IE850802L - Vaccine and serum for endotoxin associated disease - Google Patents

Description

r- c-, — 1 The present invention relates to a vaccine and serum for immunization against, and treatment for, gram negative bacteria diseases. More specifically, this invention relates to a bacterial mutant of Salmonella enteritidis and its use in a combination vaccine to immunize mammals and birds against diseases caused by endotoxin producing gram negative bacteria in the taxonomic family Enterobacteriaceae. This invention also relates to a detoxified endotoxin immune modulator useful in 10 the treatment of animals and men in combination with other antigens, and 2 methods of making and using said modulator.

In the field of animal husbandry, endotoxin associated diseases pose serious animal health problems 15 and consequently, represent an economic influence of significant proportion.

In horses, endotoxin-associated diseases include founder (i.e., laminitis), colic (i.e., abdominal crisis associated with dietary engorgement and other 20 stressful phenomena such as abdominal obstruction, intestinal ischemia, Gram negative bacterial enteritis/ diarrhea, intestinal malabsorption, transport stress, parturition, etc.) septic arthritis, and Gram negative intrautrine infections. Endotoxin-associated diseases 25 in cattle include laminitis in both dairy and feedlot cattle, sudden death syndrome in feedlot cattle, mastitis in dairy cattle, and dysentery, white scours or colibacillosis, and Salmonella diarrhea in baby calves.

Endotoxin-associated disease in swine include parturition dysagalactia (i.e., mammary gland failure related to Gram negative endometritis), intestinal edema disease, and baby pig Salmonella diarrhea. Salmonella diarrhea, 5 hemorrhagic septicemia, infection of the air sacs and sinuses; and fowl cholera and other Pasteurelloses are examples of endotoxin-associated diseases of birds.

Previous treatment for endotoxin mediated and/or associated diseases has been retrospective (i.e., 10 after development of clinical illness) .and has been limited to chemotherapeutic intervention. Prevention measures were not achieved with such treatment. Prior limited, definitive, vaccinal protection from Gram negative septicemia and/or endotoxemia has been ac-15 complished only via (a) individualized vaccines comprised of autogenous bacterial isolates expressing various antigenic epitopes (K-antigens or O-carbohydrate side chains) or (b) live vaccines comprised of attenuated or deletion-modified, live bacterial isolates. 20 The major disadvantage of the current method ologies for treating endotoxin mediated and/or associated diseases is that such treatments are initiated only after clinical illness has developed, which frequently is after the disease has attained an irreversible 25 state. The prior vaccinal protection for Gram negative septicemia and/or endotoxemia that has been reported for individualized vaccines comprised of autogenous bacterial isolates is not time, cost or production efficient because such vaccines are produced retrospective-30 ly, after disease has developed.

The primary disadvantages of the polyvalent vaccines comprised of multiple bacterial isolates expressing various antigenic epitopes (K-antigens or O-carbohydrate side chains) are that the bacterial iso-35 lates causing disease at any given time are subject to epidemiological shifts and/or drifts in antigenic epitopes causing a change in antigenic specificity and thus loss of protective efficacy. The K-antigens or O-carbohydrate side chains also are potent stimulators of immunoglubulin IgE which is responsible for undesirable anaphylactoid reactions in many animal species, especially the horse.

The primary, disadvantages of live vaccines comprised of attenuated or deletion-modified bacterial . isolates is that they have the potential for reversion to the wild-type parential strains and thus resumption of pathogenicity for vaccinated animals.

Accordingly, a long felt need exists for a vaccine and serum to immunize and treat against diseases caused by Gram negative bacteria and to overcome the deficiencies found in the prior art. One important aspect of this invention is to meet this need.

This invention provides a vaccine comprising a vaccine for immunizing against Gram negative bacterial diseases comprising: a killed suspension of a non-0-Carbohydrate side chain bacterial mutant from the taxonomic family Enterobacteriaceae, a protein or lipid binding carrier having a high lipophilic and high proteinophilic affinity to insure uniform component suspension and prolonged antigenic release, and an immune modulator having propensity for B-lymphocyte proliferation and preferably with high levels of neutralizing and opsonizing antibodies of high antigenic affinity and avidity neutralizing and opsonizing antibodies of antigenic affinity.

In a»further aspect this invention provides a method for preparing a vaccine for immunizing against Gram negative bacteria diseases which steps comprise: (1) combining a killed suspension of a non-O-carbohydrate side chain bacterial mutant from the taxonomic family Enterbacteriaceae and an immune modulator having propensity for B-lymphocyte proliferation and with high levels of neutralizing and opsonizing antibodies of high antigenic affinity and avidity; and (2) suspending said combination of bacterial mutant and immune modulator in a protein and lipid binding carrier having a high lipophilic and high proteinophilic affinity to insure uniform ' component suspension and prolong antigenic release.

Other aspects of the invention include preparation of a hyperimmune serum derived from the combination vaccine, and a method of treating animals diseased with Gram negative bacterial disease. Also, the invention provides for the combination of the mutant and carrier alone.

As indicated, the present invention involves several different concepts, namely, (1) a vaccine featuring a bacterial mutant and a protein and lipid binding carrier having a high lipophilic and proteinophilic affinity which insures uniform component suspension on prolonged antibody releases, and also preferably including an immune modulator which is a detoxified endotoxin; (2) a bacterial mutant, possessing broad protective potential, specifically a non-O-carbohydrate side chain bacterial mutant from the family Enterobacteriaceae; (3) the aforesaid immune modulator with specific propensity for B-lymphocyte proliferation and, consequently the earlier occurence of higher levels of neutralizing tand opsonizing antibodies of high antigenic affinity and avidity, and the method of preparing said immune modulator; (4) a hyperimmune serum for treating Gram negative bacterial diseases and a method of using said serum.

For immunization, the vaccine is administered intramuscularly or subcutaneously at concentrations hav- 7 ing at least 1 x 10 bacteria and 100 micrograms immune modulator. For treatment of an animal having a Gram negative bacterial caused disease, serum is pre-5 pared from a vaccinated donor and administered to provide a protective level of antibodies.

The advantage of the combination vaccine is that it is prophylactic in nature, in opposition to current treatment modes which are initiated retrospec-10 tively or only after development of disease. .4, added advantage of the combination vaccine is that immunized animals develop an earlier and higher degree of protection from many of the endotoxin associated diseases without the risks inherent in existing vaccines such 15 as (a) provoking potentially fatal anaphylaxis; (b) developing potentially fatal infections by reversion of live, non-pathogenic bacterins to pathogenic forms; or (c) losing ability to elicit protection because of relative changes in strains of bacteria causing disease. 20 The detoxified endotoxin component of the combination vaccine, as a potent immune modulator with propensity for B-lymphocytes causes not only more rapid proliferation of these antibody progenitor cells, but also their earlier occurrence in the activated functional state, 25 thus resulting in production of protective levels of antibody in the host's circulation much sooner after vaccination than observed for conventional bacterin vaccines. The bacterin component of the combination vaccine, comprised of a mutant exhibiting a naked core 30 antigen (2-Keto-3-deoxyoctonic Acid-Lipid A), devoid of any of the O-carbohydrate side chains (K Antigens) present in conventional bacterin vaccines, precludes the development of O-carbohydrate specific Immunoglobulin E (IgE, Reagin) and thus elicitation of IgE-35 mediated anaphylaxis after vaccination. Since the naked core antigen, in contrast to O-carbohydrate side chains (K-antigens serotypes), is common to many Gram negative bacteria, it elicits antibodies of broad cross-pro 6 tection, while also precluding the loss of protective efficacy because of epidemiological shifts and drifts in O-carbohydrate side chains (K-antigens, serotypes) relative to time.

Prior to the development of the combination vaccine and hyperimmune serum elicited by the combination vaccine, medical management involved primarily chemotherapy only after onset of the Endotoxin-as-sociated diseases. The advantage of hyperimmune serum, 10 is that in the non-vaccinated animal with clinically apparent disease, it may ameliorate the the disease process, thus precluding crippling and/or death in the horse or other species.

The concept of broad spectrum protection via 15 a combination vaccine per se, and/or combination vaccine-elicited hyperimmune serum against bacteremias and/or endotoxemias mediated and /or associated with a wide variety of Gram negative bacteria is also an economical breakthrough for the animal industry using 20 new molecular concepts in applied immunology. Diseases for which protection is developed by the combination vaccine includes those associated with endotoxin disseminated intravascular coagulation and, more particularly, the Gram negative bacteria Salmonella enteritidis, 25 Salmonella typhimurium, Salmonella typhosa, Salmonella minnesota, Salmonella abortus-equi, and Escherichia coli.

More specifically, the individual components of the present invention are described as follows: MUTANT The mutant is deposited in the American Type Culture Collection as ATCC No. 53000.

The parent isolate used toaprepare the genetically modified Mutant Strain R-17 was isolated from an active diarrheal infection of a horse at the University 35 of Missouri College of Veterinary Medicine. The original clinical isolate was isolated on MacConkeys Agar, exhibiting a lactose negative, smooth, mucoid glistening colony of 3.5<~4xnm diameter at 24 hours incubation at 37 C. Biochemical analysis using an API System (API Laboratory Products, 200 Express St., 5 Plainview, New York 11803) in conjunction with the API Profile Recognition System and characterization on routine laboratory media identified the original.clinical isolate as Salmonella enteritidis (Serotype B-typhimurium). This organism is described by Ewing and 10 Martin. (Ewing, W.H. and W. J. Martin: Enterobacteriaceae. In Manual of Clinical Microbiology, 2nd ed. Washington, American Society for Microbiology, 1974).

A specific embodiment of the organism of this invention relates to a deletion mutant strain of the 15 parent isolate of Salmonella enteritidis (Serotype B-typhimurium) effected by ionizing radiation. Ionizing radiation, by virtue of high energy penetrance, induces free radical formation which labilizes cytoplasmic molecules causing single-stranded breaks in the de-20 oxyribonucleic acid molecules, thus resulting in a high frequency of deletion mutations. Surviving mutants frequently phenotypically express various degrees of inability to synthesize intact lipopolysaccharide.

Such mutants are easily recognized, since they exhibit 25 smaller diameter, flat, rough (R) colonies, in contrast to large, punctate or convex, smooth (S) colonies produced by the parent bacterium.

X-ray mutagensis was accomplished on standard pour plates seeded with viable parent bacteria. Plates 30 were irradiated in 5 second increments to a maximum of 35 seconds using a Machelett OEG 60 X-ray tube with beryllium window, operated at 50 kV peak and 25 mA, to give a dose rate of 250 rad/sec. Irradiated plates were held at 4 C for 2-4 hours and then incubated in 35 the dark at 37 C to preclude Photoreactivation. At the 8 end of 24 hours incubation plates were examined for a change in colonial morphology. Colonies of equal or less than 2mm diameter exhibiting rough (R) morphology were selected, passaged at least 10 times on solid 5 plate media, and passaged at least 3 times by intraperitoneal inoculation of laboratory mice to insure stable rough (R) phenotypic expression. Mutant strain R-17 was assayed for avirulence, in comparison with the parent isolate by a standard mouse potency assay via 10 intragastric inoculation. Purified lipopolysaccharide from the mutant strain R-17 and the parent isolate were analyzed chemically by electrophoresis in 2% sodium dodecyl sulfate - 10% poly aery lamide gels (Palva, E.T & P. Helena Makela, 1980, Lipopolysaccharide 15 Heterogeneity in Salmonella typhimurium. Analyzed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, European Journal of Biochemistry 107:137-143) and biologically by the chromatogenic limulus lystate assay (Webster, C.J. 1980, Principles of a Quantitative 20 Assay for Bacterial Endotoxins in Blood That Uses Limulus Lysate and a Chromogenic Substrate, Journal of Clinical Microbiology 12(5):644-650) , and seroagglutina-tion (Lindberg, A. A. & L. Le Minor. 1984, Serology of Salmonella. Vol. 15, pp. 1-141; In Methods in Micro-25 biology, T. Bergan, Editor, Academic Press, New York, 19 84) and no O-carbohydrate antigen could be detected. Therefore, the mutant strain R-17 was presumed to be a Chemotype I or II, naked-core mutant and a novel embodiment of this invention.

TRICHLOROACETIC ACID EXTRACTION (BOIVIN METHOD) OF LIPOPOLYSACCHARIDE Lipopolysaccharide (LPS) is extracted from either acetone dried bacteria or wet bacteria suspended in 5 volumes of distilled ^0 with 0.25 N aqueous 35 Trichloroacetic Acid. The solubilized LPS (supernatant) 9 is separated from residual bacteria (pellet) by cen-trifugation (5000xg 30 min., 40°C). The pH of the • supernatant is adjusted to pH 6.8 with ION NaOH and LPS then precipitated from the supernatant by the ad-5 dition of 2 volumes of cold absolute ethyl alcohol. The precipitated LPS is collected and washed (3x) with cold absolute ethanol by centrifugation (10,000xg, lhr, 4°C), lyophilized, and stored at 4°C until use.

PREPARATION OF THE IMMUNE MODULATOR Trichloroacetic acid extracted lipopolysac- charide (LPS) is dissolved in 100 volumes of freshly prepared pyridine, 90% formic acid (2:1 v/v) by slowly increasing the temperature to the boiling point and holding for approximately 15 minutes or until apparent 15 clearing. Detoxification then is accomplished by the addition of an equal volume of distilled H2O to the LPS-pyridine-formic acid solution and refluxing for 60 minutes. The detoxified LPS is precipitated overnight (4°C) by the addition of 4 volumes of cold absolute 20 ethanol and centrifugation (10,000xg, 1 hr., 4°C), washed (3x) with cold absolute ethanol and then lyophilized detoxified LPS immune modulator was stored at 4°C or reconstituted in 0.1% aqueous triethylamine for immediate use in effecting immune potentiation.

POTENTIATION OF IMMUNE RESPONSE & HYBRIDOMA FUSION WITH IMMUNE MODULATOR Purified lipopolysaccharide is known to effect lymphocyte blastogensis in vitro, i.e., cultured tissue cells. In man and other mammals lymphocytes 30 produce the interleukins (IL-1, IL-2) which mediate the immune response and thereby the production of antibodies, via the ecosatetraenoic acid metabolites (i.e. prostanoids or prostaglandins). It is also known that purified lipopolysaccharide potentiates IL-1 and pro- 1 0 stacylin synthesis in vivo. However, purified lipopolysaccharide or in situ (associated with Gram negative bacteria per se) lipopolysaccharide possess intact O-carbohydrate side chain antigens which are toxic 5 when introduced in vivo (i.e. into mammals), causing untoward febrile responses, coagulopathies,and sometimes fatal disseminated intravascular coagulation via anaphylactoid reactions in the sensitized host.

Purified lipopolysaccharide also is cidal to mammalian 10 tissue or cells grown in vitro culture, at picagram and low nanogram concentrations.

Embodi.ments of this invention include: 1) a novel method for preparation of an immune modulator which is non-toxic to mammals and tissue cell cultures, 15 2) a novel method employing the immune modulator for immunizing mammals to either particulate or soluble antigens which enchances more rapid and greatly elevated antibody responses and also potentiates recognition of broader spectra of antigenic determinants 20 (epitopes) and consequent production of wider ranges of immunologic specificities, and 3) a novel method for enhancing the frequency of hybridization between antibody synthesizing-plasmacytoma cells and B-lymphocytes in cell culture from 15-30% to greater than 85% by 25 primary immunization of donor mammals with particulate or soluble antigens in the presence of immune modulator.

Immune modulator, when given simultaneously with antigen, enhances the primary immune response of 30 C57BL/6J mice. Enhancement occurs both with particulate antigens such as Pseudomonas aeruginosa and with soluble antigens such as keyhold limpet hemocyanin. Animals injected with antigen and immune modulator simultaneously demonstrated higher antibody titers at 7, 14, 35 and 35 days after injection than did animals receiving antigen alone. Immune modulator not only enhances 1 1 antibody titers early in the immune response, but more importantly, appers to prolong high serum antibody levels. Enhancement of the specific antibody response by immune modulator is not significantly affected by route of injection, since enhancement is observed when 5 antigen and immune modulator are injected intravenously, intraperitoneally, or subcutanesouly in incomplete Freund's Adjuvant.

These results are summarized in Table A. In these experiments 5 female animals were in each 10 group. Experimental animals received antigen and immune modulator (75 ^g/dose) and controls received an equal amount of antigen and sterile saline. Serum antibody titers were measured by an indirect ELISA assay.

Immune modulator demonstrates no toxicity as assayed by its effect on cell culture growth. When SP2/0 mouse myeloma cells were cultured with immune modulator at concentrations of 100, 10, 1, 0.1 ng per ml of culture fluid, cultures with immune modulator 20 reached cell densities equivalent to or slightly greater than the corresponding control cultures.

Myeloma cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum 2%, L-glutamine, 1% sodium pyruvate, and antibiotics. Immune modulator 25 was added at appropriate concentrations to the media as a sterile, aqueous solution. Cell densities were determined at 24, 48, 72, and 96 hours after the addition of immune modulator or and equivalent volume of sterile distilled water.

TABLE A Immunization of C57BL/6J Mice with Particulate or Soluble Antigens with and without Immune Modulator by Various Routes of Inoculation.

Antibody Titers Route of After Immunization Animal Group Antigen Inoculation 7 Days 14 Days 35 Davs Immune Modulator P. aeruginosa Intravenous *1:32,000 1:128,000 1:32,000 Control P. aeruginosa Intravenous 1:8,000 1:16,000 1:4000 Immune Modulator P. aeruginosa in incomplete Freund's Subcutaneous 1:4,000 1:16,000 1:16,000 Control P. aeruginosa in incomplete Freund's Subcutaneous 1:2,000 1:4,000 1:2,000 Immune Modulator hemocyanin Intraperitoneal 1:16,000 1:256,000 1:64,000 Control hemocyanin Intraperitoneal 1:4,000 1:64,000 1:16,000 ♦Dilution of Sera i :i The Vaccine The vaccine comprises a bacterial mutant (bacterin), an immune modulator (endotoxoid) and a protein and lipid binding carrier (adjuvant). The vaccine is administered intramuscularly or subcutaneously 7 at concentrations equal to or greater than 1 x 10 bacteria (preferably, 1 x 10^ bacteria), 100 or greater micrograms (preferably 100 to 4000 micrograms) detoxified endotoxin, in a lipophilic-proteinophilic 10 absorbent carrier.

The bacterin preferably consists of a killed suspension of a non-O-carbohydrate-side chain mutant of Salmonella enteritidis. The bacteria may be prepared by inoculation of sterile, enriched broth with a 15 subculture of the Salmonella enteritidis mutant and aerobic incubation at 37°C to obtain maximal bacterial mass. The bacteria are killed by addition of a bactericidal agent such as merthiolate. The bacteria are checked for non-viability, then washed (4X) with sterile, 20 non-pyrogenic physiologic saline, and reconstituted to the desired stock concentration for admixing with the other components of the vaccine.

The detoxified endotoxin is prepared by admixing Gram negative bacterial endotoxin to pyridine-25 formic acid (2:1) solution. The endotoxin-pyridine-formic acid mixture is thoroughly mixed in a sterile reflux condenser apparatus, the temperature increased to the boiling point and refluxed to obtain optimal methylation of endotoxin. The methylated, detoxified 30 endotoxin then is precipitated from the aqueous reflux mixture by the addition of alcohol, collected by centrifugation, washed by resuspension in alcohol and recentriguation, and, finally, dissolved in nonpyrogenic distilled water to the desired stock concentration for 35 admixing with the other components of the vaccine. 1 4 The adjuvant consists of high affinity lipophilic and proteinophilic carrier sufficient to absorb the protein-moiety of the bacterin and lipid-moiety of the detoxified endotoxin. Preferably the adjuvant is 5 a fatty acid based adjuvant, oil based adjuvant, or alum based adjuvant (^uch as dialuminum trioxide). The carrier properties of dialuminum trioxide function by maintaining uniform suspension and allow prolonged release of the bacterin and detoxified endotoxin, thus 10 insuring maximal antibody production. When dialuminum trixoide is utilized as the high affinity proteinophilic and lipophilic adjuvant, approximately 1.5% by volume is optimal.

Normal horses when immunized with the com-15 bination vaccine developed antibodies in their blood stream sufficient for protection when challenged in the laboratory by overfeeding with carbohydrate (i.e. dietary engorgement mimikcing what occurs in nature) or intravenous injection of bacterial endotoxins (i.e., 20 artifically induced disease mimicking what occurs in nature). Normal cattle when immunized with the vaccine developed antibodies; and exhibited no rise in body temperature, or abnormal increase in numbers or types of white cells. The advantages of the combination 25 vaccine include (a) the mutant bacterium which is devoid of components normally present in its cell wall which cause undesirable anaphylactoid reactions; (b) an adjuvant or carrier which insures prolonged release of bacterin and/or detoxified endotoxin and consequently 30 maximal production of neutralizing antibodies, and (c) the detoxified endotoxin which itself enhances the production of an earlier and higher level of the desirable neutralizing antibodies against intact endotoxin and bacteria. The combination vaccine, also has the quality 35 of providing broad spectrum protection against many Gram negative bacterial diseases, since the basic structure of the antigen is common to most Gram negative bacteria; yet is devoid of those components present in existing vaccines which cause undesirable anaphylaxis.

Laboratory observations indicate that carbohydrate overload causes increased concentrations of 5 acid in the gut (i.e., large bowel), which in turn damages the normally impermeable bowel lining and simultaneously decreases the number of Gram negative bacterial normally present in the contents and lining of the bowel by enhancement of migration into the blood stream 10 (septicemia) and acid killing in the gut per se. The killing of these bacteria, results in the release of endotoxin from their cell walls, which in turn, also crosses the acid-damaged gut lining into the blood stream. The endotoxin in the blood stream then causes 15 undesirable blood clots in Ihe small blood vessels (intravascular coagulation). In the case of horses and other hoofed animals, these undesirable blood clots form in the small blood vessels, ultimately causing death of the hoof tissue, permanent crippling and/or 20 death of the animal.

The specific advantages of the combination vaccine in horses is to prevent the crippling and killing effects of founder or colic, by neutralizing endotoxin and/or Gram negative bacteria that gets into 25 the blood stream of horses experiencing accidental overfeeding, grass founder, or stress. The specific advantage of the combination vaccine in cattle also is to prevent or reduce the endotoxin associated diseases by neutralizing any endotoxin and/or Gram negative bac-30 teria that gain entrance to the blood stream or other tissues. Sudden death syndrome in feedlot cattle, mediated by endotoxin from Gram negative bacteria of gut origin, is a classic example of such a disease with current, immense economic implications in the cattle 35 industry. 1 6 Potency and Safety of Vaccine Components I) Extensive testing has been conducted to establish the potency and safety for the components of the vaccine.

It was discovered that incorporation of the immunoregulator endotoxoid with bacterin would elicit an earlier, and enhanced immune responsiveness in vaccinated animals. Groups of healthy adult horses and ponies were vaccinated intramuscularly with bacterin + endotoxoid or the bacterin alone. Blood samples had been drawn on the animals a week before th e start of the experiment and immune profiles indicated all were normal (i.e., no history of chronic laminitis). Serum samples were collected at 24 hours intervals after 15 immunization, and antibody titers ascertained using the antigen-specific, solid-phase radioimmunoassay. The data presented in Fig. 1 indicates that animals vaccinated with the bacterin _ endotoxoid developed detectable antibody titers as soon as 3 days after vac-20 cination compared to 7 days for those animals receiving the bacterin alone. Examination of the immune response curves in Fig. 1 between days 3 and 14 indicated a steeper slope for the bacterin + endotoxoid groups compared to the bacterin-only groups. This indicated an 25 enhanced rate of antibody production for the former.

Consequently, it was postulated that the bacterin + endotoxoid groups developed earlier protection. Additionally, an overall greater degree of protection resulted due to higher concentrations of neutralizing 30 and/or opsonizing antibodies in their cirulation, compared to those animals receiving the bacterial alone.

Endotoxoid (i.e., detoxified endotoxin) was administered to mice, horses and ponies, and cattle to ascertain the maximal amount of endotoxoid that could 1 ? safely be incorporated with the bacterin, as an im-munoregulator in the vaccine. An for CF-1 mice usually is attained within 72-96 hours by intravenous injection of 0.3-0.6 mg of native endotoxin.

Groups of male, CF-1 mice (L5-20 grams) were inoculated in the marginal tail vein with 0.1 milliliter physiologic saline, 300 ;ug of endotoxin in 0.1 of physiologic saline, aid respective concentrations of 600 jag, 6000 jig, and 12,000 jag of endotoxoid in o.l mil-10 liliter of physiologic saline. The mice were observed at 24 hour intervals for adverse effects and mortality. Deaths were observed in the endotoxin (positive control) group by 48 hours with maximal mortality (56%) recorded at 72 hours (Table I). In comparison, no mortality was 15 observed for the group receiving twice (600 jag that amount of endotoxoid; only 17% mortality occurred in the group receiving the 20X (6000 /ug) endotoxoid; while 40X (12,000 /ug) of endotoxoid resulted in an LDg3' It was concluded that the endotoxoid was at least forty tinves 20 less toxic than its native endotoxin.

Table 1. Comparison of Endotoxoid and Endotoxin in CF-1 Mice Groups of CF-1 Concentrations of Survival Mice Receiving in 0.1 ml, IV Endotoxin 300 44 Placebo Physiologic Saline 100 Endotoxoid Cone. 1 600 100 Cone. 2 6,000 83 Cone. 3 12,000 47 i R Adult horses and ponies were inoculated intramuscularly with up to 5 mg of endotoxoid. The animals were observed twice daily for 4 days for pyro-genicity, loss of peripheral perfusion, elevated heart 5 rate, blood pressure, lethargy, and diarrhea. None of the animals receiving the 2.5 mg dose exhibited any adverse symptomology. A few of the animals inoculated with the 5 mg dose experienced transient rise in temperature to 10 3-105°F, slight elevation in heart rate 10 and mild loss of peripheral perfusion. These symptoms subsided within 8-12 hours. None of the animals developed diarrhea, blood dyscrasias or other irreversible effects. It was concluded that up to 2.5 mg (2500 fig), or 25 times the 100 JUg of endotoxoid proposed for 15 incorporation as an immunoregulator in the vaccine, could be safely used in horses and ponies.

Cattle were inoculated intramuscularly with up to 1000 Aig of endotoxoid and observed at 2 day intervals for 16 days for pyrogenicity, leukopenia 20 and/or leukocytosis, mononuclear cell abnormalicies (differential counts), erythrocyte abnormalicies, lethargy, and diarrhea. N untoward effects were observed in the cattle. It was concluded that up to 1000 ^ug of endotoxoid could be safely incorporated as an 25 immunoregulator in the vaccine for cattle.

Safety of The Vaccine The safety of using the vaccine was evaluated relative to the following criteria: 1) Large (up to 5X) doses in laboratory mice, horses and cattle relative 30 to recommended dosage for routine vaccination regimens; 2) Inoculation of horses and cattle with multiple doses within a short time span; 3) The route of inoculation, (iJe., intramuscular, subcutaneous or intraperitoneal); 4) In laboratory mice as a means of future quality control; 5) In laboratory horses and ponies, where multiple criteria could be evaluated; and 6) In field studies, where horses and ponies and cattle in large numbers and of ubiquitous gene pool could be evaluated 5 for a limited number of parameters.

Groups of adult, mixed sex mice (20-25 gm) were inoculated with 1 ml aliquots of the vaccine or components of the vaccine. The mice were observed for mortality, hair coat texture, spinal arching and 10 clustering indicative of peripheral vascular hypothermia, dehydration, lethargy, diarrhea, and abscessation for up to 96 hours.

Table 2. Inoculation of Mice with Vaccine Group 96 Hour Survival (%) vaccine/subcutaneous 100 vaccine/intramuscular *100 vaccine/intraperitoneal ** 70 bacterin/subcutaneous 100 endotoxoid/subcutaneous 100 carrier/subcutaneous 100 carrier/intraperitoneal *** 50 physiological saline 100 *Mice were inoculated intramuscularly with 25 10-0.1 ml aliquots due to small size.

** Mice died of severe dehydration due to adsorption of serum proteins by the highly lipo-protein-ophilic carrier.

♦♦♦Subsequent intraperitoneal inoculation of 30 1 ml aliquots, (equivalent to quantity of carrier in 2 ml of vaccine).

The data in Table 2 indicated that 1 ml doses of the vaccine administered either subcutaneously or intramuscularly offered no risk to laboroatry mice. 35 However, the peritoneal route of inoculation resulted in % mortality due to the high ratio of lipo-proteiLn-ophilic carrier to body mass (1:20) and consequent: protein dehydration. It was concluded that the adverse effect observed for intraperitoneal inoculation 5 of laboratory mice would be irrelevant since the target species for the vaccine would involve phenomenally greater body weight to carrier ratios, and the recommended route of inoculation would be intramuscular or subcutaneous rather than intraperitoneal. 10 Healthy, adult horses were inoculated intra muscularly with 2 consecutive 5X (5 x 10^"® bacteria, 500 /ug endotoxoid) doses of the vaccine, six days apart. The animals were observed daily for 20 days for anorexia, lethargy, diarrhea, dehydration, and tender-15 ness, swelling and/or abscessation at the injection site. The only adverse effect was transient tenderness and swelling at the injection site which subsided within 48-72 hours.

Cattle weighing 500-650 lbs. were vaccinated 20 intramuscularly with 2 consecutive 4.5X (equivalent to 4.5 x 10^ bacteria and 437 jag endotoxoid) doses of vaccine 18 days apart. One cow was vaccinated with one 11.25X (equivalent to 1.125 x 10^® bacteria and 1 mg endotoxoid) dose of the vaccine. All animals were 25 examined twice daily for anorexia, lethargy, diarrhea, dehydration, and tenderness, swelling and/or abscessation at the injection site. No adverse reactions were observed in any of the cattle.

The above data indicated that the dose range 30 and regiment recommended for use in horses and cattle offered no untoward risk to the animals.

Healthy, adult horses were inoculated intramuscularly with 2.5X (equivalent to 2.5 x 10^ bacteria and 200 jag endotoxoid) doses of the vaccine at 3 day 35 intervals for 9 days, allowed to rest for 7 days and then the 3 dose-3 day interval inoculation regimen was repeated for 5 additional times. The horses received 2 1 a total of 18 .inculations over a period of 64 days. Consecutive inoculations were alternated from the neck to buttock. The animals were observed daily for anorexia, lethargy, diarrhea, dehydration; and tenderness, 5 swelling and/or abscessation at injection sites. The only adverse reaction was localized tenderness, circumscribed swelling and abscess formation at two injection sites in one animal after ten inoculations. The abscesses, upon drainage, rapidly resolved and the 10 inoculation regimen was continued. The animals experienced no other adverse reactions.

Calves weighing between 190-650 lbs. were I inoculated subcutaneously with 4X (equivalent to 4 x 10 bacteria and 400 yug endotoxoid) doses of vaccine. 15 Seventeen days later a portion of the calves were re-inoculated intramuscularly with a IX dose of the vaccine. All animals were observed daily for 120 days for anorexia, lethargy, diarrhea and dehydration. All animals were examined at 3 day intervals* for 15 days, 20 followed by 30 day intervals for 120 days thereafter, for weight loss, body temperature, and blood dyscrasias. Five of the calves had firm, circumscribed nodules in the subcutaneous tissue which resorbed by 8-12 days after the primary (4X) subcutaneous inoculation. Sim-25 ilar nodules were inapparent after re-inoculation of the same calves with IX doses by the intramuscular route.

A herd of horses and ponies were inoculated intramuscularly with 2 doses of vaccine (equivalent to 1 x 1010 bacteria and 100 /ig endotoxoid per kg body 30 weight) 14 days apart. Animals were observed daily for 20 days for anorexia, lethargy, diarrhea, dehydration and tenderness, swelling and/or abscessation at injection site. No untoward effects, other than a mild degree of transient tenderness at the injection site in 35 a few animals, were observed.

A herd of feeder cattle weighing between 550- 650 lbs. was inoculated with 1 dose of vaccine (equivalent to 1 x 10^® bacteria and 100 ug endotoxoid per kg body weight). A portion of the cattle were inoculated intramuscularly and the balance were inoculated sub-5 cutaneously. The cattle were observed daily for 50 days for anorexia, lethargy, diarrhea, and dehydration, tenderness, swelling and/or abscessation at injection sites. No adverse reactions were observed related to the injection sites. No apparent systemic 10 abnormalicies were observed, related to the vaccination. No subcutaneous nodulation was apparent in any of the subcutaneously inoculated animals.

In summary it is apparent that intramuscular or subcutaneous inoculation of cattle, and horses or 15 ponies with the vaccine at reasonable and recommended dose-regimens offers little or no risk to animals.

EFFICACY OF VACCINE Healthy, adult horses and ponies were immunized by intramuscular inoculation of two IX (equiv-20 alent to 1 X 10^® bacteria and 100 Aig endotoxoid) doses of vaccine approximately two weeks apart. The animals were bled at approximately weekly intervals and antibody titers ascertained by radio immunoassay. Immune response curves usually reached a 25 maximum approximately 20-30 days after the primary immunization or 10-20 days after the secondary or anamnestic immunization. Protective efficacy was determined by either carbohydrate engorgement (Per Os) or administration of sublethal doses of endotoxin (in-30 travenously) to vaccinated animals at various times after the secondary immunization. Seventy to eighty percent of non-vaccinated horses or ponies developed Obel grade 3-4, acute laminitis by forty to fifty hours after carbohydrate engorgement with a cornstarch-wood 35 flour gruel administered via stomach tube at the dosage of 17.6 gram gruel per kilogram body weight. One hundred percent (100%) of non-vaccinated horses or ponies developed tachypnea, dyspnea and ataxia within 2-3 minutes and passed fluid, non-formed stools by 45 minutes after intravenous administration of endotoxin 5 at dosage of 10 ;ug per kilogram body weight.

Table 3. Challenge of Vaccinated Horses by Carbohydrate Overload and IV-Endotoxin Challenge Co nbination Placebo Vaccine CHO- (Per Os) *2/19 (10.5%) 75-85% Endotoxin (IV) *3/8 (37.5%) 100% Total *5/27 (18.5%) * Number of animals that developed Obel grade (3-4) 15 laminitis or endotoxin-mediated symptomology after challenge.

Table 3 indicates that approximately 90% of the vaccinated animals, compared with 15 to 25% of the non-vaccinated-control pool (consisting of 100 20 animals over 12-14 years) failed to develop Obel grade 3-4 laminitis after challenge by carbohydrate overload, suggesting at least a 65-75% protective efficacy.

Similar comparison of the vaccinated and non-vaccinated horses challenged with sublethal endotoxin, indicates 25 a greater than 60% protective efficacy. Consequently, it was concluded that vaccination of normal, adult horses or ponies with two IX doses of vaccine resulted in protection of up to 90% of the animals from carbohydrate-induced laminitis (i.e., founder) and greater than 60% 30 from endotoxin-induced endotoxemia.

A grous of cattle of mixed age and sex were vaccinated subcutaneously with one or two doses of vaccine. Animals were bled and sera obtained at 3 day intervals for 15 days, and then at 30 day intervals for 35up to 120 days. Antibody titers were determined by radioimmunoassay. All animals had developed 2 to 4 fold a 4 increases in antibody titer by 20 days after vaccination. Detectable titers were present in approximately 70% of the animals at 120 days after vaccination. A portion of the group was placed on a high carbohydrate 5 ration thirty days after vaccination and after 17 weeks had not developed any signs of anorexia, diarrhea, lameness or sudden death syndrome.

A herd of feeder cattle weighing between 550-680 lbs. were vaccinated with 1-(1X) dose of vac-10 cine and monitored daily for diarrheal disease, lameness and sudden death. No diarrheal disease, lameness or mortality occurred during 11 weeks of observation.

The Serum The development and therapeutic use of hy perimmune serum was based on the rationale that non-vaccinated animals with clinically apparent endotoxin-associated diseases are not afforded the time necessary for their own immune systems to build protective 20 levels of antibodies, after vaccination with the combination vaccine. Thus, passive immunization with preexisting, stored antibodies developed by another animal (hyperimmune serum) provided a means of short-term protection that could aid in amelioration of the 25 endotoxin-associated disease until the animal's own immune system was sufficiently protective. An acute laminitic episode in the non-vaccinated horse that accidentally gets into the grain bin and subsequently founders, is a classic example with current implication in 30 the horse industry.

The hyperimmune serum is comprised of clot serum or plasma, or parts thereof (gamma globulin, im-munoglobuline, or immunoglobulin IgGT) which contain 2 5 antibody<«) specific for the core component (2-Keto-3-deoxyoctonic Acid-Lipid A) in endotoxin of bacteria in the taxomonic family Enterobacteriaceae which are elicited by hyperimmunization of animals with the com-5 bination vaccine.

Hyperimmune sera is prepared by intramuscular injection of healthy adult horses with 6 consecutive 2.5 ml doses of the combination vaccine at 3 day intervals followed by 2 consecutive 2.5 ml doses at 7 day in- tervals. Serum samples are taken from the horses prior to vaccination and at 3 day intervals thereafter for serologic analyses. Concentrations of antigen- specific immunoglobulines (gG, GT, A and M) are deter- 125 mined by radio-immunoassay using I-Protein A.

When each animal's immune response has reached a high-titer plateau, 12 liter quantities of whole blood are collect ed via vena puncture. The hyperimmune sera is obtained by centrifugation after coagulation (approximately 24 hr.), and then heat inactivated (56°C., « 30 min.) and stored at 4 C until use or subsequent purification of gamma globulin or immunoglobulin.

Gamman globulin is prepared by precipitation from aliquots of hyperimmune sera with 50% saturated ammonium sulfate (SAS). The precipitate is then re-25 suspended in 0.01 M phosphate buffer (PB, NaHjPO^-Na2HPO^, pH 8) and exhaustively dialyzed against the same buffer to remove the SAS.

The gamma globulin obtained from 50 ml aliquots of hyperimmune sera is absorbed onto a column 30 (5 x 50 cm) of diethylaminoethyl (DEAE) cellulose equilibrated with PB, pH 8. The column is developed initially with equilibrating buffer (PB, pH 8) to elute IgG, followed by the addition of a NaCl gradient (00.3 M) to the PB to disassociate IgG(T). The eluate is 35 collected in 5 ml aliquots using a refrigerated fraction collector and elution peaks monitored continuously a a using a Beckman DB-GT Spectrophotometer and dual wavelength of 360 and 380 nm. Protein concentration is determined using the Warberg-Christian constant and confirmed by the Lowry method. The IgG(T) aliquots are 5 pooled, lyophilized and stored at -40 C for subsequent use in passive immunization. Horses, experimentally foundered by overfeeding with carbohydrate or intravenous injection of bacterial endotoxins showed rapid improvement upon administration of hyperimmune 10 serum, obtained from other horses vaccinated with the combination vaccine. Similarly, death from intravenous injection of bacterial endotoxins is precluded in laboara-tory mice by immunization with hyperimmune serum obtained from horses vaccinated with the combination vaccine. 15 Groups of healthy, male adult (20gm), (CF-1) mice were inoculated, intraperitoneally (IP) with 1 ml aliquots of 100%, 10% or 0.1% of whole, equine hyperimmune serum on four (4) consecutive days. The animals were observed for four days at 12 hour intervals for 20 dyspnea, rigor, nose and tail perfusion, swollen eyes, coat texture and death. No untoward effects were observed from the IP serum injections.

A healthy adult pony was inoculated with 700 ml of hyperimmune serum admixed with 1000 ml of Lac-25 tated Ringers Solution, by intravenous (IV) drip over 90 minutes. The animal was monitored for 6 1/2 hours (at 10-15 minute intervals) for change in body temperature, heart rate, peripheral perfusion; and discomfort and/or distress. The pony experienced no signs of dis-30 comfort or stress. The body temperature and heart rate exhibited slight (statistically insignificant) increases (100.4° 101.3°F; 50 beats/min.) ap proximately 60-90 minutes after initiation of the IV-drip. in a clinic trial, a 900 lb. horse with compli- 2 7 cations associated from sep:, Lc shoe, was inoculated intravenously with 1200 ml hyperimmune serum continuously over a period of 10 hours in Lactated Ringers solution. The animal exhibited no untwoard signs of 5 toxicity and indeed showed marked improvement.

To evaluate the safety or intramuscular inoculation, healthy, adult ponies were inoculated intramuscularly (IM) with 0, 10, 20, and 40 ml of hyperimmune serum. The animals were monitored at 30 10 min., 1, 2, 4, 8, 16, and 24 hours after inoculation for (1) urination; (2) diarrhea; (3) rigors; (4) peripheral perfusion; (5) temperature; (6) respiration; (7) heart rate; (8) leukocytosis (or -penia); and (9) erythrocytosis (or -penia). No untoward effects were 15 observed. A diffuse nodule was apparent in the neck of one pony administered the 40 ml dose intramuscularly which had receded by 4 hours.

Gamma globulin, containing the protective antibody, was extracted from the hyperimmune sera in 20 order to evaluate protective efficacy on a milligrams-protein basis. Pre-immune and hyperimmune sera from individual horses hyperimmunized with vaccine were compared by inoculating subsets of CF-1 mice with various concentrations of gamma globulin in divided doses on 25 two consecutive days before intravenous challenge with endotoxin. The data in Figs. 2 and 3 compare pre-immune and hyperimmune globulin from two separate horses (#23 and #24) using the passive immunization mouse model. Hyperimmune globulin was prepared from 30 horse #2 3 at two subsequent times after hyperimmunization (#2 3A, #2 3B) . Comparison of the percentage of mice surviving 96 hours after endotoxin challenge that were passively immunized with 50 /ig or more of #23 pre-immune or hyperimmune (#2 3A and #23B) globulin indicates 35 at least a 20 (for #23B) to 50 (for #2 3A) percent increase in survival for those subsets receiving hyper- immune globulin (Fig. 2). Comparison of #24 pre-immune with #24 hyperimmune also indicates similar protective efficacy but to a lesser degree (Fig. 3).

It was concluded that the hyperimmune serum contains 5 antibodies which can passively protect mice from lethal endotoxin challenge.

The efficacy of the hyperimmune gamma globulin was ascertained by challenge (endotoxin or carbohydrate engorgement) of subsets of horses and ponies after 10 intravenous inoculation with 5, 15, or 20 mg antibody protein/kg body weight. All animals received a mixture of #23A and #23B hyperimmune gamma globulin. Combination of the two preparations was necessary in order to insure adequate quantities of the known antibody protein 15 to complete the studies. Protection was defined as the marked delay and/or amelioration of the immediate vital sign changes and development of equal to or less than Obel Grade 2 disease, in sublethal endotoxin and/or carbohydrate challenged animals.

Table 4: Passive Immunization of Horses with Pre-immune and Hyperimmune Serum Challenge Passive Immunization with Hyperimmune globulin Pre-immune globulin CHO - (Per Os) 40% 100% 25 Endotoxin (IV) 0% 50% Percentage figures represent developing Obel grade (3-4) laminitis or endotoxin-mediated symptomology after challenge.

Since many possible embodiments may be made of 30 the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. 2 9

Claims (7)

CLAIMS:

1. A vaccine for immunizing against Gram negative bacterial diseases comprising: a killed suspension of a non-O-Carbohydrate side chain bacterial mutant from the taxonomic family Enterobacteriaceae, a 5 protein or lipid binding carrier having a high lipophilic and high proteinophilic affinity to insure uniform component suspension and prolonged antigenic release, and an immune modulator having propensity for B-lymphocyte proliferation and preferably with high levels of 10 neutralizing and opsonizing antibodies of high antigenic affinity and avidity.

2. The vaccine of claim 1, wherein said bacterial mutant comprises Salmonella enteritidis.

3. The vaccine of claim 1 or 2, wherein said 15 bacterial mutant comprises ATCC 53000.

4. The vaccine of claims 1, 2 or 3, wherein said immune modulator is lypophilized detoxified lipopolysaccharide.

5. A method for preparing a vaccine for 20 immunizing against Gram negative bacteria diseases which steps comprise: (1) combining a killed suspension of a non-O-carbohydrate side chain bacterial mutant from the taxonomic family Enterbacteriaceae and an immune modulator having propensity for B-lymphocyte proliferation and with 25 high levels of neutralizing and opsonizing antibodies of high antigenic affinity and avidity; and (2) suspending said combination of bacterial mutant and immune modulator in a protein and lipid binding carrier having a high lipophilic and high proteinophilic affinity to insure 30 uniform component suspension and prolong antigenic release.

6. The method of claim 5, wherein preparing said bacterial mutant comprises: (1) inoculating a sterile, enriched broth with a bacterial subcultural mutant selected from the taxonomic family Enterobacteriaceae 35 such as Salmonella enteritidis: (2) incubating said broth aerobically at approximately 37°C. for a maximal bacterial mass; (3) killing said bacteria with a 3 0 bactericidal agent, such as merthiolate; (4) washing said bacteria, preferably with a sterile, non-pyrogenic physiologic saline; and (5) reconstituting said bacteria for a preselected concentration.

7. The method of claim 6, wherein preparing said immune modulator comprises: (1) admixing Gram negative bacterial endotoxin with pyridine-formic acid solution, said acid preferably in a concentration of 2:1; (2) methylating said admixture by total reflux distillation; (3) precipitating said methylated endotoxin admixture wich alcohol; (4) centrifuging the alcohol and methylated endotoxin admixture; and (5) mixing the precipitate with distilled water to a preselected concentration of endotoxin. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.