A liposome-based vaccine immunoadjuvant composition and use thereof

Abstract

The application discloses a kind of liposome-based vaccine immunoadjuvant compositions and its application, the vaccine adjuvant composition includes: (a) liposome, the liposome includes phospholipid, solid sterol and squalene;(b) the chitosan and / or chitosan derivative of coated liposome.The application also provides the application of the above-mentioned vaccine immunoadjuvant composition and the polypeptide vaccine preparation comprising it, the vaccine immunoadjuvant composition of the application based on liposome effectively improves the immune response effect of polypeptide vaccine, especially new coronavirus polypeptide vaccine.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically, this invention relates to a liposome-based vaccine adjuvant composition and its application. Background Technology

[0002] Vaccination plays a crucial role in effectively preventing the spread of infectious diseases. Vaccines include inactivated vaccines, live attenuated vaccines, viral vector vaccines, recombinant protein vaccines, peptide vaccines, DNA vaccines, and RNA vaccines. Peptide vaccines are prepared through chemical synthesis based on the amino acid sequence of a known or predicted antigenic epitope in the pathogen's antigen gene. Because they are entirely synthetic, there are no issues with virulence reversion or incomplete inactivation, making them safer. They also meet the requirements for rapid and efficient production, are more suitable for addressing viral mutations, and reduce vaccine production costs. Furthermore, these vaccines do not contain the complete viral structure, resulting in higher safety. Different peptide antigenic epitopes can be combined, and corresponding synthetic antigenic peptides can be constructed targeting complex, discontinuous natural antigenic determinants.

[0003] Despite the numerous advantages of peptide vaccines, they also face several technical bottlenecks. The most significant of these is the small molecular weight of peptides, which results in low immunogenicity and poor immune response. Therefore, selecting adjuvants with immunomodulatory effects that can enhance the immune response is crucial for peptide vaccines to function effectively. Summary of the Invention

[0004] The present invention aims to at least partially solve the technical problems in the related art. To this end, one aspect of the present invention provides a vaccine adjuvant composition.

[0005] Another aspect of the present invention provides the use of this vaccine adjuvant composition in the preparation of vaccine adjuvants.

[0006] In another aspect, the present invention provides a polypeptide vaccine formulation prepared using the above-described vaccine adjuvant composition.

[0007] This invention provides a vaccine adjuvant composition comprising:

[0008] (a) Liposomes, the liposomes comprising phospholipids, sterols and squalene;

[0009] (b) Chitosan and / or chitosan derivatives that coat liposomes.

[0010] In some embodiments, the vaccine adjuvant composition further includes: (c) saponins.

[0011] In some embodiments, the vaccine adjuvant composition further includes: (d) monophosphoyl ester A.

[0012] In some embodiments, the liposomes further include mannose.

[0013] This invention also provides a vaccine adjuvant composition comprising:

[0014] (a) Liposomes, which include phospholipids and sterols but exclude squalene;

[0015] (b) Chitosan and / or chitosan derivatives that encapsulate liposomes;

[0016] (c) Saponins.

[0017] Furthermore, the vaccine adjuvant composition further includes: (d) monophospholipase A.

[0018] The present invention also provides the use of the composition in the preparation of vaccine adjuvants, wherein the composition is the above-mentioned vaccine adjuvant composition.

[0019] In some embodiments, the vaccine is a peptide vaccine.

[0020] In some embodiments, the peptide vaccine includes a peptide antigen, which includes a peptide associated with a coronavirus.

[0021] In some embodiments, the polypeptide antigen includes a coronavirus-related polypeptide, polypeptide derivative, or polypeptide-carrier protein conjugate.

[0022] In some embodiments, the coronavirus is SARS-CoV-2.

[0023] This invention also provides a polypeptide vaccine formulation, comprising:

[0024] (i) A polypeptide antigen that elicits an immune response against it in subjects in need of it;

[0025] (ii) the above-mentioned vaccine adjuvant composition,

[0026] Furthermore, the polypeptide antigen is encapsulated in the liposomes within the vaccine adjuvant composition.

[0027] In some embodiments, the polypeptide antigen includes polypeptides associated with coronaviruses.

[0028] In some embodiments, the polypeptide antigen includes a coronavirus-related polypeptide, polypeptide derivative, or polypeptide-carrier protein conjugate.

[0029] In some embodiments, the coronavirus is SARS-CoV-2.

[0030] In some embodiments, the polypeptide antigen includes an antigenic epitope polypeptide of the S protein of SARS-CoV-2.

[0031] In some embodiments, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole hemocyanin, or casein.

[0032] The advantages and beneficial effects of the present invention are as follows: the liposome-based vaccine adjuvant composition of the present invention has a significant immune-enhancing effect and significantly improves the effect of polypeptide antigens in stimulating the body's immunity.

[0033] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0034] Figure 1 To observe the morphology of peptide vaccines formed by antigens encapsulated in different vaccine adjuvants under a microscope. A corresponds to aluminum adjuvant, B to liposomal adjuvant L1, C to liposomal adjuvant L2, D to liposomal adjuvant L3, E to liposomal adjuvant L4, F to liposomal adjuvant L5, and G to liposomal adjuvant LX. Detailed Implementation

[0035] The embodiments of the present invention are described in detail below. These embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0036] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0037] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are standard procedures widely used in their respective fields or performed according to the manufacturer's recommendations.

[0038] The following is a description of the terminology used in this article:

[0039] The terms “comprising” and “including” are open-ended expressions, meaning they include the contents specified in this invention, but do not exclude other aspects.

[0040] The term "vaccine" refers to any drug that can specifically induce an immune response in the body against a target antigen, thereby effectively preventing or treating diseases related to that target antigen.

[0041] The term "vaccine adjuvant" refers to a substance or composition added to a vaccine to enhance and / or influence the immune response. Generally, the term "vaccine adjuvant" should be interpreted broadly, and adjuvants may include, but are not limited to, immune enhancers, antigen delivery systems, or combinations thereof.

[0042] The term "liposome" refers to a single-layer or multi-layer lipid structure encapsulating an aqueous interior. Methods for preparing liposomes are well known in the art. Any suitable method for preparing liposomes may be used in the practice of this invention.

[0043] The term "coronavirus" refers to single-stranded positive-sense RNA viruses belonging to the subfamily Orthocoronavirinae of the family Coronaviridae in the order Nidovirales. These viruses can infect a variety of species, including humans, bats, pigs, rats, cattle, horses, goats, and monkeys. Seven types of human-infecting coronaviruses (HCoVs) are known, including Middle East Respiratory Syndrome-related coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome-related coronavirus (SARS-CoV).

[0044] The terms "COVID-19," "novel coronavirus," and "SARS-CoV-2" all refer to the coronavirus named SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11, 2020. Currently known variants of the novel coronavirus SARS-CoV-2 include, but are not limited to, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, and Omicron.

[0045] The term "peptide" in this application refers to any peptide segment that is predicted or screened to specifically bind to an antibody or sensitized lymphocyte.

[0046] The term "peptide-carrier protein conjugate" in this application refers to an antigen formed by the conjugation of a peptide and a carrier protein. One carrier protein can conjugate one or more peptides, and when multiple peptides are conjugated, the peptides have the same or different amino acid sequences. Depending on the physicochemical properties of the specific conjugated peptide sequences, the type of specific carrier protein, and the conjugation method, the number of peptides conjugated to each carrier protein varies. In this application, 3 to 50 peptides are preferred; more preferably, 3 to 45, 5 to 40, 5 to 35, 5 to 30, 8 to 30, 10 to 30, 12 to 30, and 15 to 30 peptides are preferred; even more preferably, any one of 6 to 36, 8 to 32, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, and 10 to 15 peptides are preferred.

[0047] The term "antigen" refers to any substance capable of inducing an immune response in the body. Specifically, it refers to substances that can specifically bind to antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and specifically binding to these products both in vivo and in vitro. Therefore, antigens possess two important characteristics: immunogenicity and immunoreactivity. In some examples, an antigen refers to a complete, immunogenic antigen formed by the conjugation of a polypeptide hapten with a carrier protein. This can be a polypeptide-carrier protein conjugate formed by conjugating a polypeptide with a single amino acid sequence with a carrier protein; or a combination of polypeptide-carrier protein conjugates formed by conjugating polypeptides with multiple different amino acid sequences with a carrier protein.

[0048] When a value is described as a range, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical value falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0049] As used in this article, the term “about” means + / - 10% of the listed values.

[0050] This invention provides a vaccine adjuvant composition comprising:

[0051] (a) Liposomes, the liposomes comprising phospholipids, sterols and squalene;

[0052] (b) Chitosan and / or chitosan derivatives that coat liposomes.

[0053] In some embodiments, the phospholipids include, but are not limited to, at least one of soybean lecithin and egg yolk lecithin.

[0054] In some embodiments, the cholesterol includes, but is not limited to, at least one of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, or cholesterol.

[0055] In some embodiments, chitosan derivatives include, but are not limited to, chitosan salt derivatives, chitosan ester derivatives, or at least one of acylated chitosan, carboxylated chitosan, alkylated chitosan, glycosylated chitosan, and quaternized chitosan. Further, chitosan derivatives include, but are not limited to, at least one of chitosan hydrochloride, chitosan acetate, chitosan lactate, and chitosan quaternary ammonium salts.

[0056] In some embodiments, the number-average molecular weight of chitosan or chitosan derivatives ranges from 20 to 500 kDa.

[0057] In some embodiments, the degree of deacetylation of chitosan or chitosan derivatives is 85-95%.

[0058] In some embodiments, the vaccine adjuvant composition further includes: saponin (QS-21).

[0059] Furthermore, it also includes: monophospholipase A (MPLA).

[0060] In some embodiments, liposomes also include mannose.

[0061] This invention also provides a vaccine adjuvant composition, comprising:

[0062] (a) Liposomes, which include phospholipids and sterols but exclude squalene;

[0063] (b) Chitosan and / or chitosan derivatives that encapsulate liposomes;

[0064] (c) Saponins (QS-21).

[0065] Furthermore, the above-mentioned vaccine adjuvant composition also includes: (d) monophospholipase A (MPLA).

[0066] The present invention also provides the use of the above-described vaccine adjuvant composition in the preparation of vaccine adjuvants.

[0067] In some embodiments of the application, the vaccine is a peptide vaccine.

[0068] In some embodiments of the use, the peptide vaccine includes a peptide antigen, which includes a coronavirus-related peptide, peptide derivative, or peptide-carrier protein conjugate.

[0069] In some embodiments of the use, the coronavirus is severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, or novel coronavirus.

[0070] In some embodiments of the use, the coronavirus is SARS-CoV-2.

[0071] In some embodiments of the use, the peptides associated with the coronavirus SARS-CoV-2 are selected from the peptides in Table 1 of patent application publication number CN114276422A. Non-limiting examples include:

[0072] RGDEVRQIAPGQTGKIADYNYKL (SEQ ID NO: 1)

[0073] SKVGGNYNYLYRLFRKSNLKPFERDISTE(SEQ ID NO: 2)

[0074] This invention also provides a polypeptide vaccine formulation, characterized in that it comprises:

[0075] (i) A polypeptide antigen that elicits an immune response against it in subjects in need of it;

[0076] (ii) The above-described vaccine adjuvant composition.

[0077] Furthermore, the polypeptide antigen is encapsulated in liposomes within the vaccine adjuvant composition.

[0078] In some embodiments, the proportion (mass-volume ratio) of the polypeptide antigen in the polypeptide vaccine formulation is 0.01% to 5%. For example, it is between 0.01% and 0.1%, between 0.1% and 0.3%, between 0.5% and 0.8%, between 1% and 1.5%, between 2% and 3%, between 3.5% and 5%, etc., such as about 0.04%, about 0.08%, about 0.1%, about 0.5%, about 1%, about 2%, about 5%, etc.

[0079] In some embodiments, the proportion (mass-volume ratio) of squalene in the peptide vaccine formulation is 0.15% to 7.5%. For example, it is between 0.15% and 0.3%, between 0.5% and 0.7%, between 0.8% and 1%, between 1% and 2%, between 4% and 5%, between 5.5% and 7.5%, etc., such as about 0.15%, about 0.6%, about 0.7%, about 1%, about 3%, about 7.5%, etc.

[0080] In some embodiments, the proportion (mass-volume ratio) of chitosan and / or chitosan derivatives in the polypeptide vaccine formulation is 0.05% to 5.0%. For example, it is between 0.05% and 0.2%, between 0.3% and 0.5%, between 0.8% and 1%, between 1% and 2%, between 3% and 4%, between 4% and 5%, etc., such as about 0.05%, about 0.2%, about 0.3%, about 1%, about 3%, about 5%, etc.

[0081] In some embodiments, the proportion (mass-volume ratio) of saponin (QS-21) in the polypeptide vaccine formulation is 0.0001% to 0.008%. For example, it is between 0.0001% and 0.0002%, between 0.0003% and 0.0005%, between 0.001% and 0.002%, between 0.0035% and 0.005%, between 0.006% and 0.008%, etc., such as about 0.0002%, about 0.0003%, about 0.0004%, about 0.001%, about 0.002%, about 0.008%, etc.

[0082] In some embodiments, the proportion (mass-volume ratio) of monophospholipase A (MPLA) in the peptide vaccine formulation is 0.0001% to 0.008%. For example, it is between 0.0001% and 0.0002%, between 0.0003% and 0.0005%, between 0.001% and 0.002%, between 0.0035% and 0.005%, between 0.006% and 0.008%, etc., such as about 0.0002%, about 0.0003%, about 0.0004%, about 0.001%, about 0.002%, about 0.008%, etc.

[0083] In some embodiments, the proportion (mass-volume ratio) of mannose in the polypeptide vaccine formulation is 0.01% to 1%. For example, it is between 0.01% and 0.02%, between 0.05% and 0.08%, between 0.1% and 0.3%, between 0.35% and 0.4%, between 0.5% and 0.8%, between 0.9% and 1%, etc., such as about 0.01%, about 0.02%, about 0.08%, about 0.1%, about 0.3%, about 0.5%, 1%, etc.

[0084] In some embodiments of peptide vaccine formulations, the peptide antigen includes coronavirus-related peptides, peptide derivatives, or peptide-carrier protein conjugates.

[0085] In some embodiments of the peptide vaccine formulation, the coronavirus is severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, or novel coronavirus.

[0086] In some embodiments of the peptide vaccine formulation, the coronavirus is SARS-CoV-2.

[0087] In some embodiments of peptide vaccine formulations, the peptide antigen includes an epitope peptide of the S protein of SARS-CoV-2.

[0088] In some embodiments of the peptide vaccine formulation, when the peptide vaccine formulation is a SARS-CoV-2 peptide vaccine formulation, the peptide antigen includes one or more peptides from Table 1 of patent application publication number CN114276422A. The multiple peptides exist in tandem or parallel configurations. Preferably, at least one peptide in the peptide antigen is connected in tandem or parallel configurations 1 to 10 times, more preferably 1 to 6 times; more preferably, the multiple peptides are connected in tandem or parallel configurations via connecting arms; even more preferably, the connecting arms are glycine, lysine, AEA, Ava, ANP, β-alanine, GAB, or PEG. Non-limiting examples of peptides include:

[0089] RGDEVRQIAPGQTGKIADYNYKL (SEQ ID NO: 1)

[0090] SKVGGNYNYLYRLFRKSNLKPFERDISTE(SEQ ID NO: 2)

[0091] In some embodiments of the peptide vaccine formulation, when the peptide vaccine formulation is a SARS-CoV-2 peptide vaccine formulation, the peptide antigen includes one or more peptides from Table 1 of patent application publication number CN114276422A and a carrier protein coupled thereto. Non-limiting examples include:

[0092] RGDEVRQIAPGQTGKIADYNYKL (SEQ ID NO: 1)

[0093] SKVGGNYNYLYRLFRKSNLKPFERDISTE(SEQ ID NO: 2)

[0094] In some embodiments of the peptide vaccine, the carrier protein is selected from bovine serum albumin (BSA), ovalbumin (OVA), keyhole hemocyanin (KLH), or casein (CS).

[0095] In a specific embodiment of the present invention, the following immunogens are prepared:

[0096] PEP-B-KLH, PEP-C-KLH, PEP-B-MAP, PEP-C-MAP

[0097] PEP-B:S403-425(RGDEVRQIAPGQTGKIADYNYKL)(SEQ ID NO: 1)

[0098] PEP-C:S443-471(SKVGGNYNYLYRLFRKSNLKPFERDISTE)(SEQ ID NO: 2)

[0099] The immunogen was prepared using the following method (taking hemocyanin as the carrier protein as an example):

[0100] (1) Peptide synthesis: Organic chemical solid-phase synthesis method (Fmoc protected amino acids, solid-phase carrier-resin) was adopted. The three-channel automated peptide synthesizer (CS360 model) produced by CS Company, USA, was used to synthesize from the carboxyl terminus of the peptide to the amino terminus to obtain peptide resin. Then, the peptide was cut off from the resin by TFA method to obtain crude product.

[0101] (2) Peptide purification: The peptides were purified using a Waters high-performance liquid chromatograph with a C18 reversed-phase column and then freeze-dried. The purity of the synthesized peptides was all above 90%.

[0102] (3) Ligation of polypeptide fragments to carrier protein-hemocyanin (KLH): The steps are as follows:

[0103] 1) Dissolve 20 mg of KLH in PBS (pH 7) to a final concentration of 10 mg / ml. Add the coupling reagent m-maleimide benzoyl-N-hydroxysuccinimide (MBS, Thermo Fisher), and react at room temperature for 1 h to form a KLH-MBS complex. Remove the free coupling reagent by dialysis with PBS. 2) Dissolve 10 mg of the peptide (for peptide fragments lacking cysteine, introduce cysteine ​​at the amino or carboxyl terminus of its sequence) in PBS to obtain a solution with a concentration of 10 mg / ml. 3) Mix the peptide solution with the prepared KLH-MBS and react at room temperature for 2 h. 4) Dialyze the reaction mixture with PBS to terminate the reaction, obtaining the peptide antigen. 5) Quantify the protein; the obtained antigen concentration is approximately 5 mg / ml, of which the peptide is approximately 0.5 mg / ml.

[0104] MAP (Multi-antigen peptide): A polypeptide compound (uncoupled hemocyanin) synthesized using organic chemical solid-phase synthesis.

[0105] Example 1: PEP-C-MAP(LX)

[0106] prescription

[0107]

[0108] The 1% chitosan solution refers to chitosan dissolved in HAc-NaAc buffer (pH 4.5). The other examples are similar.

[0109] Preparation method: Ethanol injection-high pressure homogenization method

[0110] Weigh 300 mg of soybean lecithin, 30 mg of cholesterol, and 150 mg of squalene, dissolve them in 20 mL of anhydrous ethanol to form a clear solution, then slowly add 10 mL of phosphate buffer solution dropwise to the lipid solution while stirring magnetically at 400 rpm for 15 min to form a homogeneous and transparent emulsion. Place the emulsion in a pear-shaped flask and remove the ethanol by rotary evaporation in a 40°C water bath to obtain an intermediate product. Add 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) and shake at room temperature for 5 min to obtain a crude liposome suspension. Transfer the suspension to a beaker, add 5 mL of prepared 1% chitosan solution, and place the beaker on an ultrasonic cell disruptor. Sonicate the beaker 40 times in an ice-water bath, with a 4-second cycle and a 4-second rest. Then homogenize the sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0111] Example 2: PEP-C-MAP(LYZ) group

[0112] prescription:

[0113]

[0114] Preparation method: Thin film dispersion-high pressure homogenization method

[0115] Accurately weigh 110 mg of soybean lecithin and 20 mg of cholesterol into a 250 mL pear-shaped flask. Add 10 mL of chloroform and stir until fully dissolved to obtain a lipid solution. Place the solution in a 45 °C water bath and evaporate under reduced pressure using a rotary evaporator to remove the organic solvent, allowing the chloroform solution of phospholipids to form a phospholipid film on the wall of the pear-shaped flask. Simultaneously, take 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) and place it in the same flask in a 45 °C water bath for incubation. After the phospholipid film has formed, add 10 mL of preheated phosphate buffer to the flask containing the phospholipid film, and continue hydration under vacuum rotary evaporation in a 45 °C water bath for 15 min. Rotate the hydration solution until the gel detaches from the bottle wall. Remove the liposome solution into a beaker, add 5 mL of the prepared 1% chitosan solution, and place the beaker on an ultrasonic cell disruptor. Sonicate for 40 cycles in an ice-water bath, with a 4-second cycle followed by a 4-second rest. Then, homogenize the resulting sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes using repeated cycles to obtain the liposome sample. Add 60 μg of MPLA and 60 μg of QS-21, and then sonicate until completely dissolved.

[0116] Example 3: PEP-C-MAP(LXZ) group

[0117] prescription

[0118]

[0119]

[0120] Preparation method: Ethanol injection-high pressure homogenization method

[0121] Weigh 300 mg of soybean lecithin, 30 mg of cholesterol, and 150 mg of squalene, dissolve them in 20 mL of anhydrous ethanol to form a clear solution, then slowly add 10 mL of phosphate buffer solution dropwise to the lipid solution while stirring magnetically at 400 rpm for 15 min to form a homogeneous and transparent emulsion. Place the emulsion in a pear-shaped flask and remove the ethanol by rotary evaporation in a 40°C water bath to obtain an intermediate product. Add 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) and shake at room temperature for 5 min to obtain a crude liposome suspension. Transfer the suspension to a beaker, add 5 mL of prepared 1% chitosan solution, and place the beaker on an ultrasonic cell disruptor. Sonicate for 40 cycles in an ice-water bath, with a 4-second cycle and a 4-second interval. Homogenize the resulting sample repeatedly at 100,000 kPa for 6 minutes. Add 100 μg of QS-21 and sonicate until completely dissolved.

[0122] Example 4: PEP-C-MAP(LXYZ) group

[0123] prescription

[0124]

[0125] Preparation method: Ethanol injection-high pressure homogenization method

[0126] Weigh 300 mg of soybean lecithin, 30 mg of cholesterol, and 150 mg of squalene, dissolve them in 20 mL of anhydrous ethanol to form a clear solution, then slowly add 10 mL of phosphate buffer solution dropwise to the lipid solution while stirring magnetically at 400 rpm for 15 min to form a homogeneous and transparent emulsion. Place the emulsion in a pear-shaped flask and remove the ethanol by rotary evaporation in a 40°C water bath to obtain an intermediate product. Add 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) and shake at room temperature for 5 min to obtain a crude liposome suspension. Transfer the liposome solution to a beaker, add 5 mL of a prepared 1% chitosan solution, and place it on an ultrasonic cell disruptor. Sonicate for 40 cycles in an ice-water bath, with a 4-second working cycle and a 4-second rest. Then, homogenize the obtained sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes. Add 100 μg QS-21 and 100 μg MPLA, and disperse by ultrasonication until completely dissolved.

[0127] Example 5: PEP-C-MAP(LXD) group

[0128] prescription

[0129]

[0130] Preparation method: Ethanol injection-high pressure homogenization method

[0131] Weigh out 300 mg of soybean lecithin, 30 mg of cholesterol, 20 mg of mannose, and 150 mg of squalene. Dissolve them in 20 mL of anhydrous ethanol to form a clear solution. Then, under magnetic stirring at 400 rpm, slowly and continuously add 10 mL of phosphate buffer to the lipid solution, stirring thoroughly for 15 min to form a homogeneous and transparent emulsion. Place the emulsion in a pear-shaped flask and remove the ethanol by rotary evaporation in a 40°C water bath to obtain an intermediate product. Add 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) and shake at room temperature for 5 min to obtain a crude liposome suspension. Transfer the suspension to a beaker, add 5 mL of a prepared 1% chitosan solution, and place it on an ultrasonic cell disruptor. Sonicate for 40 cycles in an ice-water bath, with a 4-second working cycle and a 4-second rest. Then, homogenize the obtained sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0132] Comparative Example 1: Antigens encapsulated in conventional liposomes

[0133] (1) Prescription

[0134]

[0135] (2) Thin film dispersion-high pressure homogenization method

[0136] Weigh 0.5g soybean lecithin, 0.048g cholesterol, and 0.048g octadecylamine into a 250mL pear-shaped flask. Add 15mL chloroform and stir until fully dissolved to obtain a lipid solution. Evaporate under reduced pressure in a rotary evaporator at 45℃ to remove the organic solvent, allowing the chloroform solution of phospholipids to form a phospholipid film on the wall of the pear-shaped flask. Simultaneously, dissolve 20mg polypeptide antigen, 0.5mL Tween 80, and 0.048g PEG6000 in 24mL phosphate buffer in a small beaker and place both in a 45℃ water bath for incubation. Then, add the preheated 24mL phosphate buffer to the flask containing the phospholipid film and continue rotary evaporation for 15min in a 45℃ water bath. Rotate the hydration solution until the gel detaches from the bottle wall. Take out the liposome solution into a beaker, place it on an ultrasonic cell disruptor, and sonicate it 60 times in an ice-water bath with a 4-second working and 4-second rest interval. Then, homogenize the obtained sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0137] Comparative Example 2: PEP-C-MAP (L1)

[0138] prescription

[0139]

[0140] Preparation method: Thin film dispersion-high pressure homogenization method

[0141] Weigh 110 mg of soybean lecithin and 20 mg of cholesterol into a 250 mL pear-shaped flask, add 10 mL of chloroform and stir until fully dissolved to obtain a lipid solution. Place the flask in a 45 °C water bath and evaporate under reduced pressure using a rotary evaporator to remove the organic solvent, allowing the chloroform solution of phospholipids to form a phospholipid film on the wall of the pear-shaped flask. Simultaneously, take 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) into a small beaker and place it in a 45 °C water bath for incubation. After the phospholipid film has formed, take 10 mL of the preheated synthetic peptide solution and add it to the flask containing the phospholipid film. Continue to hydrate under vacuum by rotary evaporation in a 45 °C water bath for 15 min. Rotate the hydration solution until the gel detaches from the bottle wall. Take out the liposome solution into a beaker, add 5 mL of the prepared 1% chitosan solution, place it on an ultrasonic cell disruptor, and sonicate for 40 times in an ice-water bath with a 4-second working and 4-second rest interval. Then, homogenize the obtained sample repeatedly in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0142] Comparative Example 3: PEP-C-MAP(L2)

[0143] The difference between this and PEP-C-MAP(L1) is that the concentration of chitosan is increased by 5 times, meaning the formulation uses a 5% chitosan solution. The preparation method is the same as for L1 liposomes.

[0144] Comparative Example 4: PEP-C-MAP (L3)

[0145] prescription

[0146]

[0147] Preparation method: Thin film dispersion-high pressure homogenization method

[0148] Weigh 110 mg of soybean lecithin, 20 mg of cholesterol, and 14 mg of octadecylamine into a 250 mL pear-shaped flask. Add 10 mL of chloroform and stir until fully dissolved to obtain a lipid solution. Place the flask in a 45°C water bath and evaporate under reduced pressure using a rotary evaporator to remove the organic solvent, allowing the chloroform solution of phospholipids to form a phospholipid film on the wall of the pear-shaped flask. Simultaneously, take 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) into a small beaker and place it in a 45°C water bath for incubation. After the phospholipid film has formed, add 10 mL of preheated synthetic peptide solution to the flask containing the phospholipid film, and continue hydration under vacuum rotary evaporation in a 45°C water bath for 15 min. Rotate the hydration solution until the gel detaches from the bottle wall. Take out the liposome solution into a beaker, place it on an ultrasonic cell disruptor, and sonicate it 40 times in an ice-water bath with a 4-second working interval and a 4-second rest interval. Then, homogenize the obtained sample repeatedly in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0149] Comparative Example 5: PEP-C-MAP (L4)

[0150] prescription

[0151]

[0152]

[0153] Preparation method: Thin film dispersion-high pressure homogenization method

[0154] Accurately weigh 110 mg of soybean lecithin and 20 mg of cholesterol into a 250 mL pear-shaped flask. Add 10 mL of chloroform and stir until fully dissolved to obtain a lipid solution. Place the flask in a 45°C water bath and evaporate under reduced pressure using a rotary evaporator to remove the organic solvent, allowing the chloroform solution of lecithin to form a phospholipid film on the wall of the pear-shaped flask. Simultaneously, dissolve 10 mg of polypeptide antigen in 5 mL of phosphate buffer in a small beaker and place it in a 45°C water bath for incubation. After the phospholipid film has formed, add 5 mL of preheated phosphate buffer to the flask containing the phospholipid film and continue hydration under vacuum rotary evaporation for 15 min in a 45°C water bath. Continue hydration by rotation until the gel detaches from the flask wall, and remove the liposome solution into a beaker. Take 5 mL of the diluted solution... Add (6 mg / mL, diluted to 3 mg / mL) to a beaker, place it on an ultrasonic cell disruptor, sonicate for 40 times in an ice-water bath, with a 4-second working interval and a 4-second rest. Then, homogenize the obtained sample repeatedly in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0155] Comparative Example 6: PEP-C-MAP (L5)

[0156] prescription

[0157]

[0158] Preparation method: Ethanol injection-high pressure homogenization method

[0159] Weigh 300 mg of soybean lecithin, 30 mg of cholesterol, and 150 mg of squalene, dissolve them in 20 mL of anhydrous ethanol to form a clear solution, then slowly add 10 mL of phosphate buffer solution dropwise to the lipid solution while stirring magnetically at 400 rpm for 15 min to form a homogeneous and transparent emulsion. Place the emulsion in a pear-shaped flask and remove the ethanol by rotary evaporation in a 40°C water bath to obtain an intermediate product. Add 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) and shake at room temperature for 5 min to obtain a crude liposome suspension. Place the suspension on an ultrasonic cell disruptor and sonicate for 40 cycles in an ice-water bath, with a 4-second working cycle and a 4-second rest. Then, homogenize the obtained sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes.

[0160] Comparative Example 7: PEP-C-MAP(LY) group:

[0161] prescription

[0162]

[0163] Preparation method: Thin film dispersion-high pressure homogenization method

[0164] Weigh 110 mg of soybean lecithin and 20 mg of cholesterol into a 250 mL pear-shaped flask, add 10 mL of chloroform and stir until fully dissolved to obtain a lipid solution. Place the flask in a 45 °C water bath and evaporate under reduced pressure using a rotary evaporator to remove the organic solvent, allowing the chloroform solution of phospholipids to form a phospholipid film on the wall of the pear-shaped flask. Simultaneously, take 10 mL of synthetic peptide solution (10 mg of synthetic peptide antigen dissolved in 10 mL of phosphate buffer, 1 mg / mL) into a small beaker and place it in a 45 °C water bath for incubation. After the phospholipid film has formed, take 10 mL of the preheated synthetic peptide solution and add it to the flask containing the phospholipid film. Continue to hydrate under vacuum by rotary evaporation in a 45 °C water bath for 15 min. Rotate the hydration solution until the gel detaches from the bottle wall. Remove the liposome solution into a beaker, add 5 mL of the prepared 1% chitosan solution, and place the beaker on an ultrasonic cell disruptor. Sonicate for 40 cycles in an ice-water bath, with a 4-second cycle followed by a 4-second rest. Then, homogenize the resulting sample in a high-pressure homogenizer at 100,000 kPa for 6 minutes using repeated cycles to obtain the liposome sample. Add 60 μg of MPLA and sonicate until completely dissolved.

[0165] Experimental Example 1

[0166] New Zealand white rabbits were immunized subcutaneously with antigens loaded with aluminum adjuvant, Freund's adjuvant, and conventional liposome adjuvant, respectively.

[0167] Preparation of antigen loaded with aluminum adjuvant: The working concentration of aluminum adjuvant was 3 mg / mL. Before immunization, it was mixed with an equal volume of antigen solution and dispersed by ultrasonic cell disruptor. The immunization volume was 250 μL / animal.

[0168] Preparation of Freund's adjuvant-loaded antigen: Freund's adjuvant and antigen solution were mixed in equal volumes and emulsified using an ultrasonic cell disruptor. The immunization volume was 250 μL / animal.

[0169] The volume of antigen-loaded immunoliposomes injected was 0.5 mL per animal (containing 400 μg of antigen), and immunizations were performed four times at two-week intervals. Serum specific antibody titers were measured by ELISA after the fourth immunization. The results are shown in Table 1. It can be seen that this conventional liposome adjuvant group failed to elicit an effective immune response.

[0170] Table 1. Serum antibody titers after subcutaneous immunization of New Zealand rabbits with different antigen-adjuvant complexes

[0171]

[0172]

[0173] Experiment Example 2

[0174] Selection of Immunogens in Experiment Example 2

[0175] PEP-C-MAP polypeptide sequence: S 443-471 (SKVGGNYNYLYRLFRKSNLKPFERDISTE)(SEQ IDNO: 2)

[0176] Based on the failure of the conventional liposomal vaccine adjuvant group constructed in Experiment Example 1 to elicit a significant immune response in animals, and considering that antigen-presenting cells are usually negatively charged and easily adhere to cationic liposomes, thereby promoting phagocytosis and antigen presentation, the following experiments were conducted in this example:

[0177] Experimental group: PEP-C-MAP(LX)

[0178] Control group: PEP-C-MAP(A1) (aluminum adjuvant), PEP-C-MAP(L1), PEP-C-MAP(L2), PEP-C-MAP(L3), PEP-C-MAP(L4), PEP-C-MAP(L5), blank control group.

[0179] The control group, PEP-C-MAP(Al), consisted of an aluminum adjuvant at a concentration of 3 mg / mL, mixed with an equal volume of antigen solution and dispersed using an ultrasonic cell disruptor. The immunization volume was 500 μL per animal.

[0180] (I) Observation of the morphology of liposomes carrying antigens

[0181] Liposomes were diluted with phosphate buffer, and the liposome solution was dropped onto a glass slide, covered with a coverslip, and observed under a microscope. The prepared sample was observed under an inverted fluorescence microscope at 200x magnification. The results are shown below. Figure 1 ,pass Figure 1 It can be seen that the PEP-C-MAP(A1) group is generally in a state of particle aggregation, while the PEP-C-MAP(L1), PEP-C-MAP(L2), PEP-C-MAP(L3), PEP-C-MAP(L4), PEP-C-MAP(L5), and PEP-C-MAP(LX) groups are spherical small particles with uniform particle size.

[0182] (II) Particle size and distribution of liposomes carrying antigens

[0183] Dynamic light scattering (DLS) was used for determination. One mL of nanoliposome suspension was diluted 10-fold with phosphate buffer, and the particle size and distribution were measured using a nanoparticle size analyzer at 25℃. The results are shown in Table 2.

[0184] Table 2 Particle size and Zeta potential

[0185] Al (aluminum adjuvant) 9641 15.8 10.4±0.3 L1 259.5 20.7 55.1±0.7 L2 478.9 30.3 63.3±0.5 L3 238.6 17.6 -31.7±0.6 L4 288.1 19.1 37.7±0.6 L5 293.3 24.8 -31.3±0.2 LX 597.5 29.8 63.0±0.6

[0186] Table 2 shows that, except for aluminum adjuvant, the particle size of all liposome adjuvants is between 200-600 nm. Liposomes L3 and L5 have a particle size of 200-300 nm, a low polydispersity index, but are negatively charged. Liposome L4, combined with aluminum adjuvant, not only has a positive surface charge but also a much smaller particle size than the aluminum adjuvant-only group. Liposomes L1, L2, and LX, coated with chitosan solutions of different concentrations, are positively charged with absolute potentials greater than 50 mV. Among them, L2 and LX carry more positive charges, with potentials exceeding 60 mV, but their particle size and particle size dispersion are increased.

[0187] (III) Evaluation of Immunological Activity

[0188] (1) Immune process

[0189] Twenty-four New Zealand white rabbits were randomly divided into eight groups (aluminum adjuvant group, six liposome groups, and a blank control group), with three rabbits in each group. They were vaccinated on days 0, 14, 28, and 42, for a total of four immunizations, with a two-week interval between immunizations. The total injection volume per rabbit was 0.5 mL. The blank control group received no treatment.

[0190] (2) Blood collection

[0191] During the immunization period, blood was collected from the marginal ear vein on day 38 after the first vaccination and serum was separated. Blood was collected from the heart on day 52 after vaccination to detect the humoral immunity level in the collected samples.

[0192] Rabbit marginal ear vein blood collection: Place the New Zealand white rabbit in a holding box and observe the blood vessels in both ears. Select the side with good vein filling and a clear view. Remove the fur from the area to be blood collected, clean the skin surface, and keep it dry. Gently flick the vein with your finger, or wipe it with alcohol or apply local heat to make it fill and dilate. Press down firmly on the base of the ear with one hand, and hold a 2mL syringe in the other hand to slowly collect blood by inserting the needle into the marginal ear vein. The blood collection volume is 2mL per rabbit, and then immediately apply pressure with a sterile cotton ball to stop the bleeding.

[0193] Cardiac blood sampling: The rabbit is placed in a supine position on the operating table, with the heels of both forelimbs and hindlimbs also secured. The fur on the left chest is trimmed to fully expose the skin. Gently press the left anterior axillary region 2-2.5 cm above the level of the left sternal fossa (between the 3rd and 4th intercostal spaces) to locate the heartbeat and mark it as the puncture site. Disinfect the marked site with a 75% ethanol swab. Using a 7-gauge needle syringe, insert the needle vertically through the intercostal space 3 mm from the left edge of the sternum at the third intercostal space, maintaining a 45° angle with the horizontal plane. The depth should be approximately 3 cm. A pulsating sensation should be felt upon puncture, and blood should immediately flow into the syringe. The amount of blood obtained is typically 20-25 mL.

[0194] Serum separation method: After blood sample collection, the sample was placed in a 37℃ incubator for 2 hours, followed by centrifugation at 3500 rpm for 10 minutes to separate the serum. The serum was then aliquoted and frozen.

[0195] (3) Detection of serum antibody levels by indirect ELISA method

[0196] Dilute the polypeptide antigen to 2 μg / mL with coating buffer and coat the microplate overnight at 4°C. Add 200 μL of blocking buffer (5% fetal bovine serum) to each well and incubate at 37°C for 2 hours. After blocking, wash the wells three times with washing buffer. Then, starting with a 1:500 (rabbit) dilution, add serially diluted 2-fold buffers to each well of a 96-well microplate (100 μL / well) and incubate at 37°C for 1 hour. After washing three times with washing buffer, add horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:20000, ZSGB-BIO) to the plate and incubate at 37°C for 1 hour. Finally, wash the plate three times with washing buffer, add chromogenic solution, incubate at 37°C in the dark for 15 minutes, add stop solution, and read the absorbance at 450 nm using a microplate reader (Spectra Max M2).

[0197] (4) Statistical Analysis

[0198] GraphPad 8.0 software was used to compare and analyze the different immune groups. One-way ANOVA and multiple comparisons were employed. A p-value < 0.05 was considered statistically significant. (*P < 0.05, **P < 0.01)

[0199] (5) Results

[0200] After three immunizations in New Zealand white rabbits, the L1 group consistently produced antibodies, but with low titers (500, 500, 1000). The LX group had higher average antibody levels, with titers reaching (500, 8000, 16000). After four immunizations, both the L1 and LX groups consistently produced specific antibodies. The L1 group had titers of (8000, 4000, 4000), while the LX group showed a 1-3 fold increase in specific antibody titers compared to the L1 group, reaching (16000, 16000, 16000), a highly significant difference compared to the aluminum adjuvant control group (P<0.01). The aluminum adjuvant control group stimulated an immune response, but antibody levels were low and showed significant individual variability. The L3 group achieved specific antibody titers of (8000, 1000, 32000), but also exhibited considerable individual variability. The L2 and L5 groups showed poor immunization effects. The results of the specific antibody titer determination are shown in Table 3.

[0201] Table 3. ELISA method for determining rabbit serum-specific IgG antibodies.

[0202]

[0203]

[0204] Experimental Example 3

[0205] Selection of immunogens in Experiment Example 3

[0206] PEP-C-MAP polypeptide sequence: S 443-471 (SKVGGNYNYLYRLFRKSNLKPFERDISTE)(SEQ IDNO: 2)

[0207] The following experiments were conducted in this experimental example:

[0208] Experimental groups: PEP-C-MAP(LX) group, PEP-C-MAP(LYZ) group; Control groups: PEP-C-MAP group (without adjuvant), PEP-C-MAP(A1) group, PEP-C-MAP(L1) group, PEP-C-MAP(LY) group.

[0209] Immunological activity evaluation

[0210] (1) Grouping and immunization process of experimental animals

[0211] Thirty-six mice were randomly divided into six groups of six. After three days of acclimatization, the mice exhibited normal appetite, glossy fur, and good health, making them suitable for immunization. In this experiment, the polypeptide vaccine was administered subcutaneously (to both sides of the groin and abdomen), with a total injection volume of 200 μL per mouse. Half the dose (100 μL) was injected into each hind leg at each injection. Mice were immunized on days 0, 21, and 42. Serum samples were collected 14 days after the second immunization to detect specific antibodies.

[0212] (2) Blood collection

[0213] Blood was collected from the orbital cavity of the mice on day 14 after the third immunization.

[0214] (3) ELISA method for detecting serum antibodies that specifically bind to polypeptide antigens

[0215] Results of peptide-binding antibody titer assays in mice after immunization showed that the PEP-C-MAP(L1) group could stimulate antibody production in mice, but the antibody was unstable and the titers were low (0, 1000, 8000). With the addition of adjuvant MPLA, the titers in the PEP-C-MAP(LY) group increased to (500, 4000, 32000). Further addition of QS-21 resulted in PEP-C-MAP(LYZ), containing a combination of MPLA and QS-21 adjuvants, promoting a strong immune response to the peptide antigen, with serum antibody titers consistently above (32000, 32000, 32000). Under the same experimental conditions, the PEP-C-MAP(LX) group had a higher average antibody level, with titers reaching (16000, 16000, 16000). The PEP-C-MAP(A1) group showed a weaker immune response. Results of specific antibody titer assays are shown in Table 4.

[0216] Table 4. ELISA method for detecting mouse serum-specific IgG antibodies

[0217]

[0218]

[0219] Experiment Example 4

[0220] Selection of immunogens in Example 4

[0221] PEP-C-MAP polypeptide sequence: S 443-471 (SKVGGNYNYLYRLFRKSNLKPFERDISTE)(SEQ ID NO: 2)

[0222] The following experiments were conducted in this experimental example:

[0223] Experimental groups: PEP-C-MAP(LXZ) group, PEP-C-MAP(LXYZ) group, PEP-C-MAP(LXD) group; Control groups: PEP-C-MAP(Z) group, blank control group.

[0224] In the PEP-C-MAP(Z) group, the immunization volume was 200 μL / animal, the adjuvant QS-21 dosage was 10 μg / animal, added to a 1 mg / ml polypeptide antigen solution, and mixed and dispersed using an ultrasonic cell disruptor.

[0225] Immunological activity evaluation

[0226] (1) Grouping and immunization process of experimental animals

[0227] Sixty mice were randomly divided into six groups of ten each. After three days of acclimatization, the mice exhibited normal appetite, glossy fur, and good health, making them suitable for immunization. In this experiment, the polypeptide vaccine was administered subcutaneously (to both sides of the groin and abdomen), with a total injection volume of 200 μL per mouse. Half the dose (100 μL) was injected into each hind leg at each injection. Mice were immunized on days 0 and 14. Serum samples were collected 14 days after the second immunization to detect specific antibodies.

[0228] (2) Blood collection

[0229] Blood was collected from the orbital region of the mice on day 14 after the second immunization.

[0230] (3) ELISA method for detecting serum antibodies that specifically bind to polypeptide antigens

[0231] The results of peptide-binding antibody titer assays after mouse immunization showed that the PEP-C-MAP(Z) group could stimulate antibody production in mice, but the antibody was unstable and had a low titer. The PEP-C-MAP(LXZ) group, due to the addition of the LX liposome carrier with adjuvant QS-21, could promote a strong immune response to the peptide antigen, and the serum antibody titer was stable at over (32000). The titer of the PEP-C-MAP(LXYZ) group (LX liposome carrier with adjuvants QS-21 and MPLA) was not significantly higher than that of the PEP-C-MAP(LXZ) group (LX liposome carrier with adjuvant QS-21) (32000). The serum antibody titer of the PEP-C-MAP(LXD) group (LX liposome carrier with mannose) was also high. The results of specific antibody titer assays are shown in Table 5.

[0232] Table 5. ELISA method for detecting mouse serum-specific IgG antibodies

[0233]

[0234] This experiment used a 2-fold serial dilution, with an upper limit of 32,000 times, but the highest value was not measured.

[0235] (4) Results Analysis

[0236] The results from each group show that adjuvant QS-21 and mannose, individually or in combination with LX liposomes, both achieved good results. However, the combination of adjuvant QS-21 and adjuvant MPLA with LX liposomes did not show a significant improvement compared to the combination of adjuvant QS-21 and LX liposomes alone.

[0237] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0238] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of the composition in a liposome-based adjuvant for the preparation of vaccines, characterized in that: The composition is formulated as follows: 300 mg soybean lecithin, 30 mg cholesterol, 150 mg squalene, 20 mg mannose, 20 mL anhydrous ethanol, 20 mL phosphate buffer, and 5 mL 1% chitosan solution. The vaccine is a peptide vaccine, comprising 10 mg of synthetic peptide antigen, the sequence of which is as shown in SEQ ID NO:

2. The preparation method is ethanol injection-high pressure homogenization. The chitosan encapsulates the liposomes of the adjuvant. The polypeptide antigen is encapsulated in the liposomes of the adjuvant.

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