ANTIBODIES SPECIFIC TO ENDOTOXINS

Abstract: The invention is concerned with the problem of gram-­ negative bacteremia and endotoxemia. The invention pro­ vides a composition comprising one or more antibodies which are specific to one or more endotoxins, especially those produced by gram-negative bacteria, in admixture with a carrier medium. The carrier medium may be animal or human serum, milk or a synthetic material. The invention also provides a method of producing a composition compris­ ing an antibody or a mixture of antibodies specific to endotoxin(s), including the steps of inoculating a suitable animal with an antigen or mixture of antigens capable of triggering the appropriate immunological response in the animal, and removing a body fluid comprising the resulting antibody or mixture of antibodies.

Description: This invention relates to compositions of antibodies specific to endotoxins. Gram negative bacteremia and endotoxemia is an important medical problem carrying a mortality of 30-80 % and causes millions of deaths in humans and animals world wide every year. Conventional antibiotic therapy is becoming less successful with time because antibiotics kill bacteria but do not at all reduce the amount of the extremely potent and stable, endotoxin released from gram negative bacterial cells. This endotoxin is still active upon death of bacteria and can continue to cause shock and death. In addition under certain conditions endotoxin may leak out of the intestine where it is always present and enter the systemic blood causing shock and death. A notable result of this is the very high mortality of farm animals having gram negative sepsis. Endotoxin is known to be present in the intestines and under certain conditions of stress may leak out through the intestinal wall and enter the systemic blood supply, overwhelming the liver's detoxifying capacity and causing shock and death. In these cases there may not have been a stage of bacteremia prior to endotoxemia. A means of inactivating or reducing the free concentration of endotoxins is by administering appropriate antibodies specific to endotoxins. In the prior art it is known to immunise cows with the "pili" of gram negative bacteria to produce milk rich in "antipili" antibodies. Such antibodies prevent the attachment or adhesion of gram negative bacteria to the mucosal surface in the gut and in this manner reduce bacterial infection. In Experimental Studies on Cholera Immunization (Int. Archs. Allergy appl. Immun. 49 :434-452 (1975) Ann-Marie Svennerholm describes a study to investigate the antibody response in rabbits after immunization with formalin-killed vibrios or cholera lipopolysaccharide (LPS) with regard to amount, immunoglobulin class and binding qualities of the antibodies induced. The significance of these parameters and of different immunization schedules for the protective capacity against experimental cholera in rabbits of immune sera and of purified anti-LPS antibodies was analyzed. The study was concerned with cholera only. Elizabeth J. Ziegler et al (the Journal of Immunology Vol. III, No. 2, August 1973, 433-438) describes the immunization of rabbits with a UDP-galactose-deficient mutant (J5) of Escherichia coli 0111 that is unencumbered by 0 antigen since it cannot incorporate galactose into core LPS. Without 0 side chains, the incomplete lipopolysaccharide, the so-called core glycolipid, is accessible for producing antiserum to a wide range of bacteria with similar cores. In practice, however, the polysaccharide barrier has to be penetrated by the resulting anti-endotoxins before they can be effective and this has been a limiting factor against the use of such compositions. The method described in this document uses whole killed organisms as antigens. It is an object of the invention to provide a composition containing a high titre of antibodies which are specific to endotoxins produced by gram negative bacteria. It is a further object of the invention to incorporate such antibodies in products for human and veterinary use. The present invention provides a composition comprising antibodies produced in response to a mixture of endotoxins obtained from Salmonella enteriditis, Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Escherichia coli 026:B6, Escherichia coli 0128:B12, Escherichia coli 0127:B8, Escherichia coli 055:B5, Salmonella minnesota, Serratia marcescens, and Klebsiella pneumoniae, and, optionally, in addition from Shigella flexneri and/or E. coli 0111.B4; or from Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Shigella flexneri, Escherichia coli 055:B5, Escherichia coli 0127:B8, Escherichia coli 0128:B12, Escherichia coli 026:B6, Escherichia coli 0111:B4, Salmonella minnesota, Serratia marcescens and Klebsiella pneumoniae, generally in admixture with a carrier medium. The carrier medium preferably comprises serum obtained by suitable treatment of the blood of animals or it may be synthesised from suitable materials; for example, it may be physiological saline. The composition may be in a form suitable for oral or parenteral administration to animals and/or humans, or for incorporation in milk or a milk derivative. The antibodies are specific to endotoxin(s) produced by gram negative bacteria of more than one of the following genera: Salmonella, Eschericia, Pseudomonas, Shigella, Serratia, Vibrio and Yersinia. It will be appreciated that an antibody or antibodies may react with the endotoxin-producing organism itself, in addition to "free" endotoxin(s) produced by the organism. Such binding to the endotoxin coat of living bacteria may activate complement which in turn may cause lysis and death of the bacteria. According to a further aspect, the invention provides a method of producing a composition as specified above, which process includes the steps of inoculating a suitable animal with an antigen mixture capable of triggering the appropriate immunological response in the animal and removing a body fluid containing the resulting antibody or mixture of antibodies. The body fluid may be worked up in a known manner to give a preparation suitable for therapeutic and/or prophylactic use in a human and/or animal. Such methods are well known in the art. The present invention further provides milk or serum comprising a mixture of antibodies obtained from an animal that has been inoculated with endotoxins obtained from bacteria as defined above, suitable for use in producing a composition of the present invention, that is to say, by working up in a suitable manner. The invention also provides a mixture of endotoxins obtained from bacteria as defined above, suitable for use in producing a composition of the invention or milk or serum of the invention according to a method of the invention, that is to say, a mixture of endotoxins suitable for inoculating a host animal to produce the desired mixture of antibodies that can be worked up in a suitable manner. The antigen mixture comprising endotoxins produced by gram negative bacteria is generally injected into an animal to trigger an immunological response to produce the corresponding antibodies. The endotoxins may be sensitised in accordance with accepted laboratory methods and injected in suitable animals under controlled conditions. An adjuvant, for example Freud's Complete Adjuvant, is preferably present in the composition to be injected. Antibodies prepared in this manner may be type specific e.g. to endotoxins produced by Salmonella enteriditis, or may have a more general specificity, for example to endotoxins produced by many or all gram negative bacteria, or may be a mixture of the above types of antibodies. The animal to be used for the production of the desired antibodies may be a farm animal, for example a bovine, equine, porcine, ovine, canine or feline animal. The immunisation is, for example, subcutaneous, and the immunisation is generally repeated as often as necessary to maintain a high level of antibodies in the blood of the immunised animal, which may be used subsequently for the preparation in known manner of the desired antiserum for therapeutic or prophylactic use in the same species of animal or in another species. In the latter case, the serum is preferably subjected to a treatment which generally includes reacting the serum preparation with the blood, blood cells, blood fraction(s) or another tissue preparation of animals of the species to be treated, then removing the added blood or other tissue component(s), for example by centrifugation and/or column chromatography and/or any other procedure. An antibody composition of the invention preferably comprises a total of not less than 100 micrograms of antibodies per millilitre. An antibody composition of the invention may be in unit dose or multidose containers, and may be in lyophilised form, for reconstitution before use. According to one embodiment of this invention, the antibodies specific to the endotoxins may be incorporated in milk or a byproduct thereof. This is particularly useful where antibodies specific to endotoxins are not present naturally in adequate levels in human colostrum and breast milk. Pregnant or lactating cows, mares, ewes, bitches or cats may be immunised with endotoxins to provoke the production of antibodies specific to endotoxins, for example as described above. The milk obtained from such animals contains a high concentration of anti-endotoxin antibodies. This milk or products prepared from this milk may be given to human and animals neonates and children whose mothers' milk is deficient in these antibodies and/or who may benefit from the resulting prophylaxis. The following Examples illustrate the invention. Example 1 Cows and other farm animals were immunised by the sub-cutaneous injection of a solution of endotoxins obtained from about 12, for example 11, different bacterial strains and species, which solution was emulsified with an equal volume of Freud's Complete Adjuvant. The mixture of endotoxins injected above were those obtained from Salmonella enteriditis, Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Eschericia coli 026:B6, Eschericia coli 0128:B12, Eschericia coli 0127:B8, Eschericia coli 055:B5, Salmonella minnesota, Serratia marcescens, and Kleb siella pneumoniae, and, optionally, Shigella flexneri and/or E . coli 0111:B4. The above composition comprising endotoxins with Freud's Complete Adjuvant was injected as often as required to maintain a high antibody level in milk and/or serum. a) Milk obtained from the suitably immunised animals that were pregnant or lactating was further processed in the same way as normal milk. The resulting milk, and other products obtained therefrom, was suitable for administering to human and animal neonates and young. b) Serum which contains a high titre of antibodies specific to the above endotoxins was prepared by bleeding the immunised animal and working up the blood by a method known for the production of an antiserum from blood. Other bacterial species or strains may be substituted for any one or more of those named above. Example 2 1. There were obtained compositions of equine, bovine, sheep, canine, feline and porcine serum comprising antibodies which react with and bind endotoxins prepared from the following bacterial sources: Salmonella typhosa Salmonella typhimurium Salmonella abortus equii Shigella flexneri Eschericia coli 055:B5 Eschericia coli 0127:B8 Eschericia coli 0128:B12 Eschericia coli 026:B6 Eschericia coli 0111:B4 Salmonella minnesota Serratia marcescens Klebsiella pneumoniae The compositions were prepared as described in Example 1. 2. The total concentration in the composition of all the above anti-endotoxins together was not less than 100 micrograms per millilitre and there was a minimum concentration of 1 microgram per millilitre of at least ten of the about twelve species or strains. 3. The preparation had the ability to kill Klebsiella pneumoniae bacteria in the presence of complement and serum. 4. a) Serum prepared by suitably inoculating a horse was used therapeutically or prophylactically on other horses. b) Serum obtained by suitably inoculating a horse was suitably treated to render it fit to use therapeutically or prophylactically on another species of animal, e.g. a cow, sheep, pig, cat or dog. 5. The treatment required in 4b above included reacting the serum preparation with the blood, blood cells, blood fraction(s), or another tissue preparation of animals of the species to be treated, then removing the added blood or other tissue component(s) by centrifugation and/or column chromatography, although any other suitable procedure could be used instead or in addition. Other strains or species of bacteria may be used instead of or in addition to those listed above, and one or more of the listed sources may be omitted if desired. Other animals e.g. farm animals may be used for the preparation of serum for therapeutic or prophylactic use in the same or another species of animal, for example as described in 4 and 5 above for horses.

Claims: A composition comprising antibodies produced in response to a mixture of endotoxins obtained from Salmonella enteriditis, Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Escherichia coli 026:B6, Escherichia coli 0128:B12, Escherichia coli 0127:B8, Escherichia coli 055:B5, Salmonella minnesota, Serratia marcescens, and Klebsiella pneumoniae, and, optionally, in addition from Shigella flexneri and/or E. coli 0111:B4; or from Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Shigella flexneri, Escherichia coli 055:B5, Escherichia coli 0127:B8, Escherichia coli 0128:B12, Escherichia coli 026:B6, Escherichia coli 0111:B4, Salmonella minnesota, Serratia marcescens and Klebsiella pneumoniae. A composition according to claim 1 in admixture with a carrier medium, wherein the carrier medium comprises animal or human serum, or physiological saline, or milk or a derivative thereof. A composition according to claim 1 or claim 2, suitable for parenteral or oral administration to a human or an animal. A method of producing a composition as claimed in claim 1, which comprises removing a body fluid comprising a mixture of antibodies obtained from an animal that has been inoculated with a mixture of endotoxins obtained from bacteria as defined in claim 1, and working up the body fluid to give a preparation as claimed in claim 1, suitable for therapeutic and/or prophylactic use in a human and/or animal. A method of producing a mixture of antibodies as claimed in claim 4, wherein the body fluid is blood. A method of producing a mixture of antibodies according to claim 5, wherein the body fluid is milk. A method of producing a composition as claimed in claim 1, which process includes the steps of inoculating a suitable animal with a mixture of endotoxins obtained from bacteria as defined in claim 1, and removing a body fluid containing the resulting mixture of antibodies, with the proviso that no claim is made to the treatment of an animal body by therapy. Milk or serum comprising a mixture of antibodies obtained from an animal that has been inoculated with a mixture of endotoxins obtained from bacteria as defined in claim 1, suitable for use in producing a composition as claimed in claim 1, suitable for therapeutic and/or prophylactic use in a human and/or animal. A mixture of endotoxins obtained from bacteria as defined in claim 1, suitable for use in producing a composition as claimed in any one of claims 1 to 3 or milk or serum as claimed in claim 8, according to a method as claimed in any one of claims 4 to 7. A composition as claimed in any one of claims 1 to 3 for use in the treatment of gram negative bacteremia and endotoxemia. Claims for the Contracting State: AT A method of producing a composition comprising antibodies produced in response to a mixture of endotoxins obtained from Salmonella enteriditis, Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Escherichia coli 026:B6, Escherichia coli 0128:B12, Escherichia coli 0127:B8, Escherichia coli 055:B5, Salmonella minnesota, Serratia marcescens, and Klebsiella pneumoniae, and, optionally, in addition from Shigella flexneri and/or E. coli 0111:B4; or from Salmonella typhosa, Salmonella typhimurium, Salmonella abortus equii, Shigella flexneri, Escherichia coli 055:B5, Escherichia coli 0127:B8, Escherichia coli 0128:B12, Escherichia coli 026:B6, Escherichia coli 0111:B4, Salmonella minnesota, Serratia marcescens and Klebsiella pneumoniae, which comprises removing a body fluid comprising a mixture of antibodies obtained from an animal that has been inoculated with endotoxins obtained from bacteria as defined above, and working up the body fluid to give a composition suitable for therapeutic and/or prophylactic use in a human and/or animal. A method of producing a mixture of antibodies as claimed in claim 1, wherein the body fluid is blood. A method of producing a mixture of antibodies according to claim 1, wherein the body fluid is milk. A method according to claim 1, wherein the antibodies are admixed with a carrier medium comprising animal or human serum, or physiological saline, or milk or a derivative thereof. A method according to any one of claims 1 to 4, wherein the body fluid is worked up to give a composition suitable for parenteral or oral administration to a human or an animal. A method of producing a composition as claimed in claim 1, which process includes the steps of inoculating a suitable animal with a mixture of endotoxins obtained from bacteria as defined in claim 1, and removing a body fluid containing the resulting mixture of antibodies, with the proviso that no claim is made to the treatment of an animal body by therapy. A method as claimed in claim 6, wherein the body fluid is milk or serum suitable for use in producing a composition as defined in claim 1, suitable for therapeutic and/or prophylactic use in a human and/or animal. Use of a mixture of endotoxins obtained from bacteria as defined in claim 1, for producing a composition as defined in claim 1 or milk or serum as claimed in claim 7, according to a method as claimed in any one of claims 1 to 7.

Inventors: GAFFIN, STEPHEN LESLIE
Applicants: GAFFIN, STEPHEN LESLIE
Application year: 1982
Main classification: A61K39
IPC codes: A61K38, A61K39, C07K16