Composite adjuvant for mucosal immunity

Abstract

The invention discloses a composite adjuvant suitable for mucosal immunity. The mucosal composite adjuvant comprises the following components in percentage by mass: 0.25-5% of sodium carboxymethyl cellulose, 0.5-3% of microcrystalline cellulose, polyinosinic-polycytidylic acid with the final concentration of 0.5-5 mg / ml and cyclic dinucleotide with the final concentration of 100-300 mu g / ml. The composite adjuvant disclosed by the invention is combined with a recombinant novel coronavirus antigen protein or influenza virus split vaccine antigen protein, through local mucous membrane administration, a good mucous membrane immunological effect is generated locally on the mucous membrane, a high-level neutralizing antibody of the whole body can be stimulated to be generated, and meanwhile, the composite adjuvant has a good cellular immune effect and can be used for preparing the vaccine for treating the influenza virus. And the method has a good and wide application prospect in the development of vaccines or medicines for local administration through mucosa.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine, and in particular, to a novel compound adjuvant that enhances mucosal immunity. Background Technology

[0002] Mucosal surfaces constitute the largest and most important interface between the body and the external environment, primarily covering the gastrointestinal tract, urinary and reproductive tracts, and respiratory tract. They are the body's largest defense organs, possessing physical, biochemical, and immune barrier functions. Simultaneously, mucosal surfaces are also the most common site and route for pathogens to adhere and enter the body. Therefore, developing vaccines that can produce good immunological effects locally in the mucosa is crucial for controlling pathogenic microorganisms that infect via the mucosal route. Traditional vaccines are mostly administered via intramuscular injection, which can stimulate humoral immune responses, but rarely induce mucosal immune responses. Studies have shown that vaccines administered via mucosal surfaces exhibit rapid and widespread antigen distribution, as well as the ability to induce protective mucosal immunity and systemic cellular and humoral responses. Furthermore, mucosal vaccines have broader acceptability among the elderly and children, are easier to manage, increase vaccine accessibility, and facilitate large-scale vaccination during pandemics. Therefore, mucosal vaccines have promising application prospects. However, the number of currently marketed mucosal vaccines is extremely small, mainly because they require safe and effective mucosal adjuvants to achieve the desired immune protection effect, and the antigens must be protected from degradation. Therefore, developing a mucosal adjuvant with good immune support is crucial for advancing the research and development of mucosal vaccines. Summary of the Invention

[0003] Technical problems to be solved:

[0004] One aspect of this disclosure is to provide a novel composite adjuvant that enhances mucosal immunity, addressing the lack of mucosal adjuvants with good immune-adjuvant effects in the prior art.

[0005] Specifically, this disclosure creatively discovers a novel mucosal immune adjuvant formulation. Animal immunization experiments have verified that this formulation can stimulate the body to produce good humoral and cellular immunity, and stimulate the production of sIgA antibodies in the local mucosa, demonstrating excellent mucosal immune effects. Currently marketed nasal spray vaccines are mainly live attenuated vaccines; there are no nasal spray recombinant protein vaccines on the market, nor are there any adjuvants for mucosal administration. The novel mucosal immune adjuvant in this disclosure can not only stimulate the local production of high titers of specific sIgA antibodies in the mucosa, but also induce systemic humoral and cellular immunity, bringing hope for the application of vaccines, especially recombinant protein vaccines, via mucosal administration, and potentially promoting the clinical research and use of nasal spray recombinant protein vaccines.

[0006] Technical solution:

[0007] A mucosal adjuvant comprising 0.25-5% sodium carboxymethyl cellulose and 0.5-3% microcrystalline cellulose by weight, and polyinosinic-polycytidylic acid and cyclic dinucleotide at final concentrations of 0.5-5 mg / ml and 100-300 μg / ml, respectively.

[0008] In some embodiments of this disclosure, the mass ratio of sodium carboxymethyl cellulose, microcrystalline cellulose, polyinosinic-polycytidylic acid (PII) and cyclic dinucleotide can be 15~25:15~25:5~15:1.

[0009] Preferably, in one embodiment of this disclosure, the mass ratio of sodium carboxymethyl cellulose, microcrystalline cellulose, polyinosinic-polycytidylic acid (PII) and cyclic dinucleotide is 20:20:10:1.

[0010] Another aspect of this disclosure is the use of the aforementioned mucosal adjuvant in the preparation of vaccine adjuvants. In some embodiments of this disclosure, the vaccine may be an inactivated vaccine, a live attenuated vaccine, a protein vaccine, a bacterial polysaccharide and polysaccharide-protein conjugate vaccine, a genetically engineered vaccine, or a genetically reassortant vaccine.

[0011] Another aspect of this disclosure is to provide a method for preparing the above-mentioned mucosal adjuvant, comprising the following steps:

[0012] Step 1) Disperse the microcrystalline cellulose and sodium carboxymethyl cellulose in a buffer solution and mix them by high-speed rotational shearing for 10 min to 20 min to obtain suspension I;

[0013] Step 2) Perform at least one operation selected from high pressure homogenization, shear homogenization, ultrasonic emulsification, membrane emulsification or microfluidic homogenization on the suspension I obtained in step 1) to obtain suspension II;

[0014] Step 3) Autoclave the suspension II obtained in Step 2), and obtain suspension III after cooling;

[0015] Step 4) Add the polyinosinic-polycytidylic acid and the cyclic dinucleotide to the suspension III obtained in step 3) to obtain the mucosal complex adjuvant.

[0016] In some embodiments of this disclosure, the high-speed rotary shearing described above can be performed using any suitable equipment in the prior art at an appropriate rotational speed.

[0017] In some embodiments of this disclosure, in step 2) above, the suspension I is subjected to high-pressure homogenization at 1000-1200 bar, and the homogenization is performed 2-6 times.

[0018] Preferably, in some embodiments of this disclosure, the buffer solution in step 1) includes citrate or citric acid buffer, phosphate buffer, carbonate buffer, acetate buffer, borate buffer, histidine buffer, trimethylolpropane buffer, succinate buffer, or barbiturate buffer.

[0019] Preferably, in some embodiments of this disclosure, the buffer solution in step 1) is a histidine buffer solution, which may contain histidine at a final concentration of 0.01~1000mM and NaCl at a final concentration of 0.01~1000mM; the pH value of the histidine buffer solution is 6.5~7.0.

[0020] More preferably, in some embodiments of this disclosure, the histidine buffer solution is composed of histidine at a final concentration of 10 mM and NaCl at a final concentration of 150 mM, with a pH of 6.8.

[0021] Preferably, in some embodiments of this disclosure, the particle size D50 of the solid particles in the suspension III obtained in step 3) is 8.2 μm and the D90 is 29.69 μm.

[0022] Another aspect of this disclosure is to provide a vaccine comprising an immunogen and the aforementioned mucosal adjuvant.

[0023] In some embodiments of this disclosure, the vaccine may be a vaccine against viruses, bacteria, mycoplasma, rickettsia, spirochetes, fungi, or parasites.

[0024] Preferably, in some other embodiments of this disclosure, the vaccine may be a vaccine against a virus, wherein the virus is a herpesviridae, poxviridae, adenoviridae, human papillomavirus, parvovirus, reoviridae, clovenviridae, flaviviridae, coronavirusidae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, filoviridae, retroviridae, pitoviridae, astroviridae, caliciviridae, or hepatotropic virus.

[0025] More preferably, in some other embodiments of this disclosure, the virus may be herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus, smallpox virus, human adenovirus, human papillomavirus, parvovirus, Kabovir, rotavirus, rubella virus, Japanese encephalitis virus, dengue virus, yellow fever virus, SARS coronavirus, MERS coronavirus, novel coronavirus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, human metapneumovirus, rabies virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, HIV, enterovirus, Coxsackie virus, echovirus, poliovirus, astrovirus, norovirus, hepatitis B virus, hepatitis C virus, hepatitis A virus or hepatitis E virus, Nipah virus, Langya virus, Hendra virus.

[0026] More preferably, in some embodiments of this disclosure, the immunogen is a recombinant novel coronavirus antigen or an H3N2 influenza virus antigen.

[0027] In other embodiments of this disclosure, the viral vaccine is an inactivated vaccine, a live attenuated vaccine, a genetically engineered vaccine, a peptide vaccine, a genetically reassortant vaccine, a DNA nucleic acid vaccine, or an mRNA nucleic acid vaccine. A recombinant protein vaccine is preferred.

[0028] Another aspect of this disclosure is to provide a pharmaceutical composition administered via a mucosa, the pharmaceutical composition comprising the above-described mucosal adjuvant or the above-described vaccine, and a pharmaceutically acceptable carrier.

[0029] Preferably, in one embodiment of this disclosure, the pharmaceutical composition is administered via the nasal mucosa, respiratory mucosa, lung mucosa, reproductive tract mucosa, or digestive tract mucosa.

[0030] Beneficial effects:

[0031] The compound adjuvant disclosed herein, combined with recombinant novel coronavirus antigen protein or influenza virus split vaccine antigen protein, when administered locally via the mucosa, not only produces good mucosal immunological effects locally but also stimulates the production of high levels of neutralizing antibodies throughout the body, while also exhibiting good cellular immune effects. It has good and broad application prospects for the development of vaccines or drugs administered locally via the mucosa. Attached Figure Description

[0032] Figure 1 This is a transmission electron microscope (TEM) image of the sodium carboxymethyl cellulose-microcrystalline cellulose suspension in the embodiments of this disclosure.

[0033] Figure 2This is a graph showing the detection results of neutralizing antibodies against the novel coronavirus BF.7 strain after animals were immunized with the compound adjuvant in the embodiments of this disclosure;

[0034] Figure 3 This is a graph showing the detection results of sIgA antibodies against the novel coronavirus antigen in the respiratory tract lavage fluid of animals immunized with the compound adjuvant in this embodiment of the present disclosure.

[0035] Figure 4 This is a graph showing the detection results of neutralizing antibodies against the novel coronavirus BF.7 strain after immunizing animals with different adjuvant combinations of novel coronavirus antigen in the embodiments of this disclosure;

[0036] Figure 5 This is a graph showing the detection results of sIgA antibodies against novel coronavirus antigen in the respiratory tract lavage fluid of animals immunized with novel coronavirus antigen after different adjuvant combinations in the embodiments of this disclosure.

[0037] Figure 6 This is a graph showing the detection results of specific IFN-γ cytokines secreted by spleen lymphocytes after immunizing animals with different adjuvant combinations using the novel coronavirus antigen in the embodiments of this disclosure;

[0038] Figure 7 This image shows the detection results of specific IL-17A cytokine secreted by spleen lymphocytes in animals immunized with novel coronavirus antigen after different adjuvant combinations in the embodiments of this disclosure.

[0039] Figure 8 This image shows the detection results of specific IL-5 cytokine secreted by spleen lymphocytes in animals immunized with novel coronavirus antigen after different adjuvant combinations in the embodiments of this disclosure.

[0040] Figure 9 This is a graph showing the results of hemagglutination inhibition antibody detection in animals immunized with H3N2 influenza virus antigen using different adjuvant combinations in the embodiments of this disclosure;

[0041] Figure 10 This is a graph showing the detection results of influenza virus antigen-specific sIgA antibodies in the respiratory tract lavage fluid of animals immunized with H3N2 influenza virus antigen using different adjuvant combinations in the embodiments of this disclosure.

[0042] Figure 11 This is a graph showing the detection results of specific IFN-γ cytokines secreted by spleen lymphocytes in animals immunized with H3N2 influenza virus antigen after different adjuvant combinations in the embodiments of this disclosure.

[0043] Figure 12 This is a graph showing the detection results of specific IL-17A cytokine secreted by spleen lymphocytes in animals immunized with H3N2 influenza virus antigen after different adjuvant combinations in the embodiments of this disclosure.

[0044] Figure 13 This image shows the detection results of specific IL-5 cytokine secreted by spleen lymphocytes in animals immunized with H3N2 influenza virus antigen after different adjuvant combinations in the embodiments of this disclosure.

[0045] Sequence Description

[0046] SEQ ID No. 1 is the amino acid sequence of the recombinant novel coronavirus protein RBD antigen in the embodiments of this disclosure. Detailed Implementation

[0047] This invention discloses a compound adjuvant for mucosal immunity. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. Furthermore, those skilled in the art can clearly modify or appropriately alter and combine the content described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0048] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., shall be understood to include the stated elements or components without excluding other elements or other components. The term "a," "an," and "the" includes plural indicators. The term "a plurality of" means two or more. The terms "such as," "for example," etc., are intended to refer to exemplary embodiments and are not intended to limit the scope of this disclosure.

[0049] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common molecular biology terms can be found in Lewin's GENE XII, Jocelyn E. Krebs / Elliott S. Goldstein / Stephen T. Kilpatrick, published by Jones & Bartlett, 2018.

[0050] The terms "adjuvant," "vaccine adjuvant," and "immunoadjuvant" in this disclosure all refer to substances that can nonspecifically bind to or mix with antigens / immunogens / immunogenic substances to enhance the immunogenicity and immune protective effects of the antigens / immunogens / immunogenic substances, i.e., enhance humoral immune responses and / or cellular immune responses, while having no immunogenicity of their own. They can also be understood as "substances that, when bound to antigens, can produce better immunity than antigens alone."

[0051] In this disclosure, the "adjuvant," "vaccine adjuvant," "immunoadjuvant," nonspecific immune response enhancer, etc., are administered to any mammal, including but not limited to domestic and farm animals (cattle, horses, pigs, sheep, goats, dogs, cats, or rodents, etc.), primates, and humans. Preferably, the mammal is a human.

[0052] In some embodiments of this disclosure, the type of vaccine may include, for example, inactivated vaccines, live attenuated vaccines, protein vaccines, bacterial polysaccharide and polysaccharide-protein conjugate vaccines, genetically engineered vaccines, or genetically reassortant vaccines. Examples of inactivated vaccines include whole-virus vaccines or split vaccines, such as inactivated polio vaccine, DPT vaccine, influenza split vaccine, hepatitis A inactivated vaccine, meningococcal polysaccharide vaccine, etc. Live attenuated vaccines are made from treated pathogens that have lost their pathogenicity but can still elicit an immune response. Such vaccines are prepared by physical or chemical methods, or by continuous passage culture under in vitro conditions, to reduce or eliminate the pathogenicity of the pathogen while maintaining good immunogenicity. Examples include measles, mumps, and rubella (MMR) vaccines, varicella vaccines, yellow fever vaccines, rotavirus vaccines, oral polio vaccine (OPV), etc. Protein vaccines are made from specific pathogen proteins that can elicit an immune response. Such vaccines are prepared by directly delivering specific proteins of the pathogen along with an adjuvant that stimulates an immune response into human cells. An example is a recombinant novel coronavirus protein vaccine. The bacterial polysaccharide-polysaccharide-protein conjugate vaccine is formed by combining bacterial polysaccharide antigens with a protein carrier. This vaccine is prepared by chemically coupling specific bacterial polysaccharides with a protein carrier, allowing the polysaccharides to be recognized by T cells, thereby triggering a stronger immune response. Examples include Haemophilus influenzae polysaccharide conjugate vaccines, meningococcal polysaccharide conjugate vaccines, and pneumococcal polysaccharide conjugate vaccines. The genetically engineered vaccine is a vaccine prepared using genetic engineering technology. This vaccine is prepared by cloning the specific antigen gene of a pathogen into an expression vector, then inducing the expression of exogenous proteins through a prokaryotic or eukaryotic expression system, followed by purification and other process steps to produce the vaccine. Examples include recombinant hepatitis B vaccines, BCG vaccines, and DNA vaccines. The genetic reassortment vaccine is made from recombinant microorganisms obtained through genetic reassortment methods. Typically, a non-pathogenic attenuated strain is mixed with a virulent strain (mostly wild-type strains) for infection. Genomic fragment exchange occurs between the attenuated and wild-type strains, resulting in reassortment. Then, specific methods are used to screen for reassortant strains that are non-pathogenic to humans but contain immunogenic gene fragments from the wild-type strain. For example, rotavirus vaccines, influenza vaccines, etc. In one embodiment of this disclosure, the vaccine may be a protein vaccine or an inactivated vaccine.

[0053] In this disclosure, the selection of the mucosal adjuvant requires consideration of the specific anatomical and physiological characteristics of the mucosa. For example, during nasal administration, the poor permeability of the nasal vestibule and the rapid turnover of hair and mucous membranes can interfere with the absorption and delivery of the vaccine's active ingredients. Furthermore, local bioactive substances in the mucosa, such as various enzymes, local pH, and secretions, can affect the stability and delivery efficacy of the vaccine. Therefore, in developing mucosal vaccine adjuvants, in addition to examining their immunomodulatory effects, the protection and stability of the vaccine's effective antigenic components in the mucosa must also be considered. When designing mucosal adhesive formulations, selecting an excipient that imparts mucosal adhesion to the formulation and ensures its long-term retention at the site of action is also crucial. Excipients with mucosal adhesive properties must be synthetic or natural hydrophilic polymers containing functional groups that can interact with mucin glycoproteins. This interaction occurs through non-covalent bonds such as hydrogen bonds, van der Waals forces (intermolecular forces), and ionic interactions. Examples of such substances include, but are not limited to, carbomers (such as Carbopol). ® Polymers, xanthan gum, carrageenan, methyl vinyl ether and maleic anhydride copolymer (PVM / MA), hydroxypropyl cellulose (HPC), sodium carboxymethyl cellulose (Na-CMC), etc. In one embodiment of this disclosure, a mixture of sodium carboxymethyl cellulose and microcrystalline cellulose is selected as a component of the mucosal immune adjuvant. This mixture can be dispersed in water to form a colloid and used as a suspension of 8.3% to 18.8% (w / w) depending on the material specifications. Utilizing its colloidal properties and certain viscosity, it can protect the antigen protein and also delay the local residence time of the antigen on the mucosa, facilitating absorption and utilization.

[0054] Mucosal compound adjuvants:

[0055] The mucosal adjuvant of this disclosure comprises 0.25-5% sodium carboxymethyl cellulose and 0.5-3% microcrystalline cellulose by mass, with final concentrations of 0.5-5 mg / ml for polyinosinic-polycytidylic acid and 100-300 μg / ml for cyclic dinucleotides. In some embodiments of this disclosure, the mass percentage of sodium carboxymethyl cellulose may be about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. The mass percentage of microcrystalline cellulose may be about 0.5%, about 0.75%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.25%, about 2.5%, about 2.75%, or about 3%. The final concentrations of polyinosinic-polycytidylic acid (poly I:C) can be approximately 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, and 5 mg / ml. The final concentrations of cyclic dinucleotides can be approximately 100 μg / ml, 125 μg / ml, 150 μg / ml, 175 μg / ml, 200 μg / ml, 225 μg / ml, 250 μg / ml, 275 μg / ml, and 300 μg / ml.

[0056] In addition to the substances mentioned above, in some embodiments of this disclosure, the mucosal adjuvant may further include, for example, solvents, delivery systems (e.g., liposomes, exosomes, nanoparticles, etc.), and immunostimulants (e.g., cytokines, chemokine PAMPs, TLR-ligands, immunostimulatory sequences, CpG-containing DNA, dsRNA, endocytotic recognition receptor ligands, LPS, saponins, etc.). These substances may be contained in the same container as the mucosal adjuvant or in separate containers. In one embodiment of this disclosure, the mucosal adjuvant consists only of 0.25–5% sodium carboxymethyl cellulose and 0.5–3% microcrystalline cellulose by mass, with final concentrations of 0.5–5 mg / ml polyinosinic-polycytidylic acid (PIC) and 100–300 μg / ml cyclic dinucleotides.

[0057] Preparation method:

[0058] In embodiments of this disclosure, the mucosal adjuvant is combined using a specific preparation method to exert its effect. Simultaneously, since cellulose, c-di-AMP, and poly I:C all carry negative charges, they do not form flocculent precipitates in solution. Furthermore, the relatively viscous cellulose suspension increases the adhesion time of c-di-AMP and poly(I:C) to the mucosal surface, thus achieving a sustained-release effect.

[0059] In embodiments of this disclosure, a suitable dispersion buffer system may be selected. The term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical preparation. Suitable pharmaceutically acceptable buffers include, but are not limited to, acetate buffer, histidine buffer, citrate buffer, succinate buffer, Tris buffer, and phosphate buffer. In some embodiments, the concentration of the buffer solution is about 0.01 mM to about 1000 mM, about 0.1 mM to about 1000 mM, about 0.1 mM to about 500 mM, about 0.1 mM to about 200 mM, about 0.1 mM to about 100 mM, about 1 mM to about 1000 mM, about 1 mM to about 500 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 2 mM to about 60 mM, about 4 mM to about 60 mM, or about 4 mM to about 40 mM, about 5 mM to about 20 mM, or about 5 mM to about 25 mM. For better results, in some embodiments of this disclosure, histidine-hydrochloric acid is selected, comprising 10 mM HIS-Cl and 150 mM NaCl at final concentrations; the pH of the histidine-hydrochloric acid buffer solution is 6.5 to 7.0.

[0060] The term "high-pressure homogenization" refers to a method of reducing particle size by adding target particles to a device that combines pressure and mechanical forces to break them down. The mechanical forces used in high-pressure homogenization may include impact, shearing, and cavitation. Commonly used equipment includes, for example, a high-pressure homogenizer. In one embodiment of this disclosure, the homogenized solid particles have a D50 of 8.2 μm and a D90 of 29.69 μm, at which point the effect is optimal. Methods for determining particle size are known in the art. Examples include dynamic light scattering (DLS), transmission electron cryomicroscopy, and nanoparticle tracking analysis (NTA).

[0061] vaccine:

[0062] The term "vaccine" refers to a product that, when administered to a human or animal, induces an immune response, including humoral and / or cellular immunity. Vaccines can be prophylactic or therapeutic. A vaccine includes an immunogenic substance and optionally at least one immunomodulator. In some embodiments of this disclosure, the immunogenic substance is a protein of a virus, such as SARS-CoV-2. SARS-CoV-2 belongs to the order Nidovirales, family Coronaviridae, subfamily Orthocoronaviruses, genus Betacoronavirus, subgenus Sarbecovirus, species SARS-like virus, and is a single-stranded positive-sense RNA virus. It is enveloped, with a genome approximately 29.9 kb in length. The vast majority of its genome encodes non-structural proteins involved in viral replication and translation. A smaller portion encodes structural proteins, such as the spike protein (S), membrane protein (M), envelope protein (E), and nucleo protein (N). Several accessory proteins, including 3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14, are also involved in viral assembly. The S, M, and E proteins constitute the viral envelope and are the main surface antigens that elicit an immune response. The S protein is a transmembrane glycoprotein with a molecular weight of approximately 150 kDa, forming a prominent homotrimer on the viral surface. The S protein consists of two functional subunits, cleaved at the boundary between the S1 and S2 subunits (S1 / S2 cleavage point). These two subunits maintain non-covalent binding in the pre-fusion conformation. The S2 subunit also consists of multiple domains, its main function being to mediate viral fusion with host cells. The distal S1 subunit is structurally divided into four distinct domains: NTD, RBD, CTD1, and CTD2. The RBD is the receptor-binding domain, primarily responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface, thereby mediating viral infection of the host cell. Therefore, both the S protein and RBD are major targets in current genetic engineering vaccine development. For another example, the H3N2 influenza virus, a subtype of influenza A virus, is a significant cause of human influenza outbreaks. The virus is named after two proteins on its surface: hemagglutinin (HA) and neuraminidase (NA). H3N2 can exchange genes for its internal proteins through genetic recombination subtypes.

[0063] The term "mucosal immunity" refers to immunity mediated through the mucosal immune system. The mucosal immune system is composed of lymphoid tissue distributed in the mucosa of the gastrointestinal tract, lacrimal ducts, salivary ducts, respiratory tract, urinary tract, and mammary glands, comprising a large portion of the body's lymphoid tissue. The gastrointestinal mucosal immune system is particularly noteworthy, characterized by: specialized aggregated lymph nodes (also known as Peyer's patches); the presence of mucosal homing receptors, indicating a pattern of lymphocytes returning to the mucosa through recirculation; its location as the site of IAe B cells and memory T cells; and the secretion of immunoglobulins primarily S-IAe, etc. These properties, on the one hand, protect the host from pathogens, and on the other hand, enable the body to develop immune tolerance to common food antigens and normal microorganisms.

[0064] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0065] Example:

[0066] The recombinant novel coronavirus protein RBD antigen used in this embodiment is an intramolecular trimer composed of the first and last RBD sequences of three Omicron mutant strains of novel coronavirus BQ.1.1, BA.2.3.20 and XBB, tandemly linked together. The amino acid sequence is shown in SEQ ID No.1. This protein was obtained by recombinant expression in CHO cells and purified by a series of chromatographic analyses.

[0067] SEQ ID No. 1:

[0068] MRVQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPD DFTGCVIAWNSNKLDSRVGGNYDYMYRLFRKSKLKPFERDISTEIYQAGNKPCNGVRGFNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFA PFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

[0069] Example 1: Preparation of mucosal immune adjuvant

[0070] The preparation process of mucosal immune adjuvants includes the following steps:

[0071] Step 1) Disperse microcrystalline cellulose and sodium carboxymethyl cellulose in histidine-hydrochloric acid buffer, and shear mix them using a shear mixer at a speed of 4000 rpm-9000 rpm for 10 min-20 min. Visually inspect the solution for any clumping or adhesion to the wall to obtain suspension I.

[0072] Step 2) Homogenize suspension I in a high-pressure homogenizer at 1000-2000 bar for 2-6 times to obtain suspension II;

[0073] Step 3) Autoclave suspension II at 121±1℃ for 30±5 min. After cooling, obtain suspension III, which is the mucosal immune delivery system—sodium carboxymethyl cellulose-microcrystalline cellulose. The particle size of suspension III was measured using a laser particle size analyzer; the D50 was 8.2 μm and the D90 was 29.69 μm. Transmission electron microscopy was used to observe suspension III, and the results are as follows: Figure 1 As shown, electron microscopy reveals that suspension III exhibits a fibrous, stringy appearance, consistent with the properties of sodium carboxymethyl cellulose-microcrystalline cellulose.

[0074] Step 4) Add double-stranded polyinosinic-polycytidylic acid and cyclic dinucleotide to suspension III to finally prepare suspension IV, which is the compound adjuvant.

[0075] Example 2: Effect of compound adjuvant on the immunogenicity of recombinant novel coronavirus antigen protein

[0076] Six- to eight-week-old SPF-grade BALB / c mice were divided into groups of 10 and immunized according to the formulations and animal experimental protocols shown in Table 1. Recombinant novel coronavirus protein RBD antigen was mixed with different adjuvant formulations to prepare immune substances. A pure antigen control group without adjuvants was also included. Intranasal immunizations were administered twice at 0 and 2 weeks. Blood was collected from the inner canthus vein at 3 weeks, and serum was separated and inactivated at 56°C for 30 min. The neutralizing antibody titer against BF.7 novel coronavirus in the serum was detected using a live virus micro-neutralization assay. The neutralizing antibody detection results are shown below. Figure 2 As shown in Table 2, using any one of microcrystalline cellulose-sodium carboxymethyl cellulose, cyclic dinucleotide, or polyinosinic-polycytidylic acid alone increased the neutralizing antibody titer by 40-50 times compared to the antigen-only group. The combined adjuvant group showed a 6-9 fold increase in neutralizing antibody titer compared to using any one of the adjuvants alone, and a 370-fold increase compared to the antigen-only group; all differences were statistically significant.

[0077] At week 4, respiratory lavage fluid was collected from 5 mice in each group, and the secretion of specific sIgA was detected using an antigen-specific ELISA method. The sIgA detection results in the respiratory lavage fluid are as follows: Figure 3 As shown in Table 3, it can be seen that using any one of microcrystalline cellulose-carboxymethyl cellulose sodium, cyclic dinucleotide, or polyinosinic-polycytidylic acid alone can produce a certain level of novel coronavirus antigen-specific sIgA antibody. However, the sIgA titer is significantly increased when all three are used in combination. The sIgA antibody titer produced by the combined adjuvant group is 12 times that of the antigen group alone. The difference is statistically significant compared with the use of any one of them alone or without use.

[0078] Table 1. Animal Experimentation Protocol

[0079] Table 2. Neutralizing antibody titers against BF.7 novel coronavirus

[0080] Table 3. Antigen-specific sIgA titers of the novel coronavirus

[0081] Example 3: Comparison of the immunogenicity of compound adjuvants with other adjuvants

[0082] 6-8 week old SPF-grade BALB / c mice were grouped according to the formulation and animal experimental protocol shown in Table 4. Recombinant SARS-CoV-2 antigen protein was mixed with mucosal adjuvant, aluminum adjuvant, and MF59 adjuvant, and immunized according to the optimal adjuvant immunization route (aluminum adjuvant and MF59 adjuvant groups were immunized by intramuscular injection, while the combined adjuvant group was immunized by mucosal immunization). The antigen dose for both intramuscular and mucosal immunization was 5 μg / mouse. Two immunizations were performed at 0 and 2 weeks. Blood was collected from the inner canthal vein at 3 weeks, serum was separated, inactivated at 56℃ for 30 min, and neutralizing antibodies against SARS-CoV-2 were detected using a live virus micro-neutralization assay. Results are as follows: Figure 4 As shown in Table 5, the neutralizing antibody titers produced by the compound adjuvant were significantly higher than those of the aluminum adjuvant group, with statistical significance. The antibody titers were lower than those of the MF59 adjuvant group, but the difference was not statistically significant.

[0083] At 4 weeks, 5 mice from each group underwent respiratory tract lavage. The lavage fluid was used to detect sIgA antibody levels using the novel coronavirus antigen-specific ELISA method. The results are as follows: Figure 5 As shown in Table 6, sIgA was almost undetectable in the respiratory tract after intramuscular immunization in the MF59 adjuvant group and the aluminum adjuvant group, while a high level of sIgA antibody could be detected in the compound adjuvant group.

[0084] At 4 weeks, five mice were selected from each group, and spleen lymphocytes were harvested. The splenic lymphocytes were stimulated using the novel coronavirus RBD protein antigen peptide library as an experimental stimulus. The levels of cytokines such as IFN-γ, IL-17A, and IL-5 secreted by the splenic lymphocytes were detected using the multicolor fluorescence ELISPOT method. The results are shown in Tables 7, 8, and 9, respectively. Figure 6 , Figure 7 , Figure 8As shown, the number of IFN-γ and IL-17A-positive spleen cells in the combined adjuvant group was significantly higher than that in the aluminum adjuvant group and the MF59 adjuvant group, with statistically significant differences. The number of IL-5-positive cells was not significantly different from that in the aluminum adjuvant group, but lower than that in the MF59 adjuvant group. Cellular immunotherapy results indicate that, compared with intramuscular injection in the aluminum adjuvant and MF59 adjuvant groups, administration via mucosal membrane in the combined adjuvant group induced a stronger antigen-specific Th1 and Th17 cellular immune response, demonstrating good cellular immunomodulatory activity.

[0085] Table 4. Animal Experimentation Protocol

[0086] Table 5. Neutralizing antibody titers against BF.7 novel coronavirus

[0087] Table 6. Novel coronavirus antigen-specific sIgA titers

[0088] Table 7. IFN-γ cytokine detection results (ELISPOT method)

[0089] Table 8. Results of IL-17A cytokine detection (ELISPOT method)

[0090] Table 9. IL-5 cytokine detection results (ELISPOT method)

[0091] Example 4: Effect of compound adjuvants on the immunogenicity of influenza virus vaccines

[0092] The H3N2 influenza virus split vaccine antigen was mixed with either a compound adjuvant or an aluminum adjuvant, at a dose of 5 μg / dose / mouse. Animals (6-8 week old SPF-grade BALB / c mice) were immunized, 10 mice per group. Two doses were administered at 0 weeks and 2 weeks, respectively, using the optimal immunization route for each adjuvant (intramuscular injection for the aluminum adjuvant group and mucosal immunization for the compound adjuvant group). Serum was collected at 3 weeks, and hemagglutination inhibition antibodies against the H3N2 influenza virus were detected in 5 animals. The results are shown in Table 10. Figure 9 As shown, the H3N2 type hemagglutination inhibition antibody produced by the combined adjuvant group was comparable to that produced by the aluminum adjuvant group.

[0093] At 4 weeks, five mice underwent respiratory tract lavage. The lavage fluid was analyzed using an H3N2 influenza virus antigen-specific ELISA method to detect sIgA antibody levels. The results are shown in Table 11. Figure 10 As shown, the aluminum adjuvant group produced almost no mucosal sIgA antibodies, while the combined adjuvant group produced higher titers of sIgA antibodies.

[0094] At 4 weeks, splenic lymphocytes were isolated from 5 mice and stimulated with influenza virus lysis antigen. The secretion levels of IFN-γ, IL-17A, and IL-5 in splenic cells were detected using the multicolor fluorescence ELISPOT method. The results are shown in Tables 12, 13, and 14. Figure 11 , Figure 12 and Figure 13 As shown, the number of splenic cells in the compound adjuvant group that secrete IFN-γ and IL-17A was significantly higher than that in the aluminum adjuvant group, while the number of IL-5-positive cells was lower. The compound adjuvant group, when administered via mucosal administration, can induce a strong antigen-specific Th1 and Th17 cellular immune response and has good cellular immune activity.

[0095] Table 10 Hemagglutination inhibition antibody titers against H3N2 influenza virus

[0096] Table 11. Antigen-specific sIgA titers of H3N2 influenza virus

[0097] Table 12 Results of IFN-γ cytokine detection (ELISPOT method)

[0098] Table 13 Results of IL-17A cytokine detection (ELISPOT method)

[0099] Table 14. IL-5 cytokine detection results (ELISPOT method)

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mucosal compound adjuvant, characterized in that, The mucosal adjuvant comprises 0.25-5% sodium carboxymethyl cellulose and 0.5-3% microcrystalline cellulose by weight, and polyinosinic-polycytidylic acid and / or cyclic dinucleotides at a final concentration of 0.5-5 mg / ml and / or 100-300 μg / ml, respectively.

2. The mucosal composite adjuvant according to claim 1, characterized in that, The mass ratio of sodium carboxymethyl cellulose, microcrystalline cellulose, polyinosinic-polycytidylic acid, and cyclic dinucleotide is 15~25:15~25:5~15:

1.

3. The mucosal compound adjuvant according to claim 2, characterized in that, The mass ratio of sodium carboxymethyl cellulose, microcrystalline cellulose, polyinosinic-polycytidylic acid, and cyclic dinucleotide is 20:20:10:

1.

4. The method for preparing the mucosal composite adjuvant as described in claim 1, 2, or 3, characterized in that, Includes the following steps: Step 1) Disperse the microcrystalline cellulose and sodium carboxymethyl cellulose in a buffer solution and mix them by high-speed rotational shearing for 10 min to 20 min to obtain suspension I; Step 2) Perform at least one operation selected from high pressure homogenization, shear homogenization, ultrasonic emulsification, membrane emulsification or microfluidic homogenization on the suspension I obtained in step 1) to obtain suspension II; Step 3) Autoclave the suspension II obtained in step 2), and after cooling, obtain suspension III; Step 4) Add the polyinosinic-polycytidylic acid and / or the cyclic dinucleotide to the suspension III obtained in step 3) to obtain the mucosal complex adjuvant.

5. The preparation method according to claim 4, characterized in that, The buffer solution mentioned in step 1) includes citrate or citric acid buffer, phosphate buffer, carbonate buffer, acetate buffer, borate buffer, histidine buffer, trimethylolpropane buffer, succinate buffer, or barbiturate buffer.

6. The preparation method according to claim 5, characterized in that, The buffer solution is a histidine buffer solution, which contains histidine at a final concentration of 0.01~1000mM and NaCl at a final concentration of 0.01~1000mM; the pH range of the histidine buffer solution is 6.5~7.

0.

7. A vaccine, characterized in that, The vaccine includes an immunogen and a mucosal adjuvant as described in claim 1, 2 or 3.

8. The vaccine according to claim 7, characterized in that, The vaccine is a vaccine against viruses, bacteria, mycoplasma, rickettsia, spirochetes, fungi, or parasites.

9. The vaccine according to claim 7 or 8, characterized in that, The vaccine is a vaccine against a virus, which is a virus belonging to the Herpesviridae, Poxviridae, Adenoviridae, Human Papillomavirus, Parvoviridae, Reoviridae, Clonorviridae, Flaviviridae, Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Retroviridae, Picornaviridae, Astroviridae, Caliciviridae, or Hepatotropic virus.

10. The vaccine according to claim 9, characterized in that, The viruses mentioned are herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus, smallpox virus, human adenovirus, human papillomavirus, parvovirus, cabovirus, rotavirus, rubella virus, Japanese encephalitis virus, dengue virus, yellow fever virus, SARS coronavirus, MERS coronavirus, novel coronavirus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, human metapneumovirus, rabies virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, HIV, enterovirus, Coxsackie virus, echovirus, poliovirus, astrovirus, norovirus, hepatitis B virus, hepatitis C virus, hepatitis A virus or hepatitis E virus, Nipah virus, Langya virus, and Hendra virus.

11. The vaccine according to claim 7, characterized in that, The viral vaccine is an inactivated vaccine, a live attenuated vaccine, a genetically engineered vaccine, a viral vector vaccine, a polypeptide vaccine, a genetic reassortant vaccine, a DNA nucleic acid vaccine, or an mRNA nucleic acid vaccine.

12. The vaccine according to claim 7, characterized in that, The vaccine is administered via the nasal mucosa, respiratory mucosa, lung mucosa, reproductive tract mucosa, or digestive tract mucosa.

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