MXPA00003674A - Enterococcus antigens and vaccines - Google Patents

Abstract

A majority of E. faecalis and E. faecium clinical isolates fall into two groups and three groups, respectively. Distinct antigens are associated with each of the five groups. The Enterococcus antigens are readily obtained from strains of E. faecalis and E. faecium, and can elicit production of protective antibodies. Accordingly, the antigens are useful for vaccines which protect against infection by clinically significant (pathogenic) Enterococcus isolates. The antigens and antibodies generated to the antigens are also useful in diagnostic assays.

Description

ANTIGENS AND VACCINES OF ENTEROCOCCUS BACKGROUND OF THE INVENTION The present invention relates to antigens of Enterococcus that are useful as vaccines, and with methods to obtain and use these antigens. The presence of Enterococcus infection increases more and more. Enterococcus strains are now responsible for 12 percent of all nosocomial infections among hospitalized patients and are the second most common organism isolated from patients with nosocomial infections. This increased prevalence of Enterococcus is due, at least in part, to the appearance of enterococcal strains that are resistant to antimicrobial agents and therefore difficult to treat with currently available antibiotics. The increase in antibiotic resistance among enterococci has increased the importance of alternative prophylactic and therapeutic approaches against enterococcal infections. Several groups have described polysaccharides isolated from Enterococcus. For example, lipoteichoic acids containing a central structure of 1,3-linked polyglycerophosphate have been isolated from "E. faecalis", which according to the current classification is E. faecalis. Position 2 is glycosylated with disaccharides or trisaccharides of glycosyl residues that can be esterified with alañile residues, and intracellular teichoic acid is denoted due to its predominance between the cell wall and the protoplast membrane. icken et al., J. Gen. Microbiol 33: 231-39 (1963). Pazur et al., J. Biol. Chem. 246: 1793-98 (1971), have isolated two other polysaccharides from the cell wall of E. faecalis strain N. One of these polysaccharides is characterized as a diheteroglycan consisting of glucose and D-galactose, while the other polysaccharide is said to be a tetraheteroglycan of 2-acetamide-2-deoxy-galactose, galactose, rhamnose, and glucose in a molar ratio of 1: 1: 2: 4. Bleiweis et al., J. Bacteriol. 94: 1381-87 (1967), have isolated a third polysaccharide from strain D76 of group D streptococci. The sugar composition of this material includes glucose, glucosamine, galactosamine, rhamnose, ribitol, and phosphorus; however, structural information is not provided. It is postulated that this material can be ribitol phosphate teichoic acid with sugar substituents attached. It has also been hypothesized that glucose and N-acetyl glucosamine are the possible components of the antigenic site. The antigen or Enterococcus antigens capable of producing protective antibodies will provide an effective means of preventing and / or treating Enterococcus infections. Although the technique describes a variety of Enterococcus antigens, not every antigen is effective as a vaccine. Undoubtedly, none of the materials reported in the literature has been shown to be effective in protecting against Enterococcus infection. Regarding this, even a description that an antigen is immunogenic, ie, that causes the production of antibodies, provides an insufficient basis for a conclusion that the antibodies are protective and that the antigen is therefore useful in a vaccine Finally, the technique suggests that serologically Enterococcus is a very diverse genus. This serological diversity suggests that a vaccine composed of a practical number of active components was not feasible. Maekawa et al., 'Microbiol. Immunol. 36: 671-681 (1992).

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide Enterococcus antigens, particularly E. faecalis and E. faecium antigens, which are capable of producing the production of protective antibodies. It is another objective to provide a vaccine containing Enterococcus antigens, more particularly a vaccine containing antigens of both E. faecalis and E. faecium.

It is another object to provide a hyperimmune globulin composition containing antibodies directed against Enterococcus antigens, particularly antigens of E. faecalis and E. faecium. In accordance with these and other objects according to the invention, an isolated Enterococcus antigen that reacts with antibodies is provided to cells of one of ATCC 202013, ATCC 202014, ATCC 202015, ATCC 202016, and ATCC 202017. More particularly, an antigen of Enterococcus is selected from the group consisting of an E. faecalis antigen comprising 2-acetamido-2-deoxy-glucose and rhamnose in an approximate molar ratio of 1: 2, an E. faecalis antigen comprising a repeated trisaccharide which comprises a 6-deoxy sugar, and an E. faecium antigen comprising 2-acetamido-2-deoxy-galactose and galactose. The antigen can be used in diagnostic tests or in immunotherapy methods. A conjugate is provided in which the antigen is covalently linked to an immunocarrier, preferably a non-toxic mutant strain, recombinantly produced from Pseudomonas aeruginosa exotoxin A or diphtheria toxoid. Antigen-carrier conjugates are useful in a vaccine, particularly a multivalent vaccine, for active immunotherapy. The vaccine antigen can also be used to produce immunoglobulin for passive immunotherapy, or in the production of monoclonal antibodies for diagnostic or therapeutic use. Other objects, features and advantages of the present invention will be apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating the preferred embodiments of the invention, will be by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to the inventors. skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1, 2 and 3 are nuclear magnetic resonance spectra for the Enterococcus antigens according to the invention.

DESCRIPTION OF THE PREFERRED MODALITIES Subsequently it has been discovered that most of the clinical isolates of E. faecalis fall into two groups, and that the majority of human clinical isolates of E. faecium fall into three groups. The discovery that most clinical isolates are characterized by only a few common antigens has not been widely known in the art, and allows the development of multivalent vaccines comprising a minimum number of active components that are protective against most of the clinical isolates. The characteristic antigens of each of the two groups of E. faecalis and the three groups of E. faecium can be extracted, purified and identified. With respect to this, an antigen is characteristic of a group or strain of bacteria if it is expressed by the bacteria in an amount sufficient to cause a significant immune response when a whole cell vaccine of the group or strain is injected into an animal, i.e. , an animal produces protective antibodies when it is injected. The characteristic antigens of E. faecalis are denoted herein as EFS1 and EFS2, and the characteristic antigens of E. faecium are EFM3, EFM4 and EFM5. These antigens are collectively referred to herein as "Enterococcus antigens". A strain of bacteria is called an EFS1 strain if a whole-cell vaccine strain produces a significant immune response primarily towards EFS1 when injected into a subject, and only a minor response for EFS2. Similarly, a strain of bacteria is called an EFS2 strain if the total cell vaccine of a strain produces a significant immune response primarily against EFS2 when injected into a subject, and so on. Although each of the major clinical groups of E. f ecalis and E. faecium express a different characteristic antigen can be easily extracted and purified in recoverable quantity, the groups can also express antigenic characteristics of the other groups in smaller amounts. However, when immunized with whole cells of one of the groups, the rabbits have a significant immune response only against the antigen characteristic of that group, and not with all or only a little of the smaller amounts of the antigens more characteristic of the others. groups, as shown by the absence of a precipitin band between the antibodies of the immunized rabbit and the purified antigenic characteristics of the other group. The degree to which the non-characteristic antigen is expressed by several cells varies. For example, the antiserum raised against a whole cell vaccine of an EFS1 strain contains antibodies to EFS2 in detectable amounts by both slide agglutination and by opsonophagocytosis assay (infra). The antiserum raised against a whole-cell vaccine of an EFS2 strain, on the other hand, does not contain antibodies that precipitate with EFS1. Enterococcus antigens are easily obtained from strains of E. faecalis and E. faecium, according to the protocols provided herein, and are capable of eliciting the production of protective antibodies when conjugated with immunocarriers. Therefore, they can be used to prepare vaccines that provide protection from humans and other mammals, eg, horses, cattle, pigs, dogs, and cats, against infection by clinically significant Enterococcus isolates. In this regard, a "clinically significant" isolate is one that is pathogenic in humans or in other mammals. Clinical isolates of E. faecalis and E. faecium can be clustered by slide agglutination experiments, using an antibody preparation suitable for agglutination. the bacteria . Slide agglutination experiments with E. faecalis show that most clinical isolates fall into two groups, EFS1 and EFS2. The antiserum generated against an EFS1 strain of E. faecalis agglutinates both EFS1 and EFS2 strains of E. faecalis. The reactivity of the antiserum generated against an EFS1 strain of E. faecalis can be absorbed with cells of strain EFS1. The absorbed serum can then continue to bind only one strain of EFS2. The antiserum generated against an EFS2 strain of E. faecalis agglutinates only EFS2 strains, and this reactivity can not be absorbed with EFS1 bacteria. As expected, uptake with cells from an EFS2 strain removes the reactivity of this antiserum with cells from an EFS2 strain. Although not wishing to be subject to the theory, it is hypothesized that strains EFS1 and EFS2 of E. faecalis contain EFS2 antigen, but that this antigen is covered and otherwise masked by EFS1 antigens in EFS1 cells.

Slide agglutination experiments with E. faecium show that most clinical isolates fall into three groups. The antiserum grown against two of the groups gives results similar to those obtained with E. faecalis. That is, the antiserum generated against a CFM3 strain of E. faecium binds both CFM3 and EFM5 bacteria, and the reactivity of this antiserum with an EFM3 strain can be absorbed with cells of an EFM3 strain. Absorbed serum agglutinates only EFM5 strains of bacteria. This uptake also causes a reduction in reactivity with cells of EFM5 strains, indicating that small amounts of EFM5 antigen are exposed on the surface of EFM3 cells. The antiserum generated against an EFM5 strain of E. faecium agglutinates only isolates in that group, and this reactivity can not easily be absorbed with cells of an EFM3 strain. As expected, the uptake with cells of an EFM5 strain reduces the reactivity of this antiserum with cells. Similarly, both EFM3 and EFM5 strains of E. faecium contain EFM5 antigen. Again, it is hypothesized that this antigen is converted or otherwise masked by the EFM3 antigen in EFM3 cells. The antiserum grown against an EFM4 strain of E. faecium is specific only to cells of EFM4 strains in slide agglutination experiments. This antiserum does not demonstrate cross-reactivity with EFM3 and EFM5 bacteria.

Antibodies raised against the whole-cell vaccine are generally not directed towards proteins on the cell surface, as shown by treatment of formalin-cleared cells with pronase E. When the removed cells are incubated for three hours at 37 ° C with 500 micrograms / milliliter of pronase E, and then tested in slide agglutination against whole-cell serum, there is no difference in the agglutination pattern observed with E. faecium and untreated E. faecalis, ie, the treatment Pronase does not remove the surface antigen against which antibodies are directed. Representatives of each of the two strains of E. faecalis and three of E. faecium have been deposited under the Budapest Treaty with the American Collection of Type Crops and have been given accession numbers 202013 (E. faecalis EFSl ), 202014 (E. faecalis EFS2), 202015 (E. faecium EFM3), 202016 (E. faecium EFM4), and 202017 (E. faecium EFM5) respectively. The antigen according to the invention can be isolated from deposited strains, or the deposited strains can be used to identify other strains expressing antigen according to the invention, from which the antigen can be extracted and purified according to the invention. with protocols described herein. The Enterococcus antigens according to the invention can be obtained in recoverable amount, and in substantially pure form, from their respective E. faecalis and E. faecium isolates cultured according to the protocols described herein. A "recoverable" amount in this respect means that the isolated amount of the antigen is detectable by a methodology less sensitive than radio-labeling, such as immunoassay, and can be subjected to subsequent manipulations involving the transfer of antigen per se into the solution. In an illustrative approach for obtaining antigens according to the present invention, a strain of E. faecalis or E. faecium is first cultured on a blood agar plate and then transferred to a starter flask with 2 percent NaCl / Columbia. . A fermentor of 80 liters containing the same medium with added 4 percent glucose is inoculated with the starter flask. The cells are fermented for 16-24 hours. The cells are centrifuged to separate the cells from the supernatant. Each of the five antigens can be extracted from either the cell paste or the supernatant. When the cell paste is used, the antigen is extracted by stirring the paste with cold 10 percent trichloroacetic acid (TCA), and then precipitated from the trichloroacetic acid solution by one or more sequential precipitations with cold ethanol / CaCl2. When the supernatant is used, the supernatant is directly subjected to precipitation with ethanol / cold CaCl2. This produces a crude antigen extract.

The crude extract is redissolved in water, dialyzed and lyophilized. The lyophilized material is dissolved in a regulator and purified by ion exchange chromatography. Fractions containing antigen can be pooled, dialyzed, concentrated and lyophilized, and size exclusion chromatography is used to further purify the antigen by size on a convenient column. Fractions containing antigen are pooled, concentrated, dialyzed and lyophilized. The purified antigen is analyzed by means of 1 H nuclear magnetic resonance spectroscopy. A composition of the Enterococcus antigen according to the present invention "consists essentially of" the antigen (s) or a conjugate of the antigen (s), which means that the composition does not contain any material that interferes with the elicitation of an immune response to the antigen. or the antigens, when the composition is used in a therapeutic context, or with the antigen / antibody coupling characteristic of a diagnostic test. In a preferred embodiment, the composition contains both antigens of E. faecalis and E. faecium. The antigens according to the invention are useful in the production of diagnostic tests for detecting the presence of Enterococcus antigen and / or anti-Enterococcus antibody in a sample. Either the Enterococcus antigen or the antibody specific to the j? Nterococcus antigen is mixed with a sample suspected of containing Enterococcus antibody or antigen and monitored for antigen-antibody binding. The antigen or antibody is labeled with a radioactive or enzyme label. In a preferred embodiment, the antigen or antibody is immobilized on a solid matrix so that the antigen or antibody is accessible to the complementary antibody or antigen that is contacted with a surface of the matrix. The sample is contacted with the surface of the matrix, and the surface is monitored to determine the antigen-antibody binding. For example, the antigen or antibody can be used in an enzyme-linked immunosorbent assay (ELISA), in which the antigen or antibody binds to a solid phase and an enzyme-antibody or enzyme-antigen conjugate is used to detect and / or quantify the antibody or antigen present in a sample. Alternatively, a Western blot assay can be used in which the solubilized and separated antigens are bound in microcellulose paper. The antibody is detected by an enzyme or conjugated anti-immunoglobulin (Ig) tag, such as a horseradish peroxidase-immunoglobulin conjugate by incubating the filter paper in the presence of a precipitable or detectable substrate. Western spotting assays have the advantage of not requiring a purity greater than 50 percent for the desired antigen.

Descriptions of the enzyme-linked immunosorbent assay and Western blotting techniques are found in Chapters 10 and 11 of Ausubel, et al. (Editors), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons (1988), the content of which is incorporated herein by reference. In a vaccine context, it is preferable to conjugate the antigen (s) with an immunocarrier, usually a polypeptide or protein, to improve the interaction between T and B cells for the induction of an immune response against the antigen. This is particularly important for vaccines intended for use in patients with reduced resistance. An immunocarrier enhances immunogenicity for both active immunization and for preparing high titre antisera in volunteers for passive immunization. Suitable immunocarriers according to the present invention include tetanus toxoid, diphtheria toxoid, Pseudomonas aeruginosa exotoxin A or its derivatives, non-toxic mutant strains produced recombinantly from exotoxin A, as described, for example, in Fattom et al., Inf. And Im. 61: 1023-32 (1993), as well as other proteins commonly used as immunocarriers. In order to conjugate the antigen with a carrier, the antigen is first derived. Several methods can be used to derive the antigen and bind it covalently to an immunocarrier. In a preferred method, hydroxyl groups in the antigens are activated using l-cyano-4-dimethylamino-pyridinium tetrafluoroborate, and the antigen is derived with a bifunctional spacer of 6 carbon atoms dihydrazide of adipic acid (ADH), in accordance with techniques known in the art, according to the method of Kohn et al., FEBS Lett. 154: 209: 210 (1993). This material is then linked to diphtheria toxoid (DT), recombinant exoprotein A from Pseudomonas aeruginosa (rEPA), tetanus toxoid (TT) or to another convenient carrier protein by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide ( EDAC). The resulting conjugates can be separated from the unreacted antigen by size exclusion chromatography. Preferably the antigen conjugate is administered with an adjuvant that promotes IgG subtype 2 protective antibodies. Typical adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), alum and other adjuvants suitable for human and animal use. . Dextran sulfate has been shown to be a potent stimulant of the IgG2 antibody against the surface antigens of the staphylococcal cell and is also convenient as an adjuvant. The induction of bacteremia in some mammals, for example, laboratory animals, requires extremely high numbers of organisms or some previous maneuvers to lower host resistance. However, in vitro phagocytosis can be studied as a correlation of protective immunity in vivo for humans and other mammals. In this model, the ability of specific antibodies of monoclonal and polyclonal antigens to opsonize strains of Enterococcus in vi tro is measured by phagocytosis, according to the method described in Kojima et al., Infect. Dis. Im a. 58: 2367-74 (1990). In vitro opsonophagocytosis assays are recognized in the field as being predictive of efficacy as a vaccine. For example, Fischer et al. Describe a correlation between the functional antibody determined with an opsonic assay in vi tro and with in vivo activity. J. Inf. Dis. 169: 324-9 (1994). Antibodies induced by Enterococcus antigens according to the invention are opsonic and facilitate phagocytosis of the specific type. Rabbit antibodies cultured against Enterococcus antigens are specifically capable of mediating opsonophagocytosis of the strains carrying the antigens by human polymorphonuclear leukocyte (PMN) cells in the presence of a human complement. In vitro phagocytosis assays thus indicate that antibodies to Enterococcus antigens are protective against infection by E. faecalis and E. faecium. A vaccine based on a combination of E. faecalis and E. faecium antigens can be used to protect against infection from most strains of clinical Enterococcus. The in vivo results are consistent with the results of opsonophagocytosis in vi tro trials. Antibodies conjugate EFS1 base bacteremia in mice stimulated with E. faecalis. The present invention also relates to the use of Enterococcus antigens to produce polyclonal antibodies or monoclonal antibodies (mouse or human) that bind or neutralize Enterococcus. Illustrative protocols for producing these antibodies are described in Chapter 11 of MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (Cold Spring Harbor, NY); in METHODS OF HYBRIDOMA FORMATION 257-271, Humana Press (Clifton, NJ); in Vitetta et al., Immunol. Rev. 62: 159-83 (1982); and in Raso, Immunol. Rev. 62: 93-117 (1982). The inoculum for the production of polyclonal antibody is typically prepared by dispersing the antigen-immunocarrier in a physiologically tolerable diluent such as saline, to form an aqueous composition. An immunostimulant amount of the inoculum, with or without adjuvant, is administered to a mammal, and the inoculated mammal is maintained for a period of time sufficient for the antigen to elicit the protection of anti-Enterococcus antigen antibodies. Booster doses of the antigen-immunocarrier can be used in individuals who are not already initiated to respond to the antigen. The antibodies can include antibody preparations from a variety of commonly used animals, such as goats, primates, donkeys, pigs, rabbits, horses, chickens, guinea pigs, rats, and mice, and even human antibodies after appropriate selection, fractionation and purification. Animal antisera can also be cultured by inoculating the animals with strains removed with formalin from E. faecalis and / or E. faecium by conventional methods, by bleeding the animals and recovering the serum or plasma for further processing. The antibodies induced in this way can be harvested and isolated to the desired degree by well-known techniques, such as by fractionation of alcohol and column chromatography, or by immunoaffinity chromatography; that is, by binding antigens with a chromatographic column packing such as Sephadex®, passing the antiserum through the column, thereby retaining the specific antibodies and separating other immunoglobulins (IgGs) and contaminants, and then recovering the purified antibodies by elution with a chaotropic agent, optionally followed by another purification, for example, by passage through a column or by joining group antigens of blood or other non-pathogenic species. This method can be preferred when the desired antibodies are isolated from the serum or plasma of humans that have developed an antibody titer against a pathogen in question, thereby ensuring the retention of antibodies that are capable of binding to the antigen. They can be used in preparations for passive immunization against strains of E. faecalis and E. faecium. The monoclonal antibody composition contains, with detectable limits, only one species of antibody that combines with the site capable of effectively binding to the Enterococcus antigen. Suitable antibodies in the monoclonal form can be prepared using conventional hybridoma technology. To form hybridomas from which the monoclonal antibody composition of the present invention is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from peripheral blood, lymph nodes or the spleen of a mammal hyperimmunized with the Enterococcus antigen. It is preferred that the myeloma cell line be of the same species as the lymphocytes. Splenocytes are typically fused with myeloma cells using 1500 polyethylene glycol. The fused hybrids are selected for their sensitivity to hypoxanthine-aminopterin-thymidine. Hybridomas secreting the antibody molecules of this invention can be identified using an enzyme-linked immunosorbent assay.

A Balb / C mouse spleen, human peripheral blood, lymph nodes or splenocytes are the preferred materials for use in the preparation of murine or human hybridomas. Suitable mouse myelomas for use in the present invention include hypoxanthine-aminopterin-thymidine (HAT) sensitive cell lines, a preferred myeloma is P3X63-Ag8.653. The preferred fusion partner for the production of human monoclonal antibodies is SHM-D33, a heteromyeloma available from ATCC, Rockville, Md. Under the designation CRL 1668. A monoclonal antibody composition of the present invention can be produced by starting a hybridoma culture. monoclonal comprising a nutrient medium containing a hybridoma that secretes antibody molecules of appropriate specificity. The culture is maintained under conditions and for a period of time sufficient for the hybridoma to secrete the antibody molecules in the medium. The antibody-containing medium is collected. The antibody molecules can be further isolated by well-known techniques. Useful media for the preparation of these compositions are also well known in the art and are commercially available, and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium supplemented with 20 percent fetal calf serum. An exemplary inbred mouse strain is the Balb / c. Other methods for preparing monoclonal antibody compositions are also contemplated, such as interspecies fusions, since it is primarily the specificity of the antigen of the antibodies that affects their usefulness in the present invention. Human lymphocytes obtained from infected individuals can be fused with human myeloma cell line to produce hybridomas that can be analyzed for the production of antibodies that recognize the Enterococcus antigen. More preferable in this respect, however, is a process that does not entail the use of a biological sample from an infected human subject. For example, a subject immunized with a vaccine as described herein can serve as a source for antibodies conveniently used in an antibody composition within the present invention. Purified monoclonal antibodies can be characterized by bacterial agglutination assays using a collection of clinical isolates, or by ELISA using plates coated with purified antigen. The monoclonal and polyclonal antibody compositions produced according to the present disclosure can be used by passive immunization to induce an immune response for the prevention or treatment of infection by strains of E. faecalis and E. faecium. In this regard, the antibody preparation can be a polyclonal composition. This polyclonal composition includes antibodies that bind to or to Enterococcus antigens. The polyclonal antibody component can be a polyclonal antiserum, preferably affinity purified, from an animal that has been stimulated with the Enterococcus antigen (s). Alternatively, a "technically engineered oligoclonal" mixture, which is a mixture of monoclonal antibodies to the Enterococcus antigens from both E. faecalis and E. faecium, can be used. In both types of mixture, it may be advantageous to chemically bind to each other to form a single polyspecific molecule capable of binding both E. faecalis and E. faecium antigens. One way to effect this binding is to make bivalent hybrid fragments F (ab ') 2 by mixing two different F (ab') 2 fragments produced, for example, by digestion of pepsin from two different antibodies, the reductive dissociation to form a mixture of Fab 'fragments, followed by the oxidative reformation of the disulfide bonds to reduce a mixture of F (ab') 2 fragments including hybrid fragments containing a specific Fab 'portion to each of the original antigens. Methods for preparing these hybrid antibody fragments are described in Feteanu, LABELED ANTIBODIES IN BIOLOGY AND MEDICINE 321-23, McGraw-Hill Int '1 Book Co. (1978); Nisonoff et al., Arch Biochem. Biphys, 93: 470 (1961); and Hammerling et al., J ". Exp. Med. 128: 1461 (1968), and in U.S. Patent No. 4,331,647. Other methods are known in the art to make bivalent fragments that are completely heteroespecific, e.g. , the use of bifunctional linkers to link dissociated fragments Recombinant molecules that incorporate light and heavy chains of an antibody are known, see, for example, the products of a methodology described by Boss et al., United States Patent Number 4,816,397 Analogous methods for producing recombinant or synthetic binding molecules having the characteristics of the antibodies are included in the present invention More than two different monospecific antibodies or antibody fragments can be linked using various linkers known in the art. produced in accordance with the present invention may include anticu whole ers, fragments of antibodies, or subfragments. The antibodies can be whole immunoglobulin of any kind, for example, IgG, IgM, IgA, IgD, IgE, chimeric antibodies or hybrid antibodies with dual or multiple antigen or epitope specificities, or fragments, for example, F (ab ') 2 , Fab ', Fab and the like, which include hybrid fragments, and additionally includes any immunoglobulin or any genetically engineered or synthetic or natural protein that acts as an antibody by binding to a specific antigen to form a complex. In particular, Fab molecules can be expressed and assembled in a genetically transformed host such as E. coli. A lambda vector system is available so as to express a population of Fab with a potential diversity equal to or exceeding that of the subject generating the predecessor antibody. See Huse, W.D., et al., Science 246: 1275-81 (1989). The antigen conjugate (s) according to the present invention can be the active ingredient in a composition, further comprising a pharmaceutically acceptable carrier for the active ingredient, which can be used as a vaccine to induce a cellular immune response and / or the In vivo production of antibodies that fight Enterococcus infection. In this regard, a pharmaceutically acceptable carrier is a material that can be used as a vehicle for administering a medicament because the material is inert or otherwise medically acceptable, as well as compatible with the active agent, in the context of vaccine administration. In addition to a convenient excipient, a pharmaceutically acceptable carrier may contain conventional vaccine additives such as diluents, adjuvants, antioxidants, preservatives and solubilizing agents. According to the present invention, this vaccine can be administered to a subject that is not already infected with E. faecalis or E. faecium, whereby a protective immune response to Enterococcus (humoral or cellular) is induced in that subject. Alternatively, a vaccine within the present invention can be administered to a subject in which infection by E. faecalis and / or E. faecium has already occurred but is at a sufficiently early stage that the immune response produced to the The vaccine effectively inhibits further spread of the infection. By another approach, a vaccine of the present invention can be administered to a subject who then acts as a source for immunoglobulin, produced in response to the stimulus from the specific vaccine containing antibodies directed against Enterococcus. A subject thus treated would donate plasma from which the immunoglobulin could then be obtained, via the conventional plasma fractionation methodology, and administered to another subject in order to impart resistance against or treat infection with Enterococcus. The immunoglobulins according to the invention are particularly useful for individuals with a compromised immune system, for individuals who undergo invasive procedures or where time does not allow the individual to produce their own antibodies in response to vaccination. Similarly, the monoclonal or polyclonal anti-Enterococcus antibodies produced in accordance with the present invention can be conjugated to an immunotoxin, and administered to a subject to whom infection with Enterococcus has already occurred but has not spread widely. To this end, the antibody material produced according to the present disclosure would be administered in a pharmaceutically acceptable carrier, as defined herein. The present invention is further described with reference to the following illustrative examples. Example 1; Fermentation of E. faecalis and E. faecium E. faecalis and E. faecium were grown in Columbia broth supplemented with 2 percent NaCl and 4 percent glucose in an 80-liter fermenter containing 60 liters of broth medium 37 ° C. The fermentation was started with one liter of a 16-hour sown crop. Cells were grown with shaking at 200 rpm for 16-24 hours. The cells to be used as a vaccine to prepare the whole cell antiserum were fixed with formalin overnight at room temperature. The cells for purification were processed by centrifugation at 14,500 x g and stored at -70 ° C until use. Approximately 50 g, 180 g, and 350 g of cell paste (net weight) were obtained from the 80 liter fermenter for EFSl, EFS2 and EFM3, respectively. Example 2: Preparation of whole cell antiserum Dead and fixed cells with formalin from two strains of E. faecalis and three strains of E. faecium cultured as in example 1 were adjusted to OD50mn = 1 and injected intravenously into rabbits. No adjuvant was used. The rabbits received ten injections and were bled at weekly intervals after the last injection and collected and whole-cell positive serum was collected. IgG was purified from whole cell serum by a G protein affinity column. Example 3: Agglutination studies with E. faecalis and E. faecium Immune rabbit serum obtained from rabbits immunized with the two strains dead and fixed formalin from E. faecalis and the three dead and fixed strains with E. faecium formalin were used to typify E. faecalis and E. faecium isolates by agglutination on slides. The antiserum was used to typify 67 clinical isolates of E. faecalis and 85 clinical isolates of E. faecium. 60 of the 67 isolates of E. faecalis (89.5 percent) reacted with antisera obtained by immunization of rabbits with ATCC 202013 cells13. Forty-one of the 85 clinical isolates of E. faecium reacted with antiserum obtained by immunization of rabbits with ATCC 202015 cells. Example 4; Antigen Purification Based on the results reported in Example 3, the antigens were isolated from ATCC 202013, ATCC 202014, and ATCC 202015, respectively. The antigens were extracted from the cell paste or from the supernatant obtained according to Example 1. Purification of the EFSI antigen This antigen was isolated from the cell paste of ATCC 202013. The antigen was extracted from the cell surface by stirring the cell paste ( 434 grams) with 10 percent cold TCA (1735 milliliters) at 4 ° C for a period of 48 hours. A clear supernatant was obtained by centrifugation. This supernatant was concentrated to one fifth of its original volume by evaporation under reduced pressure at 40 ° C. An equal volume of 95 percent ethanol was added to this solution and the solution was incubated at 4 ° C overnight. A small amount of precipitates was separated from the supernatant by centrifugation. Four other volumes of ethanol were added to the clear supernatant and a sufficient amount of 1 M CaCl 2 was added to make a final concentration of 10 mM CaCl 2 in the solution. The mixture was incubated again at 4 ° C overnight. The precipitates were recovered by centrifugation. The precipitates were redissolved in a minimum amount of 10 percent cold TCA and the previous ethanol precipitation steps of 50 percent and 80 percent were repeated to remove more impurities. The final precipitates recovered after the precipitation step of 80 percent ethanol were dissolved in water, dialysed against distilled water and lyophilized. This material was dissolved in 0.01 M Tris-HCl buffer, pH 7.0, and loaded onto a Q-Sepharose anion exchange column. The column was eluted sequentially with 0.01 M regulator Tris-HCl containing 0.1 and 0.2 M NaCl. The 0.2 M NaCl fraction was dialyzed against cold distilled water and lyophilized. The lyophilized material was further purified on a Sephacryl S-300 column and eluted with phosphate buffered saline (PBS) to obtain 258 milligrams of the final purified antigen. Purification of the antigen EFS2 The antigen was purified from the supernatant obtained from the fermentation of ATCC 202014. Crude material was obtained from the supernatant by a precipitation of 25-75% ethanol containing 10 mM CaCl 2. The pressure obtained from the precipitation of the 75 percent ethanol was partially purified by ion exchange chromatography on a Q-Sepharose column. The column was eluted sequentially with 0.01 M Tris-HCl buffer containing 0.2 and 0.5 M NaCl. The 0.5 M NaCl fraction was treated with protease overnight to remove contaminating proteins and subsequently further purified by size exclusion chromatography on a Sephacryl S-300 column. The reaction fractions with the whole cell antiserum to ATCC 202014 were deposited and then purified by a second ion exchange step on a Q-Sepharose column. The material was eluted with a 0.2-0.5 M linear sodium chloride gradient in Tris-HCl buffer at pH 7. The same material was also isolated from the cells following similar steps after the release of this material from the surface cell by chemical or enzymatic treatment. Purification of the EFM3 antigen The antigen was extracted from ATCC 202015 by shaking the cell paste with 10 percent cold TCA at 4 ° C for 48 hours, as described for EFSl. A clear supernatant was obtained by centrifugation. This supernatant was concentrated to one fifth of its original volume by evaporation under reduced pressure below 40 ° C. An equal volume of 95 percent ethanol was added to this solution and the solution was incubated at 4 ° C overnight. A small amount of precipitates was separated from the supernatant by centrifugation. Four other volumes of ethanol were added to the clear supernatant, and a sufficient amount of 1 M CaCl 2 was added to make a final concentration of 10 mM CaCl 2 in the solution. The mixture was incubated again at 4 ° C overnight. The precipitates were recovered by centrifugation. The precipitates were redissolved in a minimum amount of 10 percent cold TCA and the previous ethanol precipitation steps of 50 percent and 80 percent were repeated to remove more impurities. The final precipitates recovered after the ethanol precipitation step 80 percent were dissolved in 0.01 M Tris-HCl buffer, pH 7.0, and loaded onto a Q-sepharose anion exchange column. The column was eluted by the above regulator containing 0.1 M NaCl. The fraction was dialyzed against cold distilled water and lyophilized. The lyophilized material was further purified on a Sephacryl S-300 column and eluted with PBS. The fractions containing antigens were deposited, dialyzed against cold distilled water and lyophilized. Example 5: Characterization of antigen The antigens isolated in Example 4 were analyzed to determine their composition. EFSl comprises significant amounts of four sugars: 2-acetamido-2-deoxy-glucose, rhamnose, glucose and 2-acetamido-2-deoxy-galactose in an approximate molar ratio calculated from 1: 2: 2: 2. A biochemical analysis of the antigen is shown in Table 1.

Table 1. EFSl The material was also analyzed by H nuclear magnetic resonance spectroscopy. The most important downfield peaks observed were in d 5 .14 (s), 5.03 (s), 5.01 (d, J12 = 7.8 Hz), 4.78-4.67 ( complex). In the high-field region the spectrum showed resonances at 2.21 and 2.18 due to the N-acetyl groups, and at 1.43 (d, J5 = 6Hz) due to the 6-methyl group of the 6-deoxy sugar. A complete spectrum of the material is shown in Figure 1. The EFS2 antigen comprises a repeat trisaccharide, as determined by nuclear magnetic resonance H (Figure 2). One of the sugars is a 6-deoxi sugar. The constituent sugars do not contain N- or O-acetyl substituents. The antigens give a positive color in the sulfuric acid phenol test, as indicated in the presence of neutral sugar residues. The antigen was eluted from an anion exchange column with buffer containing > 0.20 M NaCl and moved towards the anode in rocket immunoelectrophoresis, which means that it contains acid groups. The sugar analysis of the EFM3 antigen revealed the presence of 2-acetamido-2-deoxy-galactose and galactose as the two main sugars. A complete biochemical analysis of the antigen is given in Table 2. Table 2. EFM3 The EFM3 antigen was also analyzed by nuclear magnetic resonance H spectroscopy and the full spectrum is shown in Figure 3. The characteristic resonances observed in the low field region were in d. 5.01 (s), 4.73 (d, J = 7.8 Hz), 4.6-4.55, (complex) and 4.52 (d, J = 7.8 Hz). The protons of the N-acetyl groups resonated in the high field region in d. 2.14, 2.20 and 2.21 EFS1 and EFS2 each reacted specifically in capillarity with antiserum obtained from rabbits immunized with a whole cell vaccine from ATCC 202013 and ATCC 202014, respectively. Additionally, EFS2 reacted with whole cell antiserum for ATCC 202013 in capillarity, due to the expression of lower amounts of EFS2 by EFSl strains. EFSl did not react with whole cell antiserum for ATCC 202014 in capillarity, and more sensitive techniques such as spot spotting were required to detect the presence of specific EFSI antibodies in rabbit serum immunized EFS2. The EFM3 antigen reacted specifically with sera from rabbits immunized with ATCC 202015 cells. The EFM3 antigen did not cross-react with specific antiserum obtained from rabbits immunized with either ATCC 202013 or ATCC 202014. In an in vi tro assay, the rabbit antiserum against ATCC 202013 specifically deposited the C3b component of the human complement on plates coated with EFSI antigen, and rabbit antiserum against ATCC 202015 specifically deposited human complement component C3b on plates coated with EFM3 antigen. There was no cross-deposit of C3b. Example 6: Preparation of antigen-immunocarrier conjugates A solution of antigen in water (10 milligrams / milliliter) was cooled in a water bath with ice. A cold aqueous solution (100 milligrams / milliliter) of l-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) was added to this solution, in an amount 1.2 times the volume of the above antigen solution. A 0.2 M volume of aqueous triethyl amine solution equal to the volume of the CDAP solution added above was added dropwise. After stirring the mixture for a total of three minutes at 4 ° C, an equal volume of 0.5 M ADH of solution prepared in 0.5 M sodium hydrogen carbonate was added. The above solution was stirred at 4 ° C overnight, dialyzed against cold distilled water and lyophilized to obtain the final derivative. The amount of ADH incorporated in the antigen was determined colorimetrically by assaying trinitrobenzene sulphonic acid (TNBS). Equal amounts of ADH-derivative polysaccharide and DT were dissolved in water to obtain the final concentration of 5-10 milligrams / milliliter of each component. This solution was adjusted to pH 5.6 using 0.1 M hydrochloric acid. To this solution was added a fresh prepared solution of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) in a minimum amount of water, in an amount four times in weight that of the antigen. The solution was stirred vigorously at room temperature and the pH of the solution was maintained at 5.6 using 0.1 M HCl. The reaction was stopped after one hour bringing the pH to 7.0 with 0.1 M NaOH. The pure conjugate was obtained by size exclusion chromatography of the Sephacryl S-100 column eluted with PBS. The amount of antigen and protein in the conjugates was determined by a phenol-sulfuric acid assay and the BCA assay using the corresponding antigen or BSA as standards, respectively. Example 7: Preparation of antiserum for conjugates and antigen-immunocarrier of Enterococcus White female rabbits from New Zealand were immunized by subcutaneous injection with 50 micrograms of an antigen-immunocarrier conjugate prepared according to Example 6 on days 0, 14 and 28. The first injection was given with an equal volume of complete Freund's adjuvant (CFA) and subsequent injections were given with incomplete Freund's adjuvant (IFA). The test bleeds taken from the rabbits were monitored to determine the presence of the precipitation of rabbit antibodies specific to the antigen with which they were immunized. Other injections were given as needed to strengthen the titration. The rabbits were bled to obtain high titre rabbit antiserum containing antibodies specific to the antigen with which they were immunized. The antiserum was used to evaluate the ability of specific antibodies to mediate opsonophagocytosis of the corresponding Enterococcus bacteria by HL-60 cells in in vi tro assays. Serum obtained from rabbits immunized with E. faecalis conjugated EFS1-DT had high titration and gave precipitates with EFSl in capillarity. The antibodies were able to mediate the elimination of cells carrying EFS1 by HL 60 in the presence of complement. Rabbits immunized with E. faecium DT antigen conjugate were also able to elicit antigen-specific antibodies. These antibodies gave precipitates with E. faecium antigen in capillarity. Example 8: Opsonophagocytosis assays in vi tro Bacteria were transferred from broth beds to a new Todd Hewitt thioglycollate agar plate. The plate was incubated for 18-20 hours at 37 ° C in 5 percent C02. The bacterium was scraped off the plate and suspended in two milliliters of sterile saline. The tube was centrifuged at 2000 rpm for 10 minutes at 25-35 ° C, and the supernatant was removed. The agglomerated bacteria were resuspended in two milliliters of sterile saline, and used to prepare a suspension of bacteria of an optical density of 0.1 to 540 nm. A 1: 100 diluted sample prepared from the bacterial suspension described above in RP-5 medium was used as a working broth of the bacterial solution. This bacterial preparation was tested against corresponding antisera for positive slide agglutination. The bacterial work broth was loaded into wells of microtitre plate with the appropriate dilution of RP-5 medium. PMN from HL-60 cells adjusted to a concentration of 1.0 x 10 7 cells per milliliter in RP-5 medium were obtained. The PMN cells were centrifuged at 1000 rpm for 10 minutes at 30-35 ° C. The agglomerated cells were resuspended in five milliliters of RP-5 medium and centrifuged at 1000 rpm for 10 minutes. The agglomerated cells were resuspended in one milliliter of RP-5 medium to produce a working concentration of 1 x 10 7 / ml. A human complement prepared from human serum was diluted 1:40 in RP-5 medium. The reaction mixture in the microtitre plate wells contained 50 μl of bacteria [106-107 cells / milliliter], 50 μl of diluted serum, 50 μl PMN [lxlO7 cells / milliliter] and 50 μl of complement [1:40 ], to produce a total volume of 200 μl. At time zero, a 20 μl sample of the reaction plate was serially diluted 10"1, 10"2, 10 ~ 3 and 10" 4. A 10 μl sample of each solution was plated on a tryptic soy agar plate (TSA). The TSA plates were incubated overnight at 37 ° C, 5 percent C02. After dilution at time zero, the reaction plate was incubated at 37 ° C for 90 minutes. The samples were mixed again. A 20 μl sample of the reaction plate was serially diluted 10"1, 10 ~ 2, 10 ~ 3 and 10" 4. A 10 μl sample of each dilution was plated onto TSA plates, which were then incubated overnight at 37 ° C, 5 percent CO. Bacterial colonies were counted for each dilution / sample / plate, and the percentage of bacterial elimination was calculated by the formula:% of deaths = No. colonies in TQ - No. colonies in T90 x 100 number of colonies in T0 Whole cell antiserum from rabbits immunized with ATCC 202013 as rabbit antibodies raised against EFSI-DT conjugates mediated opsonophagocytosis of E. faecalis by HL-60 in the presence of human complement. The opsonic activity of the anti-EFSI-DT conjugated rabbit antibodies was completely absorbed by the EFSI-DT conjugate. The opsonic activity of the whole cell antiserum was only partially absorbed with EFSI-DT conjugate, indicating that part of the opsonic activity of the whole cell antiserum arises from the antibodies directed towards an antigen other than DFS1. The opsonic activity of both the anti-EFSI-DT conjugate and the whole-cell antibodies were completely absorbed by ATCC 202013. Whole-cell antibodies cultured against ATCC 202014 did not react with EFS1 in agglutination assays, clearly indicating that EFS1 and EFS2 are different antigens. The whole cell antiserum of mice immunized with ATCC 202014 mediated opsonophagocytosis of E. faecalis by HL-60 in the presence of human complement. Whole-cell rabbit antibodies were also capable of mediating opsonophagocytosis of multiple E. faecalis isolates, including isolates of EFS1, by HL-60 in the presence of human complement. This opsonic activity could be absorbed by the EFS2. The EFSI-DT conjugate failed to absorb the opsonic activity of the whole cell antiserum of rabbits immunized with ATCC 202014. This observation suggests that the immune response elicited by EFS2 isolates in rabbits is against the EFS2 antigen and that the antibodies against the EFS2 antigen opsonophagocytosis can mediate multiple isolates of E. faecalis in the presence of human complement. Example 9: In vivo protection of E. faecalis stimulation mice by EFSI-DT conjugated antibodies A total of 42 ICR mice were divided into three groups with 15 mice in each of the first two groups and 12 mice in the third group. Mice from the first two groups were immunized with an intraperitoneal injection of 0.75 milligrams of rabbit immunoglobulin purified on G column protein obtained either from conjugated immunized mice (I-IgG) or from normal mice (N-IgG). The third group was immunized with PBS. Twenty-four hours later, all animals were stimulated by 5xl07 CFU of a strain of EFSl other than ATCC 202013, mixed with 5% pig mucin. Blood samples were taken from all the mice through their eyes at 6, 24, 48, 72 and 168 hours. These samples were plated on TSA plates and the levels of bacteremia in the mice were quantified by bacterial counts in the blood. The results are shown in Table 3. After 48 hours, only 17% of the mice were bacteremic in group I-IgG, whereas in the immunized NOIgG and PBS groups the corresponding number was 60% and 79%. percent, respectively. After 7 days, all the animals were sacrificed and their livers and kidneys were isolated and these organs were sampled for bacterial colonization. Few animals in the I-IgG group (4 of 30) had detectable bacterial colonization in the kidneys compared to the N-IgG group (9/30) or the PBS group (13/24). These observations clearly demonstrate antibodies specific to the EFS1 antigen are able to protect mice from bacterial challenge by E. faecalis.

Table 3

Claims (33)

1. An isolated Enterococcus faecalis antigen comprising 2-acetamido-2-deoxy-glucose, rhamnose, glucose and 2-acetamido-2-deoxy-galactose wherein 2-acetamido-2-deoxy-glucose and rhamnose are in a molar proportion of 1: 2

2. A carrier antigen conjugate, comprising an antigen as claimed in claim 1, covalently linked to an immunocarrier.

3. A carrier antigen conjugate as claimed in claim 2, wherein the immunocarrier is diphtheria toxoid or a non-toxic mutant recombinantly produced from Pseudomonas aeruginosa exotoxin A.

4. A composition consisting essentially of an antigen as claimed in claim 1, and a sterile, pharmaceutically acceptable carrier therefor.

5. A composition consisting essentially of an antigen-immunocarrier conjugate as claimed in claim 2, and a sterile, pharmaceutically acceptable carrier therefor.

6. A composition as claimed in claim 5, wherein the immunocarrier is diphtheria toxoid or a non-toxic mutant recombinantly produced from Pseudomonas aeruginosa exotoxin A.

7. A composition according to claim 4, comprising an additional antigen E. faecalis or an E. faecium antigen.

8. A composition as claimed in claim 7, wherein each of the antigens is conjugated with an immunocarrier.

9. A composition as claimed in claim 8, wherein each of the antigens is conjugated to the same immunocarrier.

10. A composition as claimed in claim 9, wherein the immunocarrier is a non-toxic, recombinantly produced mutant of Pseudomonas aeruginosa exotoxin A.

11. A multivalent vaccine comprising a conjugate of an immunocarrier with an antigen according to claim 1, and a conjugate of an immunocarrier with an additional antigen of E. faecalis or an E. faecium antigen and a sterile, pharmaceutically acceptable carrier for the same.

12. A method of immunotherapy comprising a step of administering to a subject an amount of immunostimulant of a composition as claimed in claim 5.

13. A method of immunotherapy comprising a step of administering to a subject an amount of immunostimulant of a composition as claimed in claim 8.

14. A method for preparing an immunotherapeutic agent against Enterococcus infection, comprising the steps of immunizing subjects with a composition according to claim 5, collecting plasma from immunized subjects. , and harvesting an immune globulin containing antibodies directed against Enterococci from the collected plasma. A method for preparing an immunotherapeutic agent against Enterococcus infection, comprising the steps of immunizing subjects with a composition according to claim 8, collecting plasma from said immunized subjects, and harvesting an immune globulin containing antibodies directed against Enterococci to from the collected plasma. 16. A diagnostic assay for detecting the presence of anti-Enterococcus antibody in a sample, comprising the steps of: mixing an Enterococcus antigen according to claim 1 with a sample suspected of containing antibody specific for Enterococci; and monitoring the mixture to determine the link between said antigen and the specific antibody for Enterococci in the sample. 17. A diagnostic assay as claimed in claim 16, wherein the antigen is immobilized on a solid matrix. 18. An isolated Enterococcus antigen according to claim 1, which reacts with ATCC 202013 antibodies. 19. A set for detecting the presence of anti-Snterococcus antibody in a sample, comprising: an Enterococcus antigen isolated in accordance with claim 1, wherein the antigen is labeled with a radioisotope tag or an enzyme tag; and instructions for carrying out the diagnostic assay comprising the steps of mixing the Enterococcus antigen with a sample suspected of containing Enterococcus-specific antibody and monitoring the mixture to determine the binding between the antigen and the Enterococcus-specific antibody in the sample . 20. An isolated Enterococcus faecalis antigen according to claim 1, comprising 2-acetamido-2-deoxy-glucose, rhamnose, glucose and 2-acetamido-2-deoxy-galactose in an approximate molar ratio calculated from 1: 2 : 2: 2 21. An isolated antigen according to claim 20, having a nuclear magnetic resonance spectrum as shown in Figure 1. 22. An isolated Enterococcus antigen according to claim 1, extracted and purified by a process comprising precipitation by alcohol and chromatography. 23. An isolated Enterococcus antigen selected from the group consisting of an E. faecalis antigen comprising 2-acetamido-2-deoxy-glucose, rhamnose, glucose and 2-acetamido-2-deoxy-galactose, wherein 2-acetamido -2-deoxy-glucose and rhamnose are in a molar ratio of 1: 2, an E. faecalis antigen comprising a trisaccharide repeat comprising a 6-deoxy sugar, and an E. faecium antigen comprising 2-acetamido -2-deoxy-galactose and galactose. 24. An isolated Enterococcus antigen according to claim 23, having a nuclear magnetic resonance spectrum as shown in Figure 1, 2 or 3. 25. An isolated Enterococcus antigen according to claim 23, extracted and purified by a process comprising alcohol precipitation and chromatography. 26. An antigen-carrier conjugate, comprising an antigen as claimed in claim 23 covalently linked to an immunocarrier. 27. An immunotherapy method comprising a step of delivering to a subject a quantity of antigen-carrier conjugate immunostimulant as claimed in claim 26. 28. A method for preparing an immunotherapeutic agent against Enterococcus infection, comprising the steps of immunizing subjects with an antigen-carrier conjugate as claimed in claim 26, collecting plasma from the immunized subjects, and harvesting an immunoglobulin containing antibodies directed against Enterococcus from the collected plasma. 29. A kit for detecting the presence of an anti-Enterococcus antibody in a sample, comprising: an Enterococcus antigen isolated according to claim 23, wherein the antigen is labeled with an isotope radio label or an enzyme tag; and instructions for carrying out a diagnostic test comprising the steps of mixing the Enterococcus antigen with a sample suspected of containing Enterococcus-specific antibody and monitoring the mixture to bind between the antigen and the Enterococcus-specific antibody in the sample. 30. An immunoglobulin containing antibodies directed against an Enterococcus antigen as claimed in claim 23. 31. An antibody to an Enterococcus antigen as claimed in claim 23. 32. A monoclonal antibody to an Enterococcus antigen as claimed in claims in claim 23. 33. A method of immunotherapy comprising a step of administering to a subject an immunoglobulin as claimed in claim 30.