KR19990028383A - New Adjuvant Compositions and Vaccine Formulations Containing the Same - Google Patents

Abstract

The present invention relates to an adjuvant comprising a polysaccharide-phospholipid conjugate that is administered to a host animal to enhance an immune response, wherein the polysaccharide site in the polysaccharide-phospholipid defect is bound to phospholipids, e.g. Nitrided glucan, chitosan, or alginate polysaccharides. Adjuvants may be provided with therapeutic vaccines for various disease states and / or pathological diseases by appropriate formulation with the antigen.

Description

New Adjuvant Compositions and Vaccine Formulations Containing the Same

Injection of antigens in animals is effective to produce antisera (i.e. serum antibodies) or increase antiserum concentrations in animals in order to produce antisera for protecting (i.e. vaccination) or for separate use in other animals. It has long been considered a way.

Vaccines occupy a unique place in health care because, unlike most treatments, vaccines are given to healthy people to prevent disease. Since vaccination usage is a primary factor in suppressing many infant diseases, numerous efforts have been made to expand the use of vaccines. Vaccines against many diseases are being developed, including cholera, malaria, herpes, smallpox and pneumonia.

It has also been known for a long time that by using any adjuvant, it can increase the appropriate amount of antiserum produced in response to foreign antigens and provide long-term protection against the unwanted action of antigen readers or pathogens carrying such antigens. Coming. Such adjuvants may affect cell-mediated immunity as well as titration, duration, isotype and avidity of the antibody. Research and development efforts are focused on the development of vaccine adjuvant that enhances the body's immune response to long-term vaccines.

Effective adjuvant formulations in the form of Freund's Complete Adjuvant (FCA) or Freund's Incomplete Adjuvant (FIA) are known since the mid 1930s and have been used to increase the production of antisera that are resistant to heterologous antigens in experimental animals.

The main characteristic of Freund's complete and Freund's incomplete adjuvant is that the antigen is emulsified in mineral oil to ensure a sustained release depot of the antigen at the injection site upon injection. Freund's complete adjuvant contains killed mycobacteria and apparently acts on the denatured protein by selectively including antibodies against the epitope. The result is a high concentration of antiserum when compared to the antigen alone. However, Freund's complete and unsafe supplements are known to cause significant toxicity complications.

FCA includes chronic granulomas and / or malignant tumors as well as pain as the development of chronic pain and undesirable side effects. For this reason, FCA is not allowed at all for human or veterinary vaccines in the United States.

The main purpose in the field of vaccine development is to prepare vaccine formulations that include the efficacy of an adjuvant such as FCA while eliminating the harmful side effects. Attempts to reduce the toxicity while retaining the efficacy of Freund's adjuvant have been largely unsuccessful, in part because of a lack of understanding of the specific biological mechanisms for adjuvant efficacy.

Several adjuvant products on the market are more stable than Freund's adjuvant, but have significantly lower efficacy than Freund's adjuvant. For example, oil and water emulsions with antigens absorbed on oils with short retention are commercially available. Commercially available aluminum hydroxide can also be used to directly absorb antigen onto this aluminum hydroxide. In other experimental products not available to humans sold under the trade name Adjuvax, the antigen is physically contained in the polysaccharide matrix of glucan polysaccharides.

Early adjuvants were substances of biological origin that enhanced specific antibody responses. Because of the effective use of mycobacteria as an adjuvant, attempts have been made to separate the biologically active components of mycobacteria corresponding to immune stimulation from mycobacterial cell walls. Lipid fragments extracted from mycobacteria contain trehalose dicholate (TDM) as the active ingredient, while mycobacterial cell walls are known as muramyl dipeptide (MDP) as the active ingredient, N-acetylmuryl-L-alanine- It contains D-isoglutamine. Lipopolysaccharides obtained from the cell walls of these Gram-negative bacteria show immunostimulatory activity, but their use is hampered due to toxicity due to their lipid A sites. Saccharomyces cerevisiae , glucans by yeast, and β-1,3-polyglucose are known to induce antitumor activity, improve resistance to microbial pathogens, and stimulate antibody responses against various antigens. It has been reported.

Toxicological problems associated with supplements of microbial origin have led to studies based on non-microbial materials. Such non-microbial materials include surfactants, salts, sugars, polyribonucleotides, and natural materials of mammalian origin. Nonionic and cationic surfactants have achieved successful results as adjuvants, while more effective affinity surfactants are more effective. Since saponin has amphoteric surface activity, its mechanism, including adjuvant activity, may be similar to that of surfactants. Saponins are not used in human vaccines due to toxicological problems. Because lymphokines and monocaines have very short biological half-lives, pharmacodynamic relationships hinder their use as adjuvants.

In typical vaccine formulations comprising antigens and carriers, the difference between a carrier and an adjuvant cannot always be clearly distinguished, because a number of carriers, such as adjuvant, can form immunostimulatory action and / or sustained release of the antigen. Because it has activity. Aluminum salts, for example, are the most widely used carriers in vaccines licensed for human and veterinary use. Antigens remain in the aluminum gel and are released slowly over a period of time, forming a continuous challenge in the immune system. In addition to this apparent carrier action, aluminum salts can act as pure carriers by their chemotaxis to various immunological cells. Other examples of carriers having activity such as adjuvants include water / oil emulsions, oil / water emulsions, microencapsulating agents, liposomes.

For more than twenty years, liposomes have been known to have the ability to stimulate antibody responses, but problems still remain in the development of suitable components for liposomes as carriers for vaccines. The antigen may be encapsulated into the aqueous phase of the liposome core or attached to the outer surface of the lipid bilayer. In addition, the adjuvant properties of liposomes can be enhanced by containing any immunostimulant such as lipid A, lipopolysaccharide, or MDP. Liposomes display their adjuvant properties by being preferentially occupied by macrophages, but release of liposomes does not provide sustained release of antigen.

Nanoparticles, synthetic polymers of 10 to 1000 nm, such as polymethylmethylacrylate, solid colloidal particles are reported to be effective aids in which antigens are encapsulated within nanoparticles or adsorbed onto nanoparticle surfaces. Adjuvant action of such vaccines increases hydrophobicity and reduces particle size. Because polymethylmethacrylate nanoparticles biodegrade at a slow rate, adjuvant action can occur by continuous antigen administration to the immune system.

According to a recent study evaluating different adjuvants that have antibody-inducing performance in mice against HIV-2 split whole virus, polymethylmethacrylate nanoparticles are considered to have an immune response and observable toxic side effects. Overall it was the best supplement. However, the study data suggest that two or more different adjuvants may be needed to elicit an essential immune response against physically different antigens. According to an alternative explanation of the study data, the immunological response to each antigen is maximized by a unique adjuvant. In both cases, the development of alternative adjuvants is important for the successful development of potent vaccine formulations.

Various other attempts are disclosed in the technical and patent literature for improved adjuvants.

U. S. Patent No. 5,273, 965 discloses a compound of sponins that can be used to administer the vaccine via nasal sprays or drops.

Infection and Immunity , Sept. 1991, pp. 2978-2986 discloses poly (DL-lactide-co-glycolide) microspheres useful as an adjuvant for staphylococcal enterotoxin B toxoid.

U.S. Patent 5,057,503 (J.K.Czop et al.) Discloses biologically active small molecular weight oligosaccharides that interact with β-glucan receptors on mammalian phagocytes. This unit ligand composition, heptaglucoside, has been described as being inducible using 2-aminopyridine to enhance the ability of glucosides to stimulate β-glucan receptors and to increase the action regulated by such receptors. . Heptaglucoside has been described as being useful for the treatment of vaccines or other immunomodulatory agents, such as adjuvant.

U.S. Pat.No. 5,189,028 (LH Nikl et al.) Describes the administration of β-1,3 glucans, specifically β-1,3 glucans with β-1,3-crosslinked backbones with β-1,3 crosslinked single glucose side chains. To stimulate the immune system.

U.S. Pat. No. 4,981,684 (NM Mackenzie et al.) Discloses an immune system that is dissolved with solubilizers such as surfactants, urea or guanidine and subsequently mixed with glucosides, sterols, and optionally phospholipids to substantially eliminate solubilizers. Formulations of an adjuvant matrix comprising a water insoluble antigen to form a stimulatory complex are disclosed.

U.S. Patent No. 5,032,401 to S.James discloses pharmaceuticals containing pharmaceutically active substances such as spherical glucan particles and drugs or antigens contained within these spherical glucan particles and which are uniformly dispersed or chemically crosslinked with the glucan particles. The composition is disclosed.

U.S. Pat.Nos. 5,091,187 and 5,091,188 (DH, Haynes) disclose lipid-coated microcrystals or microparticle compositions that give injectable delivery forms for sustained release when administering a water insoluble drug to a mammalian host and have. Pharmaceutically active agents are produced in the form of solids coated with membrane-forming lipids that stabilize the active ingredient by hydrophobic and hydrophilic interactions. These active solid component-containing particles are prepared in finely divided forms into smaller sizes by other methods, including sonication or high shear. U.S. Patent No. 5,246,707 discloses sustained free release of drugs, microdrolets and high concentration liposomes using biomolecules and phospholipid-coated microcrystals. Phospholipid-coated microcrystals and phospholipid-coated microdroplets are disclosed to be unavailable vaccine adjuvants.

Accordingly, it is an object of the present invention to provide improved adjuvants that have immunostimulatory properties but no toxic side effects.

Another object of the present invention is to provide a composition comprising a safe and effective adjuvant in use.

Other objects and advantages of the present invention will become more fully apparent from the following detailed description and the appended claims.

The present invention relates to adjuvant compositions and vaccine formulations containing them as well as methods of making and using such adjuvant and vaccines.

1 shows bovine immune serum (BSA), BSA in microdroplet form (BSA-MD), BSA in microdroplet form (BSA-GMD) with β-glucan conjugate adjuvant according to one embodiment of the invention, and Freund It is a graph showing the titration of antibodies in mice when vaccinated using BSA with complete adjuvant (FCA).

Figure 2 shows bovine immune serum (BSA), BSA in microdroplet form with β-glucan conjugate adjuvant according to one embodiment of the invention (BSA-MD), and BSA with Freund's complete adjuvant (BSA-FCA) When vaccinated using a graph, it is a graph showing the appropriate amount of IgG in mouse plasma in 1: 4096 dilution 1, 2 and 3 months after injection.

Detailed Description of the Invention and Preferred Methods for Carrying Out the Invention

The present invention, when forming a conjugate with a phospholipid form adjuvant, (i) increases titer, duration, isotype and antibody binding in the host animal, and (ii) lower toxicity in the host animal compared to Freund's adjuvant. It is based on the surprising and unexpected discovery of polysaccharides with good efficacy and safety.

The polysaccharide used to prepare the adjuvant of the present invention may include any polysaccharide, for example polysaccharide with immunostimulatory activity.

Polysaccharides that can be used in the adjuvant compositions in the broad practice of the present invention are described in "Carbohydrates Chemistry" (authors John F. Kennedy, Clarendon Press, Oxford, 1988); "Carbohydrates, Chemistry and Biochemistry" (authors W. Pigman and D. Horton, Academic Press, Inc., 1970); "Chitin, Chitosan, and Related Enzymes" (author John P. Zikakis, Academic Press. Inc., 1984). Particularly preferred polysaccharides include β-glucans, chitosan, galactomanans and alginates, with β-glucans being the most preferred in recent years.

As mentioned above, the adjuvant of the present invention may be prepared from polysaccharides and phospholipids using any suitable reagent, including divalent functional groups or other polyfunctional reagents, and by any suitable synthetic method.

Most commonly, polysaccharides form complexes by binding to phospholipids by reaction, which involves the oxidation or other functional reaction of polysaccharides to produce functional polysaccharides in a form capable of binding to phospholipids. It may include.

For example, the adjuvant may react a polysaccharide with an oxidant to form an aldehyde functional group on the polysaccharide, and then react the aldehyde-functional polysaccharide with a suitable divalent functional reagent to form a functional poly having a crosslinking functional group. Generate saccharides. This crosslinking functional group reacts with the selected phospholipid to further produce a polysaccharide-phospholipid conjugate.

In one specific synthesis within the broad scope of the present invention, the divalent functional groups in the synthetic methods described above include thio hydrazide compounds, which produce thio-functional polysaccharides in the synthetic procedure. This thio-functional polysaccharide then reacts with the phospholipid to produce the polysaccharide-phospholipid conjugate as an adjuvant as described above.

As a further specific example of the synthesis of the adjuvant according to the invention, the starting polysaccharide is reacted with an oxidizing agent such as a periodate compound to convert it into an oxidative functional group of the polysaccharide corresponding to the aldehyde functional group (-CHO suspending group). This formed aldehyde-functional polysaccharide then acts in reaction with a merchitohydrazide compound such as, for example, 2-acetamido-4-mercapto-butyric acid hydrazide (AMBH), or other suitable divalent functional groups. A suitable reaction site (terminal group) is provided on the functional polysaccharide to crosslink the phospholipid conjugate to the acidic polysaccharide.

In the synthesis of the adjuvant conjugate of the present invention, reagents other than the divalent functional reagents described in the shear lock can be advantageously used, such reagents include, for example, polysaccharides and reagents that do not need to be initially oxidized. . Phospholipids can form conjugates with functional groups present on polysaccharides, such as amino groups or hydroxy groups, by methods known in the art of synthetic chemistry.

Phospholipid conjugates used to prepare the polysaccharide-phospholipid aid of the present invention may include any suitable phospholipid, which suitable phospholipids may be coordinative, eg covalent, ionic, hydrogen, associative, or other binding. The binding forms a pharmaceutically stable complex with the polysaccharide to generate the desired immunostimulatory response to make the polysaccharide bioavailable in the host system.

Within the broader scope of the present invention, the phospholipid component of the polysaccharide-phospholipid conjugate can be reacted with a suitable reagent, for example a suitable divalent functional reagent, into a binding form. In various embodiments of the present invention, it is desirable to functionalize the phospholipids so that the phospholipids can be directly bound to the polysaccharides, and that no intermediate reactions with the polysaccharides are necessary before the polysaccharides and phospholipids are bound. May be advantageous. In other embodiments, only polysaccharides can be modified to form a binding form, and in yet another embodiment, both polysaccharides and phospholipid starting materials can be modified to have binding to each other.

In one specific example of binding a polysaccharide to a phospholipid component by reacting the divalent functional reagent with a phospholipid, the divalent functional reagent is N-succinimidyl-3- (pyridyldithio) -pro Cypionate (hereinafter sometimes referred to as SPDP) and phospholipids to dipalmatoylphosphatinyl-ethanolamine (hereinafter sometimes referred to as DPPE). SPDP and DPPE can be reacted with each other in a suitable solvent medium, for example chloroform. Chloroform in the reaction vessel is evaporated under a nitrogen atmosphere using a suitable water miscible solvent such as acetonitrile to produce a DPPE-SPDP conjugate as a phospholipid component for continuous reaction with modified polysaccharides. This modified polysaccharide, for example, when it has a thio functional group (result of reaction with a mercaptohydrazide compound), reacts with SPDP-induced phospholipids to form a polysaccharide-phospholipid conjugate as an adjuvant product.

Since any suitable phospholipid component can be used in a broad practice in the present invention, one class of phospholipid compounds that may be advantageous to use includes the fatty acid phosphatidylethanolamine, which has two fatty acid moieties, each The fatty acid moiety of is selected from the group consisting of lauroyl, palmatoyl, myristyl, oleyl, and stearyl [hereafter, lauroyl (L), palmatoyl (P), myristyl (M), oleyl ( O), stearyl (S), and phosphatidylethanolamine moiety (PE), respectively. Illustrative phospholipid species of the fatty acid functional groups that can be very usefully used in the practice of the present invention include those described in Table 1 below.

compound Mark Dipalmitoylphosphatidylethanolamine DPPE Dilauroylphosphatidylethanolamine DLPE Dimyristoylphosphatidylethanolamine DMPE Dioleylphosphatidylethanolamine DOPE Distearylphosphatidylethanolamine DSPE Lauroyl palmitoyl phosphatidylethanolamine LPPE Lauroyl myristoyl phosphatidylethanolamine LMPE Lauroyl oleylphosphatidylethanolamine LOPE Lauroylstearylphosphatidylethanolamine LSPE Palmitoylmyristoylphosphatidylethanolamine PMPE Palmitoyl Oleylphosphatidylethanolamine POPE Palmitoylstearylphosphatidylethanolamine PSPE Oleylstearylphosphatidylethanolamine OSPE Oleyl myristoyl phosphatidylethanolamine OMPE Myristoyl stearyl phosphatidyl ethanolamine MSPE

Adjuvants prepared by binding phospholipids to polysaccharides may be administered to the host animal by any of a variety of routes of administration that provide for the enhancement of a slowly regulated immune response.

This formed adjuvant can then be mixed with any suitable antigen, carrier, excipient, stabilizer, additive, etc. for the desired formulation, which can be processed as needed for the final use or administration purpose. For example, adjuvant formulations are lyophilized into powder formulations, which can be administered by nebulization to the lung locus of the host animal. Alternatively, the formulation may be sonicated or otherwise sheared to produce a microparticle composition that is convenient for administration.

In further variations of the compositions of the invention, suitable antigen (s) provide an integrated vaccine formulation that crosslinks equally to the phospholipid and / or polysaccharide sites in the adjuvant to achieve enhanced immunostimulation from the host animal. can do.

Host animals usefully administering the adjuvant and adjuvant-containing vaccine formulations of the invention include human as well as non-human mammals, fish, reptiles and the like.

In adjuvant formulations of the invention, the use of antigens covalently linked to the phospholipid and / or polysaccharide sites of the polysaccharide-phospholipid conjugate may be useful for many applications. Alternatively, the antigen can be used in the form of a mixture with the adjuvant of the present invention. Accordingly, specific formulations of the therapeutically effective compositions of the invention may be carried out in any suitable manner, which will make the adjuvant bioavailable, safe and effective in the subject to which the formulation is administered.

The present invention contemplates a wide range of therapeutic adjuvant formulations, which adjuvant formulations include, for example, (i) one or more antigens or vaccines that are therapeutically effective, and (ii) one or more polysaccharide-phospholipids according to the invention. May comprise a conjugate.

Such therapeutic compositions may include, for example, one or more antigenic agents selected from the group consisting of (A), (B), (C) and (D):

(A) viruses, bacteria, mycoplasma, fungi and protozoa;

(B) segments, extracts, subunits, metabolites and recombinants of (A);

(C) segments, extracts, subunits, metabolites and recombinants of mammalian proteins and glycoproteins; And

(D) tumor-specific antigens.

Thus, the therapeutic composition can use any suitable antigen or vaccine component in combination with the polysaccharide-phospholipid conjugate of the invention, which antigen is combined with, for example, a polysaccharide-phospholipid conjugate, Selected from the group consisting of antigens, viruses, and fungi of hospital and non-hospital organisms.

As a further example, such therapeutic compositions include diseases and conditions such as smallpox, yellow fever, stamper, cholera, poultry, scarlet fever, diphtheria, tetanus, whooping cough, influenza, rabies, mumps, measles, foot and mouth disease, and gray ear Proteins, peptides, antigens and vaccines that have pharmacological activity against may be suitably included. In a formed vaccine formulation comprising (iii) an antigen and (ii) a polysaccharide-phospholipid conjugate, the antigen and the adjuvant induce an immune response when the formulation is administered to a host animal, embryo or egg to be vaccinated. Each is present in effective amounts.

The formed vaccine formulation comprising (iii) an antigen and (ii) a polysaccharide-phospholipid conjugate is useful for inducing an immune response in an animal by administering to the animal an effective amount of a vaccine formulation capable of producing an antibody response in the animal. Can be used.

The method of administration may include a method of delivering the adjuvant or adjuvant-containing vaccine to the body part of a host animal in which the adjuvant and the associated antigen are effective immunostimulating and / or using any suitable means. Delivery methods are parenteral administration methods, such as subcutaneous (SC) injection, intravenous (IV) injection, nasal, orbital, intradermal, intramuscular (IM), endothelial (ID), intraperitoneal (IP) ), Oral, as well as intravaginal, pulmonary, and rectal administration.

Without unnecessary experimentation, one of ordinary skill in the art would appreciate the use of conventional dose titration techniques and conventional bioefficiency / biocompatibility protocols in the single dose ratios and suitable single dosage forms for the adjuvant and vaccine compositions of the present invention. And the antigen or therapeutic agent used with the adjuvant, the desired therapeutic effect, and the desired duration of bioactivity.

Adjuvants of the present invention may be usefully administered to a host animal with any other suitable pharmaceutically or physiologically active agent, such as an antigen and / or other biochemically active substance.

The features and advantages of the present invention may be more fully illustrated by the examples listed below, but are not limited thereto, and all parts and percentages are by weight unless otherwise indicated in the following examples.

Summary of the Invention

The present invention relates to adjuvant compositions, which adjuvant compositions may be useful for enhancing an immunostimulatory response using antigens or antigen-based vaccines.

In a broad aspect of the composition, the present invention relates to adjuvants comprising a polysaccharide-phospholipid conjugate.

Highly preferred polysaccharides of the adjuvant of the present invention include β-glucan, chitosan, galactomanans, and alginate.

In another aspect, the present invention relates to a method for synthesizing adjuvants from polysaccharides.

Adjuvants can be synthesized from polysaccharides and phospholipids using any suitable reagent, including divalent or polyfunctional reagents.

Adjuvants can be synthesized by the steps listed below:

Reacting the polysaccharide with an oxidant to form an aldehyde functional group on the polysaccharide;

Reacting the aldehyde-functional polysaccharide with a suitable divalent functional reagent to produce a functional polysaccharide having a crosslinking functional group that reacts with the phospholipid to further produce a polysaccharide-phospholipid conjugate; And

Reacting the functional polysaccharide with a phospholipid to produce a polysaccharide-phospholipid conjugate.

The invention also encompasses vaccine compositions containing the adjuvant of the invention and antigens for producing antibodies in animals.

The present invention also relates to an immunological response in an animal comprising a method of administering a vaccine containing an amount of adjuvant sufficient to produce an antibody response in the animal.

Other aspects and features of the present invention will become more fully apparent from the following detailed description and the appended claims.

Example 1

The β-glucan-phospholipid conjugate according to the present invention was prepared according to the synthetic procedures listed below.

Modification of β-glucan

β-glucan was bound to AMBH as follows.

β-glucan was treated with sodium periodate to induce aldehyde formation in polysaccharides. 50 μl (5 μmole) of 0.1 M sodium periodate was added to a suspension of β-glucan (20 mg) in 1 ml of water. The reaction took place over 15 hours at room temperature. To the formed suspension was added 50 μl (5 μmole) of 0.1 M 2-acetamido-4-mercaptobutyric acid hydrazide (AMBH, Molecular Probes, Inc.) dissolved in acetonitrile. After stirring for 24 hours at room temperature, AMBH bound to β-glucan suspension was used to bind to SPDP induced phospholipids.

Synthesis of SPDP-linked dipalmitoylphosphatidylethanolamine

Dipalmitoylphosphatidylethanolamine (173 mg; 250 μmole) was dissolved in chloroform. Triethylamine (20 μml) and N-succinimidyl-3- (2-pyridylthio) -propionate (78 mg; 250 μmole) (Pierce Chemical Company, Rockford, Illinoid) (hereafter SPDP Are added in order). The mixture was slowly stirred at rt for 24 h. Chloroform was evaporated using nitrogen. The residue was dissolved in 4 ml of acetonitrile to provide a solution containing 62 μmole of SPDP bound dipalmitoylphosphatidylethanolamine per ml of solution.

Preparation of β-glucan-phospholipid conjugate

SPDP bound dipalmitoylphosphatidylethanolamine (100 μl with 6.2 μmole) was added to 1 ml of a suspension of AMBH-linked β-glucan. The mixture was stirred slowly at room temperature for 24 hours to produce the binder product.

Example 2

β-glucan-phospholipid conjugate BSA formulation (BSA-GL)

0.5 ml of the β-glucan-phospholipid conjugate product of Example 1, a mixture of 0.2 ml of aqueous BSA (1 mg / ml), 140 mg of egg phosphatidylcholine, 70 mg of vitamin E, 70 mg of squalane, and 0.75 ml of saline buffered with phosphate The mixture was sonicated at 4 ° C. for 15 minutes with a probe sonicator to prepare β-glucan-phospholipid-conjugate-BSA vaccine emulsion formulations.

Example 3

Microdrop Adjuvant Formulation of BSA (BSA-MD)

Microdrop emulsion formulations of BSA were performed according to the disclosure of US Pat. No. 5,246,707 (Haynes), supra. This vaccine formulation was used as a control.

A mixture of 0.2 ml of aqueous BSA (1 mg / ml), 140 mg of egg phosphatidylcholine, 70 mg of vitamin E, 70 mg of squalene, and 1.25 ml of brine buffered with phosphate were sonicated at 4 ° C. for 15 minutes with a probe sonicator. BSA-MD microdroplet vaccine formulations were prepared.

Example 4

Adjuvant Studies in Mice

Adjuvant properties of the adjuvant compositions of the invention were determined by vaccine formulations containing bovine serum albumin (BSA) antigens. Comparative studies were conducted in mice and the results included measurements of antibody titers generated by vaccination with each vaccine formulation.

The test included vaccination of each test animal using the vaccine formulations listed below: (i) bovine serum albumin (BSA) in saline, (ii) BSA in microdrop emulsion form according to Example 3 above. (BSA-MD), (iii) BSA with the β-glucan-phospholipid binder adjuvant prepared by the procedure of Example 2, and (iii) Freund's complete BSA with Adjuvant (FDA). The results are described in more detail later, and are shown in the graph of FIG.

Experimental design

CF-1 mice weighing about 25 g (Charles River) were used. Mice (5 per group) were injected intraperitoneally using 50 μl of each formulation containing the same amount of antigen (day 0), and then injected a second dose of the same amount as the initial injection (day 14) Serum was taken from the mice and analyzed (day 28).

Screening of Serum Samples

Wells of 96-well microtiter plates were coated with 0.5 mg / ml solution of BSA used as antigen (50 μl aliquots were added to the wells and dried in the freezer). The wells were washed three times with wash buffer (Tris 20 mM, NaCl 0.8 M, 0.05% Tween-80, pH 7.4), and then each well was filled with an aqueous solution containing 1 mg / 1 ml of gelatin for 30 minutes, Wash three times with wash buffer. Serial dilutions of serum (100 μl) were added to the wells and kept at 4 ° C. overnight. Wells were washed three times with wash buffer and 100 μl of a 1: 1000 dilution of goat anti-mouse IgG-alkaline phosphatase conjugate (Oganon Teknica Corporation, Charlotte, New York) was added. After standing at room temperature for 1 hour, the wells were washed three times using wash buffer. Prepared directly of 1 mg / 1 ml of substrate (p-nitrophenyl phosphate disodium) in diethanolamine buffer (pH 9.8) (97 ml ethynolamine in 1 L, 0.2 g NaN ₃ , MgCl ₃ .6H ₂ 0 100 mg) Solution (200 μl) was added and kept in the dark for 2 hours at room temperature. Color development was quantified at 405 nm of microtiter plate reader.

result

The results are shown in FIG. Non-formulated antigens (indicated by BSA) have minimal antibody titers as expected, whereas microdroplet-formed antigens (BSA-MD) produce better responses than non-formulated antigens at various concentrations of anti-serum. Provided. The immune response of the BSA-glucan preparation (BSA-GL) comprising the β-glucan-phospholipid conjugate according to the invention is much better than that of the BSA-microscopic preparation (BSA-MD) and in all dilutions of the anti-serum tested It was found that this is equivalent to the immune response of the antigen formulated in Freund's complete adjuvant.

Example 5

In the separation test comparing (1) unmodified β-glucan (not bound to phospholipid) and (2) adjuvant properties of the modified β-glucan phospholipid conjugate according to the present invention, the unmodified β-glucan Adjuvant containing did not provide fortification similar to antibody titer achieved by modified β-glucan phospholipid conjugate.

Example 6

Adjuvant properties of the adjuvant compositions of the invention were determined by vaccine formulations containing bovine serum albumin (BSA) antigens. Comparative studies were conducted in mice and the results included measurements of antibody titers generated by vaccination with each vaccine formulation.

The test included vaccination of each test animal using the vaccine formulations listed below: (iii) BSA, (iii) prepared by the procedure of Example 2, and representative BSA-containing adjuvant formulations of the invention ( BSA-MD), and (iii) BSA with Freund's complete adjuvant (BSA-FCA). The results are described in more detail later, and are shown in the graph of FIG.

Adjuvant Studies in Mice

Six-week-old BALB / c (Charles River) mice were injected subcutaneously with 200 μl of adjuvant formulation. Each administered adjuvant formulation contained 200 μg / ml BSA. In addition, standard saline of BSA was prepared at 200 μg / ml. On day 14, each animal was second vaccinated to the concentration of the initial formulation, using the adjuvant formulation prepared immediately. As in the standard practice of secondary vaccination, FIA was used instead of FCA. Blood was collected from mice every 1, 2 or 3 months after initial treatment.

Screening of Serum Samples

Wells of 96-well microtiter plates were coated with 1 mg / ml solution of BSA used as antigen (50 μl aliquots were added to the wells and left overnight at 4 ° C.). This well was washed three times with PBS / 1% Tween 20. Then 100 μl of PBS containing 10% goat serum and 1% Tween 20 were incubated in the wells at 37 ° C. for 1 hour; The plate was then washed three times with PBS / 1% Tween 20. Test serum was added to the wells serially diluted in 100 μl of PBS / 10% goat serum / 1% Tween 20 (1:32 to 1: 4096) and incubated overnight at 4 ° C. or for 2 hours at 37 ° C. ; Plates were washed six times with PBS / 1% Tween 20. HRP antibodies bound to goat anti-mouse IgG or IgM were diluted 1: 1000 in PBS / 1% Tween 20 and incubated at 37 ° C. for 2 hours in plates; Wash 6 times with PBS / 1% Tween 20. The ABTS peroxidase substrate and the peroxidase solution were used to generate the peroxidase reaction. ELISA readings were performed using a Fisher Biotech, Microkinetics BT 2000 plate reader (wavelength of 405 nm).

result

The results are shown in FIG. 2, which is 1 month, 2 months, and 3 months after injection with 1: 4096 dilution when vaccinated with the respective BSA, BSA-MD, and BSA-FCA compositions It is a graph which shows the titration amount of IgG in mouse plasma which passed. As shown, the non-formulated antigen (BSA) has a minimum antibody titer, whereas the representative microdroplet-formed antigen (BSA-MD) of the present invention is predominantly 3 months after treatment It gave a better response than complete adjuvant (BSA-FCA).

The adjuvant compositions of the present invention are useful for enhancing an immune stimulatory response of a receptor host by administering to a host animal, embryo or egg using an antigen or antigen-based vaccine.

Such therapeutic compositions include, for example, (A) viruses, bacteria, mycoplasma, fungi and protozoa; (B) segments, extracts, subunits, metabolites and recombinants of (A); (C) segments, extracts, subunits, metabolites and recombinants of mammalian proteins and glycoproteins; And (D) one or more antigenic agents, such as tumor-specific antigens, wherein such therapeutic compositions include smallpox, yellow fever, stamper, cholera, chicken pox, scarlet fever, diphtheria, tetanus, pertussis, influenza, rabies, mumps It can be pharmacologically active against disease states and diseases such as, measles, foot and mouth disease, and whitish beard, wherein in the therapeutic composition, each antigen and adjuvant is formulated in a vaccinated host animal, embryo or egg In the case of administration, each is present in an effective amount capable of inducing an immune response.

Claims (31)

An adjuvant comprising a polysaccharide-phospholipid binder in which the binder components are bound by covalent bonds, wherein the adjuvant is useful for stimulating an immune response by administering to a host animal. 2. The adjuvant of claim 1, wherein the polysaccharide-phospholipid conjugate comprises a polysaccharide selected from the group consisting of glucan, chitosan, galactomanans, and alginate. The adjuvant of Claim 1, wherein the polysaccharide-phospholipid conjugate comprises glucan polysaccharide. 2. The adjuvant of claim 1, wherein the polysaccharide-phospholipid conjugate comprises β-glucan polysaccharide. The adjuvant of Claim 1, wherein the polysaccharide-phospholipid conjugate comprises a phospholipid moiety containing a fatty acid phosphatidylethanol amine moiety. The fatty acid phosphatidyl ethanol amine according to claim 5 is dipalmitoyl phosphatidyl ethanol amine, dilauroyl phosphatidyl ethanol amine, dimyristoyl phosphatidyl ethanol amine, dioleyl phosphatidyl ethanol amine, distearyl phosphatidyl ethanol amine, lauro Yl palmitoyl phosphatidyl ethanolamine, lauroyl myristoyl phosphatidyl ethanolamine, lauroyl oleyl phosphatidyl ethanolamine, lauroyl stearyl phosphatidyl ethanolamine, palmitoyl myristoyl phosphatidyl ethanolamine, palmitoyl oleyl phosphatidyl ethanol amine An amine, palmitoylstearylphosphatidylethanolamine, oleylstearylphosphatidylethanolamine, oleylmyristoylphosphatidylethanolamine, and myristoyl stearylphosphatidylethanolamine. The adjuvant of claim 1, wherein the polysaccharide-phospholipid conjugate is in lyophilized form. The adjuvant of claim 1, wherein the polysaccharide-phospholipid binder is in the form of microparticles. The adjuvant of claim 1, comprising an antigen covalently linked to the phospholipid and / or polysaccharide site of the polysaccharide-phospholipid conjugate. (Iii) a therapeutically effective antigen or vaccine and (ii) a polysaccharide-phospholipid conjugate in which the components of the conjugate are joined by covalent bonds. The method of claim 10, wherein the antigen and the vaccine comprise (A) viruses, bacteria, mycoplasma, fungi and protozoa; (B) segments, extracts, subunits, metabolites and recombinants of (A); (C) segments, extracts, subunits, metabolites and recombinants of mammalian proteins or glycoproteins; (D) tumor-specific antigens; And (E) at least one antigenic agent selected from the group consisting of combinations thereof. The therapeutic composition of claim 10, wherein the antigen or vaccine comprises an antigenic agent selected from the group consisting of pathogens, non-pathogens, viruses, and fungi. The antigen or vaccine of claim 10, wherein the antigen or vaccine is from the group consisting of smallpox, yellow fever, stamper, cholera, chicken pox, scarlet fever, diphtheria, tetanus, pertussis, influenza, rabies, mumps, measles, foot and mouth disease, and gray ear. Therapeutic composition comprising an antigen for a disease state of choice. (Iii) at least one adjuvant and (ii) at least one adjuvant containing a polysaccharide-phospholipid conjugate in which the components of the conjugate are covalently bound, wherein the antigen and the adjuvant are each intended to be vaccinated. A vaccine formulation present in an effective amount capable of inducing an immune response when administering the formulation to a host animal. 15. The vaccine formulation of claim 14 further comprising at least one component selected from the group consisting of non-amine E, squalene, and lecithin. Adjuvants containing polysaccharide-phospholipid conjugates in which the components of the conjugate are bound by covalent bonds are administered to the animal, embryo or egg in an amount sufficient to generate an antibody response to the animal, embryo or egg. Or inducing an immune response in the egg. 17. The method of claim 16, comprising administering said adjuvant to said animal with at least one therapeutic ingredient selected from the group consisting of (i) other adjuvant and (ii) antigen. 17. The administration according to claim 16, selected from the group consisting of subcutaneous injection, intravenous injection, oral, nasal, intraorbital, intradermal, intramuscular, endothelial, intraperitoneal, intravaginal, pulmonary, and rectal administration. A method comprising administering to an animal by a method. The method of claim 16 comprising administering to said animal by parenteral administration. The method of claim 16 comprising administering to said animal by oral administration. A vaccine preparation adjuvant comprising (i) at least one antigen and (ii) at least one polysaccharide-phospholipid conjugate in which the components of the conjugate are covalently bound to generate an antibody response in an animal, embryo or egg Administering to said animal, embryo or egg an amount sufficient to induce an immune response in said animal, embryo or egg. A method of synthesizing an adjuvant from an immunostimulatoryly effective polysaccharide, comprising covalently binding the polysaccharide and phospholipid to form a polysaccharide-phospholipid conjugate. 23. The method of claim 22, wherein said at least one polysaccharide and phospholipid react with a divalent functional reagent to produce a functional conjugate component that binds and reacts with the polysaccharide and phospholipid. The method of claim 23, wherein said phospholipid is reacted with a divalent functional reagent. The method of claim 24, wherein the divalent functional reagent is N-succinimidyl-3- (2-pyridyldithio) -propionate. The method of claim 23, wherein the polysaccharide-phospholipid conjugate comprises a phospholipid moiety containing a fatty acid phosphatidylethanolamine. 27. The fatty acid phosphatidylethanolamine of claim 26 is dipalmitoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dioleylphosphatidylethanolamine, distearylphosphatidylethanolamine, lauro Yl palmitoyl phosphatidyl ethanolamine, lauroyl myristoyl phosphatidyl ethanolamine, lauroyl oleyl phosphatidyl ethanolamine, lauroyl stearyl phosphatidyl ethanolamine, palmitoyl myristoyl phosphatidyl ethanolamine, palmitoyl oleyl phosphatidyl ethanol amine Amine, palmitoylstearylphosphatidylethanolamine, oleylstearylphosphatidylethanolamine, oleylmyristoylphosphatidylethanolamine, and myristoylstearylphosphatidylethanolamine. The method of claim 22, Reacting the polysaccharide with an oxidant to form an aldehyde functional group on the polysaccharide; Reacting the aldehyde-functional polysaccharide with a thiol hydrazide compound to produce a thio-functional polysaccharide; And Reacting the thiol-functional polysaccharide with a phospholipid derived with a thio-reactive functional group comprising phosphatidylethanolamine to produce the polysaccharide-phospholipid conjugate as the adjuvant. The method of claim 22, comprising mixing the adjuvant with at least one antigen to prepare a vaccine formulation. The method of claim 29, wherein mixing the adjuvant with the antigen comprises mixing the antigen with the adjuvant such that the antigen chemically binds to the adjuvant. The method of claim 29, wherein the vaccine formulation is treated under shear conditions to form the microdroplet composition.