RU2779622C2 - Complex for enhancing immune response - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: group of inventions relates to the pharmaceutical industry, namely to a product for potentiation of an immune response. A combined product for potentiation of an immune response contains polyinosin-polycytidylic acid (hereinafter – PIC), a cationic stabilizer, and soluble calcium salt in a liquid reaction system. In this case, the cationic stabilizer is grafted copolymer of chitosan oligosaccharide and methoxypolyethylene glycol (hereinafter – COS-g-MPEG), where chitosan oligosaccharide is characterized by a molecular weight of 5 kDa or less; polyinosin-polycytidylic acid is preheated at a temperature from 88 to 90°C for 70 to 120 minutes, a ratio of PIC to COS-g-MPEG in the combined product is from 1:0.8 to 1:6.4. A complex for potentiation of an immune response contains a combined product. Non-therapeutic use of the complex as an immune adjuvant is disclosed. The use of the complex for the production of an antibody is also disclosed. The use of the complex for the production of a vaccine composition is also disclosed. The use of the complex for the production of an adjuvant for the vaccine is disclosed. A vaccine composition contains a complex and at least one antigen. The use of the complex for the regulation of the activity of immune cells is described, where the use is carried out in vivo or in vitro. The use of the complex for the production of drugs for the treatment and/or prevention of tumors is described. The use of the complex for the production of antiviral drugs is also described. The use of the complex for the production of antibacterial drugs is also described. The use of the complex for the production of antifungal drugs is also described. The use of the complex for the production of antiparasitic drugs is described. The use of the complex for the production of drugs stimulating an immune response to an antigen in the body is also described. A pharmaceutical composition for the treatment of cancer and infection contains a complex, where the pharmaceutical composition additionally contains one or more of a drug for immune cell therapy, a drug for antibody therapy, a chemical drug, substance, which contributes to the absorption of immunologically active agents by the mucous membrane or adhesion to the mucous membrane, an immunomodulator, a pathogen antigen, a ligand od a pattern-recognizing receptor, and pharmaceutically acceptable auxiliary substance, where the chemical drug is one or more of drugs selected from a group consisting of an alkylating drug, an antimetabolic drug, an antitumor antibiotic, an antitumor drug of plant origin, a hormonal drug, and another drug; where another drug is selected from a group consisting of L-asparaginase, cisplatin, carboplatin, oxaliplatin, dacarbazine, drugs based on hexamethyl melamine and derivatives of the above-mentioned drugs. A method for the stimulation of an immune response to an antigen in the body in vivo includes the administration to the body of a complex, or a vaccine composition, or a pharmaceutical composition. If the antigen is a viral, bacterial, fungal, or parasitic antigen, the complex, or the vaccine composition, or the pharmaceutical composition is administered at a dose of 1-8 mg/kg, in each administration. Alternatively, it is administered once a day, once every 2 days, once every 3 days, or once every 4 days. If the antigen is a tumor antigen, the complex, or the vaccine composition, or the pharmaceutical composition is administered at a dose of 1-10 mg kg, in each administration, and administered for at least 360 days, at least 180 days, at least 60 days, or at least 30 days. A method for the regulation and potentiation of the activity of immune cells in the body includes the administration to the body of a complex, or a vaccine composition, or a pharmaceutical composition. If the antigen is a viral, bacterial, fungal, or parasitic antigen, the complex, or the vaccine composition, or the pharmaceutical composition is administered at a dose of 1-8 mg/kg, in each administration. Alternatively, it is administered once a day, once every 2 days, once every 3 days, or once every 4 days. If the antigen is a tumor antigen, the complex, or the vaccine composition, or the pharmaceutical composition is administered at a dose of 1-10 mg/kg, in each administration, and administered for at least 360 days, at least 180 days, at least 60 days, or at least 30 days.

EFFECT: product and complex based on it have average viscosity and molecular weight, is convenient for use in pharmaceutical production; have stable chemical properties, practically do not degrade during long-term storage, are safe for use, significantly enhance a nonspecific immune response of the body, and achieve improved antitumor, antiviral and anti-(over)bacterial efficiency.

48 cl, 35 dwg, 3 tbl, 16 ex

Description

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the priority of Chinese Patent Application No. 201810700708.6 filed on June 29, 2018 with the Chinese Patent Office under the title "COMPLEXES FOR IMMUNE RESPONSE POTENTATION" and Chinese Patent Application No. 201810698033.6 filed with the Chinese Patent Office on June 29, 2018 with the title " METHOD FOR OBTAINING COMPLEXES FOR POTIGNATION OF THE IMMUNE RESPONSE”, the full content of which is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

The present invention relates to the field of biomedicine and, in particular, to a complex for potentiation of the immune response.

BACKGROUND OF THE INVENTION

Double-stranded RNA (dsRNA) adjuvants, which are currently and commonly considered to contain PIC (polyriboinosinic-polyribocytidylic acid), PICLC (PIC with poly-L-lysine and carboxymethyl cellulose), PIC ₁₂ U (PIC with uridylic acid in a certain range, commercial name ampligen) and PICKCa (PIC-kanamycin-CaCl2), are ligands of various pattern recognition receptors (PRR) and function, perhaps, on the one hand, by potentiating the immune response, and on the other hand, by changing the types of immunity to obtain therapeutic vaccines from preventive vaccines.

PIC (polyinosinic-polycytidylic acid) was developed by Merck (USA) in the 1960s. In mice, PIC is an IFN-α inducer with antiviral activity. PIC is able to protect mice from lethal infections in the nasal cavity and lungs. However, due to degradation of PIC by primate and human serum nucleases, PIC is characterized by reduced structural stability with little IFN-α production and no antitumor activity.

PICLC (polyinosine-polycytidylic acid with poly-L-lysine and carboxymethyl cellulose), conjugate of PIC, polylysine (poly-L-lysine, relative molecular weight is 27,000) and carboxymethyl cellulose (CMC, relative molecular weight is 700,000), which was developed by Levy HB in the 1970s, is characterized by a large relative molecular weight and resistance to nuclease hydrolysis, which is 5-10 times higher than that of PIC, and produces a significant amount of interferon (15) in monkeys. Preliminary clinical studies with PICLC have shown that moderate to severe reactions such as fever (100%), myalgia (50%), hypotension (50%), a significant decrease in white blood cells, etc., can only be induced by therapeutic dose. This leads to the misunderstanding that more molecular weight leads to more toxicity.

PIC ₁₂ U, in which uracil nucleotides are inserted into positions in the PIC chain, was developed at Johns Hopkins University in the mid-1970s and has similar potency to PIC but less toxicity. In August 2012, the biopharmaceutical company Hemispherx released additional original clinical trial data; however, PIC ₁₂ U has not been approved by the US Food and Drug Administration (FDA) due to insufficient safety and efficacy data.

PICKCa contains an antibiotic i.e. kanamycin. Kanamycin has moderate ototoxicity and the amount in the vaccine exceeds the standard set by the National Pharmacopoeia.

It can be seen that PIC alone cannot be used in primates and more advanced animals than primates, including humans, PIC ₁₂ U was rejected by the US FDA because of its low effect, and PICLC does have severe side effects.

In view of this, the present invention is presented herein.

SHORT DESCRIPTION

The present invention relates to a new complex, and the preparation, use, etc. of the complex has been studied.

In the inventor's previous work PIC, kanamycin and calcium chloride were used to prepare adjuvants for vaccines (commercial name: PIKA adjuvant). Kanamycin is used because it contains 4 amino groups that will bind to the phosphate group in PIC to stabilize its structure. However, the use of the product in vaccines is limited due to the inclusion of an antibiotic.

In addition, the inventors of the present invention have found that replacing kanamycin with chitosan (hydrochloride) can also act as a cationic stabilizer; however, chitosan (hydrochloride) has a large molecular weight and is poorly absorbed by humans, so it is difficult to obtain the desired efficacy.

Therefore, the present invention provides a complex for potentiating an immune response. The complex is prepared from at least the following components under suitable conditions: polyinosinic-polycytidylic acid, at least a cationic stabilizer and a soluble calcium salt.

The complex is prepared from at least the following components under suitable conditions: polyinosinic-polycytidylic acid, at least a cationic stabilizer, a soluble calcium salt and/or a manganese salt.

Wherein, the cationic stabilizer is a water-soluble amino compound other than an antibiotic having a molecular weight of 5 kDa or less, or a graft copolymer formed from a water-soluble amino compound other than an antibiotic and one or more of methoxypolyethylene glycol, polyethylene glycol, polyethyleneimine, folic acid and galactose.

Compared with the prior art, the complex has a medium viscosity and molecular weight, is easily converted into a drug, has stable chemical properties, is resistant to degradation during long-term storage, and is safe to use. The complex is able to significantly potentiate the nonspecific immune response in the body and achieve the goal of preventing and treating the disease when used alone and is able to provide better antitumor, antiviral and antibacterial efficacy, and is also easily absorbed by the patient when used in combination with other drugs.

BRIEF DESCRIPTION OF GRAPHICS

In order to more clearly illustrate the technical solutions in accordance with the embodiments of the present invention or the prior art, the accompanying drawings are briefly presented to describe the embodiments or the prior art. Obviously, the accompanying graphics in the following description are only some embodiments of the present invention, and other graphics can be obtained by those skilled in the art from the accompanying graphics without creative effort.

Fig. 1 is a schematic diagram of the structure of the Pamica complex; A: Schematic diagram of the structure of poly-I: C-COS-Ca ²⁺ complex; B: Schematic diagram of the structure of an antigen complex (Ag) + particle; C: Schematic diagram of the structure of poly-I: C-COS-Ca ²⁺ + Ag complex.

Fig. 2 is an electropherogram of the molecular weight of PIC after heating for various periods of time.

Fig. 3 is an enzymatic degradation curve of a Pamica complex according to an embodiment of the present invention.

Fig. 4 is a melting curve of a Pamica complex according to an embodiment of the present invention.

Fig. 5 is an absorption spectrum when scanning the respective substances at 240-260 nm according to an embodiment of the present invention.

Fig. 6 is a transmission electron microscope image of a PIC-COS-CaCl ₂ complex according to an embodiment of the present invention; scale bar = 500 nm.

Fig. 7 is a transmission electron microscope image of a PIC-COS-CaCl ₂ complex according to an embodiment of the present invention; scale bar = 200 nm.

Fig. 8 is a transmission electron microscope image of a PIC-COS-g-MPEG-CaCl ₂ complex according to an embodiment of the present invention; scale bar = 200 nm.

Fig. 9 is a transmission electron microscope image of a PIC-COS-g-MPEG-CaCl ₂ complex according to an embodiment of the present invention; scale bar = 100 nm.

Fig. 10 is a transmission electron microscope image of nanoparticles formed from a complex of PIC-COS-CaCl ₂ and TPP in accordance with an embodiment of the present invention; scale bar = 1000 nm.

Fig. 11 is a transmission electron microscope image of nanoparticles formed from a complex of PIC-COS-CaCl ₂ and TPP in accordance with an embodiment of the present invention; scale bar = 200 nm.

Fig. 12 is a graph of the detection of IgG antibodies detected by the Elisa method in the serum of mice on the 21st day after immunization with adjuvant aluminum/rHBsAg (CHO), ADV20/rHBsAg (CHO), and Pamica/rHBsAg (CHO), respectively, according to embodiment of the present invention.

Fig. 13 is the humoral immunity of mice at day 21 after immunization with aluminum adjuvant/rHBsAg (CHO), ADV20/rHBsAg (CHO), and Pamica/rHBsAg (CHO), respectively, according to an embodiment of the present invention; The ordinate shows swelling of the paw pads: increase in mm.

Fig. 14 is a comparison of the effects of Pamica complex and Freund's complete adjuvant in producing an anti-MYO antibody according to an embodiment of the present invention.

Fig. 15 is a comparison of the effects of Pamica complex and Freund's complete adjuvant in producing an anti-MYO antibody according to an embodiment of the present invention.

Fig. 16 is an exemplary depiction of an experiment in which the Pamica group stimulates the phagocytic function of macrophages according to an embodiment of the present invention;

Fig. 17 is an exemplary depiction of an experiment in which a control group receiving PBS does not stimulate the phagocytic function of macrophages according to an embodiment of the present invention;

Figures 18-21 are experimental results of the anti-cancer effects of the Pamica mucosal immune formulation in the tumor-bearing LL2 lung cancer mouse model according to an embodiment of the present invention.

Fig. 18: Curves of changes in tumor volume with various drugs.

Fig. 19: Tumor weight at treatments with various drugs (control group treated with vehicle medium had a tumor volume of 2201.09 ± 68.01 mm ³ on the 14th day after administration, and the experiment ended on the 14th day after introductions).

Fig. 20: A: vehicle control administered via nasal dropper once every two days; B: cisplatin (5 mg/kg) was injected into the tail vein once a week; C Pamica-1 (i.e., Pamica) was administered at a dose of 200 μg/mouse via nasal dropper once every two days; D: Pamica-1 was administered at 200 μg/mouse via nasal dropper (administered immediately after inoculation) once every two days; E: Pamica-1 was administered at a dose of 150 μg/mouse via nasal dropper once every two days; F: Pamica-1 was administered at a dose of 200 μg/mouse by intramuscular injection once every two days; G: Pamica-1 + cisplatin 200 μg/mouse + 5 mg/kg nasal drip + tail vein injection once every other day + once a week; scale bar = 1 cm.

Fig. 21: Degree of tumor suppression by mouse anti-PD-1 antibody.

In FIG. 22-35 show the in vivo antitumor effects of Pamica in a 4T1-luc in-situ mouse model of breast cancer, in accordance with an embodiment of the present invention.

Fig. 22: Curves of changes in tumor volume.

Fig. 23: Body weight curves of mice.

Fig. 24: Effects of treatments with various drugs on tumor weight.

Fig. 25: photograph of tumors under the influence of various drugs.

Fig. 26: Effects of treatments with various drugs on spleen weight.

Fig. 27: Photograph of the front of the lungs under the influence of various drugs.

Fig. 28: Photograph of the back of the lungs under the influence of various drugs.

Fig. 29-35: Photographs of the tumor site and bioluminescence intensity of metastases taken with a small animal imaging device.

Fig. 29: Bioluminescence of mice in the advanced cancer group treated with Pamica on day 7.

Fig. 30: Bioluminescence of mice in the Pamica group on day 0.

Fig. 31: Bioluminescence of mice in the vehicle medium group.

Fig. 32: Bioluminescence of mice in the Pamica group administered at 200 μg/mouse via a nasal dropper.

Fig. 33: Bioluminescence of mice in the Pamica group administered at 300 μg/mouse via a nasal dropper.

Fig. 34: Bioluminescence of mice in the Pamica group administered at a dose of 200 μg/mouse by intramuscular injection.

Fig. 35: Bioluminescence of mice in the Pamica group administered at a dose of 300 μg/mouse by intramuscular injection.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention may become more apparent through the following description of some of its embodiments and the detailed content of the examples included herein.

Before describing the present invention further, it should be understood that the present invention is not limited to specific embodiments, as these embodiments are necessarily varied. It should also be understood that the terms used in this specification are only intended to illustrate specific embodiments, and not to limit, since the scope of the present invention will be defined only in the appended claims.

Definition of terms

Before setting out the details of the present invention, several terms used in the present description should be clarified.

The term "Pamica" generally refers to a complex derived from polyinosinic-polycytidylic acid, a cationic stabilizer and a soluble calcium salt (calcium ions), regardless of the specific physical characteristics and immunogenicity of the complex.

"Polyinosinic-polycytidylic acid" is also known as polyinosine-cytidine, polycytidylic acid-polyinosinic acid, polyinosinic acid cytosine nucleotide, polyinosinic acid-polycytidylic acid, PIC or poly-I:C.

As used herein, the term “potentiation of an immune response” refers to the induction or potentiation of an immune response to an antigenic substance in an organism, or to the potentiation of immune cell function, or to the stimulation of the release of pro-inflammatory factors or cytokines from cells of the immune system, or to an increase in resistance to a pathogenic substance in body.

The term "inducing an immune response" refers to the stimulation, initiation or development of an immune response.

"Immune response potentiation" refers to the improvement, development, addition, expansion, enhancement and expansion of an existing immune response.

The expression "potentiation of the immune response" or similar expressions means that, compared with the previous state of the immune response, the immune response is enhanced, improved or increased, which is favorable to the host. The previous state of the immune response, for example, is the state of the previous immune response prior to administration of the immunogenic composition of the present invention.

The term "individual" is used interchangeably with "host", "subject" and "animal" in this document and includes humans and all farm animals (for example, domestic animals and pets), as well as wild animals and birds, including, without limitation, cattle. cattle, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock, etc.

The term "antibody" includes polyclonal antibodies and monoclonal antibodies, as well as fragments of these antibodies that bind an antigenic compound, including Fab, F(ab') ₂ , Fd, Fv, scFv, bispecific antibodies and the minimum recognition unit of antibodies, as well as single-stranded derivatives of these antibodies and fragments. The type of antibody may be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies and related synthetic isoforms. The term "antibody" may be used interchangeably with "immunoglobulin".

As used herein, the term "antigenic compound" refers to any substance that can be recognized by the immune system (eg, bind to an antibody or be processed to induce a cellular immune response) under appropriate circumstances.

As used herein, the term "antigen" includes, without limitation, cells, cell extracts, proteins, lipoproteins, glycoproteins, nucleoproteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, polysaccharide peptide mimics, fats, glycolipids, saccharides, viruses, viral extracts, bacteria, bacterial extracts, fungi, fungal extracts, multicellular organisms such as parasites, and allergens. Antigens may be exogenous (eg, from a source other than the individual receiving the antigen, such as from a different species) or endogenous (eg, from a host organism such as disease factors, cancer antigens, antigens produced by cells infected with viruses in body, etc.). Antigens can be natural (eg, naturally occurring), synthetic, or recombinant. Antigens include cell extracts, intact cells, and purified antigens, where the term "purified" means that the antigen is present in a more enriched form compared to the form in the environment in which the antigen would normally exist and/or compared to that in the form of the crude extract ( for example, the form of an antigen from a cell culture).

As used herein, the term "vaccine composition" refers to a combination of two or more substances (such as antigens and adjuvants) that will jointly stimulate an immune response when administered to a host.

The terms "polypeptide", "peptide", "oligopeptide" and "protein", etc. are used interchangeably in the present description and mean the polymeric form of amino acids of any length. The polymeric form may contain encoded and non-encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone.

The term "immune response" refers to any responses of the immune system of a vertebrate individual to an antigenic or immunogenic compound. Typical immune responses include, but are not limited to, local and systemic cellular and humoral immune responses such as cytotoxic T lymphocyte (CTL) responses, including antigen-specific CD8+CTL induction, helper T cell responses, including T proliferation responses. -cells and release of cytokines and immune responses involving B cells, including responses involving antibodies.

As used herein, the term "adjuvant" refers to any substance or mixture of substances that enhances or alters the immune response to an antigenic compound in an organism.

Used in the present description, the term "treatment" usually refers to the achievement of the desired pharmacological and/or physiological efficacy. Efficacy may be prophylactic in terms of complete and/or partial prevention of diseases or their symptoms, and/or effects may be medical in terms of complete and/or partial stabilization or treatment of diseases and/or negative effects caused by diseases. As used herein, the term "treatment" encompasses any form of treatment for a disease in an individual (especially a mammalian and in particular human individual) and includes: (a) preventing the development of a disease or symptom thereof in an individual who may be predisposed to to the development of the disease, but it has not yet been diagnosed; (b) suppressing a symptom of the disease, for example, preventing the development of a symptom of the disease; or alleviating a symptom of a disease, such as ameliorating the disease or symptoms; (c) a decrease in the level of products formed from the infectious substance produced during the disease (for example, toxins, antigens, etc.); (d) reducing the intensity of adverse physiological reactions (such as fever, tissue edema, etc.) caused by infectious substances produced during the disease.

A chemical substance that is a "pharmaceutically acceptable salt" means that the salt is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. These salts include: (1) salts formed together from inorganic acids from which salts are synthesized, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or salts formed together with organic acids and the like, such as acetic acid, propionic acid, caproic acid, cyclopentapropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2-en-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert- butyl lactic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stear nic acid, muconic acid, etc.; or (2) salts formed when acid protons present in the parent compound are replaced by alkali metal ions, alkaline earth metal ions, or aluminum ions, or coordinate with organic compounds such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, etc.

Exemplary Embodiments of the Present Invention

One aspect of the present invention relates to a combination product for potentiating the immune response, containing polyinosinic-polycytidylic acid, at least a cationic stabilizer and a soluble calcium salt;

wherein the cationic stabilizer is a water-soluble amino compound other than an antibiotic having a molecular weight of 5 kDa or less, or a graft copolymer formed from a water-soluble amino compound other than an antibiotic and one or more of methoxypolyethylene glycol, polyethylene glycol, polyethyleneimine, folic acid and galactose.

An important advantage is that Pamica used alone is able to significantly potentiate the non-specific immune response in the body and is able to more effectively initiate a specific humoral immune response and cellular immune response, enhancing protective immunity; better effects can be achieved when used in combination with an antigenic substance.

An important advantage is that Pamica is able to pass the "Anomalous Toxicity Test 1141" of 2015 PHARMACOPEIA OF THE PEOPLE'S REPUBLIC OF CHINA 4th volume, and can be administered safely in the human body. Complexes (such as PIC amino adjuvant CaCl ₂ or PIC amino compound adjuvant CaCl ₂ vaccine) made with a PIC having a molecular weight that has not been heat treated cannot pass the abnormal toxicity test.

An important advantage is that Pamica has better chemical and/or physical stability, which makes it easier to store.

An important advantage is that Pamica can promote apoptosis of tumor cells through a signaling pathway, and is also able to stimulate immune cells to express various cytokines and change the microenvironment in which tumor cells are present, allowing immune cells to attack pathogenic substances such as tumor cells, viruses. , bacteria, etc.

An important advantage is that Pamica is more easily absorbed by the host or taken up by the host cell, which in turn can further deliver more antigens to the cells, thus potentiating protein and peptide induced immune responses.

An important advantage is that Pamica has a clear analgesic effect on the patient with cancer pain.

An important advantage is that Pamica is able to change the titer of the virus in people infected with HPV from strongly positive to negative.

An important advantage is that Pamica plus an inactivated vaccine based on Bacterium burgeri has a good protective effect.

It should be noted that Pamica, as described in the present disclosure, is a complex having a completely new structure compared to a simple composition.

In some embodiments, the cationic stabilizer has a molecular weight that can be further selected from 4 kDa, 4.5 kDa, 3 kDa, 3.5 kDa, 2.5 kDa, 2 kDa, 1.5 kDa, 1 kDa, 500 Da , 400 Da, 300 Da, 200 Da and 100 Da.

In some embodiments, the water-soluble amine compound, other than an antibiotic, is one or more selected from the group consisting of chitosan oligosaccharides, chitooligosaccharides, glucosamines, cationic liposomes, DEAE-dextran, polyacrylamide, polyamines, tetraaminofulvene, and polyethyleneimine;

In some embodiments, the cationic stabilizer is selected from the group consisting of a graft copolymer (COS-g-MPEG) of chitosan oligosaccharide and methoxy polyethylene glycol, a graft copolymer (PEG-g-CS) of chitosan hydrochloride and polyethylene glycol, a graft copolymer (FA-g-CS) of folate acid and chitosan hydrochloride, graft copolymer (GAL-g-PEG-g-PEI) of galactose, polyethylene glycol and polyethyleneimine, graft copolymer (COS-g-MPEG-g-PEI) of chitosan, methoxypolyethylene glycol and polyethyleneimine, graft copolymer (CS-g- PEG-g-PEI) of chitosan, methoxy polyethylene glycol and polyethyleneimine, graft copolymer (PEI-g-PEG) of polyethylene glycol and polyethyleneimine, graft copolymer (PEI-g-COS) of chitosan oligosaccharide and polyethyleneimine, graft copolymer (PEI-g-CS) of chitosan hydrochloride and polyethyleneimine, a graft copolymer (COS-g-PEG) of chitosan oligosaccharide and polyethylene glycol, and a graft copolymer (COS-g-PEG-g-PEI) of chitosan oligosaccharide, polyethylene glycol and polyethyleneimine.

In some embodiments, the cationic stabilizer is selected from the group consisting of chitosan oligosaccharide (COS), graft copolymer (COS-g-MPEG) of chitosan oligosaccharide and methoxy polyethylene glycol, graft copolymer (COS-g-MPEG-g-PEI) of chitosan oligosaccharide, methoxy polyethylene glycol, and polyethyleneimine.

In some embodiments, the graft copolymer has a molecular weight of 50 kDa or less.

In some embodiments, the molecular weight of the cationic stabilizer can be further selected from 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa, 9 kDa, 8 kDa, 7 kDa, 8 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 500 Da, 400 Da, 300 Da, 200 Da and 100 Da.

In some embodiments, the chitosan oligosaccharide has a degree of deacetylation of 70% or more; you can also choose 80%, 85%, 90% or 95%, preferably 90-100%.

In some embodiments, the chitosan oligosaccharide monomer has a molecular weight of 161, a degree of polymerization of 2-20, and a selected molecular weight in the range of 322-3220.

In some embodiments, the molecular weight of the chitosan oligosaccharide, chitooligosaccharide, and glucosamine is 3200 or less.

In some embodiments, the methoxypolyethylene glycol, polyethylene glycol, and polyethyleneimine have a molecular weight of 40,000 or less, and optionally 30,000, 20,000, 15,000, 10,000, 8,000, 6,000, 4,000, 2,000, 1,500, 1,000, or 500 may be selected.

In some embodiments, the soluble calcium salt is selected from CaCl ₂ and/or CaNO ₃ .

In some embodiments, the polyinosinic-polyribocytidylic acid has a molecular weight of 100-3000 bp.

In some embodiments, the polyriboinosinic-polyribocytidylic acid has a molecular weight of 100-1500 bp.

In some embodiments, the combination product further comprises one or more of a pH adjuster, sodium tripolyphosphate, sodium alginate, phenylboronic acid, catechol, buffer salt/reagent, and water.

In some embodiments, the respective components in the combined product are packaged separately;

In some embodiments, at least two components in the combined product are mixed and packaged together, for example, positively charged ions and water and/or buffer salt are packaged together;

In some embodiments, polyinosinic-polycytidylic acid is packaged in the form of its raw materials, for example, polyinosinic acid (PI) and polycytidylic acid (PC).

According to an aspect of the present invention, the present invention also relates to an immune response potentiating complex which is derived from the reagents in the combination product as described above.

In some embodiments, the preparation is carried out in a system that is a solution, and in the reagent polyinosine-polycytidylic acid is characterized by a concentration of 0.1-10 mg/ml;

the concentration of polyinosinic-polycytidylic acid can theoretically reach a higher concentration due to grafting to increase solubility;

In some embodiments, the preparation is carried out in a system that is a solution, and the concentration of polyinosine in the reagent is 0.5-5 mg / ml, and you can additionally select 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg/ml, 6 mg/ml, 6.4 mg/ml, 7 mg/ml, 8 mg/ml or 9 mg/ml.

In some embodiments, the preparation is carried out in a system that is a solution, and the concentration of the cationic stabilizer in the reagent is 0.5-51.2 mg/ml;

In some embodiments, the implementation of the cationic stabilizer is characterized by a concentration of 0.8-25.6 mg/ml, and you can additionally select 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml or 20 mg/ml.

In some embodiments, the preparation is carried out in a system that is a solution, and in the reagent, the mass ratio of polyinosine-polycytidylic acid and cationic stabilizer is 1:0.8-25.6; you can additionally select 1:6.4 or 1:12.8.

In some embodiments, the preparation is carried out in a system that is a solution, and in the reagent the concentration of calcium ions in a soluble calcium salt is 0.1-1 mm, and additionally you can choose 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM or 0.9 mM.

In some embodiments, the complex is stored in solution.

The solution is preferably a buffer solution.

In some embodiments, the implementation of the solution is characterized by a pH value in the range of 5.0-7.2.

In some embodiments, the pH of the solution is 5.9-6.9, and 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, 7.4, 7, 6 or 7.8.

According to an aspect of the present invention, the present invention also relates to the non-therapeutic use of the complex described above as an immune adjuvant.

In accordance with an aspect of the present invention, the present invention also relates to the use of the complex described above for the production of an antibody, vaccine formulation or vaccine composition, or to use for the production of a vaccine excipient or vaccine adjuvant.

According to an aspect of the present invention, the present invention also relates to a vaccine composition comprising the complex described above and at least one antigen.

In some embodiments, the antigen is a virus, bacterium, protein, polypeptide, polysaccharide, nucleic acid, or small molecule protein conjugate.

In some embodiments, the vaccine composition is, for example, an attenuated vaccine (e.g., an attenuated virus or bacterial vaccine), an inactivated vaccine (e.g., an inactivated virus or bacterial vaccine), a protein-based vaccine, a polysaccharide-based vaccine, a vaccine a protein subunit vaccine, a chimeric vector vaccine, a DNA vaccine, an RNA vaccine, a polypeptide vaccine, or a small molecular weight protein conjugate vaccine.

According to an aspect of the present invention, the present invention also relates to the use of the complex described above for the regulation of immune cell activity, which is used in vivo or in vitro.

In some embodiments, regulation of immune cell activity specifically potentiates immune cell activity.

In some embodiments, the implementation of the immune cell is selected from macrophages, lymphocytes and dendritic cells.

In some embodiments, regulation or potentiation of immune cell activity promotes the release of a pro-inflammatory factor by immune cells.

In some embodiments, the pro-inflammatory factor includes IL-2, IL-6, IL-12p40, IL-18, IL-22, IFN-α, IFN-γ, and TNF-α.

In some embodiments, the implementation of the pro-inflammatory factor includes IFN-γ and TNF-α.

In accordance with an aspect of the present invention, the present invention also relates to the use of the complexes described above for the preparation of drugs for the treatment and/or prevention of tumors, antiviral, antibacterial, antifungal, antiparasitic agents, agents that reduce the intensity of side effects of chemotherapy, counteract fatigue or improve immunity, relieving pain in the body and stimulating an immune response to an antigen in the body.

In some embodiments, the drug is an injection dosage form, a respiratory dosage form, nasal drops, a transdermal dosage form, a transmucosal dosage form, or an oral dosage form.

In some embodiments, the injection dosage form is selected from, for example, injections (including multiple injection routes such as intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection).

In some embodiments, the respiratory dosage form is selected from, for example, sprays, aerosols, powder aerosols, and the like.

In some embodiments, the transdermal dosage form is selected from the group consisting of, for example, solutions, lotions, liniments, ointments, patches, pastes, dressings, and the like, which are applied externally and act locally or act systemically via transdermal absorption after administration.

In some embodiments, the transmucosal dosage form is selected from the group consisting of, for example, eye drops, nasal drops, ophthalmic ointments, mouth or throat rinses, and sublingual tablets, etc., transmucosal administration may act locally or through mucosal absorption to exhibit systemic action. ⑤ The oral dosage form includes, for example, suppositories, aerosols, etc. used for the rectum, vagina, urethra, nasal cavity, ear canal, etc. The oral administration can act locally or have a systemic effect after absorption .

In some embodiments, the antigen includes a tumor, viral, bacterial, fungal, or parasitic antigen.

In some embodiments, the organism is a mammal.

In some embodiments, the organism is a primate.

In some embodiments, the organism is a human.

In some embodiments, if the antigen is a viral, bacterial, fungal, or parasitic antigen, the drug has an amount of 1-8 mg per dose;

In some embodiments, if the antigen is a tumor antigen, the drug is in an amount of 1-10 mg per dose.

According to an aspect of the present invention, the present invention also relates to a pharmaceutical composition containing the complexes described above. The pharmaceutical composition further comprises one or more of an immune cell therapy drug, an antibody therapy drug, a chemical drug, a substance that promotes absorption of immunologically active agents by the mucosa or adhesion on the mucosa, an immunomodulator, a pathogen antigen, a pattern recognition ligand receptor and a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises the complex described above, and the pharmaceutical composition further comprises one or more of an immune cell therapy drug, an antibody therapy drug, a chemical drug, a substance that promotes mucosal absorption or adhesion of immunologically active agents. on the mucosa, an immunomodulator, a pathogen antigen, a pattern recognition receptor ligand, and a pharmaceutically acceptable salt or excipient.

In some embodiments, the immune cell therapy drug is one or more cells selected from the group consisting of tumor infiltrating lymphocytes (TIL), dendritic cells (DC), cytokine-induced killer cells (CIK), dendritic cells and cytokine-induced killer cells (DC-CIK), natural killer (NK) cells, γδT cells, CD3AK, CAR-T, and TCR-T.

In some embodiments, the antibody therapeutic drug is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, and an anti-CD antigen.

In some embodiments, the chemical drug is one or more selected from the group consisting of an alkylating agent, an antimetabolite, an antineoplastic antibiotic, an anticancer herbal drug, a hormonal drug, and another drug;

The other drug is selected from the group consisting of L-asparaginase, cisplatin, carboplatin, oxaliplatin, dacarbazine, drugs based on hexamethylmelamine and derivatives of the above drugs.

In some embodiments, the alkylating agent is selected from the group consisting of cyclophosphamide, busulfan, dacarbazine, cisplatin, mechlorethamine, melphalan, nitrosoureas, and derivatives of the foregoing drugs;

In some embodiments, the antimetabolite is selected from the group consisting of 5-fluorouracil, methotrexate, cytarabine, cyclocytidine, hydroxyurea, and derivatives of the above drugs;

In some embodiments, the anticancer antibiotic is selected from the group consisting of actinomycin, mitomycin, idarubicin, doxorubicin, daunomycin, dactinomycin, bleomycin, and derivatives of the above drugs;

In some embodiments, the hormonal drug is selected from the group consisting of a sex hormone, a corticosteroid hormone, and derivatives of the above drugs.

In some embodiments, the agent that promotes mucosal uptake of immunologically active agents or mucosal adhesion is one or more selected from the group consisting of anionic surfactants (such as carboxylates, sulfonates, sulfates, phosphates, and etc.), cationic surfactants (such as amine salts, quaternary ammonium salts, heterocycles, onium salts, etc.), zwitterionic surfactants (such as carboxylate type, sulfonate type, phosphate type , betaine type, imidazoline type, amino acid type, etc.), non-ionic surfactants (for example, alkyl polyglycoside type, polyoxyethylene type, polyol type, alkanolamide type, polyether block polymer type), special surfactants (such such as fluorine type, silicon type, boron type, resin type, etc.), chel adhesive agents (such as polyphosphate, aminocarboxylic acid, 1,3-diketone, hydroxycarboxylic acid, polyamine, etc.), adhesive agents [water-soluble adhesive agents (such as starch, dextrin, polyvinyl alcohol, carboxymethyl cellulose, etc.) , hot melt adhesives (such as polyurethane, polystyrene, polyacrylate, ethylene-vinyl acetate, etc.), solvent-based adhesives (such as shellac, butyl rubber, etc.), emulsion adhesives (such as vinyl acetate resin , acrylic resin, chlorinated rubber, etc.), solvent-free liquid adhesives (such as epoxy resin, etc.)], polylactic acid-hydroxyacetic acid copolymer, dextran and polysaccharide.

In some embodiments, the immunomodulator is one or more immunomodulators selected from the group consisting of cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSFs), interferons, erythropoietins, thrombopoietins, tumor necrosis factors (TNFs). ), interleukins (IL), granulocyte colony stimulating factors (G-CSF), granulocyte-macrophage colony stimulating factors (GM-CSF), and stem cell growth factors.

In some embodiments, the pathogen antigen is selected from the group consisting of tumor, viral, bacterial, fungal, or parasitic antigens.

In some embodiments, tumors include tumors arising from any lesions in bone, bone junction, muscle, lung, trachea, pharynx, nose, heart, spleen, artery, vein, blood, capillary, lymph node, lymph vessel, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, large intestine, rectum, anal canal, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, kidney, ureter, bladder, urethra, ovary , fallopian tube, uterus, vagina, vulva, scrotum, testicles, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid gland , parathyroid, adrenal, pituitary, pineal, pancreatic islet, thymus, gonad, sublingual, and parotid glands.

In some embodiments, the bacterium includes one or more of Staphylococcus, Streptococcus, Listeria, Erysipelothrix, Renibacterium, Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia, Corynebacterium, Rhodococcus, Bacillus anthracis, erysipelas bacillus, Bacillus tetani, listeria monocytogenes, Bacillus perfringens, Bacillus gangraenae emphysematosae, causative agent of tuberculosis, Escherichia coli, Bacterium proteus, Shigella dysenteriae, Pneumobacillus, Bacterium burgeri, Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter, Yersinia, Legionella pneumophila, Bordetella pertussis, Pastis, Shigella, Vibrio cholerae and Vibrio parahemolyticus.

In some embodiments, the parasite includes one or more of digestive tract parasites (such as roundworms, hookworms, tapeworms, endoamoeba histolytica, parasitic flagella, etc.), intracavitary parasites (such as trichomonas vaginalis), intrahepatic parasites (such as liver fluke, echinococcus), intrapulmonary parasites (such as paragonimus westermani), brain tissue parasites (such as Cysticercus cellulosae, toxoplasma gondii), intravascular parasites (such as schistosomiasis), parasites of the lymphatic system (such as filariae), parasites of muscle tissue ( such as Trichinella larvae), intracellular parasites (such as Plasmodium, Leishmania), bone tissue parasites (such as Echinococcus; skin parasites such as Sarcoptidae, ironwort), and intraocular parasites (such as Thelazia callipaeda, Cysticerecus cellulosaes).

In some embodiments, the virus includes one or more of adeniviridae, arenaviridae, astroviridae, bunyaviridae, cliciviridae, flaviviridae, delta hepatitis virus, hepeviridae, mononegavirales, nidovirales, piconaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, or togaviridae.

In some embodiments, the implementation of the virus is a human papillomavirus.

In some embodiments, the fungus comprises one or more of Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Histoplasma duboisii, Blastomyces lobo, Paracoccidiodes brasiliensis, Blastomyces dermatitis, Sporothrix schenckii, Penicillium marneffei, Candida albicans, Candida glabrata, Candida tropicalis, Candida lusitanias carinii Aspergillus incongruus, Enterocytozoon bieneusi, Encephalitozoon intestinalis, Rhinosporidium seeberi, hyalohyphomycet and phaeohyphomycete.

In some embodiments, the pattern recognition receptor ligand is selected from the group consisting of a TLR receptor ligand, an RLR receptor ligand, a CLR receptor ligand, and an NLR receptor ligand.

In some embodiments, binding of a ligand to a TLR receptor includes, for example, binding of peptidoglycan, disaccharide, mannan, lipopeptide, glycolipid, atypical lipopolysaccharide, serum amyloid protein, CPG-DNA, dsRNA, ssRNA, LPS, PGN, saturated fatty acids, lipoteichoic acid , resistin, lactoferrin, surfactant protein, flagellin, hyaluronic acid, RNA-related antigen, profilin-like molecules, etc.

In some embodiments, ligand binding to RLRs includes binding, for example, RNA, PIC, PICLC, PIC12u, and the like.

In some embodiments, ligand binding to the CLR receptor includes binding, for example, mannose and β-glucan to the surface of fungal cell walls, and the like.

In some embodiments, binding a ligand to an NLR receptor includes binding, for example, MDP, Mesϴ DAP, and the like.

According to an aspect of the present invention, the present invention also relates to a method for preparing an immune response potentiating complex, comprising:

contacting polyinosinic-polycytidylic acid, at least a cationic stabilizer, and a soluble calcium salt in a liquid reaction system;

wherein the cationic stabilizer is a water-soluble amino compound other than an antibiotic having a molecular weight of 5 kDa or less, or a graft copolymer formed from a water-soluble amino compound other than an antibiotic and one or more of methoxypolyethylene glycol, polyethylene glycol, polyethyleneimine, folic acid and galactose.

In some embodiments, polyinosinic-polycytidylic acid is derived from polycytidylic acid and polyinosinic acid via a base pairing reaction.

In some embodiments, the molecular weight values of polycytidylic acid and polyinosinic acid are greater than 23,000 daltons.

In some embodiments, the molecular weight of polycytidylic acid is in the range of 66,000-660,000 daltons.

In some embodiments, the implementation of the molecular weight of polyinosinic acid is in the range of 66,000-660,000 daltons.

In some embodiments, the base pairing reaction is carried out at a temperature of 40-50°C, optionally 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48 °C or 49°C.

In some embodiments, the base pairing reaction is carried out at a pH value of 6.8-7.6, optionally 7.0, 7.2, 7.4 can be selected.

In some embodiments, the polyinosinic-polycytidylic acid is heated at 88-92°C for 70-120 minutes before contacting;

In some embodiments, the temperature may further be selected from 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, or 98°C.

In some embodiments, the heating time may optionally be selected from 80 minutes, 90 minutes, 100 minutes, or 110 minutes.

In some embodiments, the liquid reaction system temperature is 40-50°C, and optionally 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C or 49°C.

In some embodiments, a method for making a graft copolymer comprises

first activating one or more of methoxypolyethylene glycol, polyethylene glycol, polyethyleneimine, folic acid, and galactose using carbonyldiimidazole, and then grafting the activated product onto a water-soluble amine compound other than an antibiotic in a [bmim]Cl ionic liquid.

In some embodiments, the graft copolymer is a chitosan oligosaccharide-methoxypolyethylene glycol graft copolymer, first activating methoxypolyethylene glycol (MPEG) with carbonyldiimidazole (CDI) and then grafting MPEG-activated chitosan oligosaccharide (COS) in [bmim]Cl ionic liquid.

In some embodiments, the grafting reaction is carried out at 60-80° C. in a non-oxidizing atmosphere.

In some embodiments, the method further comprises:

adding a solution of a cross-linking agent dropwise to the resulting complex with stirring until the Tyndall effect occurs in the reaction system, and then stirring to obtain nanoparticles;

the crosslinker is at least one crosslinker selected from the group consisting of sodium tripolyphosphate, sodium alginate, phenylboronic acid and catechol.

In some embodiments, the implementation of the cross-linking agent contains an antigen (pathogen).

In some embodiments, the method further comprises co-incubation of the complex or nanoparticles with the antigen.

In some embodiments, the antigen is a protein or polypeptide antigen.

According to an aspect of the present invention, the present invention also relates to a method for stimulating an immune response to an antigen in an organism in vivo, regulating and potentiating the activity of immune cells in an organism, supporting the body to reduce fatigue or relieve pain in the body, the method comprising administering to the host a complex, described above, or the vaccine composition described above, or the pharmaceutical composition described above.

In some embodiments, the host is suffering from an infectious disease and is administered an antigenic compound to stimulate an immune response against the pathogen causing the infectious disease.

In some embodiments, administration is by parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, topical injection, transdermal injection, or intradermal injection.

In some embodiments, the organism is a patient with a tumor, a patient with a viral infection, a patient with a bacterial infection, a patient with a parasitic infection, or a patient with rhinitis that did not respond to surgery, chemotherapy, radiation therapy, or immunotherapy, or was left for treatment in a medical facility .

In some embodiments, the method may be used in combination with surgery, radiation therapy, chemotherapy, and various immunotherapies, or may also be used in combination with conventional therapies for a patient with a viral infection, a patient with a bacterial infection, or a patient with a parasitic infection.

In some embodiments, the pain is caused by a microbial or parasitic infection that is caused by cancer or neuropathic pain.

In some embodiments, if the antigen is a viral, bacterial, fungal, or parasitic antigen, the drug is administered at a dose of 1-8 mg/kg at each administration, or alternatively preferably administered once a day, once at 2 days, once every 3 days or once every 4 days;

if the antigen is a tumor antigen, the drug is administered at a dose of 1-10 mg/kg at each administration, and preferably administered over a period of at least 360 days, at least 180 days, at least 60 days or at least 30 days.

Embodiments of the present invention will be described in detail below in combination with examples, but those skilled in the art will appreciate that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. Examples that are given without any specific conditions are carried out under normal conditions or conditions recommended by the manufacturer. Reagents or instruments used without manufacturer identification are standard products that are commercially available.

Example 1: Getting Pamica

I. Preparation of the Pamica Complex

1. Preparation of PBS solution (pH 7.2):

1.1 Preparation of sodium chloride solution (0.85%, 1500 ml): 12.75 g of sodium chloride were weighed, added to a 2000 ml graduated cylinder and made up to 1500 ml with water;

1.2 Preparation of a solution of disodium hydrogen phosphate (0.006 mol/l, 500 ml): 0.4259 g (0.006 x 0.5 x 141.96) disodium hydrogen phosphate was weighed, added to a 500 ml volumetric flask and diluted to 500 ml with 0.85% physiological saline;

1.3 Preparation of sodium dihydrogen phosphate solution (0.006 mol/l, 500 ml): 0.4140 g (0.006 x 0.5 x 137.99) sodium dihydrogen phosphate was weighed, added to a 500 ml volumetric flask and diluted to 500 ml with 0.85% physiological saline;

1.4 Preparation of a PBS solution with a pH of 7.2: 273.6 ml of the "solution of 1.2" are mixed with 126.4 ml of the "solution of 1.3".

2. Preparation of PIC solution (2.0 mg/ml, 100 ml):

2.1 Weighed 114.5mg (2.0mg/mL*100mL*【1.04/1.04+1)】/91.5%/(1-2.7%)) PI, added to a triangular flask 250 ml, dissolved by adding 50 ml of PBS solution and equilibrated in a water bath at 40-60°C;

2.2 Weighed 113.2mg (2.0mg/mL*100mL*【1/(1.04+1)】/90.4%/(1-4.2%)) PC, added to triangular flask 250 ml, dissolved by adding 50 ml of PBS solution and equilibrated in a water bath at 40-60°C;

2.3 Preparation of PIC solution: 50 ml of PI solution was poured into 50 ml of PC solution and the mixture was subjected to reaction in a water bath at 45°C for 30 minutes.

2.4 The PIC solution was heated at 80-99°C for 15～300 minutes.

3. Obtaining a solution of calcium chloride (0.16 mol/l, 25 ml):

0.5881 g of CaCl ₂ .2H ₂ O (MW: 147.02) was weighed, added to a 100 ml triangular flask, dissolved by adding approximately 25 ml of water for injection and diluted to 100 ml.

4. Get COS-g-MPEG:

The method for producing a graft copolymer of COS-g-MPEG is as follows: a graft copolymer of methoxypolyethylene glycol (COS-g-MPEG) with a grafted chitosan oligosaccharide was obtained and used as an auxiliary substance for the preparation of an anticancer drug.

Principle: The combination of carbonyldiimidazole (CDI) is used to produce COS-g-MPEG. First, methoxypolyethylene glycol (MPEG) is activated with carbonyldiimidazole to obtain activated MPEG, and then the activated MPEG is reacted with chitosan oligosaccharide (COS) in an ionic liquid to synthesize a COS-g-MPEG copolymer. The specific reaction includes the following three steps:

4.1 Obtaining an ionic liquid, which is the chloride salt of 1-butyl-3-methylimidazole ([BMIM]Cl)

carry out the reaction of 1-methylimidazole with chlorobutane to obtain the ionic liquid [BMIM]Cl, and the synthesis reaction equation is as follows:

.

.

4.2 Activation of methoxypolyethylene glycol (MPEG, molecular weight is 1000)

MPEG is activated by CDI and the synthesis reaction equation is as follows:

4.3 Synthesis of COS-g-MPEG copolymer;

activated MPEG is grafted and polymerized with COS in an ionic liquid, and the synthesis reaction equation is as follows:

4.4 Equipment and reagents:

methylimidazole, chlorobutane, toluene, methoxy polyethylene glycol, carbonyldiimidazole, anhydrous ether, 4A molecular sieve (2-3mm), dimethyl sulfoxide, 1,4-dioxane, chitosan oligosaccharide, collecting thermostatic heated magnetic stirrer (DF-101S type), electronic balance , electrothermal jet drying oven, circulating water vacuum pump, automatic pure water triple distillation distiller, vacuum drying oven, lyophilizer, air-flow dryer for glass instruments, single-phase capacitor start motor, rotary vane vacuum pump, six-circuit magnetic stirrer heated, cellulose dialysis bag, ready-to-use dialysis bag with 45-2000RC membrane, three-necked flask (500 ml, 1000 ml), glass stopper, magnetic stirrer, disposable paper cup, 500 ml measuring cup, 2 L measuring cup, disposable pipette, measuring spoon, reagent bottle, desiccator and etc.

4.5 How to obtain:

4.5.1 Obtaining distilled water

Distilled water was obtained using a Z-97A pure water triple distillation automatic distiller three times.

4.5.2 Washing glassware

the three-necked flask, glass stopper, petri dish, magnetic stirrer, etc. were first washed with tap water, then washed three times with distilled water, and finally dried in a glass instrument air dryer.

4.5.3 Solvent Drying: A molecular sieve of suitable mesh size was placed in a disposable beaker, then the appropriate amount of dimethyl sulfoxide, 1,4-dioxane and anhydrous ether was added, and the water was removed.

4.5.4 Pre-freeze anhydrous ether.

4.6. Operations (an indication of what you need to pay attention to at each stage):

4.6.1 Obtaining an ionic liquid

(1) 100 g of 1-methylimidazole and 148.5 ml of chlorobutane were successively added to a 500 ml three-necked flask. A condenser was placed on the flask and argon was introduced for 30 min. The solution was stirred on a magnetic stirrer, then heated to 80° C. in an oil bath and reacted for 24 hours;

(2) after completion of the reaction, the flask was taken out and the solution was cooled to room temperature and then frozen in a refrigerator at -18°C for 2 hours. Separation of the solution can be observed, and then the supernatant was removed (mainly to remove chlorobutane);

(3) the residue was placed in a jet oven at 80° C., and after the solid had completely melted, an appropriate amount of toluene was added while it was hot and shaken to thoroughly mix the toluene with the solution. The mixture was then cooled to room temperature, frozen in a refrigerator and then removed, and the supernatant was removed (1-methylimidazole and chlorobutane were dissolved in toluene, and toluene, 1-methylimidazole and chlorobutane were removed);

(4) step (3) was repeated twice to completely remove unreacted chlorobutane;

(5) the sample was placed in a vacuum drying oven and heated to 90°C. After complete melting, the sample was dried in vacuum at 90° C. for 8 hours (to remove toluene), then removed, allowed to cool to room temperature, and then placed in a desiccator for use.

4.6.2 MPEG activation

(1) 10 ml of dimethyl sulfoxide and 20 ml of 1,4-dioxane were added to a 500 ml three-necked flask and stirred with a magnetic stirrer. 20 g of MPEG was added and after complete melting of the MPEG, 3.24 g of CDI was added. The mixture was heated to 37° C. in a water bath and reacted for 18 hours;

(2) after completion of the reaction, the sample was added to pre-cooled anhydrous ether in an ice bath while stirring on a magnetic stirrer, and the opening of the beaker was covered with a preservative film, and then the sample was placed in a refrigerator for 30 minutes;

(3) the sample was removed after 30 min and the supernatant of the solution was removed. Pre-cooled anhydrous ether was added to the precipitate and stirred on a magnetic stirrer for 30 minutes, and then the mixture was placed in a refrigerator for 30 minutes.

(4) Step (3) was repeated twice to completely wash out unreacted CDI;

(5) the supernatant of the solution was removed and the residue was placed in a jet oven at 40° C. for 6 hours to preliminarily remove the ether;

(6) the residue was placed in a vacuum drying oven at 40° C., dried in vacuum for 2.5 hours to completely remove the ether, then taken out, allowed to cool to room temperature, and then placed in a desiccator for use.

4.6.3 Get COS-g-MPEG:

(1) the ionic liquid was melted in a jet oven at 80°C;

(2) 105 g of the ionic liquid was weighed, added to a three-necked flask, and heated to 70° C. in an oil bath. Argon was introduced and 9 g of COS was added slowly. After complete dissolution of COS, 6 g of activated MPEG was added and the mixture was stirred on a magnetic stirrer. After all starting materials were added, the mixture was reacted for 6 hours under argon protection. After completion of the reaction, the reaction vessel was cooled to room temperature;

(3) First, the dialysis bag was clamped on the side with a clamp, and an appropriate amount of distilled water was added to wash the dialysis bag three times and check the dialysis bag for water leaking out of it. The sample from the reaction vessel was then placed in a dialysis bag (molecular weight cutoff of 2000) and dialyzed for 72 hours with sufficient distilled water to submerge the dialysis bag. The water was renewed every 2-3 hours on the first day, then every 12 hours.

(4) After completion of dialysis, the solution was added to a 1000 ml three-necked flask and placed in a water bath. The vacuum distillation apparatus was firmly installed, and the distillation temperature was gradually increased from room temperature to 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C. Distillation was continued until approximately 50 ml of solution remained, then the distillation apparatus was removed. The sample was poured into a disposable measuring cup while it was hot. The opening of the beaker was covered with a disposable glove and the sample was frozen in a refrigerator for at least 8 hours.

(5) The lyophilizer was pre-cooled for 30 minutes so that the freezing point reached -52°C. The sample was crushed, distributed and leveled in a Petri dish. The Petri dish was placed in a pre-chilled lyophilizer and lyophilized at -52°C for 30 hours. After freezing was completed, the lyophilizer was turned off and the sample was removed, weighed, placed in a reagent bag and then stored in a desiccator.

(6) Infrared analysis: an appropriate amount of sample was taken and tableted with potassium bromide. The infrared spectrum of the sample was measured with a scan area of 400-4000 cm-1.

5. Preparation of COS-g-MPEG solution in PBS:

5.1 5.12%: weighed 0.128 g COS-g-MPEG, added to a 5 ml centrifuge tube, dissolved by adding PBS solution and diluted to 2.5 ml;

5.2 2.56%: 1.2 ml 5.12% solution + 1.2 ml PBS solution;

5.3 1.28%: 1.2 ml 2.56% solution + 1.2 ml PBS solution;

5.4 0.64%: 1.2 ml 1.28% solution + 1.2 ml PBS solution;

5.5 0.32%: 1.2 ml 0.64% solution + 1.2 ml PBS solution;

5.6 0.16%: 1.2 ml 0.32% solution + 1.2 ml PBS solution;

6. Preparation of PIC solution with Pamica, COS-g-MPEG and calcium chloride solution:

6.1 1.0 ml of PIC solution was placed in a water bath at 45°C, 1.0 ml of solution 5.1 was added dropwise, and then 0.005 ml of calcium chloride solution was added to reach a final concentration of 0.0004 mol/l.

6.2 1.0 ml of PIC solution was placed in a water bath at 45°C, 1.0 ml of 5.2 solution was added dropwise, and then 0.005 ml of calcium chloride solution was added to reach a final concentration of 0.0004 mol/l.

6.3 1.0 ml of PIC solution was placed in a water bath at 45°C, 1.0 ml of 5.3 solution was added dropwise, and then 0.005 ml of calcium chloride solution was added to reach a final concentration of 0.0004 mol/l.

6.4 1.0 ml of PIC solution was placed in a water bath at 45°C, 1.0 ml of 5.4 solution was added dropwise, and then 0.005 ml of calcium chloride solution was added to reach a final concentration of 0.0004 mol/l.

6.5 1.0 ml of PIC solution was placed in a water bath at 45°C, 1.0 ml of 5.5 solution was added dropwise, and then 0.005 ml of calcium chloride solution was added to reach a final concentration of 0.0004 mol/l.

6.6 1.0 ml of PIC solution was placed in a water bath at 45°C, 1.0 ml of 5.6 solution was added dropwise, and then 0.005 ml of calcium chloride solution was added to reach a final concentration of 0.0004 mol/l.

7. Results

MPEG, PEG, PEI, etc. are characterized by good water solubility and good compatibility with many organic components. In this example using, for example, MPEG, compatibility is greatly improved if a graft copolymer of MPEG with a cationic stabilizer (such as chitosan oligosaccharide) is prepared with PIC. Theoretically, compatibility will also increase if a PEG (etc.) grafted cationic stabilizer such as chitosan is made with PIC; after grafting the cationic stabilizer to PEG, the graft copolymer itself will also have the characteristics of PEG.

The test results showed that the solution was still clear at a PIC:COS-g-MPEG ratio of 1:25.6 mg, but the hyperchromic results showed that more graft copolymers did not mean a better result. In addition, the samples in 6.1, 6, 6.3, 6.4, 6.5 and 6.6 were left at room temperature after receiving on May 7, 2018 and on May 14, 2018 it was noted that a flocculent precipitate formed in the sample in 6.6, which cannot be dissolved again. Based on a comprehensive review, the ratio of PIC to COS-g-MPEG in the Pamica-based formulation is limited to the range of 1 mg: 6.4 mg.

II. Obtaining nanoparticles based on Pamica

Sodium tripolyphosphate (TPP) was purchased according to medical standards. The PIC-COS-g-MPEG-CaCl ₂ complex in an appropriate ratio was stirred with on a magnetic stirrer at a constant temperature at a certain speed, and aqueous solutions of TPP at various concentrations were added dropwise. The dropwise addition was stopped immediately if a clear opalescence was observed and the reaction was maintained for 30 minutes. Nanoparticles with a particle size of less than 1000 nm were obtained by self-assembly by cross-linking using ions and obtained by high-speed centrifugation. To determine the quality, the nanoparticles were verified using various tests.

III. Nanoparticles based on polypeptide or protein antigen

Scheme 1: The polypeptide or protein antigen was added during the formation of the above nanoparticles: the appropriate components were included in the PEG-COS graft copolymer or COS matrix and the polypeptide or protein antigen was included in the TPP-containing aqueous phase and bound. Appropriate components should be associated at a suitable ratio and pH with agitation with magnetic beads to form a complex and nanoparticles.

Scheme 2: the polypeptide or protein antigen was incubated with the above pre-formed complex and nanoparticles in such a way that the polypeptide or protein antigen bound on the surface of the complex and nanoparticles, or the polypeptide or protein antigen was mixed with the above complex and nanoparticles at a certain ratio. The mixture was stirred on a magnetic stirrer for 5 minutes, kept at room temperature for 1 hour and subjected to ultracentrifugation in a glycerol matrix at 20,000 rcf and 4°C for 2 hours, after which nanoparticles were obtained based on a polypeptide or protein antigen.

To determine the quality of the complexes and nanoparticles according to Scheme 1/Scheme 2, it is necessary to verify using various tests.

IV. Composition based on Pamica

The aforementioned complex/complex containing the graft copolymer/complex-based nanoparticles/nanoparticles based on a polypeptide or protein antigen was aseptically packaged in a suitable/qualified packaging material to obtain various dosage forms such as injection, spray or aerosol, and was obtained in the form of various products after verification with various product tests to determine the quality.

The aforementioned complex/complex containing the graft copolymer/nanoparticles based on the complex/nanoparticles based on the polypeptide or protein antigen was aseptically packaged in a suitable/qualified packaging material and prepared as an ointment.

Spray preparation example: Pamica was prepared according to the above method and the Pamica solution was placed in spray bottles. Detection of the spray pattern of the medical solution and detection of droplet size distribution were carried out for 20 vessels with Pamica solution, respectively.

Spray patterns are shown in the following table:

note: spray patterns were evaluated by the ratio of the largest diameter to the smallest diameter (the closer to 1.0, the better the spray patterns).

Droplet distributions are shown in the following table:

Example 2: Pamica test in combination with PEI to improve solubility

The preparation was carried out in accordance with the method of obtaining "Pamica in combination with PEG" (ie, example 1). After increasing the amount of COS in Pamica, a precipitate formed, which affected the uniformity of its introduction. Precipitation was avoided after the addition of PEI, and the dose of COS was increased to further enhance its immune effect.

The experiment clearly demonstrates that after combining Pamica with PEI, the solubility of COS in PIC increased from 1.6 mg/ml to 6.4 mg/ml, increasing at least 4-fold.

Patent Publication No. CN105396130A discloses "PIC amino compound-Cacl ₂ adjuvant and vaccine containing PIC amino compound-Cacl ₂ adjuvant" and discloses that the amino compound other than an antibiotic may optionally be chitosan.

In this comparative example, water-soluble chitosan (chitosan hydrochloride, abbreviated as CS) was used to replace chitosan oligosaccharide in Example 1 to compare the effects of water-soluble chitosan and chitosan oligosaccharide in terms of uniformity of administration. The results are shown in the following table.

Studies have shown that the addition of water-soluble chitosan leads to the formation of a precipitate that cannot be dispersed by shaking during production, which has effects on the uniformity of its introduction, while chitosan oligosaccharide can be used to solve the above problem. In addition, both PEG-grafted chitosan and water-soluble chitosan need to be degraded to low molecular weight chitosan oligosaccharide for easy absorption by the human body, but chitosan oligosaccharide can be absorbed directly.

Example 3: PIC heating and molecular weight determination

The PIC solution was prepared according to the preparation method of Example 1, wherein 260 ml of the PIC solution was divided into 13 tubes for a total of 20 ml/tube. The 12 sample tubes were placed in a constant temperature water bath when its temperature was raised to 80-99°C (preferably 90°C). After the tubes were placed in the water bath, the time was started and one tube was removed after 10 minutes (Group 3), 20 minutes (Group 4), 30 minutes (Group 5), 40 minutes (Group 6), 50 minutes ( group 7), 60 minutes (group 9), 70 minutes (group 10), 80 minutes (group 11), 90 minutes (group 12), 100 minutes (group 13), 110 minutes (group 14) and 120 minutes (group 15) respectively. The sample in group 2 is not heated.

PIC-derived complex (Pamica) and its vaccine heated for 70-120 minutes (preferably heated for 120 minutes) can pass abnormal toxicity in mice and guinea pigs according to the Abnormal Toxicity Test in mice and guinea pigs. toxicity 1141" from the 4th volume of the PHARMACOPEIA OF THE PEOPLE'S REPUBLIC OF CHINA 2015. PIC used to obtain Pamica must be heated to 90°C for at least 70 minutes, preferably to 90°C for 120 minutes prior to its use to obtain the product. Selected complexes prepared with PIC without heating (group 2) and prepared with heating for 60 minutes (group 9), as well as vaccines based on them, cannot pass the detection of abnormal toxicity in guinea pigs. The results are shown in FIG. 2. Comparison group 1 from bottom to top: 100 bp, 300 bp, 500 bp, 750 bp, 1000 bp, 1500 bp, 2000 bp, 3000 bp, 5000 bp Comparative group 8 from bottom to top: 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp . o., 900 p. o., 1000 p. o.

Example 4 Enzymatic Degradation Pamica Complex Test

Method: The Pamica complex was prepared according to the preparation method of Example 1 of the present invention. After completion of preparation, the respective samples of the Pamica complex and polyinosine-polycytidylic acid for injection (polyinosine-polycytidylic acid-kanamycin-calcium chloride) were diluted to 0.04 mg/ml, respectively. To each of 13 tubes (10 ml) was added 5 ml of sample diluent, then 25 μg of RNase (Cat. No. R4642) from Sigma was added to each tube. The tubes were placed in a water bath at 37°C. 1 tube was removed every 5 minutes to measure the OD value at 248 nm and plotted.

The result is shown in Fig. 3.

Example 5: The Pamica complex of the present invention is a complex with a novel structure

(1) Determining the peak of the melting curve

Method: Appropriate samples of Pamica-polyinosinic-polycytidylic acid complex for injection of the present invention were diluted to 0.04 mg/ml, then transferred to 250 ml reagent vials. The reagent vessels were placed in a water bath and the water bath was constantly heated. Every 5°C, 3 ml of the corresponding samples were taken and placed in a quartz cuvette. Measured the value of OD at 248 nm and built a curve.

The result is shown in Fig. four.

The test result shows that the Pamica complex (PIC-cationic stabilizer-calcium chloride) of the present invention has a melting curve peak at 85°C, and polyinosine-polycytidylic acid (PIC-kanamycin-calcium chloride) for injection has a melting curve peak at 80°C C, indicating that the Pamica complex of the present invention is a novel complex.

(2) Absorption Peak Scan

PI solution, PC solution, PIC solution, PIC-COS solution, PIC-COS-CaCl ₂ solutions were prepared according to the preparation method of Example 1, respectively. The above samples were diluted to 0.04 mg/ml with PBS buffer and the absorption spectrum was measured by scanning separately with ultraviolet light. In FIG. 5 shows that the peaks appearing at 240-260 nm, from high to low, are PI, PC, PIC, PIC-COS, PIC-COS-CaCl ₂ , from these peaks PIC-COS and PIC-COS-CaCl ₂ overlapped, demonstrating that the Pamica (PIC-COS-CaCl ₂ ) complex of the present invention is a complex with a novel structure.

Example 6: Individual nanoparticles formed with PIC, COS and calcium chloride.

Pamica was prepared according to the method of Example 1, where COS was chosen as the cationic stabilizer and calcium chloride was chosen as the metal cation. It can be seen from the transmission electron micrographs (FIG. 6 and FIG. 7) that the nanoparticles were formed in the Pamica solution. Most nanoparticles are spherical and relatively uniform, with a particle size of approximately 50 nm, and some nanoparticles are square with side lengths greater than 100 nm.

Example 7 Single Nanoparticles Formed with PIC, COS-g-MPEG and Calcium Chloride

Pamica was prepared according to the method of Example 1, where COS-g-MPEG was chosen as the cationic stabilizer and calcium chloride was chosen as the metal cation. It can be seen from the transmission electron micrographs (Fig. 8 and Fig. 9) that nanoparticles have formed in the Pamica solution, most of which are square in shape with side lengths greater than 100 nm and some are spherical.

Example 8 Single Nanoparticles Formed with PIC, COS, TPP and Calcium Chloride

COS was dissolved in phosphate buffer solution and a solution of PIC in TPP was prepared according to the method of Example 1. A solution of PIC in TPP was slowly added dropwise to the COS solution with stirring, and then a solution of calcium chloride was added dropwise. It can be seen in the micrographs obtained with a transmission electron microscope (Fig. 10, Fig. 11) that the formed nanoparticles are spindle-shaped.

In examples 5-8, it can be found that the solution of the Pamica complex simultaneously contains two states of substances, one of which is a nanoparticle (results of electron microscopy), and the other is a solution without nanoparticles (result of electrophoresis from example 3).

The advantage of nanoparticles is that they are able to directly penetrate the cell membrane without endocytosis, penetrate the cell and act quickly; a solution without nanoparticles can enter the cell only through endocytosis and act more slowly than nanoparticles. Pamica can exert its action in two ways: by endocytosis and by direct entry into cells. In addition, more importantly, the nanoparticles are characterized by a structure that can protect Pamica from PIC degradation by ribonuclease in the blood serum of primates and more advanced animals than primates, including humans, to achieve a breakthrough in antiviral and antitumor agents, and have stronger effects. These effects allow Pamica to have an outstanding anti-cancer effect in humans and mice.

Experimental Example: Evaluation of the Immune Effect of the Pamica Complex

In the following experimental examples, Pamica refers to a solution of Pamica in PIC, COS, and a calcium chloride solution prepared according to Example 1, unless otherwise indicated.

Experimental Example 1 Evaluation of the Immune Effect of Pamica Complex on Recombinant Hepatitis B Vaccine [rHBsAg (CHO)]

Materials: rHBsAg (CHO): 20 µg/ml; adjuvant based on Pamica: 1 mg/ml; adjuvant based on ADV20: 400 µg/ml; aluminum hydroxide adjuvant: 10 mg/ml; saline.

Aluminum adjuvant/rHBsAg (CHO): 0.07 ml aluminum adjuvant + 0.5 ml rHBsAg (CHO) + 0.43 ml saline

ADV20 (cytokine adjuvant)/rHBsAg (CHO): 0.25 ml ADV20 + 0.5 ml rHBsAg (CHO) + 0.25 ml saline

Pamica/rHBsAg adjuvant (CHO): 0.5 ml Pamica adjuvant + 0.5 ml rHBsAg (CHO)

Method: Mice were immunized intramuscularly with 0.1 ml aluminum adjuvant/rHBsAg (CHO), ADV20 (cytokine adjuvant)/rHBsAg (CHO), Pamica/rHBsAg (CHO) on day 0 and day 14, respectively, and were found to have cellular immunity and humoral immunity on day 21.

Results: The Pamica complex of the present invention has an outstanding immune effect; in particular, antibody-mediated immunity as measured by ELISA and cellular immunity are significantly improved, significantly better than aluminum adjuvant and ADV20 (cytokine adjuvant). Pamica is a promising immune adjuvant, see FIG. 12 and FIG. 13 for details.

Experimental Example 2: Evaluation of the immune effects of the Pamica complex and an inactivated antigen based on Bacterium burgeri

Method: Mice were immunized with PBS, polyinosine-polycytidylic acid for injection + inactivated Bacterium burgeri antigen, Pamica complex of the present invention + inactivated Bacterium burgeri antigen respectively, immunized once on day 0, and mice were exposed to a virulent Brucella strain after immunization for 45 days. After infection for 15 days, mice were sacrificed to isolate the spleens of mice and cultured with Bacterium burgeri in the spleen for 3 days, counted and then evaluated the protection efficiency of the Pamica complex of the present invention + inactivated Bacterium burgeri antigen.

Results: The Pamica complex of the present invention has outstanding immune effects and is promising for the development of inactivated Bacterium burgeri antigens. The number of isolated bacteria using the Pamica complex of the present invention + inactivated Bacterium burgeri antigen differs by 3.03 log from the number determined using the intact control group, and differs by 1.35 from the amount determined using polyinosine-polycytidylic acid for injection + inactivated Bacterium burgeri antigen. Pamica complex and inactivated Bacterium burgeri have an outstanding protective effect.

Experimental Example 3: Use of Pamica Complex and MYO Antigen (Human Myoglobin) to Generate Antibodies

Materials: MYO antigen (human myoglobin) at a concentration of 1 mg/ml

The Pamica complex of the present invention at a concentration of 1 mg/ml

Complete Freund's adjuvant (CFA, Sigma)

Method: blood was collected from rabbits weighing 2.3-2.5 kg for a negative control and blood serum was separated as a control before immunization. After subcutaneous injection of various samples into several sites of the abdomen and back, the blood serum was separated for the detection of antibodies. Antigen + adjuvant was received in an amount of 0.5 ml + 0.5 ml, and the dosage at each immunization was 1 ml.

Results:

(1) Rabbits were immunized with the Pamica complex of the present invention + MYO antigen, complete Freund's adjuvant + MYO antigen once every 7 days. Freund's complete adjuvant was added for immunization 2 times (10 days) at an ELTSA antibody titer of 13,000; Freund's complete adjuvant was added for immunization 3 times (18 days) at an ELTSA antibody titer of 39,000, Freund's complete adjuvant was added for immunization 6 times (3 months) at an ELTSA antibody titer of 25,000; the Pamica complex of the present invention was added to immunize 2 times (10 days) at an ELTSA antibody titer of 45,000, to immunize 3 times (18 days) at an ELTSA antibody titer of 51,000, and to immunize 3 times (3 months) with an ELTSA antibody titer of 36,000. The Pamica adjuvant has a relatively high early MYO antibody titer and a better duration of immunization, indicating that the Pamica complex of the present invention is significantly better than than Freund's complete adjuvant as the accepted standard of immune adjuvant with MYO antigen, see FIG. 14 for details.

(2) rabbits were immunized against the MYO antigen with the Pamica complex of the present invention in combination with Freund's complete adjuvant twice (once immunized on day 0, once immunized on day 14), antibody titer determined by ELISA (after 35 days post immunization) is 10,000 with complete Freund's adjuvant, 60,000 with the Pamica complex of the present invention, and 160,000 with the Pamica complex of the present invention + complete Freund's adjuvant (FIG. 15).

Kits were prepared with antibodies generated by immunizing rabbits with complete Freund's adjuvant + antigen, Pamica adjuvant + complete Freund's adjuvant + antigen, and Pamica adjuvant + antigen, respectively. After clinical verification, antibodies produced by the complete Freund's adjuvant group or the complete Freund's adjuvant + Pamica-based antigen adjuvanted group do not match clinical specimens and cannot be clinically tested. Only antibodies produced with adjuvant Pamica + antigen can pass the clinical trial, which will completely break the monopoly of the Dutch company Dako on the supply of antibody-based starting materials used in the human myoglobin turbidity latex test kit, and solve the serious problem of usually low immune titers, insufficient epitope diversity, and ineffective detection of clinical samples when using complete Freund's adjuvant for domestically produced products.

The kits obtained using antibodies produced using Pamica adjuvant + antigen group and clinically used kits obtained using antibodies provided by the Dutch company Dako are compared in the following table.

Experimental Example 4: NIH Evaluation of Pamica Compound Efficacy on Rabies Vaccine Antigen

Title: NIH Evaluation of Pamica Complex of the Invention for Rabies Vaccine Antigen

Method: Mice were immunized with the Pamica complex of the present invention (1 mg/ml) + antigen, polyinosine-polycytidylic acid injection (1 mg/ml) + antigen, PBS + antigen and antigen on day 0 at day 14 challenge, and activity was determined after 28 days.

Results: see table below.

The results demonstrate that:

a. the protective effect of the Pamica complex of the present invention + rabies vaccine antigen from CTN strain is 3.6 times higher than that of rabies vaccine antigen from CTN strain alone, and the antigen is retained by 1/5;

b. the protective effect of the Pamica complex of the present invention + CTN strain rabies vaccine antigen is 1.8 times higher than that of polyinosine-polycytidylic acid injection + CTN strain rabies vaccine antigen, and is outstanding.

Experimental Example 5 Pamica Complex Induces the Production of Several Cytokines in Mice.

Method: Mice were immunized with the Pamica complex of the present invention and polyinosine-polycytidylic acid injection. The eyeballs of mice were removed 1 hour, 2 hours and 5 hours after immunization and blood was collected in a sterile 2 ml centrifuge tube. The centrifuge tube was kept at room temperature for 30 minutes, centrifuged at 3500 rpm for 5 minutes. The supernatant was transferred to a new centrifuge tube and the blood serum was frozen at -20°C.

Results: TNF-α and IFN-γ cytokine yields induced by the Pamica complex of the present invention appear to be superior to those of polyinosine-polycytidylic acid for injection. All data obtained are geometric averages and specific results are shown in the following table.

Summary: TNF-α is capable of killing and suppressing tumor cells, promoting neutrophil phagocytosis, conferring resistance to infection, and is a type of cytokine that can directly induce tumor cell death; IFN-γ is able to induce cell resistance to viral infection, and by interfering with transcription of viral genes or translation of viral protein components, IFN-γ is able to prevent or limit viral infections and is currently the most important cytokine against viral infection and antitumor cytokine.

The levels of TNF-α and IFN-γ produced cytokines induced by Pamica in mice are higher than those induced by polyinosinic-polycytidylic acid injection, indicating that TNF-α and IFN-γ produced by Pamica complex are of the present invention, are characterized by a more powerful ability to synergistically kill tumor cells and counteract infections.

Experimental Example 6: Abnormal Toxicity Test

1. Test of the Pamica complex of the present invention in mice:

1.1 Test:

Sample injection: Kunming healthy SPF mice weighing 18～22g were intraperitoneally injected at 0.5ml/mouse and 5 mice/sample, and at the same time, 5 healthy mice as intact controls were weighed, the weight values were 18～22 G.

1.2 Criteria:

Each mouse is intraperitoneally injected with 0.5 ml of the test substance and observed for 7 days. During observation, all mice should survive without abnormal reactions. At the end of the time, each mouse should gain weight, and then the test substance is considered to be compliant. If the above requirements are not met, 10 mice can be used for one replicate test, the criteria being identical to those above (ip. is short for intraperitoneal injection).

1.3. results

2 Test of the Pamica complex of the present invention in guinea pigs:

2.1 Test:

Sample injection: Hartely SPF-class healthy guinea pigs weighing 250～350g were intraperitoneally injected at 5ml/guinea pig and 2 guinea pigs/sample, and at the same time, 2 healthy guinea pigs as intact controls were weighed, the weight values were 250- 350 g.

2.2 Criteria:

Each guinea pig is injected intraperitoneally with 5 ml of the test substance and observed for 7 days. During observation, all guinea pigs must survive without abnormal reactions. At the end of the time, each guinea pig must gain weight and the test substance is then considered compliant. If the above requirements are not met, 4 guinea pigs may be used for one retest, the criteria being identical to those above.

2.3. Test results:

Note: when re-tested, still did not meet the requirements.

3 Pamica complex test of the present invention + purified rabies vaccine antigen in guinea pigs:

3.1 Test:

Sample injection: Hartely SPF-class healthy guinea pigs weighing 250～350g were intraperitoneally injected at 5ml/guinea pig and 2 guinea pigs/sample, and at the same time, 2 healthy guinea pigs as intact controls were weighed, the weight values were 250～ 350 g.

3.2 Criteria:

Each guinea pig is injected intraperitoneally with 5 ml of the test substance and observed for 7 days. During observation, all guinea pigs must survive without abnormal reactions. At the end of the time, each guinea pig must gain weight and the test substance is then considered compliant. If the above requirements are not met, 4 guinea pigs may be used for one retest, the criteria being identical to those above.

3.3. Test results:

Note: 1) still failed to meet requirements when retested;

2) purified rabies vaccine antigen: cell: Vero cell; virus strain: CTN strain (not limited to Vero cells and CTN virus strain).

4 Pamica complex test of the present invention + hepatitis B vaccine antigen in guinea pigs:

4.1 Test:

Sample injection: Hartely SPF-class healthy guinea pigs weighing 250～350g were intraperitoneally injected at 5ml/guinea pig and 2 guinea pigs/sample, and at the same time, 2 healthy guinea pigs as intact controls were weighed, the weight values were 250～ 350 g.

4.2 Criteria:

Each guinea pig is injected intraperitoneally with 5 ml of the test substance and observed for 7 days. During observation, all guinea pigs must survive without abnormal reactions. At the end of the time, each guinea pig must gain weight and the test substance is then considered compliant. If the above requirements are not met, 4 guinea pigs may be used for one retest, the criteria being identical to those above.

4.3. Test results:

Note: 1) still failed to meet requirements when retested;

2) hepatitis B vaccine antigen: expressed in yeast cells (not intended to be limited when expressed in yeast cells, and recombinant engineered CHO cells can also be used to express hepatitis B virus antigen).

Brief description and analysis:

PIC (double-stranded nucleic acid) unwinds after heating at a certain temperature, and two single strands are paired by hydrogen bonding, while the temperature is slowly reduced to restore the double strand. Heating can reduce the molecular weight of PIC and reduce its toxicity. If PIC is not subjected to heating or is prepared without heating for a sufficient time, the complex obtained by this method is itself very toxic, and the vaccine obtained using this complex is also very toxic, which makes application difficult.

Experimental Example 7: Use of the Pamica Complex in Certain Patients with Advanced Cancer and to Treat an Infection.

From many advanced cancer cases, it can be seen that the product of the present invention can be administered by nasal spray in addition to injection, and has a significant effect on metastatic cancer.

(1) Good safety level, no obvious side effects; the Pamica immune formulation is not a cytotoxic drug;

(2) fewer side effects of radiotherapy and chemotherapy: increased white blood cell count and serum protein content;

(3) significant clinical effects: pain relief, increased appetite, increased physical strength, change in mental state from pessimism to optimism, confidence, improved overall survival by several months, and in some cases by 13 months;

(4) greatly reduced the size of metastatic cancer, in some cases by 1/3-1/2.

Test Case 8: Stability Tests

After completion of the preparation of the Pamica complexes of the present invention, they were stored indoors in the dark, and the samples were tested every 6 months.

Results: The samples were kept at room temperature (room temperature from August to September in Beijing was over 30 degrees) for 6 months, and the test performance of the products did not change significantly, indicating that the products were relatively stable; Pamica prepared at 1 mg/ml can be stabilized for at least 12 months; Pamica prepared at 3 mg/ml can be stabilized for at least 9 months. The pH value of the products is relatively stable and does not change significantly over 3 years. The level of bacterial endotoxin in Pamica of the present invention is less than 10 EU/ml, while the level of bacterial endotoxin in polyinosinic-polycytidylic acid for injection exceeds 100 EU/ml. Since it is a common technology in the art, bacterial endotoxin is a component of the cell wall of gram-negative bacteria, bacteria will release endotoxins after death or autolysis. Thus, bacterial endotoxin is widely distributed in nature. For example, endotoxin contained in tap water is characterized by an amount of 1-100 EU/ml. If bacterial endotoxin enters the human body through the digestive tract, it is harmless, but if endotoxin enters the bloodstream by injection or other routes, it can cause various diseases. After a small amount of endotoxin enters the bloodstream, it is inactivated by Kupffer cells of the liver and is harmless to the human body. The ingestion of a large amount of endotoxin into the blood causes a fever reaction, i.e. a “pyrogenic reaction”. Therefore, formulations such as biological products, drugs for injection, chemicals, radiopharmaceuticals, antibiotics, vaccines, dialysate, etc., as well as medical equipment (for example, disposable syringes, implantable biological materials) must be verified through a bacterial endotoxin test to determine the quality before use.

Experimental example 9: determination of the ability of Pamica to stimulate the phagocytic function of macrophages

Location: Institute of Pharmacology, Chinese Academy of Medical Sciences

Method: Collecting macrophages: 6 Kunming SPF-class mice weighing 20-25 g were randomly divided into 2 groups, 3 mice in each group. Mice were immunized with Pamica and PBS on day 0, each mouse was instilled into the nose 200 μl. After 2 hours, each mouse was injected with 5.0% chicken red blood cells in 0.85% saline suspension. After 4 hours, 3 mice in each group were sacrificed by dislocation of the cervical vertebrae. After disinfection, an incision was made in the skin and Hank's buffer was administered by peritoneal injection at a dose of 2.5 ml/mouse, and the abdomen of the mouse was gently rubbed to completely wash away macrophages in the abdominal cavity with Hank's buffer. Then, a small hole was cut in the middle of the peritoneum and approximately 2 ml of fluid from the abdominal cavity was removed using a 5 ml pipette and placed in a test tube.

Slides with drops: Aseptic peritoneal lavage was removed from the test tube and the drops were placed on a slide. A glass slide with drops was placed horizontally on wet gauze. The slide was incubated in a constant temperature incubator at 37°C for half an hour and at that time; a large number of macrophages adhered to the glass slide. Chick red blood cells and cells of other tissues on the slide that did not undergo phagocytosis were washed off with 0.85% saline, then the slide was dried in cold air.

Fixation and staining of samples: samples were fixed with methanol for 5 minutes and stained with Giemsa-Wright staining solution. Staining was carried out according to the Giemsa staining method and the pH of the PBS buffer used was adjusted to 6.5. Samples were observed and counted with an oil immersion lens after drying in cold air.

Phagocytosis percentage: total macrophages phagocytic red blood cells ÷ total macrophages × 100%.

Phagocytosis index: total number of red blood cells phagocytosed by macrophages ÷ total number of macrophages phagocytosed by red blood cells × 100% (100 macrophages were observed with an oil immersion lens and the value obtained by counting the amounts of red blood cells phagocytosed by each macrophage is summed numbers and divide the resulting sum by 100, and get the value of the phagocytosis index).

Results: In the PBS control group, the phagocytosis percentage was 12% and the phagocytosis index was 0.11; and in the Pamica group, the phagocytosis percentage is 66% and the phagocytosis index is 1.2, indicating that Pamica has a strong stimulating effect on the phagocytic function of macrophages. In FIG. 16 shows that red blood cells are phagocytosed by macrophages, and FIG. 17 shows that red blood cells are not phagocytosed by blue-colored macrophages (shown by arrows).

Experimental Example 10: Test for Protective Properties of Pamica Mucosal Immune Formula Nasal Drops Separately Against Influenza in Mice

Influenza virus: murine lung adaptive influenza subtype A strain FM1 purchased from the Institute of Viral Disease Prevention and Control of the Chinese Academy of Preventive Medicine.

Ribavirin: positive control drug, purchased from Shenyang Yanfeng Pharmaceutical Factory.

Mice: Kunming mice, mice weighing 8～10g were used for passage of the FM1 virus, and mice weighing 14～20g were used for subsequent formalized experiments.

Fatal pneumonia can be induced by nasal instillation of a suspension of lung-adaptive mouse strain FM1 of influenza virus at 5 LD ₅₀ /mouse. In the test, mice were first infected and then injected. The test was carried out in groups according to the following table.

The results of the experiment demonstrate that, in a protection test in mice, the nasal drops of the present invention, through the immune route of administration through the mucosa, have a better non-specific anti-influenza effect than ribavirin, which is a recognized antiviral drug and has a significant anti-influenza effect, demonstrated by statistical analysis.

Experimental Example 11: Effects of Pamica Mucosal Immune Formula (Nasal Drops) in Combination with Influenza Vaccine Via Nasal Mucosal Immunization and Subcutaneous Immunization Injection on IgA Humoral Antibody and Reproductive Influenza Virus Titer in Comparison with Complete Freund's Adjuvant

The scheme of the experiment is as follows:

Influenza virus: mouse lung adaptive influenza virus strain FM1 purchased from the Institute of Viral Disease Prevention and Control of the Chinese Academy of Preventive Medicine.

Influenza Vaccine: Influenza virus split vaccine purchased from Hualan Biological Products Co., Ltd.

Freund's complete adjuvant: Shanghai Via-geneprobio Technologies Co. Ltd.

Mucosal immune adjuvant of the present invention: from Xinfu (Beijing) Medical Technology Co., Ltd.

Mouse: Kunming mice, mice weighing 8～10g were used for passage of FM1 virus, and mice weighing 14～20g were used for subsequent formalized experiments.

Influenza vaccine with Freund's complete adjuvant: the vaccine and Freund's complete adjuvant were added in the same volume to a centrifuge tube, mixed homogeneously on a shaker to form a water-in-oil emulsion.

Nasal drops in combination with the influenza vaccine of the present invention: The influenza vaccine and the mucosal immune adjuvant of the present invention were mixed in equal amounts to form an aqueous solvent.

Influenza Vaccine: Influenza vaccine was mixed with PBS in equal amounts to form an aqueous solvent.

Method of immunization:

Immunization by subcutaneous injection: mice were administered by subcutaneous injection on day 0 and day 28 at a dose of 0.1 ml/mouse, on the 42nd day some mice were bled and the blood serum was separated to determine the antibody titer. Other mice were infected with a suspension of mouse lung adaptive strain FM1 influenza virus at 5 LD ₅₀ /nasal drip. On the 5th day after infection, the virus titer in the lung tissue was determined.

Nasal immunization: Mice were immunized at a dose of 0.1 ml/mouse using a nasal dropper on day 0 and day 28. On day 42, some mice were bled and serum was separated to determine antibody titer. Other mice were infected with a suspension of mouse lung adaptive strain FM1 influenza virus at 5 LD ₅₀ /nasal drip. On the 5th day after infection, the virus titer in the lung tissue was determined.

The results of the experiment for each group are shown in the following table:

Anti-influenza activity test of the mucosal immune formulation in combination with the influenza vaccine using a nasal dropper

Freund's complete adjuvant is the accepted standard for testing stimulation of the body's cellular immunity. The test results demonstrate that immunization by subcutaneous administration of the mucosal immune formulation of the present invention in combination with an influenza vaccine produces antibodies in an amount that is 10 times less than in the case of an influenza vaccine with complete Freund's adjuvant, but reduces the virus titer influenza 31.6 times compared with influenza vaccine with complete Freund's adjuvant; in particular, nasal mucosal immunization in mice demonstrates that nasal drops of the mucosal immune formulation of the present invention, in combination with an antigen, provide antibody production 31.6 times higher than that of a single influenza vaccine and reduce influenza virus titer. more than 3100 times, which has extremely important consequences.

Experimental Example 12: Pamica showed a clear effect in the preliminary test in the experiment with the action of CIK cells on target cells.

In a test conducted by Beijing Jingmeng Hi-Tech Stem Cell Technology Co., Ltd., the present invention has a clear effect in a preliminary test for determining the effect of CIK cells on target cells.

Sample number: JSCIK2016042614; test date: April 26, 2016; date of submission of the report: April 28, 2016.

Operation Method: Culturing and analysis were carried out according to the traditional experiment to determine the effect of CIK cells on target cells, the experimental method is shown, for example, in (The Immunotherapeutic Antitumor Effect of Dendritic Cells Co-cultured with Cytokine Induced Killer Cells, Jie Zhuang et al., Chinese Journal of Cell Biology, 2007, 29;237-240.

Target cell: A549, effector cell: JSCIK2016042614 cultured cell

Conclusion: The ability to kill JSCIK2016042614 cells was comprehensively analyzed in combination with the use of a fluorescent microscope and detection with a microplate reader, as well as the error calculation in the experiment itself, and is moderate at 1:10 (when the ratio of target cells to effector cells was 1: 10, the killing rate of A549 target cells was 51.4%).

Experimental Example 13: Anticancer Effect of Mucosal Immune Drug Pamica in the LL2 Lung Cancer Tumor Mouse Model

The antitumor activity of Pamica was tested in a nasal spray transplanted mouse LL2 tumor model. The tumor cells in this model grew rapidly, the tumor volume was 2201.9 ± 68.01 mm ³ on the 14th day after inoculation, and the experiment was terminated. Cisplatin as a positive control had the best effect in terms of tumor reduction, followed by the group that was administered Pamica by intramuscular injection. However, for Pamica nasal spray, except for the group administered at 0.1 mg/mouse, each group administered nasal spray at a dose of 0.2 mg/mouse had a P < 0.0001 compared to the medium group. -carrier as a negative control, showing a significant difference. In particular, in the same mouse model from previous data, the PD1 mouse type is shown to be practically inapplicable. As an explanation, the cisplatin group is more likely to show efficacy in this very rapid cell division model. Overall, Pamica has a marked effect in this tumor mouse model through a novel anti-cancer mechanism. In addition, no side effects were found in this mouse model.

The experimental groups are shown in the following table:

The specific results of the experiment are shown in Fig. 18-21.

In this experiment, the cells exhibited a high rate of tumor formation, and the tumor in the vehicle control group grew rapidly, with a tumor volume of 2201.09 ± 68.01 mm ³ at the end of the experiment. In the positive control group, cisplatin showed a clear effect of tumor suppression, which indicates the success of this experiment and the reliability of the results.

The test results show that in this LL2 tumor transplant mouse model of mouse lung cancer with C57BL/6 cells, groups except for the group administered with Pamica at a dose of 150 μg/mouse show a significant tumor growth suppression effect regardless of route of administration: nasal drop or intramuscular injection, and statistical determination demonstrates that there is an extremely significant difference in P <0.0001; at the same time, there are no obvious toxic and side effects in mice with a tumor.

Previous data from Huiyuan Biotech showed that the mouse anti-PD-1 antibody is relatively less effective in the LL2 model (tumor suppression rate is less than 10%). Pamica, which also acts on the immune system, shows a better effect than the PD-1 antibody. It is generally believed that the antitumor efficacy of an anti-PD-1 antibody depends on the level of expression of PD-L1 in tumor cells or is associated with mutation load, high microsatellite instability (MSI-H), or mismatched nucleotide repair (dMMR) defects, which in turn turn affects its medicinal effect. As an immune adjuvant, Pamica has a suppressive effect over a broader range than that of immune response checkpoints, showing great potential for development.

Experimental Example 14: In vivo study of the antitumor effect of Pamica in the 4T1-luc in-situ mouse model of breast cancer

This pharmacodynamic experiment was carried out for 9 groups: a vehicle group, a PD1 group, 6 Pamica treatment groups, and a Pamica plus PD1 group. The vehicle group received a PBS solution via nasal dropper at a dose of 66.7 μl/mouse on day 16 post-inoculation once every two days; the PD1 group received a PD1 solution by intraperitoneal injection at a dose of 100 μg/mouse on the 16th day after inoculation once a week; The 6 Pamica-treated groups were respectively administered as follows: one group was administered Pamica via a nasal dropper at a dose of 200 μg/mouse 7 days prior to inoculation once every two days; one group was administered Pamica via nasal dropper at a dose of 200 μg/mouse on the day of inoculation once every two days; one group was administered Pamica by nasal dropper at a dose of 200 μg/mouse on the 16th day after inoculation once every two days; one group was administered Pamica by nasal dropper at a dose of 300 μg/mouse on the 16th day after inoculation once every two days; one group was administered Pamica by intramuscular injection at a dose of 200 μg/mouse on the 16th day after inoculation once every two days; and one group was administered Pamica by intramuscular injection at a dose of 300 μg/mouse on the 16th day after inoculation once every two days; the Pamica group in combination with PD1 received Pamica via a nasal dropper at a dose of 200 μg/mouse on the 16th day after inoculation once every two days + administered Pamica by intraperitoneal injection at a dose of 100 μg/mouse, on the 16th day after inoculation once a week. In the experiment, female Balb/c mice were used, the tumor volume was measured once every three days and weighed for body weight every two days. The experiment was terminated when the mean tumor volume in the vehicle control group exceeded 2000 mm ³ and tumor bioluminescence was measured using an in vivo small animal imaging device. Finally, the organs of mice in each group were selected for staining with HE.

The results of the pharmacodynamic test showed that the day 7 group with advanced cancer and the day 0 group were characterized by relatively weak tumor growth and metastasis inhibition, and the two groups administered Pamica by intramuscular injection had a stronger inhibitory effect in in terms of tumor growth and metastasis than the two groups administered Pamica via nasal drip. Except for the group administered Pamica by nasal drip at a dose of 200 μg/mouse, the group administered Pamica by intramuscular injection at a dose of 300 μg/mouse, the group administered Pamica by intramuscular injection at a dose of 200 µg/mouse, and the groups that were administered Pamica by intramuscular injection at a dose of 300 µg/mouse, all groups are characterized by the ability to significantly suppress tumor growth and metastasis. At the end of the experiment, the tumor suppression rates in the group administered with Pamica by nasal dropper at a dose of 200 μg/mouse and in the group administered with Pamica by nasal dropper at a dose of 300 μg/mouse were 25% and 35% respectively. The tumor suppression ratios in the group administered Pamica by intramuscular injection at a dose of 200 μg/mouse and in the group administered with Pamica by intramuscular injection at a dose of 300 μg/mouse were 56% and 61%, respectively.

The above results indicate that Pamica has a good effect in suppressing tumor growth and metastasis in 4T1 breast cancer.

The method of carrying out the experiment and the results of the experiment are described in detail below.

1. Method of conducting the experiment

1.1 Construction of the in-situ 4T1-luc breast cancer mouse model

Female Balb/c mice were used, 4T1-luc mammary cancer cells in logarithmic growth phase were selected and inoculated under the fourth mammary gland of Balb/c mice at 1×10 ⁵ cells/0.2 ml/mouse to construct a mouse model with tumor in situ. The volume of the tumor mass was dynamically measured using a vernier caliper. Tumor volume is calculated according to the following formula: V=0.5xLxD2 (where V is tumor volume, L is tumor long axis size, and D is short axis tumor size).

1.2 Setting the injection time

For the group administered Pamica by nasal dropper 7 days prior to inoculation (designated as the group from day 7 with advanced cancer), 10 mice were randomly selected 7 days prior to tumor inoculation and Pamica was administered according to Table 1 ;

for the group administered with Pamica by nasal dropper 0 days before inoculation (designated as group 0 day), 10 mice were randomly selected on the day of tumor inoculation and Pamica was administered according to Table 1;

For the group administered with vehicle medium, when the tumor volume increased to approximately 80 mm ³ , i.e., on the 16th day after tumor inoculation, administration was started according to Table 1.

For the PD1-administered group, when the tumor volume increased to approximately 80 mm ³ , that is, on the 16th day after tumor inoculation, administration was started according to Table 1.

For the group administered Pamica via a nasal dropper at a dose of 200 μg/mouse, when the tumor volume increased to approximately 80 mm ³ , i.e. on the 16th day after tumor inoculation, administration was started according to Table 1.

For the group administered Pamica via a nasal dropper at a dose of 300 μg/mouse, when the tumor volume increased to approximately 80 mm ³ , i.e. on the 16th day after tumor inoculation, administration was started according to Table 1.

For the group administered Pamica by intramuscular injection at a dose of 200 μg/mouse, when the tumor volume increased to approximately 80 mm ³ , i.e., on the 16th day after tumor inoculation, administration was started according to Table 1.

For the group administered Pamica by intramuscular injection at a dose of 300 μg/mouse, when the tumor volume increased to approximately 80 mm ³ , i.e., on the 16th day after tumor inoculation, administration was started according to Table 1.

Table 1: dosages and groups in the experiment

1.3 Completion of the experiment

The entire experiment was terminated on day 30 post-injection as the tumor volume exceeded 2000 mm ³ .

1.4 Observed indicators

Tumor volume was measured for volume determination once every three days, and mice were weighed once every two days. At the end of the experiment, the heart, liver, spleen, lung, kidney and tumor were separated. Heart, liver, spleen, and kidney were fixed with 4% neutral formaldehyde, then paraffin-embedded sections were prepared and analyzed by staining with HE; the tumor was photographed and weighed; the lung was fixed in Bouin's fixative mixture for 16 hours, immersed in 50% alcohol for 2 hours, photographed, fixed with 4% neutral formaldehyde, paraffin-embedded sections were obtained and analyzed by staining with HE.

1.5 Small Animal Imaging Device

At the end of the administration, mice were intraperitoneally injected with 100 μl of luciferin at a concentration of 30 mg/ml as a fluorescein substrate and anesthetized with isoflurane. After 17 minutes, the mice were fixed in an in vivo small animal imaging device to observe bioluminescence. The image capture parameters are as follows: capture time: 0.2 seconds; Bin value: 4; and F value: 2. Image processing software: Living Image® software (version 4.3.1; Caliper Life Sciences Inc.).

2. Statistical analysis

All experimental data are expressed as "mean ± standard deviation" and SPSS Statistics 19 software (version 4.0.100.1124; SPSS Inc., IBM Company, USA) was used for data analysis. One-way analysis of variance ANOVA was used to compare data, and t-test was used to determine significant differences between groups: *p<0.05; **p<0.01; ***p<0.001.

3. EXPERIMENTAL RESULTS AND DISCUSSION

3.1 Tumor volume

Curves of changes in tumor volume are shown in Fig. 22.

Table 2: t-test results for tumor volume

Note: The error bar points to SD; —— indicates no data *: indicates p < 0.05 compared to vehicle group; **: indicates p<0.01 compared to vehicle group; ***: indicates p < 0.001 compared to vehicle group.

In FIG. 22 and Table 2, it can be seen that the tumor suppression effects of the day 7 group with advanced cancer and the day 0 group are relatively weak, and the two groups administered by intramuscular injection have better tumor suppression effects, than the two groups administered via nasal drip. Specific results are as follows.

The 7th day group administered with Pamica and the 0th day group have certain late-stage tumor suppression effects and generally no early-stage tumor suppression effect, indicating that late-stage administration or immediate administration does not have clear tumor-killing effect, and practically proves that Pamica is a tumor treatment vaccine and not a prophylactic vaccine. At the end of the experiment, the tumor suppression rate values in the day 7 group with advanced cancer and in the day 0 group were 36% and 26%, respectively.

Tumor volume values in the PD1 group at day 12 were significantly different from tumor volume values in the vehicle group (**p<0.01), indicating that PD1 has some suppressive effect on 4T1 breast cancer in mice.

In each treatment group of the PD1 in combination with Pamica groups, the majority of mice died on the 20th day after administration, for this reason no data were obtained regarding the later measurement for the two groups. Among them, the tumor volumes in the combined administration groups on days 6, 12, and 18 were significantly different from those in the vehicle group (*p<0.05, **p<0.01, ***p<0.001, respectively) .

The group administered with Pamica by nasal dropper at a dose of 200 μg/mouse had a significant inhibitory effect when administered at an early stage and a gradually decreasing effect of tumor suppression at a late stage of administration. Tumor volume values at days 6 and 12 were significantly different from those in the vehicle group (**p<0.01). Except for day 18, the tumor volume values in the Pamica nasal dropper group at 300 μg/mouse differed significantly from those in the vehicle group in a number of cases. This demonstrates that administration by nasal dropper is effective and the tumor suppression effect is dose-dependent. At the end of the experiment, the tumor suppression rates in the group administered with Pamica by nasal dropper at a dose of 200 μg/mouse and in the group administered with Pamica by nasal dropper at a dose of 300 μg/mouse were 25% and 35% respectively.

The tumor suppression effects of the two groups dosed with Pamica by intramuscular injection were similar throughout the administration process, and differed significantly from those of the vehicle medium group, and the tumor suppression effect of the two groups dosed with Pamica by intramuscular injection injection was better than the two groups that received Pamica via a nasal drip. At the end of the experiment, the tumor suppression ratios in the group injected with Pamica by intramuscular injection at a dose of 200 μg/mouse and in the group administered with Pamica by intramuscular injection at a dose of 300 μg/mouse were 56% and 61%. respectively.

3.2 Body weight

Curves of changes in body weight of mice are shown in Fig. 23.

Table 3: t-test statistical results for body weight of mice in each group and vehicle group

Note: The error bar points to SD; —— indicates no data *: indicates p < 0.05 compared to vehicle group; **: indicates p<0.01 compared to vehicle group; ***: indicates p < 0.001 compared to vehicle group.

In FIG. 23 and Table 3, it can be seen that the body weights of mice in the PD1 and combination groups did not differ significantly from those in the vehicle group during the effective measurement time, indicating that they had fewer side effects. Except for the later stage of body weight administration in the day 7 group with advanced cancer, the day 0 group, the group administered Pamica via nasal dropper at a dose of 200 μg/mouse, and the group administered Pamica via a nasal dropper at a dose of 300 μg/mouse were significantly lower than those in the vehicle medium group in some cases. No significant difference was observed between the Pamica intramuscular injection group at a dose of 200 μg/mouse and the vehicle group during the entire administration period. Body weights in the Pamica group administered by intramuscular injection at a dose of 300 μg/mouse were lower than body weights in the vehicle group at the intermediate stage of administration, and did not differ significantly from the body weight in the vehicle group at an early stage and a late stage of administration.

The above results indicate that intramuscular injection has little effect on the body weight of mice and has less side effect, and intranasal injection has a certain side effect in mice.

3.3 Tumor weight, spleen weight and photographs of the tumor

In FIG. 24 and FIG. 25, it can be seen that there was no significant difference in tumor weight between the day 7 group with advanced cancer or the day 0 group compared to the control group, and the two groups given Pamica by intramuscular injection had a stronger inhibitory effect. effect on tumor growth than the two groups administered Pamica via nasal drip. Except for the group administered Pamica by nasal dropper at a dose of 200 μg/mouse, the group administered Pamica by nasal dropper at a dose of 300 μg/mouse, the group administered Pamica by intramuscular injection at a dose of 200 μg/mouse, and the group administered Pamica by intramuscular injection at a dose of 300 μg/mouse can be defined as having a significant suppression of tumor growth with a statistical difference compared to the vehicle group (*p<0.05, **p<0.01, ***p<0.001 respectively).

The spleen is the largest immune organ of the body, accounting for 25% of the total lymphatic tissue of the body, contains a large number of lymphocytes and macrophages, and is the center of cellular immunity and humoral immunity of the body. In FIG. 26, it can be seen that the spleen weights in the Pamica intramuscular injection group at a dose of 200 μg/mouse and the Pamica intramuscular injection group at a dose of 300 μg/mouse were significantly higher than in vehicle group, with statistical differences (**p<0.01, *p<0.05, respectively), indicating that the immune response of the Pamica intramuscular injection group may be stronger.

3.4 Photographs of the lungs

In FIG. 27 and FIG. 28, it can be seen that there are many white tumor nodules on the surface of the lung tissue in the vehicle group, the day 7 group with advanced cancer, and the day 0 group, indicating that the day 7 group with advanced-stage cancer and the day 0 group are generally characterized by no effect on suppression of 4T1 lung metastasis. There are fewer nodules on the surface of the lung tissue in the group that was administered Pamica via a nasal dropper at a dose of 200 μg/mouse and the group that was administered Pamica via a nasal dropper at a dose of 300 μg/mouse, and in the group that received administered Pamica by intramuscular injection at a dose of 200 μg/mouse, and the group that was administered Pamica by intramuscular injection at a dose of 300 μg/mouse had the least number of nodules on the surface of the lung tissue, indicating that the two groups which were administered Pamica by nasal drip, and the two groups which were administered Pamica by intramuscular injection are able to effectively suppress 4T1 metastasis to the lungs, and the group which was administered Pamica by intramuscular injection has a stronger ability to suppress 4T1 metastasis to the lungs than the group , which was injected with Pamica using a nasal dropper.

3.5 Small animal imaging device

In FIG. 29-35, it can be seen that the bioluminescence intensities of tumor sites and metastases in the carrier medium group, the 7th day group with advanced cancer, and the 0th day group are higher. The bioluminescence intensities of the tumor site and metastases in the group administered with Pamica by nasal dropper at a dose of 200 μg/mouse and with Pamica at a dose of 300 μg/mouse were weaker, and the bioluminescence intensities of tumor sites and metastases in the group , which was administered Pamica by intramuscular injection at a dose of 200 µg/mouse and the group that was administered Pamica by intramuscular injection at a dose of 300 µg/mouse were the weakest, indicating that the group that was administered Pamica by intramuscular injection , has a stronger ability to inhibit the growth and metastasis of 4T1 breast cancer than the group administered Pamica via nasal drip, which is consistent with the above results.

4. Conclusion

In this experiment, the authors of the present invention successfully created a mouse model of 4T1-luc breast cancer in-situ. The tumor grew rapidly, and by the end of the experiment, the tumor volume exceeded 2000 mm ³ .

The test results demonstrate that in this study, using the mouse orthotopic tumor model for breast cancer with Balb/c 4T1-luc cells, except for the results for the PD1 and combination groups, a large number of mice died due to PD1, thus received only part of the experimental data, on the other hand, each group was characterized by a certain tumor suppression effect in a certain period of time. The day 7 group with advanced cancer, the day 0 group and the group administered with Pamica by nasal dropper at a dose of 200 μg/mouse did not have a clear effect of tumor inhibition, the group administered with Pamica by nasal droppers at a dose of 200 μg/mouse, the group injected with Pamica by intramuscular injection at a dose of 200 μg/mouse, and the group administered with Pamica by intramuscular injection at a dose of 300 μg/mouse showed clear inhibitory effects tumors.

Experimental Example 15: Use of Pamica to Treat Patients Infected with Human Papilloma Virus (HPV)

Experimental Example 16: Use of Pamica in the Treatment of Breast Cancer

1. Positive control drugs:

PD1, paclitaxel injection

2. Construction of an Orthotopic Animal Model of Breast Cancer in 4T1 Mice

BALB/c female mice were used; 4T1-luc breast cancer cells in logarithmic growth phase were selected and inoculated at 1×10 ⁵ cells/0.2 ml/mouse under the fourth mammary gland of BALB/c mice to construct an orthotopic mouse tumor model. The volume of the tumor mass was dynamically measured using a vernier caliper. Tumor volume was calculated according to the following formula: V=0.5×L×D2 (where V is the volume of the tumor, L is the size of the tumor on the long axis, and D is the size of the tumor on the short axis).

3. Effect on growth and spontaneous metastasis of 4T1 breast cancer in situ

3.1 Division into groups and dosing regimen

① for the PBS-injected group, a few days after tumor inoculation, PBS was administered with a nasal dropper at a dose of 100 µl/mouse once every other day;

② for the PD1-administered group, a few days after tumor inoculation, PD1 was intraperitoneally administered at a dose of 100 µg/mouse once a week;

③ For the paclitaxel-administered group, a few days after tumor inoculation, paclitaxel was administered by tail vein injection at a dose of 10 mg/kg once a week;

④ For the group injected with Pamica by nasal dropper, a few days after tumor inoculation, Pamica was administered by nasal dropper at a dose of 100μl (0.3mg)/mouse once every other day, 50μl was administered by nasal drip in the morning on the day of administration and using a nasal dropper, 50 μl was administered in the afternoon;

⑤ for the group administered Pamica by intramuscular injection, a few days after tumor inoculation, Pamica was administered by intramuscular injection at a dose of 100 µl (0.3 mg)/mouse was administered once every other day;

⑥ for the combined administration group, a few days after tumor inoculation, PD1 was administered intraperitoneally (100 μg/mouse, administered once a week) + Pamica by nasal drip (100 μL/mouse, administered once every other day);

⑦ For the combined administration group, a few days after tumor inoculation, paclitaxel was administered by tail vein injection (10mg/kg, administered once a week) + Pamica by nasal drip (100μL/mouse, administered once every other day).

3.2 Number of animals in each group: 15 animals in each group (5 reserve animals in each group).

3.3 Time to end the experiment: Depending on the actual situation (the mouse died or the difference between the treated group and the control group is obvious), the experiment can be ended earlier.

3.4 Experimental protocol

A 4T1-luc in situ breast cancer model was created and an in vivo small animal imaging device was used for early detection and grouping. The introduction was carried out in accordance with the division into groups and dosing regimen according to paragraph 6.1. Tumor volume was measured (measured once every 3 days), weight change (measured once every 3 days), tumor weight (determined at the end), spleen weight (determined at the end), TUNEL staining was performed (determined at the end). The lung was fixed with Bouin's fixative mixture and then photographed. Each tissue and organ was fixed with neutral formaldehyde, then stained with H&E (for end-detection) and the intensity of in situ tumor bioluminescence and metastases was observed at various time points and end time points using an in vivo small animal imaging device. The effects of tumor growth suppression and in situ metastasis in each group were comprehensively compared.

Experiments have proven that Pamica has clear effects of reducing tumor volume in breast cancer, controlling tumor weight and spleen weight, as well as stimulating tumor cell apoptosis and other aspects.

Finally, it should be noted that the above embodiments are only intended to illustrate and not limit the technical solutions of the present invention; although the present invention has been described in detail with reference to the above examples, those skilled in the art should understand that it is still possible to make changes to the technical solutions set forth in the above examples, or to make equivalent substitutions for some or all of the specifications; and these modifications or substitutions do not deviate from the spirit of the respective technical solutions from the scope of the technical solutions of the examples of the present invention.

Industrial Applicability

The present invention discloses a complex for potentiating an immune response. Compared with the prior art, the complex has a medium viscosity and molecular weight and is convenient for use in pharmaceutical production; it is characterized by stable chemical properties and practically does not degrade during long-term storage, and is also safe to use. The complex, when used alone, can significantly potentiate the nonspecific immune response of the body and achieve the goal of preventing and treating diseases, and when used in combination with other drugs, it has better antitumor, antiviral and anti(super)bacterial efficacy and is easily absorbed by patients.

Claims (57)

1. Combined product for potentiation of the immune response containing polyinosinic-polycytidylic acid (PIC), cationic stabilizer and soluble calcium salt in a liquid reaction system; wherein the cationic stabilizer is a graft copolymer of chitosan oligosaccharide and methoxypolyethylene glycol (COS-g-MPEG), wherein the chitosan oligosaccharide has a molecular weight of 5 kDa or less; polyinosine-polycytidylic acid is preheated at a temperature of 88 to 90°C for 70 to 120 minutes, the ratio of PIC to COS-g-MPEG in the combined product is from 1:0.8 to 1:6.4. 2. The product according to claim 1, wherein the chitosan oligosaccharide has a deacetylation degree of 70% or more. 3. The product according to claim 1, wherein the soluble calcium salt is CaCl ₂ and/or CaNO ₃ . 4. The product according to claim 1, where polyinosinic-polycytidylic acid is characterized by a molecular weight of 100-3000 p. 5. The product according to claim 1, where polyinosinic-polycytidylic acid is characterized by a molecular weight of 100-1500 p. 6. The product of claim 1, wherein the combination product further comprises one or more of a pH adjuster, sodium tripolyphosphate, sodium alginate, phenylboronic acid, catechol, buffer salt, and water. 7. Complex for potentiation of the immune response, containing a combined product according to paragraphs. 1-6. 8. The complex according to claim 7, where the concentration of polyinosinic-polycytidylic acid in the combined product is 0.1-10 mg/ml. 9. The complex according to claim 8, where the concentration of polyinosinic-polycytidylic acid in the combined product is 0.5-5 mg/ml. 10. The complex according to claim 7, where the concentration of the cationic stabilizer in the combined product is 0.5-51.2 mg/ml. 11. The complex according to claim 10, where the concentration of the cationic stabilizer in the combined product is 0.8-25.6 mg/ml. 12. The complex according to claim 10, where the concentration of calcium ions in the soluble calcium salt in the combined product is 0.1-1 mm. 13. Non-therapeutic use of the complex according to any one of paragraphs. 7-12 as an immune adjuvant. 14. The use of the complex according to any one of paragraphs. 7-12 to obtain an antibody. 15. The use of the complex according to any one of paragraphs. 7-12 to obtain a vaccine composition or vaccine composition. 16. The use of the complex according to any one of paragraphs. 7-12 to obtain an adjuvant for a vaccine. 17. Vaccine composition containing the complex according to any one of paragraphs. 7-12 and at least one antigen. 18. The vaccine composition according to claim 17, wherein the antigen is a virus, bacterium, protein, polypeptide, polysaccharide, nucleic acid, or low molecular weight protein conjugate. 19. The use of the complex according to any one of paragraphs. 7-12 for regulation of immune cell activity, where the application is carried out in vivo or in vitro. 20. Use according to claim 19, wherein regulation of immune cell activity specifically potentiates immune cell activity. 21. Use according to claim 19, wherein the immune cell is selected from macrophages, lymphocytes and dendritic cells. 22. Use according to claim 19, wherein the regulation and potentiation of immune cell activity promotes the release of pro-inflammatory factors by immune cells. 23. Use according to claim 22 wherein the pro-inflammatory factors include IL-2, IL-6, IL-12p40, IL-18, IL-22, IFN-α, IFN-γ and TNF-α. 24. The use of the complex according to any one of paragraphs. 7-12 to obtain drugs for the treatment and/or prevention of tumors. 25. The use of the complex according to any one of paragraphs. 7-12 to obtain antiviral drugs. 26. The use of the complex according to any one of paragraphs. 7-12 to obtain antibacterial drugs. 27. The use of the complex according to any one of paragraphs. 7-12 to obtain antifungal drugs. 28. The use of the complex according to any one of paragraphs. 7-12 to obtain antiparasitic drugs. 29. The use of the complex according to any one of paragraphs. 7-12 to obtain drugs that stimulate the immune response to an antigen in the body. 30. Application according to any one of paragraphs. 24-29, where the drug is an injection dosage form, a respiratory dosage form, nasal drops, a transdermal dosage form, a transmucosal dosage form, or an oral dosage form. 31. Use according to claim 29, wherein the antigen includes a tumor, viral, bacterial, fungal or parasitic antigen. 32. Use according to claim 29, wherein the organism is a mammal. 33. Use according to claim 29, where the organism is a primate. 34. Use according to claim 29, where the organism is a human. 35. Pharmaceutical composition for the treatment of cancer and infection, containing the complex according to any one of paragraphs. 7-12, wherein the pharmaceutical composition further comprises one or more of an immune cell therapy drug, an antibody therapy drug, a chemical drug, a substance that promotes mucosal uptake of immunologically active agents or mucosal adhesion, an immunomodulator, a pathogen antigen , a pattern recognition receptor ligand, and a pharmaceutically acceptable excipient, where the chemical drug is one or more selected from the group consisting of an alkylating agent, an antimetabolite agent, an antitumor antibiotic, an antitumor herbal drug, a hormonal drug, and another drug; where the other drug is selected from the group consisting of L-asparaginase, cisplatin, carboplatin, oxaliplatin, dacarbazine, drugs based on hexamethylmelamine and derivatives of the above drugs. 36. The pharmaceutical composition of claim 35, wherein the immune cell therapy drug is one or more cells selected from the group consisting of tumor infiltrating lymphocytes (TIL), dendritic cells (DC), cytokine-induced killer cells (CIK), dendritic and cytokine-induced killer cells (DC-CIK), natural killer (NK) cells, γδT cells, CD3AK, CAR-T and TCR-T. 37. The pharmaceutical composition of claim 35 or 36, wherein the antibody therapeutic drug is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, and an anti-CD antigen. 38. Pharmaceutical composition according to any one of paragraphs. 35-37, where the substance that promotes absorption of immunologically active agents by the mucosa or adhesion on the mucosa is one or more substances selected from the group consisting of anionic surfactants, cationic surfactants, zwitterionic surfactants, nonionic surfactants, specialty surfactants, chelating agents, adhesive agents, polylactic acid-glycolic acid copolymers, dextrans and polysaccharides. 39. Pharmaceutical composition according to any one of paragraphs. 35-38, wherein the immunomodulator is one or more immunomodulators selected from the group consisting of cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, column stimulating factors (CSFs), interferons, erythropoietins, thrombopoietins, tumor necrosis factors (TNF), interleukins (IL), granulocyte colony stimulating factors (G-CSF), granulocyte-macrophage colony stimulating factors (GM-CSF) and stem cell growth factors. 40. Pharmaceutical composition according to any one of paragraphs. 35-39, wherein the pathogen antigen is selected from the group consisting of a tumor, viral, bacterial, fungal, or parasitic antigen. 41. The pharmaceutical composition according to claim 40, where the tumor includes tumors resulting from any lesions of the bone, bone junction, muscle, lung, trachea, pharynx, nose, heart, spleen, artery, vein, blood, capillary, lymph node, lymph vessel, lymphatic fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, large intestine, rectum, anal canal, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, kidney, ureter, urinary bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva, scrotum, testes, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, spinal cord fluid, nerves, thyroid, parathyroid, adrenal, pituitary, pineal, pancreatic islet, thymus, gonad, sublingual, and parotid gland. 42. Pharmaceutical composition according to claim 40, where the bacterium includes one or more of Staphylococcus, Streptococcus, Listeria, Erysipelothrix, Renibacterium, Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia, Corynebacterium, Rhodococcus, Bacillus anthracis, Erysipelas bacillus, Bacillus tetani, Listeria monocytogenes, Bacillus perfringens, Bacillus gangraenae emphysematosae, tuberculosis pathogen, Escherichia coli, Bacterium proteus, Shigella dysenteriae, Pneumobacillus, Bacterium burgeri, Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter, Yersinella, Bortophilatus, persist, , Shigella, Pasteurella, Vibrio cholerae and Vibrio Parahemolyticus . 43. The pharmaceutical composition according to claim 40, wherein the parasite comprises one or more of digestive tract parasites, intracavitary parasites, intrahepatic parasites, intrapulmonary parasites, brain tissue parasites, intravascular parasites, lymphatic system parasites, muscle tissue parasites, intracellular parasites, bone parasites tissues and intraocular parasites. 44. The pharmaceutical composition according to claim 40, where the virus includes one or more of adeniviridae, arenaviridae, astroviridae, bunyaviridae, cliciviridae, flaviviridae, delta hepatitis virus, hepeviridae, mononegavirales, nidovirales, piconaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae, retroviridae or togaviridae. 45. The pharmaceutical composition according to claim 40, where the fungus includes one or more of Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Histoplasma duboisii, Blastomyces lobo, Paracoccidiodes brasiliensis, Blastomyces dermatitis, Sporothrix schenckii, Penicillium marneffei, Candida albicans, Candida glabrata tropicalis, Candida lusitaniae, Pneumocystis carinii, Aspergillus, Exophiala jeanselmei, Fonsecaea pedrosoi, Fonsecaea compacta, Chromomyces verruciformis, Pigmentation dermatitis, Geotrichum candidum, Pseudallescheria boydii, Cryptococcus neoformans, Trichosporon cutaneum, Rhizopus oryzae, Mucor indicus, Absidia corymbifera, Syncephalastrum racemosum, Basidiobolus ranarum, Conidiobolus coronatus, Conidiobolus incongruus, Enterocytozoon bieneusi, Encephalitozoon intestinalis, Rhinosporidium seeberi , hyalologithomycetes and pheohyphomycetes. 46. Pharmaceutical composition according to any one of paragraphs. 35-45, wherein the pattern recognition receptor ligand is selected from the group consisting of a TLR receptor ligand, an RLR receptor ligand, a CLR receptor ligand, and an NLR receptor ligand. 47. A method of stimulating an immune response to an antigen in the body in vivo, including the introduction into the body of the complex according to any one of paragraphs. 7-12, or a vaccine composition according to claim 17 or 18, or a pharmaceutical composition according to any one of paragraphs. 36-46, if the antigen is a viral, bacterial, fungal or parasitic antigen, the complex according to any one of paragraphs. 7-12, or a vaccine composition according to claim 17 or 18, or a pharmaceutical composition according to any one of paragraphs. 36-46 are administered at a dose of 1-8 mg/kg with each administration; alternatively administered once a day, once every 2 days, once every 3 days or once every 4 days; if the antigen is a tumor antigen, the complex according to any one of paragraphs. 7-12, or a vaccine composition according to claim 17 or 18, or a pharmaceutical composition according to any one of paragraphs. 36-46 are administered at a dose of 1-10 mg/kg at each administration and administered for at least 360 days, at least 180 days, at least 60 days, or at least 30 days. 48. The method of regulation and potentiation of the activity of immune cells in the body, including the introduction into the body of the complex according to any one of paragraphs. 7-12, or a vaccine composition according to claim 17 or 18, or a pharmaceutical composition according to any one of paragraphs. 36-46, if the antigen is a viral, bacterial, fungal or parasitic antigen, the complex according to any one of paragraphs. 7-12, or a vaccine composition according to claim 17 or 18, or a pharmaceutical composition according to any one of paragraphs. 36-46 are administered at a dose of 1-8 mg/kg with each administration; alternatively administered once a day, once every 2 days, once every 3 days or once every 4 days; if the antigen is a tumor antigen, the complex according to any one of paragraphs. 7-12, or a vaccine composition according to claim 17 or 18, or a pharmaceutical composition according to any one of paragraphs. 36-46 are administered at a dose of 1-10 mg/kg at each administration and administered for at least 360 days, at least 180 days, at least 60 days, or at least 30 days.

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