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AU2006249236B2 - A Method for Identification, Isolation and Production of Antigens to a Specific Pathogen - Google Patents

A Method for Identification, Isolation and Production of Antigens to a Specific Pathogen Download PDF

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AU2006249236B2
AU2006249236B2 AU2006249236A AU2006249236A AU2006249236B2 AU 2006249236 B2 AU2006249236 B2 AU 2006249236B2 AU 2006249236 A AU2006249236 A AU 2006249236A AU 2006249236 A AU2006249236 A AU 2006249236A AU 2006249236 B2 AU2006249236 B2 AU 2006249236B2
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antigens
protein
pathogen
screening
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Agnieszka Dryla
Hildegard Etz
Claire M. Fraser
Steven Gill
Martin Hafner
Tamas Henics
Christoph Klade
Andreas Meinke
Duc Bui Minh
Eszter Nagy
Brigitte Tempelmaier
Uwe Von Ahsen
Oresta Vytvytska
Thomas Weichhart
Wolfgang Zauner
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Valneva Austria GmbH
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Intercell Austria AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Description

AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Intercell AG Actual Inventors Andreas MEINKE Eszter NAGY Uwe VON AHSEN Christoph KLADE Tamas HENICS Wolfgang ZAUNER Duc, Bui MINH Oresta VYTVYTSKA Hildegard ETZ Agnieszka DRYLA Thomas WEICHART Martin HAFNER Address for service is: WRAY & ASSOCIATES Level 4, The Quadrant 1 William Street Perth, WA 6000 Attorney code: WR Invention Title: A Method for Identification, Isolation and Production of Antigens to a Specific Pathogen The following statement is a full description of this invention, including the best method of performing it known to me: 1/1 1/2 A method for identification, isolation and production of antigens to a specific pathogen The invention relates to a method for identification, isolation and production of antigens to a specific pathogen as well as new antigens suitable for use in a vaccine for a given type of animal or for humans. Vaccines can save more lives (and resources) than any other medi cal intervention. Owing to world-wide vaccination programmes the incidence of many fatal diseases has been decreased drastically. Although this notion is valid for a whole panel of diseases, e.g. diphtheria, pertussis, measles and tetanus, there are no effec tive vaccines for numerous infectious disease including most vi ral infections, such as HIV, HCV, CMV and many others. There are also no effective vaccines for other diseases, infectious or non infectious, claiming the lifes of millions of patients per year including malaria or cancer. In addition, the rapid emergence of antibiotic-resistant bacteria and microorganisms calls for alter native treatments with vaccines being a-logical choice. Finally, the great need for vaccines is also illustrated by the fact that infectious diseases, rather than cardiovascular disorders or can cer or injuries remain the largest cause of death and disability in the world. Several established vaccines consist of live attenuated organisms where the risk of reversion to the virulent wild-type strain exists. In particular in immunocompromised hosts this can be a live threatening scenario. Alternatively, vaccines are administe red as a combination of pathogen-derived antigens together with compounds that induce or enhance immune responses against these antigens (these compounds are commonly termed adjuvant), since these subunit vaccines on their own are generally not effective. Whilst there is no doubt that the above vaccines are valuable medical treatments, there is the disadvantage that, due to their complexity, severe side effects can be evoked, e.g. to antigens that are contained in the vaccine that display cross-reactivity with molecules expressed by cells of vaccinated individuals. In addition, existing requirements from regulatory authorities, e.g.
-2 the World Health Organization (WHO), the Food and Drug Admin istration (FDA), and their European counterparts, for exact specification of vaccine composition and mechanisms of induction of immunity, are difficult to meet. Some widely used vaccines are whole cell-vaccines (attenuated bacteria or viruses (e.g. Bacille Calmette-Guerin (BCG) (tubercu losis), Measles, Mumps, Rubella, Oral Polio Vaccine (Sabin), killed bacteria or viruses (e.g. Pertussis, Inactivated polio vaccine (Salk)), subunit-vaccines (e.g. Toxoid (Diphtheria, Teta nus)), Capsular polysaccharide (H. influenzae type B), Yeast re combinant subunit (Hepatitis B surface protein). A vaccine can contain a whole variety of different antigens. Ex amples of antigens are whole-killed organisms such as inactivated viruses or bacteria, fungi, protozoa or even cancer cells. Anti gens may also consist of subfractions of these organisms/tissues, of proteins, or, in their most simple form, of peptides. Antigens can also be recognized by the immune system in form of glycosy lated proteins or peptides and may also be or contain polysaccha rides or lipids. Short peptides can be used since for example. cytotoxic T-cells (CTL) recognize antigens in form of short usu' ally 8-11 amino acids long peptides in conjunction with major histocompatibility complex (MHC). B-cells can recognize linear epitopes as short as 4-5 amino acids, as well as three dimen sional structures (conformational epitopes). In order to obtain sustained, antigen-specific immune responses, adjuvants need to trigger immune cascades that involve all cells of the immune sys tem necessary. Primarily, adjuvants are acting, but are not re stricted in their mode of action, on so-called antigen presenting cells (APCs). These cells usually first encounter the antigen(s) followed by presentation of processed or unmodified antigen to immune effector cells. Intermediate cell types may also be in volved. Only effector cells with the appropriate specificity are activated in a productive immune response. The adjuvant- may also locally retain antigens and co-injected other factors. In addi tion the adjuvant may act as a chemoattractant for other immune cells or may act locally and/or systemically as a stimulating agent for the immune system.
-3 Antigen presenting cells belong to the innate immune system, which has evolved as a first line host defence that limits infec tion early after exposure to microorganisms. Cells of the innate immune system recognize patterns or relatively non-specific structures expressed on their targets rather than more sophisti cated, specific structures which are recognized by the adaptive immune system. Examples of cells of the innate immune system are macrophages and dendritic cells but also granulocytes (e.g. neu trophiles), natural killer cells and others. By contrast., cells of the adaptive immune system recognize specific, antigenic structures, including peptides, in the case of T-cells and pep tides as well as three-dimensional structures in the case of B cells. The adaptive immune system is much more specific and so phisticated than the innate immune system and improves upon re peated exposure to a given pathogen/antigen. Phyl6genetically, the innate immune system is much older and can be found already in very primitive organisms. Nevertheless, the innate immune sys tem is critical during the initial phase of antigenic exposure since, in addition to containing pathogens, cells of the innate immune system, i.e. APCs, prime cells of the adaptive immune sys tem and thus trigger specific immune responses leading to clear ance of the intruders. In sum, cells of the innate immune system and in particular APCs play a critical role during the induction phase of immune responses by a) containing infections by means of a primitive pattern recognition system and b) priming cells of the adaptive immune system leading to specific immune responses and memory resulting in clearance of intruding pathogens or of other targets. These mechanisms may also be important to clear or contain tumor cells. The antigens used for such vaccines have often been selected by chance or by easiness of availability. There is a demand to iden tify efficient antigens for a given pathogen or - preferably - an almost complete set of all antigens of a.given pathogen which are practically (clinically) relevant. Such antigens may be preferred antigen candidates in a vaccine. It is- therefore an object of the present invention to comply with these demands and to provide a method with which such antigens may be provided and with which a practically complete set of an- -4 tigens of e.g. a given pathogen may be identified with a given serum as antibody source. Such a method should also be suitable for rapidly changing pathogens which evolve a fast resistance against common drugs or vaccines. The method should also be ap plicable to identify and isolate tumor antigens, allergens, auto immune antigens. Therefore, the present invention provides a method for identifi cation, isolation and production of hyperimmune serum-reactive antigens from a specific pathogen, a tumor, an allergen or a tis sue or host prone to auto-immunity, especially from a specific pathogen, said antigens being suited for use in a vaccine for a given type of animal or for humans, said method being character ized by the following steps: *providing an antibody preparation from a plasma pool of said given type of animal or from a human plasma pool or individual sera with antibodies against said specific pathogen, a tumor, an allergen or a tissue or host prone to auto-immunity, *providing at least one expression library of said specific pathogen, a tumor, an allergen or a tissue or host prone to auto-immunity, *screening said at least one expression library with said anti body preparation, *identifying antigens which bind in said screening to antibod ies in said antibody preparation, *screening the identified antigens with individual antibody preparations from individual sera from individuals with anti bodies against said specific pathogen, tumor, allergen or tis sue or host prone to auto-immunity, *identifying the hyperimmune serum-reactive antigen portion of said identified antigens which hyperimmune serum-reactive anti gens bind to a relevant portion of said individual antibody preparations from said individual sera and *optionally isolating said hyperimmune serum-reactive antigens and producing said hyperimmune serum-reactive antigens by chemical or recombinant methods. This method is also suitable in general for identifying a practi cally complete set of hyperimmune serum-reactive antigens of a specific pathogen with given sera as antibody sources, if at -5 least three different expression libraries are screened in a pathogen/antigen identification programme using the method ac cording to the present invention. The present invention therefore also relates to a method for identification, isolation and pro duction of a practically complete set of hyperimmune serum-reac tive antigens of a specific pathogen, said antigens being suited for use in a vaccine for a given type of animal or for humans, which is characterized by the following steps: +providing an antibody preparation from a plasma pool of said given type of animal or from a human plasma pool or individual sera with antibodies against said specific pathogen, *providing at least three different expression libraries of said specific pathogen, *screening said at least three different expression libraries with said antibody preparation, *identifying antigens which bind in at least one of said at least three screenings to antibodies in said antibody prepara tion, *screening the identified antigens with individual antibody preparations from individual sera from individuals with anti bodies against said specific pathogen, *identifying the hyperimmune serum-reactive antigen portion of said identified antigens which hyperimmune serum-reactive anti gens bind to a relevant portion of said individual antibody preparations from said individual sera, *repeating said screening and identification steps at least once, +comparing the identified hyperimmune serum-reactive antigens identified in the repeated screening and identification steps with the identified hyperimmune serum-reactive antigens identi fied in the initial screening and identification steps, *further repeating said screening and identification steps, if at least 5% of the hyperimmune serum-reactive antigens have been identified in the repeated screening and identification steps only, until less than 5 % of the hyperimmune serum-reac-, tive antigens are identified in a further repeating step only to obtain a complete set of hyperimmune serum-reactive antigens of a specific pathogen and *optionally isolating said hyperimmune serum-reactive antigens and producing said hyperimmune serum-reactive antigens by -6 chemical or recombinant methods. The method according to the.present invention mainly consists of three essential parts, namely 1. identifying hyperimmune serum sources containing specific antibodies against a given pathogen, 2. screening of suitable expression libraries with a suitable an tibody preparation wherein candidate antigens (or antigenic frag ments of such antigens) are selected, and - 3. in a second screening round, wherein the hyperimmune serum-reactive antigens are identified by their ability to bind to a relevant portion of individual antibody preparations from individual sera in order to show that these antigens are practically relevant and not only hyperimmune serum-reactive, but also widely immunogenic (i.e. that a lot of individual sera react with a given antigen). With the present method it is possible to provide a set of antigens of a given pathogen which is practically complete with respect to the chosen pathogen 'and the chosen serum. Therefore, a bias with respect to "wrong" antigen candidates or an incomplete set of an tigens of a given pathogen is excluded by the present method. Completeness of the antigen set of a given pathogen within the meaning of the present invention is, of course, dependent on the completeness of the expression libraries used in the present method and on the quality and size of serum collections (number of individual plasmas/sera) tested , both with respect.to repre sentability of the library and usefulness of the expression sys tem. Therefore, preferred embodiments of the present method are characterized in that at least one of said expression libraries is selected from a ribosomal display library, a bacterial- surface library and a proteome. A serum collection used in the present invention should be tested against a panel of known antigenic compounds of a given pathogen, such as polysaccharide, lipid and proteinaceous components of the cell wall, cell membranes and cytoplasma, as well as secreted products. Preferably, three distinct serum collections are used: 1. With very stable antibody repertoire: normal adults, clini cally healthy people, who overcome previous encounters or cur rently carriers of e.g. a given pathogen without acute disease and symptoms, 2. With antibodies induced acutally by the presence of the pathogenic organism: patients with acute disease with dif ferent manifestations (e.g. S. aureus sepsis or wound infection, etc.), 3.. With no specific antibodies at all (as negative con trols): 5-8 months old babies who lost the maternally transmitted immunoglobulins 5-6 months after birth. Sera have to react with multiple pathogen-specific antigens in order to consider hyperim mune for a given pathogen (bacteria, fungus, worm or otherwise), and for that relevant in the screening method according to the. present invention. In the antigen identification programme for identifying a com plete set of antigens according to the present invention, it is preferred that said at least three different expression libraries are at least a ribosomal display library, a bacterial surface li brary and a proteome. It has been observed that although all ex pression libraries may be complete, using only one or two expression libraries in an antigen identification programme will not lead to a complete set of antigens due to preferential ex pression properties of each of the different expression librar ies. While it is therefore possible to obtain hyperimmune serum reactive antigens by using only one or two different expression libraries, this might in many cases not finally result in the identification of a complete set of hyperimmune serum-reactive antigens. Of course, the term "complete" according to the present invention does not indicate a theoretical maximum but is indeed a practical completeness, i.e. that at least 95% of the practically relevant antigens or antigenic determinants have been identified of a given pathogen. The practical relevance is thereby defined by the occurrence of antibodies against given antigens in the pa tient population. According to the present invention also serum pools or plasma fractions or other pooled antibody containing body fluids are "plasma pools". An expression library as used in the present invention should at least allow expression of all potential antigens, e.g. all sur face proteins of a given pathogen. With the expression libraries according to the present invention, at least one set of potential antigens of a given pathogen is provided, this set being prefera- -8 bly the complete theoretical complement of (poly-)peptides en coded by the pathogen's genome (i.e. genomic libraries as de scribed in Example 2) and expressed either in a recombinant host (see Example 3) or in vitro (see Example 4). This set of poten tial antigens can also be a protein preparation, in the case of extracellular pathogens preferably a protein preparation contain ing surface proteins of said pathogen obtained from said pathogen grown under defined physiological conditions (see Example 5). While the genomic approach has the potential to contain the com plete set of antigens, the latter one has the advantage to con tain the proteins in their naturally state i.e. including for instance post-translational modifications or processed forms of these proteins, not obvious from the DNA sequence. These or any other sets of potential antigens from a pathogen, a. tumor, an al lergen or a tissue or host prone to auto-immunity are hereafter * referred to as "expression library". Expression libraries of very different kinds may be applied in the course of the present in vention. Suitable examples are given in e.g. Ausubel et al., 1994. Especially preferred are expression libraries representing a display of the genetic set of a pathogen in recombinant form such as in vitro translation techniques, e.g. ribosomal display, or prokaryotic expression systems, e.g. bacterial surface expres sion libraries or which resemble specific physiological expres sion states of a given pathogen in a given physiological state, such as a proteome. Ribosome display is an established method in recombinant DNA technology, which is applicable for each specific pathogen for the sake of the present invention (Schaffitzel et al, 1999). Bac terial surface display libraries will be represented by a recom binant library of a bacterial host displaying a (total) set of expressed peptide sequences of a given pathogen on e.g. a se lected outer membrane protein at the bacterial host membrane (Georgiou et al., 1997). Apart from displaying peptide or protein sequences in an outer membrane protein, other bacterial display techniques, such as bacteriophage display technologies and ex pression via exported proteins are also preferred as bacterial surface expression library ( Forrer et al., 1999; Rodi and Makowski, 1993; Georgiou et al., 1997).
-9 The antigen preparation for the first round of screening in the method according to the present invention may be derived from any source containing antibodies to a given pathogen. Preferably, if a plasma pool is used as a source for the antibody preparation, a human plasma pool is selected which comprises donors which had experienced or are experiencing an infection with the given pathogen. Although such a selection of plasma or plasma pools is in principle standard technology in for example the production of hyperimmunoglobulin preparations, it was surprising that such technologies have these effects as especially shown for the pre ferred embodiments of the present invention. Preferably the expression libraries are genomic expression li braries of a given pathogen, or alternatively m-RNA, libraries. It is preferred that these genomic or m-RNA libraries are com plete genomic or m-RNA expression libraries which means that they contain at least once all possible proteins, peptides or peptide fragments of the given pathogen are expressable. Preferably the genomic expression libraries exhibit a redundancy of at least 2x,. more preferred at least 5x, especially at least lOx. Preferably, the method according to the present invention com prises screening at least a ribosomal display library, a bacte rial surface display library and a proteome with the antibody preparation and identifying antigens which bind in at least two, preferably which bind to all, of said screenings to antibodies in said antibody preparation. Such antigens may then be regarded ex tremely suited as hyperimmunogenic antigens regardless of their way of expression. Preferably'the at least two screenings should at least contain the proteome, since the proteome always repre sents the antigens as naturally expressed proteins including post-translational modifications, processing, etc. which are not obvious from the DNA sequence. The method according t o the present invention may be applied to any given pathogen. Therefore, preferred pathogens are selected from the group of bacterial, viral, fungal and protozoan patho gens. The method according to the present invention is also ap plicable to cancer, i.e. for the identification of tumor associated antigens, and for the identification of allergens or - 10 antigens involved in auto-immune diseases. Of course, especially the recombinant methods are rather simple for pathogens having a small genome or a comparatively small number of expressed pro teins (such as bacterial or viral pathogens) and are more compli cated for complex (eukaryotic) organisms having large genomes. However, also such large genomic libraries of higher organism pathogens may well be analyzed with the method according to the present invention, at least in a faster and more reliable way than with known methods for identifying suitable antigens. Preferred pathogens to beanalyzed or which antigens are to be extracted, respectively, include human immunedeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepati tis C virus (HCV), Rous sarcoma virus (RSV), Epstein-Barr vi rus (EBV), influenza virus (IV), rotavirus (RV), Staphylococcus aureus (S.aureus) , Staphylococcus epidermidis (S. epidermidis) , Chlamydia pneumoniae (C. pneumoniae), Chlamydia trachomatis (C. trachomatis), Mycobacterium tuberculosis (M. tuberculosis), Myco bacterium leprae (M. leprae), 'Streptococcus pneumoniae (S. pneu moniae), Streptococcus pyogenes (S. pyogenes), Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Bacillus anthracis (B. anthracis), Vibrio cholerae (V. cholerae), Borrelia burgdorferi (B. burgdorferi), Plasmodium sp., fungal diseases such as Pneumocystis carinii, Aspergillus sp., Crypto coccus sp., Candida albicans or parasitic infections such as as cariasis (Ascaris lumbricoides) and taeniasis (Taenia saginata). The method according to the present invention is most applicable for bacteria, worms or candida. As a model organism for the present application Staphylococcus aureus has been chosen to demonstrate the applicability and effi cacy of the method according to the present invention. Especially with respect to the examples it is clear that the invention is easily transferable to all potential pathogens, especially the ones listed above. it was surprising that the method according to the present inven tion allows an efficient and fast biological screening of a given pathogen, especially in view of the fact that only a small frac tion of a patient's antibody repertoire is directed to a given - 11 pathogen, even in a state where this pathogen is effectively de feated. It has been discovered within the course of the present invention, especially during performance of the S.aureus example that only 1-2% of the antibody repertoire of a patient having high titers against S.aureus are indeed antibodies directed against S.aureus. Moreover, over 70% of this specific 1% portion is directed against non-protein antigens, such as teichoic acid, so that only a total of 0.1% or less of the antibodies are di rected to proteinaceous antigens. One of the advantages of using recombinant expression libraries, especially ribsome display libraries and bacterial surface dis play libraries, is that the identified hyperimmune serum-reactive antigens may be instantly produced by expression of the coding sequences of the screened and selected clones expressing' the hyperimmune serum-reactive antigens without further recombinant DNA technology or cloning steps necessary. The hyperimmune serum-reactive antigens obtainable by the method according to the present invention may therefore be immediately finished to a pharmaceutical preparation, preferably by addition of a pharmaceutically acceptable carrier and/or excipient, imme diately after its production (in the course of the second selec tion step), e.g. by expression from the expression library platform. Preferably, the pharmaceutical preparation containing the hyperimmune serum-reactive antigen is a vaccine for preventing or treating an infection with the specific pathogen for which the antigens have been selected. The pharmaceutical preparation may contain any suitable auxiliary substances, such as buffer substances, stabilisers .or further ac tive ingredients, especially ingredients known in connection of vaccine production. A preferable carrier/or excipient for the hyperimmune serum-reac tive antigens according to the present invention is a immu nostimulatory compound for further stimulating the immune response to the given hyperimmune serum-reactive antigen. Pref- - 12 erably the immunostimulatory compound in the pharmaceutical preparation according to the present invention is selected from the group of polycationic substances, especially polycationic peptides, immunostimulatory deoxynucleotides, alumn, Freund's complete adjuvans, Freund's.incomplete adjuvans, neuroactive com pounds, especially human growth hormone, or combinations thereof. The polycationic compound(s) to be used according to the present invention may be any polycationic compound which shows the char acteristic effects according to the WO 97/30721. Preferred poly cationic compounds are selected from basic polypeptides, organic polycations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues (see: Tuftsin as described in Goldman et al. (1983)). Especially preferred are substances like polylysine, polyarginine and polypeptides containing more than 20%, espe cially more than 50% of basic amino acids in a range of more than 8, especially more than 20, amino acid residues or mixtures thereof. Other preferred polycations and their pharmaceutical 'compositons are described in WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 resi dues. These polycationic compounds may be produced chemically or recom binantly or may be derived from natural sources. Cationic (poly)peptides may also be anti- microbial with proper ties as reviewed in Ganz et al, 1999; Hancock, 1999. These (poly)peptides may be of prokaryotic or animal or plant origin.or may be produced chemically or recombinantly (Andreu et al., 1998; Ganz et al., 1999; Simmaco et al., 1998). Peptides may also be long to the class of defensins (Ganz, 1999; Ganz-et al., 1999). Sequences of such peptides can be, for example, be found in the Antimicrobial Sequen-ces Database under the following internet ad dress: http: //www.bbcm.univ.trieste.it/-tossi/pcag2.html Such host defence peptides or defensives are also a preferred form of the polycationic polymer according to the present inven- - 13 tion. Generally, a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycati onic polymer. Especially preferred for use as polycationic substance in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (International patent application PCT/EP01/09529, incorporated herein by reference), especially an timicrobial peptides derived from mammal cathelicidin, preferably from human, bovine or mouse. Polycationic compounds derived from natural sources include HIV REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides de rived from these peptides or proteins by biochemical or recombi nant production. Other preferred polycationic compounds are cathelin or related or derived substances from cathelin. For ex *ample, mouse cathelin is a peptide which has the amino acid se quence NH 2 -RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH. Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues. Deri vations may include the substitution or modification of the natu ral amino acids by amino acids which are not among the 20 standard amino acids. Moreover, further cationic.residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the antigen. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for a antigen without the addition of -further adjuvants. It is therefore possible to use such cathelin molecules as effi cient adjuvants in vaccine formulations with or without further immunactivating substances. Another preferred polycationic substance to be used according to the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (International patent application PCT/EP01/12041, incorpo rated herein by reference). Immunostimulatory deoxynucleotides are e.g. neutral or artificial - 14 CpG containing DNA, short stretches of DNA derived. from non-ver tebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine di-nucleotides (CpG) in a certain base context (e.g. Krieg et al., 1995) but also inosine contain ing ODNs (I-ODNs) as described in WO 01/93905. Neuroactive compounds, e.g. combined with polycationic substances are described in WO 01/24822. According to a preferred embodiment the individual antibody preparation for the second round of screening are derived from patients with have suffered from an acute infection with the given pathogen, especially from patients who show an antibody titer to the given pathogen above a certain minimum level, for example an antibody titer being higher than 80 percentile, pref erably higher than 90 percentile, especially higher than 95 per centile of the human (patient or carrier) sera tested. Using such high titer individual antibody preparations in the second screen ing round allows a very selective identification of the hyperim mune serum-reactive antigens to the given pathogen. It is important that the second screening with the individual an tibody preparations (which may also. be the selected serum) allows a selective identification of the hyperimmune serum-reactive an tigens from all the promising candidates from the first round. Therefore, preferably at least 10 individual antibody prepara tions (i.e. antibody preparations (e.g. sera) from at least 10 different individuals having suffered from an infection to the chosen pathogen) should be used in identifying these antigens in the second screening round. Of course, it is possible to use also less than 10 individual preparations, however, selectivity of the step may not be optimal with a low number. of individual antibody preparations. On the other hand, if a given hyperimmune serum-re active antigen (or an antigenic fragment thereof) is recognized in at least 10 individual antibody preparations, preferably at .least 30, especially at least 50 individual antibody prepara tions, identification of hyperimmune serum-reactive antigen is also selective enough for a proper identification. Hyperimmune serum-reactivity may of course be tested with as many individual preparations as possible (e.g. with more than 100 or even with - 15 more than 1000). Therefore, the relevant portion of the hyperimmune serum-reactive antibody preparation according to the method of the present in vention should preferably be at least 10, more preferred at least 30, especially at least 50 individual antibody preparations. Al ternatively (or in combination) hyperimmune serum-reactive anti gen may preferably be also identified with at least 20%, preferably at least 30%, especially at least 40% of all individ ual antibody preparations used in the second screening round. According to a preferred embodiment of the present invention, the sera from which the individual antibody preparations for the sec ond round of screening are prepared (or which are used as anti body preparations), are selected by their titer against the specific pathogen (e.g. against a preparation of this pathogen, such as a lysate, cell wall components and recombinant proteins). Preferably, some are selected with a total IgA titer above 4000 U, especially above 6000 U, and/or an IgG titer above 10 000 U, especially above 12 000 U (U = units, calculated from the OD 40 sn reading at a given dilution) when whole organism (total lysate or whole cells) is used as antigen in ELISA. Individual proteins with Ig titers of above 800-1000 U are specifically preferred for selecting the hyperimmune serum-reactive antigens according to the present invention only for total titer. The statement for in dividual proteins can be derived from Fig. 9. According to the demonstration example which is also a preferred embodiment of the present invention the given pathogen is a Staphylococcus pathogen, especially Staphylococcus aureus and Staphylococcus epidermidis. Staphylococci are opportunistic pathogens which can cause illnesses which range from minor infec tions to life threatening diseases. Of the large number of Staphylococci at least 3 are commonly associated with human dis ease: S. aureus, S. epidermidis and rarely S. saprophyticus (Crossley and Archer, 1997). S. aureus has been used within the course of the present invention as an illustrative example of the way the present invention functions. Besides that, it is also an important organism with respect to its severe pathogenic impacts on humans. Staphylococcal infections are imposing an increasing - 16 threat in hospitals worldwide. The appearance and disease causing capacity of Staphylococci are related to the wide-spread use of antibiotics which induced and continue to induce multi-drug re sistance. For that reason medical treatment against Staphylococ cal infections cannot rely only on antibiotics anymore. Therefore, a tactic change in the treatment of these diseases is desperately needed which aims to prevent infections. Inducing high affinity antibodies of the opsonic and neutralizing type by vaccination helps the innate immune system to eliminate bacteria and toxins. This makes the method according to the present inven tion an optimal tool for the identification of staphylococcal an tigenic proteins. Every human being is colonized with S.- epidermidis. The normal habitats of S. epidermidis are the skin and the mucous membrane. The major habitats of the most pathogenic species, S. aureus, are the anterior nares and perineum. Some individuals become perma nent S. aureus carriers, often with the same strain. The carrier stage is clinically'relevant because carriers undergoing surgery have more infections than noncarriers. Generally, the established flora of the nose prevents acquisition of new strains. However, colonization with other strains may occur when antibiotic treat ment is given that leads to elimination of the susceptible car rier strain. Because this situation occurs in the hospitals, patients may become colonized with resistant nosocomial Staphylo cocci. These bacteria have an innate adaptability which is com plemented by the widespread and sometimes inappropriate use of antimicrobial agents. Therefore hospitals provide a fertile envi ronment for drug resistance to develop (close contact among sick patients, extensive use of antimicrobials, nosocomial infec tions). Both S. aureus and S. epidermidis have become resistant to many commonly used antibiotics, most importantly to methicil lin (MRSA) and vancomycin (VISA) . Drug resistance is an increas ingly important -public health concern, and soon many infections caused by staphylococci may be untreatable by antibiotics. In ad dition to its adverse effect on public health, antimicrobial re sistance contributes to higher health care costs, since treating resistant infections often requires the use of more toxic and more expensive drugs, and can result in longer hospital stays for infected patients.
- 17 Moreover, even with the help of effective antibiotics, the most serious staphylococcal infections have 30-50 % mortality. Staphylococci become potentially pathogenic as soon as the natu ral balance between microorganisms and the immune system gets disturbed, when natural barriers (skin, mucous membrane) are breached. The coagulase-positive S. aureus is the most pathogenic staphylococcal species, feared by surgeons for a long time. Most frequently it causes surgical wound infections, and induces the formation of abscesses. This local infection might become sys temic, causing bacteraemia and sepsis. Especially after viral in fections and in elderly, it can cause severe pneumonia. S. aureus is also a frequent cause of infections related to medical de vices, such as intravascular and percutan catheters (endocardi tis, sepsis, peritonitis), prosthetic devices (septic arthritis, osteomyelitis). S. epidermidis causes diseases mostly related to the presence of foreign body and'the use of devices, such as catheter related infections, cerebrospinal fluid shunt infec tions, peritonitis in dialysed patients (mainly CAPD), endocardi tis in individuals with prosthetic valves. This is exemplified in immunocompromised individuals such as oncology patients and pre mature neonates in whom coagulase-negative staphylococcal infec tions frequently occur in association with the use of intravascular device. The increase in incidence is related to the increased used of these devices and increasing number of immuno compromised patients. Much less is known about S. saprophyticus, another coagulase negative staphylococci, which causes acute urinary tract infec tion in previously healthy people. With a few exceptions these are women aged 16-25 years. The pathogenesis of staphylococci is multifactorial. In order to initiate infection the pathogen has to gain access to the cells and tissues of the host, that is adhere. S. aureus expresses-sur face proteins that promote attachment to the host proteins such as laminin, fibronectin, elastin, vitronectin, fibrinogen and many other molecules that form part of the extracellular ma trix (extracellular matrix binding proteins, ECMBP). S. epider- - 18 midis is equipped with cell surface molecules which promote ad herence to foreign material and through that mechanism establish infection in the host. The other powerful weapons staphylococci use are the secreted products, such as enterotoxins, exotoxins, and tissue damaging enzymes. The toxins kill or misguide immune cells which are important in the host defence. The several dif ferent types of toxins are responsible for most of the symptoms during infections. Host defence against S. aureus relies mainly on innate immuno logical mechanisms. The skin and mucous membranes are formidable barriers against invasion by Staphylococci. However, once the skin or the mucous membranes are breached (wounds, percutan catheters, etc), the first line of nonadaptive cellular defence begins its co-ordinate action through complement and phagocytes, especially the polymorphonuclear leukocytes (PMNs). These cells can be regarded as the cornerstones in eliminating invading bac teria. As Staphylococci are primarily extracellular pathogens, the major anti-staphylococcal adaptive response comes from the humoral arm of the immune system, and is mediated through three major mechanisms: promotion of opsonization, toxin neutralisa tion, and inhibition of adherence. It is believed that opsoniza tion is especially important, because of its requirement for an effective phagocytosis. For efficient opsonization the microbial surface has to be coated with antibodies and complement factors for recognition by PMNs through receptors to the Fc fragment of the IgG molecule or to activated C3b. After opsonization, staphy lococci are phagocytosed and killed. Moreover, S. aureus can at tach to endothelial cells, and be internalised by a phagocytosis like process. Antibodies bound to specific antigens on the cell surface of bacteria serve as ligands for the attachment to PMNs and promote phagocytosis. The very same antibodies. bound to the adhesins and other cell surface proteins are expected to neutral ize adhesion and prevent colonization. There is little clinical evidence that cell mediated immunity has a significant contribution in the defence against Staphylococci, yet one has to admit that the question is not adequately ad dressed. It is known, however, that Staphylococcus aureus util izes an extensive array of molecular countermeasures to - 19 manipulate the defensive microenvironment of the infected host by secreting polypeptides referred to as superantigens, which target the multireceptor communication between T-cells and antigen-pre senting cells that is fundamental to initiating pathogen-specific immune clearance. Superantigens play a critical role in toxic shock syndrome and food poisoning, yet their function in routine infections is not well understood. Moreover, one cannot expect a long lasting antibody (memory) response without the involvement of T-cells. It is also known that the majority of the anti staphylococcal antibodies are against T-cell independent antigens (capsular polysacharides, lipoteichoic acid, peptidoglycan) with out a memory function. The T-cell dependent proteinaceous anti gens can elicit long-term protective antibody responses. These staphylococcal proteins and peptides have not yet been deter mined. For all these above mentioned reasons, a tactic change on the war field against staphylococcal infections is badly needed. One way of combating infections is preventing them by active immunisa tion. Vaccine development against S. aureus has been initiated by several research groups and national institutions worldwide, but there is no effective vaccine approved so far. It has been shown that an antibody deficiency state contributes to staphylococcal persistence, suggesting that anti-staphylococcal antibodies are important in host defence. Antibodies - added as passive immuni sation or induced by active vaccination - directed towards sur face components could both prevent bacterial adherence, neutralize toxins and promote phagocytosis. A vaccine based on fibronectin binding protein induces protective immunity against mastitis in cattle and suggest that this approach is likely to work in humans refss). Taking all this together it is suggestive that an effective vaccine should be composed of proteins or polypeptides, which are expressed by all strains and are able to induce high affinity, abundant antibodies against cell surface components of S. aureus. The antibodies should be IgG1 and/or IgG3 for opsonization, and any IgG subtype and IgA for neutrali sation of adherence and toxin action. A chemically defined vac cine must be definitely superior compared to a whole cell vaccine (attenuated or killed), since components of S. aureus which para lyze TH cells (superantigens) or inhibit opsonization (protein A) - 20 can be eliminated, and the individual proteins inducing protec tive antibodies can be selected. Identification of the relevant antigens help to generate effective passive immunisation (human ised monoclonal antibody therapy), which can replace human immu noglobulin administration with all its dangerous side-effects. Neonatal staphylococcal infections, severe septicemia and other life-threatening acute conditions are the primary target of pas sive immunisation. An effective vaccine offers great potential for patients facing elective surgery in general, and those re ceiving endovascular devices, in particular. Moreover, patients suffering from chronic diseases which decrease immune responses or undergoing continuous ambulatory peritoneal dialysis are likely to benefit from such a vaccine. For the illustrative example concerning Staphylococcus aureus three different approaches have been employed in parallel. All three of these methods are based on the interaction of Staphylo coc.cus proteins or peptides with the antibodies present in human sera with the method according to the present invention. This in teraction relies on the recognition of epitopes within the pro teins which can be short peptides (linear epitopes) or polypeptide domains (structural epitopes). The antigenic proteins are identified by the different methods using pools of pre-se lected sera and - in the second screening round - by individual selected sera. Following the high throughput screening, the selected. antigenic proteins are expressed as recombinant proteins or in vitro trans lated products (in case it can not be expressed in prokaryotic expression systems), and tested in a series of ELISA and Western blotting assays for the assessment of immunogeneicity with a large human serum collection (> 100 uninfected, > 50 patients sera). The preferred antigens are located on the cell surface or secreted, that is accessible extracellularly. Antibodies against the cell wall proteins (such as the Extracellular matrix binding proteins) are expected to serve double purposes: to inhibit adhe sion and promote phagocytosis. The antibodies against the se creted proteins are beneficial in toxin neutralisation. It is also known that bacteria communicate with each other through se creted proteins. Neutralizing antibodies against these proteins - 21 will interrupt growth promoting cross-talk between or within staphylococcal species. Bioinformatics (signal sequences, cell wall localisation signals, transmembrane domains) proved to be very useful in assessing cell surface localisation or secretion. The experimental approach includes the isolation of antibodies with the corresponding epitopes and proteins from human serum, and use them as reagents in the following assays: cell surface staining of staphylococci grown under different conditions (FACS, microscopy), determination of neutralizing capacity (toxin, ad herence), and promotion of opsonization and phagocytosis (in vi tro phagocytosis assay). The recognition of linear epitopes by antibodies can be based on sequences as short as 4-5 aa. Of course it does not necessarily mean that these short peptides are capable of inducing the given antibody. in vivo. For that reason the defined epitopes, polypep tides and proteins may further be tested in animals (mainly in mice) for their capacity to induce antibodies against the se lected proteins in vivo. The antigens with the proven capability to induce antibodies will be tested in animal models for the ability to prevent infections. The antibodies produced against Staphylococci' by the human immune system and present in human sera are indicative of the in vivo expression of the -antigenic proteins and their immunogenicity. Accordingly, novel hyperimmune serum-reactive antigens from Staphylococcus aureus or Staphylococcus epidermidis have been made available by the method according to the present invention. According to another aspect of the present invention the inven tion relates to a hyperimmune serum-reactive antigen selected from the group consisting of the sequences listed in any one of Tables 2a, 2b, 2c, 2d, 3, 4 and 5, especially selected from the group consisting of Seq.ID No. 56, 57, 59, 60, 67, 70, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 85, 87, 88, 89, 90, 92, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 132, 134, 138, 140, 142, 151, 152, 154, 155 and hyperimmune fragments thereof. Accordingly, the pre sent invention also relates to a hyperimmune serum-reactive anti gen obtainable by the method according to the present invention - 22 and being selected from the group consisting-of the sequences listed in any one of Tables 2a, 2b, 2c, 2d, 3, 4 and 5, espe cially selected from the group consisting of Seq.ID No. 56, 57, 59, 60, 67, 70, 72, 73, 74, 75, 76,' 77, 78, 79, 80, 81, 82, 85, 87, 88, 89, 90, 92, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 132, 134, 138, 140, 142, 151, 152, 154, 155 and hyperimmune fragments thereof. Antigens from Staphylococcus aureus and Staphylococcus epider midis have been extracted by the method according to the present invention which may be used in the manufacture of a pharmaceuti cal preparation, especially for the manufacture of a vaccine against Staphylococcus aureus and Staphylococcus epidermidis in fections. Examples of such hyperimmune serum-reactive antigens of Staphylococcus aureus and Staphylococcus epidermidis to be used in -a pharmaceutical preparation are selected from the group con sisting of the sequences listed in any one of Tables 2a, 2b, 2c, 2d, 3, 4 and 5, especially selected from the group consisting of Seq.ID No. 55, 56, 57, 58, 59, 60, 62, 66, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78,. 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 92, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 126, 128, 130, 132, 134, 138, 140, 142, 151, 152, 154, 155, 158 and hyperimmune fragments thereof for the manufacture of a pharmaceutical preparation, especially for the manufacture of a vaccine against Staphylococcus aureus and Staphylococcus epidermidis infections. A hyperimmune fragment is defined as a fragment of the identified antigen which is for itself antigenic or may be made antigenic when provided as a hapten. Therefore, also antigen or antigenic fragments showing one or (for longer fragments) only a few amino acid exchanges are enabled with the present invention, provided that the antigenic capacities of such fragments with amino acid exchanges are not severely deteriorated on the exchange(s). i.e. suited for eliciting an appropriate immune response in a individ ual vaccinated with this antigen and identified by individual an tibody preparations from individual sera. preferred examples of such hyperimmune fragments of a hyperimmune serum-reactive antigen are selected from the group consisting of - 23 peptides comprising the amino acid sequences of column "predicted immunogenic aa", "Location of identified immunogenic region" and "Serum reactivity with relevant region" of Tables 2a, 2b, 2c and 2d and the amino acid sequences of column "Putative antigenic surface areas" of Table 4 and 5, especially peptides comprising amino acid No. aa 12-29, 34-40, 63-71, 101-110, 114-122, 130-138, 140-195, 197-209, 215-229, 239-253, 255-274 and 39-94 of Seq.ID No. 55, aa 5-39, 111-117, 125-132, 134-141, 167-191, 196-202, 214-232, 236-241, 244-249, 292-297, 319-328, 336-341, 365-380, 385-391, 407-416, 420-429, 435-441, 452-461, 477-488, 491-498, 518-532, 545-556, 569-576, 581-587, 595-602, 604-609, 617-640, 643-651, 702-715, 723-731, 786-793, 805-811, 826-839, 874-889, 37-49-; 63 77 and 274-334, of Seq.ID No.56, aa 28-55, 82-100, 105-111, 125-131, 137-143, 1-49, of Seq.ID No. 57, aa 33-43, 45-51, 57-63, 65-72, 80-96, 99-110, 123-129, 161-171, 173-179, 185-191, 193-200, 208-224, 227-246, 252-258, 294-308, 321-329, 344-352, 691-707, 358-411 and 588-606, of Seq.ID No. 58, aa 16-38, 71-77, 87-94, 105-112, 124-144, 158-164, 169-177, 180 186, 194-204, 221-228, 236-245, 250-267, 336-343, 363-378, 385 394, 406-412, 423-440, 443-449, 401-494, of Seq.ID No. 59, aa 18-23, 42-55, 69-77, 85-98, 129-136, 182-188, 214-220, 229 235, 242-248, 251-258, 281-292, 309-316, 333-343, 348-354, 361-- 367, 393-407, 441-447, 481-488, 493-505, 510-515, 517-527, 530 535, 540-549, 564-583, 593-599, 608-621, 636-645, 656-670, 674 687, 697-708, 726-734, 755-760, 765-772, 785-792, 798-815, 819 824, 826-838, 846-852, 889-904, 907-913, 932-939, 956-964, 982 1000, 1008-10-15, 1017-1024., 1028-1034, 1059-1065, 1078-1084, 1122-1129, 1134-1143, 1180-1186, 1188-1194, 1205-1215, 1224-1230, 1276-1283, 1333-1339, 1377-1382, 1415-1421, 1448-1459, 1467-1472, 1537-1545, 1556-1566, 1647-1654, 1666-1675, 1683-1689, 1722-1'737, 1740-1754, 1756-1762, 1764-1773, 1775-1783, 1800-1809, 1811-1819, 1839-1851, 1859-1866, 1876-1882, 1930-1939, 1947-1954, -1978-1985, 1999-2007, 2015-2029, 2080-2086, 2094-2100, 2112-2118, 2196-2205, 2232-2243, 198-258, 646-727 and 2104-2206, of Seq.ID No. 60, aa 10-29, 46-56, 63-74, 83-105, 107-114, 138-145, 170-184, 186 193, 216-221, 242-248, 277-289, 303-311, 346-360, 379-389, 422 428, 446-453, 459-469, 479-489, 496-501, 83-156, of Seq.ID No. 62, - 24 aa 14-22, 32-40, 52-58, 61-77, 81-93, 111-117, 124-138, 151-190, 193-214, 224-244, 253-277, 287-295, 307-324, 326-332, 348-355, 357-362, 384-394, 397-434, 437-460, 489-496, 503-510, 516-522, 528-539, 541-547, 552-558, 563-573, 589-595, 602-624, 626-632, 651-667, 673-689, 694-706, 712-739, 756-790, 403-462, of Seq.ID No. 66, aa 49-56, 62-68, 83-89, 92-98, 10.9-115, 124-131, 142-159, 161 167, 169-175, 177-188, 196-224, 230-243, 246-252, 34-46, of Seq.ID No. 67, aa 11-20, 26-47, 69-75, 84-92, 102-109, 119-136, 139-147, 160 170, 178-185, 190-196, 208-215, 225-233, 245-250, 265-272, 277 .284, 300-306, 346-357, 373-379, 384-390, 429-435, 471-481, 502 507, 536-561, .663-688, 791-816, 905-910, 919-933, 977-985, 1001 1010, 1052-1057, 1070-1077, 1082-1087, 1094-1112, 493-587, 633 715 and 704-760, of Seq.ID No.70, aa.6-20, 53-63, 83-90, 135-146, 195-208, 244-259, 263-314, 319 327, 337-349, 353-362, 365-374, 380-390, 397-405, 407-415, 208 287 and 286-314, of Seq.ID No. 71, -aa 10-26, 31-43, 46-58, 61-66, 69-79, 85-92, 100-115, 120-126, 128-135, 149-155, 167-173, 178-187, 189-196, 202-222, 225-231, 233-240, 245-251, 257-263, 271-292, 314-322, 325-334, 339-345, 59-74, of Seq.ID No. 72, aa 4-9, 15-26, '65-76, 108-115, 119-128, 144-153, 38-52 and 66 114, of Seq.ID No. 73, aa 5-22, 42-50, 74-81, 139-145, 167-178, 220-230, 246-253, 255 264, 137-237 and 250-267, of Seq.ID No. 74, aa 10-26, 31-44, 60-66, 99-104, 146-153, 163-169, 197-205, 216 223, 226-238, 241-258, 271-280, 295-315, 346-351, 371-385, 396 407, 440-446, 452-457, 460-466, 492-510, 537-543, 546-551, 565 582, 590-595, 635-650, 672-678, 686-701, 705-712, 714-721, 725 731, 762-768, 800-805, 672-727, of Seq.ID No. 75, aa 5-32, 35-48, 55-76, of Seq.ID No. 76, aa 7-35, 54-59, 247-261, 263-272, 302-320, 330-339, 368-374, 382 411, 126-143 and 168-186, of Seq.ID No. 77, aa 5-24, 88-94, 102-113, 132-143, 163-173, 216-224, 254-269, 273 278, 305-313, 321-327, 334-341, 31-61 and 58-74, of Seq.ID No. 78, aa 16-24, 32-39, 43-49, 64-71, 93-99, 126-141, 144-156, 210-218, 226-233, 265-273, 276-284, 158-220, of Seq.ID No. 79, aa 49-72, 76-83, 95-105, 135-146, 148-164, 183-205, 57-128, of - 25 Seq.ID No. 80, aa 6-15, 22-32, 58-73, 82-88, 97-109, 120-131, 134-140, 151-163, 179-185, 219-230, 242-255, 271-277, 288-293, 305-319, 345-356, 368-381, 397-406, 408-420, 427-437, 448-454, 473-482,- 498-505, 529-535, 550-563, 573-580, 582-590, 600-605, 618-627, 677-685, 718-725, 729-735, 744-759, 773-784, 789-794, 820-837, 902-908, 916-921, 929-935, 949-955, 1001-1008, 1026-1032, 1074-1083, 1088 1094, 1108-1117, 1137-1142, 1159-1177, 1183-1194, 1214-1220, 1236-1252, 1261-1269, 1289-1294, 1311-1329, 1336-1341, 1406-1413, 1419-1432, 1437-1457, 1464-1503, 1519-1525, 1531-1537, 1539-1557, 1560-1567, 1611-1618, 1620-1629, 1697-1704, 1712-1719, 1726-1736, 1781-1786, 1797-1817, 1848-1854, 1879-1890, 1919-1925, 1946-1953, 1974-1979, 5'to 134, of Seq.ID No. 81, aa 6-33, 40-46, 51-59, 61-77, 84-104, 112-118, 124-187, 194-248, 252-296, 308-325, 327-361, 367-393, 396-437, 452-479, 484-520, 535-545, 558-574, 582-614, 627-633, 656-663,.671-678, 698-704, 713-722, 725-742, 744-755, 770-784, 786-800, 816-822, 827-837, .483-511, of Seq.ID No. 82, aa 4-19, 57-70, 79-88, 126-132, 144-159, 161-167, 180-198, 200 212, 233-240, 248-255, 276-286, 298-304, 309-323, 332-346, 357 366, 374-391, 394-406, 450-456, 466-473, 479-487, 498-505, 507 519, 521-530, 532-540, 555-565, 5~71-581, 600-611, 619-625, 634 642, 650-656, 658-665, 676-682, 690-699, 724-733, 740-771,.774 784, 791-797, 808-815, 821-828, 832-838, 876-881, 893-906, 922 929, 938-943, 948-953, 969-976, 1002-1008, 1015-1035, 1056-1069, 1105-i116, 1124-1135, 1144-1151, 1173-1181, 1186-1191, 1206-1215, 1225-1230, 1235-1242, 6-66, 65-124 and 590-604, of Seq.ID No. 83, aa 5-32, 66-72, 87-98, 104-112, 116-124, 128-137, 162-168, 174 183, 248-254, 261-266, 289-303, 312-331, 174-249, of Seq.ID No. 84, aa 4-21., 28-40, 45-52, 59-71, 92-107, 123-137, 159-174, 190-202, 220-229, 232-241, 282-296, 302-308, 312-331, 21-118, of Seq.ID No. 85, aa 9-28, 43-48, 56-75, 109-126, 128-141, 143-162, 164-195, 197 216, 234-242, 244-251, 168-181, of Seq.ID No. 87, aa 4-10, 20-42, 50-86, 88-98, 102-171, 176-182, 189-221, 223-244, 246-268, 276-284, 296-329, 112-188, of Seq.ID No. 88, aa 4-9, 13-24, 26-34, 37-43, 45-51, 59-73, 90-96, 99-113, 160 173, 178-184, 218-228, 233-238, 255-262, 45-105, 103-166 and 66 153, of Seq.ID No. 89, - 26 aa 13-27, 42-63, 107-191, 198-215, 218-225, 233-250, 474-367, of Seq.TD No. 90; aa 26-53, 95-123, 164-176, 189-199, 8-48, of Seq.ID No. 92, aa 7-13, 15-23, 26-33, 68-81, 84-90, 106-117, 129-137, 140-159, 165-172, 177-230, 234-240, 258-278, 295-319, 22-56, 23-99, 97 115, 233-250 and 245-265, of Seq.ID No. 94, aa 13-36, 40-49, 111-118, 134-140, 159-164, 173-183, 208-220, 232-241, 245-254, 262-271, 280-286, 295-301, 303-310, 319-324, 332-339, 1-85, 54-121 and 103-185, of Seq.ID No. 95, aa 39-44, 46-80, 92-98, 105-113, 118-123, 133-165, 176-208, 226 238, 240-255, 279-285, 298-330, 338-345, 350-357, 365-372, 397 402, 409-415, 465-473, 488-515, 517-535, 542-550, 554-590, 593 601, 603-620, 627-653, 660-665, 674-687, 698-718, 726-739, 386 402, of Seq.ID No. 96, aa 5-32, 34-49, 1-43, of Seq.ID No. 97, aa 10-27, 37-56, 64-99, 106-119, 121-136, 139-145, 148-178, 190 216, 225-249, 251-276, 292-297, 312-321, 332-399, 403-458, 183 200, of Seq.ID No. 99, aa 5-12, 15-20, 43-49, 94-106, 110-116, 119-128, 153-163, 175 180, 185-191, 198-209, 244-252, 254-264, 266-273, 280-288, 290 297, 63-126, of Seq.ID No. 100, aa 5-44, 47-55, 62-68, 70-78, 93-100, 128-151, 166-171, 176-308, 1-59, of Seq.ID No. 101, aa 18-28, 36-49, 56-62, 67-84, 86-95, 102-153, 180-195, 198-218, 254-280, 284-296, 301-325, 327-348, 353-390, 397-402, 407-414, 431-455, 328-394, of Seq.ID No. 102, aa 7-37, 56-71, 74-150, 155-162, 183-203, 211-222, 224-234, 242 272, 77-128, of Seq.ID No. 103, aa 34-58, 63-69, 74-86, 92-101, 130-138, 142-150, 158-191, 199 207, 210-221, 234-249, 252-271, 5-48, of Seq.ID No. 104, aa 12-36, 43-50, 58-65, 73-78, 80-87, 108-139, 147-153, 159-172, 190-203, 211-216, 224-232, 234-246, 256-261, 273-279, 286-293, 299-306, 340-346, 354-366, 167-181, of Seq.ID No. 106, aa 61-75, 82-87, 97-104, 113-123, 128-133, 203-216, 224-229, 236-246, 251-258, 271-286, 288-294, 301-310, 316-329, 337-346, 348-371, 394-406, 418-435, 440-452 of Seq.ID No. 112, aa 30-37, 44-55, 83-91,.101-118, 121-128, 136-149, 175-183, 185 193, 206-212, 222-229, 235-242 of Seq.ID No. 114, aa 28-38, 76-91, 102-109, 118-141, 146-153, 155-161, 165-179, 186-202, 215-221, 234-249, 262-269, 276-282, 289-302, 306-314, - 27 321-326, 338-345, 360-369, 385-391 of Seq.ID No. 116, aa 9-33, 56-62,75-84, 99-105, 122-127, 163-180, 186-192, 206 228, 233-240, 254-262, 275-283, 289-296, 322-330, 348-355, 416 424, 426-438, 441-452, 484-491, 522-528, 541-549, 563-569, 578 584, 624-641, 527-544, of Seq.ID No. 142, aa 37-42, 57-62, 121-135, 139-145, 183-190, 204-212, 220-227, 242-248, 278-288, 295-30, 304-309, 335-341, 396-404, 412-433, 443-449, 497-503, 505-513, 539-545, 552-558, 601-617, 629-649, 702-711, 736-745, 793-804, 814-829, 843-858, 864-885, 889-895, 905-913, 919-929, 937-943, 957-965, 970-986, 990-1030, 1038-1049, 1063-1072, 1080-1091, 1093-1116, 1126-1136, 1145-1157, 1163-1171, 1177-1183, 1189-1196, 1211-1218, 1225-1235, 1242-1256, 1261-1269, 624-684, of Seq.ID No. 151, aa 8-23, 31-38, 42-49, 61-77, 83-90, 99-108, 110-119, 140-147, 149-155, 159-171, 180-185, 189-209, 228-234, 245-262, 264-275, 280-302, 304-330, 343-360, 391-409, 432-437, 454-463, 467-474, 478-485, 51~5-528, 532-539, 553-567, 569-581, 586-592, 605-612, 627-635, 639-656, 671-682, 700-714, 731-747, 754-770, 775-791, 797-834, 838-848, 872-891, 927-933, 935-942, 948-968, 976-986, 1000-1007, 1029-1037, 630-700, of Seq.ID No. 152, aa 17-25, 27-55, 84-90, 95-101, 115-121, 55-101, of Seq.ID No. 154,. aa 13-28, 40-46, 69-75, 86-92, 114-120, 126-137, 155-172, 182 193, 199-206, 213-221, 232-238, 243-253, 270-276, 284-290, 22 100, of Seq.ID No. 155 and aa 7-19, 46-57, 85-91, 110-117, 125-133, 140-149, 156-163, 198 204, 236-251, 269-275, 283-290, 318-323, 347-363, 9-42 and 158 174, of Seq.ID No. 158, .aa 7-14, 21-30, 34-50, 52-63, 65-72, 77-84, 109-124, 129-152, 158-163, 175-190, 193-216, 219-234 of Seq.ID.No. 168, aa 5-24, 38-44, 100-106, 118-130, 144-154, 204-210, 218-223, 228 243, 257-264, 266-286, 292-299 of Seq.ID.No. 174, aa 29-44, 74-83, 105-113, 119-125, 130-148, 155-175, 182-190, 198-211, 238-245 of Seq.ID.No. 176,-and fragments comprising at least 6, preferably more than 8, especially more than 10 aa of said sequences . All these fragments individually and each inde pendently form a preferred selected aspect of the present inven tion. Especially suited helper epitopes may also be derived from these - 28 antigens. Especially preferred helper epitopes are peptides com prising fragments selected from the peptides mentioned in column "Putative antigenic surface areas" in Tables 4 and 5 and from the group of aa 6-40, 583-598, 620-646 and 871-896 of Seq.ID.No.56, aa 24-53 of Seq.ID.No.70, aa 240-260 of Seq.ID.No.74, aa 1660 1682 and 1746-1790 of Seq.ID.No. 81, aa 1-29, 680-709, and 878 902 of Seq.ID.No. 83, aa 96-136 of Seq.ID.No. 89, aa 1-29, 226 269 and 275-326 of Seq.ID.No. 94, aa 23-47 and 107-156 of Seq.ID.No. 114 and aa 24-53 of Seq.ID.No..142 and fragments thereof being T-cell epitopes. According t.o another aspect, the present invention relates to a vaccine comprising such a hyperimmune serum-reactive antigen or a fragment thereof as identified above for Staphylococcus aureus and Staphylococcus epidermidis. Such a vaccine may comprise one or more antigens against S. aureus or S. epidernidis. Optionally, such S. aureus or S. epidermidis antigens may also be combined with antigens against other pathogens in a combination vaccine. Preferably this vaccine further comprises an immunostimulatory~ substance, preferably selected from the group comprising polyca tionic polymers, especially polycationic peptides, immunostimula tory deoxynucleotides (ODNs), neuroactive compounds, especially human growth hormone, alumn, Freund's complete or incomplete ad juvans or combinations thereof. Such a vaccine may also comprise the-antigen displayed on a surface display protein platform on the surface of a genetically engineered microorganism such as E. coli. According to another aspect, the present invention relates to specific preparations comprising antibodies raised against at least one of the Staphylococcus aureus and Staphylococcus epider midis antigens or Staphylococcus aureus and Staphylococcus epi dermidis antigen fragments as defined above. These antibodies are preferably monoclonal antibodies. Methods for producing such antibody preparations, polyclonal or monoclonal, are well available to the man skilled in the art and properly described in the prior.art. A preferred method for pro ducing such monoclonal antibody preparation is characterized by the following steps - 29 -initiating an immune response in a non human animal by admin istering a Staphylococcus antigen or a fragment thereof, as de fined above, to said animal, -removing the spleen or spleen cells from said animal, -producing hybridoma cells of said spleen or spleen cells, -selecting and cloning hybridoma cells specific for said anti gen and -producing the antibody preparation by cultivation of said cloned hybridoma cells and optionally further purification steps. Preferably, removing of the spleen or spleen cells is connected with killing said animal. Monoclonal antibodies and fragments thereof can be chimerized or humanized (Graziano et al. 1995) to enable repeated administra tion. Alternatively human monoclonal antibodies and fragments thereof can be obtained from phage-display libraries (McGuinnes et al., 1996) or from transgenic animals (Brdggemann et al., 1996). A preferred method for producing polyclonal antibody preparations to said Staphylococcus aureus or Staphylococcus epidermidis anti gens identified with the present invention is characterized by the following steps -initiating an immune response in a non human animal by admin istering a Staphylococcus antigen or a fragment thereof, as de fined above, to said animal, -removing an antibody containing body fluid from said animal, -and -producing the antibody preparation by subjecting said antibody containing body fluid to further purification steps. These monoclonal or polyclonal antibody preparations may be used for the manufacture of a medicament for treating or preventing diseases due to staphylococcal infection. Moreover, they may be used for the diagnostic and imaging purposes. The method is further described in the following examples and in the figures, but should not be restricted thereto.
- 30 Figure 1 shows the pre-selection of sera based on anti-staphylo coccal antibody titers measured by ELISA. Figure. 2 shows the size distribution of DNA fragments in the LSA50/6 library in pMAL4.1. Figure 3 shows the MACS selection with biotinylated human serum. The LSA50/6 library in pMAL9.1 was screened with 10 ig biotiny lated, human serum in the first (A) and with 1 ig in the second selection round (B) . P.serum, patient serum; B.serum, infant se rum. Number of cells selected after the 2 "d and 3 elution are shown for each selection round. Figure 4 shows the serum reactivity with specific clones isolated by bacterial surface display as analyzed by Western blot analysis with patient serum at a dilution of 1 5000. Figure 5 shows peptide ELISA with serum from patients and healthy individuals with an epitope identified by ribosome display. Figure 6 shows representative 2D Immunoblot of S. aureus surface proteins detected with human sera. 800 ig protein from S. au reus/COL grown on B1HI were resolved by IEF (pI 4-7) and SDS-PAGE (9-16%), and subsequently transferred to PVDF membrane. After blocking, the membrane was incubated with sera IC35 (1:20,000). Binding of serum IgG was visualized by an anti-human IgG/HRPO conjugate and ECL development'. Figure 7 demonstrates a representative 2D gel showing S. aureus surface proteins stained by Coomassie Blue. 1 mg protein from S. aureus/COL were resolved by IEF (pI 4-7) and SDS-PAGE (9-16%). Spots selected for sequencing after serological proteome analysis are marked. Figures BAand 8B show the structure of LPXTG cell wall proteins. Figure 9 shows the IgG response in uninfected (N, C) and infected (P) patients to LPXTGV, a novel antigen and probable surface ad hesin of S. aureus, discovered by both the inventive bacterial - 31 surface-display and proteomics approaches. Figure 10 shows the surface staining of S. aureus with purified anti-LPXTGV IgGs. Figure 11 shows a 2D gel where S. aureus surface proteins are stained by Coomassie Blue (left). 1 mg protein from S. aureus/agr grown to early log phase was resolved by IEF (pI 6-11) and SDS PAGE (9-16%). Spots selected for sequencing after serological proteome analysis are marked. Corresponding 2D-immunoblot (right). 800 pg protein from the same preparation was resolved in parallel by 2DE, and subsequently transferred to PVDF membrane. After blocking, the membrane was incubated with the P-pool (1:10,000) . Binding of serum IgG was visualized by an anti-human IgG/HRPO conjugate and ECL development. EXAMPLES Discovery of novel Staphyloccocus aureus antigens Example 1: Preparation of antibodies from human serum The antibodies produced against staphylococci by the human immune system and present in human sera are indicative of the in vivo expression of the antigenic proteins and their immunogenicity. These molecules are essential for the identification of individ ual antigens in the approach as the present invention which is based on the interaction of the specific anti-staphylococcal an tibodies and the corresponding S. aureus peptides or proteins. To gain access to relevant antibody repertoires, human sera were collected from I. patients with acute'S. aureus infections, such as bacteriaemia, sepsis, infections of intravascular and percutan catheters and devices, wound infections, and superficial and deep soft tissue infection. S. aureus was shown to be the causative agent by medical microbiological tests. II. A collection of serum samples from uninfected adults was also included in the present analysis, since staphylococcal infections are common, and anti bodies are present as a consequence of natural immunization from - 32 previous encounters with Staphylococci from skin and soft tissue infections (furunculus, wound infection, periodontitits etc.). The sera were characterized for S. aureus antibodies by a series of ELISA assays. Several styaphylococcal antigens have been used to prove that the titer measured was not a result of the sum of cross-reactive antibodies. For that purpose not only whole cell S. aureus (protein A deficient) extracts (grown under different conditions) or whole bacteria were used in the ELISA assays, but also individual cell wall components, such as lipoteichoic acid and peptidoglycan isolated from S. aureus. More importantly, a recombinant protein collection was established representing known staphylococcal cell surface proteins for the better characteriza tion of the present human sera collections. Recently it was reported that not only IgG, but also IgA serum antibodies can be recognized by the FcRIII receptors of PMNs and promote opsonization (Phillips-Quagliata et al., 2000; Shibuya et al., 2000). The primary role of IgA antibodies is neutralization, mainly at the mucosal surface. The level of serum IgA reflects the quality, quantity and specificity of the dimeric secretory IgA. For that reason the serum collection was not only analyzed for anti-staphylococcal IgG, but also for IgA levels. In the ELISA assays highly specific secondary reagents were used to de tect antibodies from the high affinity types, such as IgG and IgA, and avoided IgM. Production of IgM antibodies occurs during the primary adaptive humoral response, and results in low affin ity antibodies, while IgG and IgA antibodies had already under gone affinity maturation, and are more valuable in fighting or preventing disease Experimental procedures Enzyme linked immune assay (ELISA). ELISA plates were coated with 2-10 pg/ml of the different antigens in coating buffer (sodium carbonate pH 9.2). Serial dilutions of sera (100-100.000) were made in TBS-BSA. Highly specific (cross-adsorbed) HRP (Horse Rad ish Peroxidase) -labeled anti-human IgG or anti-human IgA secon dary antibodies (Southern Biotech) were used according to the manufacturers' recommendations (- 2. 000x). Antigen-antibody com plexes were quantified by measuring the conversion of the sub- - 33 strate (ABTS) to colored product based on OD 40 readings in an automated ELISA reader (Wallace Victor 1420). The titers were compared at given dilution where the dilution response was linear (Table 1). The - 100 sera were ranked based on the reactivity against multiple staphylococcal components, and the highest ones (above 90 percentile) were selected for further analysis in anti gen identification. Importantly, the anti-staphylococcal antibod ies.from sera of clinically healthy individuals proved to be very stable, giving the same high ELISA titers against all the staphy lococcal antigens measured after 3, 6 and 9 months (data not shown). In contrast, anti-S. aureus antibodies in patients de crease, then disappear after a couple of weeks following the in fection (Coloque-Navarro et al, 1998). However, antibodies from patients are very important, since these are direct proof of the in vivo expression of the bacterial antigens tested in or ELISAs or identified as immunogenic during the screens according to the present invention. This comprehensive approach followed during antibody characteri zation is unique, and led to unambiguous identification of anti staphylococcal hyperimmune sera. Purification of antibodies for genomic screening. Five sera from both the patient and the noninfected group were selected based on the overall anti-staphylococcal titers. Antibodies against E. coli proteins were removed by either incubating the heat inacti vated sera with whole cell E. coli (DH5a, -transformed with pHIE11, grown under the same condition as used for bacterial dis play) or with E. coli lysate affinity chromatography for ribosome -display. Highly enriched preparations of IgG from the pooled, de pleted sera were generated by protein G affinity chromatography, according to the manufacturer's instructions (UltraLink Immobi lized Protein G, Pierce). IgA antibodies were purified also by affinity chromatography using biotin-labeled anti-human IgA (Southern Biotech) immobilized on Streptavidin-agarose (GIBCO BRL). The efficiency of depletion and purification was checked by SDS-PAGE, Western blotting, ELISA, and protein concentration measurements. For proteomics, the depletion the IgG and IgA preparation was not necessary, since the secondary reagent en sured the specificity.
- 34 Example 2: Generation of highly random, frame-selected, small fragment, genomic DNA libraries of Staphylococcus aureus Experimental procedures Preparation of staphylococcal genomic DNA. This method was devel oped as a modification of two previously published protocols (So hail, 1998, Betley et al., 1984) and originally specifically adapted for the methicillin resistant Staphylococcus aureus strain COL to obtain genomic DNA in high quality and large scale. 500 ml BHI (Brain Heart Infusion) medium supplemented with 5 ig/ml Tetracycline was inoculated with bacteria from a frozen stab and grown with aeration and shaking for 18 h at 370. The culture was then harvested in two aliquots of 250 ml each, cen trifuged with 1600 x g for 15 min and the supernatant was re moved. Bacterial pellets were carefully re-suspended in 26 ml. of 0.1 mM Tris-HCl, pH'7.6 and centrifuged again with 1600 x g for 15 min. Pellets were re-suspended in 20 ml of 1 mM Tris-HC1, pH 7.6, 0.1 mM EDTA and transferred into sterile 50 ml polypropylene tubes. 1 ml of 10 mg/ml heat treated RNase A and 200 U of RNase T1 were added to each tube and the solution mixed carefully. 250 pl of Lysostaphin (10 mg/ml stock, freshly prepared in ddH 2 0) was then added to the tubes, mixed thoroughly and incubated at 40 0 C for 10 min in a shaking water bath under continuous agita tion. After the addition of 1 ml 10 % SDS, 40 pl of Proteinase K (25 mg/ml stock) and 100 pl of Pronase (10 mg/ml), tubes were again inverted several times and incubated at 400C for 5 min in a shaking water bath. 3.75 ml of 5 M NaCl and 2.5 ml of cetyl tri methyl-ammonium bromide solution (CTAB) (10% w/v, 4% w/v NaCl) were then added and tubes were further incubated at 65 0 C in a 'shaking water bath for 10 min. Samples were cooled to room tem perature and extracted with PhOH/CHCl 3 /IAA (25:24:1) and with CHCl 3 /IAA (24:1). Aqueous phases were carefully collected and transferred to new sterile 50-ml tubes. To each tube 1.5 ml of Strataclean7 Resin was added, mixed gently but thoroughly and in cubated for one minute at room temperature. Samples were centri fuged and the upper layers containing the DNA were collected into clean 50ml-tubes. DNA was precipitated at room temperature by adding 0.6 x volume of Isopropanol, spooled from. the solutiondJ with a sterile Pasteur pipette and transferred into tubes con- - 35 taining 80% ice cold ethanol. DNA was recovered by centrifuging the precipitates with 10-12 000 x g, then dried on air and dis solved in ddH 2 0. Preparation of small genomic DNA fragments. Genomic DNA fragments were mechanically sheared into fragments ranging in size between 150 and 300 bp using a cup-horn sonicator (Bandelin Sonoplus UV 2200 sonicator equipped with a BB5 cup horn, 10 sec. pulses at 100 % power output) or into fragments of size between 50 and 70 bp by mild DNase I treatment (Novagen). It was observed that sonication yielded a much tighter fragment size distribution when breaking the DNA into fragments of the 150-300 bp size range. However, despite extensive exposure of the DNA to ultrasonic wave-induced hydromechanical shearing force, subsequent decrease in fragment size could not be efficiently and reproducibly achieved. Therefore, fragments of 50 to 70 bp in size were ob tained by mild DNase I treatment using Novagen's shotgun cleavage kit. A 1:20 dilution of DNase I provided with the kit was pre pared and the digestion was performed in the presence of MnCl 2 in a 60 p.l volume at 20 0 C for 5 min to ensure double-stranded cleav age by the enzyme. Reactions were stopped with 2 il of 0.5.M EDTA and the fragmentation efficiency was evaluated on a 2% TAE-aga rose gel. This treatment resulted in total fragmentation of ge nomic DNA into near 50-70 bp fragments. Fragments were then blunt-ended twice using T4 DNA Polymerase in the presence of 100 pM each of dNTPs to ensure efficient flushing of the ends. Fragments were used immediately in ligation reactions or frozen at -20 0 C for subsequent use. Description of the vectors. The vector pMAL4.1 was constructed on a pEH1 backbone (Hashemzadeh-Bonehi et al., 1998) with the Kana mycin resistance gene'. In addition it harbors a b-lactamase (bla) gene cloned into the multiple cloning site. The bla gene is pre ceded by the leader peptide sequence of ompA to ensure efficient secretion across the cytoplasmic membrane. A Sma I restriction site serves for library insertion. The Sma I site is flanked by an upstream FseI site and a downstream NotI site which were used for recovery of the selected fragments. The three restriction sites are inserted after the .ompA leader sequence in such a way that the bla gene is transcribed in the -1 reading frame result- - 36 ing in a stop codon 15 bp. after the NotI site. A +1 bp insertion restores the bla ORF so that b-lactamase protein is produced with a consequent gain of Ampicillin resistance. The vector pMAL4.31 was constructed on a pASK-IBA backbone (Skerra, 1994) with the b-lactamase gene exchanged with the Kana mycin resistance gene. In addition it harbors a b-lactamase (bla) gene cloned into the multiple cloning site. The sequence encoding mature b-lactamase is preceded by the leader peptide sequence of ompA to allow efficient secretion across the cytoplasmic mem brane. Furthermore a sequence encoding the first 12 amino ac ids (spacer sequence) of mature b-lactamase follows the ompA leader peptide sequence to avoid fusion of sequences immediately after the leader peptidase cleavage site, since e.g. clusters of positive charged amino acids in this region would decrease or abolish translocation across the cytoplasmic membrane (Kajava et al., 2000). A SmaI restriction site serves for library insertion. The SmaI site is flanked by an upstream FseI site and a down stream NotI site which were used for recovery of the selected fragment. The three restriction sites are inserted after the se quence encoding the 12 amino acid spacer sequence in such a way that the bla gene is transcribed in the -1 reading frame result ing in a stop codon 15 bp after the NotI site. A +1 bp. insertion restores the bla ORF so that b-lactamase protein. is produced with a consequent gain of Ampicillin resistance. The vector pMAL9.1 was constructed by cloning the lamB gene into the multiple cloning site of pEH1. Subsequently, a sequence was inserted in lamB after amino acid 154, containing the restriction sites FseI, SmaI and NotI. The reading frame for this insertion was chosen in a way that transfer of frame-selected DNA fragments excised by digestion with FseI and NotI from plasmids pMAL4.1 or pMAL4.31 to plasmid pMAL9.1 will yield a continuous reading frame of lamB and the respective insert. The vector pHIE11 was constructed by cloning the fhuA gene into the multiple cloning site of pEH1. Thereafter, a sequence was in serted in fhuA after amino acid 405, containing the restriction site FseI, XbaI and NotI. The reading frame for this insertion was chosen in a way that transfer of frame-selected DNA fragments excised by digestion with FseI and NotI from plasmids pHAL4.1 or - 37 pMAL4.31 to plasmid pHIE11 will yield a continuous reading frame of fhuA and the respective insert. Cloning and evaluation of the library for frame selection. Ge nomic S. aureus DNA fragments were ligated into the SmaI site of either the vector pMAL4.1 or pMAL4.31. Recombinant DNA was elec troporated into DH10B electrocompetent E. coli cells (GIBCO BRL) and transformants plated on LB-agar supplemented with Kanamy cin (50 ig/ml) and Ampicillin (50 ig/ml). Plates were incubated over night at 37 0 C and colonies collected for large scale DNA ex traction. A representative plate was stored and saved for col lecting colonies for colony PCR analysis and large-scale sequencing. A simple colony PCR assay was used to initially de termine the rough fragment size distribution as well as insertion efficiency..From sequencing data the precise fragment size was evaluated, junction intactness at the insertion site as well as the frame selection accuracy (3n+1 rule). Cloning and evaluation of the library for bacterial surface dis play. Genomic DNA fragments were excised from the pMAL4.1 or pMAL4.31 vector, containing the S. aureus library with the re striction enzymes FseI and NotI. The entire population of frag ments was then transferred into plasmids pMAL9.1 (LamB) or pHIE11 (FhuA) which have been digested with FseI and NotI. Using these two restriction enzymes, which recognise an 8 bp GC rich se quence, the reading frame that was selected in the pMAL4.1 or pMAL4.31 vector is maintained in each of the platform vectors. The plasmid library was then transformed into E. coli DH5a cells by electroporation. Cells were plated onto large LB-agar plates supplemented with 50 Vig/ml Kanamycin and grown over night at 379C at a density yielding clearly visible single colonies. Cells were then scraped off the surface of these plates, washed with fresh LB medium and stored in aliquots for library screening at -80 0 C. Results Libraries for frame selection. Two libraries (LSA50/6 and LSA250/1) were generated in the pMAL4.1 vector with sizes of ap proximately 50 and 250 bp, respectively. For both libraries a to tal number of clones after frame selection of 1-2x 106 was - 38 received using approximately 1 pg of pNAL4.1 plasmid DNA and 50 ng of fragmented genomic S. aureus DNA. To assess the randomness of the LSA50/6 library, 672 randomly chosen clones were se quenced. The bioinformatic analysis showed that of these clones none was present more than once. Furthermore, it was shown that 90% of the clones fell in the size range of 19 to 70 bp with an average size of 25 bp (Figure 2). All 672 sequences followed the 3n+l rule, showing that all clones were properly frame selected. Bacterial surface display libraries. The display of peptides on the surface of E. coli required the transfer of the inserts from the LSA50/6 library from the frame selection vector pMAL4.1 to the display plasmids pMAL9.1 (LamB) or pHIEll (FhuA). Genomic DNA fragments were excised by FseI and NotI restriction and ligation of 5ng inserts with 0.1ig plasmid DNA resulted in 2-5x 10' clones. The clones were scraped off the LB plates and frozen without further amplification. Example 3: identification of highly immunogenic peptide sequences from S. aureus using bacterial surface displayed genomic librar ies and human serum Experimental procedures MACS screening. Approximately 2.5x 10' cells from a given library were grown in 5 ml LB-medium supplemented with 50 pg/ml Kanamycin for 2 h at 370C. Expression was induced by the addition of 1 mM IPTG for 30 min. Cells were washed twice with fresh LB medium and approximately 2x 107 cells re-suspended in 100 jil LB medium and transferred to an Eppendorf tube. 10 ig of biotinylated, human serum was added to the cells and the suspension incubated over night at 4 0 C with gentle shaking. 900 pl of LB medium was added, the suspension mixed and subsequently centrifuged for 10 min at 6000 rpm at 40C. Cells were washed once with 1 ml LB and then re-suspended in 100 pl LB medium. 10 pl of MACS microbeads coupled to streptavidin (Miltenyi Biotech, Ger many) were added and the incubation continued for 20 min at 4 0 C. Thereafter 900 pl of LB medium was added and the MACS microbead cell suspension was loaded onto the equilibrated MS column (Mil- - 39 tenyi Biotech, Germany) which was fixed to the magnet. (The MS columns were equilibrated by washing once with 1 ml 70% EtOH and twice with 2 ml LB medium.) The column was then washed three times with 3 ml LB medium. The elution was performed by removing the magnet and washing with 2 ml LB medium. After washing the column with 3 ml LB medium, the 2 ml eluate was loaded a second time on the same column and the washing and elution process repeated. The loading, washing and elution process was performed a third time, resulting in a final eluate of 2 ml. A second round of screening was performed as follows. The cells from the final eluate were collected by centrifugation and re suspended in 1 ml LB medium supplemented with 50 pg/ml Kanamycin. The culture was incubated at 37 0 C for 90 min and then induced .with 1 mM IPTG for 30 min. Cells were subsequently collected, washed once with 1 ml LB medium and suspended in 10 il LB medium. Since the volume was reduced, 1 pig of human, biotinylated serum was added and the suspension incubated over night at 4 0 C with gentle shaking. All further steps were exactly the same as in the first selection round. Cells selected after two rounds of selec tion were plated onto LB-agar plates supplemented with 50 ig/ml Kanamycin and grown over night at 37 0 C. Evaluation of selected clones by sequencing and Western blot analysis. Selected clones were grown over night at 37 0 C in 3 ml LB medium supplemented with 50 pg/ml Kanamycin to prepare plasmid DNA using standard procedures. Sequencing was performed at MWG (Germany) or in a collaboration with TIGR (U.S.A.). For Western blot analysis approximately 10 to 20 jig of total cel lular protein was separated by 10% SDS-PAGE and blotted onto Hy bondC membrane (Amersham Pharmacia Biotech, England) . The LamB or FhuA fusion proteins were detected using human serum as the pri mary antibody at a dilution of 1:5000 and anti human IgG antibod ies coupled to HRP at a dilution of 1:5000 as secondary antibodies. Detection was performed using the ECL detection kit (Amersham Pharmacia Biotech, England). Alternatively, rabbit anti FhuA or mouse anti LamB antibodies were used as primary antibod ies in combination with the respective secondary antibodies cou- - 40 pled to HRP for the detection of the fusion proteins. Results Screening of bacterial surface display libraries by magnetic ac tivated cell sorting (MACS) using biotinylated human serum. The libraries LSA50/6 in pMAL9.1 and LSA250/1 in pHIE11 were screened with a pool of biotinylated, human patient sera (see Example 1) Preparation of antibodies from human serum) . The selection proce dure was performed as described under Experimental procedures. As a control, pooled human sera from infants that have most likely not been infected with S. aureus was used. Under the described conditions between 10 and 50 fold more cells with the patient compared to the infant serum were routinely selected (Figure 3). To evaluate the .performance of the screen, approximately 100 se lected clones were picked randomly and subjected to Western blot analysis with the same pooled patient serum. This analysis re vealed that 30 to 50% of the selected clones showed reactivity with antibodies present in patient serum whereas the control strain expressing LamB or FhuA without a S. aureus specific in sert did not react with the same serum. Colony PCR analysis showed that all selected clones contained an insert in the ex pected size range. Subsequent sequencing of a larger number of randomly picked clones (500 to 800 per screen) led to the identification of the gene and the corresponding peptide or protein sequence that was specifically recognized by the human patient serum used for screening. The frequency with which a specific clone is selected reflects at least in part the abundance and/or affinity of the specific antibodies in the serum used for selection .and recogniz ing the epitope presented by this clone. In that regard it is striking that some clones (ORF2264, ORF1951, ORF0222, lipase and IsaA) were picked up to 90 times, indicating their highly immuno genic property. All clones that are presented in Table 2 have been verified by Western blot analysis using whole cellular ex tracts from single clones to show the indicated reactivity with the pool of human serum used in the screen. It is further worth noticing that most of the genes identified by the bacterial surface display screen encode proteins that are ei- - 41 ther attached to the surface of S. aureus and/or are secreted. This is in accordance with the expected role of surface attached or secreted proteins in virulence of S. aureus. Assessment of reactivity of highly immunogenic peptide sequences with different human sera. 10 to 30 different human patient sera were subsequently used to evaluate the presence of antibodies against the selected immunogenic peptide sequences that have been discovered in the screen according to the present invention. To eliminate possible cross-reactivity with proteins expressed by E. coli, all sera were pre-adsorbed with a total cellular lysate of E. coli DHa cells expressing FhuA protein. This analysis is summarized in Table 2 and as an example shown in Figure 4 and is indicative of the validity of the present screen. It further shows that already short selected epitopes can give rise to the production of antibodies in a large number of pa tients (ORF1618, ORF1632, IsaA, Empbp, Protein A). Those peptide sequences that are not recognized by a larger set of patient sera may still be part of an highly immunogenic protein, but the re combinant protein itself may be tested for that purpose for each single case. Example 4: Identification of highly immunogenic peptide. so quences from genomic fragments. from S. aureus using ribosome display and human serum Experimental procedures Ribosome display screening: 2.4 ng of the genomic library from S. aureus LSA250/1 in pMAL4.1 (described above) was PCR amplified with oligos ICC277 and ICC202 in order to be used for ribosome display. Oligos ICC277 (CGAATAATACGACTCACTATAGGGAGACCACAACGGTTTCCCACTAGTAATAATTTTGTTTAAC TTTAAGAAGGAGATATATCCATGCAGaCCTTGGCCGGCCTCCC) and ICC202 (GGCCCACCCGTGAAGGTGAGCCGGCGTAAGATOCTTTTCTGTGACTGG) hybridize 5' and 3' of the Fse I-Not I insertion site of plasmid pMAL4.1, re spectively. ICC277 introduces a T7 phage RNA polymerase promoter, a palindromic sequence resulting in a stem-loop structure on the RNA level, a ribosome binding site (RBS) and the translation start of gene 10 of the T7 phage including the ATG start codon.
- 42 Oligo ICC202 hybridizes at nucleotide position 668 of the 9-lac tamase open reading frame and also introduces a stem-loop struc ture at the 3' end of the resulting RNA. PCR was performed with the High fidelity PCR kit (Roche Diagnostic) for 25 cycles at 500C hybridization temperature and otherwise standard conditions. The resulting PCR library was used in 5 consecutive rounds of se lection and amplification by ribosome display similar as de scribed previously (Hanes et al., 1997) but with modifications as described below. One round of ribosome display contained the following steps: In vitro transcription of 2 pg PCR product with the RiboMax kit (Promega) resulted in ca. 50 jig RNA. In vitro translation was per formed for 9 minutes at 37 0 C in 22 pl volume with 4.4 pl Premix Z (250 mM TRIS-acetate pH 7.5, 1.75 mM of each amino acid, 10 mM ATP, 2.5 mM GTP, 5 mM cAMP, 150 rnM acetylphosphate, 2.5 mg/ml E. coli tRNA, 0.1 mg/.ml folinic acid, 7.5 % PEG 8000, 200 mM potas sium glutamate, 13.8 nM Mg(Ac)2, 8 pl S30 extract (x mg/ml) and about 2 pg in vitro transcribed RNA from the pool. S30 extract was prepared as described (Chen et al, 1983). Next, the sample was transferred to an ice-cold tube containing 35.2 pl 10 % milk WBT (TRIS-acetate pH 7.5, 150 mM NaCl, 50 mM Mg(Ac)2, 0.1 % Tween-20, 10 % milk powder) and 52.8 pl WBTH (as before plus 2.5 mg/ml heparin).- Subsequently, immuno precipitation was performed by addition of 10 pg purified IgGs, incubation for 90 minutes on ice, followed .by' addition of 30 pl MAGmol Pro*tein G beads (Mil tenyi Biotec, 90 minutes on ice). The sample was applied to a pre-equilibrated p column (Miltenyi Biotec) and washed 5 times with ice-cold WBT buffer. Next 20 pl EB20 elution buffer (50 mM TRIS-acetate, 150 mM NaCl, 20 mM EDTA, 50 pg/ml S. cerevisiae RNA) was applied to the column, incubated for 5 minutes at 4 0 C. Elution was completed by adding 2 x 50 pl EB20-. The mRNA from the elution sample was purified with the High pure TRNJ isolation kit (Roche Diagnostics). Subsequent reverse transcription was per formed with Superscript II reverse transcriptase kit (Roche Diag nostics) according to the instruction of the manufacturer with 60 pmol oligo ICC202 for 1 hour at 50 0 C in 50 p1 volume. 5 pl of this mix was used for the following PCR reaction with primers ICC202 and ICC277 as described above.
- 43 Three rounds of ribosome display were performed and the resulting selected PCR pool subsequently cloned into plasmid pHIEll (de scribed above) by cleavage with restriction endonucleases NotI and FseI. Evaluation of selected clones by sequencing and peptide-ELISA analysis: Selected clones were grown over night at 370C in 3 ml LB medium supplemented with 50 pg/ml Kanamycin to prepare plasmid DNA using standard procedures. Sequencing was performed at MWG (Germany) or at the Institute of Genomic Research (TIGR; Rockville, MD, U.S.A.). Peptides corresponding to the inserts were synthesized and coated in 10 mM NaHCO pH 9.3 at a concen tration of 10 pg/ml (50 pl) onto 96-well microtiter plates (Nunc). After blocking with 1% BSA in PBS at 37 0 C, 1:200 and 1:1000 dilutions of the indicated sera were diluted in 1% BSA/PBS and applied to the wells. After washing with PBS/0.1 % Tween-20, biotin-labeled anti-human IgG secondary antibodies (SBA) were added and these were detected by subsequent adding horseradish peroxidase-coupled streptavidin according to standard procedures. Results The 250-bp genomic library (LSA250/1) as described above was used for screening. Purified IgGs from uninfected adults but with high titer against S. aureus as described above were used for selec tion of antigenic peptides. Three rounds of ribosome display selection and amplificaton were performed according to Experimental procedures; finished by clon ing and sequencing the resulting PCR pool. Sequence analyses of a large number of randomly picked clones (700) led to the identification of the gene and the corresponding peptide or protein sequence that was specifically recognized by the high titer serum used for screening. The frequency with which a- specific clone was selected reflects at least in part the abun dance and/or affinity of the specific antibodies in the serum used for selection and recognizing the epitope presented by this clone. Remarkably, some clones (ORFs) were picked up to 50 times, indicating their highly immunogenic property. Table 2 shows the ORF name, the Seq.ID No. and the number of times it was identi- - 44 fied by the inventive screen. For a number of immuno-selected ORFs peptides corresponding to the identified immunogenic region were synthesized and tested in peptide-ELISA for their reactivity towards the sera pool they were identified with and also a number of additional sera from patients -who suffered from an infection by S. aureus. The two ex amples in the graphs in figure 5 show the values of peptides from aureolysin and Pls. They are not only hyperimmune reactive against the high titer sera pool but also towards a number of in dividual patient's sera. All synthesized peptides corresponding to selected immunogenic regions showed reactivity towards the high titer sera pool and Table 2 summarizes the number of times the peptides were reactive towards individual patients sera, similar as described above. In addition, it is striking that for those ORFs that were also identified by bacterial surface display described above), very often the actual immunogenic region within the ORF was identical or overlapping with 'the one identified by ribosome display. This comparison can be seen in Table 2. Example 5: Identification of highly immunogenic -antigens from S. aureus using Serological -Proteome Analysis. Experimental procedures Surface protein preparations from S. aureus containing highly im munogenic antigens. S. aureus strains COL (Shafer and landolo, 1979) and- agr- (Recsei et al., 1986) were stored as glycerol stocks at -80 0 C' or on BHI (DIFCO) plates at 4 0 C. Single clones were used for inoculation of overnight cultures in either BHI ("standard conditions") or RPMI 1640 (GibcoBRL), last one de pleted from iron ("stress conditions") by treating o/n with imi nodiacetic acid (Sigma). Fresh medium was inoculated 1:100 the next day and bacteria were grown to O.D.
60 D between 0.3 and 0.7. Bacteria were harvested by centrifugation and washed with ice cold PBS. Surface proteins were prepared by lysostaphin treatment under isotonic conditions (Lim et al. 1998). Briefly; -3x 109 bac teria (according to O.D.
00 = 1 are about 5x10' bacteria) were re- - 45 suspended in 1 ml digestion buffer containing 35% raffinose (Ald rich Chemical Company), protease inhibitors (Roche) and 5 units lysostaphin (Sigma). After incubation at 370C for 30 min, proto plasts were carefully sedimented by low-speed centrifugation. This treatment releases surface proteins covalently linked to the pentaglycine bridge of the peptidoglycan cell wall to the super natant (in Crossley, 1997). Cell surface proteins were either precipitated with methanol/chlorophorm (Wessel, 1984) or concen trated in centrifugal filter-tubes (Millipore). Protein samples were frozen and stored at -800C or dissolved in sample buffer and used for isoelectric focusing (IEF) immediately (Pasquali et al. 1997). Serological proteome analysis of surface protein preparations from S. aureus. Samples were obtained from a) S. aureus/agr grown -under stress conditions", b) S. aureus/COL grown under "standard conditions" and c) S. aureus/COL "stress conditions". Loading onto 17 cm-strips containing immobilized pH gradients (pH 4-7, BioRad) was done using the "in-gel-reswelling procedure" (Pasquali et al., 1997). The gels for blotting were loaded with 100-800 pig protein, the preparative gels with 400-1,000 pig protein. Isoelectric focusing and SDS-PAGE (9-16% gradient gels) were performed as described (Pasquali et al., 1997). For Western blotting, proteins were transferred onto 'PVDF-membranes (BioRad) by semi-dry blotting. Transfer-efficiency was checked by amido black staining. After blocking (PBS/0.1% Tween 20/10% dry milk, 40C for 16 h), blots were incubated for two hours with serum (1:2,500-1:100,000 in *blocking solution, see Table 3). After washing, specific binding of serum IgG was visualized with a goat-anti-human-IgG / peroxidase conjugate (1:25.,000, Southern Biotech) as secondary antibody and development with - a chemiluminescence substrate (ECLm, Amersham) . A representative result is shown in Figure 6. Membranes were stripped by treatment with 2% Z-ME/Laemmli buffer for 30 min at 50-650C, immediately re-probed with a different serum, and developed as' described above. This procedure 'was repeated up to five times. Signals showing up with patient and/or healthy donor control sera but not with the infant pool, were matched to the Coomassie . (BioRad) stained preparative gels (example shown in Figure 7). The results of these serological proteome analyses of surface protein prepa rations from S. aureus are summarized in Table 3.
- 46 Sequencing of .protein spots by peptide-fingerprint MALDI-TOF-MS and tandem MS/MS. Gel pieces were washed alternately three times with 10 il digestion buffer (10mM Nn 4
HCO
3 /CAN, 1:1). Afterwards the gel pieces were shrunken with 10 il ACN and reswollen with 2 y1 protease solution (0.05 pg/il trypsin, Promega, Madison, USA). Digestion was performed for 10-12 h at 370C. For MALDI-TOF-MS peptides were extracted from the gel pieces with 10 4 digestion buffer. The supernatant was concentrated with ZipTip" (Millipore, Bedford, USA), the peptides were eluted onto the MALDI target with 0.5 pl extraction buffer (0.1% TFA/CAN, 1:1) and 0.5 1 matrix solution (HCCA in ACN/0.1% TFA, 1:1) was added. MALDI-TOF MS was done using a REFLEX III (Bruker Daltonik, Bremen, Germany) equipped with a SCOUT384 ion source. The acceleration voltage was set to 25 kV, and the reflection voltage to 28.7 kV. The mass range was set from 700 Da to 4000 Da. -Data acquisition was done on a SUN Ultra using XACQ software, version 4.0. Post-analysis data processing was done using XMASS software, version 4.02 (Bruker Daltonik, Bremen, Germany). The results are summarized in tables 3 and 4. Example 6: Characterisation of highly immunogenic proteins from S. aureus The antigens identified by the different screening methods with the IgG and IgA preparations form pre-selected sera are. further characterized, by the following ways: 1. The proteins are purified, most preferably as recombinant proteins expressed in E. coli or in a Gram+ expression system or in an in vitro translation'system, and evaluated for antigenicity by a series of human sera. The proteins are modified based on bioinformatic analysis: N-terminal sequences representing the signal peptide are removed, C-terminal regions downstream of the cell wall anchor are also removed, and extra amino acids as tags are introduced for the ease of purification (such as Strep-tagII, His-tag, etc.) A large number of sera is then used in ELISA as says to assess the fraction of human sera containing specific an tibodies against the given protein (see Fig. 9 as an example) . one of the selected antigens is a 895 aa long protein, what was called LPXTGV (see Tables 2 and 4), since. it contains the Gram+ cell wall anchor sequence LPXTG. This signature has been shown to - 47 serve as cleavage site for sortase, a trans-peptidase which cova lently links LPXTG motif containing proteins to the peptidoglycan cell wall. LPXTGV is also equipped with a typical signal peptide (Fig. 8). ELISA data using this protein as a Strep-tagged recom binant protein demonstrate that this protein is highly immuno genic (high titers relative to other recombinant proteins) in a high percentage of sera (Fig. 9). Importantly, patients with acute S. aureus infection produce significantly more of these anti-LPXTGV antibodies, than healthy normals, suggesting that the protein is expressed during in vivo infection. The overall ELISA titers of the individual antigenic proteins* are compared, and the ones inducing the highest antibody levels (highly immunogenic) in most individuals (protein is expressed by most strains in vivo) are favored. Since the antigen specificity and quality (class, subtype, functional, nonfunctional) of the antibodies against S. aureus produced in individual patients can vary depending on the site of infection, accompanying chronic diseases (e.g. diabetes) and chronic conditions (e.g. intravascular device), and the indi viduals' immune .response, -special attention was paid to the dif ferences detected among the different patient groups, since medical records belonging to each sera were available. In addi tion, each patient serum is accompanied by the pathogenic* strain isolated from the patient at the time of serum sampling. 2. Specific antibodies are purified for functional characteri zation. The purity and the integrity of the recombinant proteins are checked (e.g. detecting the N-terminal Strep-tag in Western blot analysis in comparison to silver staining in SDS-PAGE). The antigens are immobilized through the tags to create an affinity matrix', and used for the purification of specific antibodies from highly reactive sera. Using as an example strep-tagged LPXTGV as the capture antigen, 20 pig of antibody from 125 mg of IgG were purified. Based on the ELISA data a pure preparation was re ceived, not having e.g. anti-LTA and anti-peptidoglycan (both dominant with unfractionated IgG) activity. The antibodies are then used to test cell surface localization by FACS and fluores cent microscopy (Fig. 10). 3.- Gene occurrence in clinical isolates An ideal vaccine antigen would be an antigen that is present in all,. or the vast majority of, strains of the target organism to - 48 which the vaccine is directed. In order to establish whether the genes encoding the identified Staphylococcus aureus antigens oc cur ubiquitously in S. aureus strains, PCR was performed on a se ries of independent S. aureus isolates with primers specific for the gene of interest. S. aureus isolates were obtained from pa tients with various S. aureus infections. In addition several na sal isolates from healthy carriers and several lab strains were also collected and analyzed. The strains were typed according to restriction fragment length polymorphism (RFLP) of the spa and coa genes (Goh et al. 1992, Frdnay et al., 1994, vanden Bergh et al. 1999). From these results 30 different strains were identi fied - 24 patient isolates, 3 nasal isolates and 3 lab strains. To establish the gene distribution of selected antigens, the ge nomic DNA of these 30 strains was subjected to PCR with gene spe cific primers that flank the selected epitope (ORF1361: Seq.ID No. 187 and 188; ORF2268: Seq.ID No. 193 and 194; ORF1951: Seq.ID No. 195 and 196; ORF1632: Seq.ID No. 181 and 182; ORF0766: Seq.ID .No. 183 and 184; -ORF0576: Seq.ID No. 185 and 186; ORF0222: Seq.ID No. 189 and 190; ORF0360: Seq.ID No. 191 and 192). The PCR prod ucts were analyzed by gel electrophoresis to identify a product of the correct predicted size. ORFs 1361, 2268, 1951, 1632, 0766 and 0222 are present in 100% of strains tested and ORF0576 in 97%. However ORF0360 occurred in only 71% of the strains. .Thus ORFs 1361, 2268, 1951, 1632, 0766, 0576 and 0222 each have the required ubiquitous presence among S. aureus isolates. These antigens (or antigenic fragments thereof, especially the fragments identified) are especially preferred for use in a vac cination project against S. aureus. 4. Identification of highly promiscuous HLA-class II helper epitopes within the ORFs of selected antigens The ORFs corresponding to the antigens identified on the basis of recognition by antibodies in human sera, most likely also contain. linear T-cell epitopes. Especially the surprising finding in .the course of the invention that even healthy uninfected, non-colo nized individuals show extremely high antibody titers (> 100,000 for some antigens, see Example 5) which are stable for >1 year (see Example 1), suggests the existence of T-cell dependent mem ory most probably mediated by CD4+ helper-T-cells. The. molecular - 49 definition of the corresponding HLA class II helper-epitopes is usefull for the design of synthetic anti-staphylococcal vaccines, which can induce immunological memory. In this scenario the helper-epitopes derived from the staphylococcal antigens provide "cognate help" to the B-cell response against these antigens or fragments thereof. Moreover it is possible to use these helper epitopes to.induce memory to T-independent antigens like for in stance carbohydrates (conjugate vaccines). On the other hand, in tracellular occurring staphylococci can be-eliminated by CD8+ cytotoxic T-cells, which recognize HLA class I restricted epi topes. T-cell epitopes can be predicted by various public domain algo rithms: http://bimas.dcrt.nih.aov/molbio/hla bind/ (Park er et al. 1994), htto://134.2.96.221/scripts/MHCSeryer.dll/home.htm (Rammensee at al. 1999), http://mypaae.ihost.com/usinet.hamme76/ (Sturniolo et al. 1999). The latter prediction algorithm offers the possibility to identify promiscuous helper-epitopes, i.e. peptides that bind to several HLA class II molecules. In order to identify highly promiscuous helper-epitopes within staphylococcal antigens the ORFs corresponding to Seq ID 64 (IsaA), Seq ID 114 (POV2), Seq ID 89 (QRF0222), Seq ID 70 (LPXTGIV), Seq ID 56 (LPXTGV), Seq ID 142 (LPXTGVI), Seq ID 81 (ORF3200), Seq ID 74 (ORF1951), Seq ID 94 (Empbp), Seq ID 83 (autolysin) and Seq ID 58 (ORF2498) were analyzed using the TEPITOPE package http://mvpaae.ihost.com/usi net.hamme76/ (Sturniolo et al. 1999). The analysis was done for 25 prevalent DR-alleles and peptides were selected if they were predicted to be a) strong binders (1% threshold) for at least 10/25 alleles or b) intermediate (3% threshold) binders for at least 17/25 alleles. The following peptides containing one or several promiscuous h'elper-epitopes were selected (and are claimed): Seq ID 56: pos. 6-40, 583-598, 620-646, 871-896 Seq ID 58: no peptide fulfills selection criteria Seq ID 64: no peptide fulfills selection criteria Seq ID 70: pos. 24-53 Seq ID 74: pos. 240-260 Seq ID 81: pos. 1660-1682, 1746-1790 Seq ID 83: pos. 1-29, 680-709, 878-902 - 50 Seq ID 89: pos. 96-136 Seq ID 94: pos. 1-29, 226-269~, 275-326 Seq ID 114: pos. 23-47, 107-156 Seq ID 142: pos. 24-53 The corresponding peptides or fragments thereof (for instance overlapping 15-mers) can be synthesized and tested for their ability to bind to various HLA molecules in vitro. Their immuno genicity can be tested by assessing the peptide (antigen)-driven proliferation (BrdU or 3H-thymidine incorporation) or the secre tion of cytokines (ELIspot, intracellular cytokine staining) of T-cells in vitro (Mayer et al. 1996, Schmittel et al. 2000, Ses ter et al. 2000). In this regard it will be interesting to deter mine quantitative and qualitative differences in the T-cell response to the staphylococcal antigens or the selected promiscu ous peptides or fragments thereof in populations of patients with different staphylococcal infections, or colonization versus healthy individuals neither recently infected nor colonized. Moreover, a correlation between the antibody titers and the quan tity and quality of the -T-cell response observed in these popula tions is expected. Alternatively, immunogenicity of the predicted peptides can be tested in HLA-transgenic mice (Sonderstrup et al. 1999). Similar approaches can be taken for the identification of HLA class I restricted epitopes within staphylococcal antigens. Synthetic peptides representing one or more promiscuous T helper epitopes from S.aureus Partially overlapping peptides spanning the indicated regions of Seq ID 56 (LPXTGV), Seq ID 70 (LPXTGIV) , Seq ID 74 (ORFihoml), Seq ID 81 (EMBP), Seq ID 83 (Autolysin), Seq ID 89 (ORFlhom2), Seq ID 94 (EMPBP), Seq ID 114 (POV2) and Seq ID 142 (LPXTGVI) were synthesized. Sequences of the individual peptides are given in Table 5. All peptides were synthesized using Fmoc chemistry, HPLC purified and analyzed by mass spectrometry. Lyophilized pep tides were dissolved in DMSO and stored at -20 0 C at a concentra tion of 5-10 mM. binding of synthetic peptides representing promiscuous T helper - 51 epitopes to HLA molecules in vitro Binding of peptides to HLA molecules on the surface of antigen presenting cells is a prerequisite for activation of T cells. Binding was assessed in vitro by two independent biochemical as says using recombinant soluble versions of HLA class II mole cules. One assay measures the concentration dependent competitive replacement of a labeled reference peptide by the test peptides. The second assay is based on the formation of SDS-stable com plexes upon binding of high- and intermediate affinity ligands. A summary of the results obtained by the two assays is given in Table 5. Soluble HLA molecules (DRAl*0101/DRB1*0101 and DRA1*0101/DRB1*0 4 0 1) were expressed in SC-2 cells and purified as described in Aichinger et al., 1997. For the competition assay (Hammer et al. 1995) HLA molecules were applied between 50 and 200 ng/well. For DRB1*0101 biotinilated indicator peptide HA (PKYVKQNTLKLAT, Valli et al. 1993) was used at 0.008 p1M. For DRB1*0401 biotinilated indicator peptide UD4 (YPKFVKQNTLKAA, Valli et al. 1993) was used between 0.03 and 0.06 p!M. Test pep tides were used in serial dilutions from 0.02 nM to 200 pM. mole cules, indicator and test peptides were incubated overnight at 37 0 C, pH 7. HLA:peptide complexes obtained after incubation with serial dilutions of test and reference peptides (the known high affinity binders HA and UD4 were used as positive control) were captured in ELISA plates coated with antibody L243, which is known to recognize a conformational epitope formed only by cor rectly associated heterodimers. Incorporated biotin was measured by standard colorimetric detection using a streptavidin-alkaline phosphatase conjugate (Dako) with NBT/BCIP tablets (Sigma) as substrate and automated OD reading on a Victor reader (Wallac). T cell response against promiscuous T helper epitopes assessed by IFNg ELIspot assay Upon antigenic stimulation T cells start to proliferate and to secrete cytokines such as interferon gamma (IFNg) . Human T. cells specifically recognizing epitopes within S.aureus antigens were detected by IFNg-ELIspot (Schmittel et al. 2000). PBMCs from healthy individuals with a strong anti-S.aureus IgG response were isolated from 50-100 ml of venous blood by ficoll density gradi- - 52 ent centrifugation and used after freezing and thawing. Cells were seeded at- 200,000/well in 96-well plates. Peptides were added as mixtures corresponding to individual antigens, in both cases at 10 ig/ml each. Concanavalin A (Amersham) and PPD (tuber culin purified protein derivate, Statens Serum Institute) served as assay positive controls, assay medium without any peptide -as negative control. After overnight incubation in Multi Screen 96 well filtration plates (Millipore) coated with the anti-human IFNg monoclonal antibody B140 (Bender -Med Systems) the ELIspot was developed using the biotinylated anti-human IFNg monoclonal antibody B308-BT2 (Bender Med Systems), Streptavidin-alkaline phosphatase (DAKO) and BCIP/NBT alkaline phosphatase substrate (SIGMA). Spots were counted using an automatic plate reader (Bioreader 2000, BIO-SYS). Spots counted in wells with cells stimulated with assay medium only (negative control, generally below 10 spots / 100.000 cells) were regarded as background and subtracted from spot numbers counted in wells with peptides. Table 5: Promiscuous T helper epitopes contained In S.aureus antigens Amino acid sequences within S.aureus antigens containing binding IFNg highly promiscuous T helper epitopes ELIspot 2) Seq ID 56 (LPXTGV): pos. 6-40 p6- 28 >PKLRSFYSIRKSTLGVASVIVST// + p2 4
-
40 >VIVSTLFLISQHQAQA// 44;80;8 ;95;112 Seq ID 56 (LPXTGV): pos. 620-646 p620- 646 >FPYIPDKAVYNAIVKVVNIGYEGQ// Seq ID 56 (LPXTGV)i pos. 871-896 p871-896 >QSWWGLYALLGMLALFIPKFRKESK// Seq ID 70 (LPXTGIV): pos. 24-53 p24-5 3 >YSIRKFTVGTASILIGSLMYLGTQQEAEA// nd 34;14;0 ;57;16 Seq ID 74 (ORF1homl): pos. 240-260 p240- 260 >MNYGYGPGVVTSRTISASQA// + 47; 50; 0 85;92 - 53 Seq ID 81 (EMBP): pos. 1660-1682 p1660-1 6 8 2 >NEIVLETIRDINNAHTLQQVEA// nd 2;14;5; 77;26 Seq ID 81 (EM_BP) : pos. 1746-1790 p1746-1 773 >LHMRHFSNNFGNVIKNAIGVVGISGLLA// nd p 1 7 5 3
-
1 7 7 9 >NNFGNVIKNAIGVVGISGLLASFWFFI// nd p177 7
-
1 7 8 9 >FFIAKRRRKEDEE/ nd' Seq ID 83 (Autolysin) pos. 1-29 pl-29: >MAKKFNYKLPSMVALTLVGSAVTAHQVQA// nd 6;35;7; 60;49 Seq ID 83 (Autoly'sin) pos. 878-902 p878- 9 0 2 : >NGLSMVPWGTKNQVILTGNNIAQG/ nd Seq ID 89 (ORFlhom2): pos. 96-136 p96-121 >GESLNIIASRYGVSVDQLMAANNLRG// p117-136 >NNLRGYLIMPNQTLQIPNG/ / - 0; 35; 0; 29;104 Seq ID 94 (EMPBP): pos. 1-29 p 4
-
2 9 : >KLLVLTMSTLFATQIMNSNHAKASV// + Seq ID 94 (EMPBP): pos. 226-269 p 2 2 6-251 >IKINHFCVVPQINSFKVIPPYGHNS// p254-2 7 0 >MHVPSFQNNTTATHQN// + 26; 28; 1 6; 43;97 Seq ID 94 (EMPBP): pos. 275-326 p275- 2 99 >YDYKYFYSYKVVKGVKKYFSFSQS// + p 2 8 4
-
3 0 5 >YKVVKGVKKYFSFSQSNGYKIG// + p 3 06-326 >PSLNIKNVNYQYAVPSYSPT// + Seq ID 114 (POV2): pos. 23-47 p23-47 >AGGIFYNQTNQQLLVLCDGMGGHK// 49;20;4 ;77;25 Seq ID 114 (POV2): pos. 107-156 p107-124 >ALVFESVVIANVGDSRA/ p126-14 6 >RAYVINSRQIEQITSDHSFVN// nd p142-158 >SFVNHLVLTGQITPEE// nd Seq ID 142 (LPXTGVI): pos. 1-42 p6-30 >KEFKSFYSIRKSSLGVASVAISTL// ++ p18-4 2 >SSLGVASVAISTLLLIMSNGRAQA// nd 0; 41; 20 Seq ID 142 (LPXTGVI): pos. 209-244 p 2 09-233 >IKLVSYDTVKDYAYIRFSVSNGTKA// p 2 18-244 >KDYAYIRFSVSNGTKAVKIVSSTHFNN// + - Seq ID 142 (LPXTGVI): pos. 395-428 - p3 9 5
-
4 1 8 >FMVEGQRVRTISTYAINNTRCTIF// p4 16
-
428 >TIFRYVEGKSLYE/
/
- 54 Seq ID 142 (LPXTGVI): pos. 623-647 p623-647 >MTLPLMALLALSSIVAFVLPRKRKN // 1) binding to soluble DRA1*0101/DRB1*0401 molecules was determined using a com petition assay (+, ++: binding, -: no competition up to 200 Jpm test peptide; nd: not done) 2) results from 5 healthy individuals with strong anti-S.aureus IgG response. Data are represented as spots/200.000 cells (background values are subtracted 5. Antigens may be injected into mice - and the antibodies against these proteins can be measured. 6. Protective capacity of the antibodies induced by the anti gens through vaccination can be assessed in animal models. Both 5. and 6. are methods well available to the skilled man in the art. Example 7: Applications A) An effective vaccine offers great potential for patients fac ing elective surgery in general, and those receiving endovascular devices, 'in particular. Patients suffering from chronic diseases with decreased immune responses or undergoing continuous ambula tory peritoneal dialysis are likely to benefit from a vaccine with S. aureus by immunogenic serum-reactive antigens according to the present invention. Identification of the relevant antigens will -help to generate effective passive immunization (humanized monoclonal antibody therapy), which can replace human immuno globulin administration with all its dangerous side-effects. Therefore an effective vaccine offers great potential for pa tients facing elective surgery in general, and those receiving endovascular devices, in particular. S. aureus can cause many different diseases. 1. Sepsis, bacteriaemia 2. Haemodialysed patients - bacteriemia, sepsis 3. Peritoneal dialyses patients - peritonitis 4. Patients with endovascular devices (heart surgery,etc) - en docarditis, bacteriemia, sepsis - 55 5. Orthopedic patients with prosthetic devices - septic arthri tis 6. Preventive vaccination of general population B) Passive and active vaccination, both with special attention to T-cells with the latter one: It is an aim to induce a strong T helper response during vaccination to achieve efficient humoral response and also immunological memory. Up till now, there is no direct evidence that T-cells play an important role in clearing S. aureus infections, however, it was not adequately addressed, so far. An effective humoral response against proteinaceous anti gens must involve T help, and is essential for developing mem ory. Naive CD4+ cells can differentiated into Th1 or Th2 cells. Since, innate immunological responses (cytokines) will influence this decision, the involvement of T-cells might be different dur ing an acute, serious infection relative to immunization of healthy individuals with subunit vaccines, not containing compo nents which impair the immune response during the natural course of the infection. The consequences of inducing Th1 or Th2 re sponses are profound. Thl cells lead to cell-mediated immunity, whereas Th2 cells provide humoral immunity. C) Preventive and therapeutic vaccines Preventive: active vaccination/passive immunization of people in high .risk groups, before infection Therapeutic: passive vaccination of the already sick. Active vaccination to remove nasal carriage Specific example for an application Elimination of MRSA carriage and prevention of colonization of the medical staff Carriage rates of S. aureus in the nares of people outside of the hospitals varies from 10 to 40%. Hospital patients and personnel have higher carriage rates. The rates are especially high in pa tients undergoing hemodialysis and in diabetics, drug addicts and patients with a variety of dermatologic conditions. Patients at highest risk, or MRSA infection are those in large tertiary-care hospitals, particularly the elderly and immunocompromised, those - 56 in intensive care units, burn patients, those with surgical wounds, and patients .with intravenous catheters. The ELISA data strongly suggest that there is a pronounced IgA response to S. aureus, which is not obvious *or known from the literature. Since the predominant mucosal immune response is the production of IgA with neutralizing activity, it is clear that the staphylococcal epitopes and antigens identified with the highly pure IgA preparations lead to an efficient mucosal vac cine. *Clear ~indication: Everybody's threat in the departments where they perform- operation (esp. orthopedics, traumatology, gen. surgery) eWell-defined population for vaccination (doctors and nurses) *Health care workers identified as intranasal carriers of an epidemic strain of S. aureus are currently treated with mupi rocin and rifampicin until they eliminate the bacteria. Some times it is not effective, and takes time. *Available animal model: There are mice models for intranasal .carriage.
57 GO .... *1 0 44 on 0s Ue 0q 00 58 -- -T f -. cl1 0 - - -r en 0.. 4)- cf r. oC C t- 00 0% C, C*4 m Nr Ln %0 t- w 0% rg rI CL n r - 59 Table I. ELISA titers of sera from non-infected individuals against multiple staphylococcal proteins. Anti-s taphylococcal antibody levels were measured individually by standard ELISA with total lysate prepared.from S. aureus grown in BHI medium (BHI), lipoteichoic acid (LTA), peptidoglycan (PG), 13. recombinant proteins, representing cell surface and secreted pro teins, such as clumping factor A and B (ClfA, ClfB), Fibronectin binding protein (FnBPA), SD-repeat proteins (sdrC, sdrE), MHC Class II analogous protein (map-w), Elastin-binding protein (EBP), enolase (reported to be cell surface located and immuno genic), iron transport lipoproteins (LP309, LP342), sortase (srtA), coagulase (coa), extracellular fibrinogen-binding protein (fib). Two short synthetic peptides representing 2 of the five immunodominant D repeat domains fiom FnBPA was also included (D1+D3) as antigens. The individual sera were ranked based on the IgG titer, and obtained a score from 1-9. Score 1 labels the highest titer serum and score 8 or 9 labels the sera which were 8* or 9" among all the sera tested for the given antigen. It re sulted in the analyses of the top 20 percentile of sera (8-9/40) The five "best sera" meaning the most hyper reactive in terms. of anti-staphylococcal antibodies were selected based on the number of scores 1-8. **** means that the antibody reactivity against the particular antigen was exceptionally high (>2x ELISA units relative to the 2 nd most reactive serum). Table 2a: Immunogenic proteins identified by bacterial surface and ribosome display: S. aureus Bacterial surface display: A, LSA250/1 library in fhuA with pa tient sera 1 (655); B, LSA50/6 library in lamB with patient sera 1 (484)*; C, LSA250/1 library in fhuA with IC sera 1 (571); E, LSA50/6 library in lamB with IC sera 2 (454); F, LSA50/6 library in lamB with patient sera P1 (1105); G, LSA50/6 library in lamb with IC sera 1 (471) ); H, LSA250/1 library in fhuA with patient sera 1 (IgA, 708). Ribosome display: D, LSA250/1 library with IC sera (1686). *, identified 18 times of 33 screened; was therefore eliminated. from screen C. **, prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTI GENIC (Kolaskar and Tongaonkar, 1990); #, identical sequence pre sent twice in ORF; ##, clone not in database (not sequence by - 60 TIGR). S. Old Putative function predicted immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected Identified with relevant re- (DNA antigen number clones per Immuno- gion (positiveltotal) +Prot) protein ORF and genic region screen SaA0003 ORF2963P repC 5-20,37-44,52-59, 87-94, 116-132 C:3 aa 112-189 C:GSBYM94(112- 171, 172 189):26/30 SaAO003 ORF2967P rcpC 7-19,46-57,85-91, 110-117,125- C:18 aa9-42 C:GSBYI53(9- 150,158 133, 140-149, 156-163, 198-204, aa 158-174 42):1/1 236-251, 269-275, 283-290,318 323, 347-363 0093 ORF1879 SdjC 23-51,75-80,90-99, 101-107,151- kI, D:5, aa 98-182 A:GSBXL70(98- 34,86 157, 173-180,186-205,215-226, C:1, F:6, aa 684-764 182):9/30 239-263,269-274,284-304,317- G:2 aa 836-870 D:n.d. 323, 329-336,340-347,360-366, C:GSBYH73(815 372-379,391-397, 399-406,413- 870):3/16 425,430-436,444-455,499-505, 520-529,553-568,586-592, 600- - - J 617, 631-639,664-678,695-701, 891-903,906-912,926-940 0095 ORF1881 SdrE 25-45,72-77,147-155,198-211, C:12, E:2 aa 147-192 C:OSBYH31(147- 145, 153 217-223,232-238,246-261,266- 192):2/14 278, 281-294, 299-304,332-340, E:GSBZA27(144 353-360,367-380, 384-396,404- 162):23/41 409,418-429,434-440,448-460, 465-476,493-509,517-523,531 540,543-555,561-566,576-582, 584-591,603-617,633-643,647 652, 668-674, 677-683,696-704, 716-728,744-752,755-761,789 796, 809-815,826-840,854-862, 887-903,918-924, 1110-1116, 1125-1131,1145-1159 0123 ORF1909 unknown 9-28,43-48,56-75,109-126,128- B:3,E:7, aa168-181 B:GSBXF80(168- 35, 87 141, 143-162, 164-195, 197-216, G:1 181):5/27 234-242, 244-251 E:GSBZC17(168 181):25141 0160 ORFl941 unknown 4-10,20-42,50-86,88-98, 102-171, A:1 as 112-188 A:GSBXO07(112- 36,88 176-182, 189-221,223-244,246- 188):5/30 268, 276-284,296-329 1 0222 ORF1988 homology with 4-9,13-24,26-34,37-43,45-51, A:52, a 45-105 A:GSBXM63(65- 37,89 ORFI 59-73,90-96,99-113, 160-173, C:18*, aa 103-166 95):1I/1 178-184, 218-228,233-238,255- H:19 aa 66-153 A:GSBXM82(103 262 166):14/29 A:GSBXK44 bmd3(65 _153):47151 0308 ORF2077 Complement,un- 13-27,42-63,107-191,198-215, A:6, B:2, complement A:GSBXK03(bp473 38,90 known 218-225,233-250 C:47, bp 474-367 -367):28/69 E:35 B:GSBXD29(bp465 1-431):10127
-
- 61 S. Old Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) elected Identified with relevant re- (DNA antigenic number clones per immuno- gion (posItive/total) +Prot) protein ORF and genic region screen 0317 ORF2088 preprotein translo- 16-29,64-77, 87-93, 95-101, 127- A: an 1-19 A:GSBXP37(1- 39,91 case seca subunit 143, 150-161, 204-221, 225-230, 19):6/29 236-249, 263-269, 281-309,311 325,337-343,411-418,421-432, 435-448,461-467,474-480,483 489,508-516,542-550,580-589, 602-611,630-636,658-672,688 705, 717-723,738-746,775-786, 800-805,812-821,828-834 0337 ORF2110 Hypothetical pro- 26-53,95-123, 164-176, 189-199 D:12 an 8-48 Dn.d. 40, 92 tein 0358 ORF2132 Clumping factorA 8-35,41-48, 59-66,87-93, 139-144, C:1, D:2, aa 706-809 D:n.d. 41, 93 156-163, 198-209, 215-229,236- E:A 244,246-273,276-283,285-326, 328-342,349-355,362-370,372 I.] 384,396-402,405-415,423-428, 432-452,458-465,471-477,484 494,502-515,540-547,554-559, 869-875, 893-898, 907-924 0360 ORF2135 extracellular 7-13,15-23,26-33,68-81,84-90, A:46, an 22-56 A:GSBXK24(23- 42,94 Empbp matrix and plasma 106-117,129-137,140-159,165- B:21, an 23-99 55):11 binding protein 172, 177-230,234-240,258-278, C:11. E-2, an 97-115 B:GSBXB43(39 295-319 F:18, 0-7, aa 233-250 54):58/71 H: 12 an 245-265 A:GSBXK02 bmdl(22-99):59/59 B:GSBXD82 bdbl9(97-115):1/1 F:SALAIU3(233 250):15/41 0453 ORF2227 coma operon 17-25,27-55, 84-90,95-101, 115- C:3 an 55-101 C:GSBYG07(55- 146, 154 protein 2 121 101):1/1 0569 ORF1640 VS pTotease 5-32, 66-72, 87-98, 104-112, 116- A:, F:l an 174-249 A:GSBXS51(174- 32, 84 124, 128-137, 162-168,174-183, 249):11/30 248-254,261-266,289-303,312 1 1_331 - 62 S Old Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected identified with relevant re- (DNA antigenic number clones per immuno- gion (positive/total) +Prot) protein ORF and genic region screen 0576 ORF1633 autolysinadhe- 4-19, 57-70, 79-88, 126-132, 144- A:21, a 6-66 kGSBXN93(6- 31,83 Autolysin sion 159, 161-167, 180-198,200-212, B:46, aa65-124 66):5116 233-240,248-255, 276-286, 298- C:55, E:5, aa 579-592 C:GSBYH0O(45 304, 309-323,332-346,357-366, F:85, aa 590-604 144):7/8 374-391,394-406,450-456,466- H:19 A:GSBXK66 473, 479-487,498-505,507-519, bmdlg(65 521-530,532-540, 555-565, 571- 124):16130 581, 600-611, 619-625, 634-642, B:GSBXB89(108 650-656,658-665, 676-682,690- 123):i/l 699, 724-733, 740-771, 774-784, B:GSBXBO2(590 791-797, 808-815, 821-828, 832- 603):39n71 838, 876-881, 893-906,922-929, F:SAIAMiS(579 938-943,948-953, 969-976, 1002- 592):25/41 1008, 1015-1035, 1056-1069, 1105 1116, 1124-1135, 1144-1151, 1173 1181, 1186-1191, 1206-1215, 1225 ______ __________ 230, i235-1242 ____ 0657 ORF n- LPXTGVI prtein 9-33,56-62,75-84,99-105, 12- A.2, B:27, an 527-S44 B:GSBXE7- 1, 142 known 127, 163-180, 186-192,206-228, F.15 bdbl(S27 233-240,254-262,275-283,289- 542):i16 296, 322-330, 348-355,416-424, F:SALAX7O(526 426-438,441--452, 484-491, 541- 544):1 1141 _______ __________549,563-569,578-584,6244641 _________ 0149 0RF1462- Cartmsmoy-phos- 8-23,31-38,42-49,61-77,83-90, C:2 an 630-700 C:GSBYK17(630- 144,152 phate synthase 99-108, 110-119, 140-147, 149-155, 700) 5/9 159-171,180-185,189-209,228 234, 245-262,264-275,280-302, 304-330,343-360,391-409,432 437, 454-463,467-474,478-485. 515-528,C532-539,:553-567,G56 58 1, 586-592, 605-612, 627-635, 639-656, 671-682, 70D-714, 73 1 747,754-770, 775-791,797-834, 838-848, 872-891, 927-933, 935 942. 948-968,976-986, 100S-1007, 1029-1037B:GSBX 944 ORF1414 Yfix 6-33,40-46,51-59,61-77,84-104, DA4 aa483-51i Dn~d. 30,82 112-118,124-187,194-248,252 296, 308-325, 327-361, 367-393, 396-437, 452-479,484-520,535 545, 558-574, 582-614, 627-633, 656-663, 671-678, 698-704,713 722,725-742,B744-755,770-784, 76-800F 8:16-822,27-837 05 ORFu30 unknown 49-7, 6-83,95-105, 135-146, A:2, H:45 aa 57-128 A:GSBXM26(57- 28,80 148-164, 183-205 289-128):7130 - 63 S. Old Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected Identified with relevant re- (DNA antigenic number clones pe immuno- gion (positive/total) +Prot) protein ORF and genic region screen 1209 ORF3006 hemN homolog 12-36,43-50,58-65,73-78,80-87, B:7, F:8 aa 167-181 B:GSBXB76(167- 54, 106 108-139, 147-153, 159-172, 190- 179):25/71 203,211-216,224-232,234-246, F:SALBC54(169 256-261, 273-279, 286-293, 299- 183):18/41 306,340-346,354-366 1344 ORF0212 NifS protein 8-16,22-35, 49-58,70-77, 101-121, A:11 aa 34-94 kGSBXK59- 5,141 homolog 123-132, 147-161, 163-192, 203- bmd2l(34-94):6/29 209,216-234, 238-249,268-274, 280-293,298-318,328-333,339 345,355-361,372-381 1356 ORF0197 Hypotheticalpro- 28-55, 82-100, 105-111, 125-131, D:12 aa 1-49 D:n.d. 4,57 tease 137-143 1361 ORF0190 LPXTGV protein 5-39,111-117,125-132,134-141, A:, B:23, aa 37-49 B:GSBXF8I(37- 3,56 167-191, 196-202, 214-232, 236- E:3, F:31 aa 63-77 49):1/1 241,244-249,292-297,319-328, aa 274-334 B:GSBXD45 336-341, 365-380, 385-391, 407- bdb4(62-77):12/70 416,420-429,435-441,452-461, A:GSBXL77(274 477-488,491-498,518-532,545- 334):5/30 556,569-576,581-587,595-602, F:SALAP8I(62 604-609, 617-640, 643-651, 702- 77):10/411 715, 723-731, 786-793, 805-811, 826-839, 874-889 1371 ORF0175 YtpT, conserved 37-42,57-62, 121-135, 139-145, C:3, E:2, aa 624-684 C:GSBYG95(624- 143, 151 hypothetical pro- 183-190,204-212,220-227,242- G:1 aa 891-905 684):7/22 tein 248, 278-288,295-30,304-309, E:GSBZB45(891 335-341, 396-404,412-433,443- 905):10/41 449,497-503,505-513,539-545, 552-558, 601-617,629-649, 792 711,736-745,793-804,814-829, 843-858, 864-885, 889-895, 905 913, 919-929,937-943,957-965, 970-986,990-1030, 1038-1049, 1063-1072, 1080-1091,1093-1116, 1126-1136,1145-1157, 1163-1171, 1177-1183, 1189-1196, 1211-1218, 1225-1235, 1242-1256, 1261-1269 1491 ORF0053 Cmp binding fac- 12-29,34-40,63-71,101-110,114- A:7,C:2, aa39-94 A:GSBXMI3(39- 2,55 tor I homolog 122, 130-138, 140-195, 197-209, E:7, F:4 94):10/29 215-229,239-253,255-274 F:SALAY30(39 53):4/41 1616 ORFIl80 leukocidin F ho- 16-24,32-39,43-49,64-71,93-99, A:10 aa158-220 A:GSBXK06(158- 27,79 molog 126-141,144-156,210-218,226- 220):8/29 233,265-273,276-284 1618 ORF 1178 LukM bomolog 5-24,88-94,102-113, 132-143, A:13,B:3 aa3l-61 A:GSBXK60(31- 26,78 163-173, 216-224, 254-269, 273- C:36, E:4, an 58-74 61):20/29 278, 305-313, 321-327,334-341 F:12,0:2, B:GSBXB48(58 :10 74):491 F:SALAY41(58 _74):30/41 - 64 S Old Putative function predicted immunogenic as** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected Identified with relevant re- (DNA antigenic number clones per immuno- glon (positive/total) +Prot) protein ORF and genic region screen 1632 ORFI 163 SdrH homolog 7-35,54-59,247-261,263-272, B:6, E:l , aa 105-119 B:GSBXG53(168- 25, 77 302-320, 330-339,368-374,382- F:34 aa 126-143 186):39/71 411 aa 168-186 F:SALAP07(105 119):11/41 1763 ORF1024 unknown 5-32,35-48,55-76 C:3 complement C:GSBY30(98a):1 24, 76 bp 237-170 /1 1845 ORF0942 Hyaluronate lyase 10-26, 31-44, 60-66, 99-104, 146- D:5, F:2 aa208-224 D:n.d. 23, 75 153, 163-169, 197-205, 216-223, aa 672-727 226-238, 241-258, 271-280,295 315, 346-351, 371-385, 396-407, 440-446,452-457, 460-466,492 510,537-543,546-551,565-582, 590-595, 635-650, 672-678, 686 701, 705-712, 714-721, 725-731, 762-768, 800-805 1951 ORF0831 homology with 5-22,42-50,74-81, 139-145, 167- A:223, aa 137-237 B:GSBXC07(180- 22,74 ORF1 178, 220-230,246-253, 255-264 B:56, aa 250-267 190):1/1 C:167, A:GSBXK29(177 E:43, 195):15/29 P:A00, BGSBXD43(250 G:13, 267):10/71 H:102 F:SALAM13(178 191):2041 1955 ORF0826 homology with 4-9,15-26,65-76,108-115,119- A1, B:3, aa38-52. AGSBX1O(66- 21,73 ORFI 128,144-153 E.1,F:8 aa66-I14 114)-5/30 F:SALAM67(37 52):16/41 2031 ORF0749 unknown 10-26,31-43,46-58,61-66,69-79, B:2, F:2 aa 59-74 B:GSBXC0I(59- 20,72 85-92, 100-115,120-126,128-135, 71):11/26 149-155, 167-173. 178-187, 189 196, 202-222,225-231,233-240, 245-251, 257-263, 271-292,314 322, 325-334,339-345 2086 ORF0691 IgG binding 6-20,53-63, 83-90, 135-146, 195- A:1, B:8, aa 208-287 A:GSBXS55(208- 19,71 Sbi protein 208,244-259,263-314,319-327, E:24, F:9, aa 261-276 287):38/46 337-349,353-362,365-374,380- G:137 aa 286-314 B:GSBXB34(299 390,397-405,407-415 314)::11/71 F:SALAX32(261 276):21/41 - 65 S Old Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected identified with relevant re- (DNA antigen number clones per immuno- glon (positive/total) +Prot) protein ORF and genic region screen 2180 ORF0594 LPXTGIV protein 11-20,26-47,69-75,84-92,102- A:3, C:3. a493-587 A:GSBXS6I(493- 18,70 109, 119-136, 139-147, 160-170, E:6, 1:2, aa633-715 555):l11 178-185, 190-196, 208-215, 225- H: 6 a M4-760, A:GSBXaK496 233, 245-250,265-272,277-284, a,760-832 585):111 300-306,346-357, 373-379, 384- (oa 832- A.GSBXS92(760 390,429-435,471-481,502-507, 887) 841):1/1. 536-561, 663-688, 791-816, 905- A:bmd4(704 910,919-933,977-985, 1001-1010, 760):16/30 1052-1057, 1070-1077, 1082-1087, (A:bm4(830 1094-1112 ~885):1630Y F:SALB3C43(5 19 2184 ORF0590 FnbpB 5-12, 18-37, 104-124, 139-145, A2 CA, aa 701-777 A:GSBXM62(702- 17,69 154-166, 175-181, 185-190, 193- G:9 aa783-822 777):28/28 199,203-209,235-244,268-274, A:GSBXR22(783-. 278-292,299-307,309-320,356- 855):11 364, 375-384,390-404,430-440, 450-461,488-495,505-511,527 535, 551-556,567-573, 587-593, 599-609,624-631, 651-656,665 671, 714-726,754-766,799-804, 818-825, 827-833, 841-847, 855 ___________861, 876-893,895--903,927-94M____ ____ 2186 ORF0588 Fnbp 8-29996-105, 114-121,123-129, A., C:, aa 710-787 C:GSBYNS(710- 16,68 141-147, 151-165,171-183,E198- D:5, F:2 aa 63-975 787):19t25 206,222-232,253-265,267-277, aa 916-83 D:n/d. 294-300,302-312,332-338,362- A:GSBX9I(916 368,377-383,397-402,410-416, 983):17/30 451-459,473-489,497-503,537 543,549-559,581-6 23-629, 643-4(m9,655-666,680-687,694 700,C707-71:721-727,770-782, 810-822,A874-881,:883-89,897 903,911-917,925-931,933-939 61, 946-963, 965-973, 997-1010 2224 ORF058 unknown 49-56,62-68,83-89,92-98, 109- B:2 aa 34-46 :GSBX589(34- 15,67 115,124-131,142-159,161-167, 46):111 169-175, 177-188, 196-224,230 - . 90_______ 243, 246-252 -931, 933-939, - 66 & Old Putative function predicted Immunogenic aa** No. of se- ocation of Serum reactivity Seq ID no: aureas ORF (by homology) lected Identilied with relevant re- (DNA antigenic number clones per Inuuno- giort (positiveltotal) +Prot) protein ORF and genic region ________screen 2254 ORFOSI19 Conserved hypo- 14-22,32-40, 52-58,61-77,81-93, D:3 aa 403-462 D.n.d. 14,66 Ilietical protein 111-117, 124-138,1[51-190, 193 214,224-244, 253-277, 287-295, 307-324, 326-332,348-355,357 362, 384-394, 397-434,437-460, 489-496,503-510, 516-522, 528 539, 541-547, 552-558, 563-573, 589-595, 602-624, 626-63Z 651 667, 673--689, 694-706, 7 12-739, ____________756-790 2264 ORF05O9 ORFI; homology 5-31,47-55, 99-104, 133-139, 156- A:131, aa 7-87 A:GSBXP22(145- 13,65 withs putative se- 172,214-224,240-247 B:5 1, aa 133-242 196):111 ceted antigen C: 13, A.OGSBXKS precursorfHom I E:43, bmdl6(178 epidermidis F.78,0G:2, 218):6/29 M 1 B:GSBXE24 bdb20(1 67-178): 1/1 F:SAIAQ91(173 ________ 184):15/41 _____ 2268 ORF0503 IsaA, possibly ad- 7-19,26-45,60"11.94-100, 111- A:7, B:65, aa, 67-116 kO-SBKB8(67- 12,64 hesion/aggrega- 119,126-137,143-148,169-181, C:3,E2, aa 99-194 116):1/1 tion 217-228 F:53 as 182-225 A.GSBXN19(98 184):2229 AGSBXN32(182 225):34/7i B:GSBXB37I(196 209):16/29 F:SALA.L22(196 _____________ 210):16141_____ 2344 OR1F0426 Clumping fctor B 4-10,17-45,120-127,135-141, D:9,E1I, as 706-762 D:nd. 11,63 168-190, 187-208, 216-224, 244- F:3, It4 aa8lO-852 254,256-264,290-312,322-330, 356-366,374-384,391-414,421 428,430-437,442-449,455-461, 464-479, 483-492, 501-512, 548 ___________555, 862-868, 871-876, 891-904 2.351 ORF0418 aureolysin 10-29,46-56,63-74,83-105,107- A1A,C.6 aa 83-156 A:OSBXO46(83- 10,62 114, 138-145, 170-184,186-193, 156):14/29 216-221, 242-248, 277-289, 303 311,346-360,379-389, 422-428, 446-453,459-469,479-489,496 ____________501 ____ _____ ________- 67 - _ _ _____ S. Old Putative function predicted Immunogenic aa** , No.ofse- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected Identified with relevant re- (DNA antigen number clones per Immuno- glon (positive/total) +Prot) protein ORF and genic region screen 1 2359 ORF0409 ISSP, immuno- 4-29,92-99,119-130,228-236, B:4, F: II aa 168-184 B:GSBXDOI(168- 9,61 genic secreted 264-269,271-280,311-317,321- aa 206-220 184):I/l protein precursor, 331, 341-353, 357-363, 366-372, an 297-309 B:GSBXD62(205 putative 377-384, 390-396, 409-415,440- 220):1I/1 448,458-470, 504-520,544-563, B:GSBXC17(297 568-581, 584-592,594-603,610- 309):6/27 616 F:SALAL04(205 220):9141 2378 ORF0398 SrpA 18-23,42-55,69-77,85-98, 129- C:1, D:7, aa 198-258 C:GSBY73(646- 8,60 136, 182-188, 214-220,229-235, F:4, It II aa 646-727 727): 2/9 242-248,251-258,281-292,309- aa 846-857 F:SALA033(846 316,333-343,348-354,361-367, aa 2104- 857):10/41 393-407, 441-447, 481-488,493- 2206 D:n.d. 505,510-515,517-527,530-535, 540-549,564-583,593-599,608 621, 636-645, 656-670, 674-687, 697-708,726-734,755-760,765 772,785-792,798-815,819-824, 826-838,846-852,889-904,907 913,932-939, 956-964,982-1000, 1008-1015, 1017-1024, 1028-1034, 1059-1065,1078-1084, 1122-1129, 1134-1143,118D-1186,1188-1194, 1205-1215, 1224-1230, 1276-1283, 1333-1339, 1377-1382, 1415-1421, 1448-1459, 1467-1472, 1537-1545, 1556-1566, 1647-1654, 1666-1675, 1683-1689, 1722-1737, 1740-1754, 1756-1762, 1764-1773, 1775-1783, 1800-1809, 1811-1819, 1839-1851, 1859-1866, 1876-1882, 1930-1939, 1947-1954, 1978-1985, 1999-2007, 2015-2029,2080-2086,2094-2100, 2112-2118,2196-2205,2232-2243 2466 ORF0302 YycH protein 16-38,71-77,87-94,105-112, 124- D:14 aa 401-494 D:n.d. 7,59 144, 158-164, 169-177, 180-186, 194-204,221-228,236-245,250 267,336-343,363-378,385-394, 406-412,423-440,443-449 2470 ORF0299 Conserved hypc- 4-9, 17-41, 50-56, 63-69, 82-87, C:3 aa 414-455 C:GSBYH60(414- 169,170 thetical protein 108-115, 145-151,207-214,244- 455):28/31 249, 284-290, 308-316, 323-338, 348-358,361-378,410-419,445 451,512-522,527-533,540-546, 553-558,561-575, 601-608, 632 644,656-667,701-713,727-733, -766-780 - 68 - Old Putative function predicted Immunogenic aa** No.of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected identified with relevant re- (DNA antigen number clones per Immuno- gion (positive/total) +Prot) protein ORF and genic region screen 2498 ORF0267 Conserved hypo- 33-43,45-51, 57-63, 65-72, 80-96, D:12 as358-411 D:17/21 6,58 thetical protein 99-110, 123-129, 161-171, 173-179, aa588-606 185-191, 193-200, 208-224, 227 246, 252-258, 294-308, 321-329, 344-352,691-707 2548 ORF2711 ISg binding 4-16, 24-57, 65-73, 85-91, 95-102, A:55, as 1-48 A:GSBXK68(1- 53, 105 protein A 125-132,146-152,156-163,184- B:54, aa 47-143 73):21/30 190, 204-210, 214-221, 242-252, C:35, aa 219-285 AGSBXK41(47 262-268,272-279,300-311,320- F:59, aa 345-424 135):1/1 337,433-440,472-480,505-523 G:56, A:GSBXN38(219 H:38 285):19/30 A:GSBXL11(322 375):10/30 B:GSBXB22(406 418):37/71 F:SALAM17(406 418):29/41 2577 ORF2683 Hypothetical pro- 4-21, 49-56,65-74,95-112,202- C:6 aa 99-171 C:GSBYI56(99- 149, 157 tein 208, 214-235 171):1/i 2642 ORF2614 unknown 34-58,63-69,74-86,92-101,130- C:1, E:1 aa5-48 C:bhe3(5- 52,104 138, 142-150, 158-191, 199-207, 48)25/30" 210-221,234-249,252-271 2664 ORF2593 Conserved hypo- 7-37,56-71,74-150,155-162, 183- D:35 aa 77-128 D:n.d. 51,103 thetical protein 203, 211-222, 224-234, 242-272 1 2670 ORF2588 Hexose transporter 18-28,36-49,56-62, 67-84,86-95, D:16 aa 328-394 D:.d. 50, 102 102-153,180-195, 198-218,254 280,284-296,301-325,327-348, 353-390, 397-402, 407-414,431 - _ 455 2680 ORF2577 Coagulase 4-18,25-31,35-40,53-69,89-102, C:26, G:4, aa438-516 C:GSBYH16(438- 148, 156 147-154,159-165, 185-202, 215- H:8 aa505-570 516):3/5 223,284-289, 315-322,350-363, aa 569-619 C:GSBYG24(505 384-392, 447-453,473-479,517- 570):1/7 523, 544-550, 572-577,598-604, C:GSBYL82(569 617-623 619):2/7 2740 ORF2515 Hypnthetical pro- 5-44,47-55,62-68,70-78,93-100, D:4 aa 1-59 D:n.d. 49, 101 tein 128-151, 166-171, 176-308 2746 ORF2507 homology with 5-12, 15-20, 43-49, 94-106, 110- A-1, H:13 aa 63-126 A:GSBXO40(66- 48, 100 ORFI 116, 119-128, 153-163, 175-180, 123):8/29 185-191, 198-209, 244-252, 254 264,266-273,280-288,290-297 2797 ORF2470 unknown 10-27,37-56,64-99,106-119, 121- B:3, E:2, aa 183-200 B:GSBXE85(183- 47,99 136,139-145,148-178,190-216, F:13,H:3 aa349-363 200):11/27 225-249,251-276,292-297,312- F:SALAQ47(183 II321, 332-399,403-458 | |200):8/41 - 69 . Old Putative function predicted immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureur ORF (by homology) lected identified with relevant re- (DNA antigen number clones per Immuno- glon (positive/total) +Prot) protein ORF and genic region screen 2798 ORF2469 Lipase (geh) 12-35,93-99, 166-179, 217-227, AA41, aa48-136 L.GSBYGOi(48- 46,98 239-248, 269-276, 288-294, 296- B:42, C:3, aa 128-172 136):2/6 320, 322-327,334-339, 344-356, F:35,G:1, aa 201-258 AGSBXM3I 362-371, 375-384, 404-411, 433- H:i bmdi2(128 438,443-448,455-464, 480-486, 188):11/30 497-503, 516-525, 535-541, 561- B:GSBXE16(165 570, 579-585, 603-622, 633-641 177):10/30 A:GSBXN2(201 258):8/30 F:SALAW05(165 177):13/41 2815 ORF2451 Conserved hypo- 5-32,34-49 D:21 aa 1-43 D:n.d. 45,97 thetical protein 2914 ORF2351 metC 39-44,46-80,92-98, 105-113, 118- :1, C:14, aa 386-402 A:GSBXMI8(386- 44,96 123, 133-165, 176-208, 226-238, F:2 402):17/29 240-255,279-285,298-330,338 345,350-357,365-372, 397-402, 409-415, 465-473, 488-515, 517 535, 542-550, 554-590, 593-601, 603-620, 627-653, 660-665, 674 687,698-718,726-739 2960 ORF2298 putative Exotoxin 13-36, 40-49, 111-118, 134-140, C:101, an 1-85 C:GSBYG32(1- 43,95 159-164, 173-183, 208-220, 232- B:2, L58 aa 54-121 85)::6/7 241,245-254,262-271,280-286, aa 103-195 C:GSBYO61 295-301, 303-310, 319-324, 332- bhe2(54-121):26/30 339 C:GSBYN80(103 195):13/17 2963 ORF2295 putative Exotoxin 13-28, 40-46,69-75, 86-92, 114- C:3, E:3, aa 22-100 C:GSBYJ58(22- 147, 155 120,126-137,155-172, 182-193, G:1 100):9/15 199-206, 213-221, 232-238, 243 253,270-276,284-290 3002 ORF1704 homologywith 4-21,28-40,45-52,59-71,92-107, A:2,C:1, aa21-118 A:GSBXL6(21- 33,85 ORF1 . 123-137,159-174,190-202,220- HA 118):50/52 229,232-241,282-296.302-308, 1312-331 70 S. Old Putative function predicted immunogenic aa** No. of se- Location of Serum reactivity Seq ID no: aureus ORF (by homology) lected Identified with relevant re- (DNA antigenic number clones per * immuno- gion (positive/total) +Prot) protein ORF and genic region screen 3200 ORF1331 putative extracel- 6-15,22-32,58-73, 82-88,97-109, A:M, a 5-134 A:GSBXL07(5- 29,81 lular matrix bind- 120-131,134-140, 151-163,179- B:11, 134):628 ingprotcin 185, 219-230,242-255,271-277, C:36 288-293,305-319,345-356, 368 381, 397-406,408-420,427-437, 448-454,473-482,498-505,529 535, 550-563, 573-580, 582-590, 600-605,618-627,677-685,718 725,729-735,744-759,773-784, 789-794,820-837,902-908,916 921,929-935,949-955, 1001-1008, 1026-1032, 1074-1083, 1088-1094, 1108-1117, 1137-1142,1159-1177, 1183-1194, 1214-1220,1236-1252, 1261-1269,1289-1294, 1311-1329, 1336-1341, 1406-1413, 1419-1432, 1437-1457, 1464-1503, 1519-1525, 1531-1537, 1539-1557, 1560-1567, 1611-1618, 1620-1629, 1697-1704, 1712-1719, 1726-1736, 1781-1786, 1797-1817, 1848-1854, 1879-1890, 1919-1925, 1946-1953, 1974-1979 Table 2b: Additional immunogenic proteins identified by bacterial surface and ribosome display: S. aureus Bacterial surface display: A, LSA250/1 library in fhuA with pa tient sera 1 (655); B, LSA50/6 library in lamB with patient sera 1 (484); C, LSA250/1 library in fhuA with IC sera 1 (571); E, LSA50/6 library in lamB with IC sera 2 (454); F, LSA50/6 library in lamB with patient sera P1 (1105); G, LSA50/6 library in lamb with IC sera 1 (471); H, LSA250/1 library in fhuA with patient sera 1 (IgA, 708). Ribosome display: D, LSA250/1 library with IC sera (1686). **, prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolaskar and Tongaonkar, 1990). ORF, open reading frame; CRF, reading frame on complementary. strand; ARF, alternative reading frame.
- 71 & Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity with relevant Seq.1D aureus (by homology) lected Identified region (positiveltotal) no: antigens clones Immune- (DNA c protein per ORF genie region +Prot) and screen ARF028 Putative protein 7-14 F:6 aa 25-43 SALAMvt9(2S-43): 1/1 401,402 0 _ _ _ _ _ _ CP.F014 Putative protein 18-28, 31-37,40-47,51-83,86-126 F.5 an 8 l-90 SALAZ40(8 1-90): 2/12 403,404 CRF025 Putative protein 4-24,26-46,49-86 G:8 aa 60-76 SALAJ87(60-76): nd. 365, 378 CRFO30 Putative protein 40-46 A.6, 13:2, aa, 5-38 AkGSBXKO3(7-36):28/69 391,3921 8 C:47, B:OSBXD29(I0-20):10/27 ____________ __________________________E:3S CRF033 Unknown 4-17 D:3 an 1-20 I.nd. 469; 486 7 ____________ CRFD49 Putative protein 4-28, 31-53, 58-64A B: 13, 175 an 18-34 GSBXF31(19-34) in7 366,379 7 __________________ ______________________ CRF053 Unknown 4-20 D: 7 an 1-11 D-nd. 470; 487 8 ______________________ CRF075 Putative protein 4-11, 18-24,35-40 G:44 aa 25-39 SALAG92(26-39): nid., 367,380 0 1____ _____________ CRFI 14 Unknown 4-57 D:28 aa 16-32 D3nLd. 464; 481 5 ________________ CRF124 Putative protein 4-25,27-56 17:6 an 36-46 SAIAR.23(36-46). nd. 368,381 7 ___________________ CtF 125 Putative protein 19-25,38-47,55-74,77-87 0:5 an 54-67 SALAG65(54--67): mnd. 369,382 6 1_______ _____ _____ _______________ CRF 35 jUnknown 8-15; 18-24;27-38 D, 5 ana5-33 D3n4. 471; 488 6 ___________ ___________________ CRF176 IPutative protein '4-9,23-41,43-58,71-85 Q:3 a -22 C:GBYI3O(I-22): 1/1 407,408 3 _______ _____ CRFI78 lUnknown 8-161 .5 aa76-127 D3Ld. 465;482 3 _______ ____ CRF184 IUnknown 4-28; 30-36 D: 272 sa 1-17 P.nA. 472,489 5 ________ ____ ______________________ CRF186 jUnknown 6-11; 13-34; 36-SO D:8 asa4-27 Dan~d. 466;483 I ________________ CRF192 Putative protein 4-9, 17-30 F:9 aa. 13-22 SALAR41(13-22): nd. 370,383 CRF200 Putative protein 18-38 F: 13 an 16-32 SALAM7S(16-32): mnd. 371,384 CRF215 Putative protein 4-15,30-58 F:9 an 54--66 SALAQ54(54-66):1/12 372,385 5 ___________________________ CRF218 Putative protein 4-61. 65-72,79-95,97-106 E: 13 aa 86-99 GSBZEO8(86-99): nd. 373, 386 0 CRF220 Unknown 4-13 D: 3 an 17-39 D:nd. 473; 490 7 _________ CRF230 Putative protein 4-9,22-33,44--60 Q:5 an 80-1 16 GSBYL75(80-1 16): r~d. 374,387 5 C1URF234 Putative protein 4-23,30-44,49-70 F:8 an 46-55 SALAW31l(46-55). md.37,8 CR F234 Putative protein 14-32,39-46,62-69,77-83 B: 10, FA4 an 46-67 GSBXC92(52--67):2/1 I 376, 389 9 _______ - 72 S. Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity with relevant Seq ID aureus (by homology) lected Identified region (positive/total) no: antigeni clones Immuno- (DNA c protein per ORF genic region +Prot) and screen CRF235 Unknown 4-18 D: 3 sa 3-18 D:n.d. 475; 492 6 CRF245 Unknown 4-31 D: 9 aa 7-21 D:n.d. 476; 493 2 CRF249 Putative protein 4-29,31-41 G:8 aa 2-15 SALAF30(3-15): n.d. 377,390 8 CRF2$5 Unknown 4-35; 37-42 D: 4 aa 1-20 D.n.d. 474; 491 3 CRF257 Unknown 5-25; 30-39 D: 11 an 9-30 D:nLd. 467; 484 8 CRF266 Unknown 11-21 D: 17 aa 1-14 D:n.d. 477; 494 4 1 CRF272 Putative protein 10-41,50-57 F:A aa 40-56 SALAQ25(40-56): 1/1 405, 406 9 CRF286 Unknown 4-43 D: 78 aa 17-40 Dn.d. 478; 495 3/1 CRF286 Unknown 4-46 D: 78 aa 44-49 D:n.d. 479; 496 3/2 CRFAO0 Unknown 17-39;52-59 D: 3 aa 38-55 D:n.d. 463; 480 2 - CRFN1 Unknown 5-20; 37-44; 52-59;87-94; 116-132 D:4 ' aa 94-116 D:n.d. 468; 485 ORF018 UDP-N-acctyl- 11-18,43-56, 58-97,100-118,120- B:4, 1:29 an 197-210 SALAM14(198-209): n.d. 397, 398 8 D-mannosamine 148,152-171,195-203,207-214, transferase, puta- 220-227,233-244 tive ORF025 Multidrug cfflux 4-33, 35-56, 66-99, 109-124, 136- D: 3 aa 155-175 D: nd. 297,325 4 transporter 144,151-180, 188-198,201-236, 238-244,250-260,266-290,294 306,342-377 ORF030 Conserved hypo- 4-23,25-67,76-107, 109-148 D: 3 an 9 - 44 D: nd. 298,326 7 thetical protein I ORF045 Conserved hypo- 4-35,41-47,55-75, 77-89,98-113, D: 5 an 105-122 D: n.d. 299,327 2 thetical protein 116-140,144-179,194-215,232 254,260-273,280-288,290-302, 315-323, 330-369, 372-385,413-432 ORF045 Na+/H+Antiporter 4-81 D: 66 an 1-21 D. n.d. 300, 328 6 ORF055 Iron(I1I)dicitrate 5-23,50-74,92-99, 107-122, 126- D: 10 an 1-18 D: Ld. 301,329 6 binding protein 142, 152-159,172-179,188-196, 211-218,271-282 ORF062 Hypothetical 9-44,63-69,75-82,86-106, 108- D: 313 aa 13 - 37 D: n.d. 302,330 9 Protein 146, 153-161, 166-178,185-192, 1233-239,258-266,302-307 - 73 S Putative function predicted immunogenic Ra** No. of se- Location of Serum reactivity with relevant Seq ID aureus (by homology) lected Identified region (positiveltotal) no: antigen clones Immuno- (DNA c protein per ORF genic region +Prot) and screen ORF063 GTP--binding 10-19,22-32,95-105, 112-119,121- F:3 aa 107-119 F:SALAX70(107-119):l0/41 393,395 7 protein TypA 133, 140-154, 162-174, 186-200, 207-224,238-247,254-266,274 280, 288-294,296-305, 343-351, 358-364, 366-373, 382-393, 403 413, 415-422, 440-447, 499-507, 565-575,578-588 ORF071 Conserved 22-51, 53-71, 80-85, 93-99, 105- D: 3 aa 487 - 513 D: n.d. 303,331 3 hypothetical 112, 123-146,151-157, 165-222, transmembrane 226-236,247-270,290-296,301 protein, putative 324, 330-348,362-382, 384-391, 396-461,463-482,490-515 ORF078 Cell division pro- 104-111, 158-171,186-197,204- D: 4 an 152- 178 D: n.d. 304,332 8 tein 209, 230-247, 253-259,269-277, 290-314,330-340.347-367,378-388 ORF079 Conserved 11-40,56-75,83-102,112-117, 129- D:12 as 196-218 D:nd 305,333 7 hypothetical 147, 154-168, 174-191, 196-270, protein 280-344, 354-377, 380-429, 431 450,458-483,502-520,525-532, 595-602, 662-669,675-686,696 702,704-711,720-735,739-748, 750-756,770-779,793-800,813 822,834-862 0RF083, Cell Division Pro- 34-91,100-119,126-143,147-185, D:5 aa 26-56 D n.d. 306,334 6 tein 187-197,319-335,349-355,363 395,397-412, 414-422,424-440, 458-465,467-475,480-505, 507 529,531-542, 548-553,577-589, 614-632, 640-649,685-704, 730 741, 744-751,780-786 ORF131 Aminoacidper- 11-21,25-32,34-54,81-88,93-99, D:8 aa27-152 D:nd 307,335 8 mease 105-117, 122-145, 148-174, 187 193,203-218,226-260,265-298, 306-318, 325-381, 393-399, 402 421,426-448 ORF132 Pyruvatidnase 4-11,50-67,89-95, 103-109, 112- B:6 as420-432 E:GSBZEI6(420-432):5/41 197,216 1 135, 139-147,158-170, 185-204, 213-219, 229-242, 248-277, 294 300, 316-323,330-335,339-379, 390-402, 408-422, 431-439, 446 457,469-474,484-500,506-513, 517-530,38-346,548-56103, - 74 S. Putative function predicted immunogenic aa** No. of se- Location of Serum reactivity with relevant Seq ID aureus (by homology) lected Identified region (positive/total) no: antigen[ clones immuno- (DNA c protein per ORF genic region +Prot) and screen 0RF138 LPXTG ccll wall 11-31, 86-91, 103-111, 175-182, D: 3 aa 508-523 D: n.d. 308,336 8 anchor motif 205-212,218-226,242-247,260 269. 279-288, 304-313, 329-334, 355-360, 378-387, 390-399, 407 435,468-486, 510-516, 535-547, 574-581, 604-615, 635-646,653 659, 689-696, 730-737, 802-812, 879-891, 893-906. 922-931, 954 964, 997-1009, 1031-1042, 1089 1096, 1107-1120, 1123-1130, 1149 1162, 1176-1184, 1192-1207, 1209 1215, 1253-1259, 1265-1275, 1282 1295, 1304-1310, 1345-1361, 1382 1388, 1394-1400, 1412-1430, 1457 1462, 1489-1507, 1509-1515, 1535 1540, 1571-1591, 1619-1626, 1635 1641, 1647-1655, 1695-1701, 1726 1748, 1750-1757, 1767-1783, 1802 1807, 1809-1822, 1844-1875, 1883 1889, 1922-1929, 1931-1936, 1951 1967, 1978-1989,1999-2008,2023 2042, 2056-2083,2101-2136,2161 2177 ORF140 3,4-dihydrxy-2- 18-23,32-37,54-63,65-74,83-92, E:3 as 121-137 E:GSBZB68(121-137):7/41 198,217 2 butanone-4- 107-114, 123-139, 144-155, 157 phosphate syn- 164, 191-108,232-240,247-272, thase 284-290,295-301,303-309,311 321,328-341,367-376 ORF147 hemolysin I- 4-36,39-47,57-65,75-82,108-114, F:1 as 245-256 F:SALAP76(245-256):&41 199,218 3 (LAkD-Leuktoxin) 119-126, 135-143, 189-195,234 244, 250-257, 266-272, 311-316 ORFi52 Imn uptakeregu- 13-27,29-44,46-66,68-81,97-116, D3 as 120-135 D;nd. 309,337 3 lator 138-145 - ORFl70 innermembrane 4-23,57-77, 89-103, 119-125,132- F :1 as 418 F:SALBC82(104-118):7/41 200,219 7 protein, 60 kDa 172, 179-197,210-254,256-265, 281-287 ORF75 amiB 5-10, 16-24,62-69,77-96,100-115, D:3 aa 293-312 D:nrd. 310,338 4 117-126,137-156,165-183,202 211, 215-225, 229-241, 250-260, 267-273,290-300, 302-308,320 333,336-342,348-356,375-382, 1384-389
______________
- 75 S. Putative function predicted immunogenic aa** No. of se- Location of Serum reactivity with relevant Seq ID aureus (by homology) lected identified region (positive/total) no: antigen[ clones immuno- (DNA c protein per ORF genic region +Prot) and screen ORF178 Mrpprotein 5-29,46-52,70-76,81-87,155-170, F:2 aa 850-860 F:SALAQ36(850-860):8/41 201,220 3 (fintB) 192-197, 206-213, 215-220,225 231, 249-258, 273-279, 281-287, 300-306, 313-319, 323-332,335 341, 344-351, 360-382, 407-431, 443-448,459-468,475-496,513 520, 522-537, 543-550,556-565, 567-573, 580-585, 593-615, 619 631, 633-642, 670-686, 688-698, 759-766, 768-782, 799-808, 842 848, 868-877, 879-917, 945-950, 979-988,996-1002, 1025-1036, 1065-1084, 1101-1107, 1113-1119, 1125-1142, 1163-1169, 1183-1189, 1213-1219, 1289-1301, 1307-1315. 1331-1342, 1369-1378, 1385-1391, 1410-1419, 1421-1427, 1433-1447, 1468-1475, 1487-1494, 1518-1529, 1564-1570, 1592-1609, 1675-1681, 1686-1693, 1714-1725, 1740-1747, 1767-1774, 1793-1807, 1824-1841, 1920-1937. 1953-1958, 1972-1978, 1980-1986, 1997-2011, 2048-2066, 2161-2166, 2219-2224, 2252-2257, 2292-2298, 2375-2380, 2394-2399, 2435-2440,2449-2468 ORF184 Map-ND2C 4-27, 42-66, 70-76, 102-107, 113- E-5 aa 75-90 ROSBZB15(75-90):6/41 202,221 8 protein 118, 133-138 ORF189 n mbosoal protein 31-39, 48-54, 61-67, 75-83, 90-98, F:4 aa 239-257 F:SALAV36(239-257):19/ 4 1 203,222 1 L2 (rplB) 103-119, 123-145, 160-167, 169 176, 182-193, 195-206, 267-273 ORF201 Putative drug 5-27,79-85, 105-110, 138-165,183- D5 aa 205 - 224 D: n.d. 311,339 1 transporter 202, 204-225, 233-259, 272-292, 298-320, 327-336,338-345, 363 376, 383-398, 400-422, 425-470, 489-495, 506-518, 536-544, 549 554, 562-568, 584-598, 603-623 ORF202 lactase permease. 10-33,38-71,73-103, 113-125, 132- E2 aa422-436 E:GSBZF58(422-436):6/41 204,223 7 putative 147,154-163,170-216,222-248, 250-269, 271-278, 287-335, 337 355, 360-374, 384-408, 425-442, 453-465.468-476,478-501,508-529 ORF208 Hemolysin 11 8-27,52-59,73-80,90-9,104-110, D: 3 aa 126 - 147 D: n.d. 312,340 7 (putative) 1]7-124, 131-140,189-209,217 232, 265-279,287-293, 299-306 - 76 S. Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity with relevant Seq ID aureus (by homology) lected identified region (positive/total) no: antigeni clones Immuno- (DNA c protein per ORF genic region +Prot) and screen ORF209 preLukS 8-26, 75-82, 118-126,136-142, 163- F:2 aa 270-284 F:SALAQ77(270-284):23/41 205, 224 0 177, 182-189,205-215,221-236, 239-248,268-274 . . ORF209 Hemolysin II 5-22,30-47,58-65,75-81,87-92, F:3 aa 238-253 F:SALAQ67(237-252):10/41 206, 225 2 (prcLUK-F) 99-105,107-113, 119-126,189-195, 217-223,234-244,250-257,266-272 QR52I1 Multidrug I0-28,30-43,50-75,80-113,116- D: 9 aa 54 - 104 D n.d. 313,341 7 resistance protein 125, 136-167, 170-191,197-245, (putative) 253-329,345-367,375-396 ORF219 Transcriptional 20-31, 46-52, 55-69, 74-79, 89-97, D: 3 aa 15 -35 D: n.d. 314, 2 regulator GntR 108-113, 120-128, 141-171,188-214 342 family, putative ORF230 Amino acid per- 25-79,91-103, 105-127, 132-149, D: 53 aa 363 - 393 D: nd. 315, 343 5 mease 158-175,185-221,231-249,267 293,307-329,336-343,346-359, 362-405,415-442, 446-468 ORF232 Citrate dransporter 10-77, 85-96, 99-109, 111-138, 144- D: 7 aa 37 - 83 D n.d. 316,344 4 155, 167-176, 178-205,225-238, 241-247,258-280,282-294,304 309,313-327,333-383,386-402, 405-422,429-453 ORF242 Anion transporter 7-26,28-34,36-53,55-73,75-81, D 16 aa 275 - 295 D: nd. 317,345 2 family protein 87-100, 108-117, 121-138, 150-160, 175-181, 184-195,202-215,221 247, 265-271, 274-314,324-337, 341-412, 414-423, 425-440, 447 462,464-469 ORF25 SirA 5-22,54-78,97-103,113-123, 130- D:3 aa 1 -22 D: nad. 318, 346 3 148, 166-171, 173-180, 192-201, 254-261,266-272,310-322 1 ORF255 omithine cyclode- 20-35,37-50,96-102,109-120,123- E:2 aa 32-48 EGSBZB37(32-48) 11/41 207,226 5 aminase 137, 141-150, 165-182,206-224, 237-256,267-273, 277-291, 300 305,313-324 ORF255 Multidrug resis- I1-63,79-129,136-191,209-231, D: 8 aa 84- 100 D: nd. 319, 347 8 tance efflux pro- 237-250,254-276,282-306,311 ten, putative 345,352-373,376-397 ORF261 Cap5M 4-30,34-40,79-85,89-98,104-118, D: 13 aa 114 - 141 D: n.d. 320,348 0 124-139, 148-160, 167-178 ORF261 Cap5P (UDP-N- 4-9, 17-24,32-38,44-54,68-82, B:3, F:lI aa 321-341 F:SALAU27(325-337):9/41 208,227 3 acetylglucosamine 89-95, 101-120, 124-131, 136-142, 2-epimerase) 145-157, 174-181, 184-191, 196 204,215-224,228-236,243-250, 259-266,274-281, 293-301, 314 319,325-331,355-367,373-378 - 77 S Putative function predicted Immunogenic aa** No. of se- Location of Serum reactivity with relevant Seq ID aureus (by homology) lected identified region (posltive/total) no: antigeni clones immuno- (DNA e protein per ORF genic region +Prot) and screen ORF262 Hypothetical pm- 9-IS, 28-36, 44-62, 69-88, 98-104, F:6 aa 694-708 F:SALBD82(1288-1303):9/41 209,228 8 tein 111-136, 139-149, 177-186, 195- ar790-800 217,224-236,241-257,260-278, aa 1218 283-290, 292-373, 395-408, 411- 1305 443,465-472,475-496,503-520, 552-559, 569-589, 593-599, 607 613, 615-636,648-654,659-687, 689-696, 721-733, 738-759, 783 789, 795-801, 811-823, 827-836, 839-851, 867-875, 877-883, 890 898, 900-908, 912-931, 937-951, 961-992, 994-1002, 1005-1011, 1016-1060, 1062-1074, 1088-1096, 1101-1123, 1137-1153, 1169-1192, 1210-1220, 1228-1239, 1242-1251, 1268-1275, 1299-1311, 1322-1330, 1338-1361, 1378-1384, 1393-1412, 1419-1425, 1439-1459, 1469-1482, 1489-1495, 1502-1519, 1527-1544, 1548-1555, 1600-1607, 1609-1617, 1624-1657, 1667-1691, 1705-1723, 1727-1742, 1749-1770, 1773-1787, 1804-1813, 1829-1837, 1846-1852, 1854-1864, 1869-1879, 1881-1896, 1900-1909, 1922-1927, 1929-1935, 1942-1962, 1972-2005,2009-2029, 2031-2038,2055-2076,2101-2114, 2117-2124, 2147-2178, 2188-2202, 2209-2217, 2224-2230, 2255-2266, 2271-2280, 2282-2302, 2307-2316, 2319-2324,2379-2387 ORF264 PTS system, su- 8-15,24-30,49-68,80-93, 102-107, F:4 aa 106-159 F:SALAW60(106-125):3/41 210,229 4 erose-specific 126-147, 149-168, 170-180, 185 11BC component 193, 241-305, 307-339, 346-355, 358-372, 382-390, 392-415, 418 425, 427-433, 435-444, 450-472 ORF265 Oligopeptide ABC 5-61,72-84,87-99, 104-109, 124- D: S aa 182 -209 D: n.d. 321,349 4 transporter, puta- 145, 158-170, 180-188, 190-216, live 223-264,270-275,296-336,355-372 ORF266 maltose ABC 4-21, 71-79, 99-105, 110-121, 143- F:1 aa 306-323 F:SALBC05(306-323):2/41 211,230 2 transporter, puta- 161, 199-205, 219-235, 244-258, ive 265-270, 285-291,300-308, 310 318,322-328, 346-351,355-361, 409-416 - 78 S. Putative function predicted Immunogenic a** No, of se- Location of Serum reactivity with relevant Seq ID areas (by homology) lected identified region (positive/total) no: antigent clones imnuno- (DNA c protein per ORF genic region +Prot) and screen ORF271 sorbitol 4-12,19-40,61-111, 117-138, 140- B:2, F:4 a 244-257 F:SAIAX93(249-256):641 212,231 0 dehydrogenase 153, 161-180, 182-207, 226-235, 237-249, 253-264, 267-274,277 292,311-323 0RF274 Hypothetical prm- 4-41, 49-56,61-67.75-82,88-104, D 188, aa 303 - 323 D: nd. 322, 350 2 tein 114-125,129-145, 151-165,171- H-:4 178, 187-221,224-230,238-250, 252-275,277-304,306-385 0 189, 193-209,220-248,260-270, 273-299,301-326, 328-35S, 366 397, 399-428 1 ORF280 Phage related pro- 10-17,23-29,31-37,54-59,74-81, F:3 aa 104-116 F:SALBC34:1/1 213,232 6 tein 102-115, 127-137, 145-152, 158 165, 178-186, 188-196, 203-210, 221-227,232-237 ORF290 Conserved hypo- 4-27,34-43,62-73,81-90,103-116, D:24 aa 360 - 376 D: n.d. 324,352 0 thetical pmtein 125-136, 180-205, 213-218,227 235, 238-243,251-259,261-269, 275-280, 284-294, 297-308,312 342, 355-380,394-408,433-458, 470-510, 514-536, 542-567 ORF293 conserved 4-19,43-54,56-62, 84-90,96-102, E:6 aa 22-37 EGSBZA13(22-37):7/41 214,233 1 hypotheical 127-135,157-164, 181-187 . proteinI I ORF295 Exotoxin 2 7-19, 26-39,44-53, 58-69, 82-88, F:1 aa 154-168 F:SALBB59(154-168):4/41 215,234 8 91-107, 129-141, 149-155, 165-178, 188-194 ORF297 Surface protein, 9-23,38-43,55-60,69-78,93-101, H:5 a 1-70 H:GSBYU66: n.d. 399,400 0 putative 103-112, 132-148, 187-193,201 208, 216-229, 300-312, 327-352, 364-369,374-383,390-396,402 410, 419-426,463-475,482-491 Table 2c: Immunogenic proteins identified by bacterial surface and ribosome display: S. epidermidis. Bacterial surface display: A, LSE150 library in fhuA with patient sera 2 (957); B, LSE70 library in lamB with patient sera 2 (1420); C, LSE70 library in lamB with patient sera 1 (551). Ri bosome display: D, LSE150 in pMAL4.31 with P2 (1235)z **, predic tion of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolaskar and Tongaonkar, - 79 1990). ORF, open reading frame; ARF, alternative reading frame; CRF, reading frame on complementary strand. ORF, open reading frame; CRF, reading frame on complementary 'strand. S Putative function predicted immunogenic an** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected Identified region (positive/total) no: S antigenic clones immuno- (DNA protein per ORF genic region +Prot) and screen ARF0172 cation-iransport- 4-34,37-43 D:6 aa3-32 D nd 497, ing ATPase, El- 548 E2 family ARF0183 condensing en- 4-22,24-49 D:4 aal-52 D: ud 498, zyme, putative, 549 FabH-related ARF2455 NADH 4-29 D:3 aal-22 D: nd 499, dehydrogenase, 550 putative CRFO0I Unknown 4-14, 16-26 D-3 aa5-21 D: nd 500, 551 CRY002 Unknown 4-13,15-23,36-62 D.5 aa2l-70 D: nd 501, L 552 CRFOO03 Unknown 4-12,14-28 D.3 aa 4-31 D:nd 502, 553 CRF004 Unknown 5-15, 35-71, 86-94 D:4 aa3l-72 D nd 503, 554 CRF0005 Unknown 8-26,28-34 D:3 a&:9-33 Dnd 504, 555 CRF0006 Unknown 4-11,15-28 D:3 aal0-22 D-nd 505, 556 CRFOO07 Unknown 4-19,30-36 D:3 aa 7-44 D: nd 506, 557 CRF0008 Unknown 10-48 D4 aa:9-44 D: nd 507, 558 CRF0009 Unknown 41883 D:3 aa5-14 D: nd 508, 559 CRF0192 Putative protein 4-23,25-68 C:4 aa 15-34 C:GSBBM10(I 5-34): nd. 445, 446 - 80 S. Putative function predicted Immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected Identified region (positive/total) no: s antigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen CRF0275 Putative protein 4-40,49-65 B:5 aa 35-68 B:SELAK28(35-68): n.d. 447, 448 CRF0622 Putative protein 4-12, 17-57, 62-70, 75-84, 86-100 C:4 aa 75-99 C:GSBBR74(76-99): n.d. 449, 450 CRF0879 Putative protein 4-14,38-44 A:3, B:10 aa 9-40 B:SELAC39(10-40): n.d. 451, 452 CRF1004 Putative protein 4-40 A:3, B:5 aa 29-65 B:SELAI63(35-63): n.d. 453, - 454 CRF2248 Putative protein 4-10,19-40, 53-64, 74-91 C:30 aa 74-111 C:GSBBN64(16-35): n.d. 455, 456 CRF2307 Putative protein 4-19,35-41, 80-89 . A:19 aa 41-87 A:SEFAL47(41-87):n.d. 457, . . _ 458 CRF2309 Putative protein 15-21 B:6 as 4-16 B:SELALD2(4-16): nid. 459, 460 CRF2409 Putative protein 6-25 B:6 aa 2-24 B:SELAB48(5-24): n.d. 461, 462 ORFOOS hypothetical pro- 13-27,33-67,73-99, 114-129, 132- D:3 aalOS-128 D: nd 509, tein 158, 167-190, 193-234,237-267, 560 269-299,316-330,339-351,359 382,384-423 ORFOO08 Streptococcal he- 9-14, 16-24,26-32,41-50,71-79, B:2 an895-926 B:SELAF79(895-926):7/12 239, magglutinin 90-96, 177-184,232-237,271-278, 268 293-301, 322-330,332-339,349-1 354, 375-386, 390-396, 403-409, 453-459, 466-472, 478-486, 504 509, 518-525, 530-541, 546-552, 573-586, 595-600, 603-622, 643 660, 668-673, 675-681, 691-697, 699-711,713-726,732-749,753 759, 798-807, 814-826, 831-841, 846-852, 871-878, 897-904, 921 930, 997-1003, 1026-1031, 1033 1039, 1050-1057, 1069-1075, 1097 1103, 1105-1111,1134-1139,1141 1147,1168-1175, 1177-1183, 1205 1211,1213-1219,1231-1237, 1241 1247,1267-1273,1304-1309,1311 1317, 1329-1335, 1339-1345, 1347 1353, 1382-1389,1401-1407, 1411 1417, 1447-1453, 1455-1461, 1483 1489, 1491-1497, 1527-1533, 1545 1551, 1556-1561,1581-1587, 1591 1597, 1627-1638, 1661-1667, 1684 1689, 1691-1697, 1708-1715, 1719 1725, 1765-1771, 1813-1820, 1823 1830, 1835-1856 - 81 S Putative function predicted Immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected identified region (positive/total) no: s antigenic ones immuno- (DNA protein per ORF genic region +Prot) and screen ORF0038 extracellular 6-25,29-35,39-45, 64-71, 82-88, C:6 a 136-165 C:GSBBN08(136-165):1/1 353,359 elastase precursor 96-102, 107-113,119-131, 170-176, 186-192, 196-202,215-220,243 248,302-312, 345-360, 362-371, 378-384,458-470,478-489,495 504 ORF0099 hypothetical 6-18,31-37,42-49,51-67,73-85, D:5 aa218- 26 5 D. M 510, protein 87-93, 102-109, 119-126, 150-157. 561 170-179; 185-191, 204-214,217 223,237-248, 269-275,278-316, 320-340,359-365 ORF0101 hypothetical 4-10, 15-27,67-94,123-129, 167- D:18 aa26-109 D. d 511, protein 173, 179-184, 187-198,217-222, 562 229-235,238-246 ORF0121 C4-dicarboxylate 4-20, 24-62, 73-86, 89-106, 110- D:5 aa323-37 9 D ad 512, transporter, an- 122,131-164, 169-193,204-213, 563 aerobic, putative 219-236, 252-259, 263-281, 296 306,318-324,328-352,356-397, 410-429 ORF0143 amino acid per- 25-79, 91-103, 105-127, 132-150, D:35 za247-339 D nd 513, mease 157-174, 184-206, 208-219, 231- 564 249,267-294, 310-329,336-343, 346-405,417-468 ORF0162 Inmmunodominant 4-27,35-45,52-68,83-89, 113-119, A:11, an 99-227 B:SELAAI9(100-118): 1/1 240, Antigen A 133-150, 158-166, 171-176, 198- B:11; B:SELAE24170-190): 11/12 269 204,219-230 C:153 ORF0201 capa protein, 10-17, 27-53, 81-8.6, 98-105, 126-- D:9 aall-53 D:nd 514, putative 135, 170-176, 182-188, 203-217, 565 223-232, 246-252, 254-269, 274 280,308-314 ORF0207 Ribokinase(rbsK) 5-11, 15-23,47-5, 82-90,98-103, B:10 an20-45 B:SELAQ3O(20-45): 12/12 241, 108-114, 126-132, 134-156,161- 270 186, 191-197, 210-224, 228-235, 239-248,258-264,275-290 1 ORF0288 LrgB 7-28, 34-56,.68-119, 127-146,149- D.4 aaI2-149 D:nd , 2180,182-189,193-200,211-230 D:d566 - 82 S. Putative function predicted immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected identified region (positive/total) no: s antigenic clones immuno- (DNA protein per ORF genic region +Prot) and screen ORF0304 Hepesvirus 8-16, 30-36,83-106, 116-122, 135- D:8 aa69-117 D: nd 516, saimiri ORF73 143, 152-165, 177-188, 216-225 567 homolog, putative ORF0340 nitrate transporter 7-21 ,24-93,101-124,126-139, D:5 aa238-309 D:nd 517, 141-156,163-179,187-199,202- 595 242, 244-261, 267-308, 313-322, 340-353,355-376 ORF0346 hypothetical pro- 8-27, 65-73, 87-93, 95-105 D:8 aa 1-29 D:nd 518, tein 568 ORF0355 conserved 5-30, 37-43,57-66, 85-94, 103-111, C:5 aa 63-86 C:GSBBL39(63-86):1/1 354, hypothetical 118-125 360 protein ORF0356 conservedbhypo- 4-14,21-53,60-146,161-173,175- D:5 aa51-91 D:nd 519, thetical protein 182, 190-198,200-211 569 ORF0406 hypothetical pro- 12-32,35-63,68-102, 106-137, D:19 aal-48, D:nd 520, tein 139-145, 154-168, 173-185, 203- aa69-102 570 222,230-259,357-364, 366-374 . ORF0425 amino acid per- 40-58,75-86,93-110, 117-144, D:3 aa401-440 D: nd 521, mease 150-173, 199-219, 229-260, 264- 571 300, 317-323, 329-356, 360-374, 377-390,392-398,408-424,427 452 ORF0442 SceB precusor 7-22,42-48,55-66,83-90, 109-118, C38 aa 60-102 C:GSBBM60(65-84):1/1 355, 136-141 361 ORF0448 SsaA precursor 6-25,39-47,120-125, 127-135, C:170 aa 15-208 C:GSBBN58(81-105):1/1 356, 140-148, 157-168, 200-208, 210- C:GSBBL13(167-184):1/1 362 220,236-243,245-254 C:GSBBL25(22-45):1I1 ORF0503 Ribosomal protein 31-39,48-54,61-67, 75-83,90-98, A:1, B:3 aa 212-273 B:SELAA47(238-259):12112 242, L2 103-115,123-145,160-167,169- 271 176, 182-193, 195-206, 267-2731 ORF0551 Conserved hypo- 5-25, 29-36, 45-53, 62-67, 73-82, A:16, B:9 an 162-213 B:SELAL12(164-197): 8/12 243, thetical protein 84-91, 99-105, 121-142, 161-177, 272 187-193,203-224,242-251,266 271, 278-285 F ORF0556 hypothetical pro- 4-24,30-41,43-68, 82-90, 107-114, D:3 aa 1-26 D: nd 522, . tein 123-143, 155-168 596 - 83 S Putative function predicted Immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected Identified region (positive/total) no: s antigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen OEF0623 Fumble, putative 10-17, 32-38, 55-72, 77-84, 88-96, A:10, aa 95-150 B:SELAB86(95-128): 3/12 244, 126-134, 152-160, 176-185, 190- B:12; C:1 273 203, 208-214, 217-225,233-252, 257-262 ORF0740 Hypothetical pro- 18-24, 47-61, 69-83, 90-96, 125- B:3 aa 1093- B:SELAB23(1097-1114):7/12 245, tein 132, 140-163, 171-188,222-249, 1114 274 281-296, 305-315, 322-330, 335 351, 354-368, 390-397,411-422, 424-431,451-469,479-485,501 507,517-524,539-550,560-568, 588-599, 619-627, 662-673, 678 689, 735-742, 744-749,780-786, 797-814, 821-827, 839-847, 857 863, 866-876,902-911,919-924, 967-982, 1005-1015, 1020-1026, 1062-1070, 1078-1090, 1125-1131, 1145-1150, 1164-1182, 1208-1213, 1215-1234, 1239-1251, 1256-1270, 1298-1303, 1316-1325, 1339-1349, 1362-1369, 1373-1384, 1418-1427, 1440-1448, 1468-1475, 1523-1532, 1536-1542, 1566-1573, 1575-1593, 1603-1619, 1626-1636, 1657-1667, 1679-1687, 1692-1703, 1711-1718, 1740-1746, 1749-1757, 1760-1769, 1815-1849, 1884-1890, 1905-1914, 1919-1925, 1937-1947, 1955-1963, 1970-1978,2003-2032,2075-2089, 2117-2124,2133-2140,2146-2151, 2161-2167, 2173-2179, 2184-2196, 2204-2220, 2244-2254, 2259-2264, 2285-2296,2300-2318,2328-2334, 2347-2354, 2381-2388, 2396-2408, 2419-2446, 2481-2486, 2493-2500, 2506-2516, 2533-2540, 2555-2567, 2576-2592, 2599-2606, 2615-2639, 2647-2655 ORF0757 hypothetical 13-20,22-28,33-40,60-76,79-86, C:6 ma260-284 C;GSBBNOI(260-284):1/1 357, protein 90-102, 112-122, 129-147, 157-170, 363 178-185, 188-193, 200-205,218 228, 234-240, 243-250, 265-273, 285-291, 310-316, 330-348, 361 380, 399-405,427-446,453-464 .
- 84 S. Putative function predicted Immunogenic a** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected Identified region (positive/total) no: s antigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen ORF0912 DNA mismatch 9-16,28-39,47-56,69-76, 104-121, A:25 aa 242-304 SEFAT3i(242-290): nd. 441, repair protein 124-130,137-144, 185-195, 199- 442 214,238-243,293-307,317-337, 351-370,385-390, 411-428, 472 488, 498-516, 518-525,528-535, 538-545,553-559,563-568, 579 588, 592-607,615-622,632-638, 641-648,658-674,676-705, 709 720,727-739,742-750,753-760, 768-773,783-788, 811-819, 827 838 ORF0923 GTP-binding 4-10, 18-27, 42-55, 64-72, 77-92, B:13 aa 144-163 B:SELAD55(151-163): 8/12 246, protein 114-126, 132-157, 186-196, 206- 275 217, 236-243, 257-280, 287-300, 306-314 321-328, 338-351, 360 367,371-382,385-399 ORF0979 Conserved hypo- 4-28,44-51,53-84,88-107,113- A9,B:18 aa 12-51 B:SELAHO(26-49) 5/12 247, thetical protein 192 276 ORF0982 sodium/alanine 13-11,24-50, 73-84, 91-118, 126- D-3 aa277-305 D: nd 523, symporter (alsT) 133, 142-149, 156-175, 189-249, 572 251-273,294-332,339-347,358 381, 393-413,425-448,458-463 ORF1230 Signal peptidase 1 6-33, 44-59, 61-69, 74-82, 92-98, D:14 aa 1-53 D: nd 524, 133-146,163-175 573 ORF1232 Exonuclease 4-12, 16-32,36-48, 50-65,97-127, B:6 as 188-219 B:SELAA13(188-216): n.d. 443, RexA 136-142, 144-165, 176-190, 196- 444 202,211-222,231-238,245-251, 268-274,280-286,305-316,334 356,368-376,395-402,410-417, 426-440,443-449,474-486,499 508, 510-525, S40-549, 568-576, 608-617, 624-639,646-661,672 678, 688-703, 706-717, 727-734, 743-755,767-773, 783-797, 806 814, 830-839, 853-859, 863-871, 877-895, 899-918, 935-948, 976 990,998-1007,1020-1030, 1050 10 62, 1070-1077, 1111- 1125, 1137 1149, 1153-1160, 1195-1211 ORF1284 permease PerM, 10-60,72-96, 103-109, 127-133, D:27 aa55-106 D: nd 525, putative 146-177,182-189,196-271,277- 574 289, 30l-319, 323-344, 347-354 - 85 S. Putative function predicted immunogenic a&** No. of Location of Serum reactivity with relevant Seq ID epidernidi (by homology) selected identified region (positive/total) no: s antigenle clones Immuno- (DNA protein per ORF genic region +Prot) and screen ORF1319 2-oxoglutarate 9-31, 36-45, 59-67, 71-81, 86-94, B:5; C: 1 aa 400-413 B:SELAF54(404-413): 11/12 248, decarboxylase 96-107, 111-122, 127-140, 153-168, 277 (menD) 180-211,218-224, 226-251,256 270, 272-289, 299-305, 3 10-323, 334-341, 345-353, 358-364, 369 379, 384-390, 396-410, 417-423, 429-442,454-464,470-477,497 505, 540-554 ORF1326 autolysinAtlE 6-25,40-46,75-81,150-155,200- B:7;C:5 aa 1282- B:SELAD20(1282-1298): 10/12 249, (lytD) 205,237-243,288-295,297-306, 1298 278 308-320, 341-347, 356-363,384 391,417-429,440-452,465-473, 481-514,540-546, 554-560, 565 577,585-590,602-609,611-617, 625-634,636-643, 661-668, 676 684,718-724,734-742,747-754, 766-773,775-781, 785-798, 800 807, 825-832, 840-857, 859-879, 886-892,917-923,950-956,972 978, 987-1002, 1028-1035, 1049 1065, 1071-1099, 1111-1124, 1150 1172,1185-1190,1196-1207, 1234 1241, 1261-1271, 1276-1281, 1311 1320, 1325-1332 ORF1333 quinoi oxidaso 4-27,33-55,66-88 D:4 aa 3-93 D nd 526, polypeptide iv (ec 575 1.9.3.-) (quinol oxidase aa3-600, subunit qoxd) ORF1356 hypothetical pro- 9-36,44-67,74-97,99-149, 161- D.32 aaS4-95 D nd 527, tein 181, 189-198,211-224,245-253, 597 267-273,285-290,303-324,342 394, 396-427 0RF1373 dihydmipoanide 33-39,42-78, 103-109,126-136, A:3, B:A aa 124-188 A:SEFAP57(124-188): 2/12 250, acetyltransfemse 184-191,225-232,258-279,287- 279 294, 306-315, 329-334, 362-379, 381-404, 425-430 ORF1381 hypothetical pro- 21-45, 62-67, 74-106, 108-142, D:5 aa7-44 D: nd 528, tein 154-160,230-236,245-251,298- 576 305 - 86 S Putative function predicted immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected Identified region (positive/total) no: s antigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen ORF1420 Muts2 pmtein, 8-32, 34-41, 46-55, 70-76, 81-89, B:7 aa 581-608 B:SELAM40(581-604): 9/12 251, putative 97-115, 140-148, 153-159, 165-171, 280 175-188, 207-239,256-276,280 289, 297-319, 321-335,341-347, 352-360, 364-371, 384-411, 420 440,449-460,495-502, 505-516, 560-566, 573-588, 598-605,607 614, 616-624, 674-694, 702-717 ORF1443 cell division 61-66, 111-117, 148-155, 173-182, D:4 aa175-229 D: nd 529, protein (divlB) 194-224, 263-293, 297-303, 313- 577 321, 334-343, 345-356, 375-381, 384-395,408-429,448-454 ORFI500 Cell division pro- 100-107, 154-167.182-193,200- A:2, B-3 aa 77-182 B:SELAP37(139-162): 9/12 252, tein FtsY 206,223-231, 233-243, 249-257, .281 265-273, 298-310, 326-336,343 362,370-384 ORF1665 amino acid ABC 4-25, 44-55, 66-76, 82-90, 93-99, D:5 aa 1-52 D: nd 530, transporter, 104-109, 176-209,227-242,276- 578 permease pmtein 283,287-328, 331-345, 347-376, 400-407, 409-416, 418-438, 441- 474 ORF1707 putative host cell 12-31, 40-69, 129-137, 140-151, D:4 aa20-76 D:nd 531, surface-exposed 163-171,195-202,213-218 598 lipoprotein ORF1786 D-3- 4-10, 16-32, 45-55, 66-78, 87-95, D:5 aa400-442 D: nd 532, phosphoglycerate 103-115, 118-124, 135-150, 154- 579 dehydogenase, 161, 166-174, 182-193, 197-207, putative 225-231, 252-261, 266-304, 310 315, 339-347, 351-359, 387-L402, 411-423, 429-436, 439-450, 454 464,498-505,508-515 ORF1849 yhjN protein 8-51, 53-69, 73-79, 85-132, 139- D:5 aa254-301 Dnd 533, 146,148-167, 179-205,212-224, 580 231-257,264-293, 298-304, 309 317, 322-351 - - O'/ Putative function predicted Immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected identified region (positive/total) no: s antigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen ORF1877 protein-export 6-19, 26-39, 41-51, 59-67, 72-85, D:7 aa367-409 D: nd 534, membrane protein 91-98, 104-111, 120-126,147-153, 581 SecD (secD-1) 158-164, 171-178,199-209,211 218, 233-249,251-257,269-329. 362-368,370-385,392-420,424 432,454-489,506-523,534-539, 550-556, 563-573, 576-596, 603 642, 644-651, 655-666,685-704, 706-733,747-753 ORF1912 unknowncon- 23-35,37-70,75-84,90-112,129- D:4 aal31-187 D: nd 535, served protein 135, 137-151, 155-180,183-209, 582 (conserved) 211-217, 219-225,230-248, 250 269, 274-284, 289-320, 325-353, 357-371, 374-380, 384-399, 401 411, ORF2015 Trehalose-6- 8-17, 30-54, 82-89, 94-103, 157- A:3, B:8 aa 465-498 B:SELAH62(465-498): 5/12 253, phosphate 166, 178-183,196-204,212-219, 282 hydrolase 222-227, 282-289, 297-307, 345 364,380-393,399-405,434-439, 443-449,453-475,486-492,498 507,512-535,538-548 ORF2018 Glucose-6- 4-16, 21-27, 39-51, 60-69, 76-83, B:17 aa250-287 B:SELA119(250-279):3/12 254, phosphate 1-DH 97-118, 126-132, 159-167, 171-177, 283 192-204, 226-240, 247-259, 28 1 286,294-305,314-320,330-338, 353-361, 367-372, 382-392, 401 413, 427-434, 441-447, 457-463 ORF2040 LysM domain 51-56, 98-108, 128-135, 138-144, D.23 aa259-331 D: nd 536, protein protein 152-158, 177-192,217-222,232- 583 251, 283-305, 406-431, 433-439 ORF2098 PUB related 13-18, 36-43, 45-50, 73-79, 95-100, A:60 aa 1-57 A:SBFAQ50(15-57): 5/12 255, protein 111-126,133-139 ..-- 284 ORF2139 sodium:sulfate 7-12, 22-97, 105-112, 121-128, D:41 aa42-118 D:nd -- 537, symporter family 130-146, 152-164,169-189, 192- 584 protein, putative 203, 211-230, 238-246,260-281, 304-309,313-325,327-357, 367 386,398-444,447-476,491-512 - 88 S Putative function predicted immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected identified region (positive/total) no: s tigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen ORF2172 SceB precursor 4-23, 28-34, 38-43, 45-51, 63-71, A:438, aa 6-215 B:SELAH53(188-209): 3/12 256, (lytE) 85-96,98-112, 118-126, 167-174, B:40, D:4 285 179-185, 219-228,234-239,256 263 ORF2200 zinc ABC 4-31, 33-40, 48-64, 66-82,92-114, D:19 aa162-225 D: nd 538, transporter, 118-133, 137-159,173-246,248- 585 permease protein, 266 putative ORF2248 membrane protein, 4-11, 17-34,72-78,127-137,178- D:17 aal-59, D:nd 539, MmpL family, 227, 229-255,262-334, 352-380, an159-225, 586 putative 397-405,413-419, 447-454,462- aa634-674 467,478-490, 503-509,517-558, 560-568,571-576, 582-609, 623 629, 631-654, 659-710, 741-746, 762-767,771-777, 788-793, 856 867 ORF2260 Unknown oon- 5-10,18-29,31-37,66-178,196- B:4 aa 123-142 B:SELAG77(123-142): 12/12 257; served protein in 204,206-213 286 others ORF2282 conserved hypo- 16-22,41-50, 52-64,66-74, 89-95, A:4 aa5l-97 A:SEFAR88(51-97): 3/12 258, thetical protein 107-114, 123-130, 135-159,167- 287 181, 193-199, 223-231, 249-264, 279-289 ORF2376 DivIC homolog, 27-56, 102-107, 111-116 D:7 aaI5-58 and 540, putative 587 ORF2439 membrane-bound 4-9,11-26,36-56,59-73,83-100, A:459, aa 10-217 B:SELAC31(75-129): 12/12 259, lytic murein 116-130,148-163, 179-193, 264- B:2, D:53 288 transglycosidase 270,277-287,311-321 '3, ptive _____________ ORF2493 conserved hypo- 4-29,37-77, 80-119 D:6 aa69-113 D: nd 541, thetical protein 588 ORF2535 ATP-binding 5-28, 71-81, 101-107, 128-135, D:8 aal-65 D: nd 542, cassette 146-52, 178-188,209-214,224-233, 589 transporter-like 279-294, 300-306, 318-325, 342 protein, putative 347, 351-357 - 89 S. Putative function predicted immunogenic aa** No. of Location of Serum reactivity with relevant Seq ID epidermidi (by homology) selected Identified region (positive/total) no: s antigenic clones Immuno- (DNA protein per ORF genic region +Prot) and screen ORF2627 cation- 8-31, 34-80, 125-132, 143-153, D:3 aa6l-105 D: nd 543, transporting 159-165,176-189,193-198,200- 590 ATPase, El-E2 206,215-242,244-262,264-273, family, putative 281-289, 292-304, 318-325,327 338,347-371,404-416,422-429, 432-450,480-488,503-508,517 525, 539-544,551-562, 574-587, 600-631, 645-670 ORF2635 Hypothetical 4-10, 17-24, 26-42, 61-71, 90-96, A:2, B:2 aa 139-169 B:SELAB63(138-163): 7/12 260, protein 102-111, 117-125,158-164,173- 289 182, 193-201,241-255,268-283, 289-298,305-319,340-353,360 376, 384-390,394-406 ORF2669 Hypothetical 4-21,35-42,85-90,99-105, 120- A:14, B:8 aa22-81 B:SELAE27(22-51): 5/12 261, protein 125, 148-155, 175-185, 190-196, 290 205-210,217-225 L _ ORF2671 Hypothetical pro- 4-23, 43-49, 73-84, 93-98, 107-113, A.44, aa 23-68 B:SELAD21(36-61): 5/12 262, tein 156-163, 179-190, 197-204,208- B:14 291 218,225-231,248-255 ORF2673 Hypothetical 4-20,65-71,99-105, 148-155, 171- A.16, B:3 aa 23-68 B:SELAE25(23-54): 2/12 263, protein 182,190-196,204-210,221-228, 292 240-246 ORF2694 Hypothetical 4-26,93-98, 121-132, 156-163, A:19, aa 25-82 B:SE1AB26(27-60): 5/12 264, protein 179-192, 198-204,212-220,225- B:30 293 238 ORF2695 Hypothetical 4-26,43-50,93-98, 107-113, 156- A:7 aa 22-78 A:SEFAH77(22-66): 6/12 265, protein 163, 179-190, 198-204,212-218, 294 225-231,247-254 ORF2719 two-component 5-52,60-71,75-84,91-109,127- B:4 aa123-132 B:SELAA62(123-132):6/12 266, .ensor histidino 135, 141-156, 163-177, 185-193, 295 Icinase, putative 201-214,222-243,256-262,270 279,287-293,298-303,321-328, 334-384, 390-404, 411-418,427 435, 438-448,453-479, 481-498, 503-509 ORF2728 Accumulation- 4-13,36-44,76-86,122-141, 164- A:265, aa 803- B:SELAA1O(850-878): 11/12 267, associated protein 172,204-214,235-242,250-269, B:448; 1001 296 291-299, 331-337, 362-369, 377- C:4, D:9 396,419-427,459-469, 505-524, 547-555,587-597,618-625,633 652,675-683,715-727,740-753, 761-780, 803-811-, 842-853, 962 1968, 1006-1020 - 90 & Putat ive function predicted immunogenlc aa** No. of Location of Serum reactivity vith relvant SeqMI epidermidi (by homology) selected Identified region (positive/total) no, s antigenie clones immuno- (DNA protein per ORF genic region +-Prot) and screen ORF2740 lipase prectusor 4-21, 190-200,219-728,233-241, 03 an 110-177 C:GSBBL8O(110-17 7)111 359, 243-261, 276-297, 303-312, 316- 364 - 325, 346-352, 381-387,436-442, - ~ 457-462,405-505, 518-532, 543 ____________557, 574-593 0RF2764 oligopeptideA3C 14-36, 62-131, 137-147,149-162, DA4 aa 6-41 [D: rd 544, transporter, per- 164-174,181l-207,212-222,248- 591 mease Prote in, 268, 279-285 puttie _ _ _ _ ORF2767 unknown con- 7-20, 22-35, 40-50, 52-61, 63-92, DA4 aa276-316 D: zid 545, sered protein in 94-101, 103-126, 129-155, 161-178, 592 oflzes 192-198, 200-208.210-229,232 241, 246-273, 279-332,338-359, 369--383 0RP20 sodiunvsulfate 4-29,37-53,56-82,87-100, 108- D.9 aa266-3 17, D- zd 546, symporterfamily 117,121-138, 1507160, 175-180, aa357-4 0 1 593 protein 189-195, 202-214,220--247,269 315.,324-337, 341-355, 361-412, 414-423,425-440,447-467 ORF2851 putative basns- 7-13,20-32,37-90,93-103, 107- 0:11 aa137-195 D ad 547, meatbrne efflux 126,129-155, 159-173, 178-189, 594 proten 195-221,234-247, 249-255, 268 1 303,308-379 ____ ___________ - 91 Table 2d: Immunogenic proteins identified by bacterial surface and ribosome display: S. aureus (new annotation) Bacterial surface display: A, LSA250/1 library in fhuA with pa tient .sera 1.(655); B,' LSA50/6 library in lamB with patient sera 1 (484); C, LSA250/1 library in fhuA with IC sera 1 (571); E, LSA50/6 library in lamB with IC sera 2 (454); F, LSA50/6 library in lamB with patient sera P1 (1105); G, LSA50/6 library in lamb with IC sera 1 (471). Ribosome display: D, LSA250/1 library with IC sera (1686) . **, prediction of antigenic sequences longer than 5 amino acids was performed with the programme ANTIGENIC (Kolas kar and Tongaonkar, 1990); #, identical sequence present twice in ORF. S. Old Putative predicted Immunogenic aa** No. of se- Location of Serum reactivity with rele- Seq amresan ORF function - lected Identified vant region (positive/total) ID no: tigenic number (by homology) -- clones per immuno- (DNA protein ORF and genic re- +Prot) screen glon SaAO003 ORF2967 repC 7-19,46-57,85-91,110-117, 125- B:3,C:14; aa9-42 C:GSBYIS3(9-42):l/1 394, & 133, 140-149, 156-163, 198-204, F:29 aa 156-241 C:GSBYG39(156-241):1/1 396 ORF2963 236-251,269-275,283-290,318- aa 300-314 CGSBYM94(343-420):26/30 323, 347-363 aa 343-420 ORF0123 ORF1909 unknown 14-10,25-30,38-57,91-108, 110- 13:3, E-7, aa 145-163 B:OGSBXFO(150-163).5/27 409, - 18 aa at 123, 125-144, 146-177, 179-198, G:1 E:GSBZC17(150-163):2541 410 N- 216-224,226-233 tenninus ____ __________________ ORF0160 ORF1941 unknown 4-26, 34-70,72-82,86-155,160- A:l aa96-172- A:GSBXO07(96-172):5/30 411, -16 aa at 166, 173-205,207-228,230-252, 412 N- 260-268 ,280-313 IterinusI__ ORF0657 ORF un- 12XTGVI 9-33,56-62,75-84,99-105,122- A:2, B:27, aa 526-544 B:GSBXE07-bdbl(527- 413, known protein 127,163-180,186-192,206-228, F:15 542):!1/71 414 233-240,254-262,275-283,289- F:SALAX70(526-544):11/41 296,322-330,348-355,416-424, 426-438,441-452, 484-491,541 549,563-569, 578-584, 624-641 ORF1050 ORLF1307 unknown 45-68,72-79,91-101,131-142, A:1, H:4 aa 53-124 A:GSBXM26(S3-124):7/30 415, -4 aa at 144-160,179-201 46 N-temi nus ORF1344 ORF0212 NifS protein 13-26,40-49,61-68,92-112,114- A:11 aa 24-84 A:GSBXK59-bmd2l(24- 417, -10 aa at homolog 123, 138-152, 154-183,194-200, 84):6/29 418 N- 207-225,229-240,259-265,271 tenninus 284, 289-309,319-324,330-336, 346-352,363-372 - 92 S. Old Putative predicted immunogenic as** No. of se- Location.of Serum reactivity with rele- Seq aureusan ORF function lected identified vant region (positive/total) ID no: tigenic number (by homology) clones per immuno- (DNA protein ORF and genic re- +Prot) screen gion ORF1 632 ORFi 163 SdrH homolog 4-31, 50-55, 243-257, 259-268, B:6, E 11, aa 101-115 B:GSBXG53(164-182):39/71 419, -4 aa at 298-316, 326-335, 364-370,378- F:34 aa 115-139 F:SALAP07(101-I 15): 11/41 420 N- 407 aa 158-186 terminus I ORF2180 ORF0594 LPXTGIV 9-t7,24-45,67-73,82-90, 100-107, A:3, C:3, aa491-587 A:GSBXS61(491-555):i/ 421, - 2 aa at protein 117-134, 137-145, 158-168, 176- E:6, F:2, aa633-715 A:GSBXL64(494-585):1/l 422 N- 183, 188-194,206-213,223-231, H:6 aa 702- A:GSBXS92(758-841):il tenninus 243-248, 263-270, 275-282,298- 757' A:bmd4(702-757):16/30' 304, 344-355, 371-377, 382-388, aa 758-830 (A:bmd4(830-885):16/30)' 427-433,469-479,500-505, 534- - (aa 830- F:SALBC43(519-533):4/41 559, 597-607, 662-687, 790-815, 885), 918-943, 1032-1037, 1046-1060, 1104-1112,1128-1137, 1179-1184, 1197-1204, 1209-1214,1221-1239 ORF2184 ORF0590 FnbpB . 10-29, 96-116, 131-137, 146-158, A:2, C:4, aa 694-769 A:GSBXM62(694-769).28/28 423, - 8 aaat 167-173, 177-182, 185-191,195- G:9 aa 774-847 A.GSBXR22(774-847):1/1 -424 N-tenni- 201,227-236,260-266,270-284, nus 291-299,301-312,348-356,367 376,382-396,422-432,442-453, 480-487,497-503,519-527, 543 548,559-565,579-585, 591-601, 616-623, 643-648, 657-663, 706 718, 746-758, 791-796, 810-817, 819-825, 833-839, 847-853, 868 - - 885, 887-895,919-932 ORF2470 ORF0299 Conserved hy- 4-27,36-42,49-55,68-73,94-101, C:3 aa 400-441 C:GSBYH60(400-441):28/31 425, - 14 aa at pothetical 131-137,193-200,230-235,270- 426 N- protein 276,294-302,309-324,334-344, terminus 347-364,396-405,431-437,498 508,513-519,526-532,539-544, 547-561, 587-594, 618-630, 642 653, 687-699,713-719,752-766 ORF2498 ORF0267 Conserved hy-8-19,21-44,63-76,86-92,281-286, D:12, F:6 aa 358-411 D:17/21 427, ORF app. pothetical 303-322,327-338,344-354,364- aa 588-606 F:SALAT38(895-909):8/41 428 580 aa protein 373,379-394,405-412,453-460, sa 895-909 lnger at 501-506,512-518,526-542,560 N termi- 570, 577-583,585-604,622-630, nus; plus 645-673, 677-691,702-715,727 other 741, 748-753,770-785,789-796, changes 851-858, 863-869, 876-881,898 913, 917-924, 979-986, 991-997, 1004-1009, 1026-1041, 1045-1052, 1107-1114,1119-1125, 1132-1137, 1154-1169, 1173-1192, 1198-1204, 1240-1254, 1267-1274, 1290-1298, 1612-1627 - 93 S. Old Putative predicted Immunogenic aa** No. of se- Location of Serum reactivity with rele- Seq aureusan ORF function lected identified vant region (positive/lotal) ID no: tigenic number (by homology) clones per immuno- (DNA protein ORF and genie re- +Prot) screen gion _ ORF2S48 ORF2711 IgG binding 4-37,44-53,65-71,75-82, 105-112, A:55, aa 1-123 A:GJSBXK68(1-73):21130 429, -12 anat protein A 126-132,136-143, 164-170,184- B:54, aa 207-273 A:GSBXK41(35-123):1/1 430 N- 190, 194-201, 222-232, 242-248, C:35, aa 310-410 A.GSBXN38(207-273):19/30 terminus 252-259, 280-291, 300-317, 413- F:59, A:GSBXLI 1(310-363):10/30 420,452-460,485-503 G-56, B:GSBXB22(394-406):37/71 H:38 F:SALAM17(394-406):29/41 ORF2746 ORF2507 homology with 4-9,12-17, 40-46, 91-103, 106-113, A:1, H:13 aa 63-126 A:GSBXO40(66-123):8/29 431, -3 aa at ORFI 116-125, 150-160, 172-177,182- 432 N- 188,195-206,241-261,263-270, terminus 277-285,287-294 ORF2797 ORF2470 unknown 13-32,40-75,82-95,97-11,115- B:3,F2, aa 159-176 B:GSBXESS(159-176):l 27 433, -24 aa at 121, 124-154, 166-192,201-225, F.13, H3 an 325-339 F:SALAQ47(159-176):841 434 N-tcrmi- 227-252, 268-273,288-297,308 nus 1375,379-434 ORF2960 ORF2298 putative 8-31,35-44,106-113,129-135, Q10I, an 1-80 C:GSBYG32(1-80)::6n 435, - 5 an at Exotoxin 154-159,168-178,203-215,227- E:2. 1:58 a 48-121 C:GSBYG6I-bie248- 436 N- 236, 240-249, 257-266, 275-28 1, an, 98-190 116):26/30 terminus 290-296,298-305,314-319,327- C:GSBYN80C98-190).13/17 334 ORF2963 ORF2295 putative 8-23,35-41,64-70,81-87, 109-115, C.3, -3, an 17-95 C:GSBY]58(17-95):9/15 437, -S an at Exotoxin 121-132, 150-167, 177-188,194- GA1 438 N- 201, 208-216, 227-233, 238-248, terminus 265-271,279-285 8-297_308 - 94 S. Old Putative predicted immunogenic aa** No. of se- Location ol Serum reactivity with relc- Seq aureusan ORF function lected Identified vant region (positiveltotal) ID no: tigenic number (by homology) clones per inimuno- (DNA protein ORF and genic re- +Prot) screen gion ORF3200 ORF1331 putative 8-32,45-52,92-103,154-159,162- A:11, aa8543- A:GSBXL07(8543-8601):6/28 439, +8506 aa extracellular 168, 207-214,232-248,274-280, B:11, 8601 440 at N- matrix binding 297-303, 343-349, 362-375,425- C:36, aa 8461 terminus protein 442,477-487,493-498,505-512, 11:32 8475 522-533, 543-550,558-564,568 574, 580-600, 6 18
-
63 0 , 647-652, 658-672,692-705,711-727, 765 771, 788-798,812-836,847-858, 870-898, 903-910, 1005-1015, 1018-1025, 1028-1036, 1058-1069, 1075-1080,1095-1109,1111-1117, 1119-1133,1166-1172, 1183-1194, 1200-1205, 1215-1222, 1248-1254, 1274-1280, 1307-1317, 1334-1340, 1381-1391, 1414-1420, 1429-1439, 1445-1467, 1478-1495, 1499-1505, 1519-1528, 1538-1550, 1557-1562, 1572-1583, 1593-1599, 1654-1662, 1668-1692, 1701-1707, 1718-1724, 1738-1746, 1757-1783, 1786-1793, 1806-1812, 1815-1829, 1838-1848, 1853-1860, 1875-1881, 1887-1893, 1899-1908, 1933-1940, 1952-1961, 1964-1970, 1977-1983, 1990-1996, 2011-2018, 2025-2038, 2086-2101, 2103-2117, 2177-2191, 2195-2213, 2220-2225, 4*2237-2249, 2273 2279,2298-2305,2319-2327,2349 2354,2375-2381,2391-2398,2426 2433,2436-2444,2449-2454,2463 2469,2493-2499,2574-2589,2593 2599,2605-2611,2615-2624,2670 2684, 2687-2698, 2720-2727, 2734 2754, 2762-2774, 2846-2866, 2903 2923,2950-2956,2985-2998,3011 3031, 3057-3064, 2"3102-3117, 3137-3143,3186-3195,3211-3219, 3255-3270,3290-3300,3327-3334, 3337-3343,3390-3396,3412-3419, 3439-3446,3465-3470,3492-3500, 3504-3510,3565-3573,3642-3650, 3691-3698, 3766-3775, 3777-3788, 3822-3828,3837-3847,3859-3864, 3868-3879,3895-3902, 3943-3951, 3963-3971, 3991-3997,4018-4030, 4054-4060,4074-4099,4123-4129, 4147-4153, 4195-4201,4250-4255, 4262-4267,4270-4277,4303-4310, 95 4321-4330,4343-4352,4396-4408, 4446-4451, 4471-4481,4503-4509, 4516-4534,4596-4604, 4638-4658, 4698-4710, 4719-4732,4776-4783, 4825-4833, 4851-4862, 4882-4888, 4894-4909, 4937-4942, 5047-5054, 5094-5100, 5102-5112, 5120-5125, 5146-5153, 5155-5164, 5203-5214, 5226-5236, 5278-5284, 5315-5321, 5328-5342, 5348-5359, 5410-5420, 5454-5466, 5481-5489, 5522-5538, 5597-5602, 5607-5614, 0"5623 5629, 5650-5665, 5707-5719, 5734 5742, 5772-5778, 5785-5790, 5833 5845, 5857-5863, 5899-5904, 5908 5921, 5959-5971, 5981-5989, 6010 6017, 6034-6043, 6058-6064, 6112 6120, 6154-6169, 6210-6217, 6231 6240, 6261-6268, 6288-6294, 6318 6324, 6340-6349, 6358-6369, 6402 6407, 6433-6438, 6483-6493, 6513 6519, 6527-6546, 6561-6574, 6599 6608, 6610-6616, 6662-6673, 6696 6705, 6729-6743, 6769-6775, 6792 6801, 6819-6828,6840-6846,6860 6870, 6915-6928, 6966-6972, 7021 7028, 7032-7047, 7096-7101, 7109 7117, 7138-7149,7157-7162,7201 7206, 7238-7253, 7283-7294, 7296 7302, 7344-7365, 7367-7376, 7389 7404, 7413-7433, 7475-7482, 7493 7500, 7535-7549, 7596-7608, 7646 7651, 7661-7678, 7722-7731, 7741 7754, 7764-7769, 7776-7782, 7791 7806, 7825-7837, 7862-7875, 789 1 7897, 7922-7931,-7974-7981, 7999 8005, 8039-8045, 8049-8065, 8070 8075, 8099-8112, 8119-8125, 8151 8158, 8169-8181, 8226-8232, 8258 8264, 8291-8299, 8301-8310, 8325 8335, 8375-8389, 8394-8400, 8405 8412, 8421-8436, 8478-8485, 8512 8521, 8528-8538,8564-8579, 8587 8594, 8603-8615,8626-8637, 8640 8646, 8657-8672, 8684-8691, 8725 8736, 8748-8761, 8777-8783, 8794 8799, 8810-8825, 8851-8862, 8874 8887, 8903-8912, 8914-8926, 8933 8943, 8954-8960, 8979-8988,9004 9011,9035-9041, 9056-9069,9077 9086, 9088-9096, 9106-9111,9124 9133, 9183-9191, 9224-9231, 9235 9241, 9250-9265, 9279-9290, 9295- 96 9300,9326-9343, 9408-9414, 9422 9427, 9435-9441, 9455-9461,9507 9517, 9532-9538, 9580-9589, 9594 9600, 9614-9623, 9643-9648, 9665 9683, 9688-9700, 9720-9726, 9742- 9758, 9767-9775, 9795-9800, 9817 9835, 9842-9847, 9912-9919, 9925 9938, 9943-9963, 9970-10009, 10025-10031; 10037-10043, 10045 10063, 10066-10073, 10117-10124, 10126-10136, 10203-10210, 10218 10225, 10232-10242, 10287-10292, 10303-10323, 10352-10360, 10385 10396, 10425-10431, 10452-10459, 10480-10485 -97 Table 3. Serological proteome analysis of S. aureus surface proteins using human sera a) S. aureus/agr "stress conditions" Spot ID/sera IC40 IC35, N26, C4 Infant pool N22 1:10.000 1:20,000 1:50,000 each C2,5,6,10,12 IC40 1:50,000 1:10,000 PCK2 + + - + PCK4 + +++ PCK5 - (+) - + PCK6 + + - + Spot ID/sera IC35, 40 P-pool Infant pool 1:50,000 (P6,18,25,28,29) C2,5,6,10,12 N22 1:10,000 1:50,000 each 1:10,000 PAC1 ++ L ++ PAC2 ++ _ +++ PAC3 - + PAC5 - ++ Spot ID/sera P-pool Infant 14 IC pool / IgG IC pool / IgA (PG,18,25,28,29) 1:10,000 (N26, IC34,35) (N26, IC34,35) 1:50,000 each 1:30,000 each 1:30,000 each PAC11 ++ ++ ++ PAC12 ++ ++ ++ PAC13
-
- ++ PAC14 - + + PAC15 - +++ +++ PAC16 + + + PAC17 + + + PAC18 ++ _ PAC19 - 4+ ++ PAC20 + POV31 ++.
POV32 + POV33 + - - POV34 + POV35 + - - P OV36 + - P OV37 ++_-_
-
-
- 98 P0OV38 1++ P 0V39 P 0V40 b) S. aureus/COL "standard conditions"______ Spot IDlsera IC pool IC35 P18 P25 P1 P29 Infant 18 (N26,IC34,35) 1:20,000 1:10,000 1:10,000 1:5,000 1:2,500 1:10,000 1:30,000 each ___ __ ____ POV2 -i+++ -- ++ 4++ POV3.1 .. + +++ +++ .. ++ POV3.2 +++ +++ +++ .4+ ++4 POV4 + 44 POW-- +++ - POV1O - +4- W+ W+ W + POV12 I ++ POV13 44 +++ +4+ +.4 44 44 POV14 44 .+++ .+44 44 44 44 POV15 + 1+ - +(+
-
CS. aureus/COL "stress conditions" _____ Spot IDlsera P-pool lC3444C35 pi8 P29 Infant 14 (P6,18,25,28,29) 1:20,000 each 1:10,000 1:10,000 1:10,000 1:50,000 each ________ ___________ POV16- t-.-- POV17 4---*+- POV18 + +4- POV19 W+
+++
POV21
+
P0V23
-+--
P0V24 -+- P0V25_
+---
- 99 Table 4. S. aureus antigens identified by MALDI-TOF-MS sequencing (ORFs in bold were also identified by bacterial surface display) Prediction of antigenic regions in selected antigens identified by serological proteome analysis using human sera spot ID S. aureus pro- Putative function (by homology) Seq ID no: Putative local tein (DNA, Prot) Ization (ORF no. / ab brev.) PCK2 ORF0599 Glycinamide-ibosyl synthase 107, 108 cytoplasmic PCK5 ORF0484 yitU conserved hypoth. protein (yitU) 109, 110 cytoplasmic PCK6 ORF2309 membrane-associated malate-quinone 111, 112 peripheral mem mqo oxidase brane POV2 ORF0766 aux1 protein phosphatase contributing to me- 113, 114 trans-membrane thicilin resistance POV4, 17 ORF0078 EF- C-terminal part of 44 kDa protein similar 115, 116 cytoplasmic/ se PACI 4, 19 Tu to elongation factor Tu - creted POV5 1) ORF0782 3-ketoacyl-acyl carrier protein reduc- 117, 118 cytoplasmic tase (fabG) POW ORF0317 SecA protein transport across the membrane 39,91 cytoplasmic SecA POV10 ORF1252 yrzC hypothetical BACSU 11.9 kd protein 119,120 cytoplasmic (upfOO74 (ff2) family) POV12 ORF0621 pdhB dihydrolipoamide acetyltransferase 121, 122 cytoplasmic (pdhB) POV14 ORF0072 rpoB DNA-directed RNA polymerase B 125, 126 cytoplasmic POV15 ORF0077 EF- 85 kD vitronectin binding protein 127, 128 cytoplasmic G POV18 not found general stress protein YLY1 129,130 cytoplasmic YLY1 POV30 ) ORF0069 RL7 ribosomal protein L7 131, 132 cytoplasmic POV21 ORF0103 probable hexulose-6-phosphate syn- 133, 134 cytoplasmic yckG thase (yckG) ,POV24 ORF0419 conserved hypothetical protein (yurX) 137, 138 cytoplasmic yurX - 100 spot ID S. aureus pro- Putative function (by homology) Seq ID no: Putative local tein (DNA, Prot) Ization (ORF no. / ab brev.) POV25 ORF2441 glucose inhibited division protein a (gidA) 139, 140 cytoplasmic gidA PACI ORF1490 protein export protein prsa precursor 173, 174 periplasmic prsA (prsA) PAC2 ORF1931 periplasmic molybdate binding protein 175, 176 surface ModA (ModA) PAC3 ORF2053 heavy metal dependent transcriptional 177, 178 cytoplasmic activator, putative regulator of multidrug resistance efflux pump pmrA PAC5 ORF2233 pyruvate oxidase (ydaP) 179, 180 cytoplasmic ydaP_ _ PACI I ORF1361 LPXTGV, extracellulanatrix-bdg. 3, 56 surface PAC12 ORF1244 alanyl-tRNA synthetase 159, 160 cytoplasmic alaS PAC13 ORF0835 RNA processing enzyme/ATP-bdg. 161, 162 cytoplasmic ymfA PAC15 ORF1 124 lipoamid acyltransferase component of 163, 164 cytoplasmic bfmBB branched-chain alpha-keto acid dehy drogenase complex * PAO16 ORF0340 glyceraldehydes-3-phosphate 165, 166 cytoplasmic GAPDH dehydrogenase PAC17 not found 5'-methylthloadenosine nucleosidase I. cytoplasmic Contig83 S-adenosylhomo-cysteine nucleosidase PAC20 ORF2711 75% Identity to ORF2715 167, 168 unknown similar to hypothetical proteins* POV31 ORF0659 29 kDa surface protein 236, 238 surface POV32 ORF0659 29 kDa surface protein 236, 238 surface POV33 ORF0659 29 kDa surface protein 236, 238 surface POV34 ORF0659 29 kDa surface protein 236, 238 surface POV35 ORF0659 29 kDa surface protein 236, 238 surface P OV36 ORF00661 LPXTG-motlf cell wall anchor domain 235, 237 surface protein P OV37 ORF0659 29 kDa surface protein 236; 238 surface - 101 spot ID S. aureus pro- Putative function (by homology) Seq ID no: Putative local tein (DNA, Prot) lzation (ORF no. I ab brev.) P 0V38 ORF0659 29 kDa surface protein 236, 238 surface P OV39 ORF0657 LPXTG-anchored surface protein 1, 142 surface P OV40 not identified Seq ID no: spot ID S. aureus.ORF Putative local- Putative antigenic surface areas (Protein) no. / abbrev. Izatlon (Antigenic package) 112 PCK6 ORF2309 peripheral 61-75,82-87,97-104,113-123,128-133, mqo membrane 203-216, 224-229, 236-246,251-258, 271 286, 288-294, 301-310, 316-329, 337-346, 348-371, 394-406, 418-435,440-452 114 POV2 ORF766 aux1 trans-mem- 30-37,44-55,83-91,101-118,121-128, brane 136-149,175-183,185-193,206-212, 222 116 POV4 ORF078 EF-Tu cytoplasmic/ 28-38,76-91,102-109,118-141,146-153, secreted 155-161,165-179,186-202,215-221, 234 249, 262-269, 276-282, 289-302, 306-31 4, 321-326, 338-345, 360--369,385-391 176 PAC2 ORF1931 periplasmic 29-44,74-83,105-113,119-125,130-148, ModA 155-175,182-190,198-211,238-245 174 PAC ORF 49 pe I plasmic 174 PACI ORF1490 perplasm6c 5-24,38-44, 100-106,-118-130, 144-154, prsA 204-210, 218-223, 228-243, 257-264, 266 286, 288-294,313091-2,3736 168 PAC20 ORF2711 unknown 7-14,21-30,34-0, 52-63,65-72,77-84, 109-124,129-152,158-163,175-190 193 216, 219-234 spot ID GI no. or S. aureus pro- Putative function (by homology) Seq I D no: TIGR no. teln (DNA, Prot) (ORF no.,1 ab brev.) = PCK2 ITIGR1280O0RF0599 Glyclnamlde-ribosyl synthase 107,108 - 102 PCK4 7672993 ORF2268 IsaA possibly adhesion/aggregation 12,64 PCK5 TIGR6209 ORF0484 yitU conserved hypoth. protein (yitU) 109, 110 PCK6 TIGR6182 ORF2309 membrane-associated malate-quinione 111,112 oxidase POV2 6434044 ORF0766 auxI protein phosphatase contributing to methl- 113, 114 cilin resistance _ _ _ POV3.1 7672993 ORF2268 lsaA possibly adhesion/aggregation 12, 64 POV3.2 7672993 ORF2268 IsaA possibly adhesion/aggregation 12, 64 POV4 TIGR8079 ORF0078 EF- C-terminal part of 44 kDa protein similar 115,116 Tu to elongation factor Tu POV5 I TIGR8091 ORF0782 3-ketoacyl-acyl carrier protein reductase 117, 118 (fabG) POV7 2500720 ORF0317 SecA protein transport across the membrane 39, 91 SecA POVIO TGR8097 ORF1252 yrzC hypothetical BACSU 11.9 kd protein 119, 120 (upfOO74 (rff2) family) POV12 2499415 ORF0621 pdhB dihydrolipoamide acetyltransferase (pdhB) 121, 122 POV13 7470965 ORF0094 SdrD fibrinogen-bdg. (LPXTG) protein homolog 123, 124 (SdrD). POV14 1350849 ORF0072 rpoB DNA-directed RNA polymerase B 125, 126 POV1I5 6920067 ORF0077 EF-G 85 kD vitronectin binding protein 127, 128 POV17 TIGR8079 ORF0078 C-terminal part of 44 kDa protein similar 115, 116 to elongation factor Tu POVI 8 3025223 not found general stress protein YLY1 129,130 POV30 1 350771 ORF0069 RL7 ribosomal protein L7 131, 132 POV21 ORF0103 probable hexulose-6-phosphate synthase 133, 134 (yckG) POV23 ORF0182 lipoprotein (S.epidermis) 135, 136 1 identified from a total lysate from S. aureus 8325-4 spa- grown under standard conditions. Seroreactivity with 1/1 patient and 2/4 normal sera but not with infant serum (C5).
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Claims (40)

1. A method for identification, isolation and production of hyperimmune serum reactive antigens from a pathogen, said antigens being suited for use in a vaccine for a given type of animal or for humans, the method comprising the 5 following steps: (i) providing an antibody preparation from a plasma pool of said given type of animal or from a human plasma pool or individual sera with antibodies against said specific pathogen; (ii) providing at least one bacterial surface expression library of said 10 specific pathogen; (iii) screening said at least one bacterial surface expression library with said antibody preparation; (iv) identifying antigens which bind in said screening to antibodies in said antibody preparation; 15 (v) screening the identified antigens with individual antibody preparations from individual sera from individuals with antibodies against said specific pathogen; and (vi) identifying the hyperimmune serum-reactive antigen portion of said identified antigens and which hyperimmune serum-reactive antigens 20 bind to a relevant portion of said individual antibody preparations from said individual sera.
2. A method according to claim 1 further comprising the step of isolating said hyperimmune serum-reactive antigens and producing said hyperimmune serum-reactive antigens by chemical or recombinant methods, provided that 25 said individual sera are from patients showing an antibody titre against said specific pathogen being higher than 80 percentile and an IgG titre above 10000 U. - 109
3. A method for identification, isolation and production of a practically complete set of hyperimmune serum-reactive antigens of a specific pathogen, said antigens being suited for use in a vaccine for a given type of animal or for humans, the method comprising the following steps: 5 (i) providing an antibody preparation from a plasma pool of said given type of animal or from a human plasma pool or individual sera with antibodies against said specific pathogen; (ii) providing at least three different expression libraries of said specific pathogen at least one being a bacterial surface expression library; 10 (iii) screening said at least three different expression libraries with said antibody preparation; (iv) identifying antigens which bind in at least one of said at least three screenings to antibodies in said antibody preparation; (v) screening the identified antigens with individual antibody 15 preparations from individual sera from individuals with antibodies against said specific pathogen; (vi) identifying the hyperimmune serum-reactive antigen portion of said identified antigens which hyperimmune serum-reactive antigens bind to a relevant portion of said individual antibody preparations 20 from said individual sera; (vii) repeating said screening and identification steps at least once; (viii) comparing the hyperimmune serum-reactive antigens identified in the repeated screening and identification steps with the hyperimmune serum-reactive antigens identified in the initial 25 screening and identification steps; and -110 (ix) further repeating said screening and identification steps, if at least 5% of the hyperimmune serum-reactive antigens have been identified in the repeated screening and identification steps only, until less than 5% of the hyperimmune serum-reactive antigens are 5 identified in a further repeating step only to obtain a complete set of hyperimmune serum-reactive antigens of a specific pathogen.
4. A method according to claim 3 further comprising the step of isolating said hyperimmune serum-reactive antigens and producing said hyperimmune serum-reactive antigens by chemical or recombinant methods, provided that 10 said individual sera are from patients showing an antibody titre against said specific pathogen being higher than 80 percentile and an IgG titre above 1000 U.
5. A method according to any one of the preceding claims wherein at least one expression library is selected from the group consisting of a ribosomal display 15 library and a proteome.
6. A method according to claim 3 or 4 wherein said at least three different expression libraries are at least a ribosomal display library, a bacterial surface library and a proteome.
7. A method according to any one of claims 1 to 6 wherein said plasma pool is a 20 human plasma pool taken from individuals having experienced or are experiencing an infection with said pathogen.
8. A method according to any one of claims 1 to 7, wherein said expression libraries are genomic expression libraries of said pathogen.
9. A method according to any one of claims 1 to 8, wherein said expression 25 libraries are complete genomic expression libraries.
10.A method according to claim 9 wherein the complete genomic expression libraries have a redundancy of at least 2x. - 111
11 .A method according to claim 9 wherein the complete genomic expression libraries have a redundancy of at least 5x.
12.A method according to claim 9 wherein the complete genomic expression libraries have a redundancy of at least lOx. 5
13.A method according to any one of claims 1 to 12, comprising the steps of screening at least a ribosomal display library, a bacterial surface display library and a proteome with said antibody preparation and identifying antigens which bind in at least two, preferably which bind to all, of said screening to antibodies in said antibody preparation. 10
14.A method according to any one of claims 1 to 13, wherein said pathogen is selected from the group of bacterial, viral, fungal and protozoan pathogens.
15.A method according to any one of claims 1 to 14, wherein said pathogen is selected from the group of human immunodeficiency virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, Rous sarcoma virus, Epstein-Barr virus, 15 influenza virus, rotavirus, Staphylococcus aureus, Staphylococcus epidermis, Chlamydia pneumoniae, Chlamydia trachomatis, Myco-bacterium tuberculosis, Mycobacterium leprae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Bacillus anthracis, Vibrio cholerae, Borrelia burgdorferi, Plasmodium sp., Aspergillus sp. or Candida 20 albicans.
16. A method according to any one of claims 1 to 15, wherein at least one expression library is a ribosomal display library and said hyperimmune serum reactive antigens are produced by expression of the coding sequences of said hyperimmune serum-reactive antigens contained in said library. 25
17. A method according to any one of claims 1 to 16, further comprising the step of formulating the produced hyperimmune serum-reactive antigens into a pharmaceutical preparation. -112
18. A method of claim 17 wherein the pharmaceutical preparation is produced by addition of a pharmaceutically acceptable carrier and/or excipient to the antigens.
19. A method according to claim 17 or 18 wherein said pharmaceutical preparation 5 is a vaccine.
20. A method according to claim 18 or 19 wherein said pharmaceutically acceptable carrier and/or excipient is an immunostimulatory compound.
21. A method according to claim 20, wherein said immunostimulatory compound is a polycationic substance. 10
22. A method according to claim 21 wherein the polycationic substance is a polycationic peptide.
23. A method according to claim 20 wherein the immunostimulatory compound is selected from the groups consisting of: immunostimulatory deoxynucleotides, alum, Freund's complete adjuvant, Freund's incomplete adjuvant, neuroactive 15 compounds, especially human growth hormone, or combinations thereof.
24. A method according to any one of claims 1 to 23, wherein said individual antibody preparations are derived from patients with acute infection with said pathogen with an antibody titre to said pathogen being higher than 90% of human patient or carrier sera tested. 20
25. A method according to claim 24 wherein the antibody titre to said pathogen is higher than 95% of human patient or carrier sera tested.
26. A method according to any one of claims 1 to 25, wherein at least 10, individual antibody preparations are used in identifying said hyperimmune serum-reactive antigens. -113
27. A method according to claim 26 wherein at least 30 individual antibody preparations are used in identifying said hyperimmune serum-reactive antigens.
28. A method according to claim 26 wherein at least 50 individual antibody 5 preparations are used in identifying said hyperimmune serum-reactive antigens.
29. A method according to any one of said claims 1 to 28, wherein said relevant portion of said individual antibody preparations from said individual sera are at least 10 individual antibody preparations used in said screening. 10
30. A method according to claim 29 wherein at least 30 individual antibody preparations are used in said screening.
31. A method according to claim 29 wherein at least 50 individual antibody preparations are used in said screening.
32. A method according to any one of claims 1 to 31 wherein at least 20% of all 15 individual antibody preparations are used in said screening.
33. A method according to claim 32 wherein at least 30% of all individual antibody preparations are used in said screening.
34. A method according to claim 32 wherein at least 40% of all individual antibody preparations are used in said screening. 20
35. A method according to any one of claims 1 to 34, wherein said individual sera are selected by having an IgA titre against a lysate, cell wall components or recombinant proteins of said pathogen being above 4000 U.
36. A method according to claim 35 wherein the IgA titre is above 6000 U.
37. A method according to claim 35 wherein the IgA titre is above 12000 U. -114
38. A method according to any one of claims 1 to 37, wherein said pathogen is a Staphylococcus pathogen.
39. A method according to claim 38 wherein the Staphylococcal pathogen is a Staphylococcus aureus. 5
40. A method according to claim 38 wherein the Staphylococcal pathogen is a Staphylococcus epidermis.
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