inventors led to the identification of different types and numbers of fragments from the same polypeptides for each of the fractions. For example, as is described in Example 1, five Cssl peptides were identified in the cell-wall fraction while only one was identified in diffusate and cell-surface-exposed fractions. This difference may indicate structural features of the protein. Without being limited to a specific theory, a possible explanation for this is that a portion of Cssl can be cleaved from the cell wall, releasing one part of the protein into the surrounding milieu, while the remainder of the protein remains embedded in the cell wall. Similarly, although other explanations are possible, the fact that only one peptide is detected in cell-surface fractions may suggest that an area of the protein comprising that peptide is exposed while the remainder of the protein is not. Even regions of a polypeptide which are not embedded in other cellular structures such as the cell wall, may still contain parts that are more accessible than other parts. For instance, the surface of a polypeptide may be more accessible than parts of the polypeptide which are buried within a tertiary protein structure. Protease treatment may also identify such protein surface regions.
Thus, the inventors have identified protein regions of particular interest.
Exposed domains are, due to their accessibility, particularly attractive targets for diagnosis or for antifungal treatment. Moreover, exposed polypeptide fragments or domains are likely to contribute to, or comprise epitopes, and thus be highly suitable for antibody recognition. For many of the applications described below, it can be advantageous to work with fragments that are larger than the ones that were identified by the inventors. This can in particular be the case for methods of identifying binding partners and methods for raising antibodies, such as immunisation, which sometimes do not work well with small fragments.

In a main aspect, the invention relates to fragments of extracellular Aspergillus polypeptides that comprise exposed domains and/or epitopes. The invention also relates to the full-length GAPDH-B polypeptide (SEQ ID N0:3) and to the full-length isopropylmalate dehydrogenase B polypeptide (SEQ ID N0:36).

Accordingly, in a main aspect, the invention relates to an Aspergillus polypeptide selected from the group of fragments of SEQ ID N0:1 of less than 259 amino-acid residues in length, such as 10 less than 200, preferably less than 150, such as less than 100, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:7,8,17,26,28,29 and/or 30 and variants of said fragments;

15 fragments of SEQ ID N0:2 of less than 106 amino-acid residues in length, such as less than 75, preferably less than 50, such as less than 25 residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID
N0:9,10;18 and/or 19 and variants of said fragments;
polypeptides comprising SEQ ID NO:3, fragments thereof and variants thereof, with the proviso that if the polypeptide is a fragment of SEQ ID N0:3, that this fragment is not the fragment set forth in SEQ ID N0:35;
fragments of SEQ ID N0:4 of less than 437 amino-acid residues in length, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:13,14,23,24 and/or 25 and variants of said fragments;
fragments of SEQ ID N0:5 of less than 727 amino-acid residues in length, e.g.
less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g.
less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:15,16 and/or and variants of said fragments;

fragments of SEQ ID N0:6 of less than 748 amino-acid residues in length, e.g.
less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g.
less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:34 and variants of said fragments;
and polypeptides comprising SEQ ID N0:36, fragments thereof and variants thereof.
In a preferred embodiment, the above fragments comprise one or more residues of the amino-acid sequences set forth in SEQ ID NOs: 7-27 and 37. In a more preferred embodiment, the fragments comprise one or more residues of the amino-acid sequences set forth in SEQ ID NOs: 7-16. In another more preferred embodiment, the fragments comprise one or more residues of the amino-acid sequences set forth in SEQ ID NOs: 17-25 and/or SEQ ID N0:14. In yet another more preferred embodiment, the fragments comprise one or more residues of the amino-acid sequences set forth in SEQ ID NO: 18, 19, 26, 27, and/or 37.
Further preferred polypeptides are fragments of SEQ ID N0:1 of less than 259 amino-acid residues in length, such as less than 200, preferably less than 150, such as less than 100, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:7,8,17,26,28,29 and/or 30, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:7, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:8, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:17, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:26, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:28, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:29, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:30.
Further preferred polypeptides are fragments of SEQ ID N0:2 of less than 106 amino-acid residues in length, such as less than 75, preferably less than 50, such as less than 25 residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID NO:9,10,18 and/or 19, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:9, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:10, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:18, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:19.
Preferred polypeptides include fragments of SEQ ID N0:3, with the proviso that if the polypeptide is a fragment of SEQ ID N0:3, that this fragment is not the fragment set forth in SEQ ID N0:35. Preferred are fragments of SEQ ID N0:3 of less than 171 amino acids in length, such as less than 150, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID
N0:11,12,20,21,22,31,32 and/or 33, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:11, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:12, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:20, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID NO:21, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:22, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:31, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:32, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:33. Other preferred fragments of SEQ ID N0:3 are fragment between 173 residues and 317 residues in length, comprising one or more residues of the amino-acid sequences set forth in SEQ
ID
N0:11,12,20,21 and/or 22 or variants of said fragments.
Further preferred polypeptides are fragments of SEQ ID N0:4 of less than 437 amino-acid residues in length, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues, in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:13,14,23,24 and/or 25, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:13, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:14, such as one or more residues of the amino acid sequences set forth in SEQ ID N0:23, e.g. one or more residues of the amino acid sequence set forth in SEQ ID N0:24, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:25.

Further preferred polypeptides are fragments of SEQ ID N0:5 of less than 727 amino-acid residues in length, e.g. less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid. residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:1'S,16 and/or 27, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:15, e.g. one or more residues of the amino-acid sequence set forth in SEQ ID N0:16, such as one or more residues of the amino-acid sequences set forth in SEQ ID N0:27.
Further preferred polypeptides are fragments of SEQ ID N0:6 of less than 748 amino-acid residues in length, e.g. less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:34.
Preferred polypeptides include polypeptides comprising or consisting of SEQ ID
N0:36. Further preferred are fragments of SEQ ID N0:36, of less than 367 amino acid residues in length, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID
N0:37. In one embodiment, X~ in SEQ ID N0:36 and SEQ ID NO: 37 is a serine. In another embodiment, X~ in SEQ ID N0:36 and SEQ ID NO: 37 is an alanine. In a further embodiment, XZ in SEQ ID N0:36 and SEQ ID NO: 37 is a leucine. In another embodiment, X2 in SEQ ID NO:36 and SEQ ID NO: 37 is an isoleucine.
Thus, different sequence embodiments for SEQ ID NO: 37 and the equivalent part of SEQ ID N0:36 include LAAELALR, LSAELALR, LAAEIALR, LSAEIALR.
Preferably, the above defined polypeptide fragments of SEQ ID NOs:1-6 and 36 comprise more than one residue of the specified amino-acid sequence, such as 2,3,4,5,6,7,8 or 9 residues of the specified amino-acid sequence. A non-limiting example of such a preferred fragment is a fragment of SEQ ID N0:1 comprising 9 residues of the amino-acid sequence set forth in SEQ ID N0:7. Most preferably, the above polypeptide fragments comprise all residues of the specified amino-acid sequence. A non-limiting example of a most preferred fragment is a fragment of SEQ ID N0:1 comprising all 16 residues of the amino-acid sequence set forth in SEQ ID N0:7.
In one embodiment, the polypeptide of the invention, preferably consists of an exposed domain, such as domains comprising an amino-acid sequence selected from the group of SEQ ID NOs: 7-34 and 37, preferably the group of SEQ ID NOs:
7-27 and 37, more preferably the group of SEQ ID NOs:17-27, SEQ ID N0:14 and SEQ ID N0:37, or variants thereof. An exposed domain may be determined as described above in the definition section.
Further preferred polypeptides consist of an epitope of a polypeptide selected from the group of SEQ ID N0:1-6 and 36, comprising at least one amino acid from a peptide selected from the group of SEQ ID NO: 7-27 and 37, and fragments or variants of said epitope. In one preferred embodiment, the amino acid residues of the epitope are consecutive residues from the polypeptide. In another preferred embodiment, the amino acid residues of the epitope are non-consecutive residues from the polypeptide. Further preferred embodiments include more than 1, such as more than 2, preferably more than 3, such as more than 4 consecutive or non-consecutive amino acids of the sequences of SEQ ID NO: 7-27 and 37. The invention also relates to use of such epitopes in any of the methods or preferred methods of the invention.
Fragments that consist or essentially consist of an amino-acid seguence selected from the Group of SEQ ID NO: 7-34 and 37.
Preferred polypeptides of the invention are fragments of one of the polypeptides set forth in SEQ ID N0:1-6 and 36 that essentially consist of one of the fragments set forth in SEQ ID N0:7-34 and 37. 'Essentially consists of is meant to indicate that the fragment comprises a substantial part of an amino-acid sequence selected from the group of SEQ ID N0:7-34 and 37 and in addition to that contains 10 or fewer flanking residues from the polypeptide on either or both (N-terminal and/or C-terminal) sides of the smaller fragment. A 'substantial part' herein means at least 2, such as at least 5 amino acids of any of the amino acid sequence set forth in SEQ
ID N0:7-34 and 37. Such a fragment thus overlaps with the corresponding fragment selected from the group of SEQ ID N0:7-34 and 37. Preferably, the fragment that essentially consists of any of the amino-acid sequences set forth iri SEQ ID
N0:7-34 and 37 comprises the entire amino-acid sequence of that sequence. Thus, a preferred fragment of the invention is a fragment of one of the polypeptides set forth in SEQ ID N0:1-6 and 36 that comprises and essentially consists of one of the fragments set forth in SEQ ID N0:7-34 and 37. Such a fragment is thus larger than 5 the corresponding fragment selected from the group of SEQ ID N0:7-34 and 37.
'Comprises and essentially consists of is meant to indicate that the larger fragment comprises a smaller peptide selected from the group of SEQ ID N0:7-34 and 37 and in addition to that contains 10 or fewer flanking residues from the polypeptide on either or both (N-terminal and/or C-terminal) sides of the smaller fragment.
10 Preferably, the larger fragment contains fewer than 8, such as fewer than 6, e.g.
fewer than 4, e.g. fewer than 3, such as 2 or only 1 residue on one or both sides of the smaller fragment.
Most preferred polypeptides of the invention are fragments selected from the group 15 of SEQ ID NO: 7-34 and 37. Thus, such most preferred polypeptides include any of the fragments from the group of fragments set forth in SEQ ID N0:7-34 and 37, such as the fragment set forth in SEQ ID N0:7, or the fragment set forth in SEQ ID
N0:8, or the fragment set forth in SEQ ID N0:9, or the fragment set forth in SEQ ID
N0:10, or the fragment set forth in SEQ ID N0:11, or the fragment set forth in SEQ

20 ID N0:12, or the fragment set forth in SEQ ID N0:13, or the fragment set forth in SEQ ID N0:14, or the fragment set forth in SEQ ID N0:15, or the fragment set forth in SEQ ID N0:16, or the fragment set forth in SEQ ID N0:17, or the fragment set forth in SEQ ID N0:18, or the fragment set forth in SEQ ID N0:19, or the fragment set forth in SEQ ID N0:20, or the fragment set forth in SEQ ID N0:21, or the fragment set forth in SEQ ID N0:22, or the fragment set forth in SEQ ID N0:23, or the fragment set forth in SEQ ID N0:24, or the fragment set forth in SEQ ID
N0:25, or the fragment set forth in SEQ ID N0:26, or the fragment set forth in SEQ ID
N0:27, or the fragment set forth in SEQ ID N0:28, or the fragment set forth in SEQ
ID N0:29, or the fragment set forth in SEQ ID N0:30, or the fragment set forth in SEQ ID NO: 31, or the fragment set forth in SEQ ID N0:32, or the fragment set forth in SEQ ID N0:33, or the fragment set forth in SEQ ID NO:34, or the fragment set forth in SEQ ID N0:37. The invention also relates to a variant of any of the above fragments or any other fragment described herein.
Preferably, the fragment is selected from the group of fragments set forth in SEQ ID
N0:7-16 and 37, such as the fragment set forth in SEQ ID N0:7, or the fragment set forth in SEQ ID N0:8, or the fragment set forth in SEQ ID N0:9, or the fragment set forth in SEQ ID NO:10, or the fragment set forth in SEQ ID N0:11, or the fragment set forth in SEQ ID N0:12, or the fragment set forth in SEQ ID N0:13, or.the fragment set forth in SEQ ID N0:14, or the fragment set forth in SEQ ID N0:15, or the fragment set forth in SEQ ID N0:16, or the fragment set forth in SEQ ID
NO:37, or a variant of any of these fragments.
Compositions of the invention Compositions of the invention comprising one or more of polypeptides of the invention can be used in various methods and for various applications as described below. Having more than one polypeptide of the invention in such a composition can have important advantages. For instance, immunisation or vaccination may be more effective when several polypeptides or fragments are introduced at the same time.
Thus, in a main aspect the invention relates to a composition comprising one or more extracellular Aspergillus polypepiides or polypeptide fragments of the invention. Preferred compositions of the invention ara ones that comprise one or more preferred polypeptides of the invention, i.e. the polypeptides described above.
Thus, any preferred polypeptide of the invention can be used to generate a preferred composition of the invention. A preferred composition of the invention is a pharmaceutical composition comprising one or more polypeptide(s) and/or one or more polypeptide fragments of the invention and a pharmaceutically-acceptable carrier.
Cssl, isopropylmalate dehydroaenase B and GAPDH-B
Three extracellular polypeptides that were identified by the inventors are of particular interest, namely Cssl, isopropylmalate dehydrogenase B, and GAPDH-B.
Css I
This document presents data indicating the first identifications of Cssl, a novel cell-surface-exposed/secreted protein. This protein had previously been hypothesised based on the output of a gene prediction programme. However, the inventors' studies have confirmed the existence of this protein and have revealed it to be a conidial cell-wall-associated protein that is exposed on the surface while also being secreted/released into the surrounding milieu. The function of this protein is yet to be determined. However, its location within the diffusate is interesting in light of the documented abilities of diffusate to suppress the immune responses (Hobson RP

(2000) Med. Mycol. 38, 133-141 ). Attempts to identify the proteins) responsible for this suppressing activity have to date been unsuccessful. Without being limited to any specific theory, it is possible that Cssl is responsible for these functions, but that they have not been attributed to it due to the basic difficulties in performing molecular biology studies in Aspergillus fumigatus. It is interesting to note that the protein displays homology to LANA, a transcriptional regulator of Herpes virus (see below under Examples). Again without limitation to a specific theory, the possibility exists that Cssl possesses a similar function. If so, one could envisage it functioning as an extracellular sensor that transmits signals into the interior of the fungus.
Alternatively, this protein may become active upon uptake into the host cell, where it utilises its transcriptional activities to interfere with host processes, to the benefit of the fungus. Other possible functions of this protein may include roles in adhesion, invasion, conidial cell-wall processing or enzymatic digestion of host proteins.
Isopropylmalate dehydro4enase B
Isopropylmalate dehydrogenase B (IMDH B) is an enzyme involved in the biosynthesis of leucine. It has previously been found intracellularly in other microorganisms. The inventors have now identified this protein in cell surface fractions of A. fumigatus. The primary sequence of the enzyme does not reveal a traditional signal sequence and thus the question arises as to how the enzyme is transported to the cell surface. Without being limited to a particular theory, it is possible that the protein interacts with a heat shock protein and that the heat shock protein mediates translocation of IMDH B across the membrane. Similar mechanisms have been described for other proteins in Young et al. (2003) Cell 112:41-50.
GAPDH-B
The inventors have been the first to identify GAPDH-B, the polypeptide of SEQ
ID
N0:3. In one aspect, the invention relates to the sequence set forth in SEQ ID
NO:3, and variants thereof. Furthermore, the invention relates to use of the polypeptide set forth in SEQ ID N0:3 in any of the methods or preferred methods of the invention.
GAPDHs are documented to function in glycolysis. Without being limited to a particular theory, the cell-surface localisation of GAPDH-B might suggest a role for this protein in the initiation of germination. It would seem logical to assume that dormant conidia are more prone to germination when environmental conditions become more favourable for growth and propagation of the species. One requirement for growth is a carbon source, e.g. glucose. However, the dormant conidia must have some way of detecting external environmental conditions while in its state of low metabolic activity. It is possible that the presence of glycolytic enzymes on the cell surface could result in the production of glucose by-products that may communicate to the cell that the external environment is of sufficient status to support propagation of the species. The protein may alternatively or additionally function in other processes such as adhesion, invasion, intracellular motility or intracellular survival. It is interesting to note that GAPDH proteins possess the capability to bind to cytoskeletal components (see e.g. Tisdale (2002) J.
Biol. Chem.
277, 3334-3341). This feature may provide conidia with a mechanism by which it can traverse host cells in order to reach the basal membranes and cause invasive disease.
Production of polypeptide and fragments The polypeptides and fragments of the invention can be produced synthetically by conventional techniques known in the art. Alternatively, they can be produced recombinantly in heterologous host cells. Thus, the invention also encompasses polynucleotide sequences encoding polypeptides and fragments of the invention, expression vectors comprising such polynucleotides, and host cells transformed or transfected with such polynucleotides or expression vectors. Non-exclusive examples of polynucleotides of the invention are the polynucleotides of SEQ ID
N0:38 and SEQ ID N0:39. Suitable host cells can be mammalian cells, e.g. CHO, COS or HEK293 cells. Alternatively, insect cells, bacterial cells or fungal cells can be used. In preferred embodiments, yeast cells or cells from other Aspergillus species than A. fumigates are used. Methods for heterologous expression of polynucleotide sequences in the cell types listed above and subsequent purification of the produced polypeptides are well-known to those skilled in the art.
Preferably, polypeptides, fragments and polynucleotides of the invention are isolated.

Vaccination Exposure of a fungal polypeptide or a fragment thereof to the extracellular space often allows it to be detected by the immune system of a host organism. If such a polypeptide furthermore has a relatively high copy number, such as is the case for the extracellular polypeptides of this invention, such a polypeptide or a fragment thereof becomes particularly suitable as a target for antibodies.
In an important aspect, the invention relates to use of any one or more of the polypeptides, polynucleotides or compositions as defined herein for the manufacture of a medicament, preferably a vaccine. Such a medicament can preferably be used for prevention (i.e. prophylactic treatment) of Aspergillus infections in a mammal. In such use the polypeptide, polynucleotide or composfion is used for active immunisation or vaccination. Accordingly, the invention also relates to a medicament for treating Aspergillus infections comprising a polypeptide, po(ynucleotide or composition of the invention as an active ingredient.
Thus, the invention relates to a vaccine comprising a pharmaceutically-acceptable carrier and - a polypeptide comprising a sequence selected from the group of extracellular Aspergillus fumigatus sequences of SEQ ID N0:1-6, or an antigenic fragment of any of said sequences, or - a polynucleotide comprising a sequence encoding said polypeptide or fragment.
Furthermore, the invention relates to a method of treatment comprising the step of administering to an individual a pharmaceutically effective amount of any of the polypeptides, polynucleotides or compositions of the invention. Preferably, the treatment generates a protective immune response. Preferably, the medicament is used for the treatment or prophylactic treatment of a human being. Preferred embodiments include the use of any of the polypeptides set forth in SEQ ID
N0:1,2,3 or 36 or fragments of these polypeptides for said manufacture of said medicament or said method of treatment, preferably any of the preferred polypeptide fragments defined herein.

In preferred embodiments of this method, said polypeptide is selected from the group of SEQ ID NOs:1, 2, 3, 5, 6, and 36. In a more preferred embodiment, the polypeptide that is provided is Cssl (SEQ ID N0:1) or a fragment thereof, preferably a fragment comprising one or more residues of the amino-acid sequences set forth 5 in SEQ ID N0:7,8,17,26,28,29 and/or 30, or a variant of said polypeptide. In another more preferred embodiment, the polypeptide that is provided is hydrophobin (SEQ
ID N0:2) or a fragment thereof, preferably a fragment comprising one or more residues of the amino-acid sequences set forth in SEQ ID N0:9,10,18 and/or 19, or a variant of said polypeptide: In a further more preferred embodiment GAPDH-B
10 (SEQ ID N0:3) or a fragment thereof, preferably a fragment comprising one or more residues of the amino-acid sequences set forth in SEQ ID
N0:11,12,20,21,22,31,32 and/or 33, or a variant of GAPDH-3 or the fragment is provided. In a still further more preferred embodiment, the polypeptide that is provided is catalase A (SEQ
ID
N0:6) or a fragment thereof, preferably a fragment comprising one or more residues 15 of the amino-acid sequence set forth in SEQ ID N0:34, or a variant of said polypeptide. In a further more preferred embodiment, the polypeptide that is provided is isopropylmalate dehydrogenase B (SEQ ID NO:36) or a variant or fragment thereof, preferably a fragment comprising one or more residues of the amino-acid sequence set forth in SEQ ID N0:37.
In other preferred embodiments, a fragment selected from the group of SEQ N0:7-34 and 37 is provided, preferably a fragment selected from the group of SEQ ID
N0:7-16, such as the fragment set forth in SEQ ID N0:7, or the fragment set forth in SEQ ID N0:8, or the fragment set forth in SEQ ID NO:9, or the fragment set forth in SEQ ID N0:10, or the fragment set forth in SEQ ID N0:11, or the fragment set forth in SEQ ID N0:12, or the fragment set forth in SEQ ID N0:13, or the fragment set forth in SEQ ID N0:14, or the fragment set forth in SEQ ID N0:15, or the fragment set forth in SEQ ID N0:16.
Active immunisation or vaccination may be done in different ways, such as raising anti-protein antibodies indirectly using DNA immunisation techniques or directly using the polypeptide or a fragment thereof. The polypeptide may be administrated to said mammal more than once, such as twice, for example 3 times, such as 3 to 5 times, for example 5 to 10 times, such as 10 to 20 times, for example 20 to 50 times, such as more than 50 times. It is also possible that different polypeptides or fragments are administered to the same mammal, either simultaneously of sequentially in any order. Administration may be done by any suitable method, for example parenterally, orally or topically. Preferably, however it is administered by injection, for example intramuscular, intradermal, intravenous or subcutaneous injection, more preferably by subcutaneous or intravenous injection.
Methods for determining suitable protocols for active immunisation, such as determining dosage, use of adjuvants and/or pharmaceutically acceptable carriers are known to those skilled in the art. Various adjuvants can be used to increase the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as fysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
Antibodies In another main aspect, the invention relates to isolated antibodies capable of binding an extracellular Aspergillus fumigatus polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID N0:36), Cssl (SEQ ID
N0:1), hydrophobin (SEQ ID N0:2), GAPDH-B (SEQ ID N0:3), and catalase A
(SEQ ID N0:6).
In preferred embodiments, the antibody is capable of binding a polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID N0:36), Cssl (SEQ ID N0:1) and catalase A (SEQ ID N0:6). More preferably, the antibody is capable of binding a polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID N0:36) and Cssl (SEQ ID N0:1). Most preferably, the antibody is capable of binding isopropylmalate dehydrogenase B
(SEQ ID N0:36).
Preferred affinities of the binding of the antibody to the target polypeptide include those with a dissociation constant or Kd of less than 5 X 10~'M, such as less than 10~M, e.g. less than 5 X 10-5M, such as less than 10-5M, e.g. less than 5 X 10-6M, such as less than 10-sM, e.g. less than 5 X 10-'M, such as less than 10-'M, e.g. less than 5 X 10-8M, such as less than 10-$M, e.g. less than 5 X 10-9M, such as less than 10-9M, e.g. less than 5 X 10-'°M, such as less than 10-'°M, e.g.
less than 5 X 10-"M, such as less than 10-~~M, e.g. less than 5 X 10-~2M, such as less than 10-'2M, e.g.
less than 5 X 10-'3M, such as less than 10-'3M, e.g. less than 5 X 10-'4M, such as less than 10-'4M, e.g. less than 5 X 10-'5M, or less than 10-'5M. Binding .constants can be determined using methods well-known in the art, such as ELISA (e.g. as described in Orosz and Ovadi (2002) J. Immunol. Methods 270:155-162) or surface plasmon resonance analysis.
In preferred embodiments, the antibody of the invention is capable of binding an intact Aspergillus fumigatus cell, i.e. capable of binding a living or a dead Aspergillus cell which has maintained its structural integrity, preferably a cell that has maintained the integrity of the plasma membrane (i.e. wherein the plasma membrane is not permeabilised). Binding of antibodies to intact cells can e.g.
be tested as described in the Examples herein.
In further preferred embodiments, the antibody of the invention, or at least an Fab fragment thereof, is capable of reducing the adhesion of Aspergillus fumigatus conidia to lung epithelia in an in vitro assay set-up as described herein in the Examples, preferably reducing said adhesion with at least 20%, such as at least 40%, e.g. at least 60% or at least 80%.
Furthermore, it is preferred that the antibody of the invention, or at least an Fab fragment thereof, is capable of reducing the germination of Aspergillus fumigatus conidia in an in vitro assay set-up as described herein in the Examples, preferably reducing said adhesion with at least 20%, such as at least 40%, e.g. at least 60% or at least 80%.
The antibodies of the invention are capable of specifically recognising and binding an Aspergillus fumigatus target polypeptide selected from the group of SEQ ID
N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:6 and SEQ ID N0:36. 'specifically' is, in this content, not intended to mean absolute specificity. Thus, 'species-specific' is used when it is intended to indicate that the antibody cannot bind to homologous polypeptides from other fungi.

In some embodiments, the antibody of the invention is, in addition to being capable of binding an Aspergillus fumigatus polypeptide, capable of binding a homologous polypeptide from another fungus. E.g. in these embodiments, the antibody of the invention is further capable of binding a homologous polypeptide, wherein the homologous polypeptide has a sequence identity of 39% or more, such as 42% or more, e.g. 48% or more, such as 68% or more, e.g. 80% or more, such as 90% or more, to a polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID N0:36), Cssl (SEQ ID N0:1), hydrophobin (SEQ ID
N0:2), GAPDH-B (SEQ ID N0:3), and catalase A (SEQ ID N0:6). Preferred are antibodies that are capable of binding a homologous polypeptide originating from one of the following species:
- an Aspergillus species, such as Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzea, - Neurospora crassa, - Saccharomyces cerevisiae, - a Candida species such as Candida albicans, - a Coccidioides species, such as Coccidioides posadasii, or Coccidioides immitis, - a Cryptococcus species, such as Cryptococcus neoformans var. neoformans, - a Fusarium species, - a Pneumocystis species, - a Penicillium species, - Histoplasma capsulatum.
More preferably, the homologous polypeptide originates from - an Aspergillus species, such as Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzea, - Candida albicans, - Coccidioides posadasii, or - Cryptococcus neoformans var. neoformans.
In one specific embodiment, the antibody of the invention further recognises a homologous polypeptide which also originates from Aspergillus fumigatus, such as the polypeptide of SEQ ID N0:41.
In preferred examples of the type of embodiments described above, said homologous polypeptide is also extracellular. Thus, the antibody capable of binding both an Aspergillus fumigatus polypeptide as well as a homologous polypeptide, will be capable of binding an intact cell of any one or more of the species from which the homologous polypeptide originates, i.e. one of - an Aspergillus species other than Aspergillus fumigatus, such as Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzea, - Neurospora crassa, - Saccharomyces cerevisiae, - a Candida species such as Candida albicans, - a Coccidioides species, such as Coccidioides posadasii, or Coccidioides immitis, - a Cryptococcus species, such as Cryptococcus neoformans var. neoformans, - a Fusarium species, - a Pneumocystis species, - a Peniciflium species, and - Histoplasma capsulatum.
Binding of the antibodies of the invention to homologous polypeptides and/or other intact cells of other fungi can be tested by the method described herein in the Examples or by other standard methods known in the art.
In preferred embodiment, the antibody of the invention is capable of binding one or more amino acid residues comprised within a region of SEQ ID NO:36 that has significant identity to homologous polypeptides from other fungi, such as regions of SEQ ID N0:36 that have identity with a homologous polypeptide of 4,5,6,7,8 or more consecutive amino acids. Most preferably, antibody of the invention recognises an epitope which is entirely consisting of residues comprised within one of these regions of SEQ ID N0:36. In particular the following regions are preferred:
Ser67-Leu71, A1a74-Trp80, Ser191-Arg205, Leu268-Leu273, His292-Pro296, G1u355-I1e360, Asp193-GIu209, Asp193-A1a199, I1e15-Va119, Va175-Trp80, and Pro11-GIu18. Further preferred are antibodies that bind to an epitope of SEQ
ID
N0:36 -which comprises one or more residues of SEQ ID N0:37. Methods for epitope mapping are well known in the art.

In a different set of embodiments, the antibody of the invention is not capable of binding intact cells from one or more another fungi. For instance, in one such embodiment, the antibody is not capable of binding an intact cell of any of - Neurospora crassa, 5 - Saccharomyces cerevisiae, - Candida albicans, - Coccidioides posadasii, or Coccidioides immitis, - Cryptococcus neoformans var. neoformans, or 10 - Histoplasma capsulatum.
More preferably, the antibody of the invention is not capable of binding an intact cell of any of - Aspergillus nidulans - Aspergillus niger 15 - Aspergillus oryzea, - Neurospora crassa, - Saccharomyces cerevisiae, - Candida albicans, - Coccidioides posadasii, or Coccidioides immitis, 20 - Cryptococcus neoformans var. neoformans, or - Histoplasma capsulatum.
In one specific embodiment, the antibody of the invention is species-specific, i.e. not capable of binding homologous polypeptides or intact cells from other fungi than 25 Aspergillus fumigatus.
The invention also relates to pharmaceutical compositions comprising an antibody of the invention and a pharmaceutically-acceptable carrier.
30 Raising antibodies and functional eguivalents In another aspect, the invention relates to a method for raising specific antibodies to a polypeptide selected from the group of polypeptides set forth in SEQ ID NOs:

and 36 in a non-human animal comprising the steps of a. providing a polypeptide selected from the group of polypeptides set forth in SEQ
ID NOs: 1-6 and 36 or a polypeptide selected from the group of the polypeptide fragments as defined in the present application, or a cell expressing any of these polypeptides, , b. introducing a composition comprising said polypeptide or said cell into said animal, c. raising antibodies in said animal, and d. isolating and optionally purifying the antibodies.
In one embodiment of the above method, the polypepti.de that is provided is Cssl (SEQ ID N0:1) or a fragment thereof, or a variant of said polypeptide. In another embodiment of the above method, the polypeptide that is provided is hydrophobin (SEQ ID N0:2) or a fragment thereof, or a variant of said polypeptide. In yet another embodiment of the above method, the polypeptide that is provided is GAPDH-B
(SEQ ID NO:3) or a ffagment thereof, or a variant of said polypeptide. In a yet further embodiment of the above method, wherein the polypeptide that is provided is catalase A (SEQ ID N0:6) or a fragment thereof, or a variant of said polypeptide.
And in an even further embodiment of the above method, the polypeptide that is provided is isopropylmalate dehydrogenase B (SEQ ID N0:36) or a fragment thereof, or a variant of said poiypeptide.
Antibodies include polyclonal antibodies, monoclonal antibodies, human, humanised or chimeric antibodies, single-chain antibodies, and also Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies, hybrids comprising antibody fragments, and epitope-binding fragments of any of the these. The term also includes mixtures of monoclonal antibodies.
In some embodiments, the antibody of the invention is polyclonal. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunised with an antigen, such as one of the extracellular polypeptides identified by the inventors, or a fragment, epitope or variant thereof.
For the production of polyclonal antibodies, host animals can be immunised by injection with the polypeptide supplemented with adjuvants. The antibody titer in the immunised animal can be monitored over time by standard techniques, such as ELISA using immobilised polypeptide. If desired, the antibody molecules can be isolated from the animal, for instance from the blood, and further purified by well-known techniques, such as protein-A chromatography, to obtain the IgG
fraction.

Thus, in a preferred embodiment, the above described method for generating an immune response comprises a step d. of isolating and purifying antibodies generated in said immune response.
In other embodiments, the antibody of the invention is monoclonal. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular epitope, can be obtained by any technique which provides for the production of antibody molecules by continuous cell-lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein ((1975) Nature 256, 497; and U.S. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4, 72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA
80, 2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. A preferred class is IgG 1. The hybridoma producing the monoclonal antibody of this invention can be cultivated in vitro or in vivo.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage-display library) with the polypeptide of interest or a fragment thereof.
Kits for generating .and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene Surf2APTM Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S.
5,223,409; WO 92/18619; WO 91/17271; WO 92/20791 ; WO 92/15679 ; WO
93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al. (1991 ) Bio/Technology 9: 1370-1372; Hay et a1.(1992) Hum. Antibod. Hybridomas 3, 81-85;
Huse et al. (1989) Science 246, 1275-1281; and Griffiths et al. (1993) EMBO J.
12, 725-734.
Additionally, recombinant antibodies, such as chimeric and humanised monoclonal antibodies comprising both human ~~and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.

A chimeric antibody is a molecule in which different portions are derived from . different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., U.S.
4,816,567; and U.S. 4,816,397, which are incorporated herein by reference in their entirety.) Humanised antibodies are antibody molecules from non-human species having one or more complementarity-determining regions from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g.
U.S. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanised monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO
87/02671 ; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; WO 86/01533; U.S. 4,816,567;
European Patent Application 125,023. Better et al. (1988) Science 240:1041-1043;
Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.
Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;
Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559);
Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BiolTechniques 4:214; U.S. Pat.
No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534-1536; Beidler et al. (1988) J. Immunol. 141:4053-4060; and Westin Kwon et al. (2002) Clin. Diagn. Lab. Immunol. 9, 201-204.
In a highly preferred embodiment, the antibody of the invention is a human antibody.
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and kappa light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunised in the normal fashion with a selected antigen, e.g., all or a fragment of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can e.g. be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harboured by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13: 65-93).

For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g.
WO 02/43478; U.S. 5,625,126; U.S. 5,633,425; U.S. 5,569,825; U.S. 5,661,016;
and U.S. 5,545,806. Completely human antibodies which recognise a selected epitope can be generated using a technique referred to as "guided selection".
In this approach a selected non-human monoclonal antibody, e.g. a mouse antibody, is used to guide the selection of a completely human antibody recognising the same epitope (see Jespers et al. (1994) Bio/Technology 12, 899-903).
Highly suitable methods for the production of human monoclonal antibodies have been described in WO 04/035607 (Genmab) and WO 04/043989 (Medarex). Further similar methods have been described in WO 03/017935 (Genmab), WO 02/100348 (Genmab), WO 02/064634. (Medarex) and WO 03/040169 (Medarex).
Antibody fragments which recognise specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to:
F(ab')z fragments which can be produced by pepsin digestion of the antibody molecule and Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al. (1989) Science 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
Antibodies of the invention also include bispecific antibodies having two binding specificities, of which at least one is a specificity for a polypeptide selected from the group of SEQ ID N0:1-6 and 36, preferably selected from the group of SEQ ID
NOs:1-4 and 36.
In preferred embodiments, the antibody of the invention is purified.
Antibody treatment Antibodies can be used for passive immunisation of mammals, preferably human beings, more preferably immunocompromised patients. A treatment with antibodies can be done to cure or to prevent Aspergillus infections. ~ Thus, the invention relates to use of an antibody as defined herein for the manufacture of a medicament, preferably a medicament for the treatment of fungal infections or the prophylactic treatment (prevention) of fungal infections, preferably Asperg%illus infections, such as Aspergillus fumigatus infections. Examples of fungal infections are invasive aspergillosis, aspergilloma, and allergic aspergillosis, such as allergic bronchopulmonary aspergillosis.

Formulated in another way, the invention relates to an antibody as defined herein or a composition as defined herein for use as a medicament. The invention also relates to a method of treatment comprising the step of administering to an individual a 10 pharmaceutically-effective amount of an antibody of the invention as defined herein, and to a medicament for treating Aspergillus infections comprising an antibody of the invention as an active ingredient.
Antibodies of the invention may be mechanistically divided into the following 15 preferred groups:
1. Function-inhibiting antibodies that work as an antifungal (i.e. affect the viability of the fungus, including both fungicidal and fungistatic effects). Such antibodies should be efFective regardless of the immune status of the patient. This category of antibodies includes pathogenesis-inhibiting antibodies, which block a protein 20 required for disease (Adhesion/Invasion), and growth-inhibiting antibodies, which block a protein required for germination andlor sporulation.
2. Opsonising antibodies that are designed to enhance phagocytic killing.
Effectiveness of such antibodies may depend on the immune status of the patient, but it is very well possible that they will enhance phagocytic killing even in 25 compromised patients. Opsonising antibodies also comprise antibodies which enhance clearance by the immune system via complement and phagocytosis.
3. Antibodies conjugated to a therapeutic moiety such as a toxin or fungicidal agent, a g. ricin or radioisotopes, directed against fungal surface components.
Techniques for conjugating a therapeutic moiety to antibodies are well known, see, e.g.
Thorpe 30 et a1.(1982) Immunol. Rev. 62, 119-158. These antibodies should also be effective regardless of the immune status of the patient.
Validation of targets and antibodies can be done by the following methods known in the art:
35 - Invasion assays - test whether invasion of Aspergillus into lung cells is prevented - Adhesion assays - test whether adhesion to and colonisation of lung cells is prevented - Germination assays - test whether growth and germination is prevented - Opsonisation assay - test whether function is inhibited, and clearance is eased - Aggregation assays - test whether clumping is prevented, and whether clearance is eased - Invasive disease animal models - test whether disease is prevented In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising an antibody, e.g. a human monoclonal antibody of the present invention. The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.
The pharmaceutical composition may be administered by any suitable route and mode. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
Formulations of the present invention which are suitable for vaginal administration include. pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches -and inhalants.
The pharmaceutical composition is preferably administered parenterally. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In one embodiment the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion. In one embodiment the antibodies of the invention are administered in crystalline form by subcutaneous injection, cf. Yang et al.
(2003) PNAS, 100(12):6934-6939. - ~ - .
Regardless of the route of administration selected, the compounds of the present invention, which may be used in the form of a pharmaceutically acceptable salt or in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.
Preferably, the carrier is suitable for parenteral administration, e.g. intravenous or subcutaneous injection or infusion. Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surFactants. The pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonicity agents, such as sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
Pharmaceutically-acceptable antioxidants may also be included, for example (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g.
as enumerated above, as required, followed by sterilization microfiltration.
Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
If appropriate, the antibody may be used in a suitable hydrated form or in the form of a pharmaceutically acceptable salt. A "pharmaceutically acceptable salt"
refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al.
(1977) J.
Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
Depending on the route of administration, the active compound, i.e., antibody, and bispecific/multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J.
Neuroimmunol. 7:27). The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
The pharmaceutical compositions can be administered with medical devices known in the art. For example, in a prefer-ed embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in US 5,399,163, US 5,383,851, US 5,312,335, US
5,064,413, US 4,941,880, US 4,790,824, or US 4,596,556. Examples of well-known implants and modules useful in the present invention include: US 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate;
US
4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in 5 liposomes. For methods of manufacturing liposomes, see, e.g., US 4,522,811;
US
5,374,548; and US 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685).
Exemplary targeting moieties include folate or biotin (see, e.g., US 5,416,016 to Low et al.);
10 mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.
153:1038);
antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al.
(1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al.
(1995) Am. J. Physiol. 1233:134), different species of which may comprise the formulations of the inventions, as welt as components of the invented molecules;
15 p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K_ Keinanen; M.L.
Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; LJ. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred 20 embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area, e.g., the site of infection.
The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
In a further embodiment, the antibodies of the invention can be formulated to prevent or reduce their transport across the placenta. This can be done by methods known in the art, e.g., by PEGylation of the antibodies or by use of F(ab')2 fragments. Further references can be made to "Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation. J Immunol Methods. 152:177-190; and to "Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann Allergy Asthma Immunol 74:279-283.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to-formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. Actual dosage levels of the active ingredients in the pharmaceutical compositibns of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
In one embodiment, the antibodies according to the invention can be administered by infusion in a weekly or daily dosage of from 10 to 500 mg/m2, such as of from 200 to 400 mg/m2. Such administration can be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. In another embodiment, the human monoclonal antibodies can be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.
In still another embodiment the human monoclonal antibodies can be administered in a weekly dosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4 to times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. In yet another embodiment, the human monoclonal antibodies can be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
Combination treatment The pharmaceutical composition of the invention may contain one or a combination of antibodies of the invention. Thus, in a further embodiment, the pharmaceutical compositions include a combination of multiple (e.g., two or more) isolated antibodies of the invention which act by different mechanisms.
Treatment of antifungal infections as defined herein, for example passive immunisation with antibodies specifically recognising and binding the extracellular Aspergillus polypeptides as defined herein, can also be combined with any other type of therapy, in particular other antifungal therapy. Thus, an antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with antifungal chemotherapeutics or other therapeutic agents. For instance, treatment with an antibody as defined herein can be combined with treatment with antifungal compounds, such as azoles (e.g.
fluconazole, itraconazole, ketoconazole, miconazole), amphotericin B, flucytosine, or echinocandins, such as Caspofungin. Alternatively, or in addition, treatment with antibodies as defined herein can also be combined with treatment with other antifungal antibodies, for instance antibodies directed against HSP90, such as Mycograb (Matthews et al. (2003) Antimicr. Agents and Chemotherapy 47:2208-2216).
Combination therapy can in some circumstances be more effective than single component therapy. Combination therapy can be particularly useful for patients that suffer from infection with multiple fungal species, for example patients having both a Candida and an Aspergillus infections.
Binding partners and inhibitors of extracellular polypeptides In addition to antibodies, it is of interest to identify other types of binding partners to extracellular polypeptides. Extracellular polypeptides of a pathogenic fungus often interact with the host organism. Any type of binding partner of an extracellular polypeptide may interfere with host-pathogen interaction. Binding partners may thus antagonise the pathogenicity of the fungus.
Identification of binding partners of the extracellular polypeptides set forth in SEQ ID
NO:1-6 and 36, or fragments thereof, is another main aspect of this invention.
This may be done using biochemical or cell-based methods.
Biochemical methods In a main aspect, the invention relates to a method for identifying a binding partner of a polypeptide of the .invention and/or a polypeptide selected from the group of SEQ ID NOs:1-6 and 36, comprising the steps of a. providing a polypeptide of the invention as defined herein or a polypeptide selected from the group of SEQ ID NOs:1-6 and 36, b. contacting said polypeptide with a putative binding partner, and c. determining whether said putative binding partner is capable of binding to said polypeptide.

Thus, in one embodiment of the above method, the polypeptide that is provided is Cssl (SEQ ID N0:1) or a fragment thereof, or a variant of said polypeptide. In another embodiment, the polypeptide that'is provided is hydrophobin (SEQ ID
N0:2) or a fragment thereof, or a variant of said polypeptide. In another embodiment, the polypeptide that is provided is GAPDH-B (SEQ.ID N0:3) or a fragment thereof, or a variant of said polypeptide. In a yet other embodiment, the polypeptide that is provided is catalase B (SEQ ID N0:5) or a fragment thereof, or a variant of said polypeptide. In a further embodiment, wherein the polypeptide that is provided is catalase A (SEQ ID N0:6) or a fragment thereof, or a variant of said polypeptide. In an even further embodiment, wherein the polypeptide that is provided is isopropylmalate dehydrogenase B (SEQ ID N0:36) or a fragment thereof, or a variant of said polypeptide.
In a preferred embodiment of this method of the invention, said polypeptide is selected from the group of SEQ ID NOs:1,2,3,5,6, and 36 such as the polypeptide set forth in SEQ ID N0:1, or the polypeptide set forth in SEQ ID N0:2, or polypeptide set forth in SEQ ID N0:3, or the polypeptide set forth in SEQ ID
N0:5, or the polypeptide set forth in SEQ ID N0:6 or the polypeptide set forth in SEQ ID
N0:36. In other preferred embodiments, an exposed domain, an epitope or a fragment of one of the polypeptides set forth in SEQ ID NOs:1-6 and 36 comprising one or more amino-acid residues of the sequences set forth in SEQ ID NO: 7-34 and 37 is provided in step a. In further preferred embodiments, a fragment selected from the group of SEQ N0:7-34 and 37 is provided, preferably a fragment selected from the group of SEQ ID N0:7-27 and 37, or a variant or fragment of any of the amino-acid sequences set forth in SEQ ID N0:7-27 and 37.
In preferred embodiments of this method, the polypeptide or fragment thereof is provided immobilised on a solid support, such as e.g. a column or microtiter plate, and, after the contacting step, it is determined whether or not the putative binding partner has bound to the solid support. Immobilisation of the polypeptide or fragment thereof may be through direct binding to the solid support, or through indirect binding e.g. using a specific antibody. In preferred embodiments, a washing step is performed between the contacting step and the determination step, in order to improve the specificity of detection. In further preferred embodiments, the putative binding partner is labelled. The putative partner may be labelled before the contacting takes place. Alternatively, labelling may also be performed after the contacting step. Furthermore, in some embodiments of this method, immobilisation may be performed after the polypeptide or fragment thereof has been bound to the binding partner. In preferred embodiments, the method is repeated for a plurality of 5 putative binding partners. Putative binding partners include host-derived molecules.
Alternatively, a binding partner of a polypeptide of the invention or of a polypeptide selected from the group of SEQ ID N0:1,2,4,5, 6 and 36 may be identified as follows: purified host membranes are electrophoretically separated, blotted over to a 10 membrane and incubated with the polypeptide of interest or fragment thereof.
Binding can then be detected using antibodies specific for the polypeptide of interest or fragment thereof. The host binding partner to which the polypeptide or fragment thereof has bound can subsequently be identified by elution from the blot and subsequent analysis by mass spectrometry, or by any other technique known in the 15 art.
If the binding partner of an extracellular polypeptide of a pathogenic organism is a host-derived molecule, then such an interaction between the extracellular polypeptide and the host may be important for the virulence of the fungus.
20 Compounds that interfere with the interaction of the extracellular polypeptide and the host binding partner may thus be suitable for prevention or treatment of fungal infections. Accordingly, another method of the invention relates to a method of identifying an inhibitor of the interaction of an extracellular Aspergillus polypeptide selected from the group of SEQ ID N0:1-6 and 36 or fragment thereof with a host-25 derived binding partner comprising the steps of:
a. providing a polypeptide selected from the group of SEQ ID N0:1-6 and 36, or a fragment thereof, b. providing a host-derived binding partner of said polypeptide c. contacting said polypeptide with said host-derived binding partner in the 30 absence of a putative inhibitor of said interaction d. contacting said polypeptide with said host-derived binding partner in the presence of said putative inhibitor e. determining whether the strength of the binding of said polypeptide to said host-derived binding partner resulting from step d. is reduced as compared to that 35 resulting from step c.

In some embodiments, step c. and d. may be performed in two different sample compartments. In other embodiments, step d. may be performed by adding the putative inhibitor to the mixture of step c. In preferred embodiments, a fragment selected from the group of SEQ ID N0:7-34 and 37 is provided in step a. In other preferred embodiments, the polypeptide of SEQ ID N0:1,2,3,5, 6, or 36 is provided.
In further preferred embodiments, the method is repeated for a plurality of putative inhibitors. Of further particular interest are binding partners that inhibit an activity of an extracellular polypeptide. Such activity may be enzymatic activity, transport activity, or any type of other biochemical or cellular activity, preferably enzymatic activity. Inhibitors of IMDH B, GAPDH, enolase or catalase may be screened for using known biochemical assays of the enzymes, such as the catalase assay kit of CALBIOCHEM, cat. no. 219263, and e.g. the assays described in Pirrung et al.
(1996) J Org Chem 61, 4527-4531; Bartolini et al. (2003) J. Chromatogr. 987, 340; Lal et a1.(1991) Plant Mol. Biol. 16, 787-795; Machida et al. (1996) Biosci Biotechnol Biochem 60, 161-163; and Maitra and Lobo (1971) J Biol Chem 246, 475-88.
Cell based methods Reducing the level of an extracellular polypeptide, by deletion or disruption of the structural gene for it or by down-regulating gene expression (see below), may affect a fungal cell. The cell may become more sensitive to cytotoxic compounds.
Especially for extracellular polypeptides, a reduction of their level may affect the function of the cell's exterior parts, such as the plasma membrane or cell wall, in preventing compounds of entering the cell. Thus, reduction of the level of an extracellular polypeptide can make a cell more 'permeable' for various compounds.
An aspect of the present invention relates to a method for identifying a compound with anti-Aspergillus fumigatus activity comprising the steps of a. providing a sensitised cell which has a reduced level of a polypeptide selected from the group of SEQ ID N0:1-6 and 36, and b. determining the sensitivity of said cell to a putative inhibitor, for instance by a growth assay. .
In a preferred embodiment, a sensitised cell which. has a reduced level of a polypeptide selected from the group of SEQ ID N0:1,2,3,5, 6, and 36 is provided in step a. In an even more preferred embodiment of the method, a sensitised cell which has a reduced level of Cssl (SEQ ID N0:1), . hydrophobia (SEQ ID N0:2), GAPDH (SEQ ID N0:3), catalase A (SEQ ID N0:6), or isopropylmalate dehydrogenase B (SEQ ID N0:36) is provided.
The rationale behind this approach is that a cell with a lower level of the extracellular polypeptide will exhibit increased sensitivity to cytotoxic compounds, allowing identification of antifungal compounds with low potency that are missed when using wild-type cells for the assay. Compounds identified by this method will be often need to be modified in order to improve potency. This can be done by chemical modification. In preferred embodiments, the method is repeated for a plurality of putative binding partners.
Inhibition of the activity of an extrace(lular polypeptide may affect the viability (i.e.
survival, growth and/or proliferation) of the fungus. Of particular interest is inhibition of extracellular polypeptides that are essential for viability of A.
fumigatus.
Essentiality of an Aspergillus gene may be ~ investigated e.g. using regulatable expression as. described in WO 02/086090. Inhibitors of essential extracellular polypeptides may not need to enter the fungal cell to be able to affect its viability.
Thus, generally fewer requirements are posed on the structure of an inhibitor of essential extracellular target polypeptide than on .an inhibitor of an intracellular target, to be effective as an antifungal agent.
Thus, the invention relates to a method for identifying an inhibitor of an extracellular Aspergillus fumigatus polypeptide selected from the group of SEQ ID NO:1-6 and comprising the steps of a. providing two cells which differ in the level of a polypeptide selected from the group of SEQ ID N0:1-6 and 36, b. determining the sensitivity of said cells to a putative inhibitor, for instance by a growth assay, and c. determining whether said two cells are differently affected by the presence of said putative inhibitor.
The rationale behind this approach is that the viability of a cell with a lower activity of the essential polypeptide will be more affected by an inhibitor of the polypeptide than the viability of the cell with a higher level. If the two cells are differently affected, this is an indication that the inhibitor acts on the target or in the same biochemical pathway. In a preferred embodiment of the method, said polypeptide is Cssl (SEQ
ID N0:1), GAPDH (SEQ ID N0:3), catalase A (SEQ ID N0:6), or isopropylmalate dehydrogenase B (SEQ ID N0:36).
In some embodiments of the method, the two cells with different activity of the polypeptide of interest are a wild-type cell (or other cell with wild-type activity of the gene of interest) and a sensitised cell with a reduced activity of the polypeptide of interest. In some embodiments, the different or reduced level in the sensitised cell can be a different or reduced expression level of the gene of interest (resulting in a different or reduced copy number of the polypeptide). This can be accomplished by putting the gene under control of a regulatable promoter or by regulatable expression of an antisense RNA which inhibits translation of an mRNA encoding the essential polypeptide. In other embodiments, the different or reduced activity can be a different or reduced activity of the polypeptide of interest, e.g. due to a mutation, such as a temperature-sensitive mutation. In preferred embodiments, the method is repeated for a plurality.of putative binding partners.
Suitable ways of generating sensitised cells and of using these in screening for inhibitors have been described in WO 02/086090. Sensitised cells may be obtained by growing a conditional-expression A. fumigatus mutant strain in the presence of a concentration of inducer or repressor which provides a level of a gene product required for fungal viability such that the presence or absence of its function becomes a rate-determining step for viability. A number of suitable regulatable promoters for constructing such conditional-expression mutants of Aspergillus is described in WO 02!086090, page 76, line 34 through page 85, line 4. For example, if the regulatable promoter is repressed by tetracycline, the conditional-expression Aspergillus fumigatus mutant strain may be grown in the presence of partially repressing concentrations of tetracyline. The sub-lethal concentration of inducer or repressor may be any concentration consistent with the intended use of the assay.
For example, the sub-lethal concentration of the inducer or repressor may be such that growth inhibition is at least about 10%, such as at least about 25%, e.g.
at least about 50%, such as at least about 75%, e.g. at least 90%, such as at least 95%.
Similarly, the virulence or pathogenicity of cells exposed to a candidate compound which express a rate-limiting amount of a gene product required for virulence or-pathogenicity may be compared to the virulence or pathogenicity of cells exposed to the candidate compound in which the level of expression of the gene product required for virulence or pathogenicity is not rate-limiting. In such methods, test animals are challenged with the conditional-expression A. fumigatus mutant strain and fed a diet containing the desired amount of tetracycline and the candidate compound. Thus, the conditional-expression mutant strain infecting the test animals expresses a rate limiting amount of a gene product required for virulence or pathogenicity (i. e. the conditional-expression mutant cells in the test animals are sensitised). Control animals are challenged with the conditional-expression mutant strain and are fed a diet containing the candidate compound but lacking tetracycline.
The virulence or pathogenicity of the conditional-expression A. fumigatus mutant strain in the test animals is compared to that in the control animals. For example, if a significant difference in growth is observed between the sensitised conditional-expression mutant cells (i. e. the cells in animals whose diet included tetracycline) and the non-sensitised cells (i. e. the conditional-expression mutant cells animals whose diet did not include tetracycline), the candidate compound may be used to inhibit the virulence or pathogenicity of the organism or may be further optimised to identify compounds which have an even greater ability to inhibit the virulence or pathogenicity of the organism. Virulence or pathogenicity may be measured using the techniques known in the art.
In another embodiment of the cell-based assays of the present invention, sensitised cells are obtained by reduction of the level activity of a polypeptide required for fungal viability using a mutation, such as a temperature-sensitive mutation, in the polypeptide. Growing such cells at an intermediate temperature between the permissive and restrictive temperatures produces cells with reduced activity of the gene product. It will be appreciated that the above method may be performed with any mutation which reduces but does not eliminate the activity or level of the gene product which is required for fungal viability. This approach may also be combined with the conditional-expression approach. In this combined approach, cells are created in which there is a temperature-sensitive mutation in the gene of interest and in which this gene is also conditionally-expressed.
When screening for inhibitors of an essential polypeptide, growth inhibition can be measured by directly comparing the amount of growth, measured by the optical density of the culture relative to uninoculated growth medium, in an experimental sample with that of a control sample. Alternative methods for assaying cell proliferation include measuring green fluorescent protein (GFP) reporter construct emissions, various enzymatic activity assays, and other methods well known in the 5 art. Other parameters used to measure viability include e.g. colony forming units.
The above method may be performed in solid phase, liquid phase, a combination of the two preceding media, or in vivo. Multiple compounds may be transferred to agar plates and simultaneously tested using automated and semi-automated equipment.
10 Cell-based assays of the present invention are capable of detecting compounds exhibiting low or moderate potency against the target molecule of interest because such compounds are substantially more potent on sensitised cells than on non-sensitised cells. The effect may be such that a test compound may be two to several times more potent, e.g. at least 10 times more potent, such as at least 20 times 15 more potent, e.g. at least 50 times more potent, such as at least 100 times more potent, e.g: at least 1000 times more potent, or even more than 1000 times more potent when tested on the sensitised cells as compared to non-sensitised cells.
A mutant A. fumigatus strain that overexpresses an extracellular polypeptide can 20 also be used to identify a compound that inhibits such a polypeptide. If the compound is cytotoxic, overexpression of the target polypeptide can make cells more resistant. Thus, the invention also relates to a method for identifying an inhibitor of an extracellular Aspergiilus polypeptide selected from the group of SEQ
ID N0:1-6 and 36 comprising the steps of 25 a. providing two cells which differ in the activity of a polypeptide selected from the group of SEQ ID N0:1-6 and 36, wherein one cell contains a substantially wild-type copy number of said polypeptide and the other cell contains higher than wild-type activity of said polypeptide b. determining the sensitivity of said cells to a putative inhibitor, for instance by a 30 growth assay, and c. determining whether or not said two cells are differently affected by the presence of said putative inhibitor.
Preferably, the two cells differ in the activity of a polypeptide selected from the group 35 of SEQ ID N0:1,2,3,5,6, and 36, such as the polypeptide of SEQ ID N0:1, or the polypeptide of SEQ ID N0:2, or the polypeptide of,SEQ ID N0:3, or the polypeptide of SEQ ID N0:5, or the polypeptide of SEQ ID N0:6 or the polypeptide of SEQ ID
N0:36 As also overexpression of polypeptides that are not the cellular target of an inhibitor can make cells resistance to an inhibitor, inhibition of the target polypeptide of interest by said inhibitor will need to be verified by other means, such as e.g. a biochemical assay.
Overexpression may be achieved using strong promoters, e.g. the A. niger Pgla A
promoter, the A. nidulans promoter alcA(p), or the constitutive promoters PGK-(phosphoglycero-kinase), GPD-(glucose-6-phosphate dehydrogenase) or ENO
(enolase) promoters or regulated promoters such as ADH2, PH05, GAL1, GAL10, CUP1 or HSP70. Other useful promoters include the ones described in Adams et al.
(1998) Microbiol. Mol. Biol. Rev. 62, 35-54; Adams et al. (1988) Cell 54, 353-362;
Andrianopoulos and Timberlake (1991) Plant Cell 3, 747-748; Gwynne et al.
(1987) Gene 51:205-216; Lockington et al. (1985) Gene 33:137-149.
In addition to inhibitors of a biochemical or other cellular activity of an extracellular polypeptide, the cellular methods described above may identify compounds that reduce the expression level of a target, and thereby its copy number, e.g. by interfering with gene regulation.
In preferred embodiments of the any of the cell-based- or biochemical methods for identifying binding partners or inhibitors, the method is repeated for a plurality of candidate compounds.
In a further aspect, the invention relates to the mutant A. fumigates strains used in the cell-based methods described herein, such as strains in which the gene encoding the extracellular polypeptide is placed under the control of a heterologous regulatable promoter, strains carrying temperature-sensitive alleles of the extracellular polypeptides, and strains overexpressing the extracellular polypeptides.
Other methods of interfering with fungal growth by targeting essential extracellular ' polypeptides include suppression of gene expression using specific antisense molecules, such antisense RNA or DNA, and using ribozyme molecules specific for mRNA encoding the essential extracellular polypeptides.
Diagnosis In a further main aspect, the invention relates to a method of diagnosing Aspergillus infection comprising the steps of a. providing a sample from an individual, b. contacting said sample with an indicator moiety specific for a polypeptide of the invention as defined herein, or specific for a polypeptide selected from the group of Cssl (SEQ ID N0:1), hydrophobia (SEQ ID N0:2), GAPDH (SEQ ID N0:3), catalase A (SEQ ID NO:6) and isopropylmalate dehydrogenase B (SEQ ID
N0:36), and c. determining whether a signal has been generated by the indicator moiety.
In a preferred embodiment of this method, the polypeptide of the invention is a polypeptide selected from the group of SEQ ID N0:1,2,3,5,6, and 36 such as the polypeptide of SEQ ID N0:1, or the polypeptide of SEQ ID N0:2, or the polypeptide of SEQ ID N0:3, or the polypeptide of SEQ ID N0:5, or the polypeptide of SEQ
ID
N0:6, or the polypeptide of SEQ ID N0:36. In other preferred embodiments of this method, the indicator moiety is specific for a fragment selected from the group of fragments set forth in SEQ ID N0:7-34 and 37.
The indicator moiety is capable of binding to the target polypeptide of interest. In preferred embodiments, said indicator moiety is or comprises an antibody.
Antibodies directed against a target extracellular polypeptide or fragment thereof can be used to detect the polypeptide in order to evaluate the abundance and pattern of expression of the polypeptide under various environmental conditions, in different morphological forms (mycelium, yeast, spores) and stages of an organism's life cycle.
Preferably, however, antibodies directed against a target polypeptide or fragment thereof can be used diagnostically to monitor levels of a target gene product in the tissue of an infected host as part of a clinical testing procedure, e. g., to, for example, diagnose a patient for Aspergillus infection or determine the efficacy of a given treatment regimen. In particular Cssl is of considerable interest for diagnostic purposes. It appears that the protein is unique to A. fumigates as no significant homologues to the protein have yet been detected through the use of immunological or sequence-based procedures. Furthermore, the cell-surface and secreted nature of the protein is also favourable feature from the point of view of detecting the protein in human fluids.
Detection using antibodies can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase ; examples of suitable prosthetic group complexes include Streptavidinibiotin and avidinibiotin ; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin ; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ~251~ 1311 ssS or 3H.
Various diagnostic assays employing the above indicator moieties can be set up to test samples for Aspergillus. Exemplary assays are described in detail in Antibodies:
A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: countercurrent immuno-electrophoresis (CIEP), radioimmunoassays, radioimmunoprecipitations, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, inhibition or competition assays, and sandwich assays, immunostick (dipstick) assays, simultaneous immunoassays, immunochromatographic assays, immunofiltration assays, latex bead agglutination assays, immunofluorescent assays, biosensor assays, and low-light detection assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988.

Detailed description of the tables and figures Table 1 shows the sequences of the peptides that were identified in the AfC
fractions. Shown are, for each of the polypeptides (Cssl, Hydrophobin, GAPDH, enolase, ,catalase (A+B) and isopropylmalate dehydrogenase B), the peptides that were identified in the three different fractions (difPusate, cell-surface-exposed, and cell wall), and the sequences of the peptides that were used for antibody production.
X~ is serine or alanine, X~ is leucine or isoleucine.
Table 2 shows some biochemical characteristics for the full-length Cssl polypeptide and for its N-terminal and its C-terminal half. 'MW' indicates molecular weight.
'Residues' indicates the number of residues.
Table 3. Sequence of peptides chosen for the production of multiple antigenic peptides and antisera against selected target proteins Table 4. Analysis of the ability of anti-IMDH B IgG to bind the surface of clinical isolates.
Figure 1 shows the predicted full-length polypeptide sequences of Cssl (A) (SEQ ID
N0:1), hydrophobin (B) (SEQ ID N0:2), GAPDH-B (C) (SEQ ID N0:3), enolase (D) (SEQ ID N0:4), catalase B (E) (SEQ ID N0:5), catalase A (F) (SEQ ID NO:6), and isopropylmalate dehydrogenase B (G) (SEQ ID N0:36).
Figure 2 shows the predicted antigenicity indices of Cssl (A) and hydrophobin (B) residues, predicted according to Jameson and Wolf (1988).
Figure 3. Alignment of the predicted protein sequences for GAPDH-A (AfA), GAPDH-B (AfB), and GAPDH-C (AfC) from Aspergilius fumigatus. Residues that are identical in all three proteins are presented on a dark background.
Peptides of GAPDH-B that have been identified by MS have been underlined.
Figure 4. Silver stained SDS-PAGE of AfM lysate samples (lanes 2 and 7) added to affigel immunoaffinity columns prepared with (lanes 2-5) or without (lanes 6-10) anti-AfM IgG. Lanes 1 and 6 indicate the samples that were eluted from these columns after extensive washing steps (lanes 3-5 and 8-10). The proteins that are eluting at approximate weights of 97, 64 and 51 were all identified as IMDH B via mass spectrometry.
Figure 5. Phase contrast (A) and immunofluorescent micrographs (B) of AfM
5 labelled with anti-AfM sera (C) and with secondary antibody alone (D).
Figure 6. Adhesion of AfC to lung epithelia demonstrating the ability of anti-AfM Fab fragments to reduce adhesion of AfC to A549 cells.
10 Figure 7. A silver stained SDS-PAGE illustrating the steps involved in purification of IMDH B. Lane 1, MW standard; lane 2, recombinant IMDH B purified via a nickel sepharose column; lame 3, recombinant protein following purification via an S200 gel filtration column; lane 4, as with lane 4 but ten times the quantity of protein is added.
15 Figure 8. Coomasie blue stained SDS-PAGE indicating the nickel sepharose purified recombinant IMDH B that was used to immunize rabbits (1); and the reactivity of preimmune serum (2), the first bleed post immunization (3) and the second bleed post immunization against the recombinant protein as detected via Western blotting.
20 Figure 9. Immunofluorescent micrographs of AfC and AfM from ATCC 46640 stained with Pre and Post immune IgG isolated from a rabbit immunized with IMDH
B.
Figure 10. Pre-incubation of AfC with anti-IMDH B reactive post immune Fab 25 fragments reduces AfC adhesion to A549 cells.
Figure 11. Pre-incubation of rIMDH B with A549 cells, followed by washing, reduces subsequent adherence of AfC in a linear manner.
30 Figure 12. Incubation of AfC in the presence of anti-IMDH B sera and normal complement (sample 2) results in reduced germination as compared to samples incubated in the presence of preimmune or unrelated sera (samples 5 and 8, respectively).

Figure 13. Incubation of AfC in the presence of anti-IMDH B IgG and normal complement results in reduced germination as compared to samples incubated in the presence of preimmune or anti-KLH IgG.
Figure 14. An alignment of IMDH B 1 versus IMDH B 2.
Figure 15. Immunofluoi-escent microscopy analysis of A. fumigatus ATCC 46640 performed with antisera raised against MAP molecules. AfC and AfM were stained with both pre- and post-immunisation sera.
Figure 16. Immunofluorescent microscopy analysis perFormed with a clinical isolate using antisera raised against GAP-B-2. AfC were stained with both pre- and post immunisation sera.
Figure 17. Western blot experiments illustrating the detection of (A) recombinant enolase with anti-his (lane 1 ) and anti-enolase (lane 3; ENO-2, see Table 3) antibodies, but not with pre immune serum from the animal used to produce anti-enolase sera (Lane 4); and (B) native enolase in the cell membrane of AfC by anti-enolase (ENO-2, see Table 3) antibodies (lane 6).
Figure 18. An alignment of part of SEQ ID N0:36 with a homologous sequence from Candida albicans, derived from publicly available nucleotide sequences (contig19-10262 in the Ca-Assembly19.contigs).
Figure 19. An alignment of part of SEQ ID N0:36 with Aspergillus nidulans homolog (ACCESSION: AnrP4374925 - DESCRIPTION: LE3B ASPNG 3-isopropylmalate dehydrogenase B (Beta-IPM dehydrogenase B) (IMDH B) (3-IPM-DH B) [Aspergillus nidulans FGSC A4] DBXREF: gi~40744045~gb~EAA63227.1) Figure 20. An alignment of part of SEQ ID N0:36 with Aspergillus oryzae homolog (ACCESSION: AnrP3711474 - DESCRIPTION: hypothetical protein [Aspergillus oryzae] DBXREF: gi~27901558~dbj~BAC55906.1 ).

Figure 21. An alignment of part of SEQ ID N0:36 with Aspergillus nidulans homolog (ACCESSION: AnrP4379986 - DESCRIPTION conserved hypothetical protein [Aspergillus nidulans FGSCA4] DBXREF: gi~40741202~gb~EAA60392.1).
Figure 22. An alignment of part of SEQ ID N0:36 with a Coccidioides posadasii homolog: TIGR 222929/contig 1772 C. posadasii C735.
Figure 23. An alignment of part of SEQ ID N0:36 with a Cryptococcus homolog.
Ref.nr.: chr01b.b3501.031220.c11.
Figure 24. ClustalW of IMDH B homologs from figures 18-22.
Figure 25. Continuation of figure 24.
Examples Example 1. Identification of peptides in extracts of A. fumigates.
A number of protein purification procedures were used to facilitate identification of A.
fumigates proteins that are secreted, cell-surFace exposed or cell-wall associated.
Proteins were then identified from these extracts via mass spectrometry techniques.
Culture of A. fumigates. A, fumigates conidia (AfC) of strain NCPF 2140 or ATCC
46640 were routinely prepared by inoculation of malt agar plates with AfC and subsequent growth at 30°C for 10 days.
Preparation of a diffusible extract from A. fumigates conidia. A. fumigates Diffusate (AfD) was routinely prepared as follows. AfC (2 x 108) were added to water (0.5 ml) containing protease inhibitors (Roche, cat. no. 1 697 498) and the mixture was vortexed and then sonicated to solubilise the AfC. The resultant solution was incubated for 1 hour at 37°C, with shaleing. AfD was then separated from washed spores by passage through a 0.2 ~,m filter, or, by centrifugation (3000 x g) of washed spores and passage of the AfD through a 0.2 ~.m filter.
Preparation of surface-exposed protein extracts from A. fumigates conidia.
Washed AfC (2.0 x 10'°) were resuspended in 1 ml of PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) containing a reducing agent (10 mM Tris 2-carboxy-ethyl phosphine (TCEP)) and incubated for 20 min at room temperature. AfC were pelleted by centrifugation (20000 x g, 30 min) and washed in PBS to remove TCEP before being resuspended in a trypsin-solution (seq. grade modified porcine trypsin, Promega cat. no. V5111, 20 p.g/ml PBS) and incubated for 30 min at room temperature. AfC were then removed by centrifugation and filtration.
To get rid of any conidia in the supernatant, the supernatant was purified using a YM-10 column (from Millipore, cat. no. 4206, 5000xg, 4°C for 30 min.) and the supernatant was incubated over night at 37°C with shaking at 40 rpm.
The supernatant was concentrated using a SpeedVac concentrator, 1 p,1 was added to ~,I 5% formic acid, and the resultant solution was analysed via mass spectrometry.
Preparation of cell wall extracts from A. fumigatus conidia. AfC solutions (20 ml; 1.8 x 108 conidia/ml) were prepared in both PYG (C rich) (Peptone-yeast extract and glucose: 0.1 % peptone, 0.1 % g yeast extract and 0.3% glucose) and HBSS (C
poor) (HANKS 1X from Gibco, Invitrogen (cat. no. 24020-083)) media. These solutions were vortexed for 3 min, sonicated for 5 min and then incubated for 4 hours at 37°C
with shaking (160 rpm). AfC were pelleted by centrifugation (6000 x g, 30 min) and the supernatant from the HBSS incubation was collected and passed through a 0.2 ~.m filter. The supernatant from the PYG was discarded. Both AfC pellets were washed with 5 ml HBSS and pelleted as before. To each pellet 1 ml of lysis buffer (2% Triton, 1 % SDS, 10 mM Tris (pH=2), 1 mM EDTA, 100 mM NaCI, 1 proteinase inhibitor tablet (Roche, cat. no. 1 697 498) and approx. 500 p.1 glass beads (200-300 microns) were added. The resultant solution was then incubated in a water bath sonicator for 40 min, vortexed for 30 min, chilled on ice for 5 min, and finally vortexed for another 30 min. Glass beads were then removed from the sample and conidial walls were sedimented by centrifugation at 1200 x g for 10 min. The supernatant was removed and stored for future use.
Conidial wall enriched pellets were washed three times with 1 ml of cold distilled water, resuspended in 250 p,1 of 2% (w/v) SDS, 1 % (w/v) 2-mercaptoethanol solution and boiled for 5 min. The resultant solution was centrifuged (10,000 x g for 15 min), the supernatant was transferred to a new tube, and added to 1 ml ice-cold acetone prior to an overnight incubation at -30°C. Precipitated proteins were pelleted (20,810 x g for 45 min) and dried in a SpeedVac for 15 min to remove residual acetone.

Pellets were resuspended in ddH20, and proteins were separated -on an SDS-PAGE according to standard procedures. Resultant gels were then visualised via silver staining Analysis of A. fumigatus protein extracts by mass spectrometry. Analysis of ~
A.
fumigatus proteins separated by SDS-PAGE was performed as follows. Fragments of SDS-PAGE gels, corresponding to specific protein bands, were extracted and placed in sodium bicarbonate solution (50 mM NH4HC03). These gel plugs were then washed twice with 50 mM NH4HC03 in 50% ethanol for 30 min and dehydrated by incubation in 96% ethanol for 10 min. Reduction and alkylation was performed by incubating in reducing solution (50 mM DTT, 50 mM NH4HC03) for 45 min at 56°C
followed by a 30 min room temperature incubation in alkylation solution (55 mM
iodoacetamide, 50 mM NH4HC03) in the dark. Two cycles of washing and dehydration were then performed prior to the addition of 10 p.1 trypsin solution (12.5 ng/p.l trypsin in 50 mM NH4HC03 (seq. grade modified porcine trypsin, Promega batch no. V511X 14755007)). After 15 min an additional 20 p,1 of sodium bicarbonate solution was added and the digests were incubated overnight at 37°C.
Samples were then extracted twice by 30 min incubations, with shaking, iri 3 p.1 of 20%
trifluoroacetic acid, and 20 p.1 of a solution containing acetonitrile (10%) and trifluoroacetic acid (1 %). Both extracts were pooled, dried down, and resuspended in 9% of 5% formic acid prior to analysis via LC-MS.
Peptide and fragment mass tolerance was set to 200 ppm and 0.5 Da, respectively.
Search parameters were adjusted to include oxidation of Met, the addition of alkyl of polyacrylamide groups to Cys, and trypsin was allowed to miss one cleavage site per peptide.
Search parameters for analysis of cell surface peptide fragments were adjusted to include oxidation of Met, trypsin was allowed to miss one cleavage site on each peptide; and, peptide and fragment mass tolerance was set to 100 ppm and 0.3 Da, respectively.
Following the identification of a peptide sequence, a TBLASTN was performed against A. fumigatus shotgun sequences in the public domain. This' identified all shotgun sequences capable of encoding the peptide fragment. These shotgun sequences were then used to extract all other shotgun sequences that shared regions of homology of at least 40 by in length with no less than 90%
identity. All appropriate shotgun sequences were then formed into a contiguous sequence using Seqman. Resulting contigs were submitted to a GenScan search using maize, 5 arabidopsis, and human parameters. Output predicted protein sequences were then compared with the encoding nucleotide sequence and with the sequences of protein homologues to facilitate the prediction of a potentially more accurate protein sequence. Resulting predicted nucleotide and peptide sequences were then entered into appropriate in-house databases and MASCOT searches were then rerun 10 against a database containing these newly predicted proteins. For isopropylmalate dehydrogenase B, mismatches were found between the peptide found by peptide sequencing and the corresponding polypeptides predicted from the nucleotide sequences in the database. The mismatches may be due to differences between strains due to mutation or to sequencing errors. Furthermore, an MS instrument 15 does not differentiate between a leucine and an isoleucine. Mutations in this region may have significant structural implications and alter the thermostability of the enzyme, as has been described for a homologous enzyme from Thermos thermophilus (Qu et al. 1997 Protein Eng. 10, 45-52).
20 The peptides identified in the Diffusate, Cell-surface exposed, and Cell-wall fractions are shown in Table 1. The corresponding predicted protein sequences are given in Figure 1. SEQ ID N0:3i3 and SEQ ID N0:39 are predicted polynucleotide sequences encoding isopropylmalate dehydrogenase B (SEQ ID N0:36).
25 Peptides from both hydrophobin and the hypothetical protein were identified in all three fractions indicating both to be cell-wall-associated proteins that are exposed on the surface of the AfC while also being secreted/released into the surrounding milieu. Based on these data we propose to name the newly identified, former hypothetical, protein Conidial Surface and Secreted protein I, Cssl. It was also 30 interesting to note the presence of GAPDH in the AfD and cell wall;
.enolase in the AfD; IMDH B in the cell-surface-exposed fraction and of cell wall located, and surface exposed variants of catalase. Since the procedures used to purify and identify these peptides are biased for proteins of high abundance, one can also conclude that they are expressed in relatively high copy numbers.

Example 2. Bioinformatic analyses SignaIP predictions were performed using the parameters recommended for a eukaryotic protein, while Antigenicity index studies were performed using the default parameters determined by DNAStar. BLAST searches were performed using default parameters.
Analysis of Cssl for the presence of a signal peptide. The group who reported the original hypothetical sequence predicted an N-terminal signal peptide of 24 residues (NCBI entry CAD29600. Protein AfA35G10.07). However, a repeat of these studies using the SignaIP program with default parameters (Nielsen et al. (1997) Protein Engineering 10, 1-6) indicates the presence of a 47-residue signal peptide with predicted signal cleavage occurring between A47 and R48.
Analysis of the predicted protein sequence of Cssl.
A brief overview of the sequence of this protein reveals the two most abundant residues to be E and Q, which comprise 9.62% and 8.64%, respectively, of all residues in the protein. A closer analysis revealed that 67% of charged residues (D, E, K, R) are located in the C-terminal half of the protein (see Table 2), and 62% of hydrophobic residues (A, I, L, F, W, V) in the N-terminal half.
The sequence of Cssl was analysed via the antigenicity index programme of Jameson and Wolf (1988). This programme predicted the C-terminal half of the protein to be most antigenic (see Figure 2).
BLAST analysis of Cssl revealed the absence of a protein with high homology.
However, a number of proteins displayed low, yet significant, levels of homology.
One such protein, ORF73 of Human herpesvirus 8, is the Latency associated nuclear antigen (LAN/LANA) that is used as a marker for Kaposi's sarcoma. It displays 26% identity and 46% similarity to the C-terminal half of Cssl. This region of LANA is rich in Q and E repeats and is located in the middle of the protein.
It has been suggested that similar regions of acidic repeats often function in transcriptional activation in viral and cellular transcription factors (Struhl, 1995, Annu.
Rev. Genet.
29, 651-674). LANA has been shown to be capable of modulating both viral and cellular gene expression (Renne et al., 2001, J. Virol. 75, 458-468).

GAPDH sequences. An attempt to construct a gene sequence for this protein revealed the presence of at least three genes in Aspergillus fumigatus that are capable of expressing a GAPDH-related proteiri. These predicted proteins have been labelled GAPDH-A, GAPDH-B, and GAPDH-C. A number of differences exist between these two proteins (see Fig 3). However, it is possible to conclude that only GAPDH-B has been identified to date. An inability to identify GAPDH-A or -C, to date, could be due to a number of reasons, e.g., a failure to be expressed under laboratory conditions; or, to the absence of an appropriate predicted protein sequence in the databases. The fact that only GAPDH-B was identified in cell wall and secreted preparations indicates that this version of the protein is likely to be primarily a cell-wall variant, and perhaps GAPDH-A and -C the cytoplasmic variants.
GAPDH A and GAPDH-B share 73% identity and 85% similarity over a over a stretch of 269 residues. The more divergent GAPDH-C shares only 43% identity with both GAPDH-A and GAPDH-B. An analysis of all three sequences via an InterProScan revealed all three to be GAPDH sequences. However, only proteins A
and B had sequences that matched to the active-site motif ([ASV]-S-C-[NT]-T-x(2)-[LIM]). This could imply that C does not function 'as a true GAPDH protein.
Upon closer analysis of the sequences it is apparent that C contains a V residue, instead of [LIM], at the last position in the motif. Considering that V, L, M, and I
are all hydrophobic residues, it is unlikely that the difference will result in a non-functional GAPDH active site.
Isopropylmalate dehydrogenase 8 sequence.
The closest homologues of the predicted isopropylmalate dehydrogenase sequence were previously described enzymes from A. niger, (accession number in NCBI
database P87257 (77% identity over 363 aa)) and A. oryzae, (accession number in NCBI database BAC55906 (52% identity over 367 aa)).
Homology to human proteins and essentiality to A. fumigatus. Of the protein mentioned above, neither Cssl, IMDH B nor Hydrophobin have any significant human homologues. Both enolase (61 % identical, 77% similar) and GAPDH (77%
identical/ 83% similar), on the other hand, do have human homologues over the full length of the protein. However, due to the small size of any given epitope, and to the specificity of antibodies in general, it is likely that a suitable antibody can be found to distinguish A. fumigatus versions from the human versions.
Peptides for antibody.production The peptides found in the mass spectrometry analysis were used for antibody production. Some of them were extended with flanking residues from the predicted or known sequences.
Example 3. Generation and properties of anti-AfM and anti-IMDH antibodies Methods Phosphate buffered saline PBS tablets (Sigma) were used to produce a final solution of 0.01 M phosphate buffer, 0.0027 M KCI and 0.137 NaCt, pH 7.4 at 25°C.
Generation of anti-Aspergillus fumigatus mycelia (anti-AfM) antibodies AfM-rich preparations were grown as follows: 10E5 AfC were added to 10 ml RPMI
and incubated for approximately 10 hours at 37°C. AfM were then harvested by centrifugation and washed twice in PBS. These preparations were fixed by incubation in 3% formaldehyde for 30 min at room temperature, washed and 100 p,g quantities injected into rabbits according to the following protocol.
Null sera was collected from New Zealand White female rabbits (4-6 months old, approximately 3 kg) prior to immunisation with 100 p.g AfM and Freunds complete adjuvant. A booster was administered on day 14 in conjunction with Freunds incomplete adjuvant and again on day 28. The first and second bleeds were harvested on days 42 and 72, respectively, before the final bleed was taken on day 93. Subsequent analysis via immunofluorescent microscopy and western blotting demonstrated that the rabbit raised an Ab-based immune response against AfM.
IgG purification IgG was then purified from this sera using the MabTrap kit (Amersham) in accordance with the manufacturers instructions. Fab fragments were purified from IgG via Immunopure Fab kit (Pierce) again in accordance with the manufacturers instructions.
Adhesion assay protocol A549 cells (1 x 105) (DeHart et al. (1997) J. Infect Dis. 175(1):146-150) were seeded into the well of a Lab-Tek II 8 well chamber slide (Nalge Nunc International) and grown overnight. A solution of AfC (1 x 10°lml) was prepared in RPMI
medium, vortexed for 10 min and sonicated for 10 sec to suspend the AfC (in house studies have demonstrated that greater than 99% of AfC are viable after this step).
The AfC
population was then aliquoted, IgG preparations added where appropriate, and the samples incubated with shaking for 30 min at 37°C. A549 cells were prepared by washing three times with 400 ~.I F12K media. If required, purified protein was pre-incubated with A549 cells for 30 mins at 37°C after which the cells were washed three times with 400 ~.I F12K media. Finally, 190 p.1 of F12K was added to each well, prior to the addition of 10 ~.I (1 x 106 AfC) of the appropriate AfC
solution_ The samples were then incubated at 37°C for 60 min. Unbound AfC were removed by washing 4 times with F12K media and A549 cells were detached using 400 ~.I
trypsin EDTA. Following detachment the solutions were removed from the wells, stored in an eppendorf, and sonicated at medium intensity for 10 s to lyse mammalian cells. Finally, 0.03% Triton-X-100 was added to each solution to ensure that the AfC existed in a single cell form. The quantity of AfC in each sample was then determined by counting using a haemocytometer. All assays were run in triplicate.
Antigen identification via IP with anti-AfM antibodies Resultant IgG preparations were used, in conjunction with the Affigel kit (Biorad), to prepare an anti-AfM affinity column. This was done in accordance with manufacturers instructions. Thus, 20 mg of anti-AfM IgG was coupled to 200 ~.I
affi-gel and the resultant column was rotated overnight with 100 mg of whole AfM
lysate prepared in PBS. A column containing 200 p.1 affi-gel without IgG was also treated in the same manner. The supernatant was then removed and the gel washed twice with 0.5 ml PBS adjusted to 0.5 M NaCI. Washing was then performed with 0.5 ml PBS adjusted to 0.25 M NaCI and samples were eluted with 100 ~.I 0.2 M Glycin-HCI
pH 2.5. Following storage on ice for 5 mins the samples were neutralised via the addition of 24 ~,I of 1 M Tris-HCI pH 8.5 and separated via SDS-PAGE.

Amplification and cloning of the cDNA copy of IMDH 8 Total RNA was purified from 1 x1 O9 AfC using the RNAEasy Kit (Qiagen) in accordance with manufacturers instructions. In order to design oligonucleotide 5 primers to assist in the amplification of the cDNA, the sequence of the IMDFi B was predicted using a number of bioinformatic steps. First, peptides identified via LCMS
were BLASTED against the A. fumigatus shotgun database. All sequences matching the peptide were then aligned to produce a large contig sequence that was then input into the gene prediction program Genscan. This produced a number of 10 predicted gene sequences and those that were predicted to encode the peptide identified via LCMS were selected for further study. IMDH B genes from other organisms that displayed homology to the A. fumigatus sequence were also used to assist in the selection of the most likely STOP and START codon_ Thus, 18-mer oligos (F: 5'-ATGGTAACTACTTACAAC-3' (SEQ ID N0:44); R: 5'-15 TGAACTACCCTGCAACGC-3' (SEQ ID N0:45)) were designed and used to amplify a cDNA copy of the Af IMDH B gene using the Superscript One Step kit from Invitrogen in accordance with manufacturers instructions. Following amplification the product was cloned into pBAD using the TOPO TA cloning kit (Invitrogen) and sequencing was used to confirm the sequence and orientation of the insert.
Seguencing Sequence reactions were performed using the BigDye terminatorv3.1 (Applied Biosystems) kit in accordance with the manufacturers instructions.
Expression of heterologous proteins Upon identification of a clone containing the desired sequence a number of experiments were performed to identify the optimal expression conditions for the heterologous protein. These revealed optimal expression when induced with 0.02%
arabinose for 4 hours. Proteins were then purified via utilisation of pBAD
encoded his-tag sequence.
Preparation of bacterial lysates Each liter of E. coli culture was induced for four hours with 0.02% arabinose and bacterial cells were harvested by centrifugation (5000 rpm, 15 min). The bacterial pellet was then resuspended in 25 ml cold native buffer (20 mM NaP04, 500 mM

NaCI, _25 mM imidazole; pH, 7.4), and the solution supplemented with 2 protease inhibitor tablets (ROCHE, complete-EDTA free Protease ~ inhibitors) and 625 p.1 lysozyme (25 p,g/p.l) before incubation on ice for 1 h. The solution was then sonicated for 4 min, subjected to a cycle of freeze thaw, and 7.5 p,1 benzonase (362 units/p.l) added prior to incubation on ice for 20 minutes. In order to assist in the removal of insoluble components the solution was centrifuged at 5000 x g for 20 min and the supernatant harvested. This step was repeated twice more and the cleared lysate then analysed via SDS-PAGE.
Preparation of nickel sepharose columns for purification of his-tagged proteins Purification of recombinant proteins was performed using Probond resin (invitrogen),, a niclele-sepharose based resin that utilises the pBAD-encoded his-tag on the heterologously-expressed recombinant protein. AI( centrifugation steps were performed at 800 x g for 2 min. The resin was resuspended by inverting and gently tapping the container and 2 ml slurry of resin was added to an Econo-Pac Chromatography Columns (BioRad, Cat. No. 732-1010) for equilibration. The column was centrifuged and the supernatant discarded. The resin was then washed with 10 ml sterile water, and again the supernatant discarded following - centrifugation. Next, the resin was resuspended in 10 ml Native buffer (20 mM
NaP04, 500 mM NaCI, 25 mM imidazole, pH 7.4), centrifuged and the supernatant discarded. This process was repeated twice. Finally the resin was resuspended in 1 ml native bufFer giving a final volume of 2 ml.
Application of Iysate, washing and elution of purified protein The bacterial lysate containing the recombinant protein was added to the column containing the equilibrated resin and the mixture incubated on a roller for 100 min at 4°C. The mixture was then centrifuged at 800 x g for 2 min and the supernatant poured off and saved. The resin was then allowed to settle, the column plug was removed and the liquid allowed to flow through. The run through from this step, and all subsequent steps, was collected for SDS-PAGE analysis. The column was then washed with 20 ml Native buffer (20 mM NaP04, 500 mM NaCI, 25 mM imidazole, pH 7.4) a total of 5 times and the protein eluted protein by applying 20 ml Native elution buffer (20 mM NaP04, 500 mM NaCI, 250 mM imidazole, pH 7.4) and collecting ten 2 ml aliquots. SDS-PAGE analysis was then performed to determine the outcome of the procedure.
Gel filtration purification of nickel sepharose purified IMDH B
Probond purified IMDH B was first desalted via dialysis in PBS (dialysis tubing 12-14000 Daltons, Visking). Gel filtration was performed using a HiPrep 16/60 Sephacryl S-200 High Resolution column (Amersham Biosciences). Preparation, equilibration and cleaning of the column was completed according to the manufacturers instructions. Protein purification was performed with a Tris running buffer (10 mM Tris + 0.15 M NaCI, pH 8) at an approximate flow rate of 0.25 mllmin.
Batches of his-tagged protein (10-20 mg) were added to the column and were eluted using 80 ml running buffer, a void volume of approximately 40 ml was calculated and 18 x 2 ml fractions were collected.
Generation of anti-IMDH B antisera.
Probond-purified protein was used to immunise New Zealand White female rabbits (4-6 months old, ~3 kg). Pre-immune sera were harvested on day 1 prior to immunisation with 100 pg of protein in the presence of Freunds complete adjuvant.
Further immunisations were also carried out on days 28 and 49 in the presence of Freunds incomplete adjuvant. Blood samples were taken on days 28, 42, 69 and finally on 87. IgG and Fab fragments were prepared as previously described.
Immunofluorescent microscopy Immunofluorescence microscopy slides were first cleaned in a detergent solution and rinsed in distilled water. The slides were then incubated in coating solution (0.1 % gelatin [w/v], 0.01 % [w/v] chrome alum) and allowed dry at room temperature.
Harvested AfC spores were placed onto the well of a treated immunofluorescence microscopy slide and the slide was washed three times with PBS to remove unbound AfC. If AfM were required 30 p.1 RPMI was added to the surface of the well and the slide incubated at 37°C for 12-14 hours before washing with PBS. IgG (30 p,1, 1:500 dilution in PBS) was then added to each well and incubated at 37°C for 30 min in a moist environment. The wells were washed three times with PBS before the addition of Alexa Fluor 488 goat-anti-rabbit IgG (30 p,1 of a 1:400 dilution prepared in PBS) to each well. Again the slides were incubated at 37°C for 30 min in a moist environment, washed three times with PBS and then fixed in 3% formaldehyde for 30 min at room temperature. The slides were washed three times in PBS, allowed to dry and then a drop of Sigma oil was added to the surface and a coverslip applied.
This was sealed with nail polish and the slides stored at 4°C in the dark until use.
All wells were scanned in both bright and fluorescent fields.
Determination of the ability of IgG to inhibit conidial germination AfC were resuspended in RPMI medium and the concentration adjusted to give a solution containing 1 x 106 AfC ml. 0.5 ml of this solution was then added to an eppendorf and 10 ~.I of each IgG/serum sample added to 3 aliquots of AfC.
Normal fresh rabbit serum was added to one aliquot, heat-inactivated (60°C for 30 min) serum to the second, and 10 ~I PBS to the third. The samples were then incubated for 7 hours at 200 rpm. The total number of cells was then counted and the number of conidia that have germinated, or started to produce a germ tube, determined.
Percent filamentation equals the number of cells with germ tubes divided by the total number of cells.
Generation of sera against predicted antigenic peptides of target proteins Analysis of target proteins was performed with the aim of identifying a number of antigenic peptides within each target sequence. The peptides chosen (see table below) correspond to those that were identified during mass spectrometry procedures used to identify surface exposed peptides (see table 1) and to those predicted to be most antigenic (Jameson, B.A. & Wolf, H (1988) Comput Appl Biosci 4, 181-186).
Peptides and antisera were produced in accordance with Sigma's standard Custom Peptide Synthesis and rabbit immunisation protocols. The peptides were produced as part of their multiple antigenic peptides (MAP) service.The immunisation protocol used is as follows: on day 0 pre-immune sera was collected from New Zealand white rabbits which were then immunised with 200 ~.g of MAP in complete freunds adjuvant (CFA). A second immunisation was performed on day 14 with 100 ~,g in Incomplete freunds adjuvant (IFA). This immunisation was repeated on days 28, 42, 56 and 70. Bleeds were taken on days 35, 49, 63, and finally on day 77.

MS.analysis In-gel digestion The gel bands are washed two times with 25mM NH4HC03/50% ethanol. The cysteine residues are reduced (DTT) and alkylated (iodoacetamide), followed by cycles of wash and dehydration of the gel bands. Washing buffer is 50mM

and dehydrating agent is ethanol. After the last dehydration step, the digestion is started by addition of protease, dissolved in suitable digestion buffer.
Default enzyme is trypsin for which a 50mM NH4HC03/10% Acetonitrile digestion buffer is used. The digestion takes place over night at 37°C. The resulting peptide pool are extracted with TFA and formic acid and analysed by mass spectrometry. The handling of all samples takes place in a dust free environment with a minimum of manual handling to avoid keratin contamination.
MALDI TOF sample preparation and analysis The peptide mixture extracted after in-gel digestion is analysed by a fully automated procedure on an Ultraflex Bruker MALDI-TOF mass spectrometer. A peptide mass fingerprint is recorded and each spectrum is annotated and internally calibrated using trypsin auto-digest peaks. Sample preparation:
1. A 96 tip robot is used to prepare a Bruker 384 polished steel target with a nitrocellulose/0-cyano-4-hydroxy-cinnamid acid (HCCA) matrix.
2. Approximately 5% of the sample is added to the matrix (1,5 01).
3. When the sample is completely dry, it is washed with 0.1 % TFA and is now ready to be analysed in the Bruker mass spectrometer.
Database search The resulting peak list is used in a database search that is perFormed using Mascot software (MATRIX SCIENCE).
The database is a non-redundant database (NRDB) based on data from NCBI.
Results Antigen identification via IP with anti AfM antibodies An affinity column consisting of anti-AfM IgG was used to immunoprecipitate the most antigenic proteins from an AfM lysate. Eluted samples were separated via SDS-PAGE and then analysed via LC MSMS analysis, which revealed 3-isopropylmalate dehydrogenase B (IMDH B) to be the prime target of anti-AfM
antibodies, suggesting IMDH B to be the main antigen of AfM (figure 4).
5 Anti-AfM antibodies bind to the surface of AfM giving a considerably higher signal than the null sera.
IFM studies revealed that anti-AfM antibodies bound to both AfM and AfC giving a higher signal than was observed with null sera. This again suggests IMDH B to be a major surface antigen of both conidia and mycelia (figure 5).
Anti-AfM Fab fragments reduce the proportion of AfC that adhere to A549 cells Having substantiated that IMDH B is a major surface antigen of both AfC and AfM it was decided to analyse the potential ability of anti-IMDH B antibodies (in the form of anfi-AfM antisera) to interFere with the pathogenicity of AfC. Thus, adhesion assays were performed where the ability of AfC, pre-incubated in IgG, to bind to lung epithelia was measured. These studies indicated that antibodies, in the form of Fab fragments, were indeed capable of reducing adhesion of the organism to lung epithelia (figure 6). Without being bound by any theory, it is believed that the Fabs possess the ability to reduce adhesion to lung epithelia by interfering with the interaction between a receptor and its ligand, either by binding an adhesin and preventing its function, or by binding to the AfC surface and sterically inhibiting the function of an underlying adhesin. While IgG molecules may also possess such properties, the Fc region is capable of binding Fc receptors on the surface of the mammalian cells, an event which has the potential to elevate adhesion of AfC.
Cloning, expression and purification of recombinant IMDH B
A copy of cDNA encoding IMDH B was successfully cloned into pBAD and the sequence of the insert confirmed (see SEQ ID N0:46 and SEQ ~ID N0:47). The protein was expressed following induction with 0.02% arabinose for 4 h and purified to a high degree of purity using nickel speharose and gel filtration columns (figure 7). The nickel sepharose purified fraction was used to immunise rabbits and this procedure was successful in generating anti-IMDH B antibodies (figure 8). IFM
analysis also confirmed that anti-IMDH B IgG bound to the surFace of AfC and AfM
(figure 9), thus confirming the surface localisation of IMDH B.

IFM analysis of clinical isolates ~IFM analysis was performed on a number of clinical isolates to confirm. the presence or absence of surface expressed IMDH B. All clinical isolates were purchased from the Belgian Co-ordinated Collections of Micro-organisms (BCCMTM). Their features and reactivity to anti-IMDH B IgG are listed in Table 4.
Post-translational modifications A number of studies were performed with the aim of identifying the presence of post-translational modifications in the recombinant IMDH B protein. These studies took a number of forms. First, MALDI analysis was performed with the aim of identifying all of the predicted tryptic fragments of the protein. Thus the protein was digested with trypsin and analysed via MS. Using this method it was possible to get 84%
sequence coverage: (SEQ ID N0:47 (vector-derived sequences in italics, sequences identified by MALDI analysis underlined))_ MGSGSGDDDDKLALMVTTYNILVLPGDGIGPEVNlTEAVKVLKVFEDTEHR_R'~~RQELIGGCSIDAHGKSVTEEVKK

AALESDAVLFAAVGGPKWDHIRRGLDGPEGGLLQT~RTtAMnIyAI~7LgpCSASSPSASIAKEFSPFRQEVIEGVDFV
V
VRENCGGAYFGKKIEEEDYAMDEWGYSEREIQRITRLSAEIALRHNPPWPVISLDKANVLASSRLWRRWEKTMTT
EYPQVKLVHQLADSASLILATNPRALNGVILADNTFGDMISDQAGSIVGTLGVLPSASLDGLPSETRKRTNGLYEP

THGSAPTIAGQNIANPVAMILCVALMFRYSLDMETEAQRIEKAVQGVLDAGIRTPDLGGKSGTNEVGDAIVAALQG
SSKGELEGKPIPNPLLGLDSTRTGHHHHHH.
SDS-PAGE and time of flight analyses of the predicted 44233 Da Protein revealed apparent masses of 45000 Da and 44235 Da, respectively. In total, these studies did not provide any evidence indicating the presence of post-translational modifications.
Adhesion assays results A number of studies support the theory that IMDH B acts as an adhesin. Thus, pre-incubation of AfC with anti-IMDH B Fab fragments produced a decline in the number of adherent AfC in comparison to AfC that had been pre-incubated in the presence of Fab fragments isolated from pre-immune sera (see figure 10). Furthermore, pre-incubation of A549 epithelia with varying quantities of recombinant IMDH B, followed by washing to remove unbound protein, suggested that the protein had sequestered the receptor on the surFace of the A549 cells and thus prevented the AfC from adhering (see figure 11).

Germination inhibition experiments A number of experiments were performed to determine the ability of anti-IMDH B
antibodies to reduce germination of AfC in the presence of normal or heat-s inactivated rabbit serum. These experiments revealed that only anti-IMDH B
heat-inactivated sera in the presence of fresh rabbit sera possessed the ability to reduce the rate of germination of AfC (see figure 12). Further studies revealed that purified anti-IMDH B IgG in the presence of normal rabbit serum also possessed the ability to reduce germination of AfC in comparison to null IgG (see figure 13).
Identification of a second copy of IMDH B
Further bioinformatic analysis revealed the presence of a second region on the Af genome that displays homology to the IMDH B gene and that is predicted to encode a protein that is 50% ID and 63% similar to IMDH B 1 (for alignment, see figure 14)_ The sequence of the respective gene and protein were predicted by BLAST
analysis. Features of this protein are displayed below. -Predicted Features of geneiprotein compared to ACE5033 (IMDH B 1) Number of exons 5 3 Size of ORF 1093 by 1107 by Protein length 363 residues 368 residues Protein size 38723 Da 39773 Da p1 5.26 5.32 Identity to ACE5033 53% (over full length)---Similarity to ACE5033 69% (over full length)---IMDH B 2 contains the final predicted ORF sequence (SEQ ID NO: 40):
atgccgtcatataacattgtcgttttcgctggggaccactgtggtccggaggtaagttcggtcctgcgcgtcatcgaga a gtgccgtgacgatgctaccttcaacctccaggatcaattgctcggtggtgtaagttcgatcgatgctaccggatctccc c ttaccgacgaagctcttaacgccgcaaagaacgccgatgccgttctcctcggtgccattggcggtcccaaatggggcac t ggcgccgtccgccccgaacagggcctcctccgtctgcgcaaggagatgggcacattcggtaacctccgcccctgcaact t cgccgccccgtcgctggtcgacggctcccctctccgccccgaagtctgccgcggcgtcgacttcaacattatccgcgaa c tgaccggtggcatctacttcggcgaccgcaaggaggacgacggcagcggcttcgccatggacacggagccgtactcccg c gcggagatcgagcgcatcacccgccttgcggcccacctcgctctgcagcacaacccccctcttcccgtgtggagcttgg a caaggccaacgtcctcgcgacgagccggctgtggcggaagaccgtgacggaggtcatggccaaggagttcccccagctc a aggtggagcaccagctcattgactccgcggccatgatcatggtcaaggagcctagaaagcttaacggtattgttgtcac t agcaacctgttcggtgacatcatcagtgatgaagccagcgttatccctggttctctgggactcttgcccagcgcaagct t gagcggcattcctgacggaaagaccaaggtcaatggtatctatgagcctattcacggttctgcccctgacattgccggc a agggcatcgttaaccccgtcgccgccattctctctgtcgccatgatgatgcagtactccctgaaccgtatggatgacgc c agggccatcgagacggccgtccgcaatgtgatcgaggccggtatccgcactgccgatattggcggcaagtcgacaacta g cgaggtcggtgacgctgttgctgccgagctggagaagctgttgaagcaatagt This encodes the following protein (SEQ ID NO: 41):
MPSYNIWFAGDHCGPEVSSVLRVIEKCRDDATFNLQDQLLGGVSSIDATGSPLTDEALNAAKNADAVLLGAIGGPKWGT

GAVRPEQGLLRLRKEMGTFGNLRPCNFAAPSLVDGSPLRPEVCRGVDFNIIRELTGGIYFGDRKEDDGSGFAMDTEPYS
R
AEIERITRLAAHLALQHNPPLPVWSLDKANVLATSRLWRKTVTEVMAKEFPQLKVEHQLIDSAAMIMVKEPRKLNGIWT

SNLFGDIISDEASVIPGSLGLLPSASLSGIPDGKTKVNGIYEPIHGSAPDIAGKGIVNPVAAILSVAMMMQYSLNRMDD
A
RAIETAVRNVIEAGIRTADIGGKSTTSEVGDAVAAELEKLLKQ
Example 4. Studies performed on additional ACE targets Generation of sera against predicted antigenic peptides of target proteins Following the idenffication of a number of novel proteins on the surface of the AfC it was decided to further these studies through the production of antisera that had been raised against multiple antigenic peptides (MAP) of the novel molecules.
Thus, two peptides from each chosen target were selected (see table below) and the information relayed to Sigma who produced the peptides and antisera in accordance with their Custom Peptide Synthesis and rabbit immunisation protocols.
The peptides chosen (see table 3) correspond to those that were identified during mass spectrometry procedures (see table 1) and to those predicted to be most antigenic using the guidelines supplied by Sigma.
IFM analysis with sera raised actainst MAP molecules Following the production of MAPs and the corresponding antisera, IgG was purified and used in IFM experiments to assist in the confirmation of the surface expression of the various target molecules. The results of these IFM experiments are summarised in table 3 and exemplified for GAP-B-2 in figure 15. Sera raised against the GAP-B-2 molecule of GAPDH2 were also tested against a number of the clinical isolates. Reactivity of this sera against the surface of the clinical isolates (exemplified in figure 16, other not shown) supports the surface localisation of GAPDH2 within isolates other than ATCC 46640.
Cloning, expression and purification of recombinant enolase; plus detection of native enolase.
The enolase gene of A. fumigatus was cloned and the protein expressed using the same protocols detailed for IMDH B, the only difference being the sequence of the forward (5'-ATGCCTATCTCCAAGATC-3' (SEQ ID N0:42)) and reverse primers (5'-CAGGTTGACGGCAGT-3' (SEQ ID N0:43)). The sequence of the cloned cDNA

molecule was confirmed using standard procedures) (SEQ ID N0:48 and SEQ ID
N0:49). Expression of the recombinant protein was confirmed using anti-his antibodies (figure 17(A)). Furthermore, the protein was shown to react with anti-MAP sera raised against ENO-2 (figure 17(A)). Having confirmed that the anti-MAP
sera binds recombinant enolase it was decided to test the sera against isolated membrane and wall fractions from AfC. These studies revealed the protein to exist in the cell membrane of AfC (figure 17(B)).
Example 5. Alianment of Asoeraillus fumiaatus IMDH B with homoloaous polypeptides from other functi The sequence of Aspergillus fumigatus IMDH B (SEQ 1D N0:36) was compared with homologous sequences from other fungi in order to find areas of high sequence identity. Figures 18-25 show alignments of SEQ ID N0:36 with homologous polypeptides.

SEQUENCE LISTING
<110> ACE Biosciences A/S
<120> Extracellular fungal polypeptides <130> P758PC00 <160> 49 <170> PatentIn version 3.1 <210> 1 <211> 260 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:1 - Css2) <400> 1 Met Leu Ala Ser Phe Gln Phe Cys Ile Leu Pro Arg Thr Tyr Arg Thr Leu Leu Cys Ser Ala Gly Ala Gly Pro Leu Leu Ile Ile Gln Phe Val Thr Val Ala Ser Ala Leu Ala Leu Ala Pro Thr Ala Va1 Val Ala Arg Gln Gly Ala Ala Ala Phe Val Thr Val Asn Ser Ile Asp Val Cys Pro Lys Lys Val Ala Gln Glu Ile Ile Asn Pro Gly Pro Lys Val Val Thr Thr Pro Tyr Thr Cys Asp Gln Val Lys Leu Gly His Gly Leu Asp Val Ser Tyr Tyr Asn Phe Asp Ile Glu Pro Leu Thr Lys Asp Thr Phe Pro Tyr Cys Lys Ala Leu Lys Val Phe Asp Asn Glu Gly Cys Leu Gly Phe Pro Thr Leu Trp I1e Pro Leu Glu Ser Pro Leu Glu Asp Lys Cys Ile Pro Glu His Tyr Phe Ser Asp Glu Val Lys Ser Ile Ser Phe Gln Leu 145 150 155 160 _ Asp Cys Arg Glu Asp Ala Pro Val Lys Lys Glu Pro Tyr Gly Pro Lys Glu Gly Ala Glu Gln Ser Ala Pro Gln Ala Glu His Ser Thr Lys Gln Asp Ala Gln Gln Gly Ser His Gln Gly Gln Glu Val Gln Asn Ser Pro Lys Gln Glu Ala Arg Gln Gly Ser Arg Pro Ala Glu Ala Ala Pro Lys Gln Glu Gln Glu Ala Glu Gln Ala Ser Glu Ala Ala Pro Glu Lys Lys Ala Ser Asn Pro Ala Asp Ser Leu Gly Leu Gly Glu Leu Thr Lys Val Leu Gly Phe Arg <210> 2 <211> 107 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:2 - hydrophobin) <400> 2 Val Arg Phe Pro Val Pro Asp Asp Ile Thr Val Lys Gln Ala Thr Glu Lys Cys Gly Asp Gln Ala Gln Leu Ser Cys Cys Asn Lys Ala Thr Tyr Ala Gly Asp Val Thr Asp Ile Asp Glu Gly Ile Leu Ala Gly Thr Leu Lys Asn Leu Ile Gly Gly Gly Ser Gly Thr Glu Gly Leu Gly Leu Phe Asn Gln Cys Ser Lys Leu Asp Leu Gln Ser Pro Ile Ile Gly Ile Pro Ile Gln Asp Leu Val Asn Gln Lys Cys Lys Gln Asn Ile Ala Cys Cys Gln Asn Ser Pro Ser Asp Ala Val Arg Phe Pro <210> 3 <211> 318 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:3 -GAPDH-B) <400> 3 Met Ala Thr Pro Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Ile Val Gly Leu Asn Ser Leu Ser His Gly Val Asp Val Val Ala Val Asn Asp Pro Phe Ile Glu Val His Tyr Ala Ala Tyr Met Leu Lys Tyr Asp Thr Thr His Gly Gln Phe Lys Gly Thr Ile Glu Thr Tyr Asp Gln Gly Leu Ile Val Asn Gly Lys Lys Ile Arg Phe Tyr Ala Glu Lys Asp Pro Ser Gln Ile Pro Trp Ser Glu Thr Gly Ala Ala Tyr Ile Val Glu Ser Thr Gly Val Phe Thr Thr Lys Glu Lys Ala Ser Ala His Leu Lys Gly Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ser Ala Asp Ala Pro Met Phe Val Met Gly Val Asn Asn Thr Thr Tyr Thr Ser Asp Ile Gln Val Leu Ser Asn A1a Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val Ile Asn Asp Lys Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val His Ser Tyr Thr Ala Thr Gln Lys Val Val Asp Ala Pro Ser Asn Lys Asp Trp Arg Gly Gly Arg Thr Ala Ala Gln Asn Ile Ile Pro Ser Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Ser Leu Asn Gly Lys Leu Thr Gly Met Ala Met Arg Val Pro Thr Ser Asn Val Ser Val Val Asp Leu Thr Cys Arg Leu Glu Lys Gly Ala Ser Tyr Asp Glu Ile Lys Gln Ala Ile Lys Ala Ala Ser Glu Glu Gly Glu Leu Lys Asn Ile Leu Gly Tyr Thr Glu Asp Asp Val Val Ser Ser Asp Leu Asn Gly Asp Glu Arg Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile Ser Leu Asn Pro Asn Phe Val Lys Leu Val Ala Trp Tyr Asp Asn Glu Trp 305 310 , 315 <210> 4 <211> 438 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:4 - enolase) <400> 4 Met Pro Ile Ser Lys Ile His Ala Arg Ser Val Tyr Asp Ser Arg Gly Asn Pro Thr Val Glu Val Asp Val Ala Thr Glu Thr Gly Leu His Arg Ala Ile Val Pro Ser Gly Ala Ser Thr Gly Gln His Glu Ala His Glu Leu Arg Asp Gly Asp Lys Thr Gln Trp Gly Gly Lys Gly Val Leu Lys Ala Val Lys Asn Val Asn Glu Thr Ile Gly Pro Ala Leu Ile Lys Glu Asn Ile Asp Val Lys Asp Gln Ser Lys Val Asp Glu Phe Leu Asn Lys Leu Asp Gly Thr Ala Asn Lys Ser Asn Leu Gly Ala Asn Ala Ile Leu Gly Val Ser Leu Ala Val Ala Lys Ala Gly Ala Ala Glu Lys Gly Val Pro Leu Tyr Ala His Ile Ser Asp Leu Ala Gly Thr Lys Lys Pro Tyr Val Leu Pro Val Pro Phe Gln Asn Val Leu Asn Gly Gly Ser His A1a Gly Gly Arg Leu Ala Phe Gln Glu Phe Met Ile Val Pro Asp Ser Ala Pro Ser Phe Ser Glu Ala Leu Arg Gln Gly Ala Glu Val Tyr Gln Lys Leu Lys Ala Leu Ala Lys Lys Lys Tyr Gly Gln Ser Ala Gly Asn Val Gly Asp Glu Gly Gly Val Ala Pro Asp Ile Gln Thr Ala Glu Glu Ala Leu Asp Leu Ile Thr Glu Ala Ile Glu Gln Ala Gly Tyr Thr Gly Lys Ile Lys Ile Ala Met Asp Val Ala Ser Ser Glu Phe Tyr Lys Ala Asp Val Lys Lys Tyr Asp Leu Asp Phe Lys Asn Pro Glu Ser Asp Pro Ser Lys Trp Leu Thr Tyr Glu Gln Leu Ala Asp Leu Tyr Lys Ser Leu A1a Ala Lys Tyr Pro Ile Val Ser Ile Glu Asp Pro Phe Ala Glu Asp Asp Trp Glu Ala Trp Ser Tyr Phe Tyr Lys Thr Ser Asp Phe Gln Ile Val Gly Asp Asp Leu Thr Val Thr Asn Pro Gly Arg Ile Lys Lys Ala Ile Glu Leu Lys Ser Cys Asn Ala Leu Leu Leu Lys Val Asn Gln Ile Gly Thr Leu Thr Glu Ser Ile Gln Ala Ala Lys Asp Ser Tyr Ala Asp Asn Trp Gly Val Met Val Ser His Arg Ser Gly Glu Thr Glu Asp Val Thr Ile Ala Asp Ile Ala Val Gly Leu Arg Ser Gly Gln Ile Lys Thr Gly Ala Pro Cys Arg Ser Glu Arg Leu Ala Lys Leu Asn Gln Ile Leu Arg Ile Glu Glu Glu Leu Gly Glu Asn Thr Val Tyr Ala Gly Ser Lys Phe Arg Thr Ala Val Asn Leu <210> 5 <211> 728 <212> PRT
<213> Aspergillus fumigatus (SEA ID N0:5 - catalase B) <400> 5 Met Arg Leu Thr Phe Ile Pro Ser Leu Ile Gly Val Ala Asn Ala Val Cys Pro Tyr Met Thr Gly Glu Leu Asn Arg Arg Asp Glu Ile Ser Asp Gly Asp Ala Ala Ala Ala Thr Glu Glu Phe Leu Ser Gln Tyr Tyr Leu Asn Asp Asn Asp Ala Phe Met Thr Ser Asp Val Gly Gly Pro Ile Glu Asp Gln Asn Ser Leu Ser Ala Gly Glu Arg Gly Pro Thr Leu Leu Glu Asp Phe Ile Phe Arg Gln Lys Ile Gln Arg Phe Asp His Glu Arg Val Pro Glu Arg Ala Val His Ala Arg Gly Ala Gly Ala His Gly Val Phe Thr Ser Tyr Gly Asp Phe Ser Asn Ile Thr Ala Ala Ser Phe Leu Ala Lys Glu Gly Lys Gln Thr Pro Val Phe Val Arg Phe Ser Thr Val Ala Gly Ser Arg Gly Ser Ser Asp Leu Ala Arg Asp Val His Gly Phe Ala Thr Arg Phe Tyr Thr Asp Glu Gly Asn Phe Asp Ile Val Gly Asn Asn Ile Pro Val Phe Phe Ile Gln Asp Ala Ile Leu Phe Pro Asp Leu Ile His A1a Val Lys Pro Arg Gly Asp Asn Glu Ile Pro Gln Ala Ala Thr Ala His Asp Ser Ala Trp Asp Phe Phe Ser Gln Gln Pro Ser Thr Met His Thr Leu Leu Trp Ala Met Ser Gly His G1y Ile Pro Arg Ser Phe Arg His Val Asp Gly Phe Gly Val His Thr Phe Arg Phe Val Thr Asp Asp Gly Ala Ser Lys Leu Val Lys Phe His Trp Lys Ser Leu Gln Gly Lys Ala Ser Met Val Trp Glu Glu Ala Gln Gln Thr Ser Gly Lys Asn Pro Asp Phe Met Arg Gln Asp Leu His Asp Ala Ile Glu Ala Gly Arg Tyr Pro Glu Trp Glu Leu Gly Val Gln Ile Met Asp Glu Glu Asp Gln Leu Arg Phe Gly Phe Asp Leu Leu Asp Pro Thr Lys Ile Val Pro Glu Glu Phe Val Pro Ile Thr Lys Leu Gly Lys Met Gln Leu Asn Arg Asn Pro Arg Asn Tyr Phe Ala Glu Thr Glu Gln Val Met Phe Gln Pro Gly His Ile Val Arg Gly Val Asp Phe Thr Glu Asp Pro Leu Leu Gln Gly Arg Leu Phe Ser Tyr Leu Asp Thr Gln Leu Asn Arg His Gly Gly Pro Asn Phe Glu Gln Leu Pro Ile Asn Gln Pro Arg Val Pro Val His Asn Asn Asn Arg Asp Gly Ala Gly Gln Met Phe Ile Pro Leu Asn Pro His Ala Tyr Ser Pro Lys Thr Ser Val Asn Gly Ser Pro Lys Gln Ala Asn Gln Thr Val Gly Asp Gly Phe Phe Thr Ala Pro Gly Arg Thr Thr Ser Gly Lys Leu Val Arg Ala Val Ser Ser Ser Phe Glu Asp Val Trp Ser Gln Pro Arg Leu Phe Tyr Asn Ser Leu Val Pro Ala Glu Lys Gln Phe Val Ile Asp Ala Ile Arg Phe Glu Asn Ala Asn Val Lys Ser Pro Val Val Lys Asn Asn Val Ile Ile Gln Leu Asn Arg Ile Asp Asn Asp Leu Ala Arg Arg Val Ala Arg Ala Ile Gly Val Ala Glu Pro Glu Pro Asp Pro Thr Phe Tyr His Asn Asn Lys Thr Ala Asp Val Gly Thr Phe Gly Thr Lys Leu Lys Lys Leu Asp Gly Leu Lys Val Gly Val Leu Gly Ser Val G1n His Pro Gly Ser Val Glu Gly Ala Ser Thr Leu Arg Asp Arg Leu Lys Asp Asp Gly Val Asp Val Val Leu Val Ala Glu Arg Leu Ala Asp Gly Val Asp Gln Thr Tyr Ser Thr Ser Asp Ala Ile Gln Phe Asp Ala Val Val Val Ala Ala Gly Ala Glu Ser Leu Phe Ala Ala Ser Ser Phe Thr Gly Gly Ser Ala Asn Ser Ala Ser Gly Ala Ser Ser Leu Tyr Pro Thr Gly Arg Pro Leu Gln Ile Leu Ile Asp Gly Phe Arg Phe Gly Lys Thr Val Gly Ala Leu Gly Ser Gly Thr Ala Ala Leu Arg Asn Ala Gly Ile Ala Thr Ser Arg Asp Gly Val Tyr Val Ala Gln Ser Val Thr Asp Asp Phe Ala Asn Asp Leu Lys Glu Gly Leu Arg Thr Phe Lys Phe Leu Asp Arg Phe Pro Val Asp His <2l0> 6 <211> 749 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:6 - catalase A) <400> 6 Met Ala Thr Lys Ile Ala Gly Gly Leu His Arg Ala Gln Glu Val Leu Gln Asn Thr Ser Ser Lys Ser Lys Lys Leu Val Asp Leu Glu Arg Asp Thr Ala Asp Ala His Thr Gln Gln Pro Leu Thr Thr Asp His Gly Val Arg Val Ser Asn Thr Asp Gln Trp Leu Arg Val Thr Asn Asp Arg Arg Thr Gly Pro Ser Leu Leu Glu Asp Gln Ile Ala Arg Glu Lys Ile His Arg Phe Asp His Glu Arg Ile Pro Glu Arg Val Val His Ala Arg Gly Thr Gly Ala Phe Gly Asn Phe Lys Leu Lys Glu Ser Ile Glu Asp Leu Thr Tyr Ala Gly Val Leu Thr Asp Thr Ser Arg Asn Thr Pro Val Phe Val Arg Phe Ser Thr Val Gln Gly Ser Arg Gly Ser Ala Asp Thr Val Arg Asp Va1 Arg Gly Phe Ala Val Lys Phe Tyr Thr Asp Glu Gly Asn Trp Asp Ile Val Gly Asn Asn Ile Pro Val Phe Phe Ile Gln Asp Ala Val Lys Phe Pro Asp Phe Val His Ala Val Lys Pro Glu Pro His Asn Glu Val Pro Gln Ala Gln Thr Ala His Asn Asn Phe Trp Asp Phe Val Tyr Leu His Pro Glu Ala Thr His Met Phe Met Trp Ala Met Ser Asp Arg Ala Ile Pro Arg Ser Tyr Arg Met Met Gln Gly Phe Gly Val Asn Thr Phe Ala Leu Val Asn Lys Glu Gly Lys Arg His Phe Val Lys Phe His Trp Ile Pro His Leu Gly Val His Ser Leu Val Trp Asp Glu Ala Leu Lys Leu Gly Gly Gln Asp Pro Asp Phe His Arg Lys Asp Leu Met Glu Ala Ile Asp Asn Lys Ala Tyr Pro Lys Trp Asp Phe Ala Ile Gln Val Ile Pro Glu Glu Lys Gln Asp Asp Phe Glu Phe Asp Ile Leu Asp Ala Thr Lys Ile Trp Pro Glu Asn Leu Val Pro Leu Arg Val Ile Gly Glu Leu Glu Leu Asn Arg Asn Val Asp Glu Phe Phe Pro Gln Thr Glu Gln Val Ala Phe Cys Thr Ser His Ile Val Pro Gly Ile Asp Phe Thr Asp Asp Pro Leu Leu Gln Gly Arg Asn Phe Ser Tyr Phe Asp Thr Gln Ile Ser Arg Leu Gly Ile Asn Trp Glu Glu Leu Pro Ile Asn Arg Pro Val Cys Pro Val Leu Asn His Asn Arg Asp Gly Gln Met Arg His Arg Ile Thr Gln Gly Thr Va1 Asn Tyr Trp Pro Asn Arg Phe Glu Ala Val Pro Pro Thr Gly Thr Lys Gly Ser Gly Val Gly Gly Gly Phe Thr Thr Tyr Pro Gln Arg Val Glu Gly Ile Lys Asn Arg Ala Leu Asn Asp Lys Phe Arg Glu His His Asn Gln Ala Gln Leu Phe Tyr Asn Ser Met Ser Glu His Glu Lys Leu His Met Lys Lys Ala Phe Ser Phe Glu Leu Asp His Cys Asp Asp Pro Thr Val Tyr Glu Arg Leu Ala Gly His Arg Leu Ala Glu Ile Asp Leu Glu Leu Ala Gln Lys Val Ala Glu Met Val Gly Ala Pro Ile Pro Ala Lys Ala Leu Lys Gln Asn His Gly Arg Arg Ala Pro His Leu Ser Gln Thr Glu Phe Ile Pro Lys Asn Pro Thr Ile Ala Ser Arg Arg Ile Ala Ile Ile Ile Gly Asp Gly Tyr Asp Pro Val Ala Ser Thr Gly Leu Lys Thr Ala Ile Lys Ala Ala Ser Ala Leu Pro Phe Ile Ile Gly Thr Lys Arg Ser Ala Ile Tyr Ala Thr Glu Asp Lys Thr Ser Ser Lys Gly Ile Ile Pro Asp His His Tyr Asp Gly Gln Arg Ser Thr Met Phe Asp Ala Thr Phe Ile Pro Gly Gly Pro His Val Ala Thr Leu Arg Gln Asn Gly Gln Ile Lys Tyr Trp Ile Ser Glu Thr Phe Gly His Leu Lys Ala Leu Gly Ala Thr Gly Glu Ala Val Asp Leu Val Lys Glu Thr Leu Ser Gly Thr Leu His Val Gln Val Ala Ser Ser Gln Ser Pro Glu Pro Val Glu Trp Tyr Gly Val Val Thr Ala Gly Gly Lys Gln Lys Pro Glu Ser Phe Lys Glu Ser Val Gln Ile Leu Lys Gly Ala Thr Asp Phe Val Gly Lys Phe Phe Tyr Gln Ile Ser Gln His Arg Asn Tyr Gln Arg Glu Leu Asp Gly Leu Ala Ser Thr Ile Ala Phe <210> 7 <211> 16 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:7- CssI fragment) <400> 7 Lys Val Ala Gln Glu Ile Ile Asn Pro Gly Pro Lys Val Val Thr Thr <210> 8 <211> 16 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:8 - CssI fragment) <400> 8 Lys Glu Gly Ala Glu Gln Ser Ala Pro Gln Ala Glu His Ser Thr Lys <210> 9 <211> 17 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:9 - hydrophobin fragment) <400> 9 Pro Val Pro Asp Asp Ile Thr Val Lys Gln Ala Thr Glu Lys Cys Gly Asp <210> 10 <211> 15 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:10 - hydrophobin fragment) <400> 10 Ala Thr Tyr Ala Gly Asp Val Thr Asp Ile Asp Glu Gly Ile Leu <210> 11 <211> 16 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:11 - GAPDH-B fragment) <400> 11 Thr Glu Asp Asp Val Val Ser Ser Asp Leu Asn Gly Asp Glu Arg Ser <210> 12 <211> 18 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:12 - GAPDH-B fragment) <400> 12 Phe Lys Gly Thr Ile Glu Thr Tyr Asp Gln Gly Leu Ile Val Asn Gly Lys Lys <210> 13 <211> 17 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:13 - enolase fragment) <400> 13 Lys Asn Val Asn Glu Thr Ile Gly Pro Ala Leu Ile Lys Glu Asn Ile Asp <210> 14 <211> 18 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:14 - enolase fragment) <400> 14 Thr Ser Asp Phe Gln Ile Val Gly Asp Asp Leu Thr Val Thr Asn Pro Gly Arg <210> 15 <211> 20 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:15 - Catalase B fragment) <400> 15 Asp Glu Glu Asp Gln Leu Arg Phe Gly Phe Asp Leu Leu Asp Pro Thr Lys Ile Val Pro <210> 16 <211> 16 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:16 - catalase B fragment) <400> 16 Arg Ile Asp Asn Asp Leu Ala Arg Arg Val Ala Arg Ala Ile Gly Val <210> 17 <211> 12 <212> PRT

<213> Aspergillus fumigatus (SEQ ID N0:17 - CssI fragment) <400> 17 Lys Val Ala Gln Glu Ile Ile Asn Pro Gly Pro Lys <210> 18 <211> 10 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:18 - hydrophobin fragment) <400> 18 Phe Pro Val Pro Asp Asp Ile Thr Val Lys <210> 19 <211> 20 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:19 - hydrophobin fragment) <400> 19 Ala Thr Tyr Ala Gly Asp Val Thr Asp Ile Asp Glu Gly Ile Leu Ala Gly Thr Leu Lys <210> 20 <211> 11 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:20 - GAPDH-B fragment) <400> 20 Ala Gly Ile Ser Leu Asn Pro Asn Phe Val Lys <210> 21 <211> 15 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:21 - GAPDH-B fragment) <400> 21 Thr Ala Ala Gln Asn Ile Ile Pro Ser Ser Thr Gly Ala Ala Lys <210> 22 <211> 20 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:22 - a GAPDH-B fragment) <400> 22 Asn Ile Leu Gly Tyr Thr Glu Asp Asp Val Val Ser Ser Asp Leu Asn Gly Asp Glu Arg <210> 23 <211> 12 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:23 - enolase fragment) <400> 23 Asn Val Asn Glu Thr Ile Gly Pro Ala Leu Ile Lys <210> 24 <211> 15 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:24 - enolase fragment) <400> 24 Val Asn Gln Ile Gly Thr Leu Thr Glu Ser Ile Gln Ala Ala Lys <210> 25 <211> 12 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:25 - enolase fragment) <400> 25 Trp Leu Thr Tyr Glu Gln Leu Ala Asp Leu Tyr Lys <210> 26 <211> 11 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:26 - CssI fragment) <400> 26 Val Ala Gln Glu Ile Ile Asn Pro Gly Pro Lys <210> 27 <211> 10 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:27 - Catalase B fragment) <400> 27 Phe Gly Phe Asp Leu Leu Asp Pro Thr Lys <210> 28 <211> 9 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:28 - CssI fragment) <400> 28 Ser Ile Ser Phe Gln Leu Asp Cys Arg <210> 29 <211> 15 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:29 - CssI fragment) <400> 29 Glu Gly Ala Glu Gln Ser Ala Pro Gln Ala Glu His Ser Thr Lys <210> 30 <211> 12 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:30 - CssI fragment) <400> 30 Val Val Thr Thr Pro Tyr Thr Cys Asp Gln Val Lys <210> 31 <211> 14 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:31 - GAPDH-B fragment) <400> 31 Val Pro Thr Ser Asn Val Ser Val Val Asp Leu Thr Cys Arg <210> 32 <211> 9 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:32 - GAPDH-B fragment) <400> 32 Tyr Asp Thr Thr His Gly Gln Phe Lys <210> 33 <211> 15 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:33 - GAPDH-B fragment) <400> 33 Gly Thr Ile Glu Thr Tyr Asp Gln Gly Leu Ile Val Asn Gly Lys <210> 34 <211> 12 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:34 - catalase A fragment) <400> 34 Thr Gly Pro Ser Leu Leu Glu Asp Gln Ile Ala Arg <210> 35 <211> 172 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:35 - GAPDH-B fragment) <400> 35 Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val Ile Asn Asp Lys Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val His Ser Tyr Thr Ala Thr Gln Lys Val Val Asp Ala Pro Ser Asn Lys Asp Trp Arg Gly Gly Arg Thr Ala Ala Gln Asn Ile Ile Pro Ser Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Ser Leu Asn Gly Lys Leu Thr Gly Met Ala Met Arg Val Pro Thr Ser Asn Val Ser Val Val Asp Leu Thr Cys Arg Leu Glu Lys Gly Ala Ser Tyr Asp Glu Ile Lys Gln Ala Ile Lys Ala Ala Ser Glu Glu Gly Glu Leu Lys Asn Ile Leu Gly Tyr Thr Glu Asp Asp Val Val Ser Ser Asp Leu Asn Gly Asp Glu Arg Ser Ser Ile Phe Asp Ala Lys Ala Gly Ile Ser Leu Asn Pro Asn Phe Val Lys Leu Val Ala Trp Tyr Asp Asn Glu Trp <210> 36 <211> 368 <212> PRT

<213> Aspergillus fumigatus (SEQ ID N0:36 - IMDH B) <220>
<221> MISC FEATURE
<222> (176)..(176) <223> the amino acid at position 176 is Ala or Ser <220>
<221> MISC FEATURE
<222> (179)..(179) <223> the amino acid at position 179 is Leu or Ile <400> 36 Met Val Thr Thr Tyr Asn Ile Leu Val Leu Pro Gly Asp Gly Ile Gly Pro Glu Val Met Thr Glu Ala Val Lys Val Leu Lys Val Phe Glu Asn Glu His Arg Lys Phe Asn Leu Arg Gln Glu Leu Ile Gly Gly Cys Ser Ile Asp Ala His Gly Lys Ser Val Thr Glu Glu Val Lys Lys Ala Ala Leu Glu Ser Asp Ala Val Leu Phe Ala Ala Val Gly Gly Pro Lys Trp Asp His Ile Arg Arg Gly Leu Asp Gly Pro Glu Gly Gly Leu Leu Gln Leu Arg Lys Ala Met Asp Ile Tyr Ala Asn Leu Arg Pro Cys Ser Ala Ser Ser Pro Ser Ala Ser Ile Ala Lys Glu Phe Ser Pro Phe Arg Gln Glu Val Ile Glu Gly Val Asp Phe Val Val Val Arg Glu Asn Cys Gly Gly Ala Tyr Phe Gly Lys Lys Ile Glu Glu Glu Asp Tyr Ala Met Asp Glu Trp Gly Tyr Ser Glu Arg Glu Ile Gln Arg Ile Thr Arg Leu Xaa Ala Glu Xaa Ala Leu Arg His Asn Pro Pro Trp Pro Val Ile Ser Leu Asp Lys Ala Asn Val Leu Ala Ser Ser Arg Leu Trp Arg Arg Val Val Glu Lys Thr Met Thr Thr Glu Tyr Pro Gln Val Lys Leu Val His Gln Leu Ala Asp Ser Ala Ser Leu Ile Leu Ala Thr Asn Pro Arg Ala Leu Asn Gly Val Ile Leu Ala Asp Asn Thr Phe Gly Asp Met Ile Ser Asp Gln Ala Gly Ser Ile Val Gly Thr Leu Gly Val Leu Pro Ser Ala Ser Leu Asp Gly Leu Pro Ser Glu Thr Arg Lys Arg Thr Asn Gly Leu Tyr Glu Pro Thr His Gly Ser Ala Pro Thr Ile Ala Gly Gln Asn Ile Ala Asn Pro Val Ala Met Ile Leu Cys Val Ala Leu Met Phe Arg Tyr Ser Leu Asp Met Glu Thr Glu Ala Gln Arg Ile Glu Lys Ala Val Gln Gly Val Leu Asp Ala Gly Ile Arg Thr Pro Asp Leu Gly Gly Lys Ser Gly Thr Asn Glu Val Gly Asp A1a Ile Val Ala Ala Leu Gln Gly Ser Ser <210> 37 <211> 8 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:37 - IMDH B fragment) <220>
<221> MISC FEATURE
<222> (2)..(2) <223> the amino acid at position 2 is Ala or Ser <220>
<221> MISC FEATURE
<222> (5)..(5) <223> the amino acid at position 5 is Leu or Ile <400> 37 Leu Xaa Ala Glu Xaa Ala Leu Arg <210> 38 <211> 1226 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:38 - IMDH B incl introns) <220>
<221> mist feature <222> (579)..(581) <223> 579+580+581 enccode an Alanine or a Serine <220>

<221> misc feature <222> (588)..(590) <223> 588+589+590 enccode a Leucine or an Isoleucine <400>

atggtaactacttacaacatcctcgtcctccccggcgatgggatcggtcccgaggtcatg60 accgaagcggtcaaggtgctaaaggtctttgagaacgagcaccgaaagttcaacctccgg120 caagagctcatcggcggttgcagcatcgatgcgcacggaaaatccgtcacagaagaagtg180 aaaaaggccgctctggaatccgacgccgtgctcttcgcagcagtcggaggtcccaaatgg240 gaccatatccgtcgtggtcttgacgggccggagggaggcctgctgcagctccgcaaggcg300 atggacatctacgcgaatctcaggccgtgctcggccagttcgccgagtgcgtcgatcgcg360 aaggagtttagcccattccgccaggaagtgatcgagggcgtagatttcgtcgtggtgagg420 gagaactgcgggggagcgtatttcgggaagaagatcgaagaagaagattatggtacgtcg480 tttttaacaagcagtatgctttcgagactgactgtgttatttcagcgatggacgaatggg540 gctatagcgagcgcgagatccagcgcatcacccgcctcnnngcggaannngccctccgtc600 acaaccccccctggcccgtcatctccctggacaaagccaatgtgctcgcctcgtcgcggc660 tctggcggcgcgtcgttgaaaagaccatgaccactgagtatccccaggtgaagctcgtgc720 accagctggcagactcagcatcgctgattctagcgaccaacccgcgggcattgaacggtg780 tcatcttggctgacaacacattcggcgacatgatttctgaccaggccggttccatcgtcg840 ggacattgggcgtgcttcccagtgccagtctcgatggactacccagtgaaacaagaaagc900 ggacaaatggtctgtacgagccgacccatggatctgcaccgacgtacgtttcttcctttg960 ttacccgaattatcatgtttcactgaagcaagctgacaatcatctgcagaattgcgggcc1020 agaacatcgccaaccccgttgccatgatcctctgtgtggctctcatgttccgctattcgc1080 tagacatggagaccgaggcgcaacggatcgaaaaagcagtgcagggtgttcttgatgccg1140 ggatccgcacccctgatctgggtgggaaatcggggacgaatgaagttggggatgcaattg1200 ttgctgcgttgcagggtagttcataa 1226 <210> 39 <211> 1107 <212> DNA

<213> Aspergillus fumigatus (SEQ ID N0:39 - IMDH B coding) <220>
<221> misc feature <222> (526)..(528) <223> 527+527+528 encode an Alanine or a Serine <220>
<221> misc feature <222> (535)..(537) <223> 535+536+537 encode a Leucine or an Isoleucine <400>

atggtaactacttacaacatcctcgtcctccccggcgatgggatcggtcccgaggtcatg60 accgaagcggtcaaggtgctaaaggtctttgagaacgagcaccgaaagttcaacctccgg120 caagagctcatcggcggttgcagcatcgatgcgcacggaaaatccgtcacagaagaagtg180 aaaaaggccgctctggaatccgacgccgtgctcttcgcagcagtcggaggtcccaaatgg240 gaccatatccgtcgtggtcttgacgggccggagggaggcctgctgcagctccgcaaggcg300 atggacatctacgcgaatctcaggccgtgctcggccagttcgccgagtgcgtcgatcgcg360 aaggagtttagcccattccgccaggaagtgatcgagggcgtagatttcgtcgtggtgagg420 gagaactgcgggggagcgtatttcgggaagaagatcgaagaagaagattatgcgatggac480 gaatggggctatagcgagcgcgagatccagcgcatcacccgcctcnnngcggaannngcc540 ctccgtcacaaccccccctggcccgtcatctccctggacaaagccaatgtgctcgcctcg600 tcgcggctctggcggcgcgtcgttgaaaagaccatgaccactgagtatccccaggtgaag660 ctcgtgcaccagctggcagactcagcatcgctgattctagcgaccaacccgcgggcattg720 aacggtgtcatcttggctgacaacacattcggcgacatgatttctgaccaggccggttcc780 atcgtcgggacattgggcgtgcttcccagtgccagtctcgatggactacccagtgaaaca840 agaaagcggacaaatggtctgtacgagccgacccatggatctgcaccgacgattgcgggc900 cagaacatcgccaaccccgttgccatgatcctctgtgtggctctcatgttccgctattcg960 ctagacatggagaccgaggcgcaacggatcgaaaaagcagtgcagggtgttcttgatgcc1020 gggatccgca cccctgatct gggtgggaaa tcggggacga atgaagttgg ggatgcaatt 1080 gttgctgcgt tgcagggtag ttcataa 1107 <210> 40 <211> 1093 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:40 - IMDH B 2 - predicted 0RF) <400>

atgccgtcatataacattgtcgttttcgctggggaccactgtggtccggaggtaagttcg60 gtcctgcgcgtcatcgagaagtgccgtgacgatgctaccttcaacctccaggatcaattg120 ctcggtggtgtaagttcgatcgatgctaccggatctccccttaccgacgaagctcttaac180 gccgcaaagaacgccgatgccgttctcctcggtgccattggcggtcccaaatggggcact240 ggcgccgtccgccccgaacagggcctcctccgtctgcgcaaggagatgggcacattcggt30'0 aacctccgcccctgcaacttcgccgccccgtcgctggtcgacggctcccctctccgcccc360 gaagtctgccgcggcgtcgaCttcaacattatccgcgaactgaccggtggcatctacttc420 ggcgaccgcaaggaggacgacggcagcggcttcgccatggacacggagccgtactcccgc480 gcggagatcgagcgcatcacccgccttgcggcccacctcgctctgcagcacaacccccct540 cttcccgtgtggagcttggacaaggccaacgtcctcgcgacgagccggctgtggcggaag60'0 accgtgacggaggtcatggccaaggagttcccccagctcaaggtggagcaccagctcatt660 gactccgcggccatgatcatggtcaaggagcctagaaagcttaacggtattgttgtcact720 agcaacctgttcggtgacatcatcagtgatgaagccagcgttatccctggttctctggga780 ctcttgcccagcgcaagcttgagcggcattcctgacggaaagaccaaggtcaatggtatc840 tatgagcctattcacggttctgcccctgacattgccggcaagggcatcgttaaccccgtc900 gccgccattctctctgtcgccatgatgatgcagtactccctgaaccgtatggatgacgcc960 agggccatcgagacggccgtccgcaatgtgatcgaggccggtatccgcactgccgatatt1020 ggcggcaagtcgacaactagcgaggtcggtgacgctgttgctgccgagctggagaagctg1080 ttgaagcaatagt 1093 <210> 41 <211> 363 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:41 - IMDH B 2 - aa) <400> 41 Met Pro Ser Tyr Asn Ile Val Val Phe Ala Gly Asp His Cys Gly Pro Glu Val Ser Ser Val Leu Arg Val Ile Glu Lys Cys Arg Asp Asp Ala Thr Phe Asn Leu Gln Asp Gln Leu Leu Gly Gly Val Ser Ser Ile Asp Ala Thr Gly Ser Pro Leu Thr Asp Glu Ala Leu Asn Ala Ala Lys Asn Ala Asp Ala Val Leu Leu Gly Ala Ile Gly Gly Pro Lys Trp Gly Thr Gly Ala Val Arg Pro Glu Gln Gly Leu Leu Arg Leu Arg Lys Glu Met Gly Thr Phe Gly Asn Leu Arg Pro Cys Asn Phe Ala Ala Pro Ser Leu Val Asp Gly Ser Pro Leu Arg Pro Glu Val Cys Arg Gly Val Asp Phe Asn Ile Ile Arg Glu Leu Thr Gly Gly Ile Tyr Phe Gly Asp Arg Lys Glu Asp Asp Gly Ser Gly Phe Ala Met Asp Thr Glu Pro Tyr Ser Arg Ala Glu Ile Glu Arg Ile Thr Arg Leu Ala Ala His Leu Ala Leu Gln His Asn Pro Pro Leu Pro Val Trp Ser Leu Asp Lys Ala Asn Val Leu Ala Thr Ser Arg Leu Trp Arg Lys Thr Val Thr Glu Val Met Ala Lys Glu Phe Pro Gln Leu Lys Val Glu His Gln Leu Ile Asp Ser Ala Ala Met Ile Met Val Lys Glu Pro Arg Lys Leu Asn Gly Ile Val Val Thr Ser Asn Leu Phe Gly Asp Ile Ile Ser Asp Glu Ala Ser Val Ile Pro Gly Ser Leu Gly Leu Leu Pro Ser Ala Ser Leu Ser Gly Ile Pro Asp Gly Lys Thr Lys Val Asn Gly Ile Tyr Glu Pro Ile His Gly Ser A1a Pro Asp Ile Ala Gly Lys Gly Ile Val Asn Pro Val Ala Ala Ile Leu Ser Val Ala Met Met Met Gln Tyr Ser Leu Asn Arg Met Asp Asp Ala Arg Ala Ile Glu Thr Ala Val Arg Asn Val Ile Glu Ala Gly Ile Arg Thr Ala Asp Ile Gly Gly Lys Ser Thr Thr Ser Glu Val Gly Asp Ala Val Ala Ala Glu Leu Glu Lys Leu Leu Lys Gln <210> 42 <211> 18 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:42 - enolase primer) <400> 42 atgcctatct ccaagatc 18 <210> 43 <211> 15 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:43 - en0lase primer) <400> 43 caggttgacg gcagt 15 <210> 44 <211> 18 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:44 - IMDH B primer) <400> 44 atggtaacta cttacaac 18 <210> 45 <211> 18 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:45 - IMDH B primer) <400> 45 tgaactaccc tgcaacgc 18 <210> 46 <211> 1233 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:46 - IMDH B insert in pBAD) <400> 46 atgggttctg gatccggtga tgacgatgac aagctcgccc ttatggtaac tacttacaac 60 atcctcgtcc tccccggcga tgggatcggt cccgaggtca tgaccgaagc ggtcaaggtg 120 ctaaaggtct ttgagaacga gcaccgaaag ttcaacctcc ggcaagagct catcggcggt 180 tgcagcatcg atgcgcacgg aaaatccgtc acagaagaag tgaaaaaggc cgctctggaa 240 tccgacgccg tgctcttcgc agcagtcgga ggtcccaaat gggaccatat ccgtcgtggt 300 cttgacgggccggagggaggcctgctgcagctccgcaaggcgatggacatctacgcgaat360 ctcaggccgtgctcggccagttcgccgagtgcgtcgatcgcgaaggagtttagcccattc420 cgccaggaagtgatcgagggcgtagatttcgtcgtggtgagggagaactgcgggggagcg480 tatttcgggaagaagatcgaagaagaagattatgcgatggacgaatggggctatagcgag540 cgcgagatccagcgcatcacccgcctctcggcggaaattgCCCtCCgtCaCaaCCCCCCC600 tggcccgtcatctccctggacaaagccaatgtgctcgcctcgtcgcggctctggcggcgc660 gtcgttgaaaagaccatgaccactgagtatccccaggtgaagctcgtgcaccagctggca720 gactcagcatcgctgattctagcgaccaacccgcgggcattgaacggtgtcatcttggct780 gacaacacattcggcgacatgatttctgaccaggccggttccatcgtcgggacattgggc840 gtgcttcccagtgccagtctcgatggactacccagtgaaacaagaaagcggacaaatggt900 ctgtacgagccgacccatggatctgcaccgacaattgcgggccagaacatcgccaacccc960 gttgccatgatcctctgtgtggctctcatgttccgctattcgctagacatggagaccgag1020 gcgcaacggatcgaaaaagcagtgcagggtgttcttgatgccgggatccgcacccctgat1080 ctgggtgggaaatcggggacgaatgaagttggggatgcaattgttgctgcgttgcagggt1140 agttcaaagggcgagcttgaaggtaagcctatccctaaccctctcctcggtctcgattct1200 acgcgtaccggtcatcatcaccatcaccattga 1233 <210> 47 <211> 410 <212> PRT
<213> Aspergillus fumigatus (SEQ ID N0:47 - IMDH B insert in pBAD) <400> 47 Met G1y Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala Leu Met Val Thr Thr Tyr Asn Ile Leu Val Leu Pro Gly Asp Gly Ile Gly Pro Glu Val Met Thr Glu Ala Val Lys Val Leu Lys Val Phe Glu Asn Glu His Arg Lys Phe Asn Leu Arg Gln Glu Leu Ile Gly Gly Cys Ser Ile Asp Ala His Gly Lys Ser Val Thr Glu Glu Val Lys Lys Ala Ala.Leu Glu Ser Asp Ala Val Leu Phe Ala Ala Val Gly Gly Pro Lys Trp Asp His Ile Arg Arg Gly Leu Asp Gly Pro Glu Gly Gly Leu Leu Gln Leu Arg Lys Ala Met Asp Ile Tyr Ala Asn Leu Arg Pro Cys Ser Ala Ser Ser Pro Ser Ala Ser Ile Ala Lys Glu Phe Ser Pro Phe Arg Gln Glu Val Ile Glu G1y Val Asp Phe Val Val Val Arg Glu Asn Cys Gly Gly Ala Tyr Phe Gly Lys Lys Ile Glu Glu Glu Asp Tyr Ala Met Asp Glu Trp Gly Tyr Ser Glu Arg Glu Ile Gln Arg Ile Thr Arg Leu Ser Ala Glu Ile Ala Leu Arg His Asn Pro Pro Trp Pro Val Ile Ser Leu Asp Lys Ala Asn Val Leu Ala Ser Ser Arg Leu Trp Arg Arg Val Val Glu Lys Thr Met Thr Thr Glu Tyr Pro Gln Val Lys Leu Val His Gln Leu Ala Asp Ser Ala Ser Leu Ile Leu Ala Thr Asn Pro Arg Ala Leu Asn Gly Val Ile Leu Ala Asp Asn Thr Phe Gly Asp Met Ile Ser Asp Gln Ala Gly Ser Ile Val Gly Thr Leu Gly Val Leu Pro Ser Ala Ser Leu Asp Gly Leu Pro Ser Glu Thr Arg Lys Arg Thr Asn Gly Leu Tyr Glu Pro Thr His Gly Ser Ala Pro Thr Ile Ala Gly Gln Asn Ile Ala Asn Pro Val Ala Met Ile Leu Cys Val Ala Leu Met Phe Arg Tyr Ser Leu Asp Met Glu Thr Glu Ala Gln Arg Ile Glu Lys Ala Val Gln Gly Val Leu Asp Ala Gly Ile Arg Thr Pro Asp Leu Gly Gly Lys Ser Gly Thr Asn Glu Val Gly Asp Ala Ile Val Ala Ala Leu Gln Gly Ser Ser Lys Gly Glu Leu Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His His His His <210> 48 <211> 1443 <212> DNA
<213> Aspergillus fumigatus (SEQ ID N0:48 - enolase insert in pBAD) <400>

atgggctctggatccggtgatgacgatgacaagctcgcccttatgcctatctccaagatc60 cacgctcgttccgtgtacgactctcgcggtaaccccaccgttgaggtggacgttgtcacc120 gagaccggtttgcaccgtgctattgttccttctggagcttccaccggccagcacgaggct180 cacgagctccgtgacggtgataagacccagtggggcggcaagggtgtcctcaaggctgtc240 aagaatgtcaacgagaccattggccctgctctcatcaaggagaacatcgatgtgaaggac300 cagtctaaggtcgacgagttccttaacaagcttgacgggactgccaacaagtccaacctc360 ggtgctaatgccatcctcggtgtcagcttggctgttgccaaggctggtgctgctgagaag420 ggtgtccctctctacgctcacatctccgaccttgccggtaccaagaagccctatgtcctt480 cccgttcccttccagaacgtcctgaacggcggctctcacgccggtggtcgcctcgctttc540 caggagttcatgatcgtccctgactccgctccctctttctccgaggccctccgccagggt600 gctgaggtctaccagaagctcaaggctctggccaagaagaagtacggccagtccgctggc660 aacgttggtgacgagggtggtgttgctcccgatattcagaccgccgaggaggctctcgac720 ctgatcaccgaggccatcgagcaggccggctacaccggcaagatcaagatcgctatggac780 gttgcctccagcgagttctacaaggccgacgtcaagaagtacgaccttgacttcaagaac840 cccgagagcgacccctccaagtggctcacctacgagcagcttgccgacctctacaagtcc900 cttgctgccaagtaccccattgtcagcattgaggaccccttcgctgaggatgattgggag960 gcctggagctacttctacaagacctccgacttccagattgttggtgatgacctgactgtt1020 actaaccctgggcgtatcaagaaggccatcgagctcaagtcctgcaacgccctcctgctc1080 aaggtcaaccagatcggtaccctcaccgagtccatccaggccgccaaggactcctacgcc1140 gacaactggggtgtcatggtctcccaccgctctggtgagactgaggacgtcaccattgcc1200 gacattgctgtcggtctgcgctctggccagatcaagaccggtgctccttgccgttccgag1260 cgtctggctaagctgaaccagatcctccgtatcgaggaggagctcggcgagaatgccgtc1320 tacgctggttccaagttccgcactgccgtcaacctgaagggcgagcttgaaggtaagcct1380 atccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccat1440 tga 1f.43 <210> 49 <211> 480 <212> PRT
<213> Aspergillus fumigatus (enolase insert in pBAD) <400> 49 Met Gly Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala Leu Met Pro Ile Ser Lys Ile His Ala Arg Ser Val Tyr Asp Ser Arg Gly Asn Pro Thr Val Glu Val Asp Val Val Thr Glu Thr Gly Leu His Arg Ala Ile Val Pro Ser Gly Ala Ser Thr Gly Gln His Glu Ala His Glu Leu Arg Asp Gly Asp Lys Thr Gln Trp Gly Gly Lys Gly Val Leu Lys Ala Val Lys Asn Val Asn Glu Thr Ile Gly Pro Ala Leu Ile Lys Glu Asn Ile Asp Val Lys Asp Gln Ser Lys Val Asp Glu Phe Leu Asn Lys Leu Asp Gly Thr Ala Asn Lys Ser Asn Leu Gly Ala Asn Ala Ile Leu Gly Val Ser Leu Ala Val Ala Lys Ala Gly A1a Ala Glu Lys Gly Val Pro Leu Tyr Ala His Ile Ser Asp Leu Ala Gly Thr Lys Lys Pro Tyr Val Leu Pro Val Pro Phe Gln Asn Val Leu Asn Gly Gly Ser His Ala Gly Gly Arg Leu Ala Phe Gln Glu Phe Met Ile Val Pro Asp Ser Ala Pro Ser Phe Ser Glu Ala Leu Arg Gln Gly Ala Glu Val Tyr Gln Lys Leu Lys Ala Leu Ala Lys Lys Lys Tyr Gly Gln Ser Ala Gly Asn Val Gly Asp Glu Gly Gly Val Ala Pro Asp Ile Gln Thr Ala Glu Glu Ala Leu Asp Leu Ile Thr Glu Ala Ile Glu Gln Ala Gly Tyr Thr Gly Lys Ile Lys Ile Ala Met Asp Val Ala Ser Ser Glu Phe Tyr Lys Ala Asp Val Lys Lys Tyr Asp Leu Asp Phe Lys Asn Pro Glu Ser Asp Pro Ser Lys Trp Leu Thr Tyr Glu Gln Leu Ala Asp Leu Tyr Lys Ser Leu Ala Ala Lys Tyr Pro Ile Val Ser Ile Glu Asp Pro Phe Ala Glu Asp Asp Trp Glu Ala Trp Ser Tyr Phe Tyr Lys Thr Ser Asp Phe Gln Ile Val Gly Asp Asp Leu Thr Val Thr Asn Pro Gly Arg Ile Lys Lys Ala Ile Glu Leu Lys Ser Cys Asn Ala Leu Leu Leu Lys Val Asn Gln Ile Gly Thr Leu Thr Glu Ser Ile Gln Ala Ala Lys Asp Ser Tyr Ala Asp Asn Trp Gly Val Met Val Ser His Arg Ser Gly Glu Thr Glu Asp Val Thr Ile Ala Asp Ile Ala Val Gly Leu Arg Ser Gly Gln Ile Lys Thr Gly Ala Pro Cys Arg Ser Glu Arg Leu Ala Lys Leu Asn Gln Tle Leu Arg Ile Glu Glu Glu Leu Gly Glu Asn Ala Val Tyr Ala Gly Ser Lys Phe Arg Thr Ala Val Asn Leu Lys Gly Glu Leu Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His His His His

Claims

1. An isolated antibody capable of binding an extracellular Aspergillus fumigatus polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1), hydrophobin (SEQ ID
NO:2), GAPDH-B (SEQ ID NO:3), and catalase A (SEQ ID NO:6).

2. The antibody of claim 1, wherein the antibody binds said polypeptide with a dissociation constant of less than 10 -7M, e.g. less than 5 × 10 -8M, such as less than 10 -8M, e.g. less than 5 × 10 -9M, such as less than 10 -9M, e.g.
less than 5 ×
10 -10M, such as less than 10 -10M, e.g. less than 5 × 10 -11M, such as less than 10-11M, e.g. less than 5 × 10 -12M, such as less than 10 -12M, e.g. less than 5 × 10-13M, such as less than 10 -13M, e.g. less than 5 × 10 -14M, such as less than 10-14M, e.g. less than 5 × 10 -15M, or less than 10 -15M.

3. The antibody of any of the preceding claims, wherein the antibody is selected from the group consisting of IgG, IgA, IgE, IgM and IgD, wherein IgG
preferably is IgG1.

4. The antibody of any of the preceding claims, wherein the antibody is capable of binding an intact Aspergillus fumigatus cell.

5. The antibody of any of the preceding claims, wherein the antibody, or at least an Fab fragment thereof, is capable of reducing the adhesion of Aspergillus fumigatus conidia to lung epithelia in an in vitro assay, preferably reducing said adhesion with at least 20%, e.g. at least 40%, or at least 60%.

6. The antibody of any of the preceding claims, wherein the antibody or at least an Fab. fragment thereof, is capable of reducing the germination of Aspergillus fumigatus conidia in an in vitro assay, preferably reducing said adhesion with at least 20%, e.g. at least 40%, or at least 60%.

7. The antibody of any of claims 1-6, wherein the antibody is polyclonal.

8. The antibody of any of claims 1-6, wherein the antibody is monoclonal.

9. The antibody of claim 8, wherein the antibody is a chimeric, human or humanised antibody.

10. The antibody of claim 8, wherein the antibody is a human antibody.

11. The antibody of any of the preceding claims, wherein the antibody is purified.

12. The antibody of any of the preceding claims, wherein the antibody is further capable of binding a homologous polypeptide, wherein the homologous polypeptide has a sequence identity of 39% or more, such as 42% or more, e.g.
48% or more, such as 68% or more, e.g. 80% or more, such as 90% or more, to a polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1), hydrophobin (SEQ ID
NO:2), GAPDH-B (SEQ ID NO:3), and catalase A (SEQ ID NO:6).

13. The antibody of claim 12, wherein said homologous polypeptide originates from - an Aspergillus species, such as Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzea, - Neurospora crassa, - Saccharomyces cerevisiae, - a Candida species such as Candida albicans, - a Coccidioides species, such as Coccidioides posadasii, or Coccidioides immitis, - a Cryptococcus species, such as Cryptococcus neoformans var. neoformans, - a Fusarium species, - a Pneumocystis species, - a Penicillium species, or - Histoplasma capsulatum.

14. The antibody of claim 13, wherein said homologous polypeptide originates from - an Aspergillus species, such as Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzea, - Candida albicans, - Coccidioides posadasii, or - Cryptococcus neoformans var. neoformans.

15. The antibody of claim 14, wherein said homologous polypeptide originates from an Aspergillus species, such as Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzea,

16. The antibody of claim 15, wherein said homologous polypeptide originates from Aspergillus fumigatus.

17. The antibody of claim 16, wherein the said homologous polypeptide is the polypeptide of SEQ ID NO:41.

18. The antibody of any of claims 12-17, wherein said homologous polypeptide is extracellular.

19. The antibody of any of the preceding claims, wherein the antibody further is capable of binding an intact cell of any one or more of - an Aspergillus species other than Aspergillus fumigatus, such as Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzea, - Neurospora crassa, - Saccharomyces cerevisiae, - a Candida species such as Candida albicans, - a Coccidioides species, such as Coccidioides posadasii, or Coccidioides immitis, - a Cryptococcus species, such as Cryptococcus neoformans var. neoformans, - a Fusarium species, - a Pneumocystis species, - a Penicillium species, or - Histoplasma capsulatum.

20. The antibody of any of claims 1-11, wherein the antibody is not capable of binding an intact cell of any of - Neurospora crassa, - Saccharomyces cerevisiae, - Candida albicans, - Coccidioides posadasii, or Coccidioides immitis, - Cryptococcus neoformans var. neoformans, or - Histoplasma capsulatum.

21. The antibody of any of the preceding claims, wherein the antibody is capable of binding a polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1) and catalase A (SEQ
ID NO:6).

22. The antibody of any of the preceding claims, wherein the antibody is capable of binding a polypeptide selected from the group consisting of isopropylmalate dehydrogenase B (SEQ ID NO:36) and Cssl (SEQ ID NO:1).

23. The antibody of any of the preceding claims, wherein the antibody is capable of binding isopropylmalate dehydrogenase B (SEQ ID NO:36).

24. The antibody of claim 23, wherein the antibody is capable of binding an epitope which comprises one or more of the residues of a region of SEQ ID NO:36 selected from the group consisting of: Ser67- Leu71, A1a74-Trp80, Ser191-Arg205, Leu268-Leu273, His292-Pro296, Glu355=Ile360, Asp193-Glu209, Asp193-Ala199, Ile15-Val19, Val75-Trp80, Pro11-Glu18 and the region defined by SEQ ID NO:37, preferably an epitope which is entirely consisting of residues comprised within said region.

25. A pharmaceutical composition comprising an antibody as defined in any of claims 1-24 and a pharmaceutically-acceptable carrier.

26. An antibody as defined in any of claims 1-24 or a composition as defined in claim 25 for use as a medicament.

27. Use of an antibody as defined in any of claims 1-24 or a composition as defined in claim 25 for the manufacture of a medicament for the treatment or prevention of fungal infections.

28. Use of claim 27, wherein the medicament is a medicament for the treatment or prevention of Aspergillus infections, preferably Aspergillus fumigatus infections.

29. Use of claim 27, wherein the medicament is a medicament for the treatment or prevention of a fungal disease selected from the group consisting of: invasive aspergillosis, aspergilloma, and allergic aspergillosis, such as allergic bronchopulmonary aspergillosis.

30. A composition comprising one or more Aspergillus fumigatus polypeptides selected from the group of polypeptides comprising SEQ ID NO:36, fragments thereof and variants thereof, fragments of SEQ ID NO:1 of less than 259 amino-acid residues in length, such as less than 200, preferably less than 150, such as less than 100, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID NO:7,8,17,26,28,29 and/or 30 and variants of said fragments;
fragments of SEQ ID NO:2 of less than 106 amino-acid residues in length, such as less than 75, preferably less than 50, such as less than 25 residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID NO:9,10,18 and/or 19 and variants of said fragments;
polypeptides comprising SEQ ID NO:3, fragments thereof and variants thereof, with the proviso that if the polypeptide is a fragment of SEQ ID NO:3, that this fragment is not the fragment set forth in SEQ ID NO:35;
fragments of SEQ ID NO:4 of less than 437 amino-acid residues in length, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID NO:13,14,23,24 and/or 25 and variants of said fragments;
fragments of SEQ ID NO:5 of less than 727 amino-acid residues in length, e.g.
less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID NO:15,16 and/or 27 and variants of said fragments;
and fragments of SEQ ID NO:6 of less than 748 amino-acid residues in length, e.g.
less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID NO:34 and variants of said fragments.

31. An Aspergillus fumigates polypeptide selected from the group of polypeptides comprising SEQ ID NO:36, fragments thereof and variants thereof, fragments of SEQ ID NO:1 of less than 259 amino-acid residues in length, such as less than 200, preferably less than 150, such as less than 100, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID NO:7,8,17,26,28,29 and/or 30 and variants of said fragments;
fragments of SEQ ID NO:2 of less than 106 amino-acid residues in length, such as less than 75, preferably less than 50, such as less than 25 residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID NO:9,10,18 and/or 19 and variants of said fragments;
polypeptides comprising SEQ ID NO:3, fragments thereof and variants thereof, with the proviso that if the polypeptide is a fragment of SEQ ID NO:3, that this fragment is not the fragment set forth in SEQ ID NO:35;

fragments of SEQ ID NO:4 of less than 437 amino-acid residues in length, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ ID NO:13,14,23,24 and/or 25 and variants of said fragments;
fragments of SEQ ID NO:5 of less than 727 amino-acid residues in length, e.g.
less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID NO:15,16 and/or 27 and variants of said fragments;
and fragments of SEQ 1D NO:6 of less than 748 amino-acid residues in length, e.g.
less than 400, such as less than 200, preferably less than 100, such as less than 75, e.g. less than 50, such as less than 25 amino-acid residues in length comprising one or more residues of the amino-acid sequences set forth in SEQ
ID NO:34 and variants of said fragments.

32. The polypeptide of claim 31, wherein the polypeptide is a fragment comprising one or more residues of the amino-acid sequences set forth in SEQ ID NOs: 7-27 and/or 37, or a variant of said fragment.

33. The polypeptide of claim 32, wherein the polypeptide is a fragment comprising one or more residues of the amino-acid sequences set forth in SEQ ID NOs: 7-16, or a variant of said fragment.

34. The polypeptide of claim 32, wherein the polypeptide is a fragment comprising one or more residues of the amino-acid sequences set forth in SEQ ID NOs: 17-25 and/or SEQ ID NO:14, or a variant of said fragment.

35. The polypeptide of claim 32, wherein the polypeptide is a fragment comprising one or more residues of the amino-acid sequences set forth in SEQ ID NO: 18, 19, 26, 27, and/or 37, or a variant of said fragment.

36. A polynucleotide encoding a polypeptide as defined in any of claims 31-35.

37. An expression vector comprising a polynucleotide as defined in claim 36.

38. A host cell transformed or transfected with a polynucleotide as defined in claim 36 and/or an expression vector as defined in claim 37.

39. A pharmaceutical composition comprising a polypeptide as defined in any of claims 31-35 or a polynucleotide as defined in claim 36 and a pharmaceutically-acceptable carrier.

40. A polypeptide as defined in any of claims 31-35, or a polynucleotide as defined in claim 36 for use as a medicament.

41. Use of a polypeptide as defined in any of claims 31-35, a polynucleotide as defined in claim 36 for the manufacture of a medicament for the immunisation of a mammal against fungal infections.

42. The use of claim 41, wherein said mammal is a human being.

43. A method for raising specific antibodies to a polypeptide selected from the group of polypeptides set forth in SEQ ID NO:1,2,3,6 and 36 in a non-human mammal comprising the steps of a. providing a polypeptide selected from the group of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1), hydrophobin (SEQ ID
NO:2), GAPDH (SEQ ID NO:3), and catalase A (SEQ ID NO:6), or a polypeptide as defined in any of claims 31-35, or a cell expressing any of these polypeptides, b. introducing a composition comprising said polypeptide or said cell into said animal, c. raising antibodies in said animal, and d. isolating and optionally purifying the antibodies.

44. The method of claim 43, wherein the raising of antibodies is done in a transgenic animal which is capable of producing human antibodies.

45. The method of claim 43 or 44, wherein the polypeptide that is provided is isopropylmalate dehydrogenase B (SEQ ID NO:36) or a fragment thereof, or a variant of said polypeptide.

46. The method of claim 43 or 44, wherein the polypeptide that is provided is Cssl (SEQ ID NO:1) or a fragment thereof, or a variant of said polypeptide.

47. The method of claim 43 or 44, wherein the polypeptide that is provided is hydrophobin (SEQ ID NO:2) or a fragment thereof, or a variant of said polypeptide.

48. The method of claim 43 or 44, wherein the polypeptide that is provided is GAPDH-B (SEQ ID NO:3) or a fragment thereof, or a variant of said polypeptide.

49. The method of claim 43 or 44, wherein the polypeptide that is provided is catalase A (SEQ ID NO:6) or a fragment thereof, or a variant of said polypeptide.

50. A method for identifying a binding partner of a polypeptide selected from the group of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID
NO:1), hydrophobin (SEQ ID NO:2), GAPDH-B (SEQ ID NO:3), enolase (SEQ
ID NO:4), catalase B (SEQ ID NO:5) and catalase A (SEQ ID NO:6), comprising the steps of a. providing a polypeptide as defined in any of claims 31-35 or a polypeptide selected from the group of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1), hydrophobin (SEQ ID NO:2), GAPDH-B (SEQ ID NO:3), catalase B (SEQ ID NO:5), and catalase A (SEQ ID NO:6), b. contacting said polypeptide with a putative binding partner, and c. determining whether said putative binding partner is capable of binding to said polypeptide.

51. The method of claim 50, wherein the putative binding partner is a host-derived molecule.

52. The method of any of claims 50-51, wherein said method is repeated for a plurality of putative binding partners.

53. A method for identifying a compound with antifungal activity comprising the steps of a. providing a sensitised cell which has a reduced level of a polypeptide selected from the group of SEQ ID NOs:1,2,3,5,6, and 36 and b. determining the sensitivity of said cell to a putative antifungal compound, for instance by a growth assay.

54. A method for identifying an inhibitor of an extracellular Aspergillus polypeptide selected from the group of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1), GAPDH (SEQ ID NO:3), and catalase A (SEQ ID NO:6), comprising the steps of a. providing two cells which differ in the level of a polypeptide selected from the group of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID
NO:1), GAPDH (SEQ ID NO:3), and catalase A (SEQ ID NO:6), b. determining the sensitivity of said cells to a putative inhibitor, for instance by a growth assay, and c. determining whether said two cells are differently affected by the presence of said putative inhibitor.

55. The method of claim 54, wherein the two cells differ in the copy number of said polypeptide.

56. The method of claim 54, wherein the two cells differ in the activity of said polypeptide.

57. A method of diagnosing fungal, preferably Aspergillus fumigatus, infection comprising the steps of a. providing a sample from an individual, b. contacting said sample with an indicator moiety capable of specifically recognising and binding a polypeptide selected from the group of isopropylmalate dehydrogenase B (SEQ ID NO:36), Cssl (SEQ ID NO:1), hydrophobin (SEQ ID NO:2), GAPDH-B (SEQ ID NO:3), and catalase A (SEQ ID
NO:6), and c. determining whether a signal has been generated by the indicator moiety.

58. The method of the preceding claim, wherein said indicator moiety is or comprises an antibody, such as an antibody as defined in any of claims 1-24.

59. A kit for the detection of fungal material, preferably intact fungal cells, most preferably intact Aspergillus fumigatus cells, in a biological sample comprising a. an indicator moiety capable of specifically recognising and binding a polypeptide selected from the group of isopropylmalate dehydrogenase B
(SEQ ID NO:36), Cssl (SEQ ID NO:1), hydrophobin (SEQ ID NO:2), GAPDH-B (SEQ ID NO:3), and catalase A (SEQ ID NO:6), and b. one or more of: a buffer for promoting binding of the indicator moiety to the fungal material; a reagent for generating a detectable signal; and written instructions to the user.

60. The kit of claim 59, wherein said indicator is or comprises an antibody, such as an antibody as defined in any of claims 1-24.