A novel vaccine for immunizing animals against Staphylococcus aureus-induced mastitis is disclosed. The vaccine is comprised of whole killed cells of S. aureus in a dosage effective to immunize an animal against the organism, in combination with a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a vaccine comprised of a mixture of killed whole cells from a particular combination of S. aureus strains, the combination having been found to both protect and to ameliorate bovine mastitis, a method of producing that vaccine and a method of using the vaccine to protect individuals from mastitis and to ameliorate the disease.

A serotyping system for S. aureus capsule antigens was developed by Karakawa and Vann and consists of eight serotypes (1982. In: Seminars in Infectious Diseases, Bacterial Vaccines, Vol. IV, Weinstein et al, Eds., Thieme-Stratton, NY, Pages 285-293). Poutrel et al. have reported on the prevalence of capsular polysaccharide Types 5 and 8 among S. aureus isolates from cow, goat, and ewe milk in France. In their study, the primary serotypes found were Types 5 (51%) and 8 (18%); 31% were non-typeable (1988. J. Clin. Microbiol. 26: 38-40). In an attempt to produce a universal S. aureus vaccine, our laboratory surveyed dairy herds in the major milk producing areas of the United States through the cooperation of the state diagnostic laboratories in the major dairy producing states. We characterized S. aureus serotypes isolated from cases of bovine mastitis obtained from veterinary diagnostic laboratories that service 44% of the dairy cattle in the United States (Guidry et al., 1997, supra). In our studies, serotype 5 accounted for 18% of the isolates and serotype 8 for 23%; however, 59% of the isolates were not typeable with either Type 5 or 8 antisera. This survey revealed that three serotypes accounted for 100% of the S. aureus isolated from cases of mastitis in dairy cattle. A similar survey revealed that these three serotypes accounted for 95% of the S. aureus isolated from cases of bovine mastitis in Korea (Han et al. 2000. J. Vet Med. Sci. 62: 1331-1333). Later studies (Guidry et al. 1998. Am. J. Vet. Res. 59:1537-1539) confirmed that all strains not typeable with either Type 5 or Type 8 antisera were found to be Type 336 by use of direct cell agglutination and immunoprecipitation of cell extracts with antiserum 336. The current vaccine is composed of representatives of each of these three serotypes. The vaccine of this invention is effective for controlling Staphylococcal infections in a variety of animals when administered thereto. Without being limited thereto, the vaccine is especially beneficial for the treatment of ruminants, particularly bovine, sheep, and goats. To our knowledge there is no comparable vaccine available.

Fattom (2001. U.S. Pat. No. 6,294,177) typed human clinical isolates obtained from various sources and reported that approximately 60% of human isolates were Type 8, approximately 30% were Type 5, and nearly all of the remaining 10% were Type 336. Fattom further obtained an additional 27 human isolates that were not typeable as either Type 5 or Type 8 and determined that they too were typeable as Type 336. Utilizing a representative strain of S. aureus isolated from human clinical samples, a strain that carries the 336 antigen and that has been deposited with the ATCC as accession number 55804, Fattom reported that 28.4% of 102 bovine mastitis isolates from Europe were Type 336 and 61.6% of bovine mastitis isolates from the United States were 336 positive. U.S. Pat. No. 6,294,177 (Fattom) describes a vaccine that comprises cells, cell lysates, or cell derivatives which have the characteristic of specifically binding with antibodies to S. aureus Type 336 deposited under ATCC 55804 and a vaccine which additionally comprises cells, cell lysates, or cell derivatives of S. aureus which carry Type 5 antigen and cells, cell lysates, or cell derivative of S. aureus which carry Type 8 antigen. However, in the Fattom studies, treatment and prevention of veterinary S. aureus infections with the '177 vaccines comprising ATCC 55804-based Type 336 were not reported.

The present invention relates to our strategy for generating an effective vaccine for preventing and treating veterinary S. aureus infections; namely, that the vaccine comprise capsule antigens and, importantly, adherence antigens. As described above, cell wall antigens of S. aureus are involved in adherence to epithelial cells. Adherence is followed by encapsulation and toxin production. The majority of unencapsulated strains of S. aureus express the 336 antigen as detected by direct cell agglutination. Capsulated strains of S. aureus also express the 336 antigen on their cell wall; however, different growth conditions are required for detection of the 336 antigen on capsulated strains because the capsule impedes the agglutination reaction. These facts, taken together, indicate that most S. aureus strains, whether capsulated or unencapsulated, can be shown to be positive for the 336 cell wall antigen when grown under conditions where expression of cell wall antigens can be detected.

Strains identified as unencapsulated strains are usually found to have the characteristic of adhering to epithelial cells. Interestingly, the unencapsulated S. aureus isolate identified as ATCC 55804 and typed as 336 positive exhibits little adherence to bovine mammary epithelial cells. However, the present invention teaches a trivalent whole cell vaccine comprising cells of strains expressing Type 5 and Type 8 capsular antigens and cells of a 336 positive unencapsulated S. aureus strain which is adherent, i.e., Smith Compact, deposited at the American Type Culture Collection, ATCC, 10801 University Blvd., Manassas, Va. 20110 on Mar. 5, 2004 and identified as ATCC BAA-934. The unencapsulated strain of the trivalent vaccine of the invention, being adherent, is therefore not the unencapsulated strain ATCC 55804. Because the trivalent vaccine of the invention encompasses adherent unencapsulated cells together with cells of strains expressing Type 5 and Type 8 capsular antigens, the trivalent vaccine of the invention has the property of being an effective vaccine for preventing and treating mastitis. Thus, the vaccine of the invention comprises three types of cells, cell lysates, or cell derivatives of Staphylococcus aureus: (1) a type which expresses S. aureus Type 5 capsular antigen, (2) a type which expresses S. aureus Type 8 capsular antigen, and (3) a type which is unencapsulated, which adheres to epithelial cells, which carries an antigen that specifically binds with antibodies to S. aureus Type 336, and which is not a cell, cell lysate, or cell derivative identified as a ATCC 55804, and a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient, and/or adjuvant.

The cells of this invention can be used as immunogens in vaccines for vaccination against S. aureus. The vaccines can be used to prevent or reduce susceptibility to diseases caused by S. aureus. While the vaccine is effective for eliciting antibody production in a variety of animals, such as bovine, ovine and caprine animals, the vaccine is particularly preferred for the treatment of bovine animals.

The inventive vaccine is a killed cell preparation comprising a mixture of three cell types. Propagation of the three cell types comprising the vaccine in preparation for inactivation by formalin may be accomplished using conventional techniques and culture media known in the art. A variety of both solid and liquid culture media may be suitable for use herein. Following culture on solid media, the cells may be harvested by suspension in a small amount of a suitable carrier, such as PBS. When using liquid culture media, the cells may be optionally concentrated, for example, by filtration or centrifugation to obtain a high density suspension of cells.

Following inactivation, the killed cells of the vaccine can be mixed, conjugated or fused with other antigens, including B or T cell epitopes of other antigens. In formulating the vaccine compositions with the cells, alone or in the various combinations described, the immunogen is adjusted to an appropriate concentration and formulated in a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient, and/or with any suitable vaccine adjuvant and/or vaccine stabilizer. Typical pharmaceutically acceptable carriers are physiological saline, mineral oil, vegetable oils, aqueous sodium caroboxymethyl cellulose, or aqueous polyvinylppyrrolidone. Typical stabilizers are, for example, sucrose, an alkali metal hydrogen phosphate salt, glutamate, serum albumin, gelatin, or casein. The stabilizer may be any one or more of the foregoing. Suitable adjuvants include, but are not limited to: surface active substances, e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, methoxyhexadecylgylcerol, and pluronic polyols; polyamines, e.g., pyran, dextran-sulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum hydroxide, aluminum phosphate, etc. and immune stimulating complexes. The adjuvant may be, for example, alum or a composition containing a vegetable oil, isomannide monooleate and aluminum mono-stearate. Other preferred adjuvants include microparticles or beads of biocompatible matrix materials. The killed cells may be incorporated into microparticles or microcapsules to prolong the exposure of the antigenic material to the subject animal and hence protect the animal against infection for long periods of time. The immunogen may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation.

Also part of this invention is a composition that comprises the cells of this invention; and a carrier, preferably a biologically-acceptable carrier, and more preferably a pharmaceutically-acceptable carrier. Typical carriers are aqueous carriers such as water, buffered aqueous solutions, aqueous alcoholic mixtures, and the like. Compositions comprising carriers that are for pharmaceutical use, particularly for use in humans, comprise a carrier that is pharmaceutically-acceptable. Examples of such carriers are known in the art and need therefore not be provided herein.

Typically, such vaccines are prepared as injectables: either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The vaccine may be administered to a target animal by any convenient route, such as subcutaneously, intraperitoneally, intramuscularly, intradermally, intravenously, orally, intranasally, or intramammarily, in the presence of a physiologically acceptable diluent. The antigens may be administered in a single dose or in a plurality of doses. The vaccine of the present invention may be stored under refrigeration or in frozen or lyophilized form. The vaccine is administered to the target animal in an amount effective to elicit a protective immune response against S. aureus as compared to a control. The effective amount will vary with the particular target animal, its age and size, and may be readily determined by the practitioner skilled in the art. Suitable regimes for initial administration and booster shots will also be variable, but may be typified by an initial administration followed by subsequent inoculations or other administrations. Further, the vaccine of the invention may be combined with vaccines of other genera of bacteria to provide a single broad spectrum vaccine.

The antigens of the present invention are used to immunize animals against S. aureus. While the vaccine is effective for eliciting antibody production in a variety of animals, the vaccine is particularly preferred for the treatment of bovine animals.

The term "antibody," as used herein, includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric, humanized, CDR-grafted, and single chain antibodies, and the like. Fragments of immunoglobulins, include Fab fragments and fragments produced by an expression library, including phage display. See, e.g., Paul, Fundamental Immunology, Third Ed., 1993, Raven Press, New York, for antibody structure and terminology.

The phrases "specifically binds to" or "specifically immunoreactive with", when referring to an antibody or other binding moiety refers to a binding reaction which is determinative of the presence of the target analyte in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target analyte and do not bind in a significant amount to other components present in a test sample. Specific binding to a target analyte under such conditions may require a binding moiety that is selected for its specificity for a particular target analyte. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immuno-reactivity. Typically a specific or selective reaction will be at least twice background signal to noise and more typically more than 10 to 100 times background.

The vaccine of this invention can be used as immunogens to generate antibodies that are selectively specific for strains of S. aureus. Thus, the vaccine can be used to generate monoclonal and polyclonal antibodies.

To prepare antibodies, a host animal is immunized using the cells of the vaccine, individually or together, as the immunogen. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the immunogen. Methods of antibody (polyclonal and monoclonal) production and isolation are well known in the art. See, for example, Harlow et al. 1988, supra. Purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-antibody.

In another embodiment, the monoclonal antibody of the invention is a chimeric monoclonal antibody or a humanized monoclonal antibody, produced by techniques well-known in the art.

Still part of this invention is a kit that comprises the vaccine of this invention; and instructions for use of the kit. In addition to the above, the kits may also comprise a control, antibodies, and the like, suitable for conducting the different assays referred to above.
 

Claim 1 of 7 Claims

1. A vaccine composition comprising (a) cells or cell lysates of a Type 5 Staphylococcus aureus strain wherein the cells express S. aureus Type 5 capsular antigen, (b) cells or cell lysates of a Type 8 S. aureus strain wherein the cells express S. aureus Type 8 capsular antigen, and (c) cells or cell lysates of an unencapsulated S. aureus strain, identified as ATCC BAA-934, wherein the cells of said unencapsulated strain adhere to epithelial cells and carry an antigen that specifically binds with antibodies to S. aureus Type 336, and wherein the cells of said unencapsulated strain are not cells of S. aureus strain identified as ATCC55804, and a pharmaceutically or veterinarally acceptable carrier, diluent or excipient, and/or adjuvant.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.