Patent 6,756,361

A majority of E. faecalis and E. faecium clinical isolates fall into two groups and three groups, respectively. Distinct antigens are associate 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.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide Enterococcus antigens, particularly antigens from E. faecalis and E. faecium, that are capable of eliciting the production of protective antibodies.

It is a further object to provide a vaccine that contains Enterococcus antigens, more particularly a vaccine that contains antigens from both E. faecalis and E. faecium.

It is another object to provide a hyperimmune globulin composition that contains antibodies directed against Enterococcus antigens, particularly antigens from E. faecalis and E. faecium.

In accordance with these and other objects according to the invention, there is provided an isolated Enterococcus antigen that reacts with antibodies to cells from one of ATCC 202013, ATCC 202014, ATCC 202015, ATCC 202016, and ATCC 202017. More particularly, an isolated Enterococcus antigen is selected from the group consisting of an E. faecalis antigen comprising 2-acetamido-2-deoxy-glucose and rhamnose in an approximate 1:2 molar ratio, an E. faecalis antigen comprising a trisaccharide repeat 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 assays or in immunotherapy methods. A conjugate in which the antigen is covalently bonded to an immunocarrier, preferably a recombinantly-produced, non-toxic mutant strain of Pseudomonas aeruginosa exotoxin A or diphtheria toxoid, is provided. The antigen-carrier conjugates are useful in a vaccine, particularly a multivalent vaccine, for active immunotherapy. The antigen or vaccine also can be used to produce immune globulin 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 become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF PREFERRED EMBODIMENTS

It surprisingly has been discovered that the majority of E. faecalis clinical isolates fall into two groups, and that the majority of E. faecium human clinical isolates fall into three groups. The discovery that the majority of clinical isolates are characterized by only a few common antigens is unheralded in the art, and permits development of multivalent vaccines that comprise a minimal number of active components yet are protective against the majority of clinical isolates.

Antigens characteristic of each of the two groups of E. faecalis and three groups of E. faecium can be extracted, purified and identified. In this regard, an antigen is characteristic of a group or strain of bacteria if it is expressed by the bacteria in a quantity 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 so injected.

The E. faecalis characteristic antigens are denoted herein as EFS1 and EFS2, and the E. faecium characteristic antigens as EFM3, EFM4 and EFM5. These antigens are referred to collectively herein as "Enterococcus antigens." A strain of bacteria is called an EFS1 strain if a whole cell vaccine of the strain produces a significant immune response primarily toward EFS1 when injected into a subject, and only a minor response to EFS2. Similarly, a strain of bacteria is called an EFS2 strain if a whole cell vaccine of the strain produces a significant immune response primarily toward EFS2 when injected into a subject, and so forth.

While each of the major clinical groups of E. faecalis and E. faecium expresses a different characteristic antigen that may be readily extracted and purified in recoverable amount, the groups also may express antigen characteristic of the other group(s) in minor amounts. However, when immunized with whole cells from one of the groups, rabbits mount a significant immune response only toward the characteristic antigen of that group, and not at all or only poorly to the minor amounts of the antigen most characteristic of the other group(s), as shown by the absence of a precipitin band between antibodies from the immunized rabbit and purified antigen characteristic of the other group.

The degree to which a non-characteristic antigen is expressed by cells varies. For example, antisera generated against a whole cell vaccine of an EFS1 strain contains antibodies to EFS2 in amounts, detectable both by slide agglutination and by opsonophagocytosis assay (infra). Antisera generated against a whole cell vaccine of an EFS2 strain, on the other hand, does not contain antibodies that precipitate with EFS1.

The Enterococcus antigens are readily obtained from strains of E. faecalis and E. faecium, pursuant to protocols provided herein, and are capable of eliciting production of protective antibodies when conjugated to immunocarriers. They therefore can be used to prepare vaccines that provide protection of humans and other mammals, e.g., horses, cattle, swine, dogs, and cats, against infection by clinically significant isolates of Enterococci. In this regard, a "clinically significant" isolate is one that is pathogenic in humans or other mammals.

E. faecalis and E. faecium clinical isolates can be grouped by slide agglutination experiments, using an appropriate antibody preparation for agglutination of bacteria. Slide agglutination experiments with E. faecalis show that the majority of clinical isolates fall into two groups, EFS1 and EFS2. Antisera generated against an EFS1 strain of E. faecalis agglutinates both EFS1 and EFS2 strains of E. faecalis . The reactivity of antisera generated against an EFS1 strain of E. faecalis can be absorbed out with cells from the EFS1 strain. The absorbed sera may then continue to agglutinate only an EFS2 strain.

Antisera generated against an EFS2 strain of E. faecalis agglutinates only EFS2 strains, and this reactivity cannot be absorbed out with EFS1 bacteria. As expected, absorption with cells from an EFS2 strain removes the reactivity of this antisera with cells from an EFS2 strain. While not wishing to be bound by theory, it is hypothesized that EFS1 and EFS2 strains of E. faecalis contain EFS2 antigen, but that this antigen is covered or otherwise masked by EFS1 antigens on EFS1 cells.

Slide agglutination experiments with E. faecium show that the majority of clinical isolates fall into three groups. Antisera raised against two of the groups give results similar to that obtained with E. faecalis . That is, antisera generated against a EFM3 strain of E. faecium agglutinates both EFM3 and EFM5 bacteria, and the reactivity of this antisera with an EFM3 strain can be absorbed out with cells from an EFM3 strain. The absorbed sera then agglutinates only EFM5 strains of bacteria. This absorption also causes a reduction in reactivity with cells from EFM5 strains, indicating that small amounts of EFM5 antigen is exposed on the surface of EFM3 cells.

Antisera generated against a EFM5 strain of E. faecium agglutinates only isolates in that group, and this reactivity cannot readily be absorbed out with cells of an EFM3 strain. As expected, absorption with cells from an EFM5 strain reduces the reactivity of this antisera with cells. Similarly EFM3 and EFM5 strains of E. faecium both contain EFM5 antigen. Again, this antigen is hypothesized to be covered or otherwise masked by EFM3 antigen on EFM3 cells.

Antisera raised against an EFM4 strain of E. faecium is specific only to cells of EFM4 strains in slide agglutination experiments. This antisera demonstrates no cross reactivity with EFM3 and EFM5 bacteria.

Antibodies generated against the whole cell vaccine generally are not directed toward proteins on the cell surface, as shown by treatment of formalin-killed cells with pronase E. When killed cells are incubated for 3 hours at 37^oC. with 500 .mu./ml pronase E, and then tested in slide agglutination against whole cell sera, there is no difference in the agglutination pattern from that observed with untreated E. faecium or E. faecalis , i.e., the pronase treatment does not remove the surface antigen against which the antibodies are directed.

Representatives of each of the two E. faecalis and three E. faecium strains have been deposited under the Budapest Treaty with the American Type Culture Collection, and have been given Accession Nos. 202013 (E. faecalis EFS1), 202014 (E. faecalis EFS2), 202015 (E. faecium EFM3), 202016 (E. faecium EFM4), and 202017 (E. faecium EFM5) respectively. Antigen according to the invention can be isolated from the deposited strains, or the deposited strains can be used to identify other strains which express antigen according to the invention, from which antigen may be extracted and purified in accordance with protocols described herein.

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 pursuant to the protocols described herein. A "recoverable" amount in this regard means that the isolated amount of the antigen is detectable by a methodology less sensitive than radiolabeling, such as immunoassay, and can be subjected to further manipulations involving transfer of the antigen per se into solution.

In an illustrative approach to obtaining antigen according to the present invention, a strain of E. faecalis or E. faecium first is grown on a blood agar plate and then transferred to a 2% NaCl/Columbia starter flask. An 80-liter fermentor that contains the same medium with added 4% glucose is inoculated with the starter flask. Cells are fermented for 16-24 hours. The cells were centrifuged to separate the cells from the supernatant. Each of the five antigens can be extracted from either cell paste or supernatant.

When cell paste is used, antigen is extracted by stirring the paste with cold 10% trichloroacetic acid (TCA), and then precipitated from the TCA solution by one or more sequential precipitations with cold ethanol/CaCl₂. When supernatant is used, the supernatant is subjected directly to precipitation with cold ethanol/CaCl₂. This produces a crude antigen extract.

The crude extract is redissolved in water, dialyzed and lyophilized. The lyophilized material is dissolved in buffer and purified by ion exchange chromatography. Fractions containing antigen can be pooled, dialyzed, concentrated, and lyophilized, and size exclusion chromatography is used to purify the antigen further by size on a suitable column. Antigen-containing fractions are pooled, concentrated, dialyzed and lyophilized. Purified antigen is analyzed by ¹ H-NMR 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 elicitation of an immune response to the antigen(s) when the composition is used in a therapeutic context, or with the antigen-antibody coupling characteristic of a diagnostic assay. In a preferred embodiment, the composition contains both E. faecalis and E. faecium antigens.

The antigens according to the invention are useful in the production of diagnostic assays for detecting the presence of Enterococcus antigen and/or anti-Enterococcus antibody in a sample. Either the Enterococcus antigen or antibody specific to the Enterococcus antigen is mixed with a sample suspected of containing Enterococcus antibody or antigen and monitored for antigen-antibody binding. The antigen or antibody is labelled with a radioactive or enzyme label. In a preferred embodiment, the antigen or antibody is immobilized on a solid matrix such that the antigen or antibody is accessible to complementary antibody or antigen contacting a surface of the matrix. The sample then is brought into contact with the surface of the matrix, and the surface is monitored for antigen-antibody binding.

For example, the antigen or antibody can be used in an enzyme-linked immunosorbent assay (ELISA), in which antigen or antibody is bound to a solid phase and an enzyme-antibody or enzyme-antigen conjugate is used to detect and/or quantify antibody or antigen present in a sample. Alternatively, a western blot assay can be used in which solubilized and separated antigens are bound to nitrocellulose paper. The antibody then is detected by an enzyme or label-conjugated anti-immunoglobulin (Ig), such as horseradish peroxidase-Ig conjugate by incubating the filter paper in the presence of a precipitable or detectable substrate. Western blot assays have the advantage of not requiring purity greater than 50% for the desired antigen. Descriptions of ELISA and western blot techniques are found in Chapters 10 and 11 of Ausubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons (1988), the contents of which are hereby incorporated by reference.

In a vaccine context, it is preferable to conjugate the antigen(s) to 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 both for active immunization and for preparing high-titered 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, recombinantly-produced non-toxic mutant strains of exotoxin A, as described, for example, in Fattom et al., Inf. and Imm. 61: 1023-32 (1993), as well as other proteins commonly used as immunocarriers.

In order to conjugate the antigen to a carrier, the antigen is first derivatized. Various methods can be used to derivatize antigen and covalently link it to an immunocarrier. In a preferred method, hydroxyl groups on the antigen are activated using 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate, and the antigen is then derivatized with a six carbon bifunctional spacer adipic acid dihydrazide (ADH), according to 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 another suitable carrier protein by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The resulting conjugates can be separated from unreacted antigen by size exclusion chromatography.

Preferably the antigen conjugate is administered with an adjuvant which promotes the protective IgG subtype 2 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 stimulator of IgG₂ antibody against staphylococcal cell surface antigens, and also is suitable as an adjuvant.

Induction of bacteremia in some mammals, e.g., laboratory animals, requires extremely high numbers of organisms or some previous maneuver to lower the host resistance. In vitro phagocytosis, however, can be studied as a correlate of protective immunity in vivo for humans and other mammals. In this model, the ability of antigen-specific monoclonal and polyclonal antibodies to opsonize Enterococcus strains in vitro is measured by phagocytosis, according to the method described in Kojima et al., Infect. Dis. Immun. 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. discloses a correlation between functional antibody determined with an in vitro opsonic assay and in vivo activity. J. Inf. Dis. 169: 324-9 (1994).

Antibodies induced by the Enterococcus antigens according to the invention are opsonic and facilitate type-specific phagocytosis. Rabbit antibodies raised against the Enterococcus antigens are able specifically to mediate the opsonophagocytosis of the strains carrying the antigens by human polymorphonuclear leukocytes (PMN) cells in the presence of human complement. The in vitro phagocytosis assays thus indicate that antibodies to the 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 the majority of clinical Enterococcus strains.

In vivo results are consistent with results of in vitro opsonophagocytosis assays. Antibodies to EFS1 conjugate lowered bacteremia in mice challenged with E. faecalis.

The present invention also relates to the use of the Enterococcus antigen(s) to produce polyclonal antibodies or monoclonal antibodies (mouse or human) that bind to 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, N.Y.); in METHODS OF HYBRIDOMA FORMATION 257-271, Humana Press (Clifton, N.J.); in Vitetta et al., Immunol. Rev. 62: 159-83 (1982); and in Raso, Immunol. Rev. 62: 93-117 (1982).

Inoculum for polyclonal antibody production typically is prepared by dispersing the antigen-immunocarrier in a physiologically-tolerable diluent such as saline, to form an aqueous composition. An immunostimulatory amount of inoculum, with or without adjuvant, is administered to a mammal, and the inoculated mammal then is maintained for a time period sufficient for the antigen to induce protecting anti-Enterococcus antigen antibodies. Boosting doses of the antigen-immunocarrier may be used in individuals that are not already primed to respond to the antigen.

Antibodies can include antibody preparations from a variety of commonly used animals, such goats, primates, donkeys, swine, rabbits, horses, hens, guinea pigs, rats, and mice, and even human antibodies after appropriate selection, fractionation and purification. Animal antisera may also be raised by inoculating the animals with formalin-killed strains of E. faecalis and/or E. faecium by conventional methods, bleeding the animals and recovering serum or plasma for further processing.

The antibodies induced in this fashion can be harvested and isolated to the extent desired by well known techniques, such as by alcohol fractionation and column chromatography, or by immunoaffinity chromatography; that is, by binding antigen to a chromatographic column packing like Sephadex.TM., passing the antiserum through the column, thereby retaining specific antibodies and separating out other immunoglobulins (IgGs) and contaminants, and then recovering purified antibodies by elution with a chaotropic agent, optionally followed by further purification, for example, by passage through a column of bound blood group antigens or other non-pathogen species. This procedure may be preferred when isolating the desired antibodies from the sera or plasma of humans that have developed an antibody titer against the pathogen in question, thus assuring the retention of antibodies that are capable of binding to the antigen. They can then be used in preparations for passive immunization against strains of E. faecalis and E. faecium.

A monoclonal antibody composition contains, within detectable limits, only one species of antibody combining site capable of effectively binding to the Enterococcus antigen. Suitable antibodies in monoclonal form can be prepared using conventional hybridoma technology.

To form hybridomas from which a 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 from the same species as the lymphocytes. Splenocytes are typically fused with myeloma cells using polyethylene glycol 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas secreting the antibody molecules of this invention can be identified using an ELISA.

A Balb/C mouse spleen, human peripheral blood, lymph nodes or splenocytes are the preferred materials for use in preparing murine or human hybridomas. Suitable mouse myelomas for use in the present invention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT) cell lines, a preferred myeloma being P3X63-Ag8.653. The preferred fusion partner for human monoclonal antibody production 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 initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules then can be isolated further by well known techniques.

Media useful for the preparation of these compositions are both well known in the art and 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% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of preparing monoclonal antibody compositions are also contemplated, such as interspecies fusions, since it is primarily the antigen specificity of the antibodies that affects their utility in the present invention. Human lymphocytes obtained from infected individuals can be fused with a human myeloma cell line to produce hybridomas which can be screened for the production of antibodies that recognize the Enterococcus antigen. More preferable in this regard, 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 suitably 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 description 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. Such a polyclonal composition includes antibodies that bind to the Enterococcus antigen(s). The polyclonal antibody component can be a polyclonal antiserum, preferably affinity purified, from an animal which has been challenged with the Enterococcus antigen (s). Alternatively, an "engineered oligoclonal" mixture may be used, which is a mixture of monoclonal antibodies to the Enterococcus antigens from both E. faecalis and E. faecium.

In both types of mixtures, it can be advantageous to link antibodies together chemically to form a single polyspecific molecule capable of binding to both E. faecalis and E. faecium antigens. One way of effecting such a linkage is to make bivalent F(ab')₂ hybrid fragments by mixing two different F (ab')₂ fragment s produced, e.g., by pepsin digestion of two different antibodies, reductive cleavage to form a mixture of two fragments, followed by oxidative reformation of the disulfide linkages to produce a mixture of F(ab')₂ fragments including hybrid fragments containing a Fab' portion specific to each of the original antigens. Methods of preparing such hybrid antibody fragments are disclosed in Feteanu, LABELED ANTIBODIES IN BIOLOGY AND MEDICINE 321-23, McGraw-Hill Int'l Book Co. (1978); Nisonoff et al., Arch Biochem. Biophys. 93: 470 (1961); and Hammerling et al., J. Exp. Med. 128: 1461 (1968); and in U.S. Pat. No. 4,331,647.

Other methods are known in the art to make bivalent fragments that are entirely heterospecific, for example, use of bifunctional linkers to join cleaved fragments. Recombinant molecules are known that incorporate the light and heavy chains of an antibody. See, for instance, the products of a methodology described by Boss et al., U.S. Pat. No. 4,816,397. Analogous methods of producing recombinant or synthetic binding molecules having the characteristics of 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.

An antibody component produced in accordance with the present invention can include whole antibodies, antibody fragments, or subfragments. Antibodies can be whole immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD, IgE, chimeric antibodies or hybrid antibodies with dual or multiple antigen or epitope specificities, or fragments, e.g., F(ab')₂, Fab', Fab and the like, including hybrid fragments, and additionally includes any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like 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 like E. coli. A lambda vector system is available thus to express a population of Fab's with a potential diversity equal to or exceeding that of subject generating the predecessor antibody. See Huse, W. D., et al., Science 246: 1275-81 (1989).

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 production in vivo of antibodies which combat 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 suitable excipient, a pharmaceutically acceptable carrier can contain conventional vaccine additives like diluents, adjuvants, antioxidants, preservatives and solubilizing agents.

Pursuant to the present invention, such a vaccine can be administered to a subject not already infected with E. faecalis or E. faecium, thereby to induce a Enterococcus-protective immune response (humoral or cellular) in that subject. Alternatively, a vaccine within the present invention can be administered to a subject in which E. faecalis and/or E. faecium infection already has occurred but is at a sufficiently early stage that the immune response produced to the vaccine effectively inhibits further spread of infection.

By another approach, a vaccine of the present invention can be administered to a subject who then acts as a source for immune globulin, produced in response to challenge from the specific vaccine that contains antibodies directed against Enterococcus. A subject thus treated would donate plasma from which immune globulin would then be obtained, via conventional plasma-fractionation methodology, and administered to another subject in order to impart resistance against or to treat Enterococcus infection. Immune globulins according to the invention are particularly useful for immune-compromised individuals, for individuals undergoing invasive procedures or where time does not permit the individual to produce his own antibodies in response to vaccination.

Similarly, monoclonal or polyclonal anti-Enterococcus antibodies produced according to the present invention can be conjugated to an immunotoxin, and administered to a subject in whom Enterococcus infection has already occurred but has not become widely spread. To this end, antibody material produced pursuant to the present description would be administered in a pharmaceutically acceptable carrier, as defined herein.

Claim 1 of 17 Claims

What we claim is:

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 1:2 molar ratio.

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