WO1993004672A1

WO1993004672A1 - Method for enhancing the immune system in a host employing liposome-encapsulated polypeptides

Info

Publication number: WO1993004672A1
Application number: PCT/US1992/007562
Authority: WO
Inventors: Carl K. Edwards, Iii; Aleksander Blum
Original assignee: Pitman-Moore, Inc.
Priority date: 1991-09-11
Filing date: 1992-09-04
Publication date: 1993-03-18
Also published as: AU2583892A

Abstract

A method for enhancing host protection against a pathogen by encapsulating a polypeptide into liposomes and administering a sufficient quantity of liposome encapsulated polypeptide to an animal such that the polypeptide enhances the ability of the phagocytic cells of the animal's immune system to kill a pathogenic microorganism.

Description

METHOD FOR ENHANCING THE IMMUNE SYSTEM IN A HOST EMPLOYING LIPOSOME-ENCAPSULATED POLYPEPTIDES

Background of the Invention This invention relates to a method for enhancing the immune system of an animal by encapsulating a polypeptide into liposomes and administering the encapsulated polypeptide to an animal in sufficient quantity to enhance the animal's immune system against a pathogen. More specifically, the invention relates to a method of encapsulating a polypeptide into multilamellar, unilamellar or any other suitable liposome and parenterally administering the liposome- encapsulated polypeptide to an animal in sufficient quantity to enhance the animal's immune system against pathogenic gram negative bacteria.

Some gram-negative bacteria, such as the Salmonella species, are facultative intracellular bacteria that are usually pathogenic to man and other mammals. In humans, the most common diseases caused by the Salmonella species are typhoid fever and gastroenteritis. Salmonella bacteria are capable of surviving within the phagocytic cells of the immune system. However, phagocytic cells such as macrophages (M ) long have been known to be particularly essential for host defense against Salmonella species. MΦ must be exposed to T cell-derived lymphokines such as interferon-c (IFN-c) and granulocyte-macrophage colony stimulating factor (GM-CSF) to fully exert their antibacterial activity. Both these substances prime phagocytic cells, such as MΦ, for release of reactive oxygen intermediates which are the most important oxygen-dependent pathway by which phagocytic cells kill intracellular microbes. U.S. Patent No. 4,842,862 discloses that Resorcylic Acid Lactone (RAL) derivatives zearalenone, zearalanone, zearalene, zearalanel, zearalenol, zearalanol and dideoxyzearalane nonspecifically stimulate an animal's immune system against various pathogens. The RAL derivatives can be administered to an animal orally or parenterally in aqueous or nonaqueous form. The RAL derivatives also can be administered to an animal in the form of a slow release implant. In addition, growth hormone, i.e., somatotropin, and prolactin also have been discovered to enhance a host's immune system against various pathogens by increasing the number of macrophages and by priming the macrophages to release reactive oxygen intermediates (see U.S. Patent No. 4,837,202). The immune enhancing effect of somatotropin has been tested in laboratory rats infected with the bacterium Salmonella typhimurlum. The ability of hypophysectomized rats, i.e., rats that have had their pituitary gland (source of growth hormone) experimentally excised, to survive Salmonella typhimurlum infections has been compared to that of rats having their pituitary intact. The hypophysectomized rats all die within a few days, while the pituitary-intact rats survive for a significantly (as assessed by chi square analyses) longer period of time. However, when the dose of Salmonella typhimurlum is reduced and the hypophysectomized rats are treated with somatotropin, IFN-c or tetracycline, the survival rate is enhanced. Further, it has been discovered that peritoneal MΦ from hypophysectomized rats that were infected in vitro with Salmonella typhimurium only killed half the number of extracellular bacteria as compared to pituitary-intact rats. This killing capacity was significantly augmented by 75% to 90% by pretreating the hypophysectomized rats with either somatotropin or IFN-c. These data establish that somatotropin augments the host's immune system against a facultative intracellular pathogen (C.K. Edwards, III, et al., "The Pituitary Gland is Required for Protection Against Lethal Effects of Salmonella typhimurium, " Proc. Natl. Acad. Sci. (in press)).

In addition to Salmonella species, somatotropin also has been shown to induce alveolar MΦ against respiratory pathogens such as Pasteurella multoclda , a gram negative bacterium. Porcine alveolar MΦ treated in vitro for 18 hours with either native porcine somatotropin or recombinant porcine IFN-c killed opsonized extracellular Pasteurella multoclda after a 4 hour period. Addition of 10⁸ viable Pasteurella multocida to untreated porcine alveolar MΦ resulted in 14 ± 5xl0⁶ colony forming units (CFU). Treatment of the porcine alveolar MΦ with native porcine somatotropin or recombinant porcine IFN-c reduced the CFU by 95% and 94% respectively (C.K. Edwards, III, et al., "Growth Hormone Enhances Bacterial Killing By Alveolar Macrophages", 1990 ASBMB/AA1, NIH AG06246, USDA 89- 37265-4536). These results imply a potential clinical application of somatotropin in enhancing a host's immune system against gram-negative bacterial infections.

Somatotropin is a polypeptide which upon oral ingestion is readily digested by the acids and enzymes of an animal's stomach. Also, large amounts of somatotropin can be degraded upon parenteral administration as well before the somatotropin can act on the immune system. In order for the somatotropin to be clinically effective, it desirably is incorporated into an appropriate vehicle.

Recently, it has been disclosed that liposomes, i.e., multilamellar or unilamellar concentric lipid bilayer vesicles with layer(s) of aqueous media separating the lipid bilayer(s) , have been used as carriers for therapeutic agents, anticancer drugs, antifungal agents and immunomodulators. Gabriel Lopez- Berestein and Isaiah J. Fidler, "Liposomes in the Therapy of Infectious Disease and Cancer, " UCLA Symposia on Molecular and Cellular Biology, New Series, Volume 89, (1989). It also has been implied that liposomes can be useful in improving the immune response to vaccines. Biotechnology News, Vol. 10, No. 13, p.3 (1989). Intravenous administration of liposomes containing muramyl tripeptide phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide that activates macrophages to a cytolytic state iii situ, have significantly protected mice against lethal challenges with herpes simplex virus type 2 (Wayne C. Koff, et al., "Protection of Mice Against Fatal Herpes Simplex Type 2 Infection by Liposomes Containing Muramyl Tripeptide," Science, Vol. 228, 1985). Antimonials, amphotericin B, and pentamide encapsulated in liposomes were found to be more effective than the free drugs for the treatment of leishmaniasis in hamsters (Amitabha Mukhopadhyay, et al. "Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis," Science, Vol. 244, 1989). Liposome encapsulated muramyl dipeptide and MΦ- activating factor have been shown to greatly enhance the immune system in tumor-bearing mice by activating MΦ against the tumorous cells (Charles Pidgeon, et al., "Macrophage Activation: Synergism Between Hybridoma MAF And Poly (1) I Poly (C) Delivered By Liposomes," The Journal of Immunology, Vol. 131, No. 1, 1983; Isaiah J. Fidler and Alan J. Schroit, "Synergism Between Lymphokines And Muramyl Dipeptide Encapsulated In Liposomes: In Situ Activation of Macrophages And Therapy of Spontaneous Cancer Metastases, " The Journal of Immunology. Vol. 133, No. 1, 1984). The type(s) of liposomes employed as vehicles as well as the methods of making them and the types of drugs or pharmaceutical agents encapsulated within the liposomes vary widely. U.S. Patent No. 4,394,448 discloses liposomes composed of phospholipid bilayers into which DNA material is encapsulated. The liposome-encapsulated DNA then is used to insert the DNA into a host cell by contacting the membrane of the target cell with the liposome. The liposome is taken up by the cell through fusion of the liposome with the cellular membrane or by endocytosis. The liposome protects the DNA from degradation during the insertion process.

U.S. Patent Nos. 4,721,612 and 4,891,208 disclose liposomes composed of bilayers comprising the salt form of an organic acid derivative of a sterol such as the tris-salt form of a sterol hemisuccinate. The sterol hemisuccinate liposome can entrap bioactive agents of limited solubility, such as growth hormone. Bovine growth hormone encapsulated in the sterol hemisuccinate liposome can be administered intramuscularly to cows to initiate growth or increase milk production.

U.S. Patent No. 4,708,861 discloses liposomes containing a bioactive agent wherein the liposomes are dispersed within a gel-matrix. The gel-matrix provides for prolonged release of the liposome-entrapped bioactive agent. The liposome bilayer can be composed of phospholipids and related chemical structures as well as steroids, such as cholesterol. The gel-matrix is composed of carbohydrates such as cellulosics. Bioactive agents such as somatotropin and other peptides and the like can be encapsulated in the liposome gel-matrix. However, the disclosure does not teach or suggest that the liposome incorporated polypeptides can enhance an animal's immune system.

None of the current literature discloses a method by which polypeptides can be optimally utilized to enhance a host's immune system against a pathogen. Therefore, there is still a need for a method which can further enhance the effect of polypeptides on a host's immune system.

Summary of the Invention In accordance with the present invention, a polypeptide is encapsulated into liposomes, and the liposome-encapsulated polypeptide is administered to a host in sufficient quantities to enhance the host's immune system against a pathogen. The liposome- encapsulated polypeptide enhances the host's immune system to a greater degree than if the polypeptide is administered to the host in non-liposome-encapsulated form. The liposome-encapsulated polypeptide is especially effective in enhancing the immune system against gram-negative bacteria, particularly against Salmonella and Pasteurella species. Brief Description of the Drawings Figure 1 illustrates the survival rate of hypophysectomized (Hypox) rats infected with S. typhimurium (% survival vs. days after injection with S. typhimurium) after being pretreated with different compositions of recombinant rat IFN-c (rRalFN-c), native, pituitary-derived porcine somatotropin, recombinant porcine somatotropin, native, pituitary- derived bovine somatotropin and tetracycline. Figure 2 illustrates the survival rate of hypophysectomized (Hypox) rats (% survival vs. days after injection with S. typhimurium) infected with S. typhimurium from an experiment indicating that rats pretreated with increasing amounts of recombinant porcine somatotropin have enhanced host-protection against S. typhimurium .in vivo.

Figure 3 illustrates that hypophysectomized rats treated with liposomes encapsulated with recombinant porcine somatotropin have enhanced host-protection against S. typhimurium when compared to somatotropin- free liposomes or recombinant porcine somatotropin given in saline.

Detailed Description of the Invention The present invention relates to a method of enhancing a host's immune system against a pathogen by administering a sufficient amount of liposome- encapsulated polypeptide to enhance phagocytic cell activity against the pathogen. Specifically, the liposome-encapsulated polypeptide is internalized by the phagocytic cells of the immune system, such as the polymorphonuclear leukocytes and/or the MΦ, when the liposome contacts the phagocytic cell's membrane and the lipid components of the liposome and the cell membrane fuse or by the process of endocytosis. The polypeptide then assists the phagocytic cell to produce reactive oxygen intermediates which are toxic to many types of intracellular microbial pathogens such as Salmonella and Pasteurella species.

The liposomes employed to practice this invention are completely closed bilayer membranes containing an aqueous phase. The liposomes may be any variety of mult la eliar vesicles (elliptical-like structures characterized by concentric membrane bilayers each separated by an aqueous layer) or unilamellar vesicles (possessing a single membrane bilayer) . The size of the liposomes can vary widely. Typically, the size of the liposomes prepared according to the method of the present invention will be less than 1 micron in size. Any chemical compound capable of forming a completely closed bilayer, i.e., liposome, in aqueous solutions can be used to practice this invention. Most amphipathic lipids can be used as constituents of the liposome bilayer. Suitable hydrophilic groups include, but are not limited to, phosphatidic , sulfatidic, carboxylic and amino groups. Suitable hydrophobic groups include, but are not limited to, saturated and unsaturated aliphatic hydrocarbon groups and aliphatic hydrocarbon groups substituted by at least one aromatic and/or cycloaliphatic group. The preferred amphipathic compounds are phospholipids and closely related chemical structures. Examples of these include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, lysolecithin, lyso- phosphatidylethanolamine, sphingomyelin, cardiolipin, phosphatidic acid, the cerebrosides, natural lecithins (e.g., egg lecithin or soybean lecithin) and synthetic lecithins such as saturated synthetic lecithins (e.g., dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoyl- phosphatidylcholine) and unsaturated synthetic lecithins (e.g., dialoylphosphatidylcholine or dilinoloyl-phosphatidylcholine) .

In addition to amphipathic compounds, a steroid component can be incorporated into the lipid bilayer to increase the amount of water insoluble or sparingly- water soluble bioactive agents such as somatotropin which can be encapsulated by the liposomes

("encapsulation" is defined as th^'e entrapment of the bioactive agent within the aqueous compartment and/or within the membrane bilayer). This enables the administration iji vivo of water-insoluble compounds, and it allows for the administration in vivo of high concentrations of water insoluble compounds or sparingly water-soluble compounds because it increases the dose:volume ratio of the compound. The cholesterol also helps form a more closely packed bilayer system during preparation and inhibits the premature break¬ down of the liposomes by the intersticial and serum proteins of the host animal. The steroids employed to practice this invention include, but are not limited to, cholesterol, coprostanol, cholestanol, cholestane, coprostane or epicholesterol and the like.

The liposomes used to practice the present invention can be prepared by any number of methods which are currently practiced in the art. Some examples of the methods that can be employed to prepare the liposomes include, but are not limited to, the methods of Bangham et al. (1965, J. Mol. Biol. 13:238- 252), Popahadjopoulos and Miller (1967, Biochem. Biophvs. Acta. 135:624-638), and Szoka and Popahadjopoulos in 1980, Ann. Rev. Biophvs. Bioen . , 9:467-508, and U.S. Patent Nos. 4,708,861, 4,235,871 and 4,891,208.

The preferred method involves preparing a methanol solution of phospholipids and mixing it with a solution of a steroid, such as cholesterol, in an organic solvent. Many organic solvents are suitable, but halogenated hydrocarbons, diethyl ether or mixtures of halogenated hydrocarbons and diethyl ether are preferred. Other organic solvents which can be employed as the solvent in the phospholipid or steroid mixture include, but are not limited to, ethanol, 2- propanol, isopropyl alcohol or combinations thereof. Preferably, the mole ratio of phospholipid to steroid is about 2:1 respectively, but it can range from as low as about 1:1 to as high as about 3:1. The mixture is stirred to form a uniform solution and then the solvent is removed by evaporation. Evaporation can be accomplished by any evaporative technique, e.g., by passing a stream of inert gas over the mixture, by heating, by vacuum or any combination thereof. The dried lipid layer then is reconstituted with a neutral to slightly basic buffer solution (pH of about 7.0 to about 7.8) such as Tris buffer or HEPES buffer and the like. The liposomes are formed by sonicating the solution in any appropriate sonicator. Sonicating the solution for about 5 minutes to about 15 minutes forms multila ellar vesicles (MLVs) while sonicating the solution for about 2 hours to about 4 hours forms unilamellar vesicles (UVs). An aqueous solution containing from about 30 mg/ml to about 40 mg/ml of a polypeptide, such as somatotropin, from about 250 mg/ml to about 400 mg/ml of the chloride salt of an amino acid and from about 65 mg/ml to about 85 mg/ml of a carbohydrate is mixed with the reconstituted liposomes. It is not necessary to employ a carbohydrate in preparing the liposomes of the present invention. However, it is preferable to incorporate a carbohydrate into the liposomes since a carbohydrate can enhance the long-term stability of the liposomes. Suitable carbohydrates include, but are not limited to, trehalose, sucrose, glucose, lactose, dextran and the like. A preferred carbohydrate is trehalose. The mixture then is either sonicated or microfluidized. Preferably, the mixture is microfluidized. Microfluidization is a process which separates fluids into two phases and then allows the phases to rejoin to form lipophilic particles. Microfluidization is performed at from about 10,000 psi to about 20,000 psi for about 5 to about 15 cycles at room temperature (about 18°C to about 23°C).

The solution of liposome-encapsulated polypeptide then is added to a dilute aqueous mixture of a suspending agent (about a 1:3 ratio respectively) to disperse the liposome-encapsulated polypeptide to form a uniform suspension. The dilute aqueous mixture of the suspending agent ranges from about 1% w/v to about 5% w/v of suspending agent to water. The suspending agents employed to practice this invention include, but are not limited to, carbohydrates such as cellulosics, methylcellulose, starch and modified starch, agarose, gum arabic, ghatti, karay, tragacanth, guar, locust bean gum, tamarind, carrageenan, alginic acid, sodium alginate, xanthan, chickle, collagen, polyacrylamide, polysiloxanes, polyanhydrides, polyacrylates and amino acid polymers such as gelled albumin and other organic or inorganic polymers which can be mixed with liposomes in vitro. The solution containing the liposome-encapsulated polypeptide is mixed with the suspending agent for about 2-5 minutes. The resulting suspension can be divided up into desired individual volumes, lyophilized and then stored in a freezer for later use.

The amount of polypeptide encapsulated ranges from about 25% to about 45% of the mass of the lipids comprising the liposomes. Encapsulated mass is the mass of the substance encapsulated per unit mass of the lipid. The term is commonly used to express the effectiveness of encapsulation and is expressed as a percentage. Any polypeptide which stimulates an animal's immune system and can be incorporated into liposomes can be employed to practice this invention. Examples of preferred polypeptides include, but are not limited to, human somatotropin, bovine somatotropin, porcine somatotropin, ovine somatotropin, and the like. Recombinant forms of the foregoing somatotropins also can be employed, as well as any physiologically active fragment or analog thereof. The preferred somatotropin used to practice this invention is a recombinant porcine somatotropin, such as delta-7 recombinant porcine somatotropin, described in European Patent Application Publication No. 104,920 (Biogen) , incorporated herein by reference.

Other suitable polypeptides which can be employed to practice this invention include, but are not limited to, growth factors and any physiologically active fragments and analogs thereof. Growth factors which can be employed to practice this invention include, but are not limited to, prolactin (PRL) , insulin-like growth factor I (IGF-I) and insulin like growth factor II (IGF-II), nerve growth factor (NGF), platelet- derived growth factor (PDGF), transforming growth factor- (TGF-α), transforming growth factor-β (TGF- β), fibroblast growth factor (FGF), epidermal growth factor (EGF), vaccinia growth factor (VGF) and the like. Recombinant forms of the foregoing growth factors also can be employed as well as any physiologically active fragment thereof. Preferred growth factors include recombinant human insulin-like growth factor I (rHuIGF-I) and recombinant human insulin-like growth factor II (rHuIGF-II). The lyophilized liposome-encapsulated polypeptide can be reconstituted with sterile water for injection, bacteriostatic water for injection or any suitable aqueous preparation that can be safely administered parenterally to an animal. Such routes of administration include, but are not limited to, inoculation or injection, (e.g., intraperitoneal, intramuscular, subcutaneous, intra-articular, intra- mammary, etc.). A sufficient amount of the liposome- encapsulated polypeptide administered to an animal, the sufficient amount varying from one animal species to another, enhances the animal's immune system against many types of gram-negative bacteria such as various Salmonella species and Pasteurella species. The amount of liposome-encapsulated polypeptide that can be administered to the host typically ranges from about 1 mg/kg to about 100 mg/kg of body weight. Preferably, from about 5 mg/kg of body weight to about 50 mg/kg of body weight is administered. In particular, the liposome encapsulated somatotropin is especially effective in enhancing an animal's immune system against Salmonella typhimurium, Salmonella typhosa, Salmonella paratyphosa, and Pasteurella multocida .

The invention is further illustrated by the following examples. Various modifications can be made without departure from the spirit and scope of the invention. Accordingly, it is not intended that the invention be limited except as by the claims.

Example I Preparation of EPC/Cholesterol Liposomes and Encapsulation of Somatotropin

A sufficient quantity of a solution of egg phosphatidylcholine (EPC) in methanol was mixed with a sufficient quantity of a solution of cholesterol in chloroform to form a uniform mixture of EPC/cholesterol having a molar ratio of 2:1 respectively. The solution then was dried under nitrogen with vacuum desiccation for about twelve hours. The dried lipid layer was reconstituted with a sufficient amount of 30mM Tris buffer to form a concentration of solute to solvent of 75 mg/ml. The solution was sonicated for 2 hours in a Branson 5200 sonicator forming liposomes with unilamellar vesicles (UVs). A solution containing 35 mg/ml zinc-complexed recombinant porcine somatotropin (ZnrpST) (Pitman-Moore Inc., lot pST D 148 010/011-

802, obtained in accordance with the general procedures disclosed in European Publication No. 216,485), 300 mg/ml of arginine hydrochloride and 75 mg/ml of trehalose was added to the liposome solution for encapsulation of rpST into the liposomes. The solutions were mixed and microfluidized at 13,000 psi for 10 cycles at 21°C to incorporate the rpST into the liposomes. The liposome solution then was added to a 4% w/v (40 mg/ml) sodium alginate aqueous composition in a ratio of 1:3, respectively, and mixed for 5 minutes. The resulting uniform suspension then was aliquoted into 5 ml portions and lyophilized. The resulting liposome encapsulation (encapsulated mass) of rpST was approximately 35% of the mass of lipids in the liposomes.

Example 2

Enhanced Host Protection in Hypophysectomized (Hypox) Rats Treated with Porcine or Bovine Somatotropin

One-hundred thirty-two female hypophysectomized rats were employed to determine the effectiveness of somatotropin in enhancing host protection against Salmonella typhimurium. The intact rats were obtained from Holtzman Company, Madison, Wisconsin, and they were hypophysectomized at Johnson Laboratories, Bridgeview, Illinois, before they were delivered to Pitman-Moore, Inc., Terre Haute, Indiana, for study. The rats were divided into five groups, each group comprising at least twelve rats. Each group was treated with a different preparation. The first group of thirty-six rats was treated with a placebo composed of Parlow's Buffer at a dose of 200 μl/rat/day (negative control); the second group of thirty-six rats was treated with recombinant rat interferon gamma (rRalFN-c) at a dose of 500 U/rat/day with a specific activity of 4xl0⁶ U/mg of protein (positive control) (obtained from AMGEN, La Jolla, CA) ; the third group of twenty-four rats was treated with pituitary-derived porcine somatotropin (npST) at a dose of 48 μg/rat/day (obtained from A.F. Parlow, UCLA Medical Center, Torrence, CA ); the fourth group of twenty-four rats was treated with bovine pituitary-derived somatotropin (nbST) at a dose of 48 μg/rat/day (obtained from A.F. Parlow, UCLA Medical Center, Torrence, CA) ; and the fifth group of twelve rats was treated with recombinant delta-7 porcine somatotropin (rpST) at a dose of 48 μg/rat/day (obtained from Pitman-Moore, Terre Haute, IN). Each rat was treated subcutaneously (S.C.) with the respective preparation for nine consecutive days. After the nine day treatment period, each rat was infected intraperitoneally (i.p.) with an infectious dose of Salmonella typhimurium, 7.5xl0⁷ viable colony forming units (CFU), (strain 84-4728, supplied by B.O. Blackburn, U.S.D.A. Infectious Disease Center, Ames IA) . Treatment regimens were continued for an additional six days after i.p. administration of the infectious bacteria.

Survivability of the hypophysectomized rats was followed for 14 days. Table I discloses the percent survivability of the hypophysectomized rats treated with the six preparations. Chi square analysis was performed as pre-planned comparison on day 7 after infection. By the seventh day, all rats treated with buffer had died (survivor/total = 0/36, 0%), whereas npST (10/24, 42%), nbST (9/24, 38%), rpST (8/12, 67%) and rRalFN-c (20/36, 56%) all significantly increased (P<0.05) the survival rates. It is apparent from the results in Table I that treatment with somatotropin for the additional six days after i.p. administration of the infectious bacteria significantly enhanced the rats' immune system against Salmonella typhimurium.

Figure 1 is a plot of the data from Table I, and more clearly illustrates the increased survival rates of rats treated with the rRalFN-c and somatotropin versus the placebo. TABLE I Percent Survival

Example 3

Enhanced Host Protection in Hypophysectomized (Hypox) Rats Treated with Increasing Concentrations of Recombinant Porcine Somatotropin (rpST)

This experiment was performed to verify that the effect of npST was not due to contaminating proteins.

Graded doses of rpST, were administered to a completely different group of hypophysectomized female rats (obtained from Holtzman Company, Madison, Wisconsin, and they were hypophysectomized at Johnson

Laboratories, Bridgeview, Illinois) according to the protocol outlined in Example 2. Fifteen rats were treated with placebo at a dose of 200 μl/rat/day; fifteen rats were treated with rRalFN-c at a dose of

500 U/rat/day; and the remaining rats were treated with graded doses of rpST. A total of 84 hyposectomized female rates were treated. Seven days after infecting the rats with 7xl0⁶ CFU of Salmonella typhimurium, survival rates of rats injected with 24, 48 and 96 μg/rat/day of rpST were 33% (4/12), 67% (10/15) and 67%

(8/12), respectively. The data from this experiment is tallied in Table II. All doses of rpST, as well as npST at 48 μg/rat/day (8/15 for 53% survival) and rRalFN-c at 500 U/rat/day (8/15 for 53% survival), significantly increased (P<0.01) survival rates compared to placebo-treated rats (0/15 for 0% survival) . Consequently, since the survival rates of rats treated with npST fall within the statistical range (p<0.01) of rats treated with rpST and rRalFN-c, it is clear that the effect of npST was not due to contaminating proteins. Figure 2 is a plot of the data from Table II which more clearly illustrates the increased survival rates of hypophysectomized rats treated with somatotropin or interferon-gamma versus the placebo.

TABLE II Percent Survival

Example 4

Enhanced Host Protection in Hypophysectomized (Hypox) Rats Treated With Recombinant Porcine Somatotropin This experiment demonstrates that liposome- encapsulated rpST improves host protection against Salmonella typhimurium over that of a host treated with placebo, rRalFN-c, unencapsulated rpST or "free liposomes. " Sixty female rats were studied to determine the effectiveness of liposome-encapsulated recombinant porcine somatotropin (rpST) in enhancing host protection against Salmonella typhimurium. The rats were obtained from the same source and hypophysectomized as the rats in Examples 2 and 3. The rats were divided up into five groups, each group comprising twelve rats. Each group was treated with a different preparation. The first group of twelve rats was treated with a placebo composed of Parlow's Buffer (negative control) at a dose of 200 μl/rat/day; the second group of rats was treated with rRalFN-c

(obtained from AMGEN, La Jolla, CA) (positive control) at a dose of 500 units/rat/day; the third group received rpST (Pitman-Moore) at a dose of 48 μg/rat/day; the fourth group was treated with "free liposomes" (free of rpST) at a dosage of 0.2 ml/rat/day; and the fifth group was treated with liposome-encapsulated rpST at a dosage of 48 μg/rat/day. The liposome-encapsulated rpST was prepared according to the method disclosed in Example 1. The free liposomes were prepared by the same method as the liposomes in Example 1 except rpST was not employed in the preparation.

Each rat was infected intraperitoneally (i.p.) with an infectious dose of Salmonella typhimurium (6.7xl0⁷ CFU). After infection with the bacteria, each rat was treated subcutaneously (s.c.) with the respective preparation for six consecutive days. Survivability of the hypophysectomized rats was followed for 14 days. Table III discloses the survivability of the hypophysectomized rats treated with the five preparations. All rats which were treated with placebo died by the seventh day (0/12, 0%) whereas rpST (8/12, 67%), rpST/liposomes (10/12, 83%) and rRalFN-c (8/12, 67%) all significantly increased (P<0.05) host survival rates. There were no significant (P>0.05) host protection differences (by chi square analysis) between the liposome (6/12, 50%), rpST and rpST/liposome-treated rats until day twelve. By the twelfth day, as can be seen by the data in Table III, there was a significant (P<0.05) difference in survival rates between the rpST/liposome (9/12, 75%) and rpST (4/12, 33%) treated rats. Figure 3 is a plot of the data from Table III, and more clearly illustrates the increased survival rates of the rats treated with rpST or rRalFN-c, particularly of liposome-encapsulated rpST, versus the rats treated with placebo.

TABLE III Percent Survival

Claims

I Claim:

1. A method for enhancing host protection against a gram-negative bacterium comprising:

(a) encapsulating a polypeptide which stimulates an animal's immune system into liposomes; and

(b) administering a sufficient quantity of the liposome-encapsulated polypeptide to the animal to enhance the animal's immune system against a gram- negative bacterium.

2. The method according to claim 1, wherein the liposome comprises a bilayer composed of one or more phospholipids and one or more steroids.

3. The method according the claim 2, wherein the phospholipids comprise phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, cardiolipin, lecithin, lysolecithin, lysophatidylethanolamine, phosphatidic acid or mixtures thereof.

4. The method according to claim 2, wherein the steroid comprises cholesterol, coprostanol, cholestanol, cholestane, coprostane or epicholesterol or mixtures thereof.

5. The method according to claim 1, wherein a stabilizing amount of a carbohydrate is encapsulated within the liposomes along with the polypeptide.

6. The method according to claim 5, wherein the carbohydrate comprises trehalose, sucrose, lactose, glucose or dextran.

7. The method according to claim 1, 2, 3 4, 5 or 6, wherein the liposomes are unilamellar.

8. The method according to claim 1, 2, 3 4, 5, or 6, wherein the liposomes are multilamellar.

9. The method according to claim 1, wherein the polypeptide comprises a somatotropin or a growth factor.

10. The method according to claim 9, wherein the somatotropin comprises natural or recombinant human somatotropin, bovine somatotropin, porcine somatotropin, ovine somatotropin or a bio-active fragment or analog thereof.

11. The method according to claim 10, wherein the somatotropin is recombinant porcine somatotropin.

12. The method according to claim 9, wherein the growth factor comprises prolactin, insulin-like growth factor I, insulin-like growth factor II, nerve growth factor, platelet-derived growth factor, transforming growth factor-α, transforming growth factor-β, epidermal growth factor, vaccinia growth factor, fibroblast growth factor or physiologically active fragments or analogs thereof.

13. The method according to claim 1, wherein the liposome-encapsulated polypeptide is dispersed in an aqueous composition comprising a suspending agent.

14. The method according to claim 13, wherein the suspending agent is sodium alginate.

15. The method according to claim 1, wherein the liposome encapsulated polypeptide is administered to the animal subcutaneously, intravenously, intraperitoneally, intramuscularly or intra-arterially.

16. The method according to claim 1, wherein the liposome encapsulated polypeptide enhances the animal's immune system against a gram-negative bacterium selected from Salmonella typhimurium, Salmonella typhosa , Salmonella paratyphosa or Pasteurella multocida .

17. The method according to claim 1, wherein the amount of the liposome-encapsulated polypeptide administered to an animal ranges from about 1 mg/kg to about 100 mg/kg of the animal's body weight.

18. The method according to claim 9, wherein the amount of the liposome-encapsulated somatotropin preferably ranges from about 5 mg/kg to about 50 mg/kg of the animal's body weight.

19. A method for enhancing host protection against a gram-negative bacterium comprising:

(a) encapsulating recombinant porcine somatotropin and a stabilizing amount of trehalose into liposomes having a bilayer composed of phosphatidyl- choline and cholesterol;

(b) dispersing the liposome encapsulated somatotropin into a mixture of sodium alginate in water to form a uniform suspension; (c) administering a sufficient quantity of the suspension of the liposome encapsulated recombinant porcine somatotropin to an animal subcutaneously to enhance the animal's immune system against the gram- negative bacterium.

20. A method for enhancing host protection against a gram-negative bacterium comprising:

(a) encapsulating one or more growth factors into liposomes; (b) dispersing the liposome encapsulated growth factor into a mixture of sodium alginate in water to form a uniform suspension; and

(c) administering a sufficient quantity of liposome-encapsulated growth factor to an animal to enhance the animal's immune system against a gram- negative bacterium.

21. The method according to claim 20, wherein the growth factor comprises recombinant human insulin-like growth factor I or recombinant human insulin-like growth factor II.