WO2022228560A1 - 人工合成的含CpG单链脱氧寡核苷酸在疫苗中的应用 - Google Patents

人工合成的含CpG单链脱氧寡核苷酸在疫苗中的应用 Download PDF

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WO2022228560A1
WO2022228560A1 PCT/CN2022/090392 CN2022090392W WO2022228560A1 WO 2022228560 A1 WO2022228560 A1 WO 2022228560A1 CN 2022090392 W CN2022090392 W CN 2022090392W WO 2022228560 A1 WO2022228560 A1 WO 2022228560A1
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vaccine
odn
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vaccines
antigen
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王立公
陈燕
曾婷
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华普生物技术(江苏)股份有限公司
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Priority to EP22795029.2A priority patent/EP4332225A1/en
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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Definitions

  • the present invention relates to the application of a series of artificially synthesized CpG-containing single-chain deoxy oligonucleotides in vaccines. Further, the present invention relates to the application of the artificially synthesized CpG-containing single-chain deoxyoligonucleotide as a vaccine adjuvant. Further, the present invention relates to the application of the artificially synthesized CpG-containing single-chain deoxyoligonucleotide in genetic engineering vaccines, subunit vaccines and inactivated vaccines.
  • a vaccine is an antigen that, when injected into the blood, stimulates the immune system to synthesize antibodies.
  • the application of vaccines plays a very important role in controlling the epidemic of human and animal diseases.
  • vaccines can be divided into inactivated vaccines, attenuated vaccines, subunit vaccines and genetically engineered vaccines.
  • Genetically engineered vaccines mainly include genetically engineered subunit vaccines, genetically engineered vector vaccines, nucleic acid vaccines, live gene deletion vaccines, and protein engineered vaccines.
  • Vaccines in a broad sense also include genetic recombinant vaccines, synthetic peptide vaccines, anti-idiotype antibody vaccines, and microcapsule controlled-release vaccines.
  • Recombinant vaccines refer to vaccines produced by genetic recombination mechanisms.
  • SIT method immune recombinant vaccine
  • the vaccine clones and amplifies the hepatitis B surface antigen HBsAg, and uses recombinant DNA technology to produce the vaccine from yeast.
  • the second is to prepare vaccines by eliminating and modifying known pathogenic genes on pathogenic microorganisms.
  • the first-generation recombinant vaccine against rotavirus developed in this way has been clinically tested in the United States and Finland, and the results suggest that the vaccine has strong protection against rotavirus-induced diarrhea in children.
  • the third is to induce an immune response by inserting a gene of a pathogenic microorganism into a non-pathogenic microorganism such as a virus, and then the modified virus acts as a carrier or carrier to express the foreign gene.
  • This technology is being used in the development of HIV vaccines. It is also used in the development of a new coronavirus vaccine.
  • new vaccines such as synthetic peptide vaccines, genetically engineered subunit vaccines, anti-idiotype antibody vaccines and nucleic acid vaccines have the advantages of good antigenicity and low toxicity, but their immunogenicity is weak, and it is necessary to use with efficient adjuvants .
  • adjuvants include antigen micronized adjuvants (insoluble aluminum salt colloids, immunostimulatory complexes, liposomes), slow-release antigen adjuvants (oil emulsion adjuvants, microencapsulation of antigens), microbial adjuvants (bacterial toxins, Corynebacterium pumilus vaccine, mycobacteria and their components, peptidoglycan), molecular adjuvants (cytokines, C3d molecules, costimulatory molecules, superantigens, heat shock proteins, CpG sequences), and other adjuvants (vitamin E and selenium, antibiotics, thymosin-like drugs, propolis, traditional Chinese medicine polysaccharides). These adjuvants mainly play their roles through immune regulation, participation in antigen presentation, induction of CD8 + T cell responses, and antigen storage.
  • antigen micronized adjuvants insoluble aluminum salt colloids, immunostimulatory complexes, liposomes
  • CpG oligodeoxynucleotide containing cytosine-guanine dinucleotide motif
  • cytosine-guanine dinucleotide motif can stimulate the natural immune system of higher animals, activate B cells and macrophages Immune cells such as dendritic cells (DC) and NK cells, and induce the secretion of cytokines such as IL-2, IL-6, IL-12, and IFN- ⁇ .
  • DC dendritic cells
  • NK cells cytokines
  • cytokines such as IL-2, IL-6, IL-12, and IFN- ⁇ .
  • CpG ODN has been well validated in humans and mice to induce the desired strong Th1-type immune responses in therapeutic vaccines for various diseases.
  • CpG ODN can be used alone as a vaccine adjuvant, and studies have reported that CpG DNA can be used as an effective enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen (Heather L.
  • CpG ODN as a vaccine adjuvant can also be used in combination with other adjuvants, and clinical trials have reported that the immunostimulatory TLR9 agonist oligodeoxynucleotide CPG 7909 as an adjuvant for Engerix-B enhanced vaccine immunogenicity (CLCOOPER, etc., Journal of Clinical Immunology, Vol. 24, No. 6, November 2004); the antibody response was significantly enhanced when adjuvant CPG 7909 was added to the blood-stage malaria vaccine candidate AMA1-C1/Alhydrogel (Vaccine. 2009 June 24; 27(31):4104-4109.).
  • CpG-ODNs alone or in combination with other adjuvants (such as aluminum hydroxide, ISA-51, MF59, ISCOMATRIX, and IL-2, etc.) could significantly enhance antigen-specific humoral and cellular immune responses.
  • CpG-ODNs can not only enhance the "quantity" of the antigen-specific immune response, but also change the "quality” of the antigen-specific immune response.
  • humoral immunity CpG-ODNs can not only accelerate antibody production, increase antibody titer, improve antibody avidity, and enhance immunity persistence, but also change antibody subtypes (Chinese Journal of New Drugs 2014, 23(1)).
  • Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that is spreading globally. According to the WHO official website, as of December 15, 2020, there have been 71,581,532 confirmed cases of new coronary pneumonia and 1,618,374 deaths. It is caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the S1 subunit contains a receptor-binding domain that recognizes and binds to the host receptor angiotensin-converting enzyme 2, while the S2 subunit mediates viral cell membrane fusion by forming a six-helix bundle through two heptapeptide repeat domains.
  • Potential drugs targeting the S protein include SARS-CoV-2 S protein-based antibodies, fusion inhibitors, and protease inhibitors targeting the SARS-CoV-2 S cleavage site [Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARSCoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020;5:562–9.].
  • the adjuvant technology has been further confirmed and applied in the research and development of the new crown vaccine.
  • the adjuvant objectively reduces the amount of antigen required by a unit of vaccine, thereby increasing the production capacity of the vaccine and expanding the immunized population. It will play an immeasurable role in the battle between humans and viruses.
  • at least 10 drug developers have said they plan to develop an adjuvanted vaccine against Covid-19, including England's GlaxoSmithKline (GSK), Australia's Seqirus and U.S. Dynavax, which have pledged to develop approved vaccines Adjuvants (AS03, MF59 and CpG 1018, respectively), are provided to others developing COVID-19 vaccines.
  • Recombinant Spike protein vaccine with doses molecular clamp stabilized Spike protein vaccine containing MF59 adjuvant jointly developed by University of Queensland/CSL/Seqirus, S-2P protein + CpG 1018 jointly developed by Medigen Vaccine Biologics Corporation/NIAID/Dynavax Vaccine, RBD+adjuvanted vaccine developed by Instituto Finlay de Vacuns in Cuba.
  • CN 111956797 A discloses the application of new vaccine adjuvant SF (chemically modified cyclic dinucleotide) in new coronary pneumonia vaccines
  • CN 111991556 A discloses Sigma Adjuvant Systerm and/ Or AddaVax's SARS-CoV-2 RBD conjugated nanoparticle vaccine
  • CN 111603556 A discloses a novel coronavirus subunit nanovaccine containing CpG oligodeoxynucleotides.
  • Phase III data for the Pfizer-BioNTech COVID-19 vaccine showed higher rates of lymphadenopathy in the vaccine group compared to placebo.
  • Bell's palsy is also imbalanced among vaccine recipients. There were 4 people in the vaccine group and 0 people in the placebo group. The causal relationship is unknown. It can be seen that the safety issues of vaccines need further research to solve.
  • the present invention provides CpG ODNs with immunomodulatory functions.
  • the structure of these CpG ODNs is novel, and it is even more rare that they have immunostimulatory effects on mice and humans and have synergistic effects when used in combination with aluminum adjuvants, so they have great clinical application value.
  • the present invention solves the problems existing in this area through the following technical solutions:
  • a composition comprising CpG ODN and one or more other adjuvants that work together with the immunomodulatory CpG ODN, such as aluminium adjuvants, such as aluminium hydroxide and insoluble aluminium salt colloids, oil and water Emulsions, microorganisms and metabolites, nucleic acids and analogs thereof, cytokines, immunostimulatory complexes, propolis, and liposomes, preferably aluminum adjuvants, wherein the CpG ODN comprises or consists of being selected from the group consisting of SEQ ID NO: 1-
  • the nucleotide sequence of 4 wherein at least one nucleotide in the nucleotide sequence is a chemically modified nucleotide having the structure shown in formula I:
  • Y is S or O
  • R is H or a positively charged counterion
  • B is independently an unmodified or modified nucleobase
  • R 1 is H, F, Cl, OH, OMe, Me, O- Ethoxymethyl.
  • composition of item 1 or 2 wherein all nucleotides of the nucleotide sequence of the CpG ODN are chemically modified nucleotides having the structure shown in general formula I.
  • composition of item 3 wherein the sequence of the CpG ODN is selected from: SEQ ID NOs: 1-4, preferably all phosphorothioated SEQ ID NOs: 1-4.
  • the vaccines are hepatitis B vaccines, rabies vaccines, and new coronary pneumonia vaccines, such as anti-new coronavirus Alpha strains, Beta strains, Gamma strains, Delta strains, and Omicron strains.
  • New coronary pneumonia vaccine including but not limited to new coronary pneumonia recombinant subunit vaccine and new coronary pneumonia inactivated vaccine, herpes zoster vaccine, influenza vaccine, such as quadrivalent influenza vaccine.
  • a pharmaceutical composition comprising an antigen and the composition of any one of items 1-5.
  • composition of item 8 wherein the content of the antigen is 5-200 ⁇ g/mL, preferably 5-80 ⁇ g/mL.
  • the pharmaceutical composition is for administration to a subject in an effective amount, preferably the pharmaceutical composition is for administration to a subject twice, wherein the The time interval is 2-24 weeks.
  • a method of preparing the pharmaceutical composition of any one of items 8-12 comprising mixing the composition of any one of items 1-5, an antigen and optionally a pharmaceutically acceptable carrier, adsorbing, coupling Combined and/or emulsified reaction, and formulated into injection or oral preparation, nasal spray.
  • the CpG ODN used in the present invention includes ODN 1-ODN 4, which respectively have the following nucleotide sequences:
  • ODN 1 (5'-tcgacgttcgtcgttcgtcgtcgttcgtcgtttc-3');
  • ODN 2 (5'-tcgcgacgttcgccgacgttcgta-3');
  • ODN 4 (5'-tcgcgaacgttcgccgcgtacgtacgtacgcgg-3').
  • the nucleotides of ODN 1-ODN 4 are all phosphorothioated nucleotides.
  • the method includes administering to the subject an effective amount of a pharmaceutical composition of the present invention.
  • the method may further comprise administering the pharmaceutical composition to the subject in an effective amount, preferably the pharmaceutical composition is administered to the subject twice, wherein the time interval between the two administrations is 2-24 weeks.
  • an immune response against the antigen can be induced in the subject, resulting in a protective antibody response level, which is relative to the current method of inducing an immune response by administering three times. , reducing the number of administrations, and shortening the whole immune time for inducing the immune response in the subject. This provides convenience and ensures subject compliance when rapid protection is required or subsequent doses cannot be guaranteed;
  • the CpG adjuvant provided by the present invention can reduce the antigen dosage, thereby reducing the potential safety hazard of viral antigens in production, and reducing the volume of the subunit recombinant vaccine antigen source, thereby expanding production capacity, reducing costs, and improving safety;
  • Figure 1 shows the effect of different ODNs on hepatitis B vaccine.
  • Figure 2 shows the effect of ODN 1 on rabies vaccine.
  • Figure 3 shows the effect of ODN 1 on the 2019-nCoV RBD antibody titer in the serum of mice immunized with the new coronary pneumonia recombinant subunit vaccine.
  • Figure 4 shows the effect of ODN 1 on the titer of SARS-CoV-2 S1 antibody in the serum of mice immunized with SARS-CoV-2 recombinant subunit vaccine.
  • Figure 5 shows the effect of ODN 1 on the titers of SARS-CoV-2 neutralizing antibodies in the serum of mice immunized with SARS-CoV-2 recombinant subunit vaccine.
  • Figure 6 shows the effect of different concentrations of ODN 2 on the titer of SARS-CoV-2 S1 antibody in the serum of mice immunized with SARS-CoV-2 inactivated vaccine.
  • Figure 7 shows the effect of ODN 2 on the titers of 2019-nCoV S1 antibody in the serum of mice immunized with 2019-nCoV inactivated vaccine containing different concentrations of antigens.
  • Figure 8 shows the specific titer of the new coronavirus S1 protein in the serum of mice after immunization with the new coronary pneumonia recombinant subunit vaccine containing different ODNs.
  • Fig. 9 is a graph of the experimental results of Example 7, indicating that there is a synergistic effect between ODN 1 and aluminum hydroxide adjuvant. Compared with the single aluminum adjuvant and the single ODN 1 adjuvant group, the double adjuvant group can significantly improve the Anti-gE protein titers.
  • Figure 10 is a graph of the experimental results of Example 8, including the relationship between the anti-A1 antigen antibody titers in the serum of each group and the number of immunization days (a), the relationship between the anti-A3 antigen antibody titers in the serum of each group and the number of immunization days (b), The relationship between the anti-Bv antigen antibody titers in the serum of each group and the days of immunization (c), and the relationship between the titers of anti-By antigen antibodies in the serum of each group and the days of immunization (d).
  • the corresponding ODN 1 adjuvant vaccine group can rapidly induce the production of protective antibodies; significantly improve the geometric mean of antibodies to the four antigens after immunization with quadrivalent split influenza vaccine titer.
  • Fig. 11 is the experimental result figure of embodiment 9, and the experiment is divided into five groups: rabies vaccine, rabies vaccine+ODN 3, 1/2 rabies vaccine+ODN 3, rabies vaccine+ODN 4, 1/2 rabies vaccine+ODN 4, The results showed that compared with the vaccine groups without ODN 3 or ODN 4, the corresponding ODN 3 or ODN 4 adjuvant vaccine groups could significantly increase the neutralizing antibody titers of rabies vaccine. In addition, the ODN 3 or ODN 4 adjuvant vaccine group could obtain an immune effect higher than that of the original fold antigen under the condition of 1/2 antigen.
  • Oligonucleotide is a molecule formed by the linking of a single nucleotide consisting of a sugar (such as deoxyribose or ribose), a phosphate group and a base, where the sugar molecule and the base are linked to form a nucleoside (nucleoside), nucleosides are connected by phosphate groups to form nucleotides, the bases forming nucleosides are pyrimidine and purine, pyrimidines are thymine (T or t for abbreviation) and cytosine (cytosine, abbreviated) C or c), purines are adenine (adenine, abbreviated as A or a) and guanine (guanine, abbreviated as G or g).
  • Oligonucleotides can be single-stranded or double-stranded.
  • "oligodeoxynucleotide” (Oligodeoxynucleotide, ODN) can be replaced by its English abbreviation ODN.
  • “Chemical modification” Compared with natural DNA, the oligonucleotides of the present invention can undergo various chemical modifications, and the modified sites can occur in phosphodiester bonds between nucleosides, ribose units or/and organic bases Base (A, T, C, G, uracil, uridine, abbreviated as U or u). Modifications can be made during or after synthesis of the oligonucleotide. Chemical modifications during synthesis can be done either internally or at the 5' end of the oligonucleotide. The synthesized oligonucleotides can be chemically modified, but not limited to, reactive groups (such as phosphates or hydroxyl groups at the 5' or 3' ends).
  • Chemical modifications in the present invention include backbone modifications of oligonucleotides. wherein the non-bridging phosphate oxygen atom in at least one internucleotide linkage is replaced by a sulfur atom.
  • the backbone of oligonucleotides can also be modified with non-ionic DNA analogs, such as alkyl, aryl-carbophosphate compounds (charged carbophosphate compound oxygen atoms are replaced by alkyl, aryl groups), such as phosphodiester and the oxygen atoms in alkyl phosphotriesters are modified by alkylation.
  • Oligonucleotides can also be chimeras of phosphorothioate and phosphodiester.
  • Chemical modifications also include base substitutions such as C-5 propynylpyrimidine and 7-deaza-7 instead of purine substitutions. Chemical modifications also include base modifications. Modified bases are chemically different from typical natural bases, but have their basic chemical structure.
  • the oligonucleotides of the present invention can also be modified with cytosine derivatives or thymidine derivatives. Cytosine derivatives here refer to cytosine-like nucleosides (other than cytosine). Thymidine derivatives refer to thymidine-like nucleosides (excluding thymine).
  • modification of the oligonucleotide of the present invention may be the attachment of a dihydric alcohol, such as tetraethylene glycol or hexaethylene glycol, to both or one end of the oligonucleotide.
  • a dihydric alcohol such as tetraethylene glycol or hexaethylene glycol
  • the present invention provides a CpG ODN comprising or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-4, wherein at least one nucleotide in the nucleotide sequence is a Chemically modified nucleotides of the structure shown in general formula I:
  • Y is S or O
  • R is H or a positively charged counterion
  • B is independently an unmodified or modified nucleobase
  • R 1 is H, F, Cl, OH, OMe, Me, O- Ethoxymethyl.
  • Y is S.
  • all nucleotides of the nucleotide sequence of the CpG ODN are chemically modified nucleotides having the structure shown in general formula I.
  • the sequence of the CpG ODN is selected from: SEQ ID NOs: 1-4, preferably all phosphorothioated SEQ ID NOs: 1-4.
  • the present invention provides a composition comprising a CpG ODN described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is a pharmaceutically acceptable carrier known in the art for use in compositions or vaccine compositions, such as phosphate buffered saline, etc.
  • Stabilizers for antigens such as one or more of maltose, human albumin, vitamin C, cysteine, urea, gelatin, etc.
  • the composition further comprises one or more other adjuvants that act in conjunction with the immunomodulatory CpG ODN, such as aluminum adjuvants such as aluminum hydroxide and insoluble aluminum salt colloids, Oil-water emulsions, microorganisms and metabolites, nucleic acids and their analogs, cytokines, immunostimulatory complexes, propolis, and liposomes, preferably aluminum adjuvants.
  • aluminum adjuvants such as aluminum hydroxide and insoluble aluminum salt colloids, Oil-water emulsions, microorganisms and metabolites, nucleic acids and their analogs, cytokines, immunostimulatory complexes, propolis, and liposomes, preferably aluminum adjuvants.
  • the present invention discloses the use of a CpG ODN described herein in the manufacture of an adjuvant.
  • the adjuvant is used for the preparation of vaccines, preferably for the preparation of hepatitis B vaccines, rabies vaccines, recombinant subunit vaccines for COVID-19, and inactivated vaccines for COVID-19.
  • the present invention provides a pharmaceutical composition comprising an antigen and a composition described herein.
  • the content of the antigen is 5-200 ⁇ g/mL, preferably 5-80 ⁇ g/mL.
  • the content of the CpG ODN is preferably 100-300 ⁇ g/mL.
  • the content of aluminum adjuvant is preferably 300-600 ⁇ g/mL.
  • the pharmaceutical composition is a hepatitis B vaccine, a rabies vaccine, a recombinant subunit vaccine for COVID-19, and an inactivated vaccine for COVID-19.
  • the present invention discloses the use of the pharmaceutical composition of the present invention in the preparation of a medicament for inducing an immune response against an antigen in a subject.
  • the use further comprises re-administering an effective dose of the pharmaceutical composition to the subject, wherein the re-administration interval is 2-24 weeks.
  • the present invention discloses a method for preparing the pharmaceutical composition described herein, which comprises adding the CpG ODN and other adjuvants, antigens and pharmaceutically acceptable carriers to a container in sequence according to the dosage ratio , adsorption, coupling and/or emulsification reaction occurs, and it is formulated into injection or oral preparation, nasal spray.
  • the other adjuvant is an aluminum adjuvant, such as aluminum hydroxide
  • the aluminum adjuvant is first mixed with the antigen for adsorption, and then mixed with CpG ODN for adsorption.
  • CpG C stands for cytosine, G stands for guanine, and p stands for phosphodiester bond.
  • CpGs are unmethylated dinucleotides composed of cytosine and guanine linked by phosphodiester bonds.
  • adjuvant is a substance that is injected into animals before or at the same time as the antigen, which can enhance the immunogenicity of the antigen. In animal vaccines, adjuvants can enhance their immune efficacy.
  • the aluminum adjuvant described in the present invention is a well-known and commonly used immunoadjuvant in the art, which can strongly adsorb protein antigens from solution to form a precipitate. When it is inoculated into the body, it can form an "antigen library", slowly release the antigen, and fully prolong the action time of the antigen. It also promotes local (injection site) macrophage responses.
  • the aluminium adjuvant of the present invention can be selected from: aluminium hydroxide, aluminium phosphate and aluminium sulfate.
  • the antigen mentioned in the present invention refers to a substance that can induce an immune response in the immune system of the body, and can combine with the product of the immune response (antibody and/or effector cell) in vivo or in vitro to generate a specific reaction, which may include Proteins, lipids, carbohydrates, nucleic acids, compounds, etc., wherein proteins that are antigens include proteins that are modified or unmodified, such as glycosylation or methylation, for example, the protein is cyclic or linked to lipids.
  • Antigens associated with an infectious agent or disease include antigens that are part of an infectious agent, eg, envelope proteins, capsid proteins, surface proteins, toxins, cell walls, antigenic lipids, and the like. Other suitable antigens may include host antigens, including those that are induced, modified or overexpressed as markers of infection or disease. All such antigens derived from or associated with an infectious agent, infection, disorder or disease are suitable for use in the present invention.
  • the antigen is an antigen related to a virus
  • the virus includes but is not limited to: hepatitis virus, rabies virus, new coronavirus pneumonia virus, and the like.
  • Vaccine the "vaccine” of the present invention is a vaccine well-known to those of ordinary skill in the art, and generally refers to all vaccines that can induce the body to produce specific antibodies and/or cellular immunity against specific pathogens after inoculation by injection or mucosal route, Thereby, the body can obtain biological products capable of protecting or eliminating the pathogen, including proteins, polysaccharides, nucleic acids, live vectors or infectious agents.
  • Vaccines are autoimmune preparations for the prevention of infectious diseases, which are prepared by artificially attenuating, inactivating or using genetic engineering and other methods of pathogenic microorganisms (such as bacteria, rickettsia, viruses, etc.) and their metabolites.
  • Vaccines retain the properties of pathogenic bacteria to stimulate the immune system of the animal body.
  • the immune system will produce certain protective substances, such as immune hormones, active physiological substances, special antibodies, etc.; when the animal is exposed to this pathogen again, the immune system of the animal will The system will follow its original memory and create more protective substances to prevent the damage of pathogenic bacteria.
  • hepatitis B vaccine (CpG ODN adjuvant) produced by Yunnan Watson Biotechnology Co., Ltd., vaccine specification: 0.5mL/bottle, containing hepatitis B virus surface antigen 10 ⁇ g, ODN 1 250 ⁇ g, The aluminum content is 0.45-0.60mg/mL (batch number: 20151201);
  • hepatitis B vaccine produced by Dalian Hanxin Bio-Pharmaceutical Co., Ltd., vaccine specification: 0.5mL/bottle, containing 10 ⁇ g of hepatitis B virus surface antigen, and aluminum content of 0.35-0.62mg/mL (batch number : 201505082).
  • Hansenula hepatitis B vaccine
  • CpG ODN adjuvant produced by Yunnan Watson Biotechnology Co., Ltd.
  • Hansenula hepatitis B vaccine
  • All groups were vaccinated according to the immunization schedule inoculated at weeks 0, 4 and 24, with one dose of 0.5 mL each.
  • the inoculation site was the skin at the attachment of the outer deltoid muscle of the upper arm, and the inoculation route was intramuscular injection.
  • the post-inoculation conditions were observed and recorded in different time periods, and the differences in the incidence of various adverse reactions between the two groups were compared.
  • Blood samples were collected from all subjects before the first dose of vaccination and 30 days after the second and third doses of vaccination, and the serum hepatitis B surface antibody (anti-HBs) was detected by chemiluminescence immunoassay.
  • the blood samples before the first dose of vaccination were checked for HBsAg and anti-HBc.
  • the blood samples were tested for anti-HBs concentration.
  • the antibody concentration was ⁇ 10.0mIU/mL as antibody positive, and ⁇ 1000.0mIU/mL was defined as strong antibody positive.
  • the positive rates of hepatitis B surface antibody (anti-HBs) were compared between the two groups. and geometric mean concentration (GMC).
  • Table 1 the results of antibody GMC after the whole course of immunization show that the antibody GMC of the test group in the full analysis data set (FAS set) is 2598.56 (95% CI: 1127.90-5986.90) mIU/mL, which is higher than that of the control group by 371.97 (164.54-840.91 ) mIU/mL; the protocol data set (PPS set) test group antibody GMC was 7808.21 (3377.00-18052.00) mIU/mL, higher than the control group [843.22 (95%CI: 213.80-3325.90) mIU/mL].
  • the CpG hepatitis B vaccine has better safety; moreover, 30 days after the 2nd and 3rd doses of immunization, the antibody GMC of the experimental group was significantly higher than that of the control group, which indicated that CpG adjuvant and aluminum
  • the adjuvanted double-adjuvanted hepatitis B vaccine (10 ⁇ g dose) showed better immunogenicity than the single aluminum adjuvant hepatitis B vaccine (10 ⁇ g dose), which was the most immunized commercially available; in addition, the second dose of immunization After the test group, the positive rate of 95% can be achieved, and the effect of 3 doses of the control vaccine can be achieved after 2 doses.
  • Hepatitis antibody production, rapid induction of protective antibodies has many advantages, especially when rapid protection is required or subsequent doses cannot be guaranteed, and in terms of vaccine cost and manpower, the implementation cost of a two-dose vaccine program will be less than Three doses of the vaccine.
  • ODN 1, ODN 2 and positive control ODN 5 (5'-TGACTGTGAACGTTCGAGATGA-3', wherein the nucleotides are fully phosphorothioated nucleotides), were provided by Jiangsu Taipu Rui Biotechnology Co., Ltd.;
  • hepatitis B vaccine (CHO cells), specification: 1.0mL, 20 ⁇ g/piece, purchased from North China Pharmaceutical Jintan Biotechnology Co., Ltd.;
  • mice (SPF grade) were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice with no history of administration were selected and divided into 5 groups. Each group was immunized once on D1 and D28 according to the designed immunization program.
  • the immunization route was the left anterior tibial muscle administration, each time 0.1 mL, mice were 5-6 weeks old at first dose.
  • blood samples were collected by cardiac puncture or orbital blood collection into blood collection tubes. The samples were centrifuged for about 30-60 minutes at room temperature, and all samples were centrifuged at 1800g at 4°C for 15 minutes to obtain serum. Anti-HBs antibody levels in serum samples were detected with a commercial HBV surface antibody detection kit.
  • the antibody titers produced in the 1/2 antigen group were significantly higher than those in the non-ODN group (group 2); 4 weeks after 1 immunization , 6 weeks, and 8 weeks, the antibody titer level continued to increase, and the 8th week was the highest antibody titer level in this experiment.
  • the antibody titers of the mice in groups 4 and 5 increased at 8 weeks. Strong growth, indicating that it can maintain antibody concentration for a long time.
  • Vero cell rabies vaccine the specification is 1mL/vial (including 2.5IU), collected from Changchun Institute of Biological Products;
  • mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice with a body weight of 18-22 g and no history of drug administration were selected and divided into 8 groups, with 8 mice in each group, half male and half male.
  • the specific groups are: rabies vaccine, rabies vaccine + aluminum hydroxide, rabies vaccine + 1.25 ⁇ g ODN 1, rabies vaccine + 5 ⁇ g ODN 1, rabies vaccine + 20 ⁇ g ODN 1, rabies vaccine + aluminum hydroxide + 1.25 ⁇ g ODN 1, rabies vaccine +aluminum hydroxide+5 ⁇ g ODN 1, rabies vaccine+aluminum hydroxide+20 ⁇ g ODN 1, wherein the aluminum hydroxide concentration is 0.45mg/mL (calculated by aluminum content).
  • mice On day 0, day 7, and day 21, mice were immunized according to different groups. The immunization method was intraperitoneal injection of mice, each injection of 0.5 mL. Blood was collected from the tail vein of the mice on the 4th, 10th and 31st days, and the serum was separated. The rabies vaccine antibody titer in the serum of the mice was detected by the rapid rabies vaccine fluorescence foci inhibition test (RFFIT). Blood was drawn from the tail vein of mice two days before immunization, and the obtained serum was used as a negative control.
  • RFFIT rapid rabies vaccine fluorescence foci inhibition test
  • rabies vaccine + aluminum hydroxide rabies vaccine + 1.25 ⁇ g ODN 1, rabies vaccine + aluminum hydroxide + 1.25 ⁇ g ODN 1, rabies vaccine + 5 ⁇ g ODN 1, rabies vaccine + aluminum hydroxide + 5 ⁇ g
  • the rabies vaccine antibody titers in the serum of mice in each group of ODN 1 were 72.13, 35.79, 186.28, 63.1, and 197.63 IU, respectively, which indicated that there was a synergistic effect between ODN 1 and aluminum hydroxide adjuvant, and it had a synergistic effect with single aluminum adjuvant and Compared with the single ODN 1 adjuvant group, the double adjuvant group could significantly increase the titer of rabies vaccine antibodies.
  • ODN 1 (Jiangsu Taipu Rui Biotechnology Co., Ltd.), specification: 8.3mg/mL, batch: 202010001;
  • mice SPF grade
  • Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice with no history of drug administration and divide them into 10 groups, group 1 is the PBS vehicle control group, and groups 2-10 are the experimental group; each group is immunized according to the designed immunization program on D0 and D28 days
  • the mice were immunized by intraperitoneal injection, 0.2 mL each time, and the mice were 5-6 weeks old at the time of the first administration.
  • blood samples were collected by cardiac puncture or orbital blood collection on D7, D14, D21, D28, D56, and D84. The samples were centrifuged for about 30-60 minutes at room temperature, and all samples were centrifuged at 1800g at 4°C for 15 minutes to obtain serum. S1 antibody and RBD antibody levels in serum samples were detected by sandwich ELISA.
  • the RBD antibody GMT of mice in group 3, group 7 and group 5 were 50, 50 and 1270 on the 14th day, respectively; the RBD antibody GMT on the 21st day were 200, 50, and 10159, respectively; the RBD antibody GMTs on day 28 were 79, 50, and 26390, respectively; the RBD antibody GMTs on day 84 were 270, 52, and 51962, respectively, indicating that the double-adjuvant vaccine group with RBD as the antigen Compared with the corresponding single-adjuvant vaccine group, the RBD antibody GMT can be significantly improved, which proves that ODN 1 has a synergistic effect with aluminum hydroxide.
  • mice in groups 8 and 9 had RBD antibody GMTs of 141 and 800 on day 7, 2425 and 6400 on day 14, and 5702 and 45948 on day 21, respectively.
  • the RBD antibody GMTs on day 28 were 4525 and 25600, respectively; the RBD antibody GMTs on day 84 were 90000 and 467654, respectively, which indicated that the double-adjuvant vaccine group with S protein as the antigen was comparable to the corresponding single-Al adjuvant vaccine group.
  • RBD antibody GMT can be significantly improved.
  • the GMT of the S1 antibody of the mice in groups 8 and 9 on the 7th day was 50 and 400, respectively; the GMT of the S1 antibody on the 14th day was 3200 and 7184, respectively; the GMT of the S1 antibody on the 21st day were 6400 and 52780, respectively; the S1 antibody GMTs on the 28th day were 12800 and 81275, respectively; the S1 antibody GMTs on the 84th day were 224825 and 810000, respectively, indicating that the double-adjuvant vaccine group with the S protein as the antigen was significantly different from the corresponding single Al Compared with the adjuvant vaccine group, the S1 antibody GMT can be significantly improved.
  • the neutralizing antibody titers of the mice in groups 8 and 9 were 40 and 240 on the 21st day, respectively; the neutralizing antibody titers on the 28th day were 20 and 360, respectively; on the 84th day
  • the neutralizing antibody titers were 3840 and 5120, respectively, which indicated that the double adjuvant vaccine group with S protein as the antigen could significantly increase the neutralizing antibody titer compared with the corresponding single Al adjuvant vaccine group.
  • CpG adjuvant and aluminum adjuvant have synergistic effect, can significantly increase vaccine RBD antibody titer, S1 antibody titer and neutralizing antibody titer, and the protective effect is lasting; 5-100 ⁇ g/mL antigen and CpG adjuvant
  • the combination of CpG adjuvant (100 ⁇ g/mL) and aluminum hydroxide (Al content of 0.45 mg/mL) produced a better effect; CpG adjuvant induced S1 antibody in the group whose antigen was S full-length protein than the group whose antigen was RBD.
  • the increase in titer and RBD antibody titer was more pronounced.
  • Antigens of inactivated vaccines against COVID-19 provided by Zhejiang Tianyuan Bio-Pharmaceutical Co., Ltd.;
  • Aluminum hydroxide adjuvant provided by Zhejiang Tianyuan Bio-Pharmaceutical Co., Ltd.;
  • mice purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice 18-20g BALB/c mice were selected, 9 or 10 mice in each group, half male and half male; each group was immunized by intraperitoneal injection on D0 and D14 according to the designed immunization program, with 0.5 mL each time. .
  • blood was collected at D0, D6, D13, D21 and D28 to separate serum, and all sera were tested for S1 protein-specific IgG and virus-neutralizing antibodies; the geometric mean of serum IgG and virus-neutralizing antibodies of each group of animals was calculated according to statistics potency. The measurement results are shown in Figure 6-7.
  • SARA-CoV-2 S protein (Acro), the specification is 500 ⁇ g/support, batch: C591P1-20B2F1-UF;
  • ODN 2, ODN 3, ODN 4 and the positive control ODN 5 were provided by Jiangsu Taipu Rui Biotechnology Co., Ltd.;
  • mice purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice with no drug history were selected and divided into 11 groups with about 30 animals in each group.
  • D0 and D28 the mice were immunized by intraperitoneal injection, 0.2 mL each time, and the mice were 5-6 weeks old at the time of the first administration.
  • blood samples were collected by cardiac puncture or orbital blood collection at D7, D14, D28, D56, D84 after administration. The samples were centrifuged for about 30-60 minutes at room temperature, and all samples were centrifuged at 1800g at 4°C for 15 minutes to obtain serum. S1 antibody levels in serum samples were detected by sandwich ELISA. The results are shown in Figure 8.
  • the results show that the S1 antibody GMT of mice in groups 3 to 11 on day 7 were 111, 80, 700, 73, 550, 30, 317, 70, and 467, respectively; on day 28
  • the S1 antibody GMTs were 1120, 2133, 50667, 1813, 48316, 960, 29333, 1600, and 40333, respectively;
  • the S1 antibody GMTs on day 84 were 28223, 34666, 709338, 22667, 641229, 10000, 373330, 16000, and 605329, respectively.
  • the double adjuvant group can significantly increase the level of antibody titers, and the protective effect is durable.
  • Example 7 The effect of ODN 1 on herpes zoster vaccine
  • PBS phosphate buffered saline dry powder purchased from Solarbio
  • Aluminum hydroxide adjuvant purchased from Thermo;
  • Herpes zoster virus gE protein purchased from SinoBiological;
  • mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice with a body weight of 18-22 g and no history of drug administration were selected and divided into 4 groups, with 8 mice in each group, half male and half male.
  • the specific groups were: 10 ⁇ g gE protein group, 10 ⁇ g gE protein+40 ⁇ g aluminum hydroxide, 10 ⁇ g gE protein+5 ⁇ g ODN 1 group, 10 ⁇ g gE protein+40 ⁇ g aluminum hydroxide+5 ⁇ g ODN 1 group, where aluminum hydroxide was calculated as aluminum content.
  • mice in each group were immunized by intraperitoneal injection on D0, D28, and D56, and each injection was 0.1 mL.
  • blood was collected from the tail vein of mice at D0, D7, D14, D21, D28, D35, D42, D49, D56, D63, D70, D77, the serum was separated, and the anti-gE protein droplets in the mouse serum were detected by ELISA.
  • the serum obtained 7 days before immunization was used as a negative control.
  • the protein titers were 190207, 1116680, 430539, and 4870992, respectively, which indicated that there was a synergistic effect between ODN 1 and aluminum hydroxide adjuvant.
  • the double adjuvant group Can significantly increase the anti-gE protein titer.
  • PBS phosphate buffered saline dry powder purchased from Solarbio
  • Influenza A1 antigen purchased from SinoBiological;
  • Influenza A3 antigen purchased from SinoBiological;
  • influenza antigen purchased from SinoBiological;
  • Influenza Bv antigen purchased from SinoBiological;
  • mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • Groups 1 to 9 are PBS group, tetravalent split seedling group (QIV), and quadrivalent split seedling + CpG group (QIV + CpG group).
  • influenza antigens A1, A3, By, Bv
  • ODN 1 ODN 1
  • the 0.1 mL solution used to immunize each mouse in the QIV group contained 3.5 ⁇ g each of the 4 antigens; the 0.1 mL solution used to immunize each mouse in the QIV+CpG group contained 3.5 ⁇ g each of the 4 antigens and 5 ⁇ g ODN 1;
  • the 0.1 mL solution of each mouse in the immunized 1/2QIV group contained 1.75 ⁇ g of each of the four antigens;
  • the 0.1 mL of the solution used to immunize each mouse in the 1/2QIV+CpG group contained 1.75 ⁇ g of each of the four antigens and 5 ⁇ g of ODN 1;
  • the 0.1 mL solution used to immunize each mouse in the 1/4QIV group contained 0.875 ⁇ g of each of the 4 antigens;
  • the 0.1 mL solution used to immunize each mouse in the 1/4QIV+CpG group contained 0.875 ⁇ g of each of the 4 antigens and 5 ⁇ g of ODN
  • mice in each experimental group were collected on D4, D5, D6, D7, D14, D21, D28, and D49, and mice in the control group (group 2 to 9) were collected on D7 and D28. 1 group) for blood collection.
  • mice in the control group were collected on D7 and D28. 1 group for blood collection.
  • Five mice were collected from each blood collection point in each group.
  • the orbital venous plexus was used for blood collection in groups 2-9 on D4, D5, D6 and D7, and the eyeballs were collected for blood collection on D14, D21, D28 and D49. Blood was collected from the eyeballs, and the animals died after planned bloodletting. The whole blood was placed at room temperature for about 30-60 minutes and then centrifuged.
  • the geometric mean titers of the four antibodies in mouse serum were detected by ELISA, and 10 mice were taken one day before immunization.
  • the pre-immune sera were collected from mice by enucleation of the eyeballs as a negative control.
  • the corresponding ODN 1 adjuvant vaccine group can rapidly induce protective antibodies;
  • the immune effect of the antigen is not lower than that of the original antigen.
  • ODN 1 adjuvant can rapidly induce the production of protective antibodies; significantly increase the antibody titer of quadrivalent influenza vaccine; reduce the amount of antigen and improve the safety of the vaccine.
  • ODN 3 and ODN 4 are provided by Jiangsu Taipu Rui Biotechnology Co., Ltd.;
  • Vero cell rabies vaccine the specification is 1mL/vial (including 2.5IU), collected from Changchun Institute of Biological Products;
  • mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • mice with a body weight of 18-22 g and no history of drug administration were selected and divided into 5 groups, with 8 mice in each group, half male and half male.
  • Groups 1-5 were: rabies vaccine, rabies vaccine + ODN 3, 1/2 rabies vaccine + ODN 3, rabies vaccine + ODN 4, 1/2 rabies vaccine + ODN 4.
  • the above-mentioned Vero cell rabies vaccine, ODN 3 and ODN 4 were diluted and dissolved in PBS in proportion, wherein the 1st, 2nd, and 4th groups of Vero cell rabies vaccine were diluted 100 times; in immunized mice. According to the designed procedure, the mice in each group were immunized once on the 0th, 3rd and 7th days respectively.
  • the immunization method was intramuscular injection in the hind legs of mice, the injection volume was 0.1 mL, and the content of ODN 3 or ODN 4 in the 0.1 mL solution used to immunize mice in groups 2-5 was 5 ⁇ g.
  • rabies vaccine, rabies vaccine + ODN 3, 1/2 rabies vaccine + ODN 3, rabies vaccine + ODN 4, 1/2 rabies vaccine + ODN 4 anti-rabies virus in the serum of each group of mice and antibody titers were 15.19, 38.33, 19.63, 34.07, and 29.83, respectively, which indicated that compared with the vaccine group without ODN 3 or ODN 4, the corresponding ODN 3 or ODN 4 adjuvant vaccine groups could significantly improve the rabies vaccine.
  • the ODN 3 or ODN 4 adjuvant vaccine group could obtain an immune effect higher than that of the original fold antigen under the condition of 1/2 antigen.

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Abstract

一种组合物，其包含CpG ODN和一种或多种与所述免疫调节性CpG ODN共同发挥作用的其他佐剂，例如铝佐剂，所述CpG ODN包含或组成为选自SEQ ID NO:1-4的核苷酸序列，其中所述核苷酸序列中至少一个核苷酸为具有通式(I)所示结构的化学修饰的核苷酸：其中，Y为S或O，R为H或带正电的抗衡离子，B独立地为未修饰或经修饰的核碱基，R 1为H、F、Cl、OH、OMe、Me、O-乙基氧甲基。组合物的用途，所述包含所述组合物的药物组合物，其制备方法，以及所述药物组合物的用途。

Description

人工合成的含CpG单链脱氧寡核苷酸在疫苗中的应用 技术领域
本发明涉及系列的人工合成的含CpG单链脱氧寡核苷酸在疫苗中的应用。进一步而言，本发明涉及所述人工合成的含CpG单链脱氧寡核苷酸作为疫苗佐剂的应用。进一步而言，本发明涉及所述人工合成的含CpG单链脱氧寡核苷酸在基因工程疫苗、亚单位疫苗及灭活疫苗中的应用。
背景技术
疫苗是一种抗原，注入血液后刺激免疫系统合成抗体。疫苗的应用对于控制人类和动物疾病的流行起到了非常重要的作用。按疫苗的制备原理，疫苗可分为灭活疫苗、减毒疫苗、亚单位疫苗和基因工程疫苗。基因工程疫苗主要包括基因工程亚单位疫苗、基因工程载体疫苗、核酸疫苗、基因缺失活疫苗、蛋白工程疫苗等等。广义的疫苗还包括遗传重组疫苗、合成肽疫苗、抗独特型抗体疫苗以及微胶囊可控缓释疫苗等。
重组疫苗(recombinant vaccines)是指通过遗传学重组机制来生产的疫苗。为了解决传统疫苗存在的问题，降低免疫原性，提高安全性，减少治疗时间，以一种新型的SIT方法———免疫重组疫苗。包括三种：一是DNA重组疫苗，以这一方式面世的第一种疫苗是乙型肝炎疫苗。该疫苗对乙型肝炎表面抗原HBsAg进行克隆扩增，应用重组DNA技术从酵母菌生产疫苗。二是通过消除和修饰病原微生物上已知的导致致病性基因来制备疫苗。以此方法研制的针对轮状病毒的第一代重组疫苗已在美国和芬兰进行临床试验，研究结果提示该疫苗对由轮状病毒所致的儿童腹泻具有很强的保护性。三是通过在一个非致病性微生物如病毒体内插入病原微生物的某个基因，然后被修饰的病毒作为一个携带者或载体来表达该外来基因，从而诱导免疫反应。这一技术正被应用于HIV疫苗的研制。同样也被用于新冠病毒疫苗的研制。
如今合成肽疫苗、基因工程亚单位疫苗、抗独特型抗体疫苗以及核酸疫苗等新型疫苗虽具有良好的抗原性和低毒等优点，但其免疫原性较弱，有必要配合高效的佐剂使用。这些佐剂包括抗原微粒化佐剂(不溶性铝盐胶体、免疫刺激复合物、脂质体)、缓慢释放抗原佐剂(油乳佐剂、抗原的微型包囊)、微生物佐剂(细菌毒素、短小棒状杆菌菌苗、分枝杆菌及其成分、肽聚糖)、分子佐剂(细胞因子、C3d分子、共刺激分子、超抗原、热休克蛋白、CpG序列)，以及其他佐剂(维生素E和硒、抗生素类、拟胸腺素药物、蜂胶、中药多糖)。这些佐剂主要通过免疫调节、参与抗原递呈、诱导CD8 +T细胞应答、抗原贮存等方式发挥作用。
人工合成的含有胞嘧啶-鸟嘌呤二核苷酸基序的寡脱氧核苷酸(CpG oligodeoxynucleotide，CpG ODN)能刺激高等动物的天然免疫系统，活化B细胞、巨噬细胞
Figure PCTCN2022090392-appb-000001
树突状细胞(DC)及NK细胞等免疫细胞，并诱导IL-2、IL-6、IL-12、IFN-γ等细胞因子的分泌。CpG ODN作为疫苗佐剂，在多种疾病的治疗性疫苗中诱导所需的强Th1型免疫应答，已在人和小鼠上得到很好的验证。动物中的研究已证明仅由CpG ODN建立的免疫防御或作为疫苗佐剂的免疫防御可防御多种病毒性疾病、细菌性疾病和寄生性疾病。CpG ODN作为疫苗佐剂可以单独使用，有研究报道CpG DNA可作为用重组乙型肝炎表面抗原免疫小鼠的特异性免疫的有效增强剂(Heather L.Davis,etc.,J Immunol 1998；160:870-876；)；含CpG单链脱氧核苷酸作为狂犬疫苗的佐剂，能够显著增强狂犬疫苗的免疫效果，且能够减少狂犬疫苗的注射次数(CN1781929A)；Dynavax公司研制的CpG ODN 1018 ISS增强型乙型肝炎病毒疫苗(HEPLISAV)的Ⅲ期临床试验结果表明，HEPLISAV组的受试者与葛兰素史克公司的乙肝疫苗Engerix-B组产生了水平相当的保护性抗体(赵玉娇，等，中国生物制品学杂志2016年7月第29卷第7期)。此外，CpG ODN作为疫苗佐剂还可以与其他佐剂联合使用，有临床试验研究报道免疫刺激TLR9激动剂寡脱氧核苷酸CPG 7909作为Engerix-B的佐剂增强了疫苗免疫原性(C.L.COOPER,etc.,Journal of Clinical Immunology,Vol.24,No.6, November 2004)；佐剂CPG 7909添加到血液阶段疟疾候选疫苗AMA1-C1/Alhydrogel中后，抗体反应显著增强(Vaccine.2009 June 24；27(31):4104-4109.)。这些研究发现CpG-ODNs单独使用或与其他佐剂(如氢氧化铝、ISA-51、MF59、ISCOMATRIX和IL-2等)联合均能够显著增强抗原特异性体液免疫和细胞免疫应答。CpG-ODNs不但能够增强抗原特异性免疫应答的“量”，还能改变抗原特异性免疫应答的“质”。就体液免疫而言，CpG-ODNs不但能够加速抗体产生、增加抗体滴度、提高抗体亲合力和增强免疫持久性，还能够改变抗体亚型(Chinese Journal of New Drugs 2014,23(1))。
冠状病毒病2019(COVID-19)是一种正在全球蔓延的新发传染病。WHO官网显示，截止2020年12月15日，已有71,581,532例新冠肺炎确诊病例，1,618,374例死亡。它是由一种新型冠状病毒(严重急性呼吸综合征冠状病毒2(SARS-CoV-2))引起的。SARS-CoV-2的刺突(S)蛋白，由S1和S2两个亚基组成，在受体识别和细胞膜融合过程中起关键作用。S1亚基包含识别并结合宿主受体血管紧张素转换酶2的受体结合域，而S2亚基通过两个七肽重复结构域形成六螺旋束来介导病毒细胞膜融合。S蛋白在病毒感染中的基础性作用表明它是疫苗开发，抗体阻断疗法和小分子抑制剂的潜在靶标。以S蛋白为靶点的潜在药物包括基于SARS-CoV-2 S蛋白的抗体、融合抑制剂、靶向SARS-CoV-2 S裂解位点的蛋白酶抑制剂[Letko M,Marzi A,Munster V.Functional assessment of cell entry and receptor usage for SARSCoV-2 and other lineage B betacoronaviruses.Nat Microbiol.2020；5:562–9.]。
佐剂技术在新冠疫苗的研发中得到了进一步证实与应用。佐剂通过增强免疫应答，提高免疫强度，客观上减少了单位疫苗所需的抗原量，从而达到增加疫苗产能，扩大被免疫人群的作用，在人类与病毒的战役中将发挥不可估量的作用。迄今为止，至少有10家药品开发商表示计划开发针对新冠肺炎的佐剂疫苗，包括英国葛兰素史克(GSK)公司，澳大利亚Seqirus公司和美国Dynavax公司等企业，上述企业已承诺研发获批的佐剂(分别为AS03，MF59和CpG 1018)，提供给其他研发新冠肺炎疫苗的人员。目前有多个佐剂型疫 苗处于临床试验阶段，例如Novavax公司研发的含有Matrix-M佐剂的全长重组SARS CoV-2糖蛋白纳米颗粒疫苗，安徽智飞龙科马生物制药有限公司/中国科学院微生物研究所共同研发的含有佐剂的CHO细胞表达的重组蛋白(RBD二聚体)疫苗，Medicago公司研发的含有AS03佐剂的植物源VLP疫苗，大阪大学/AnGes/Takara Bio共同研发的含有佐剂的DNA质粒疫苗，Biological E Ltd研发的含有佐剂的蛋白亚单位(RBD)疫苗，Vaxart公司研发的5型腺病毒(Ad5)佐剂型口服疫苗平台，Vaxine Pty Ltd/Medytox共同研发的含有Advax佐剂的重组刺突蛋白疫苗，昆士兰大学/CSL/Seqirus共同研发的含有MF59佐剂的分子钳稳定的刺突蛋白疫苗，Medigen Vaccine Biologics Corporation/NIAID/Dynavax共同研发的的S-2P蛋白+CpG 1018疫苗，古巴Instituto Finlay de Vacuns研发的RBD+佐剂疫苗。有专利文件公开了加佐剂的疫苗，如CN 111956797 A公开了新型疫苗佐剂SF(化学修饰的环二核苷酸)在新冠肺炎疫苗中的应用，CN 111991556 A公开了加Sigma Adjuvant Systerm和/或AddaVax的SARS-CoV-2 RBD共轭纳米颗粒疫苗，CN 111603556 A公开了含有CpG寡脱氧核苷酸的新型冠状病毒亚单位纳米疫苗。
辉瑞-BioNTech COVID-19疫苗的III期数据显示，与安慰剂相比，疫苗组淋巴结病的发生率更高。疫苗组64人，安慰剂组6人，由于局部淋巴结参与疫苗反应，因此这可能与疫苗有关。另外，贝尔面瘫在疫苗接种者之间也存在失衡。疫苗组4人，安慰剂组0人，因果关系未知。由此可见，疫苗的安全性问题需要进一步研究解决。
发明内容
本发明提供了具有免疫调节功能的CpG ODN。这些CpG ODN的结构新颖，更为难得的是对小鼠和人都有免疫刺激作用并且与铝佐剂联合使用时具有协同作用，因此在临床上有很大的应用价值。具体而言，本发明通过以下技术方案解决了本领域中存在的问题：
1.一种组合物，其包含CpG ODN和一种或多种与所述免疫调节性CpG ODN共同发挥作用的其他佐剂，例如铝佐剂，如氢氧化铝和 不溶性铝盐类胶体、油水乳剂、微生物及代谢产物、核酸及其类似物、细胞因子、免疫刺激复合物、蜂胶、和脂质体，优选铝佐剂，其中所述CpG ODN包含或组成为选自SEQ ID NO:1-4的核苷酸序列，其中所述核苷酸序列中至少一个核苷酸为具有通式I所示结构的化学修饰的核苷酸：
Figure PCTCN2022090392-appb-000002
其中，Y为S或O，R为H或带正电的抗衡离子，B独立地为未修饰或经修饰的核碱基，R 1为H、F、Cl、OH、OMe、Me、O-乙基氧甲基。
2.项目1所述的组合物，其中Y为S。
3.项目1或2所述的组合物，其中所述CpG ODN的核苷酸序列的全部核苷酸均为具有通式I所示的结构的化学修饰的核苷酸。
4.项目3所述的组合物，其中所述CpG ODN的序列选自：SEQ ID NO:1-4，优选地，全部硫代磷酸酯化的SEQ ID NO:1-4。
5.项目1-4任一项所述的组合物，其中所述CpG ODN的含量为10-500μg/mL，其中铝佐剂中铝的含量为300-600μg/mL。
6.项目1-5任一项所述的组合物在制备疫苗中的用途。
7.项目6所述的用途，其中所述疫苗为乙型肝炎疫苗、狂犬病疫苗、新冠肺炎疫苗，例如抗新冠病毒Alpha毒株、Beta毒株、Gamma毒株、Delta毒株、Omicron毒株的新冠肺炎疫苗，包括但不限于新冠肺炎重组亚单位疫苗和新冠肺炎灭活疫苗、带状疱疹疫苗、流感疫苗，如四价流感疫苗。
8.一种药物组合物，其包含抗原和项目1-5任一项的组合物。
9.项目8所述的药物组合物，其中所述抗原的含量为5-200μg/mL，优选5-80μg/mL。
10项目8-9任一项所述的药物组合物，其中所述的CpG ODN含量为100-300μg/mL。
11.项目8-9任一项所述的药物组合物，其中所述铝佐剂中铝含量为300-600μg/mL。
12.项目8-11任一项所述的药物组合物，其为乙型肝炎疫苗、狂犬疫苗、新冠肺炎重组亚单位疫苗、新冠肺炎灭活疫苗、带状疱疹疫苗、流感疫苗，如四价流感疫苗。
13.项目8-12任一项所述的药物组合物在制备用于诱导受试者体内针对抗原的免疫反应的药物中的应用。
14.如项目13所述的应用，其中所述药物组合物用于以有效量施用给受试者，优选所述药物组合物用于给药于受试者两次，其中两次给药的时间间隔为2-24周。
15.制备项目8-12任一项所述的药物组合物的方法，其包括将项目1-5任一项的组合物、抗原和任选地药学上可接受的载体混合，发生吸附、偶联和/或乳化反应，并配制为注射剂或口服制剂、喷鼻剂。
本发明所用的CpG ODN包括ODN 1-ODN 4，它们分别具有如下核苷酸序列：
ODN 1(5'-tcgacgttcgtcgttcgtcgttc-3')；
ODN 2(5'-tcgcgacgttcgccgacgttcgta-3')；
ODN 3(5'-tcgtcgacgtcgttcgttctc-3')；
ODN 4(5'-tcgcgaacgttcgccgcgtacgtacgcgg-3')。
其中ODN 1-ODN 4的核苷酸均为硫代磷酸酯化的核苷酸。
本发明具有如下的有益技术效果：
(1)本发明提供的CpG佐剂与铝佐剂联用时产生了协同增效作用，双佐剂疫苗比单一佐剂疫苗显示出更好的免疫原性；
(2)本发明提供的CpG佐剂与铝佐剂联用时，可以快速诱导产生保护性抗体，能减少免疫次数。所述方法包括：将有效量的本发明的 药物组合物给药于所述受试者。所述方法还可包括将所述药物组合物以有效量施用给受试者，优选所述药物组合物给药于受试者两次，其中两次给药的时间间隔为2-24周。通过向受试者给药两次本发明的药物组合物即可诱导受试者体内针对抗原的免疫反应，产生保护性抗体应答水平，这相对于目前给药三次来诱导免疫反应的方法而言，减少了给药次数，缩短了诱导受试者体内的免疫反应的全程免疫时间。这在需要快速保护或后续剂次无法保证的情况下，提供了方便，保证了受试者依从性；
(3)本发明提供的CpG佐剂能降低抗原用量，从而减少生产中病毒性抗原的安全隐患，降低亚单位重组疫苗抗原来源的体量，从而扩大产能、降低成本、提升安全性；
(4)本发明提供的CpG佐剂与铝佐剂联用时，可以产生强而持久的保护作用。
附图说明
图1表示不同ODN对乙型肝炎疫苗的作用。
图2表示ODN 1对狂犬病疫苗的作用。
图3表示ODN 1对新冠肺炎重组亚单位疫苗免疫小鼠血清中新冠病毒RBD抗体滴度的影响。
图4表示ODN 1对新冠肺炎重组亚单位疫苗免疫小鼠血清中新冠病毒S1抗体滴度的影响。
图5表示ODN 1对新冠肺炎重组亚单位疫苗免疫小鼠血清中新冠病毒中和抗体滴度的影响。
图6表示不同浓度ODN 2对新冠肺炎灭活疫苗免疫小鼠血清中新冠病毒S1抗体滴度的影响。
图7表示ODN 2对含有不同浓度抗原的新冠肺炎灭活疫苗免疫小鼠血清中新冠病毒S1抗体滴度的影响。
图8表示含不同ODN的新冠肺炎重组亚单位疫苗免疫后小鼠血清中新冠病毒S1蛋白特异性效价。
图9为实施例7的实验结果图，表明ODN 1与氢氧化铝佐剂之间存在协同增效作用，与单铝佐剂及单ODN 1佐剂组相比，双佐剂组能显著提高抗gE蛋白滴度。
图10为实施例8的实验结果图，包括各组血清中抗A1抗原抗体滴度与免疫天数关系图(a)、各组血清中抗A3抗原抗体滴度与免疫天数关系图(b)、各组血清中抗Bv抗原抗体滴度与免疫天数关系图(c)、各组血清中抗By抗原抗体滴度与免疫天数关系图(d)。从图10可以看出，与无ODN 1佐剂疫苗组相比，相应的ODN 1佐剂疫苗组可以快速诱导产生保护性抗体；显著提高四价裂解流感疫苗免疫后4种抗原的抗体几何平均滴度。
图11为实施例9的实验结果图，实验分为五组：狂犬疫苗、狂犬疫苗+ODN 3、1/2狂犬疫苗+ODN 3、狂犬疫苗+ODN 4，1/2狂犬疫苗+ODN 4，结果表明与不加ODN 3或ODN 4的疫苗组相比，相应的ODN 3或ODN 4佐剂疫苗组均能显著提高狂犬疫苗中和抗体滴度。此外，ODN 3或ODN 4佐剂疫苗组可以在1/2抗原条件下获得高于原倍抗原的免疫效果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
“寡核苷酸”：寡核苷酸是由糖(如脱氧核糖或核糖)，磷酸基团和碱基组成的单核苷酸连接而形成的分子，其中糖分子和碱基连接成核苷(nucleoside)，核苷由磷酸基团连接形成核苷酸(nucleotide)，形成核苷的碱基有嘧啶和嘌呤，嘧啶有胸腺嘧啶(thymine，缩写为T或t)和胞嘧啶(cytosine，缩写为C或c)，嘌呤有腺嘌呤(adenine，缩写为A或a)和鸟嘌呤(guanine，缩写为G或g)。寡核苷酸可以是单链的也可以是双链的。在本发明中，“寡脱氧核苷酸”(Oligodeoxynucleotide，ODN)可以用它的英文缩写ODN代替。
“化学修饰”：与自然的DNA相比，本发明中的寡核苷酸可以经各种化学修饰，修饰的部位可发生在核苷之间的磷酸二酯键，核糖单 位或/和有机碱基(A、T、C、G，尿嘧啶，uridine，缩写为U或u)。在寡核苷酸的合成期间或合成后都可以进行修饰。在合成期间的化学修饰可以在寡核苷酸的内部或5'端进行修饰。合成后的寡核苷酸可以但不限于在活性基团(如5'或3'端的磷酸或羟基)进行化学修饰。专业人员可以了解这些化学修饰具体方式。本发明中的化学修饰包括寡核苷酸的骨架修饰。其中，至少一个核苷酸间键合中的非桥性磷酸氧原子被硫原子取代。寡核苷酸的骨架也可发生非离子DNA类似物，如烷基、芳香-碳磷酸盐化合物(带电荷的碳磷酸盐化合物氧原子被烷基、芳香基取代)修饰，再如磷酸二酯和烷基磷酸三酯中的氧原子部分被烷基化修饰。寡核苷酸还可以是磷硫酰和磷酸二酯的嵌合体。化学修饰还包括碱基取代，如C-5丙炔嘧啶和7-脱氮杂-7替代嘌呤取代。化学修饰还包括碱基修饰。修饰的碱基在化学上不同于典型的自然中的碱基，但具有它们基本的化学结构。本发明中的寡核苷酸还可以用胞嘧啶衍生物或胸腺嘧啶核苷衍生物修饰。这里胞嘧啶衍生物是指胞嘧啶样核苷(除胞嘧啶外)。胸腺嘧啶核苷衍生物是指胸腺嘧样核苷(不包括胸腺嘧啶)。另外，本发明中的寡核苷酸的修饰可以是在寡核苷酸的两个或一个末端连接一个二元醇，如四乙二醇或六乙二醇。
在一个实施方案中，本发明提供了一种CpG ODN，其包含或组成为选自SEQ ID NO:1-4的核苷酸序列，其中所述核苷酸序列中至少一个核苷酸为具有通式I所示结构的化学修饰的核苷酸：
Figure PCTCN2022090392-appb-000003
其中，Y为S或O，R为H或带正电的抗衡离子，B独立地为未修饰或经修饰的核碱基，R 1为H、F、Cl、OH、OMe、Me、O-乙基氧甲基。
在另一个实施方案中，Y为S。在另一个实施方案中，所述CpG ODN的核苷酸序列的全部核苷酸均为具有通式I所示的结构的化学修饰的核苷酸。在另一个实施方案中，CpG ODN的序列选自：SEQ ID NO:1-4，优选地，全部硫代磷酸酯化的SEQ ID NO:1-4。
在一个实施方案中，本发明提供了一种组合物，其包含本文所述的CpG ODN和药学上可接受的载体。药学上可接受的载体是本领域中已知的用于组合物或疫苗组合物中的药学上可接受的载体，如磷酸缓冲液等，所述药学上可接受的载体还可以包含用于稳定抗原的稳定剂，如麦芽糖，人血白蛋白，维生素C，半胱氨酸，尿素，明胶等的一种或多种。
在一个实施方案中，所述的组合物还包含一种或多种与所述免疫调节性CpG ODN共同发挥作用的其他佐剂，例如铝佐剂，如氢氧化铝和不溶性铝盐类胶体、油水乳剂、微生物及代谢产物、核酸及其类似物、细胞因子、免疫刺激复合物、蜂胶、和脂质体，优选铝佐剂。
在一个实施方案中，本发明公开了本文所述的CpG ODN在制备佐剂中的用途。在一个实施方案中，所述佐剂用于疫苗的制备，优选用于乙型肝炎疫苗、狂犬疫苗、新冠肺炎重组亚单位疫苗、新冠肺炎灭活疫苗的制备。
在一个实施方案中，本发明提供了一种药物组合物，其包含抗原和本文所述的组合物。
在所述的药物组合物的一个实施方案中，所述抗原的含量为5-200μg/mL，优选5-80μg/mL。
在所述的药物组合物的一个实施方案中，所述CpG ODN的含量优选为100-300μg/mL。
在所述的药物组合物的一个实施方案中，铝佐剂的含量优选为300-600μg/mL。
在所述的药物组合物的一个实施方案中，所述药物组合物为乙型肝炎疫苗、狂犬疫苗、新冠肺炎重组亚单位疫苗、新冠肺炎灭活疫苗。
在一个实施方案中，本发明公开了本发明所述的药物组合物在制备用于诱导受试者体内针对抗原的免疫反应的药物中的应用。在进一步的实施方案中，所述的应用还包括将有效剂量的所述的药物组合物再次给药于受试者，其中所述再次给药的时间间隔为2-24周。
在一个实施方案中，本发明公开了制备本文所述的药物组合物的方法，其包括将所述CpG ODN和其他佐剂、抗原和药学上可接受的载体按照用量配比，顺序加入容器中，发生吸附、偶联和/或乳化反应，并配制为注射剂或口服制剂、喷鼻剂。
在所述方法的一个实施方案中，在其他佐剂为铝佐剂，如氢氧化铝时，将所述铝佐剂先与抗原混合发生吸附作用，然后再与CpG ODN混合发生吸附作用。
CpG：C代表胞嘧啶，G代表鸟嘌呤，p代表磷酸二酯键。CpG是由胞嘧啶和鸟嘌呤通过磷酸二酯键连接成的非甲基化的二核苷酸。
“佐剂”：是一些先于抗原或同时与抗原混合注射动物的物质，可以起到增强抗原免疫原性的作用。在动物疫苗中，佐剂可以增强其免疫效力。
铝佐剂
本发明中所述的铝佐剂是本领域所公知的且常用的免疫佐剂，其能从溶液中强烈吸附蛋白质抗原，形成沉淀。当将其接种到机体内后可形成一个“抗原库”，缓慢释放出抗原，充分延长了抗原的作用时间。同时还能促进局部(注射部位)巨噬细胞的应答。本发明的铝佐剂可选自：氢氧化铝，磷酸铝和硫酸铝。
抗原
本发明中所述的抗原是指能够诱导机体的免疫系统发生免疫应答，并能与免疫应答的产物(抗体和/或效应细胞)在体内或体外结合，发生特异性反应的物质，其可包括蛋白质、脂质、糖类、核酸、化合物，等等，其中，作为抗原的蛋白质包括被修饰或未被修饰的蛋白质，所述修饰例如糖基化或甲基化，例如，所述蛋白质被环化或连接至脂质。 与感染原或疾病有关的抗原包括作为感染原的一部分的抗原，例如，包膜蛋白、衣壳蛋白、表面蛋白、毒素、细胞壁、抗原脂质，等等。其他合适的抗原可包括宿主的抗原，其包括被诱导、修饰或过表达为感染或疾病的标志物的那些抗原。从感染原、感染、病症或疾病衍生得到的所有这些抗原或者与感染原、感染、病症或疾病有关的所有这些抗原适用于本发明。
在本发明的一些实施方式中，所述抗原为与病毒相关的抗原，所述病毒包括但不限于：肝炎病毒，狂犬病病毒、新冠肺炎病毒等。
“疫苗”：本发明的“疫苗”是本领域普通技术人员熟知的疫苗，泛指一切通过注射或黏膜途径接种后，可以诱导机体产生针对特定致病原的特异性抗体和/或细胞免疫，从而使机体获得保护或消灭该致病原能力的生物制品，包括蛋白质、多糖、核酸、活载体或感染因子等。疫苗是将病原微生物(如细菌、立克次氏体、病毒等)及其代谢产物，经过人工减毒、灭活或利用基因工程等方法制成的用于预防传染病的自动免疫制剂。疫苗保留了病原菌刺激动物体免疫系统的特性。当动物体接触到这种不具伤害力的病原菌后，免疫系统便会产生一定的保护物质，如免疫激素、活性生理物质、特殊抗体等；当动物再次接触到这种病原菌时，动物体的免疫系统便会依循其原有的记忆，制造更多的保护物质来阻止病原菌的伤害。
实施例
实施例1 ODN 1乙型肝炎疫苗对健康成人免疫效果的影响
一.试剂
1.云南沃森生物技术股份有限公司生产的重组(汉逊酵母)乙型肝炎疫苗(CpG ODN佐剂)，疫苗规格：0.5mL/瓶，含乙型肝炎病毒表面抗原10μg，ODN 1 250μg，铝含量为0.45-0.60mg/mL(批号：20151201)；
2.大连汉信生物制药有限公司生产的重组(汉逊酵母)乙型肝炎疫苗，疫苗规格：0.5mL/瓶，含乙型肝炎病毒表面抗原10μg，铝含量为0.35-0.62mg/mL(批号：201505082)。
二.方法
选取48名18-60岁乙型肝炎病毒(HBV)筛查指标阴性、肝肾功能筛选判定正常且符合纳入和排除标准的志愿者为研究对象，采用随机、盲法、对照的设计方法将研究对象按1∶1分为试验组和对照组，每组24名。试验组接种云南沃森生物技术股份有限公司生产的重组(汉逊酵母)乙型肝炎疫苗(CpG ODN佐剂)；对照组接种疫苗为大连汉信生物制药有限公司生产的重组(汉逊酵母)乙型肝炎疫苗。各组均按照在第0周、4周和24周接种的免疫程序进行疫苗接种，每次接种1剂，每剂0.5mL。接种部位均为上臂外侧三角肌附着处皮肤，接种途径为肌肉注射。分时段观察并记录其接种后情况，比较两组间各类不良反应发生率的差异。采集所有受试者首剂免前及第2、3剂接种后30d血样，采用化学发光免疫分析法进行血清乙型肝炎表面抗体(抗HBs)检测，其中首剂免前血样复核HBsAg和抗HBc，每剂次免后血样均检测抗HBs浓度，以抗体浓度≥10.0mIU/mL为抗体阳性，≥1000.0mIU/mL定义为抗体强阳性，比较两组乙型肝炎表面抗体(抗HBs)阳性率和几何平均浓度(GMC)的差异。
三.结果
观察期间，试验组不良事件发生率为66.67％(16例)，对照组为54.17％(13例)(P＝0.556)。不良事件均为1级或者2级，未出现3级及以上不良反应。如表1所示，全程免疫后抗体GMC结果显示，全分析数据集(FAS集)试验组抗体GMC为2598.56(95％CI：1127.90-5986.90)mIU/mL，高于对照组371.97(164.54-840.91)mIU/mL；方案数据集(PPS集)试验组抗体GMC为7808.21(3377.00-18052.00)mIU/mL，高于对照组[843.22(95％CI：213.80-3325.90)mIU/mL]。FAS(PPS)集抗HBs阳性率结果显示，试验组和对照组均为95.83％(100.00％)；FAS(PPS)集抗HBs强阳性率结果显示，试验组为79.17％(90.00％)，对照组为33.33％(50.00％)，FAS集组间差异有统计学意义(P＝0.003)，PPS集组间差异无统计学意义(P＝0.074)。
结论：与对照疫苗相比，CpG乙型肝炎疫苗有较好的安全性；而且，第2、3剂免疫后30天，试验组抗体GMC显著高于对照组，这表明含CpG佐剂和铝佐剂的双佐剂型乙型肝炎疫苗(10μg剂量)比市售的免疫效果最好的单一铝佐剂型乙型肝炎疫苗(10μg剂量)显示出更好的免疫原性；此外，第2剂免疫后，试验组可达到95％的阳性率，2剂次即可达到对照疫苗3剂次效果，这提示我们接种含CpG佐剂和铝佐剂的双佐剂型乙型肝炎疫苗可加快免疫后乙型肝炎抗体产生，快速诱导产生保护性抗体具有许多优点，特别是在需要快速保护或后续剂次无法保证的情况下，同时就疫苗成本和人力而言，两剂疫苗计划的实施成本将低于三剂疫苗。
表1 试验组和对照组接种乙型肝炎疫苗后抗体阳转率和GMC比较
Figure PCTCN2022090392-appb-000004
注： amIU/mL(95％CI)，采用独立样本t检验比较两组的差异； b％(例)，采用Fisher确切概率法比较两组的差异；GMC：几何平均浓度；FAS：全分析数据集；PPS：符合方案集；强阳性：乙型肝炎表 面抗体≥1000mIU/mL；试验组：接种含CpG佐剂的乙型肝炎疫苗；对照组：接种市售乙型肝炎疫苗
实施例2 不同ODN对乙型肝炎疫苗的作用
一.材料
ODN 1、ODN 2和阳性对照ODN 5(5'-TGACTGTGAACGTTCGAGATGA-3'，其中核苷酸为全硫代磷酸酯化的核苷酸)，由江苏泰普瑞生物技术有限公司提供；
重组乙型肝炎疫苗(CHO细胞)，规格：1.0mL，20μg/支，购自华北制药金坦生物技术股份有限公司；
BALB/c小鼠(SPF级)，购自浙江维通利华实验动物技术有限公司。
二.方法
选取15-18g无给药史的雌性BALB/c小鼠，分为5组，每组按设计的免疫程序，在D1和D28天各免疫一次，免疫途径为左胫前肌肉给药，每次0.1mL，首次给药时小鼠为5-6周龄。按实验设计时间，经心脏穿刺或眼眶采血采集血液样本至采血管中。室温条件下放置约30-60分钟后离心，所有样品在4℃1800g条件下离心15分钟获取血清。用商品化的乙肝表面抗体检测试剂盒检测血清样本中的anti-HBs抗体水平。
具体分组如下：
Figure PCTCN2022090392-appb-000005
Figure PCTCN2022090392-appb-000006
注：a，PBS；F，雌性；每个采血点6只小鼠。
三.结果
如图1所示，2针免疫后，1/2抗原组(第3、4、5组)产生的抗体滴度均显著高于未加ODN组(第2组)；1次免疫后4周、6周、8周，抗体滴度水平持续升高，并且8周为本次实验中最高抗体滴度的水平，其中第4组和第5组的小鼠抗体滴度在8周的时候有强效增长，说明其能长效保持抗体浓度。
结论：不同的ODN能降低乙肝疫苗抗原的用量；且与乙肝疫苗联用时，能产生强而持久的保护作用。
实施例3 ODN 1对狂犬病疫苗的作用
一.材料
ODN 1，由长春华普生物技术有限公司提供；
Vero细胞狂犬病疫苗，规格为1mL/支(含2.5IU)，采自长春生物制品研究所；
氢氧化铝佐剂，由长春生物制品研究所提供；
BALB/c小鼠，购自维通利华实验动物技术有限公司。
二.方法
选取体重为18-22g无给药史的小白鼠，分为8组，每组8只，雌雄各半。具体分组为：狂犬疫苗、狂犬疫苗+氢氧化铝、狂犬疫苗+1.25μg ODN 1、狂犬疫苗+5μg ODN 1，狂犬疫苗+20μg ODN 1、狂犬疫苗+氢氧化铝+1.25μg ODN 1、狂犬疫苗+氢氧化铝+5μg ODN 1、狂犬疫苗+氢氧化铝+20μg ODN 1，其中氢氧化铝浓度为0.45mg/mL(以铝含量计)。
将上述狂犬病疫苗、ODN 1和氢氧化铝佐剂按比例稀释溶解于PBS中。小白鼠免疫：于0天、7天、21天，按不同的分组分别给小白鼠进行免疫。免疫方式是小鼠腹腔注射，每次注射0.5mL。在第4天、10天、31天从小白鼠尾静脉采血，分离血清，用快速狂犬疫苗荧光灶抑制实验(RFFIT)方法检测小白鼠血清中狂犬疫苗抗体的效价。于免疫的前两天小鼠尾静脉取血，获得的血清作阴性对照。
三.结果
见图2，第31天时，狂犬疫苗+氢氧化铝、狂犬疫苗+1.25μg ODN 1、狂犬疫苗+氢氧化铝+1.25μg ODN 1、狂犬疫苗+5μg ODN 1、狂犬疫苗+氢氧化铝+5μg ODN 1各组小鼠血清中狂犬疫苗抗体效价分别为72.13、35.79、186.28、63.1、197.63IU，这表明ODN 1与氢氧化铝佐剂之间存在协同增效作用，与单铝佐剂及单ODN 1佐剂组相比，双佐剂组能显著提高狂犬病疫苗抗体的效价。
实施例4 ODN 1对新冠重组亚单位疫苗的作用
一.材料
1.PBS磷酸盐缓冲液干粉(Solarbio)；
2.SARA-CoV-2 S protein(Acro)，规格为500μg/支，批次：C591P1-20B2F1-UF；
3.SARA-CoV-2(COVID-19)S protein RBD(Acro)，规格为100μg/支、100mg/支，批次：C497P1-207AF1-TJ；
4.ODN 1(江苏泰普瑞生物技术有限公司)，规格：8.3mg/mL，批次：202010001；
5.BALB/c小鼠(SPF级)，浙江维通利华实验动物技术有限公司提供；
6.氢氧化铝，由浙江天元生物药业有限公司提供。
二.方法
选取15-18g无给药史的雌性BALB/c小鼠，分为10组，组1为PBS溶媒对照组，组2-10为试验组；每组按设计的免疫程序，在D0和D28天分别以腹腔注射的方式免疫小鼠，每次0.2mL，首次给药时小鼠为5-6周龄。按实验设计时间，在D7、D14、D21、D28、D56、D84经心脏穿刺或眼眶采血采集血液样本。室温条件下放置约30-60分钟后离心，所有样品在4℃1800g条件下离心15分钟获取血清。用夹心法ELISA检测血清样本中的S1抗体和RBD抗体水平。
具体分组如下：
Figure PCTCN2022090392-appb-000007
三.结果
见图3-图5，由图3可知，第3组、第7组和第5组中的小鼠在第14天的RBD抗体GMT分别为50、50和1270；第21天的RBD抗体GMT分别为200、50和10159；第28天的RBD抗体GMT分别为79、50和26390；第84天的RBD抗体GMT分别为270、52和51962，这表明以RBD为抗原的双佐剂疫苗组与相应的单佐剂疫苗组相比，均能显著提高RBD抗体GMT，证明了ODN 1与氢氧化铝具有协同增 效作用。第8组和第9组中的小鼠在第7天的RBD抗体GMT分别为141和800；第14天的RBD抗体GMT分别为2425和6400；第21天的RBD抗体GMT分别为5702和45948；第28天的RBD抗体GMT分别为4525和25600；第84天的RBD抗体GMT分别为90000和467654，这表明以S蛋白为抗原的双佐剂疫苗组与相应的单一Al佐剂疫苗组相比，能显著提高RBD抗体GMT。
由图4可知，第8组和第9组中的小鼠在第7天的S1抗体GMT分别为50和400；第14天的S1抗体GMT分别为3200和7184；第21天的S1抗体GMT分别为6400和52780；第28天的S1抗体GMT分别为12800和81275；第84天的S1抗体GMT分别为224825和810000，这表明以S蛋白为抗原的双佐剂疫苗组与相应的单一Al佐剂疫苗组相比，能显著提高S1抗体GMT。
由图5可知，第8组和第9组中的小鼠在第21天的中和抗体滴度分别为40和240；第28天的中和抗体滴度分别为20和360；第84天的中和抗体滴度分别为3840和5120，这表明以S蛋白为抗原的双佐剂疫苗组与相应的单一Al佐剂疫苗组相比，能显著提高中和抗体滴度。
当铝含量和ODN 1含量分别为0.45mg/mL和100μg/mL不变时，(1)随着抗原RBD含量的增加(组4-6)，RBD抗体滴度先增加后降低，这表明：RBD为抗原的疫苗组产生的抗体显示抗原剂量依赖性增加，但是当抗原浓度高过一定范围，抗体则反而降低。本次实验表明高于70μg/mL，则抗体产生浓度下降。抗原浓度在25μg/mL时最佳。(2)随着抗原S全长蛋白剂量的增加(组9-10)，S1抗体滴度增加，而中和抗体滴度保持平稳，这表明：S1抗体随抗原剂量依赖性增加，而中和抗体随抗原剂量增加变化不明显。
结论：CpG佐剂与铝佐剂具有协同增效作用，能显著提高疫苗RBD抗体滴度、S1抗体滴度和中和抗体滴度，且保护作用持久；5-100μg/mL的抗原与CpG佐剂(100μg/mL)以及氢氧化铝(Al含量为0.45mg/mL)联用产生较佳的效果；CpG佐剂在抗原为S全长蛋白 的组比抗原为RBD的组所诱导的S1抗体滴度和RBD抗体滴度的增加更显著。
实施例5 ODN 2对新冠肺炎灭活疫苗的作用
一.材料
1.新冠肺炎灭活疫苗抗原，由浙江天元生物药业有限公司提供；
2.ODN 2，由长春华普生物技术股份有限公司提供；
3.氢氧化铝佐剂，由浙江天元生物药业有限公司提供；
4.BALB/c小鼠，购自维通利华实验动物技术有限公司。
二.方法
选取18-20g的BALB/c小鼠，每组9或10只，雌雄各半；每组按设计的免疫程序，在D0和D14天分别以腹腔注射的方式免疫小鼠，每次注射0.5mL。按实验设计时间，分别在D0、D6、D13、D21和D28采血分离血清，所有血清检测S1蛋白特异性IgG和病毒中和抗体；按统计学计算每组动物血清IgG和病毒中和抗体几何平均效价。测定结果见图6-7。
具体分组情况如下：
Figure PCTCN2022090392-appb-000008
Figure PCTCN2022090392-appb-000009
结果：由图6可知，当新冠肺炎灭活疫苗抗原含量为4μg/mL时，5-80μg/mL的ODN 2与氢氧化铝(Al含量为0.45mg/mL)联用，相对于无ODN 2的组，S1抗体GMT显著增加；由图7可知，20μg/mL ODN 2与氢氧化铝(Al含量为0.45mg/mL)用于抗原含量为2-8μg/mL的新冠肺炎灭活疫苗时，S1抗体GMT显著增加。
结论：ODN 2与氢氧化铝联合用于新冠肺炎灭活疫苗，S1抗体滴度显著增加；当新冠肺炎灭活疫苗含5-80μg/mL ODN 2、氢氧化铝(Al含量为0.45mg/mL)、2-8μg/mL抗原时，效果最佳。
实施例6 不同ODN对新冠重组亚单位疫苗的作用
一.材料
1.PBS磷酸盐缓冲液干粉(Solarbio)；
2.SARA-CoV-2 S蛋白(Acro)，规格为500μg/支，批次：C591P1-20B2F1-UF；
3.ODN 2、ODN 3、ODN 4以及阳性对照ODN 5(5'-TGACTGTGAACGTTCGAGATGA-3')，由江苏泰普瑞生物技术有限公司提供；
4.氢氧化铝佐剂，购自国药集团；
5.BALB/c小鼠，购自浙江维通利华实验动物技术有限公司。
二.方法
选取15-18g无给药史的雌性BALB/c小鼠，分为11组，每组约30只动物。在D0和D28天分别以腹腔注射的方式免疫小鼠，每次0.2mL，首次给药时小鼠为5-6周龄。按实验设计时间，给药后在D7、D14、D28、D56、D84经心脏穿刺或眼眶采血采集血液样本。室温条件下放置约30-60分钟后离心，所有样品在4℃1800g条件下离心15分钟获取血清。用夹心法ELISA检测血清样本中的S1抗体水平。结果见图8。
具体分组情况如下：
Figure PCTCN2022090392-appb-000010
三.结果
见图8，由结果可知，第3组至第11组中的小鼠在第7天的S1抗体GMT分别为111、80、700、73、550、30、317、70和467；第28天的S1抗体GMT分别为1120、2133、50667、1813、48316、960、 29333、1600和40333；第84天的S1抗体GMT分别为28223、34666、709338、22667、641229、10000、373330、16000和605329，这表明不同CpG ODN与铝佐剂之间存在协同增效作用，与单铝佐剂组或单CpG ODN组相比，双佐剂组能显著提高抗体滴度的水平，而且保护作用持久。
实施例7 ODN 1对带状疱疹疫苗的作用
一、材料
PBS磷酸盐缓冲液干粉，从Solarbio购买；
ODN 1，由江苏泰普瑞生物技术有限公司提供；
氢氧化铝佐剂，从Thermo购买；
带状疱疹病毒gE蛋白，从SinoBiological购买；
BALB/c小鼠，购自维通利华实验动物技术有限公司。
二、方法
选取体重为18-22g无给药史的小鼠，分为4组，每组8只，雌雄各半。具体分组为：10μg gE蛋白组、10μg gE蛋白+40μg氢氧化铝、10μg gE蛋白+5μg ODN 1组、10μg gE蛋白+40μg氢氧化铝+5μg ODN 1组，其中氢氧化铝以铝含量计。
将gE抗原、氢氧化铝和ODN 1按比例稀释溶解在PBS中。按设计的免疫程序，在D0、D28、D56通过腹腔注射分别免疫各组小鼠，每次注射0.1mL。按实验设计时间，在D0、D7、D14、D21、D28、D35、D42、D49、D56、D63、D70、D77从小鼠尾静脉采血，分离血清，用ELISA方法检测小鼠血清中抗gE蛋白滴度，使用免疫前7天获得的血清作阴性对照。
三.结果
见图9，第35天时，10μg gE蛋白组、10μg gE蛋白+40μg氢氧化铝、10μg gE蛋白+5μg ODN 1组、10μg gE蛋白+40μg氢氧化铝+5μg ODN 1组小鼠血清中抗gE蛋白滴度分别为190207、 1116680、430539、4870992，这表明ODN 1与氢氧化铝佐剂之间存在协同增效作用，与单铝佐剂及单ODN 1佐剂组相比，双佐剂组能显著提高抗gE蛋白滴度。
实施例8 ODN 1对四价流感疫苗的作用
一、材料
PBS磷酸盐缓冲液干粉，从Solarbio购买；
ODN 1，由江苏泰普瑞生物技术有限公司提供；
A1型流感抗原，从SinoBiological购买；
A3型流感抗原，从SinoBiological购买；
By型流感抗原，从SinoBiological购买；
Bv型流感抗原，从SinoBiological购买；
BALB/c小鼠，购自维通利华实验动物技术有限公司。
二、方法
选取体重为15-20g无给药史的雌性小鼠，分为9组，第1至9组分别为PBS组、四价裂解苗组(QIV)、四价裂解苗+CpG组(QIV+CpG)、1/2四价裂解苗组(1/2QIV)、1/2四价裂解苗+CpG组(1/2QIV+CpG)、1/4四价裂解苗组(1/4QIV)、1/4四价裂解苗+CpG组(1/4QIV+CpG)、1/8四价裂解苗组(1/8QIV)、1/8四价裂解苗+CpG组(1/8QIV+CpG)，每组20只小鼠(PBS组10只)。
按比例将上述4种流感抗原(A1、A3、By、Bv)和ODN 1先后加入PBS中并溶解，用于免疫小鼠。在D0通过肌肉注射在各组小鼠后肢大腿内侧进行一次免疫，注射体积为0.1mL。用于免疫QIV组每只小鼠的0.1mL溶液含4种抗原各3.5μg；用于免疫QIV+CpG组每只小鼠的0.1mL溶液含4种抗原各3.5μg以及5μg ODN 1；用于免疫1/2QIV组每只小鼠的0.1mL溶液含4种抗原各1.75μg；用于免疫1/2QIV+CpG组每只小鼠的0.1mL溶液含4种抗原各1.75μg以及5μg ODN 1；用于免疫1/4QIV组每只小鼠的0.1mL溶液含4种抗原各0.875μg；用于免疫1/4QIV+CpG组每只小鼠的0.1mL溶液含4种 抗原各0.875μg以及5μg ODN 1；用于免疫1/8QIV组每只小鼠的0.1mL溶液含4种抗原各0.44μg；用于免疫1/8QIV+CpG组每只小鼠的0.1mL溶液含4种抗原各0.44μg以及5μg ODN 1。免疫后持续观察动物至D49。
按实验设计时间，在D4、D5、D6、D7、D14、D21、D28、D49对各试验组小鼠(第2组至第9组)进行采血，在D7和D28对对照组小鼠(第1组)进行采血。每组每个采血点采集5只小鼠，其中第2-9组在D4、D5、D6和D7采用眼眶静脉丛采血，D14、D21、D28和D49采用摘眼球取血，第1组均采用摘眼球取血，摘眼球取血后的动物经计划性放血后死亡。将全血在室温下放置约30-60分钟后离心，所有样品在4℃1800g条件下离心15分钟获取血清，用ELISA检测小鼠血清中4种抗体几何平均滴度，免疫前一天取10只小鼠经摘眼球采免疫前血清作阴性对照。
三.结果
见图10，由免疫后D4至D49小鼠血清中4种抗体几何平均滴度可知，与无ODN 1佐剂疫苗组相比，相应的ODN 1佐剂疫苗组可以快速诱导产生保护性抗体；显著提高四价裂解流感疫苗免疫后4种抗原的抗体几何平均滴度，加CpG组相对于不加CpG组抗体几何平均滴度提高约1-9倍；此外，加CpG组可以在1/8抗原条件下获得不低于原倍抗原的免疫效果。
综上所述，ODN 1佐剂能快速诱导产生保护性抗体；显著提高四价流感疫苗的抗体滴度；减少抗原用量，提高疫苗的安全性。
实施例9 不同ODN对狂犬病疫苗的作用
一、材料
ODN 3、ODN 4由江苏泰普瑞生物技术有限公司提供；
Vero细胞狂犬病疫苗，规格为1mL/支(含2.5IU)，采自长春生物制品研究所；
BALB/c小鼠，购自维通利华实验动物技术有限公司。
二、方法
选取体重为18-22g无给药史的小白鼠，分为5组，每组8只，雌雄各半。第1-5组分别为：狂犬疫苗、狂犬疫苗+ODN 3、1/2狂犬疫苗+ODN 3、狂犬疫苗+ODN 4，1/2狂犬疫苗+ODN 4。
将上述Vero细胞狂犬病疫苗、ODN 3和ODN 4按比例稀释溶解于PBS中，其中第1、2、4组Vero细胞狂犬病疫苗稀释100倍；第3、5组Vero细胞狂犬病疫苗稀释200倍，用于免疫小鼠。按设计的程序，在第0天、3天、7天，分别免疫各组小白鼠一次。免疫方式是小鼠后腿肌肉注射，注射体积为0.1mL，用于免疫第2-5组小鼠的0.1mL溶液中ODN 3或ODN 4含量均为5μg。在第4天、6天、8天、10天、14天、28天、56天从小白鼠尾静脉采血，分离血清，用快速狂犬疫苗荧光灶抑制实验(RFFIT)方法检测小白鼠血清中抗狂犬病毒中和抗体的效价。在免疫前两天从小鼠尾静脉取血，获得的血清作阴性对照。
三、结果
见图11，第14天时，狂犬疫苗、狂犬疫苗+ODN 3、1/2狂犬疫苗+ODN 3、狂犬疫苗+ODN 4，1/2狂犬疫苗+ODN 4各组小鼠血清中抗狂犬病毒中和抗体滴度均值分别为15.19、38.33、19.63、34.07、29.83，这表明与不加ODN 3或ODN 4的疫苗组相比，相应的ODN 3或ODN 4佐剂疫苗组均能显著提高狂犬疫苗中和抗体滴度。此外，ODN 3或ODN 4佐剂疫苗组可以在1/2抗原条件下获得高于原倍抗原的免疫效果。
等同方案
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (15)

  1. 一种组合物，其包含CpG ODN和一种或多种与所述免疫调节性CpG ODN共同发挥作用的其他佐剂，例如铝佐剂，如氢氧化铝和不溶性铝盐类胶体、油水乳剂、微生物及代谢产物、核酸及其类似物、细胞因子、免疫刺激复合物、蜂胶、和脂质体，优选铝佐剂，其中所述CpG ODN包含或组成为选自SEQ ID NO:1-4的核苷酸序列，其中所述核苷酸序列中至少一个核苷酸为具有通式I所示结构的化学修饰的核苷酸：
    Figure PCTCN2022090392-appb-100001
    其中，Y为S或O，R为H或带正电的抗衡离子，B独立地为未修饰或经修饰的核碱基，R 1为H、F、Cl、OH、OMe、Me、O-乙基氧甲基。
  2. 权利要求1所述的组合物，其中Y为S。
  3. 权利要求1或2所述的组合物，其中所述CpG ODN的核苷酸序列的全部核苷酸均为具有通式I所示的结构的化学修饰的核苷酸。
  4. 权利要求3所述的组合物，其中所述CpG ODN的序列选自：SEQ ID NO:1-4，优选地，全部硫代磷酸酯化的SEQ ID NO:1-4。
  5. 权利要求1-4任一项所述的组合物，其中所述CpG ODN的含量为10-500μg/mL，其中铝佐剂中铝的含量为300-600μg/mL。
  6. 权利要求1-5任一项所述的组合物在制备疫苗中的用途。
  7. 权利要求6所述的用途，其中所述疫苗为乙型肝炎疫苗、狂犬病疫苗、新冠肺炎疫苗，例如抗新冠病毒Alpha毒株、Beta毒株、 Gamma毒株、Delta毒株、Omicron毒株的新冠肺炎疫苗，包括但不限于新冠肺炎重组亚单位疫苗和新冠肺炎灭活疫苗、带状疱疹疫苗、流感疫苗，如四价流感疫苗。
  8. 一种药物组合物，其包含抗原和权利要求1-5任一项的组合物。
  9. 权利要求8所述的药物组合物，其中所述抗原的含量为5-200μg/mL，优选5-80μg/mL。
  10. 权利要求8-9任一项所述的药物组合物，其中所述的CpG ODN含量为100-300μg/mL。
  11. 权利要求8-9任一项所述的药物组合物，其中所述铝佐剂中铝含量为300-600μg/mL。
  12. 权利要求8-11任一项所述的药物组合物，其为乙型肝炎疫苗、狂犬疫苗、新冠肺炎重组亚单位疫苗、新冠肺炎灭活疫苗、带状疱疹疫苗、流感疫苗，如四价流感疫苗。
  13. 权利要求8-12任一项所述的药物组合物在制备用于诱导受试者体内针对抗原的免疫反应的药物中的应用。
  14. 如权利要求13所述的应用，其中所述药物组合物用于以有效量施用给受试者，优选所述药物组合物用于给药于受试者两次，其中两次给药的时间间隔为2-24周。
  15. 制备权利要求8-12任一项所述的药物组合物的方法，其包括将权利要求1-5任一项的组合物、抗原和任选地药学上可接受的载体混合，发生吸附、偶联和/或乳化反应，并配制为注射剂或口服制剂、喷鼻剂。
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