JP2003012545A - Immunopotentiation inducible with chitosan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new low toxic adjuvant for latent application to human being. SOLUTION: A composition used for reinforcing immune responses in animals is prepared by a method for comprising (a) a process for preparing a chitosan solution, (b) a process for preparing a sodium hydroxide solution, (c) a process for preparing an oil/surfactant solution, wherein the oil can metabolically be degraded, and (d) a process for mixing the chitosan solution with the sodium hydroxide solution, the oil/surfactant solution, and an antigen to form an emulsion. The composition can be administered to an animal through an administration route making it possible to impart the immune response against the antigen to the animal.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates generally to methods for enhancing an immune response in an animal, compositions for achieving the enhancement, and methods of making the compositions. More particularly, the invention relates to methods involving the use of an antigen / chitosan mixture or an antigen / chitosan / oil / surfactant emulsion to enhance an immune response, an antigen / chitosan mixture or an antigen / chitosan that achieves the enhancement. / Oil / surfactant emulsions and methods for preparing antigen / chitosan mixtures or antigen / chitosan / oil emulsions.

[0002]

BACKGROUND OF THE INVENTION Recent advances in biotechnology have facilitated the identification of components in complex antigens, which is expected to lead to the development of safe and practical vaccines. However, often these isolated selection components are not as immunogenic as the complete complex antigen from which they were derived. Adjuvants are often administered with the immunogen to enhance the immune response to the weakly antigenic immunogen in the recipient animal. However, although adjuvants are generally well tolerated, the number of those suitable for use in humans is limited.

Ideally, adjuvants enhance long-term expression of functionally active antibodies, induce cell-mediated immunity (CMI), and have a high degree of specific immunoreactivity to invading antigens. The production of memory T and B lymphocytes should be enhanced. In addition to providing protection upon immediate challenge with foreign antigens, these responses should provide protection against any future encounter of the host with a particular antigen. More important is the ability of the adjuvant to enhance the immune response with minimal adverse side effects. Thus, the efficacy of an adjuvant is explained in terms of how an adjuvant balances positive (immunity enhancing) and negative (toxic) effects.

Controlled immunization for the purpose of stimulating antibody production by B cells depends on a myriad of factors that are unique to both the antigen itself and the animal immunized. Generally, the more evolutionarily distant the antigen or its source is from the invading host, the more effective is the immune response elicited by that antigen. Antigens from closely related species have little ability to induce antibody production due to the fact that the host's immune system cannot clearly distinguish foreign antigens from endogenous or self antigens. Furthermore, the dose of antigen, the purity of antigen, and the frequency of administration of antigen are also factors that significantly contribute to the antibody titer obtained and the specificity of the antibody obtained. Still other factors include the morphology or complexity of the antigen and the method of administration of the antigen. Finally, the genetic constitution and general physiological condition of the immunized animal contribute to the extent to which the immune response is triggered. Among these factors, the morphology or degree of complexation of the antigen is directly influenced by immunization with an adjuvant.

Current understanding suggests that adjuvants act to enhance the immune response by a variety of different mechanisms. In one mechanism, the adjuvant is T
Directly stimulate any one of the CD4 ⁺ helper T cell subpopulations called _H 1 or T _H 2 [Mosmann and Coffman, Ann. Rev. Immuno
l. 7: 145-173 (1989)]. Helper T
The cells are required for the B cell antibody response to most antigens. In the proper immune response, the antigen is captured and processed by antigen presenting cells (APC), eg, circulating or tissue macrophages, and presented on the surface of the APC in association with class II major histocompatibility (MHC) molecules. It In this form, the antigen can interact with receptors on the surface of helper T cells, thus activating specific subpopulations of cells to express and secrete any of a number of cytokines. The nature of cytokine production depends on a subset of activated helper T cells, a consequence of which can be regulated in part by the choice of adjuvant. For example, alum adjuvants is aluminum salts is observed clinical use in humans, T _H in mice
It has been reported to selectively activate 2 cells [G
run and Maurer, Cell. Immuno
l. 121: 134-145 (1989)]. On the other hand,
Freund's Complete Adjuvant (FCA), an emulsion of mineral oil containing killed mycobacteria [Freund
Et al., Proc. Soc. Exp. Biol. Med. Three
7: 509 (1937)] preferentially activate mouse T _H 1 cells [Grun and Maurer, Cel.
l. Immunol. 121: 134-145 (19
89)].

Another mechanism by which the immune response is enhanced is, for example:
B cells with lipopolysaccharide (LPS) derived from Gram-negative bacteria
With direct stimulation of [Gery et al., J. Chem. Immunol.
  108: 1088 (1972)]. LPS is also
Stimulating the secretion of interferon gamma (INF-γ)
[Tomai and Johnson,
J. Biol. Resp. Med. 8: 625-64
3 (1989)]. This is T_H2 inhibition of cell growth and
T_HBoth stimulate the differentiation of one cell [Gajewsk
i, J. et al. Immunol. 143: 15-22 (19
89); Gajewski et al. Immunol. 1
46: 1750-1758 (1991)]. Therefore, L
The mechanism by which PS enhances the immune response is the direct stimulation of B cells.
Geki, and T _H1 cell population and T_HBoth indirects of two cell populations
Adjustment.

Yet other modes of immune enhancement have been reported for other adjuvants. Oil emulsions (ie, Freund's Complete Adjuvant [FCA], Incomplete Freund's Adjuvant [FIA]) and liposomes, like alum, act by forming a depot, thus slowing release of antigen.
let) Delayed release of antigen can prolong exposure of the antigen to the immune system, and also allows initial immunization with a dose of antigen that, when administered once, is usually ineffective against antibody formation. . Higher initial doses of antigen produce higher immediate titer antibodies, but increased antibody titer and increased antibody specificity as a function of time are associated with lower and more frequent doses of antigen. It has already been reported that it is not as large as observed [Siskind,
G. , Pharm. Rev. 25: 319-32
4 (1973)]. Thus, adjuvants that control the presentation of antigens to the immune system, in addition to altering the morphology or degree of complexation of the antigen, modulate antigen dose.

To date, the only adjuvant, alum [ALK (SO ₄ ) ₂ .H ₂ O], has proven to be sufficiently non-toxic for use in humans. Alum induces T _H 2 cell activation, depot formation of antigen, and delayed release of antigen following immunization [Edelman,
Rev. Infect. Dis. 2: 370-383
(1980); Warren et al., Ann. Rev. I
mmunol. 4: 369-388 (1986)] as well as by granuloma formation by attracting immunocompetent cells [White et al., J. Chem. Exp. Me
d. 102: 73-82 (1955)], and activation of complement [Ramanathan et al., Immunol.
37: 881-888 (1979)]. However, alum is not without its negative side effects, including erythema, subcutaneous nodules, contact hypersensitivity, and granulomatous inflammation. Other widely used adjuvants besides human applications have also been the focus of ongoing research to develop acceptable alternatives for use in humans. Included in this are the oil emulsions (ie CFA and F
IA), bacterial products (ie LPS, cholera toxin, mycobacterial components, and completely killed Corynebac)
terium parvum, Corynebacte
rum granulosum, and Bordet
ella pertussis), liposomes, immunostimulatory complexes (ISCOMs), and naturally-occurring and derivatized polysaccharides from sources other than bacterial sources.

The immunopotentiating ability of polysaccharides has been the focus of research over the past few years, as these compounds are widely naturally present (for example, as structural components of the cell wall of bacteria, and as exoskeletons of insects and crustaceans). Has become. The adjuvant properties of lipopolysaccharide (LPS) are primarily derived from the lipid A region of the molecule, and LPS isolated from certain Gram-negative bacteria, although not from the o-specific polysaccharide or core oligosaccharide regions of the molecule, One of such polysaccharides
Is one. LPS is a humoral immune system [Johnson et al.,
J. Exp. Med. 103: 225-246 (19
56)] and cell-mediated immunity [Ohta et al., Immu.
nobiology 53: 827 (1984)], and has many biological activities.
a et al., Vaccine 11: 291-306 (199).
As reviewed by 3), it is not practical for human use due to its inherent toxicity. Therefore, other polysaccharides, including chitosan among others, have received attention.

Chitosan [β- (1-4) -2-amino-
2-Deoxy-D-glucan] is a derivative of chitin and is widely used in biomedical applications due in part to its biodegradability by lysozyme and low toxicity in humans. These same properties have led to increased interest in chitosan as an immunopotentiator. For example, Matu
hashi et al., in US Pat. No. 4,372,883, conjugates of soluble polysaccharides, including chitosan, to commonly toxic antigens, thereby detoxifying the antigen and allowing use as an immunogen. ) Was disclosed. But,
Matuhashi et al. Do not mention the use of an insoluble form of chitosan, nor do Matuhashi compare the serum antibody titers obtained with those obtained from immunization with other known adjuvants.

Similarly, Suzuki et al., In US Pat. No. 4,971,956, disclosed the use of water-soluble chitosan oligomers as therapeutic agents for the treatment of bacterial and fungal infections as well as for the treatment of tumors. Suzuki
i et al. discussed the difficulty of modifying chitosan to produce a suitable water soluble form and disclosed that the water insoluble form is not practical for therapeutic use. In addition, Suzu
Ki et al. do not disclose that conjugation of antigen to chitosan achieves an enhanced immune response.

Mituhashi et al., US Pat.
No. 814,169, disclosed the use of human proteins conjugated to soluble polysaccharides, including chitosan, for the production of antibodies to human proteins in non-human animals. Administration of human protein / polysaccharide solutions was by intravenous, intraperitoneal, or subcutaneous injection. Other routes, including oral and rectal administration, were not mentioned in this disclosure.

Nishimura et al. [Vaccine
2: 93-99 (1984)] reported the immunological properties of chitin derivatives in terms of activation of peritoneal macrophages in vivo, suppression of tumor growth in mice, and protection against bacterial infection. The results showed that neither chitin nor chitosan were effective stimulators of host resistance to tumor cell or bacterial challenge.
It was suggested that chitosan moderately induced cytotoxic macrophages. The results with the modified deacetylated chitosan forming a gel in an aqueous environment showed that it activated macrophages more effectively, suppressed tumor growth, and stimulated resistance to bacterial infection.

Markinkiewicz et al. [Arc
h. Immunol. Ther. Exp. 39:12
7-132 (1991)] test the immunoadjuvant activity of water-insoluble chitosan and significantly enhance the T-dependent humoral response, but only moderately enhance the T-independent humoral response. I reported that. An enhanced humoral response was detected with chitosan administered either intravenously or intraperitoneally at the 100 mg / kg dose. Subcutaneous and oral administration have been specifically reported to be ineffective.
Furthermore, Markinkiewicz et al. Did not suggest the conjugation of the antigen to insoluble chitosan and stated that chitosan "provides a similar response, either with or without antigen, regardless of the site of administration". .

[0015]

Given the fact that there is only one adjuvant acceptable for use in humans, the art is new and less toxic for potential applications in humans. There is a need to provide an adjuvant. The improved adjuvant allows the production of more effective vaccines and enhances the production of therapeutically competent monoclonal antibodies.

[0016]

Methods and compositions for enhancing the immune response are disclosed which incorporate chitosan as an immune enhancing adjuvant. Administration of the compositions of the present invention can be provided by a variety of routes.

In the present invention, a composition used in enhancing an immune response in an animal, which composition comprises: a) preparing a chitosan solution; b) preparing a sodium hydroxide solution; c D) preparing an oil / surfactant solution, wherein the oil can be metabolically degraded; and d) adding a chitosan solution to the sodium hydroxide solution, the oil / surfactant solution, and A composition is prepared by a method that comprises mixing with an antigen to form an emulsion, wherein the composition is administered to the animal by a route of administration that allows the animal to mount an immune response to the antigen. Provided.

In one aspect, the oil is squalene and in another aspect, the pH of the chitosan solution is about 5.
It is 0. In a further aspect, the route of administration is selected from the group consisting of intramuscular injection, intraperitoneal injection, and subcutaneous injection, eg, the animal is human.

Further, in the present invention, there is provided a composition for enhancing an immune response, the composition comprising an antigen, sodium hydroxide, an oil, a surfactant, and a chitosan solution, wherein the oil is metabolically A composition is provided that can be decomposed into. In one aspect the oil is squalene and / or the pH of the chitosan solution is about 5.0.
Is.

In the present invention, an immunogen produced by the following process, which comprises: a) preparing a chitosan solution; b) preparing a sodium hydroxide solution; c) oil / surfactant. Preparing a solution, wherein the oil can be metabolically degraded; and d) the chitosan solution is added with sodium hydroxide solution, oil /
An immunogen is provided that includes a detergent solution and a step of mixing with an antigen to form an emulsion.

[0021] In one aspect, the oil is squalene and in another aspect, the pH of the chitosan solution is about 5.
It is 0.

Further in the present invention, a method for producing an immunogen, comprising: a) preparing a chitosan solution; b) preparing a sodium hydroxide solution; c) preparing an oil / surfactant solution. Wherein the oil can be metabolically degraded; and d) mixing a chitosan solution with the sodium hydroxide solution, the oil / surfactant solution, and an antigen to form an emulsion. , Are provided. In one aspect, the oil is squalene and in another aspect, the pH of the chitosan solution is about 5.0.

The kit according to the present invention further comprises: a) a chitosan solution; b) a sodium hydroxide solution; and c) an oil / surfactant solution, wherein the oil is metabolically degradable. A kit is provided that includes.

[0024] In one aspect, the oil is squalene and in another aspect, the pH of the chitosan solution is about 5.
It is 0.

An adjuvant prepared by the process of mixing a chitosan solution with a sodium hydroxide solution and an oil / surfactant solution in the present invention to form an emulsion, wherein the oil is metabolically degradable. Will be provided. In one aspect,
The oil is squalene and in another aspect, the pH of the chitosan solution is about 5.0.

[0026]

DETAILED DESCRIPTION OF THE INVENTION In all its aspects, the present invention relates to the use of chitosan formulations to enhance an immune response in a host.

[0027] In one aspect, the present invention relates to a method for enhancing an immune response, which comprises the steps of preparing a chitosan solution, incorporating an antigen into a phosphate buffer to form an antigen / phosphate buffer solution. Lyophilizing the antigen / phosphate buffer solution to form a lyophilized mixture, reconstituting the lyophilized mixture with a chitosan solution to form an antigen / chitosan mixture, and administering the mixture to animals, including humans The step of performing is included. The antigen / chitosan mixture may be administered to the animal via the oral, rectal, vaginal routes and via intraperitoneal, intramuscular or subcutaneous injections;
Administration can include a single route or multiple routes.

[0028] In another aspect of the invention there is provided a composition comprising lyophilized antigen / phosphate buffer and chitosan solution in combination. The antigen / chitosan mixture is
Via the oral, rectal, vaginal routes, as well as intraperitoneal injections,
Animals can be administered via intramuscular or subcutaneous injection; administration can involve a single route or multiple routes.

Also provided according to the invention is an immunogen comprising a lyophilized antigen / phosphate buffer and a chitosan solution. The antigen / chitosan mixture can be administered orally, rectally,
Via the vaginal route, as well as intraperitoneal injection, intramuscular injection,
Alternatively, it may be administered to the animal via subcutaneous injection; administration may involve a single route or multiple routes.

In another aspect of the invention, there is provided a method for preparing an immunogen, which comprises the steps of preparing a chitosan solution, incorporating the antigen into a phosphate buffer,
Forming a phosphate buffer solution, lyophilizing the antigen / phosphate buffer solution to form a lyophilized mixture, and reconstituting the lyophilized mixture with a chitosan solution to form an antigen / chitosan mixture .

As another aspect of the present invention, the present invention provides a method for enhancing an immune response, which comprises the steps of preparing a chitosan solution, preparing a sodium hydroxide solution, oil / interface. Preparing an activator solution, where the oil can be metabolically degraded, mixing the chitosan solution with sodium hydroxide solution, the oil / surfactant solution, and the antigen to form an emulsion, and an emulsion Is administered to the animal. Antigens can be, but are not limited to, proteins, carbohydrates, lipids, glycoproteins, or combinations thereof. Preferably, the pH of the chitosan solution is about 5.0. The emulsion may be administered to the animal via intraperitoneal injection, intramuscular injection, or subcutaneous injection. The emulsion can also be administered alone or in combination with any of a number of other adjuvants. Immunization can include single or multiple doses. In a more preferred embodiment, the oil is squalene.

In yet another aspect of the present invention there is provided a composition for enhancing an immune response when administered to an animal,
The composition comprises an antigen, sodium hydroxide, an oil, a detergent, and a chitosan solution, where the oil can be metabolically degraded.

Also provided by the present invention is an immunogen comprising an antigen, sodium hydroxide solution, oil, detergent, and chitosan solution, wherein the oil can be metabolically degraded.

In another aspect of the invention, a method for preparing an immunogen is provided, which comprises preparing a chitosan solution, preparing a sodium hydroxide solution, preparing an oil / surfactant solution. (Wherein the oil can be metabolically degraded), and the step of mixing the chitosan solution with the sodium hydroxide solution, the oil / surfactant solution, and the antigen to form an emulsion.

Also provided in another aspect of the invention is a kit containing a chitosan solution, a sodium hydroxide solution, and an oil / surfactant solution.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions and methods for using an immunopotentiating composition comprising an antigen / chitosan mixture or an antigen / chitosan / oil / surfactant emulsion, and an antigen / chitosan mixture. And the following examples of methods for preparing antigen / chitosan / oil / surfactant emulsions. In particular, Example 1 demonstrates the preparation of antigen incorporated in phosphate buffer and lyophilized. It is subsequently reconstituted in chitosan solution. Example 2 is
It provides a comparison of the ability of the antigen incorporated in phosphate buffer and reconstituted in chitosan solution to stimulate the immune response with the ability of currently available adjuvants. Example 3 demonstrates the preparation of antigen incorporated in chitosan / oil emulsion. Examples 4 and 5 provide a comparison of the ability of different antigens incorporated in chitosan / oil emulsions to stimulate the immune response with the ability of currently available adjuvants.

[0037]

EXAMPLE 1 Preparation of Antigen Incorporated in Phosphate Buffer, Lyophilized and Reconstituted in Chitosan Solution The following is illustrated by using chicken ovalbumin as the antigen. Those of skill in the art will readily understand that any number of other antigens may be employed.

15.6 ml of phosphoric acid (16M; Mall
inkrodt Chemical, Paris,
KY) with 400 ml of deionized (18 mOhm; DI)
A 0.5M phosphate buffer was prepared by diluting in water. 10N Sodium Hydroxide (Sigma Chem
ical Co. , St. Louis, MO)
The pH of the solution was adjusted to 7.3. The total volume of the solution was adjusted to 500 ml by adding DI water.

First, a dilute solution of chitosan was prepared by adjusting 1% chitosan to a 2% acetic acid solution (10).
0 ml of 2% glacial acetic acid (Mallinkrodt Che
1 g chitosan (commercial grade; Sigma Chemical Co., M.C., Paris, KY).
St. Louis, MO)). The resulting 1% chitosan in 2% acetic acid solution was then added to 7.4 m of the solution.
Further dilution was done by adding 1 to 2.6 ml DI water to obtain a chitosan working solution. The pH of the final chitosan solution was between 6 and 7.

50 μL in phosphate buffered saline
Of 10 mg / ml ovalbumin (Sigma Che
medical Co. , St. Louis, MO)
The solution was added to a 10 ml vial containing 5 ml 0.5 M phosphate buffer. As a result, it became transparent and cotton-like.
0.5 g of d-sorbitol (Sigma Chemi
cal Co. , St. (Louis, MO), the solution was immediately frozen in liquid nitrogen and lyophilized.

The lyophilized sample was reconstituted with 5 ml working chitosan solution, mixed by vortexing to form a cloudy solution containing white particles, and for immunization as described in Example 2. used.

Example 2 Comparative Immunity Enhancement Using Antigen Incorporated in Phosphate Buffer, Lyophilized, and Reconstituted in Chitosan Solution To measure the relative degree of chitosan enhancement of the response to the antigen. , A comparison between groups of mice was made (see Table 1). The mouse group was CFA (Sigma Chemical Co.,
St. Louis, MO) containing 25 μg of ovalbumin, or incorporated in phosphate buffer as prepared in Example 1, lyophilized and subsequently reconstituted in chitosan solution.
Pre-immunization (individually) with any of the vaccines (test group) containing 5 μg ovalbumin.

On day 0, 8 week old female Balb / c mice were immunized with a single intraperitoneal injection of the vaccine. The ovalbumin CFA-treated group contained 3 mice, while the test group (ovalbumin treated uptaken in phosphate buffer, lyophilized and reconstituted in chitosan solution) contained 4 mice. Was included. Both experimental groups were bled 7 days after injection. The group using CFA as an adjuvant was 21, 28, 35 after immunization,
Blood was also drawn on days 42 and 48. The test group is
26, 38, 38, 52, 70, 83, 102 after immunization
Blood was also collected on days 123 and 159. ELI
The titer of anti-ovalbumin serum antibody was measured by SA.

[0044]

[Table 1] The results showed that the composition containing the antigen taken up in phosphate buffer, lyophilized and reconstituted in chitosan solution was clearly non-toxic to recipient animals. Animals in the test group developed high antibody titers by day 26 (10,000). High titers persisted 83 days post-immunization, following booster vaccination on day 42. Immediately after booster vaccination, the titer increased to approximately 64,000 (day 52), and 10,000 until approximately 123 days (original) post vaccination.
Persisted above. The test group values obtained were comparable to those of Freund's complete adjuvant, a standard adjuvant commonly used by those skilled in the art. Furthermore, the mean titers in the test group animals were comparable to those observed with the antigen cross-linked to chitosan with glutaraldehyde. Glutaraldehyde generally improves immune enhancement over other commercially available adjuvants (P
CT / US95 / 12189; WO 96/0980
5). Given that glutaraldehyde is unacceptable in commercial and current vaccines, the antigen is administered via uptake in phosphate buffer and lyophilization and reconstitution in chitosan solution. , And an antigen cross-linked to chitosan via glutaraldehyde are alternative adjuvants.

Example 3 Preparation of Antigen Entrapped in Chitosan / Oil Emulsion The HIV peptide keyhole limpet hemocyanin conjugate (Example 4) or human zona pellucida B peptide ovalbumin (Example 5) was used as the antigen. The following is illustrated by using
One of ordinary skill in the art will readily appreciate that any number of other antigens may be employed. Furthermore, although the following is exemplified by the use of squalene, one of ordinary skill in the art will understand that any oil that is easily metabolized by the recipient animal (eg, corn, rapeseed, peanut) may be used.

4.1 g of sodium acetate (Sigma
Chemical Co. , St. Louis,
MO) was dissolved in 50 ml of deionized (18 mOhm: DI) water with stirring to prepare a 2% chitosan solution in 0.5 M sodium acetate. About 7 ml of glacial acetic acid (Mallinkrodt Chemical, Pa
ris, KY) was used to adjust the pH of the solution to 4.5, and an additional 1.5 ml glacial acetic acid was added to correct the effect of chitosan addition on the pH of the solution. The total volume of the solution was adjusted to 100 ml by adding DI water. 2 g of chitosan (Sigma Chemical Co., St.
(Louis, MO) was slowly added to the sodium acetate solution with stirring and the mixture was stirred for 2-3 hours until the chitosan had dissolved. Then, the chitosan solution was sterilized by an autoclave with a 25-minute cycle. The solution was cooled to room temperature in a biosafety cabinet. Then, the IEC clinical centrifuge (International Equipment)
Co. , Needham Hts. , MA) was clarified by centrifugation at setting 7 for 5 minutes. The supernatant was decanted from the pellet (insoluble chitosan / chitin and contaminants). 87-90% (by weight) of the added chitosan was retained in the supernatant.

50 g of sodium hydroxide (Sigma)
Chemical Co. , St. Louis,
A 50% sodium hydroxide solution was prepared by dissolving (MO) in 100 ml of deionized water with stirring. 1500 μL of squalene (2, 6, 10, 15,
19,23-Hexamethyl-2,6,10,14,1
8,22-Tetracosahexaene; Sigma Che
medical Co. , St. Louis, MO) and 600 μL of surfactant Pluronic ^{& reg ;} L12
1 (BASF Corp., Parsippany,
NJ) was combined and vortexed to homogeneity to prepare a squalene / surfactant solution.

About 420 μL of antigen (ie, HIV peptide keyhole limpet hemocyanin conjugate, Table 2; human zona pellucida B peptide ovalbumin conjugate, Table 3) in water or urea was added to about 370 μL of 2% chitosan (0. ．
A chitosan / squalene / surfactant / antigen emulsion was prepared by adding (in 5M sodium acetate) and vortexing. The actual amount of antigen used (ie protein or peptide carrier conjugate) can range from 1 μg to several milligrams. The antigen / chitosan was then added with 10 μL of 50% sodium hydroxide and the sample vortexed. 50 in 10 μL aliquots until a stable cloudy precipitate formed
% Sodium hydroxide was added. To the above solution of antigen and chitosan was added approximately 140 μL of the squalene / surfactant solution prepared above. The resulting solution was vortexed until a cloudy emulsion formed.
The resulting chitosan / squalene / surfactant / antigen solution was mixed by vortex or syringe aspiration just prior to administration in immunization studies as described in Examples 4 and 5.

Example 4 Antigen Incorporated in Chitosan / Squalene Emulsion Comparative Immunity Using (HIV Peptide KLH Conjugate) Objective To Evaluate Immune Response to Antigen Incorporated in Chitosan / Squalene Emulsion Then, the following experiment was conducted. Specifically, groups of mice were treated with varying amounts of HIV peptide KLH conjugate [Sarin et al. Vaccine Re.
s. , 3 (1): 49-57 (1994); incorporated herein by reference] with a chitosan / squalene / surfactant emulsion or 20μ
A comparative study was performed in which each of the vaccines containing g of HIV peptide KLH conjugate with CFA was individually immunized.

Regarding Tables 2 and 3, on day 0,
8 postnatal days by a single 200 μL intraperitoneal injection of vaccine
Weekly female Balb / c mice were immunized. In group 1,
A second immunization was performed at 18 weeks (126 days after the first immunization). In groups 2 and 3, a second immunization was given at week 24 (168 days after the first immunization).
The second immunization consisted of chitosan / squalene / surfactant emulsions in groups 1-3 with unconjugated HIV peptide at the doses indicated. The CFA group did not receive a second immunization. From the target animals 22, 35,
49, 63, 77, 91, 119 (excluding the first group), 1
Blood was drawn on days 40 and 149. By ELISA,
Serum antibody titer was measured.

[0051]

[Table 2]

[0052]

[Table 3] The results showed that the chitosan / squalene / surfactant emulsion adjuvant was clearly non-toxic to recipient animals. Results are further in Group 3 (20 μg peptide with chitosan / squalene / surfactant as adjuvant)
Acted similarly to Group 4 (20 μg peptide adjuvanted with CFA) and also showed an improved immune response to HIV peptide KLH conjugates at 5 and 7 weeks. Furthermore, Group 3 (3 μg peptide adjuvanted with chitosan / squalene / surfactant) produced results very similar to Group 4 (or worse if not) during the 11th week. Surprisingly, a second immunization with the "unconjugated" peptide in the group that received the chitosan / squalene emulsion produced a very strong boosting response (18 weeks after group 1 and See groups 2-3 after 24 weeks). Overall, the results shown in Table 3 indicate that the chitosan / squalene emulsion is comparable to CFA.
In some cases it is demonstrated to induce a better humoral immune response than CFA. Furthermore, the chitosan / squalene / surfactant emulsions acted as immunopotentiators, as shown by the very strong boosting response obtained with the unconjugated HIV peptide.

Example 4 Antigen Incorporated in Chitosan / Squalene Emulsion Comparative Immunity Enhancement Using (Human Zona B-Peptide Ovalbumin Conjugate) The following experiment was also performed in a chitosan / squalene / surfactant emulsion. This was done to assess the immune response to the incorporated antigen. In detail, 6 different human zona pellucida B (ZPB) with chitosan / squalene / surfactant emulsion or CFA as adjuvant in a group of mice
A comparative study was performed in which individuals were immunized (intraperitoneally) with a vaccine containing synthetic peptides [SEQ ID NOs: 1-6].

Vaccines (20 μg each of 6 different human ZPB synthetic peptides in combination with either chitosan / squalene / surfactant emulsion (Group I) or CFA (Group II)) were administered on days 0 and 28. 200
Female B at 8 weeks after birth by intraperitoneal injection of μL
Alb / c mice were immunized. Group II mice received the CFA vaccine as a boost. Serum antibody titers were determined by ELISA using plates coated with 1 μg of 6 peptide mixture per well. Full-length purified recombinant human ZPB protein produced in Chinese hamster ovary cells [Harris et al.
J. Seq. and Mapping, 4: 3
61-393, 1994; incorporated herein by reference], antibody titers were also determined by ELISA on plates coated with 50 ng of purified protein.

[0055]

[Table 4] The results in Table 4 show that animals immunized with peptide conjugates with chitosan / squalene / surfactant emulsions were more sensitive to peptide and full length protein than were immunized with peptide conjugates with CFA as an adjuvant. We demonstrate that it elicited an excellent humoral response to both.

Although the present invention has been described in terms of a preferred embodiment, it is intended that all modifications and alterations that occur to those skilled in the art in light of the disclosure herein, and particularly in the claims and their requirements. It is intended to include those embodiments that fall within the broadest appropriate interpretation.

[0057]

[Sequence Listing] (1) General information: Applicants: Podolski, Joseph S. Mitzi L. Martinez (ii) Title of invention: Chitosan-induced immunity enhancement (iii) Number of sequences: 6 (iv) Contact address: (A) Name: Marshall, Ottor, Garstein, Murray & Born (B) Address: South Wacker Drive 233 /
Sears Tower 6300 (C) City: Chicago (D) State: Illinois (E) Country: United States (F) Zip Code: 60606-6402 (v) Computer Readout Form: (A) Media Type: Floppy Disk ( B) Computer: IBM PC compatible (C) OS: PC-DOS / MS-DOS (D) Software: Patent-in-release # 1.
0, version # 1.30 (vi) Current application data: (A) Application number: (B) Application date: (C) Classification: (viii) Agent / office information: (A) Name: Crow, David W. (B) Registration number: 36,107 (C) Inquiry / record number: 27779/33753 (ix) Telephone line information: (A) Telephone: 312-474-6300 (B) Telefax: 312-474-0048

[0058]

[Chemical 1]

[0059]

[Chemical 2]

[0060]

EFFECTS OF THE INVENTION By providing an improved adjuvant, more efficient production of vaccines is possible and the production of therapeutically effective monoclonal antibodies is improved.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Mitzielle. Martinez             United States Texas 77306, CO             Narrow, Sea Terry Road 10731 F-term (reference) 4C085 AA38 DD86 EE06 FF01 FF14                       FF21 GG03 GG04 GG06

Claims (20)

[Claims] 1. A composition for use in enhancing an immune response in an animal, the composition comprising: a) preparing a chitosan solution; b) preparing a sodium hydroxide solution; c) oil. / Preparing a surfactant solution, wherein the oil can be metabolically decomposed;
And d) prepared by a method comprising the steps of mixing the chitosan solution with the sodium hydroxide solution, the oil / surfactant solution, and an antigen to form an emulsion, wherein the composition is directed against the antigen. A composition, which is administered to an animal by a route of administration that allows the immune response to be imparted to the animal. 2. The composition of claim 1, wherein the oil is squalene. 3. The composition of claim 1, wherein the pH of the chitosan solution is about 5.0. 4. The composition of claim 1, wherein the route of administration is selected from the group consisting of intramuscular injection, intraperitoneal injection, and subcutaneous injection. 5. The composition according to claim 4, wherein the animal is a human. 6. A composition for enhancing an immune response, the composition comprising an antigen, sodium hydroxide, an oil, a surfactant, and a chitosan solution, wherein the oil is metabolically degraded. A composition that can be. 7. The composition of claim 6, wherein the oil is squalene. 8. The composition of claim 6, wherein the pH of the chitosan solution is about 5.0. 9. An immunogen produced by the following process, which comprises: a) preparing a chitosan solution; b) preparing a sodium hydroxide solution; c) an oil / surfactant solution. Preparing the oil, wherein the oil can be metabolically degraded; and d) mixing the chitosan solution with the sodium hydroxide solution, the oil / surfactant solution, and the antigen, and emulsion Forming the immunogen. 10. The immunogen of claim 9, wherein the oil is squalene. 11. The immunogen of claim 9, wherein the chitosan solution has a pH of about 5.0. 12. A method for producing an immunogen, comprising: a) preparing a chitosan solution; b) preparing a sodium hydroxide solution; c) an oil / surfactant solution. Preparing, wherein the oil can be metabolically degraded; and d) mixing the chitosan solution with the sodium hydroxide solution, the oil / surfactant solution, and an antigen to form an emulsion. Forming. 13. The method of claim 12, wherein the oil is squalene. 14. The method of claim 13, wherein the pH of the chitosan solution is about 5.0. 15. A kit comprising: a) a chitosan solution; b) a sodium hydroxide solution; and c) an oil / surfactant solution, wherein the oil is metabolically degradable. kit. 16. The kit of claim 15, wherein the oil is squalene. 17. The kit according to claim 15, wherein the pH of the chitosan solution is about 5.0. 18. An adjuvant prepared by the process of mixing a chitosan solution with a sodium hydroxide solution and an oil / surfactant solution to form an emulsion, wherein the oil can be metabolically degraded. Adjuvant. 19. The adjuvant according to claim 18, wherein the oil is squalene. 20. The adjuvant of claim 18, wherein the pH of the chitosan solution is about 5.0.