WO2024027171A1 - 一种包含或由核酸水凝胶组成的佐剂及其用途 - Google Patents

一种包含或由核酸水凝胶组成的佐剂及其用途 Download PDF

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WO2024027171A1
WO2024027171A1 PCT/CN2023/083880 CN2023083880W WO2024027171A1 WO 2024027171 A1 WO2024027171 A1 WO 2024027171A1 CN 2023083880 W CN2023083880 W CN 2023083880W WO 2024027171 A1 WO2024027171 A1 WO 2024027171A1
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modification
adjuvant
nucleic acid
cross
group
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PCT/CN2023/083880
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French (fr)
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刘冬生
刘万里
李翠峰
周碧妮
李雨欣
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs

Definitions

  • the present invention relates to an adjuvant containing or consisting of nucleic acid hydrogel and its use, and belongs to the field of vaccines or adjuvants.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the intensity and speed of SARS-CoV-2 transmission have resulted in morbidity and mortality in large numbers of populations and placed considerable stress on public health systems and the global economy around the world.
  • the spread of the virus can be mitigated through methods such as controlling distance between people, wearing masks, and testing and tracing, the risk of large-scale outbreaks and disrupting economic and social life remains until most of the world's population is vaccinated with an effective vaccine to stop the spread of the virus. exist.
  • Vaccines can prevent disease and outbreaks in large numbers of people at relatively low cost, making them a powerful tool in mitigating the impact of COVID-19.
  • Attenuated pathogens usually do not require the addition of adjuvants to be sufficiently immunogenic.
  • attenuated pathogens usually have shortcomings such as difficulty in obtaining, high acquisition costs, and poor safety.
  • vaccines based on containing limited amounts of purified antigens have huge advantages in terms of safety and production costs, but are often accompanied by the problem of limited immunogenicity due to the removal of the pathogenic properties of the organism. This requires the addition of adjuvants to induce a protective and durable immune response.
  • Adding adjuvants to vaccines can enhance, maintain and guide the immunogenicity of antigens, effectively modulate appropriate immune responses, reduce the amount of antigen or number of immunizations required, and improve the effectiveness of vaccines in newborns, the elderly, or immunocompromised individuals. effectiveness.
  • nucleic acid hydrogels such as the nucleic acid hydrogels described in CN107773527B and CN107779427B
  • nucleic acid hydrogels themselves as adjuvants. Research.
  • nucleic acid hydrogel itself can serve as an adjuvant to significantly promote the increase of IgG antibodies specific to the model antigen OVA.
  • nucleic acid hydrogel Compared with traditional aluminum adjuvants, nucleic acid hydrogel not only increased the expression level of SARS-CoV-2 RBD protein-specific antibodies, but also produced neutralizing antibodies with stronger ability to neutralize the virus.
  • Nucleic acid hydrogel has the effect of sustaining the release of antigens in the body.
  • the three-dimensional cross-linked network structure of nucleic acid hydrogel slows down the degradation rate of nucleic acid hydrogel itself in the body.
  • Nucleic acid hydrogel can induce the enrichment and differentiation of antibody response-related immune cells in the immune site and draining lymph nodes, promoting antibody production; nucleic acid hydrogel can also promote antibody response-related cytokines (IL-1b, CCL2, CCL3 , CCL4)mRNA is highly expressed.
  • IL-1b IL-1b
  • CCL2 CCL2
  • CCL3 antibody response-related cytokines
  • the invention provides an adjuvant comprising or consisting of a nucleic acid hydrogel comprising:
  • a stent unit having at least three stent adhesive ends
  • cross-linking unit having at least two cross-linking sticky ends
  • the scaffold unit and the cross-linking unit are both formed by complementary base pairing of nucleic acids
  • the scaffold unit and the cross-linking unit are cross-linked in a complementary base pairing manner through the scaffold sticky end and the cross-linking sticky end, thereby forming a three-dimensional spatial network structure
  • the scaffold unit or the cross-linking unit contains or does not contain a CpG sequence
  • the nucleic acid contains or does not contain modifications.
  • the modification includes linking group modification, fluorescent group modification, quenching group modification, inter-arm modification, nucleotide variant modification, and degenerate base modification.
  • the linking group modification includes amino modification, carboxyl modification, aldehyde modification, acrylamide modification, azide modification, alkynyl modification, diphenylcyclooctyne modification, maleyl modification Imine modification, thiol modification, dithiol modification, ferrocene modification, biotin modification, digoxin modification.
  • the fluorophore modification includes Pacific Blue, ROX, Texas red.
  • the quenching group modification includes BHQ1 and BHQ2.
  • the inter-arm modification includes C3/C6 Spacer, Spacer 9/Spacer 18, PC Spacer/PC linker, and tetrahydrofuran modification.
  • the nucleotide variant modifications include phosphorylation, thio, 2-aminopurine, 5-bromodeoxyuracil, deoxyuridine, inverted dT/dG, dideoxycytosine Pyrimidine nucleoside, 5-methylcytosine deoxynucleoside, 5-hydroxymethyldC, N6 methyladenine nucleotide, deoxyinosine nucleoside, locked nucleic acid, 5-nitroindole, 2-methyl Oxygen modification, RNA base, 2-fluoro modification, 2-fluoroRNA, 2'-O-(2-methoxy)ethyl, morpholino, bridged nucleotide, BNA, pyrrole-deoxycytosine.
  • the adjuvant is used in a vaccine.
  • the length of the scaffold sticky end or cross-linked sticky end is 4nt-150nt.
  • the scaffold unit is formed by three single-stranded nucleic acids in a complementary base pairing manner, and each single-stranded nucleic acid has one sticky end of the scaffold.
  • the cross-linking unit is formed by two single-stranded nucleic acids in a complementary base pairing manner, and each single-stranded nucleic acid has one of the cross-linked sticky ends.
  • the scaffold unit and the cross-linking unit are in a stable cross-linking state under physiological conditions.
  • the scaffold unit and the cross-linking unit are in a stable cross-linked state at 37°C, pH 7.2-7.4, 0.9wt% NaCl, and isotonic conditions.
  • the adjuvant is used in a vaccine selected from the group consisting of mRNA vaccines, inactivated vaccines, attenuated vaccines and recombinant protein vaccines.
  • the vaccine used in the adjuvant is a coronavirus vaccine, and the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or COVID-19. Virus (COVID-19).
  • the coronavirus vaccine is selected from the group consisting of mRNA-1273 (Moderna, Inc.), AZD-1222 (AstraZeneca and University of Oxford), BNT162 (Pfizer and BioNTech), CoronaVac (Sinovac), COVILO , NVX-CoV 2372 (NovoVax), SCB-2019 (Sanofi and GSK), ZyCoV-D (Zydus Cadila) and CoVaxin (Bharat Biotech).
  • the vaccine is a COVID-19 vaccine, such as Alpha COVID-19 vaccine, Delta COVID-19 vaccine, Omicron COVID-19 vaccine and its mutant strains, and more.
  • the vaccine is used for the Omicron coronavirus and its mutant strains.
  • the Omicron coronavirus mutant strain is selected from the group consisting of BA.1, BA.2, xe, xl, and BA.4. , BA.5 and its branches. More preferably, in some embodiments, vaccines for the novel coronavirus and mutant strains thereof.
  • the Omicron coronavirus mutant strain is selected from the group consisting of BA.1, BA.2, xe, xl, BA.4, BA.5 and branches thereof.
  • the invention provides a use of a combination comprising a nucleic acid for forming the scaffold unit, a nucleic acid for forming the cross-linking unit, and an aqueous medium in the preparation of an adjuvant as described herein, wherein
  • the scaffold unit or the cross-linking unit may or may not contain CpG sequences.
  • the invention provides the use of a kit for preparing the adjuvant of the invention, which includes: a nucleic acid used to form the scaffold unit and a nucleic acid used to form the cross-linking unit; wherein, The scaffold unit or the cross-linking unit may or may not contain CpG sequences; optionally the kit further includes instructions.
  • the invention provides use of an adjuvant as described herein in the manufacture of a medicament for inducing or enhancing an immune response in a subject.
  • the invention provides an adjuvant for eliciting or enhancing an immune response in a subject.
  • the present invention provides a pharmaceutical combination for inducing or enhancing an immune response in a subject, comprising (1) an adjuvant as described herein; (2) an antigen; preferably the antigen is a coronavirus Viral antigens, preferably COVID-19.
  • the antigen is selected from the group consisting of SARS-CoV-2 S protein or fragments thereof, COVID-19 RBD protein or dimers or trimers thereof, etc.
  • the sequence of the new coronavirus RBD protein has at least 30%, 40%, 50%, 60%, 70%, 80%, 90% with any sequence in SEQ ID NO: 1-3 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, more preferably 100% identity.
  • the adjuvant described herein is not in physical contact with the antigen outside the subject's body.
  • the invention also provides a method of inducing or enhancing an immune response in a subject, which comprises administering to the subject an adjuvant of the invention or a pharmaceutical combination of the invention.
  • the adjuvant and the antigen in the pharmaceutical combination of the present invention can be administered to the subject together.
  • the adjuvant and the antigen can be administered to the subject simultaneously or at intervals. The interval can be 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours or more or any time in between any of the above time points.
  • the amounts of adjuvant and antigen administered are immunologically effective amounts.
  • the invention also provides an adjuvant described herein for promoting the enrichment and differentiation of immune cells in draining lymph nodes, promoting the enrichment of immune cells at the adjuvant administration site of a subject, inducing Increased expression of IL-1b, CCL-2, CCL-3, CCL-4 and other cytokines at the adjuvant administration site, increased expression of bone marrow-derived macrophage (BMDM) cytokines, or induced bone marrow-derived trees The expression of cytokines in BMDCs increased.
  • BMDM bone marrow-derived macrophage
  • the adjuvant containing or consisting of nucleic acid hydrogel of the present invention can effectively induce or enhance the immune response of a subject to a specific antigen
  • the adjuvant containing or consisting of nucleic acid hydrogel of the present invention can generate better immune responses to specific antigens;
  • the adjuvant containing or consisting of nucleic acid hydrogel of the present invention not only improves the expression level of new coronavirus-specific antibodies, but also significantly improves the ability of the neutralizing antibodies produced to neutralize the virus. , causing and enhancing the subject's immune response to the new coronavirus or new coronavirus antigen;
  • nucleic acid hydrogels herein containing or not containing CpG sequences can be used as adjuvants to enhance the body's immune response to specific antigens.
  • Figure 1 ELISA detection of OVA-specific IgG antibodies and IgG antibody subtypes.
  • ELISA detects the OVA-specific antibody IgG antibody titer in the serum of immunized mice on day 14. Blue is the PBS group, red is the DNA hydrogel group;
  • ELISA detects the antibody titers of OVA-specific IgG antibody subtypes IgG1, IgG2b, IgG2c and IgG3 in the serum of immunized mice on day 14. Blue is the PBS group and red is the DNA hydrogel group.
  • Figure 2 ELISA detection of nucleic acid hydrogel-specific IgG antibodies.
  • ELISA detects the anti-DNA-specific IgG antibody titer in the serum of immunized mice on day 14.
  • the blue color is the PBS group and the red color is the DNA hydrogel group.
  • FIG. 3 ELISA detection of RBD-specific IgG antibodies.
  • Black is the PBS group
  • blue is the aluminum adjuvant group
  • red is the RBD-specific IgG antibody titer of the DNA hydrogel group.
  • the left picture shows the mice immunized with RBD monomer combined with different adjuvants
  • the middle picture shows the mice immunized with RBD dimer and different adjuvants
  • the right picture shows the mice immunized with RBD trimer with different adjuvants.
  • Figure 4 Detection of neutralizing antibody titer of new coronavirus pseudovirus.
  • Black is the PBS group
  • blue is the aluminum adjuvant group
  • red is the pseudovirus neutralizing antibody gradient of the DNA hydrogel group.
  • Figure 5 Correlation analysis between the neutralizing antibody titer of the new coronavirus pseudovirus and the RBD antibody titer.
  • Figure 6 ELISA detection of OVA-specific antibody titers induced by different immune pathways.
  • the purple group was immunized with OVA (100 ⁇ g) on the left foot pad, and the right foot pad was immunized with 30 ⁇ L of DNA hydrogel at the same time; the orange group was immunized with OVA (100 ⁇ g) on the left foot pad, and the left leg muscle was immunized with 30 ⁇ L of DNA hydrogel at the same time ;
  • the blue group was immunized with OVA (100 ⁇ g) on the left leg muscle and 30 ⁇ L of DNA hydrogel on the left foot pad;
  • the red group was vaccinated with OVA (100 ⁇ g) + 30 ⁇ L of DNA hydrogel on the left foot pad; on the 7th day respectively.
  • blood was collected from the orbit to collect mouse serum, and ELISA was performed to detect OVA-specific IgG antibody titers.
  • Figure 7 In vivo imaging detects antigen degradation at the immune site.
  • C57 B6/L mouse footpads were immunized with OVA-Cy5+PBS or OVA-Cy5+DNA hydrogel or IgG-Cy5 at 0h, 2h, 6h, 12h, 24h, 36h, 60h, and 84h respectively in IVIS Spectrum Animal Optics In vivo imager detects Cy5 fluorescence.
  • the picture above is a picture display at different time points, and the picture below is a statistical curve chart of Cy5 fluorescence intensity. Black is the IgG-Cy5 group, blue is the OVA-Cy5+PBS group, and red is the OVA-Cy5+DNA hydrogel group.
  • Figure 8 ELISA detection of OVA-specific antibody titers induced by different gels.
  • ELISA detects the OVA-specific IgG antibody titers in the serum of mice immunized with OVA combined with different gels 14 days later.
  • Blue is the OVA+PBS group
  • purple is the OVA+Poloxamer gel (2520#) group
  • black is the OVA+Poloxamer gel.
  • red is OVA+DNA hydrogel group.
  • the elastic modulus of Poloxamer gel (2520#) is similar to that of DNA hydrogel
  • the visual gel state of Poloxamer gel (2525#) is similar to that of DNA hydrogel.
  • Figure 9 In vivo imaging detects DNA metabolism in immune sites.
  • C57B6/L mouse footpads were immunized with DNA hydrogel-Cy5.5 and soluble component DNA-Cy5.5 respectively at 0h, 2h, 6h, 12h, 24h, 36h, 60h, 84h in IVIS Spectrum animal optical live imaging
  • the instrument detects Cy5.5 fluorescence.
  • the upper picture shows the imaging pictures at different time points, and the lower picture shows the statistical curve of Cy5.5 fluorescence intensity.
  • the red color is the DNA hydrogel-Cy5.5 group
  • the blue color is the soluble component DNA-Cy5.5 group.
  • Figure 10 Immune cell profile in draining lymph nodes detected by flow cytometry.
  • mice were immunized with OVA (100 ⁇ g) antigen + PBS, OVA (100 ⁇ g) antigen + aluminum adjuvant (1:1), and OVA (100 ⁇ g) antigen + DNA hydrogel.
  • OVA 100 ⁇ g antigen + PBS
  • OVA 100 ⁇ g antigen + aluminum adjuvant
  • OVA 100 ⁇ g antigen + DNA hydrogel.
  • the mice were collected Flow cytometry was performed on the ipsilateral popliteal lymph node to detect plasma cells (Plasma), germinal center B cells (GCB), follicular helper cells (Tfh), macrophages (M ⁇ ) and dendritic cells (DC) in the popliteal lymph node.
  • Plasma cells Plasma
  • GCB germinal center B cells
  • Tfh follicular helper cells
  • M ⁇ macrophages
  • DC dendritic cells
  • Figure 11 Flow cytometry technology detects the enrichment pattern of immune cells in immune sites.
  • C57B6/L mouse footpads were immunized with OVA (100 ⁇ g) antigen + PBS, OVA (100 ⁇ g) antigen + aluminum adjuvant (1:1), and OVA (100 ⁇ g) antigen + DNA hydrogel, respectively at 0, 2, On days 3, 5, 6, 8, 9, 10, and 11, the contents of the immune site were taken for flow cytometry experiments to detect the proportions of macrophages (M ⁇ ), dendritic cells (DC), B cells, and T cells in the contents. Black is the OVA+PBS group, blue is the OVA+aluminum adjuvant group, and red is the OVA+DNA hydrogel group.
  • Figure 12 qPCR detection of cytokine mRNA expression profile in footpad tissue.
  • the footpads of C57B6/L mice were not immunized or immunized with DNA hydrogel for 6 h, and the footpad tissue was removed to extract RNA.
  • qPCR real-time fluorescence quantitative PCR
  • Figure 13 qPCR detection of cytokine mRNA expression in BMDM.
  • BMDM are primary macrophages induced by mouse bone marrow cells.
  • DNA hydrogel (1mg/mL) or Poloxamer gel (2520#) (1mg/mL) were added in vitro to stimulate for 0, 1, 3, and 6 hours respectively, and then collected.
  • RNA was used for qPCR to detect the expression of IL1 ⁇ , IL6, IFN ⁇ and CCL4mRNA.
  • the blue color is the Poloxamer gel (2520#) (1mg/mL) stimulation group, and the red color is the DNA hydrogel stimulation group.
  • Figure 14 qPCR detection of cytokine mRNA expression in BMDC.
  • BMDC are primary dendritic cells induced by mouse bone marrow cells.
  • DNA hydrogel (1mg/mL) or Poloxamer gel (2520#) (1mg/mL) were added in vitro to stimulate for 0, 1, 3, and 6 hours respectively, and then collected.
  • RNA was used for qPCR to detect the expression of IL1 ⁇ , IL6, IFN ⁇ and CCL4mRNA.
  • the blue color is the Poloxamer gel (2520#) (1mg/mL) stimulation group, and the red color is the DNA hydrogel stimulation group.
  • the term “about” or “approximately” means a change of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of quantity, level, value, quantity, frequency, percentage, scale, size, quantity, weight or length.
  • the term “about” or “approximately” means ⁇ 15%, ⁇ 10%, ⁇ 9% around a reference quantity, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length , ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2% or ⁇ 1% of quantity, level, value, quantity, frequency, percentage, scale, size, amount, Weight or length range.
  • Consisting essentially of is meant to include any of the elements listed after the phrase “consisting essentially of” and is limited to those elements that do not interfere with or contribute to the activities or actions specified in the disclosure of the listed elements other elements. Thus, the phrase “consisting essentially of” is an indication that the listed elements are required or mandatory, but that no other elements are optional and depend on whether they affect the activities or actions of the listed elements And can exist or not exist.
  • Subject refers to any animal to which a pharmaceutical combination is administered. It includes mammals and non-mammals, including primates, domestic animals, companion animals, laboratory test animals, captive wild animals, birds (including eggs), reptiles and fish. Thus, the term includes, but is not limited to, monkeys, humans, pigs; cattle, sheep, goats, horses, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, chickens, turkeys, ducks, other poultry, Frogs and lizards.
  • Immunologically effective amount means an amount that induces in a subject receiving an adjuvant, antigen, and/or vaccine sufficient to prevent or lessen signs or symptoms of disease (including adverse health effects) caused by infection with a pathogen, such as a virus or bacteria.
  • Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity can be induced.
  • the animal's immune response to the vaccine can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring for signs or symptoms after challenge with the wild-type strain.
  • the protective immunity provided by a vaccine can be assessed by measuring, for example, reduction of clinical signs such as mortality, morbidity, temperature values, general physiological condition, and general health and performance of the subject.
  • the therapeutically effective amount of a vaccine may vary depending on the specific adjuvant used, the specific antigen used, or the condition of the subject, and can be determined by one skilled in the art.
  • Adjuvant refers to a substance that can elicit or enhance an immune response to an antigen. Substances that act directly on the subject's immune system to elicit or enhance an immune response to an antigen are preferred.
  • Identity or “sequence identity” in the context of two nucleic acid sequences or polypeptide sequences or protein sequences means the residues in the two sequences that are identical when aligned for maximum correspondence within a specified comparison window.
  • percent sequence identity When percent sequence identity is used for proteins, recognize that residue positions that are not identical often differ due to conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue with similar chemical properties (such as charge or hydrophobicity) and thus does not alter the functional properties of the molecule.
  • sequences differ due to conservative substitutions the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequence alignment methods for comparison are well known in the art. Therefore, the determination of percent identity between any two sequences can be achieved using mathematical algorithms. Preferably, the measurement can be performed by the method in the embodiments herein.
  • Enrichment refers to an increase in the number of specific cells (eg, immune cells, such as dendritic cells) at a specific site in a subject administered an adjuvant relative to a subject not administered the adjuvant.
  • the number of specific cells (eg, immune cells, eg, dendritic cells) in a specific part of a subject can be measured according to various methods known in the art (eg, flow cytometry). Preferably, the measurement can be performed by the method in the embodiments herein.
  • “Differentiation” refers to a change in cell (eg, immune cell) type, which can be determined according to methods known in the art.
  • “Elevated expression” refers to an increase in the gene expression (such as mRNA expression or protein expression) of a related substance (such as a cytokine) in a specific part of a subject who is administered an adjuvant, relative to a subject who is not administered an adjuvant.
  • the gene expression (e.g., mRNA expression or protein expression) of related substances (e.g., cytokines) in a specific part of the subject administered the adjuvant can be measured according to various methods known in the art (e.g., qPCR, western blot).
  • references are made to "one embodiment”, “some embodiments”, “implementations”, “particular embodiments”, “related embodiments”, “a certain embodiment”, “another embodiment” or “Further embodiments” or combinations thereof mean that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention.
  • the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment.
  • the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
  • the nucleic acid hydrogel described herein can be used as an adjuvant to significantly promote the increase of antigen-specific antibodies, and it can also be used in the preparation of drugs that induce or enhance immune responses.
  • nucleic acid hydrogels herein can refer to those known in the prior art, for example, see Y.Xing, E.Cheng, Y.Yang, P.Chen, Z.Yang, D.Liu.Adv.Mater., 2011 , 23, 1117 and J. Jin, Y. The above non-patent and patent texts are incorporated by reference in their entirety.
  • the nucleic acid hydrogel includes: a scaffold unit equipped with at least three complementary sticky ends; a cross-linking unit equipped with at least two complementary sticky ends, and an aqueous medium; said The scaffold unit and the cross-linking unit are both formed by complementary base pairing of nucleic acids, and the scaffold unit and the cross-linking unit are formed by complementary base pairing of the scaffold sticky end and the cross-linking sticky end. cross-linking to form a three-dimensional network structure.
  • Nucleic acid hydrogels herein may also be referred to as “DNA hydrogels” or “hydrogels.”
  • nucleic acid refers to a polymer formed from ribonucleotides and/or deoxyribonucleotides, preferably deoxyribonucleic acid (DNA).
  • nucleic acids in the nucleic acid hydrogels described herein contain or do not contain modifications.
  • the modification includes linking group modification, fluorescent group modification, quenching group modification, inter-arm modification, nucleotide variant modification, and degenerate base modification.
  • the linking group modification includes amino modification, carboxyl modification, aldehyde modification, acrylamide modification, azide modification, alkynyl modification, diphenylcyclooctyne modification, maleyl modification Imine modification, thiol modification, dithiol modification, ferrocene modification, biotin modification, digoxin modification.
  • the fluorescent group modification includes Pacific Blue, ROX, and Texas red.
  • the quenching group modification includes BHQ1 and BHQ2.
  • the inter-arm modification includes C3/C6 Spacer, Spacer 9/Spacer 18, PC Spacer/PC linker, and tetrahydrofuran modification.
  • the nucleotide variant modifications include phosphorylation, thio, 2-aminopurine, 5-bromodeoxyuracil, deoxyuridine, inverted dT/dG, dideoxycytosine Pyrimidine nucleoside, 5-methylcytosine deoxynucleoside, 5-hydroxymethyldC, N6 methyladenine nucleotide, deoxyinosine nucleoside, locked nucleic acid, 5-nitroindole, 2-methyl Oxygen modification, RNA base, 2-fluoro modification, 2-fluoroRNA, 2'-O-(2-methoxy)ethyl, morpholino, bridged nucleotide, BNA, pyrrole-deoxycytosine.
  • aqueous medium refers to water or an aqueous solution.
  • a buffer solution containing a buffer salt is preferred.
  • the aqueous solution is preferably capable of forming an environment similar to the in vivo environment, such as physiological conditions (37°C, pH 7.2-7.4, 0.9wt% NaCl, isotonic).
  • the scaffold unit may be formed, for example, from three single-stranded nucleic acids, each single-stranded nucleic acid having one sticky end of the scaffold.
  • two of these three nucleic acids are combined through complementary base pairing to form a "Y"-shaped structure, and the sticky ends of the scaffold are respectively located at the three vertices of the "Y".
  • complementary pairing regions are formed between the three nucleic acids, and the length of the complementary pairing regions can be 4 to 150 bp, preferably 5 to 50 bp, more preferably 6 to 30 bp, and more preferably 8 to 20 bp.
  • the cross-linking unit may be formed, for example, from two single-stranded nucleic acids, which are combined through complementary base pairing and each has a cross-linked sticky end.
  • a complementary pairing region is formed between the two nucleic acids, and the length of the complementary pairing region can be 4 to 150 bp, preferably 5 to 100 bp, more preferably 8 to 80 bp, more preferably 10 to 60 bp, more preferably 15 to 50 bp, more preferably 20 to 40 bp.
  • any one of the two single-stranded nucleic acids can be broken into two The above single-stranded nucleic acid.
  • the scaffold unit and the cross-linking unit are cross-linked in a complementary base pairing manner through the scaffold sticky end and the cross-linking sticky end, thereby forming a three-dimensional spatial network structure.
  • the scaffold unit, the cross-linking unit and the three-dimensional spatial network structure are in a stable cross-section under physiological conditions (37°C, pH 7.2-7.4, 0.9wt% NaCl, isotonic). connection status.
  • the length of the scaffold's sticky end or cross-linked sticky end is 4 nt or more, which is beneficial to its stable cross-linked state under physiological conditions.
  • the length of the scaffold adhesive end or the cross-linked adhesive end is 150 nt or less, preferably 50 nt or less, more preferably 30 nt or less, more preferably 20 nt or less.
  • the length of the scaffold sticky end or cross-linked sticky end ranges from 4nt to 150nt.
  • the scaffold unit is formed by three single-stranded nucleic acids in a complementary base pairing manner, and each single-stranded nucleic acid has one sticky end of the scaffold.
  • the cross-linking unit is formed by complementary base pairing of two single-stranded nucleic acids, and each single-stranded nucleic acid has one cross-linked sticky end.
  • the scaffold unit and the cross-linking unit are in a stable cross-linking state under physiological conditions. In some embodiments, the scaffold unit and the cross-linked unit are in a stable cross-linked state under physiological conditions (37°C, pH 7.2-7.4, 0.9 wt% NaCl, isotonic).
  • hydrogels of the invention may or may not contain CpG sequences.
  • the CpG sequence is a palindromic sequence with cytosine-guanine dinucleotide (CpG) as the core, with two purines at the 5' end and two pyrimidines at the 3' end, namely 5'-PurPur-CG-PyrPyr-3' .
  • CpG sequences can be recognized by mammalian cells, thereby triggering a series of body defense mechanisms, including complement activation, phagocytosis, and expression of pro-inflammatory cytokine genes.
  • CpG sequences known to have strong immunostimulatory effects include, for example, 5’-TCCATGACGTTCCTGACGTT-3’, etc.
  • the hydrogel can have appropriate mechanical strength, for example, its mechanical strength can be 0.1 Pa or more, preferably 1 Pa or more, more preferably 10 Pa or more, preferably 10000 Pa or less, more preferably 1000 Pa or less. .
  • nucleic acid hydrogel of the present invention can be constructed from the following DNA sequence:
  • sequences Y1, Y2, and Y3 form a Y-scaffold unit (also known as a "scaffold unit”); the sequences L1 and L2 form an L-cross-linking unit (also known as a "cross-linking unit”); the underline indicates the sticky end of the DNA sequence, Bold font indicates the EcoRI restriction endonuclease recognition sequence; italics indicate mismatch sites formed by exchanging bases.
  • L1C and L2C are used to form the cross-linking units, the resulting hydrogel is called a hard hydrogel, and when L1M and L2M are used to form the cross-linking units, the resulting hydrogel is called a soft hydrogel.
  • nucleic acid hydrogel of the present invention can be constructed from the following DNA sequence:
  • sequences Y1, Y2, and Y3 form a Y-scaffold unit (also known as a "scaffold unit”); the sequences L1 and L2 form an L-cross-linking unit (also known as a "cross-linking unit”); the underline indicates the sticky end of the DNA sequence.
  • the nucleic acid hydrogel is the nucleic acid hydrogel described in the examples.
  • the nucleic acid hydrogel is a nucleic acid hydrogel prepared according to the method described in the embodiments.
  • nucleic acid hydrogel of the present invention there is no particular limitation on the construction of the nucleic acid hydrogel of the present invention.
  • the aqueous medium solution of the scaffold unit and the cross-linked unit can be prepared separately, and then the two are mixed to obtain the nucleic acid hydrogel of the present invention.
  • nucleic acid hydrogel described herein can be used as an adjuvant to enhance the immune response of a subject.
  • nucleic acid hydrogel adjuvant described herein can also be used in combination with other adjuvants such as aluminum adjuvant and CpG adjuvant.
  • an immune response can be any positive change in the immune status of the host, which can include one or more tissues, organs, cells or molecules involved in the maintenance and/or regulation of the immune status of the host any changes in structure or function.
  • the immune response can be detected by any of a variety of well-known parameters, including but not limited to the following detection in vivo or in vitro: detection of soluble immunoglobulins or antibodies; such as cytokines, lymphokines, chemokines Detection of soluble mediators such as hormones, growth factors, and other soluble small peptides, carbohydrates, nucleosides, and/or lipid mediators; changes in cellular activation status determined by altered functional or structural properties of cells in the immune system, such as cell proliferation , altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cellular differentiation of cells of the immune system, including altered surface antigen expression profiles or initiation of apoptosis (programmed cell death); or any Other criteria that can detect the presence of an immune response.
  • soluble immunoglobulins or antibodies such as cytokines, lymphokines, chemokines Detection of soluble mediators such as hormones, growth factors, and other soluble small peptides,
  • Immune responses are generally considered, for example, to differentiate between self and foreign structures at the molecular and cellular levels by the cells and tissues of the host's immune system, but the invention should not be limited in this regard.
  • an immune response may also include changes in the status of the immune system resulting from immune recognition of self-molecules, cells, or tissues, which may accompany many normal states such as typical regulation of immune system components, or may be present in conditions such as in autoimmunity and degeneration. Pathological state of inappropriate autoimmune response observed in disease.
  • the immune response may also include detectable suppression, attenuation, or any other down-regulation, which may be caused by the selected antigen, route of antigen administration, induction of specific tolerance, or other factors.
  • Detection of immune responses induced by the pharmaceutical combinations or nucleic acid hydrogels of the invention can be established by any of a number of well-known immunological assays readily familiar to those of ordinary skill in the art. These assays include, but are not necessarily limited to, the following in vivo or in vitro assays: detection of soluble antibodies; soluble mediators such as cytokines, lymphokines, chemokines, hormones, growth factors, and other soluble small peptides, carbohydrates, nucleosides, and /or detection of lipid mediators; changes in cellular activation status determined by altered functional or structural properties of cells in the immune system, such as cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior ;Cellular differentiation of cells of the immune system, including altered surface antigen expression profiles or initiation of apoptosis (programmed cell death).
  • the adjuvants described herein can be used in combination with an immunologically effective amount of one or more antigens to enhance a subject's immune response to the one or more antigens.
  • the antigen may be any of a variety of substances capable of producing a desired immune response in a subject.
  • Antigens may include nucleotides, polynucleotides, peptides, immunogenic fragments of polypeptides, which may be isolated from the organisms mentioned herein.
  • antigens used in combination with the adjuvants described herein can be from avian herpesvirus, bovine herpesvirus, canine herpesvirus, equine herpesvirus, feline viral rhinotracheitis virus, Marek's disease virus, ovine herpesvirus virus, porcine herpes virus, pseudo Rabies virus, avian paramyxovirus, bovine respiratory syncytial virus, canine distemper virus, canine parainfluenza virus, canine adenovirus, canine parvovirus, bovine parainfluenza virus 3, ovine parainfluenza virus 3, rinderpest virus, border disease virus (Borderdiseasevirus), bovine viral diarrhea virus (BVDV), type I BVDV, type II BVDV, classical swine fever virus, avian leukemia virus, bovine immunodeficiency virus, bovine leukemia virus, bovine tuberculosis virus, porcine infectious anemia Viruses, feline immunodeficiency
  • the antigen is a coronavirus antigen, preferably COVID-19.
  • the antigen is selected from the group consisting of SARS-CoV-2 S protein or fragments thereof, COVID-19 RBD protein or dimers or trimers thereof, etc.
  • the novel coronavirus RBD antigen sequence has at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, more preferably 100% identity.
  • the adjuvant of the present invention can be used in a variety of different vaccines to enhance the effect of different vaccines in eliciting or enhancing immune responses.
  • the adjuvant can be used in the vaccine by administering the adjuvant and the vaccine to the subject at the same time or at intervals, or by mixing the adjuvant and the vaccine and then administering it to the subject, or by other methods suitable in the field. way for vaccines.
  • the adjuvant of the present invention can be used in vaccines selected from the group consisting of mRNA vaccines, inactivated vaccines, attenuated vaccines and recombinant protein vaccines.
  • the adjuvant of the present invention can be used in a coronavirus vaccine selected from HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or new coronavirus (COVID-19).
  • the coronavirus vaccine is selected from the group consisting of mRNA-1273 (Moderna, Inc.), AZD-1222 (AstraZeneca and University of Oxford), BNT162 (Pfizer and BioNTech), CoronaVac (Sinovac), COVILO, NVX -CoV 2372 (NovoVax), SCB-2019 (Sanofi and GSK), ZyCoV-D (Zydus Cadila) and CoVaxin (Bharat Biotech).
  • the adjuvant of the present invention can be used for new coronavirus vaccines, such as Alpha new coronavirus vaccine, Delta new coronavirus vaccine, Omicron new coronavirus vaccine and vaccines of their mutant strains. More preferably, in some embodiments, the vaccine for the Omicron coronavirus and its mutant strains, optionally, the Omicron coronavirus mutant strain is selected from BA.1, BA.2, xe, xl, BA.4, BA.5 and their branches.
  • the administration routes of the adjuvant of the present invention include parenteral, oral, oronasal, intranasal, intratracheal, topical, intravenous (such as intravenous injection), subcutaneous (such as subcutaneous injection), intramuscular (such as intramuscular injection) and other routes.
  • intravenous such as intravenous injection
  • subcutaneous such as subcutaneous injection
  • intramuscular such as intramuscular injection
  • Any suitable device may be used to administer the drug combination, including syringes, droppers, needle-free injection devices, patches, and the like.
  • the route and device chosen will depend on the drug combination, antigen and subject, and are well known to those skilled in the art.
  • Administration is preferably by subcutaneous or intramuscular injection.
  • the adjuvant and antigen of the invention can be administered to a subject simultaneously or at intervals.
  • the adjuvant and antigen are administered to the same site or to different sites on the subject.
  • the adjuvant and the antigen are administered to the same part of the subject; more preferably, the adjuvant and the antigen are administered to the same part of the subject in the same administration method.
  • the adjuvant is mixed with the antigen and administered to the subject.
  • the adjuvant and vaccine of the present invention can be administered to a subject simultaneously or at intervals.
  • the adjuvant and vaccine are administered to the same site or to different sites on the subject.
  • the adjuvant and the vaccine are administered to the same part of the subject; more preferably, the adjuvant and the vaccine are administered to the same part of the subject in the same administration method.
  • the adjuvant is mixed with the vaccine and administered to the subject.
  • the dosage of the adjuvant of the present invention varies depending on the subject, administration method, administration method, etc., but is usually in the range of 1 ⁇ g to 1000 ⁇ g, preferably in the range of 20 ⁇ g to 100 ⁇ g, per adult subject. Apply 2 to 3 times every 4 to 12 weeks.
  • the amount of adjuvant administered is determined by one skilled in the art based on the condition of the subject. In some embodiments, the amount of adjuvant administered is determined by those skilled in the art according to routine methods in the art.
  • DNA hydrogel and “DNA supramolecular hydrogel” described in the following examples all belong to the “nucleic acid hydrogel” of the present invention.
  • Example 1 DNA supramolecular hydrogel promotes the production of IgG antibodies specific for the model antigen OVA.
  • the main induced IgG antibody subtypes are: IgG1, IgG2c, and IgG2b.
  • the preparation method of the DNA hydrogel is as follows: using DNA solid-phase synthesis method to synthesize the DNA single strand required for the hydrogel composition (as shown in Table 3), then purifying the DNA single strand through high performance liquid chromatography, and obtaining the DNA after desalting Single chain aqueous solution. After determining the DNA concentration using UV-visible spectroscopy, the Y1, Y2, and Y3 DNA single strands in an equal molar ratio (or approximately an equal molar ratio) are mixed and lyophilized to form a scaffold unit, and the L1 and sL2 DNA single strands in an equal molar ratio are mixed and frozen. dry to form cross-linked units.
  • aqueous medium is added to the freeze-dried powder of the scaffold unit and the cross-linked unit respectively, and the DNA single strands are assembled in the aqueous medium to form the scaffold unit and the cross-linked unit respectively.
  • the two are then mixed to form a hydrogel, in which the molar ratio of the scaffold unit and the cross-linking unit is 1:1.5, and the solid content of the hydrogel is 3.6%.
  • sequences Y1, Y2, and Y3 form a Y-scaffold unit (also known as a "scaffold unit”); the sequences L1 and L2 form an L-cross-linking unit (also known as a "cross-linking unit”); the underline indicates the sticky end of the DNA sequence.
  • This example uses enzyme-linked immunosorbent assay (ELISA) to measure antibody titers to evaluate the adjuvant effect.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA experiment Coat the enzyme plate with 2 ⁇ g/mL OVA, 50 ⁇ L per well, and coat overnight at 4°C. Discard the liquid in the plate, add 200 ⁇ L of 0.05% PBST buffer to each well and wash for 3 minutes each time, repeat 3 times.
  • DNA hydrogel can significantly promote the production of OVA-specific IgG antibodies (Figure 1B). At the same time, DNA hydrogel can significantly promote the production of OVA-specific IgG1, IgG2c, and IgG2b antibody subtypes, but has no promoting effect on IgG3. ( Figure 1C).
  • This example uses enzyme-linked immunosorbent assay (ELISA) to measure antibody titers to evaluate the adjuvant effect.
  • ELISA enzyme-linked immunosorbent assay
  • each mouse footpad was injected with 30 ⁇ L OVA+PBS, and in the experimental group, each mouse footpad was injected with 30 ⁇ L OVA+DNA hydrogel. 14 days after immunization, 100 ⁇ L of blood was taken from the orbit of the mice, and the blood was taken out and left to stand at room temperature for 2 hours, centrifuged at 800g for 8 minutes, and the upper serum was aspirated out for subsequent ELISA experiments, or the serum was stored in a -80°C refrigerator.
  • ELISA experiment Coat the enzyme plate with 6 ⁇ g/mL DNA hydrogel, 50 ⁇ L per well, and coat overnight at 4°C. Discard the liquid in the plate, add 200 ⁇ L of 0.05% PBST buffer to each well and wash for 3 minutes each time, repeat 3 times.
  • Example 3 Nucleic acid hydrogel promotes the production of new coronavirus antigen RBD-specific antibodies.
  • This example uses enzyme-linked immunosorbent assay (ELISA) to measure antibody titers to evaluate the adjuvant effect.
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 3A different types of RBD antigens (5 ⁇ g) were mixed with PBS, aluminum adjuvant (1:1) or DNA hydrogel respectively to a final volume of 30 ⁇ L. Mice were immunized on days 0, 14, and 28.
  • each mouse was injected with 30 ⁇ L RBD+PBS into the footpad; in the experimental group 1, each mouse was injected with 30 ⁇ L RBD+aluminum adjuvant into the footpad; in the experimental group 2, each mouse Mouse foot pads were injected with 30 ⁇ L RBD+DNA hydrogel.
  • ELISA experiment Coat the enzyme plate with 2 ⁇ g/mL monomeric RBD, 50 ⁇ L per well, and coat overnight at 4°C. Discard the liquid in the plate, add 200 ⁇ L of 0.05% PBST buffer to each well and wash for 3 minutes each time, repeat 3 times.
  • DNA hydrogel can significantly promote the production of RBD-specific IgG antibodies, and the antibody promotion effect is much stronger than that of aluminum adjuvant. This shows that DNA hydrogel can promote the production of antibodies specific to the new coronavirus RBD protein.
  • Example 4 Nucleic acid hydrogel promotes the production of novel coronavirus pseudovirus neutralizing antibodies (NT50).
  • the experimental sample is the serum obtained on day 38 in Example 3.
  • DNA hydrogel can significantly promote the increase in the neutralizing antibody titer of the new coronavirus pseudovirus.
  • Example 5 There is a positive correlation between the neutralizing antibody titer of the new coronavirus pseudovirus and the RBD antibody titer.
  • the experimental data comes from the RBD-specific IgG antibody titer on day 38 in Example 3, and the new coronavirus pseudovirus neutralizing antibody titer on day 38 in Example 4. Correlation analysis was performed on the RBD-specific IgG antibody titer of each mouse serum and the neutralizing antibody titer of the new coronavirus pseudovirus.
  • Example 6 The macroscopic mode of action of nucleic acid hydrogel in regulating antibody response is local effect.
  • This example uses enzyme-linked immunosorbent assay (ELISA) to measure antibody titers to evaluate the adjuvant effect.
  • ELISA enzyme-linked immunosorbent assay
  • experimental group three was immunized with OVA (100 ⁇ g) on the left foot pad, and 30 ⁇ L of DNA hydrogel was immunized on the right foot pad at the same time;
  • experimental group four was immunized with OVA (100 ⁇ g) on the left foot pad, and the left leg muscle was immunized with DNA at the same time 30 ⁇ L of hydrogel;
  • experimental group 5 was immunized with OVA (100 ⁇ g) in the left leg muscle and 30 ⁇ L of DNA hydrogel was immunized with the left foot pad;
  • experimental group 6 was immunized with 30 ⁇ L of OVA (100 ⁇ g) + DNA hydrogel on the left foot pad;
  • 100 ⁇ L of blood was taken from the orbit of the mice, and the blood was taken out and left to stand at room temperature for 2 hours, centrifuged at 800g for 8 minutes, and the upper serum was sucked out for subsequent ELISA experiments, or the serum was stored in
  • ELISA experiment Coat the enzyme plate with 2 ⁇ g/mL OVA, 50 ⁇ L per well, and coat overnight at 4°C. Discard the liquid in the plate, add 200 ⁇ L of 0.05% PBST buffer to each well and wash for 3 minutes each time, repeat 3 times.
  • Example 7 Nucleic acid hydrogel has sustained release effect on antigen in vivo.
  • Cy5 fluorescence was detected on the IVIS Spectrum animal optical live imaging system to characterize the metabolism of OVA-Cy5 antigen at the immune site. This experiment is divided into three groups. Control group one: C57B6/L mouse footpads are immunized with IgG-Cy5 (10 ⁇ g) + PBS; control group two: C57B6/L mouse footpads are immunized with OVA-Cy5 (10 ⁇ g) + PBS; experimental group One C57B6/L mouse footpad was immunized with OVA-Cy5 (10 ⁇ g) + DNA hydrogel. Detect Cy5 fluorescence on the IVIS Spectrun animal optical live imager at 0h, 2h, 6h, 12h, 24h, 36h, 60h, and 84h respectively, and take photos and records.
  • the experimental results are shown in Figure 7.
  • the picture above is a representative graph of the Cy5 fluorescence intensity of the immune site of mice in each group at each time point.
  • the picture below shows the statistical results of fluorescence intensity (Total Radiant efficiency). It can be seen from the results that the OVA-Cy5 fluorescence intensity in the DNA hydrogel group maintained at a high level from 0 to 6 hours, and did not begin to significantly decline until 6 to 12 hours. In the control group, the intensity of Cy5 continued to decrease from 0 to 12 hours.
  • the above experiments illustrate that DNA hydrogel has a sustained-release effect on OVA-Cy5 antigen and delays the metabolism time of OVA-Cy5 at the immune site.
  • Example 8 Nucleic acid hydrogels promote antibody responses independent of their gel-like physical properties.
  • the Poloxamer gel used in this article is prepared according to the mass-volume ratio of two different components to obtain two gels with different Young's modulus.
  • the two Poloxamer gels are named as follows:
  • Poloxamer gel (2520#) is a mixture of 2.5% Poloxamer 188 and 20% Poloxamer 407;
  • Poloxamer gel (2525#) is a mixture of 2.5% Poloxamer 188 and 25% Poloxamer 407.
  • mice were subjected to immunization experiments and divided into 4 groups: mice in the control group were immunized with OVA antigen (100 ⁇ g) + PBS on their left foot pads; mice in the experimental group were immunized with OVA antigen (100 ⁇ g) on their left foot pads.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA experiment Coat the enzyme plate with 2 ⁇ g/mL OVA, 50 ⁇ L per well, and coat overnight at 4°C. Discard the liquid in the plate, add 200 ⁇ L of 0.05% PBST buffer to each well and wash for 3 minutes each time, repeat 3 times.
  • the experimental results are shown in Figure 7.
  • the elastic modulus of Poloxamer gel (2520#) is similar to that of DNA hydrogel.
  • the visual status of Poloxamer gel (2525#) is similar to that of DNA hydrogel.
  • the antibody titer of the DNA hydrogel group is significantly higher than that of DNA hydrogel.
  • Poloxamer gel (2520#) and Poloxamer gel (2525#) groups indicating that DNA hydrogel-induced antibody production does not depend on the physical properties of its gel state
  • Example 9 The gel state of nucleic acid hydrogel delays its own degradation in the body.
  • Cy5.5 fluorescence was detected on the IVIS Spectrum animal optical live imaging system to characterize the metabolism of DNA at the immune site. This experiment is divided into two groups, experimental group one: C57B6/L mouse footpad immune soluble DNA-Cy5.5 (L component-Cy5.5+L component) + PBS, experimental group two C57B6/L mouse footpads Pad immune DNA hydrogel-Cy5 (L component-Cy5.5+Y component)+PBS.
  • the mass of Cy5.5-conjugated DNA in experimental group 1 and experimental group 2 was the same at 0.52 mg, and the total DNA mass of the immunization was also the same at 1.07 mg. Detect Cy5.5 fluorescence on the IVIS Spectrun animal optical live imager at 0h, 2h, 6h, 12h, 24h, 36h, 60h, and 84h, and take photos and records.
  • the experimental results are shown in Figure 7.
  • the picture above is a representative graph of Cy5.5 fluorescence intensity in the immune site of mice in each group at each time point.
  • the picture below shows the statistical results of fluorescence intensity (Total Radiant efficiency). It can be seen from the results that the fluorescence intensity of Cy5.5 in the DNA hydrogel group showed a slow decline process and could be maintained at a high level at 60h or even 80h, while the Cy5 in the soluble DNA group showed a sharp decline at the beginning, with 24h The left and right become almost 0.
  • the above experiments illustrate that the gel state of DNA hydrogel can delay the metabolism of DNA itself in mice, allowing it to remain at the immune site for a longer period of time.
  • Example 10 Nucleic acid hydrogel promotes the enrichment and differentiation of immune cells in draining lymph nodes.
  • This example uses flow cytometry technology to detect the proportion of each cell subpopulation in lymph nodes.
  • OVA antigen 100 ⁇ g
  • PBS PBS
  • aluminum adjuvant 1:1
  • DNA hydrogel DNA hydrogel respectively
  • each mouse in the control group was injected with 30 ⁇ L OVA+PBS into its footpad
  • each mouse in the experimental group was injected with 30 ⁇ L OVA+aluminum adjuvant into its footpad
  • each mouse in the experimental group 2 was injected with 30 ⁇ L OVA+DNA water into its footpad. gel.
  • the popliteal lymph nodes on the ipsilateral side of the mouse immunization site were collected for flow cytometry experiments.
  • Flow cytometry experiment detect plasma cells (B220 low CD138 + Plasma), germinal center B cells (B220 + GL7 + GCB), follicular helper cells (CD4 + PD1 + CXCR5 + Tfh), macrophages ( F4/80 + M ⁇ ) and dendritic cells (CD11c + DC).
  • Example 11 Nucleic acid hydrogel promotes the enrichment of immune cells at immune sites.
  • This example uses flow cytometry technology to detect the proportion of each cell subpopulation in the immune site.
  • OVA antigen 100 ⁇ g
  • PBS PBS
  • aluminum adjuvant 1:1
  • DNA hydrogel DNA hydrogel
  • Example 12 Nucleic acid hydrogel induces high expression of cytokines such as IL-1b, CCL-2, CCL-3, CCL-4 and other mRNAs at immune sites.
  • cytokines such as IL-1b, CCL-2, CCL-3, CCL-4 and other mRNAs at immune sites.
  • Experimental method In this example, real-time fluorescence quantitative PCR (qPCR) was used to detect changes in the mRNA of cytokines related to immune sites.
  • Experimental group DNA hydrogel was injected into mouse footpads for 6 hours. The footpad skin tissues of the control group and the experimental group were collected, RNA was extracted and reverse transcribed into cDNA, and then qPCR was performed to detect the expression of relevant mRNA in the footpad tissues.
  • Example 13 Nucleic acid hydrogel induces high expression of cytokine mRNA in bone marrow-derived macrophages (BMDM).
  • BMDM bone marrow-derived macrophages
  • qPCR real-time fluorescence quantification
  • DNA hydrogel can significantly promote the expression of IL1 ⁇ , IL6, IFN ⁇ and CCL4 mRNA in BMDM, with IL1 ⁇ reaching the highest value at 3 hours and IL6, IFN ⁇ and CCL4 reaching the highest value at 6h.
  • DNA hydrogel has a stronger ability to induce the production of relevant cytokines.
  • Example 14 Nucleic acid hydrogel induces high expression of cytokine mRNA in bone marrow-derived dendritic cells (BMDC).
  • BMDC bone marrow-derived dendritic cells
  • qPCR real-time fluorescence quantification
  • DNA hydrogel can significantly promote the expression of IL1 ⁇ , IL6, IFN ⁇ and CCL4 mRNA in BMDCs, with IL1 ⁇ and IL6 reaching the highest value at 3 hours, and IFN ⁇ and CCL4 reaching the highest value at 6h.
  • DNA hydrogel has a stronger ability to induce the production of relevant cytokines.

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Abstract

本发明提供了一种包含或由核酸水凝胶组成的佐剂及其用途。与传统铝佐剂相比，本发明的佐剂不仅显著诱导新冠RBD蛋白特异性抗体产生，还能够显著提高该特异性抗体的表达水平。

Description

一种包含或由核酸水凝胶组成的佐剂及其用途 技术领域
本发明涉及一种包含或由核酸水凝胶组成的佐剂及其用途，属于疫苗或佐剂领域。
背景技术
自2019年12月以来，严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)在全球迅速传播。SARS-CoV-2传播的强度和速度已经导致了大量人口的发病和死亡，并给世界各地的公共卫生系统和全球经济带来了相当大的压力。虽然可以通过控制人的接触距离，戴口罩，检测和追踪等方法来缓解病毒的传播，但在全球大部分人口接种有效疫苗阻止病毒传播之前，大规模爆发疫情，扰乱经济和社会生活的风险依然存在。疫苗能以相对较低的成本在大量人群中预防疾病和疫情，从而成为缓解新冠肺炎影响的有力工具。因此，开发针对新冠肺炎的疫苗和疗法(佐剂)成为当务之急，疫苗和相应佐剂的研发也重新成为一个非常活跃的研究领域。作为人类有史以来最成功的公共卫生干预措施之一，疫苗接种将在未来全球预防传染病引起死亡方面继续产生巨大的影响。
基于减毒病原体的传统疫苗通常不需要添加佐剂，就具有足够的免疫原性。但减毒病原体通常有获取难度大，获取成本大，安全性差等缺点。相比之下，基于含有有限数量纯化抗原的疫苗在安全性和生产成本方面有巨大的优势，但是由于去除了生物体的致病特性，常常伴随着免疫原性有限的问题。这就需要添加佐剂来诱导有保护性和持久性的免疫应答。在疫苗中添加佐剂可以增强、维持和引导抗原的免疫原性，有效地调节适当的免疫应答，减少所需的抗原量或免疫接种次数，并提高疫苗对新生儿、老年人或免疫缺陷个体的效力。
现有技术报道了核酸水凝胶(如：CN107773527B和CN107779427B中记载的核酸水凝胶)在疫苗组合物中作为抗原的递送载体的用途，但是针对核酸水凝胶自身作为佐剂的用途尚未有研究。
发明内容
发明人发现，核酸水凝胶自身可以作为一种佐剂显著的促进模式抗原OVA特异性的IgG抗体增加。与传统铝佐剂相比，核酸水凝胶不仅提高了新冠RBD蛋白特异性抗体的表达水平，而且产生中和病毒能力更强的中和抗体。
核酸水凝胶在体内具有缓释抗原的作用。核酸水凝胶的三维交联网络结构延缓了核酸水凝胶自身在体内的降解速度。核酸水凝胶能够诱导抗体反应相关免疫细胞在免疫部位和引流淋巴结中的富集及分化，促进抗体产生；核酸水凝胶还能够促进免疫部位抗体反应相关细胞因子(IL-1b，CCL2，CCL3，CCL4)mRNA高表达。在细胞层面，核酸水凝胶促进骨髓来源巨噬细胞和树突细胞相关细胞因子mRNA高表达。
在一方面，本发明提供了一种佐剂，其包含或由核酸水凝胶组成，所述核酸水凝胶包含：
支架单元，该支架单元具备至少三个支架粘性末端，
交联单元，该交联单元具备至少两个交联粘性末端，以及
水性介质；
所述支架单元和所述交联单元均由核酸以碱基互补配对的方式形成，
所述支架单元与所述交联单元通过所述支架粘性末端与所述交联粘性末端以碱基互补配对的方式交联，从而形成三维空间网络结构，
其中，所述支架单元或所述交联单元包含或不包含CpG序列；
其中，所述核酸包含或不包含修饰。在一些实施方式中，所述修饰包括连接基团修饰、荧光基团修饰、淬灭基团修饰、间臂修饰、核苷酸变体修饰、简并碱基修饰。
优选地，在一些实施方式中，所述连接基团修饰包括氨基修饰、羧基修饰、醛基修饰、丙烯酰胺基修饰、叠氮修饰、炔基修饰、二苯基环辛炔修饰、马来酰亚胺修饰、巯基修饰、二硫醇修饰、二茂铁修饰、生物素修饰、地高辛修饰。
优选地，在一些实施方式中，所述荧光基团修饰包括Pacific Blue、ROX、Texas red。
优选地，在一些实施方式中，所述淬灭基团修饰包括BHQ1、BHQ2。
优选地，在一些实施方式中，所述间臂修饰包括C3/C6 Spacer、Spacer 9/Spacer 18、PC Spacer/PC linker、四氢呋喃修饰。
优选地，在一些实施方式中，所述核苷酸变体修饰包括磷酸化、硫代、2-氨基嘌呤、5-溴脱氧尿嘧啶、脱氧尿嘧啶核苷、倒置dT/dG、双脱氧胞嘧啶核苷、5-甲基胞嘧啶脱氧核苷、5-羟甲基dC、N6甲基腺嘌呤核苷酸、脱氧次黄嘌呤核苷、锁核酸、5-硝基吲哚、2-甲氧基修饰、RNA碱基、2-氟修饰、2-氟RNA、2'-O-(2-甲氧基)乙基、吗啉代、桥接核苷酸、BNA、吡咯-脱氧胞嘧啶。
优选地，在一些实施方式中，所述佐剂用于疫苗。
优选地，在一些实施方式中，所述支架粘性末端或交联粘性末端的长度为4nt-150nt。
优选地，在一些实施方式中，所述支架单元由三条单链核酸以碱基互补配对的方式形成，且每一条单链核酸具有一个所述支架粘性末端。
优选地，在一些实施方式中，所述交联单元由两条单链核酸以碱基互补配对的方式形成，且每一条单链核酸具有一个所述交联粘性末端。
优选地，在一些实施方式中，所述支架单元与所述交联单元在生理条件下处于稳定交联状态。
优选地，在一些实施方式中，所述支架单元与所述交联单元在37℃，pH 7.2～7.4,0.9wt％NaCl，等渗的条件下处于稳定交联状态。
在一些实施方式中，所述佐剂用于的疫苗选自mRNA疫苗、灭活疫苗、减毒疫苗和重组蛋白疫苗。在一些实施方式中，所述佐剂用于的疫苗是冠状病毒疫苗，所述冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV或新冠病毒(COVID-19)。优选地，在一些实施方式中，所述冠状病毒疫苗选自mRNA-1273(Moderna,Inc.)、AZD-1222(AstraZeneca and University of Oxford)、BNT162(辉瑞和BioNTech)、CoronaVac(Sinovac)、COVILO、NVX-CoV 2372(NovoVax)、SCB-2019(赛诺菲和GSK)、ZyCoV-D(Zydus Cadila)和CoVaxin(Bharat Biotech)。优选地，在一些实施方式中，所述疫苗是新冠病毒(COVID-19)疫苗，例如阿尔法新冠病毒疫苗、德尔塔新冠病毒疫苗、奥密克戎新冠病毒疫苗及其突变毒株的疫苗，更优选地，用于奥密克戎新冠病毒及其突变毒株的疫苗，任选地，所述奥密克戎新冠突变毒株选自BA.1、BA.2、xe、xl、BA.4、BA.5及其分支。更优选地，在一些实施方式中，用于奥密克戎新冠病毒及其突变毒株的疫苗。在一些实施方式中，所述奥密克戎新冠突变毒株选自BA.1、BA.2、xe、xl、BA.4、BA.5及其分支。
在一方面，本发明提供了一种包括用于形成所述支架单元的核酸，用于形成所述交联单元的核酸，以及水性介质的组合在制备本文所述的佐剂中的用途，其中所述支架单元或所述交联单元包含或不包含CpG序列。
在一方面，本发明提供了一种试剂盒在制备本发明所述的佐剂的用途，其包括：用于形成所述支架单元的核酸和用于形成所述交联单元的核酸；其中，所述支架单元或所述交联单元包含或不包含CpG序列；任选地所述试剂盒还包括说明书。
在一方面，本发明提供了一种本文所述的佐剂在制备用于在受试者中引起或增强免疫应答的药物中的用途。
在一方面，本发明提供了一种用于在受试者中引起或增强免疫应答的佐剂。
在一方面，本发明提供了一种用于在受试者中引起或增强免疫应答的药物组合，其包含(1)本文所述的佐剂；(2)抗原；优选地所述抗原是冠状病毒抗原，所述冠状病毒优选COVID-19。优选地，在一些实施方式中，所述抗原选自新冠病毒S蛋白或其片段、新冠病毒RBD蛋白或其二聚体或其三聚体等。优选地，在一些实施方式中，新冠病毒RBD蛋白的序列与SEQ ID NO：1-3中的任一序列具有至少30％、40％、50％、60％、70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，更优选地具有100％的同一性。在一些实施方式中，本文所述佐剂与抗原在受试者体外不存在物理接触。
在一方面，本发明还提供了一种在受试者中引起或增强免疫应答的方法，其包括将本发明的佐剂或本发明的药物组合施用于受试者。在一些实施方式中，可将本发明的药物组合中的佐剂和抗原共同施用于受试者，在一些实施方式中，可将佐剂和抗原同时或间隔一段时间施用于受试者。间隔一段时间可以是间隔1分钟，2分钟，5分钟，10分钟，30分钟，1小时，2小时或更多或上述任一时间点之间的任一时间。优选地，施用的佐剂和抗原的量是免疫有效量。
在一方面，本发明还提供了一种本文所述的佐剂用于促进免疫细胞在引流淋巴结的富集和分化、促进免疫细胞在受试者的佐剂施用部位的富集、诱导受试者的佐剂施用部位的IL-1b、CCL-2、CCL-3、CCL-4等细胞因子的表达升高、诱导骨髓来源巨噬细胞(BMDM)细胞因子的表达升高或诱导骨髓来源树突细胞(BMDC)细胞因子的表达升高。
本发明的优异技术效果包括但不限于：
(1)本发明的包含或由核酸水凝胶组成的佐剂，能够有效引起或增强受试者对特定抗原的免疫应答；
(2)与传统铝佐剂相比，本发明的包含或由核酸水凝胶组成的佐剂能够产生更好的对特定抗原的免疫应答；
(3)与传统铝佐剂相比，本发明的包含或由核酸水凝胶组成的佐剂不仅提高了新冠病毒特异性抗体的表达水平，而且产生的中和抗体中和病毒的能力显著提高，引起并增强受试者对新冠病毒或新冠病毒抗原的免疫应答；
(4)本发明还发现，本文的包含或不包含CpG序列的核酸水凝胶均可以作为佐剂使用，用于增强机体对特定抗原的免疫应答。
附图说明
图1：ELISA检测OVA特异性IgG抗体及IgG抗体亚型。
(A)，免疫小鼠及样本收集流程示意图：在第0天C57BL/6小鼠脚垫免疫OVA抗原(100μg)+PBS或者OVA(100μg)+DNA水凝胶，第14天眼眶取血收集小鼠血清，进行酶联免疫吸附实验(ELISA)；
(B)，ELISA检测免疫小鼠第14天血清中OVA特异性抗体IgG抗体滴度。蓝色为PBS组，红色为DNA水凝胶组；
(C)，ELISA检测免疫小鼠第14天血清中OVA特异性IgG抗体亚型IgG1，IgG2b，IgG2c以及IgG3的抗体滴度，蓝色为PBS组，红色为DNA水凝胶组。
图2：ELISA检测核酸水凝胶特异性IgG抗体。
ELISA检测免疫小鼠第14天血清中抗DNA特异性的IgG抗体滴度，蓝色为PBS组，红色为DNA水凝胶组。
图3：ELISA检测RBD特异性IgG抗体。
(A)，免疫小鼠及样本收集流程示意图：在第0天，14天以及28天，C57BL/6小鼠分别脚垫免疫各类(单体，二聚体或三聚体)RBD抗原(5μg)+PBS，各类RBD抗原(5μg)+铝佐剂(1:1混合)，各类RBD抗原(5μg)+DNA水凝胶，免疫的总体积30μL，分别在第14天，21天，28天以及38天眼眶取血，收集小鼠血清，进行酶联免疫吸附实验(ELISA)。并用第38天血清进行假病毒中和实验。
(B)，用单体RBD蛋白包被后进行ELISA，检测免疫小鼠各时间点血清中RBD特异性抗体IgG抗体滴度。黑色为PBS组，蓝色为铝佐剂组，红色为DNA水凝胶组的RBD特异性IgG抗体滴度。左图为RBD单体联合不同佐剂免疫小鼠，中图为RBD二聚体不同佐剂免疫小鼠，右图为RBD三聚体不同佐剂免疫小鼠。
图4：新冠假病毒中和抗体滴度检测。
收集RBD二聚体和RBD三聚体联合不同佐剂免疫小鼠的第38天的血清进行假病毒中和实验。黑色为PBS组，蓝色为铝佐剂组，红色为DNA水凝胶组的假病毒中和抗体梯度。
图5：新冠假病毒中和抗体滴度和RBD抗体滴度相关性分析。
对免疫第38天的小鼠血清的RBD特异性IgG抗体滴度和假病毒中和抗体滴度进行相关性分析。黑色为PBS组，蓝色为铝佐剂组，红色为DNA水凝胶组。左图为RBD二聚体联合不同免疫佐剂的结果，右图为RBD三聚体联合不同免疫佐剂的结果。
图6：ELISA检测不同免疫途径诱导OVA特异性抗体滴度。
在第0天C57BL/6小鼠进行免疫实验，黑色组为空白对照；棕色组只在左脚脚垫免疫OVA抗原(100μg)+PBS；绿色组只在左脚脚垫免疫DNA水凝胶30μL；紫色组在左脚脚垫免疫OVA(100μg)，同时右脚脚垫免疫DNA水凝胶30μL；橘色组在左脚脚垫免疫OVA(100μg)，同时左腿肌肉免疫DNA水凝胶30μL；蓝色组在左腿肌肉免疫OVA(100μg)，同时左脚脚垫免疫DNA水凝胶30μL；红色组在左脚脚垫免疫OVA(100μg)+DNA水凝胶30μL；分别在第7天和第14天眼眶取血收集小鼠血清，进行ELISA检测OVA特异性IgG抗体滴度。
图7：活体成像检测抗原在免疫部位降解情况。
C57 B6/L小鼠脚垫免疫OVA-Cy5+PBS或OVA-Cy5+DNA水凝胶或者IgG-Cy5，分别在0h，2h，6h，12h，24h，36h，60h，84h在IVIS Spectrum动物光学活体成像仪检测Cy5荧光。上图为不同时间点的图片展示，下图为Cy5荧光强度统计曲线图。黑色为IgG-Cy5组别，蓝色为OVA-Cy5+PBS组别，红色为OVA-Cy5+DNA水凝胶组别。
图8：ELISA检测不同凝胶诱导OVA特异性抗体滴度。
ELISA检测OVA联合不同凝胶免疫小鼠14天后血清中OVA特异性IgG的抗体滴度，蓝色为OVA+PBS组，紫色为OVA+Poloxamer凝胶(2520#)组，黑色为OVA+Poloxamer凝胶(2525#)组，红色为OVA+DNA水凝胶组。Poloxamer凝胶(2520#)弹性模量和DNA水凝胶近似，Poloxamer凝胶(2525#)视觉凝胶状态和DNA水凝胶近似。
图9：活体成像检测DNA在免疫部位的代谢状况。
C57B6/L小鼠脚垫分别免疫DNA水凝胶-Cy5.5和可溶性组分DNA-Cy5.5，在0h，2h，6h，12h，24h，36h，60h，84h在IVIS Spectrum动物光学活体成像仪检测Cy5.5荧光，上图为不同时间点成像图片展示，下图为Cy5.5荧光强度统计曲线。红色为DNA水凝胶-Cy5.5组，蓝色为可溶性组分DNA-Cy5.5组。
图10：流式细胞技术检测引流淋巴结中免疫细胞图谱。
C57B6/L小鼠脚垫分别免疫OVA(100μg)抗原+PBS，OVA(100μg)抗原+铝佐剂(1:1)，OVA(100μg)抗原+DNA水凝胶，第10天，取小鼠同侧腘窝淋巴结进行流式实验，检测腘窝淋巴结中浆细胞(Plasma)，生发中心B细胞(GCB)，滤泡辅助细胞(Tfh)，巨噬细胞(Mφ)以及树突细胞(DC)的比例。黑色为OVA+PBS组，蓝色为OVA+铝佐剂组，红色为OVA+DNA水凝胶组。
图11：流式细胞技术检测免疫细胞在免疫部位富集图谱。
C57B6/L小鼠脚垫分别免疫OVA(100μg)抗原+PBS，OVA(100μg)抗原+铝佐剂(1:1)，OVA(100μg)抗原+DNA水凝胶，分别在第0,2,3,5,6,8,9,10,11天取免疫部位内容物进行流式实验，检测内容物中巨噬细胞(Mφ)，树突细胞(DC)，B细胞以及T细胞的比例。黑色为OVA+PBS组，蓝色为OVA+铝佐剂组，红色为OVA+DNA水凝胶组。
图12：qPCR检测脚垫组织细胞因子mRNA表达图谱。
C57B6/L小鼠脚垫不免疫或免疫DNA水凝胶6h，去脚垫组织抽提RNA，反转录后进行实时荧光定量PCR(qPCR)检测脚垫组织中不同基因mRNA的表达量。热图中，左侧3列为对照组(0h)，右侧3列为DNA水凝胶免疫6h组。如图所示，热图中不同行代表不同的基因mRNA表达水平。
图13：qPCR检测BMDM细胞因子mRNA表达。
BMDM为小鼠骨髓细胞诱导的原代巨噬细胞，体外分别加入DNA水凝胶(1mg/mL)或Poloxamer凝胶(2520#)(1mg/mL)刺激0,1,3,6小时，收集RNA进行qPCR检测IL1β，IL6，IFNβ以及CCL4mRNA的表达情况。蓝色为Poloxamer凝胶(2520#)(1mg/mL)刺激组，红色为DNA水凝胶刺激组。
图14：qPCR检测BMDC细胞因子mRNA表达。
BMDC为小鼠骨髓细胞诱导的原代树突细胞，体外分别加入DNA水凝胶(1mg/mL)或Poloxamer凝胶(2520#)(1mg/mL)刺激0,1,3,6小时，收集RNA进行qPCR检测IL1β，IL6，IFNβ以及CCL4mRNA的表达情况。蓝色为Poloxamer凝胶(2520#)(1mg/mL)刺激组，红色为DNA水凝胶刺激组。
具体实施方式
本文引用的所有公开文件、专利和专利申请通过引入全文并入本文。
除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域普通技术人员通常理解的相同的含义。为了本发明的目的，下文定义了下述术语。
在本说明书全文，除非上下文另有要求，否则术语“包含”，“包括”、“含有”和“具有”应理解为暗示包括所述步骤或要素或者步骤或要素组，但不排除任何其他步骤或要素或者步骤或要素组。在特定实施方式中，术语“包含”、“包括”、“含有”和“具有”同义使用。
如本文使用的，术语"约"或"大约”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较，改变多达15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。在一个实施方式中，术语"约"或"大约”是指围绕参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度±15％、±10％、±9％、 ±8％、±7％、±6％、±5％、±4％、±3％、±2％或±1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。
“由……组成”意指包括但限于在短语“由……组成”后的任何。因此，短语“由……组成”是指示所列出的要素是需要的或强制性的，并且没有其他要素是可以存在的。
“基本上由……组成”意指包括在短语“基本上由……组成”后列出的任何要素，并且限于不干扰或贡献于所列出的要素的公开内容中指定的活动或动作的其他要素。因此，短语“基本上由……组成”是指示所列出的要素是需要的或强制性的，但没有其他要素是任选的，并且取决于它们是否影响所列出的要素的活动或动作而可以存在或不存在。
“受试者”是指任何需要施用药物组合的动物。其包括哺乳动物和非哺乳动物，包括灵长类、家畜、伴侣动物、实验室试验动物、圈养的野生动物、鸟(包括卵)、爬行动物和鱼。因此，该术语包括，但不限于猴子、人、猪；牛、绵羊、山羊、马、小鼠、大鼠、荷兰猪、仓鼠、兔子、猫、犬、鸡、火鸡、鸭、其它禽类、青蛙和蜥蜴。
“免疫有效量”是指可在接受佐剂、抗原和/或疫苗的受试者内诱导足以预防或减轻由于病原体(诸如病毒或细菌)感染所引起的疾病的征兆或症状(包括不利的健康影响或其并发症)的免疫应答的抗原或疫苗量。可以诱导体液免疫力或细胞介导的免疫力或体液与细胞介导的免疫力二者。动物对疫苗的免疫应答可通过例如测量抗体效价、淋巴细胞增殖分析来间接评估，或在以野生型菌株攻击后通过监控征兆或症状来直接评估。由疫苗提供的保护性免疫力可通过测量例如受试者的临床征兆诸如死亡率、发病率的减少，温度数值，总体生理状况及总体健康和效能来评估。疫苗的治疗有效量可根据所使用的具体佐剂、所使用的具体抗原或受试者的状况而有不同，且可由本领域技术人员决定。
“佐剂”在本文是指可以引起或增强对抗原的免疫应答的物质。优选地直接作用于受试者的免疫系统以引起或增强对抗原的免疫应答的物质。
“同一性”或“序列同一性”在两个核酸序列或多肽序列或蛋白序列的情况中意指在指定比较窗口范围内为最大对应进行比对时，这两个序列中相同的残基。当序列同一性百分数用于蛋白质时，认识到不相同的残基位置经常因保守性氨基酸置换而不同，其中氨基酸残基被替换为具有相似化学特性(例如电荷或疏水性)的其他氨基酸残基并且因而并不改变该分子的功能特性。当序列因保守性置换而不同时，序列同一性百分数可以上调以校正置换的保守性本质。用于比较的序列比对方法是本领域熟知的。因此，使用数学算法可以实现任意两个序列之间同一性百分数的确定。优选地，可以通过本文实施例中的方法进行测量。
“富集”是指相对于未施用佐剂的受试者，施用佐剂的受试者的特定部位的特定细胞(如免疫细胞，例如树突状细胞)的数量增多。受试者的特定部位的特定细胞(如免疫细胞，例如树突状细胞)的数量可以根据本领域已知的各种方法(如：流式细胞术)进行测量。优选地，可以通过本文实施例中的方法进行测量。
“分化”是指细胞(如免疫细胞)类型发生转变，其可根据本领域已知的方法进行测定。
“表达升高”是指相对于未施用佐剂的受试者，施用佐剂的受试者的特定部位的相关物质(如细胞因子)的基因表达(如：mRNA表达或蛋白质表达)升高。施用佐剂的受试者的特定部位的相关物质(如细胞因子)的基因表达(如：mRNA表达或蛋白质表达)可以根据本领域已知的各种方法(如qPCR、western blot)进行测量。
在本说明书全文，提到“一个实施方式”、“一些实施方式”、“实施方式”、“特定实施方式”、“相关实施方式”、“某个实施方式”、“另外的实施方式”或“进一步的实施方式”或其组合意指与所述实施方式结合描述的特定特征、结构或特性被包括在本发明的至少一个实施方式中。因此，前述短语在本说明书全文的各个地方的出现不一定全部指相同实施方式。此外，特定特征、结构或特性可以以任何合适方式在一个或多个实施方式中组合。
核酸水凝胶
本文所述核酸水凝胶能够作为一种佐剂显著的促进抗原特异性抗体的增加，其也能够用于制备引起或增强免疫应答的药物中的用途。
本文中的核酸水凝胶可以参照现有技术中已知的那些，例如参见Y.Xing,E.Cheng,Y.Yang,P.Chen,Z.Yang,D.Liu.Adv.Mater.,2011,23，1117以及J.Jin,Y.Xing,Y.Xi,X.Liu,Z.Yang,S.Wang,D.Liu.Adv.Mater.,2013,257,4714，CN107773527B和CN107779427B，现将上述非专利和专利文本以引用的方式全文并入本文中。
在一些实施方式中，核酸水凝胶包括：支架单元，该支架单元具备至少三个互补的粘性末端；交联单元，该交联单元具备至少两个互补的粘性末端，以及水性介质；所述支架单元和所述交联单元均由核酸以碱基互补配对的方式形成，所述支架单元与所述交联单元通过所述支架粘性末端与所述交联粘性末端以碱基互补配对的方式交联，从而形成三维空间网络结构。
本文中的核酸水凝胶也可称为“DNA水凝胶”或“水凝胶”。
在本文中，核酸是指由核糖核苷酸和/或脱氧核糖核苷酸形成的聚合物，优选为脱氧核糖核酸(DNA)。
在一些实施方式中，本文所述的核酸水凝胶中的核酸包含或不包含修饰。
在一些实施方式中，所述修饰包括连接基团修饰、荧光基团修饰、淬灭基团修饰、间臂修饰、核苷酸变体修饰、简并碱基修饰。
优选地，在一些实施方式中，所述连接基团修饰包括氨基修饰、羧基修饰、醛基修饰、丙烯酰胺基修饰、叠氮修饰、炔基修饰、二苯基环辛炔修饰、马来酰亚胺修饰、巯基修饰、二硫醇修饰、二茂铁修饰、生物素修饰、地高辛修饰。
优选地，在一些实施方式中，所述荧光基团修饰包括Pacific Blue、ROX、Texas red。
优选地，在一些实施方式中，所述淬灭基团修饰包括BHQ1、BHQ2。
优选地，在一些实施方式中，所述间臂修饰包括C3/C6 Spacer、Spacer 9/Spacer 18、PC Spacer/PC linker、四氢呋喃修饰。
优选地，在一些实施方式中，所述核苷酸变体修饰包括磷酸化、硫代、2-氨基嘌呤、5-溴脱氧尿嘧啶、脱氧尿嘧啶核苷、倒置dT/dG、双脱氧胞嘧啶核苷、5-甲基胞嘧啶脱氧核苷、5-羟甲基dC、N6甲基腺嘌呤核苷酸、脱氧次黄嘌呤核苷、锁核酸、5-硝基吲哚、2-甲氧基修饰、RNA碱基、2-氟修饰、2-氟RNA、2'-O-(2-甲氧基)乙基、吗啉代、桥接核苷酸、BNA、吡咯-脱氧胞嘧啶。
在本文中，水性介质是指水或水溶液。作为所述水溶液，优选包含缓冲盐的缓冲液。所述水溶液优选能够形成与体内环境相似的环境，例如生理条件(37℃，pH 7.2～7.4,0.9wt％NaCl,等渗)。
在一些实施方式中，所述支架单元可以例如由三条单链核酸形成，且每一条单链核酸具有一个所述支架粘性末端。在一些实施方式中，这三条核酸之间两两通过碱基互补配对的方式结合，形成“Y”字型结构，所述支架粘性末端分别处于“Y”字的三个顶点。在一些实施方式中，所述三条核酸之间两两形成有互补配对区，该互补配对区的长度可以是4～150bp、优选5～50bp、更优选6～30bp、更优选8～20bp。
在一些实施方式中，所述交联单元可以例如由两条单链核酸形成，这两条核酸之间通过碱基互补配对的方式结合，且各具有一个交联粘性末端。在一些实施方式中，所述两条核酸之间形成有互补配对区，该互补配对区的长度可以是4～150bp、优选5～100bp、更优选8～80bp、更优选10～60bp、更优选15～50bp、更优选20～40bp。而且，在一些实施方式中，在所述交联单元能够具有所述互补配对区和所述交联粘性末端的前提下，所述两条单链核酸中的任意一条可以被断开成两条以上的单链核酸。
在一些实施方式中，所述支架单元与所述交联单元通过所述支架粘性末端与所述交联粘性末端以碱基互补配对的方式交联，从而形成三维空间网络结构。
优选地，在一些实施方式中，所述支架单元、所述交联单元以及所述三维空间网络结构在生理条件下(37℃，pH 7.2～7.4,0.9wt％NaCl,等渗)处于稳定交联状态。
优选地，在一些实施方式中，所述支架粘性末端或交联粘性末端的长度为4nt以上，这样有利于其在生理条件下处于稳定交联状态。优选地，所述支架粘性末端或交联粘性末端的长度为150nt以下、优选50nt以下、更优选30nt以下、更优选20nt以下。在一些实施方式中，支架粘性末端或交联粘性末端的长度为4nt-150nt。
在一些实施方式中，支架单元由三条单链核酸以碱基互补配对的方式形成，且每一条单链核酸具有一个所述支架粘性末端。在一些实施方式中，交联单元由两条单链核酸以碱基互补配对的方式形成，且每一条单链核酸具有一个所述交联粘性末端。
在一些实施方式中，所述支架单元与所述交联单元在生理条件下处于稳定交联状态。在一些实施方式中，所述支架单元与所述交联单元在生理条件(37℃，pH7.2～7.4,0.9wt％NaCl，等渗)条件下处于稳定交联状态。
在一些实施方式中，本发明的水凝胶可以包含或不包含CpG序列。CpG序列是以胞嘧啶鸟嘌呤二核苷酸(CpG)为核心的回文序列，5’端为2个嘌呤，3’端为2个嘧啶，即5’-PurPur-CG-PyrPyr-3’。CpG序列可被哺乳动物细胞识别，从而触发一系列机体防御机制，包括补体激活、吞噬作用和致炎细胞因子基因的表达等。目前已知具有较强免疫刺激作用的CpG序列有例如5’-TCCATGACGTTCCTGACGTT-3’等。
优选地，在一些实施方式中，所述水凝胶可以具有适宜的机械强度，例如其机械强度可以为0.1Pa以上、优选1Pa以上、更优选10Pa以上，优选为10000Pa以下，更优选为1000Pa以下。
在一个具体的例子中，本发明的核酸水凝胶可由以下DNA序列构建：
表1：构建DNA水凝胶所用的DNA信息-例子1
序列Y1,Y2,Y3形成Y-支架单元(又称为“支架单元”)；序列L1和L2形成L-交联单元(又称为“交联单元”)；下划线表示DNA序列的粘性末端，粗体表示EcoR I限制性内切酶识别序列；斜体表示通过交换碱基形成的错配位点。当使用L1C和L2C形成交联单元时，所得水凝胶称为硬水凝胶，当使用L1M和L2M形成交联单元时，所得水凝胶称为软水凝胶。
在另一个具体的例子中，本发明的核酸水凝胶可由以下DNA序列构建：
表2：构建DNA水凝胶所用的DNA信息-例子2
序列Y1,Y2,Y3形成Y-支架单元(又称为“支架单元”)；序列L1和L2形成L-交联单元(又称为“交联单元”)；下划线表示DNA序列的粘性末端。
优选地，在一些实施方式中，核酸水凝胶是实施例中记载的核酸水凝胶。优选地，在一些实施方式中，核酸水凝胶是根据实施例中记载的方法制备得到的核酸水凝胶。
对于本发明的核酸水凝胶的构建没有特殊限制，例如可以分别制备所述支架单元和所述交联单元的水性介质溶液，然后将两者混合，得到本发明的核酸水凝胶。
本文所述的核酸水凝胶可以作用佐剂使用，用以增强受试者的免疫应答。在一些实施方式中，本文所述的核酸水凝胶佐剂还可以与铝佐剂、CpG佐剂等其他佐剂联合使用。
免疫应答
本发明因此提供了在能够引起或增强免疫应答的(即，例如，相对于本领域的技术人员熟悉的适当对照，以统计学上显著的方式引起或增强)药物组合。正如本领域的普通技术人员已知的，免疫应答可以是宿主免疫状态的任何积极的改变，其可以包括参与宿主免疫状态的维持和/或调节的一种或多种组织、器官、细胞或分子的结构或功能的任何改变。通常，可以通过多种公知的参数的任一种检测免疫应答，这些参数包括但不限于体内或体外的下述检测:可溶性免疫球蛋白或抗体的检测；诸如细胞因子、淋巴因子、趋化因子、激素、生长因子等可溶性介质以及其它可溶性小肽、碳水化合物、核苷和/或脂质介质的检测；由免疫系统中细胞改变的功能或结构特性确定的细胞激活状态的变化，例如细胞增殖、改变的运动性、诸如特异性基因表达或溶细胞行为的专门活性诱导；通过免疫系统的细胞的细胞分化，包括改变的表面抗原表达谱或凋亡的启动(程序性细胞死亡)；或者任何其它可以检测免疫应答存在的标准。
免疫应答通常被认为是，例如，通过宿主免疫系统的细胞和组织在分子和细胞水平上区分自身结构和异已结构，但本发明不应局限于此。例如，免疫应答还可以包括由自身分子、细胞或组织的免疫识别导致的免疫系统状态变化，这可以伴随诸如免疫系统组分的典型调节的许多正常状态，或者可以存在于诸如在自身免疫和退行性疾病中观测到的不适当的自身免疫应答的病理状态。作为另一个实例，除了诱导特定免疫系统活性的上调(诸如抗体和/或细胞因子产生，或者细胞介导的免疫激活)外，免疫应答还可以包括可检测的免疫的抑制、消弱或任何其它的下调，这可以是由选择的抗原、抗原给药途径、特异耐受诱导或其它因素导致的。
可以通过本领域的普通技术人员易于熟悉的许多公知的免疫学测定的任一种来建立由本发明的药物组合或核酸水凝胶诱导的免疫应答的检测。这些测定包括，但不必限于体内或体外的下述检测：可溶性抗体的检测；诸如细胞因子、淋巴因子、趋化因子、激素、生长因子等可溶性介质以及其它可溶性小肽、碳水化合物、核苷和/或脂质介质的检测；由免疫系统中细胞改变的功能或结构特性来确定的细胞激活状态的变化，例如细胞增殖、改变的运动性、诸如特异性基因表达或溶细胞行为的专门活性诱导；通过免疫系统的细胞的细胞分化，包括改变的表面抗原表达谱或凋亡的启动(程序性细胞死亡)。进行这些和类似测定的操作步骤是公知的，并且可以在例如Lefkovits(Immunology Methods Manual:The Comprehensive Sourcebook of Techniques(免疫学方法手册：全面的技术读物资料)，1998；也可参见Current Protocols in Immunology(免疫学实验指南)；还可参见，例如,Weir,Handbook of Experimental Immunology(实验免疫学手册)，1986Blackwell Scientific,Boston，MA；Mishell and Shigii(eds.)Selected Methods in Cellular Immunology(细胞免疫学方法精选)，1979Freeman Pub I i sh ing,San Francisco,CA；Green and Reed,1998Science281:1309和其中引用的参考文件)中找到。
抗原
本文所述的佐剂可与一种或多种免疫有效量的抗原联合使用，以增强受试者对该一种或多种抗原的免疫应答。所述抗原可为能在受试者中产生期望的免疫应答的多种物质中的任一一种。抗原可包括核苷酸、多核苷酸、肽、多肽的免疫原性片段，其可分离自本文中所提及的生物。
一些与本文所述的佐剂联合使用的抗原可以来自鸟疱疹病毒、牛疱疹病毒、犬疱疹病毒、马疱疹病毒、猫病毒性鼻气管炎病毒、马立克氏病病毒(Marek'sdiseasevirus)、羊疱疹病毒、猪疱疹病毒、伪 狂犬病病毒、鸟副黏病毒、牛呼吸道合胞体病毒、犬瘟热病毒、犬副流感病毒、犬腺病毒、犬细小病毒、牛副流感病毒3、羊副流感病毒3、牛疫病毒、边界病病毒(Borderdiseasevirus)、牛病毒性腹泻病毒(BVDV)、I型BVDV、II型BVDV、古典猪痕病毒(Classicalswinefevervirus)、鸟白血病病毒、牛免疫缺陷病毒、牛白血病病毒、牛结核病病毒、猪感染性贫血病毒、猫免疫缺陷病毒、猫白血病病毒(FeLV)、新城疫病毒(NewcastleDiseasevirus)、羊进行性肺炎病毒、羊肺腺癌病毒、犬冠状病毒(CCV)、泛嗜性CCV(pantropicCCV)、犬呼吸道冠状病毒、牛冠状病毒、猫杯状病毒、猫肠冠状病毒、猫感染性腹膜炎病毒、猪流行性腹泻病毒、猪凝血性脑骨髓炎病毒、猪细小病毒、I型猪环病毒(PCV)、II型PCV、猪生殖及呼吸综合征(PRRS)病毒、传染性胃肠炎病毒、火鸡冠状病毒、牛流行热病毒、狂犬病轮状病毒、水泡性口炎病毒、慢病毒、鸟流感病毒、鼻病毒、马流感病毒、猪流感病毒、犬流感病毒、猫流感病毒、人流感病毒、东方马脑炎病毒(EEE)、委内瑞拉马脑炎病毒、西尼罗河病毒、西方马脑炎病毒、人免疫缺陷病毒、人乳头状瘤病毒、水痘带状疱疹病毒、乙型肝炎病毒、鼻病毒和麻疹病毒、新冠病毒及上述任何组合。
优选地，抗原是冠状病毒抗原，所述冠状病毒优选COVID-19。优选地，在一些实施方式中，所述抗原选自新冠病毒S蛋白或其片段、新冠病毒RBD蛋白或其二聚体或其三聚体等。
优选地，新冠病毒RBD抗原序列与SEQ ID NO：1-3中的任一序列具有至少30％、40％、50％、60％、70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，更优选地具有100％同一性。
疫苗
本发明的佐剂可以用于各种不同的疫苗，以增强不同疫苗的引起或增强免疫应答的效果。将佐剂用于疫苗的方式可以是将佐剂与疫苗同时或间隔一段时间施用于受试者，也可以是将佐剂与疫苗混合后施用于受试者，还可以是以其他本领域合适的方式用于疫苗。
在一些实施方式中，本发明的佐剂可以用于的疫苗选自mRNA疫苗、灭活疫苗、减毒疫苗和重组蛋白疫苗。在一些实施方式中，本发明的佐剂可以用于冠状病毒疫苗，所述冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV或新冠病毒(COVID-19)。优选地，在一些实施方式中，冠状病毒疫苗选自mRNA-1273(Moderna,Inc.)、AZD-1222(AstraZeneca and University of Oxford)、BNT162(辉瑞和BioNTech)、CoronaVac(Sinovac)、COVILO、NVX-CoV 2372(NovoVax)、SCB-2019(赛诺菲和GSK)、ZyCoV-D(Zydus Cadila)和CoVaxin(Bharat Biotech)。
优选地，在一些实施方式中，本发明的佐剂可以用于新冠病毒疫苗，例如阿尔法新冠病毒疫苗、德尔塔新冠病毒疫苗、奥密克戎新冠病毒疫苗及其突变毒株的疫苗。更优选地，在一些实施方式中，用于奥密克戎新冠病毒及其突变毒株的疫苗，任选地，所述奥密克戎新冠突变毒株选自BA.1、BA.2、xe、xl、BA.4、BA.5及其分支。
药物的施用
本发明所述的佐剂的施用途径包括肠胃道外、口、口鼻、鼻内、气管内、局部、静脉(如静脉注射)、皮下(如皮下注射)、肌肉(如肌肉注射)等途径。任何合适的装置均可用来施用药物组合，包括注射器、滴管、无针注射装置、贴片等等。被选用的途径及装置取决于药物组合、抗原及受试者，这些是本领域技术人员公知的。优选地以皮下注射或肌肉注射的方式施用。
在一些实施方式中，可将本发明的佐剂和抗原同时或间隔一段时间施用于受试者。在一些实施方式中，将佐剂和抗原施用于受试者的同一部位或不同部位。优选地，将佐剂和抗原施用于受试者的同一部位；更优选地，将佐剂和抗原以同一种给药方式施用于受试者的同一部位。在一些实施方式中，将佐剂与抗原混合后施用于受试者。
在一些实施方式中，可将本发明的佐剂和疫苗同时或间隔一段时间施用于受试者。在一些实施方式中，将佐剂和疫苗施用于受试者的同一部位或不同部位。优选地，将佐剂和疫苗施用于受试者的同一部位；更优选地，将佐剂和疫苗以同一种给药方式施用于受试者的同一部位。在一些实施方式中，将佐剂与疫苗混合后施用于受试者。
本发明的佐剂的施用量因受试者、施用方法、施用方式等而异，通常对于1个成人受试者，在每一次1μg至1000μg的范围施用、优选20μg至100μg的范围施用，通常4～12周施用2次～3次。在一些实施方式中，佐剂的施用量由本领域技术人员根据受试者的状态确定。在一些实施方式中，佐剂的施用量由本领域技术人员根据本领域的常规方法来确定。
实施例
本发明通过下述实施例进行了说明，但应当理解的是，下述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。对于本领域技术人员来说，本发明可以有各种更改和变化，包括技术特征的组合、重组。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
以下实施例中所述的“DNA水凝胶”、“DNA超分子水凝胶”均属于本发明所述的“核酸水凝胶”。
实施例1：DNA超分子水凝胶促进模式抗原OVA特异性IgG抗体产生。主要诱导的IgG抗体亚型为：IgG1，IgG2c，IgG2b。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，OVA抗原(购自Sigma公司)，DNA水凝胶。
所述DNA水凝胶的制备方法为：使用DNA固相合成法合成水凝胶组成所需的DNA单链(如表3)，之后通过高效液相色谱纯化DNA单链，除盐后得到DNA单链水溶液。使用紫外-可见光谱法确定DNA浓度后，将等摩尔比(或约等摩尔比)的Y1,Y2,Y3 DNA单链混合冻干形成支架单元，等摩尔比的L1和sL2 DNA单链混合冻干形成交联单元。分别在所述支架单元和所述交联单元的冻干粉中加入水性介质，DNA单链在水性介质中组装分别形成所述支架单元和所述交联单元。然后将两者混合即形成水凝胶，其中支架单元和交联单元的摩尔比为1:1.5，水凝胶固含量为3.6％。
表3：构建DNA水凝胶所用的DNA序列信息
序列Y1,Y2,Y3形成Y-支架单元(又称为“支架单元”)；序列L1和L2形成L-交联单元(又称为“交联单元”)；下划线表示DNA序列的粘性末端。
实验方法：本实施例采用酶联免疫吸附实验(ELISA)测定抗体滴度来评估佐剂作用。将OVA抗原(100μg)分别与PBS或者DNA水凝胶混成终体积为30μL。对照组每只小鼠脚垫注射30μL OVA+PBS，实验组每只小鼠脚垫注射30μL OVA+DNA水凝胶。免疫14天后，对小鼠进行眼眶取血100μL，取出血液室温静置2小时，800g离心8分钟，将上层血清吸出，进行后续ELISA实验，或将血清保存在-80℃冰箱。
ELISA实验：用2μg/mL OVA包被酶标板，每孔50μL，4℃包被过夜。弃去板中液体，每孔加入200μL 0.05％PBST缓冲液洗涤，每次3min，重复3次。每孔加入120μL 2％BSA封闭液，37℃封闭2h；PBST洗3次；每孔中加入用1％BSA+0.05％PBST稀释的血清50μL，室温孵育2h；PBST洗涤5次；每孔加入Anti-mouse IgG(或相应抗体亚型)-HRP抗体50μL，室温孵育1h；PBST洗涤5次；每孔加入TMB底物显色液50μL，显色(蓝色)；每孔加入50μL稀硫酸终止液(亮黄色)，在酶标仪上读出OD450值。测定抗体滴度(将大于阴性对照OD450数值的两倍的最大稀释倍数定义为抗体滴度)。
实验步骤及结果参照图1。DNA水凝胶可以显著的促进OVA特异性IgG抗体的产生(图1B)，同时，DNA水凝胶可以明显的促进OVA特异性IgG1，IgG2c，IgG2b抗体亚型的产生，但对IgG3没有促进作用(图1C)。
实施例2：核酸水凝胶不诱导抗自身DNA序列抗体产生
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，OVA抗原(购自Sigma公司)，DNA水凝胶(制备方法同实施例1)
实验方法：本实施例采用酶联免疫吸附实验(ELISA)测定抗体滴度来评估佐剂作用。
将OVA抗原(100μg)分别与PBS或者DNA水凝胶混成终体积为30μL。对照组每只小鼠脚垫注射30μL OVA+PBS，实验组每只小鼠脚垫注射30μL OVA+DNA水凝胶。免疫14天后，对小鼠进行眼眶取血100μL，取出血液室温静置2小时，800g离心8分钟，将上层血清吸出，进行后续ELISA实验，或将血清保存在-80℃冰箱。
ELISA实验：用6μg/mL DNA水凝胶包被酶标板，每孔50μL，4℃包被过夜。弃去板中液体，每孔加入200μL 0.05％PBST缓冲液洗涤，每次3min，重复3次。每孔加入120μL 2％BSA封闭液，37℃封闭2h；PBST洗3次；每孔中加入用1％BSA+0.05％PBST稀释的血清50μL，室温孵育2h；PBST洗涤5次；每孔加入Anti-mouse IgG-HRP抗体50μL，室温孵育1h；PBST洗涤5次；每孔加入TMB底物显色液50μL，显色(蓝色)；每孔加入50μL稀硫酸终止液(亮黄色)，在酶标仪上读出OD450值。测定抗体滴度(将大于阴性对照OD450数值的两倍的最大稀释倍数定义为抗体滴度)。
实验结果参照图2。PBS组和DNA水凝胶组的DNA特异性IgG抗体滴度水平相当，即DNA水凝胶不诱导DNA特异性IgG抗体的产生
实施例3：核酸水凝胶促进新冠抗原RBD特异性抗体产生。
实验材料：野生型C57BL/6小鼠，RBD抗原(序列如SEQ ID NO:1所示)、RBD抗原二聚体，RBD抗原三聚体，DNA水凝胶(制备方法同实施例1)，铝佐剂(购自Thermo公司)
实验方法：本实施例采用酶联免疫吸附实验(ELISA)测定抗体滴度来评估佐剂作用。如图3A所示，不同类别RBD抗原(5μg)分别与PBS、铝佐剂(1:1)或DNA水凝胶混成终体积为30μL。在第0,14,28天对小鼠进行免疫，对照组每只小鼠脚垫注射30μL RBD+PBS；实验组一每只小鼠脚垫注射30μL RBD+铝佐剂；实验组二每只小鼠脚垫注射30μL RBD+DNA水凝胶。分别在第14，21，28，38天对小鼠进行眼眶取血100μL，取出血液室温静置2小时，800g离心8分钟，将上层血清吸出，进行后续ELISA实验，或将血清保存在-80℃冰箱。
ELISA实验：用2μg/mL单体RBD包被酶标板，每孔50μL，4℃包被过夜。弃去板中液体，每孔加入200μL 0.05％PBST缓冲液洗涤，每次3min，重复3次。每孔加入120μL 2％BSA封闭液，37℃封闭2h；PBST洗3次；每孔中加入用1％BSA+0.05％PBST稀释的血清50μL，室温孵育2h；PBST洗涤5次；每孔加入Anti-mouse IgG-HRP抗体50μL，室温孵育1h；PBST洗涤5次；每孔加入TMB底物显色液50μL，显色(蓝色)；每孔加入50μL稀硫酸终止液(亮黄色)，在酶标仪上读出OD450值。测定抗体滴度(将大于阴性对照OD450数值的两倍的最大稀释倍数定义为抗体滴度)。
实验步骤及结果参照图3。在RBD单体，RBD二聚体和RBD三聚体免疫组别中，DNA水凝胶能显著促进RBD特异性IgG抗体的产生，抗体促进作用远强于铝佐剂。说明DNA水凝胶可以促进新冠RBD蛋白特异性抗体产生。
实施例4：核酸水凝胶促进新冠假病毒中和抗体(NT50)产生。
实验样品为实施例3中第38天获取的血清。
实验方法：96孔细胞培养板中加入150μL不同稀释倍数血清，每孔中加入50μL新冠假病毒(带荧光素酶)，37℃孵育1小时，然后每孔中加入适当浓度的100μL过表达ACE2的Huh-7细胞，培养2-3天，弃去上清，裂解细胞，加入荧光素酶反应底物，测定吸光值。计算获得假病毒中和抗体滴度。
实验结果参照图4。在二聚体RBD免疫组以及三聚体RBD免疫组中，与对照组以及铝佐剂组相比，DNA水凝胶都可以显著促进新冠假病毒中和抗体滴度的增加。
实施例5：新冠假病毒中和抗体滴度和RBD抗体滴度呈正相关。
实验数据来自实施例3中第38天的RBD特异性IgG抗体滴度，以及实施例4中第38天的新冠假病毒中和抗体滴度。将每一只小鼠血清的RBD特异性IgG抗体滴度和新冠假病毒中和抗体滴度一一对应进行相关性分析。
实验结果参照图5。新冠假病毒中和抗体滴度与RBD特异性IgG抗体滴度呈显著正相关
实施例6：核酸水凝胶调控抗体应答的宏观作用模式为局部效应。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，OVA抗原(购自Sigma公司)，DNA水凝胶(制备方法同实施例1)
实验方法：本实施例采用酶联免疫吸附实验(ELISA)测定抗体滴度来评估佐剂作用。在第0天C57BL/6小鼠进行免疫实验，分为7组：空白对照组；实验组一左脚脚垫免疫OVA抗原(100μg)+PBS；实验组二只在左脚脚垫免疫DNA水凝胶30μL；实验组三在左脚脚垫免疫OVA(100μg)，同时右脚脚垫免疫DNA水凝胶30μL；实验组四在左脚脚垫免疫OVA(100μg)，同时左腿肌肉免疫DNA水凝胶30μL；实验组五在左腿肌肉免疫OVA(100μg)，同时左脚脚垫免疫DNA水凝胶30μL；实验组六在左脚脚垫免疫OVA(100μg)+DNA水凝胶30μL；分别在免疫后第7天和第14天，对小鼠进行眼眶取血100μL，取出血液室温静置2小时，800g离心8分钟，将上层血清吸出，进行后续ELISA实验，或将血清保存在-80℃冰箱。
ELISA实验：用2μg/mL OVA包被酶标板，每孔50μL，4℃包被过夜。弃去板中液体，每孔加入200μL 0.05％PBST缓冲液洗涤，每次3min，重复3次。每孔加入120μL 2％BSA封闭液，37℃封闭2h；PBST洗3次；每孔中加入用1％BSA+0.05％PBST稀释的血清50μL，室温孵育2h；PBST洗涤5次；每孔加入Anti-mouse IgG-HRP抗体50μL，室温孵育1h；PBST洗涤5次；每孔加入TMB底物显色液50μL，显色(蓝色)；每孔加入50μL稀硫酸终止液(亮黄色)，在酶标仪上读出OD450值。测定抗体滴度(将大于阴性对照OD450数值的两倍的最大稀释倍数定义为抗体滴度)。
实验步骤及结果参照图6。当DNA水凝胶和OVA抗原同时注射在脚垫部位可以产生最高的OVA特异性IgG抗体滴度。说明DNA水凝胶调控抗体应答具有局部效应。不是系统性的全局性的作用。
实施例7：核酸水凝胶在体内对抗原具有缓释作用。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)DNA水凝胶以及IgG-Cy5(制备方法同实施例1)；OVA-Cy5(购自飞默生物)
实验方法：本实施例在IVIS Spectrum动物光学活体成像系统检测Cy5荧光来表征OVA-Cy5抗原在免疫部位的代谢。本实验分为三组，对照组一：C57B6/L小鼠脚垫免疫IgG-Cy5(10μg)+PBS，对照组二C57B6/L小鼠脚垫免疫OVA-Cy5(10μg)+PBS；实验组一C57B6/L小鼠脚垫免疫OVA-Cy5(10μg)+DNA水凝胶。分别在第0h，2h，6h，12h，24h，36h，60h，84h在IVIS Spectrun动物光学活体成像仪检测Cy5荧光，并拍照记录。
实验结果参照图7。上图为各组各时间点小鼠免疫部位Cy5荧光强度代表图。下图为荧光强度(Total Radiant efficiency)统计结果。从结果中可以看出DNA水凝胶组中OVA-Cy5荧光强度在0-6h维持在一个高水平，6-12小时才开始出现显著的下降过程。而对照组中Cy5的强度在0-12小时过程中一直持续下降。以上实验说明了DNA水凝胶对OVA-Cy5抗原具有缓释作用，延缓了OVA-Cy5在免疫部位的代谢时间。
实施例8：核酸水凝胶促进抗体反应不依赖于其凝胶状物理性质。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，OVA抗原(购自Sigma公司)，DNA水凝胶(制备方法同实施例1)，Poloxamer凝胶(成分Poloxamer 188购自Sigma公司；成分Poloxamer 407购自碧云天公司)
本文中所用的Poloxamer凝胶是根据两种不同组分的质量体积比配制得到两种不同杨氏模量的凝胶，两种Poloxamer凝胶的命名如下：
Poloxamer凝胶(2520#)为2.5％Poloxamer188和20％Poloxamer 407混合而成；
Poloxamer凝胶(2525#)为2.5％Poloxamer188和25％Poloxamer 407混合而成。
实验方法：本实施例采用酶联免疫吸附实验(ELISA)测定抗体滴度来评估佐剂作用。在第0天C57BL/6小鼠进行免疫实验，分为4组：对照组小鼠左脚脚垫免疫OVA抗原(100μg)+PBS；实验组一小鼠左脚脚垫免疫OVA抗原(100μg)+Poloxamer凝胶(2520#)30μL；实验组二小鼠左脚脚垫免疫OVA抗原(100μg)+Poloxamer凝胶(2525#)30μL，；实验组三小鼠左脚脚垫免疫OVA(100μg) +DNA水凝胶30μL；在免疫后第14天，对小鼠进行眼眶取血100μL，取出血液室温静置2小时，800g离心8分钟，将上层血清吸出，进行后续ELISA实验，或将血清保存在-80℃冰箱。
ELISA实验：用2μg/mL OVA包被酶标板，每孔50μL，4℃包被过夜。弃去板中液体，每孔加入200μL 0.05％PBST缓冲液洗涤，每次3min，重复3次。每孔加入120μL 2％BSA封闭液，37℃封闭2h；PBST洗3次；每孔中加入用1％BSA+0.05％PBST稀释的血清50μL，室温孵育2h；PBST洗涤5次；每孔加入Anti-mouse IgG-HRP抗体50μL，室温孵育1h；PBST洗涤5次；每孔加入TMB底物显色液50μL，显色(蓝色)；每孔加入50μL稀硫酸终止液(亮黄色)，在酶标仪上读出OD450值。测定抗体滴度(将大于阴性对照OD450数值的两倍的最大稀释倍数定义为抗体滴度)。
实验结果参照图7，Poloxamer凝胶(2520#)弹性模量和DNA水凝胶近似，Poloxamer凝胶(2525#)视觉状态和DNA水凝胶近似，DNA水凝胶组抗体滴度显著高于Poloxamer凝胶(2520#)和Poloxamer凝胶(2525#)组，说明DNA水凝胶诱导抗体产生不依赖于其凝胶状态的物理性质
实施例9：核酸水凝胶的凝胶状态延缓其自身在体内的降解。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)；DNA水凝胶-Cy5.5以及可溶性DNA-Cy5.5(制备方法同实施例1)，
实验方法：本实施例在IVIS Spectrum动物光学活体成像系统检测Cy5.5荧光来表征DNA在免疫部位的代谢。本实验分为两组，实验组一：C57B6/L小鼠脚垫免疫可溶性DNA-Cy5.5(L组分-Cy5.5+L组分)+PBS，实验组二C57B6/L小鼠脚垫免疫DNA水凝胶-Cy5(L组分-Cy5.5+Y组分)+PBS。实验组一和实验组二中偶联Cy5.5的DNA质量相同为0.52mg，免疫的总DNA质量也相同为1.07mg。分别在第0h，2h，6h，12h，24h，36h，60h，84h在IVIS Spectrun动物光学活体成像仪检测Cy5.5荧光，并拍照记录。
实验结果参照图7。上图为各组各时间点小鼠免疫部位Cy5.5荧光强度代表图。下图为荧光强度(Total Radiant efficiency)统计结果。从结果中可以看出DNA水凝胶组中Cy5.5荧光强度呈现一个缓慢下降的过程，在60h甚至80h都可以维持在一个高水平，而可溶性DNA组中Cy5一开始就呈现急剧下降，24h左右几乎变为0。以上实验说明了DNA水凝胶的凝胶状态能延缓DNA自身在小鼠体内的代谢，让其在免疫部位维持更长的时间。
实施例10：核酸水凝胶促进免疫细胞在引流淋巴结的富集及分化。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，OVA抗原(购自Sigma公司)，DNA水凝胶(制备方法同实施例1)，铝佐剂(购自Thermo公司)
实验方法：本实施例采用流式细胞技术检测淋巴结中各细胞亚群的比例。将OVA抗原(100μg)分别与PBS、铝佐剂(1:1)或DNA水凝胶混成终体积为30μL。在第0天，对照组每只小鼠脚垫注射30μL OVA+PBS，实验组一每只小鼠脚垫注射30μL OVA+铝佐剂，实验组二每只小鼠脚垫注射30μL OVA+DNA水凝胶。在第10天取小鼠免疫部位同侧腘窝淋巴结进行流式细胞实验。
流式细胞实验：检测腘窝淋巴结中浆细胞(B220lowCD138+Plasma)，生发中心B细胞(B220+GL7+GCB)，滤泡辅助细胞(CD4+PD1+CXCR5+Tfh)，巨噬细胞(F4/80+Mφ)以及树突细胞(CD11c+DC)的比例。
实验结果参见图10，与PBS组相比，DNA水凝胶显著促进引流淋巴结中细胞数的增加。DNA水凝胶显著促进淋巴结中浆细胞，生发中心细胞，滤泡辅助细胞，树突细胞比例增加，但不影响巨噬细胞比例。
实施例11：核酸水凝胶促进免疫细胞在免疫部位的富集。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，OVA抗原(购自Sigma公司)，DNA水凝胶(制备方法同实施例1)，铝佐剂(购自Thermo公司)
实验方法：本实施例采用流式细胞技术检测免疫部位各细胞亚群的比例。将OVA抗原(100μg)分别与PBS、铝佐剂(1:1)或DNA水凝胶混成终体积为30μL。在第0天，对照组每只小鼠脚垫注射30μL OVA+PBS，实验组一每只小鼠脚垫注射30μL OVA+铝佐剂，实验组二每只小鼠脚垫注射30μL OVA+DNA水凝胶。分别在第0,2,3,5,6,8,9,10,11天取脚垫内容物进行流式实验，检测脚垫内容物中B细胞(B220+B)，T细胞(CD3+T)，树突细胞(CD11c+DC)以及巨噬细胞(F4/80+Mφ)的比例。
实验结果参见图11，在第3天，DNA水凝胶显著促进脚垫内容物种树突细胞和巨噬细胞的比例增加，在第8天，DNA水凝胶显著促进脚垫内容物中B细胞和T细胞比例的增加。
实施例12：核酸水凝胶诱导免疫部位IL-1b、CCL-2、CCL-3、CCL-4等细胞因子mRNA高表达。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)DNA水凝胶(制备方法同实施例1)。
实验方法：本实施例实时荧光定量(qPCR)检测免疫部位相关细胞因子mRNA的变化。实验组：DNA水凝胶注射小鼠脚垫6小时，收取对照组以及实验组脚垫皮肤组织，抽提RNA并反转录成cDNA，然后进行qPCR检测脚垫组织中相关mRNA的表达情况。
实验结果参照图12，从热图中可以看出DNA水凝胶可以显著促进IL1b，CCL2，CCL3以及CCL4等mRNA高表达。
实施例13：核酸水凝胶诱导骨髓来源巨噬细胞(BMDM)细胞因子mRNA高表达。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，DNA水凝胶(制备方法同实施例1)，Poloxamer凝胶(2520#)(购自Sigma公司和碧云天公司)
实验方法：本实施例实时荧光定量(qPCR)检测骨髓来源巨噬细胞中相关基因mRNA的变化。获取小鼠骨髓细胞，利用L929细胞培养上清诱导成骨髓来源巨噬细胞，加入DNA水凝胶或Poloxamer凝胶(2520#)刺激不同时间(0，1,3,6小时)，收取细胞，抽提RNA并反转录成cDNA，然后进行qPCR检不同刺激组中相关mRNA的表达情况。
实验结果参照图13，DNA水凝胶可以显著促进BMDM中IL1β，IL6，IFNβ以及CCL4mRNA的表达，其中IL1β在3小时到最高值，IL6，IFNβ以及CCL4在6h到最高值。相对于Poloxamer凝胶(2520#)，DNA水凝胶诱导相关细胞因子产生能力更强。
实施例14：核酸水凝胶诱导骨髓来源树突细胞(BMDC)细胞因子mRNA高表达。
实验材料：野生型C57BL/6小鼠(购买自集萃药康，由清华大学动物中心饲养)，DNA水凝胶(制备方法同实施例1)，Poloxamer凝胶(2520#)(购自Sigma公司和碧云天公司)
实验方法：本实施例实时荧光定量(qPCR)检测骨髓来源树突细胞中相关基因mRNA的变化。获取小鼠骨髓细胞，利用IL4和CM-CSF细胞因子诱导成骨髓来源树突细胞，加入DNA水凝胶或Poloxamer凝胶(2520#)刺激不同时间(0，1,3,6小时)，收取细胞，抽提RNA并反转录成cDNA，然后进行qPCR检不同刺激组中相关mRNA的表达情况。
实验结果参照图14，DNA水凝胶可以显著促进BMDC中IL1β，IL6，IFNβ以及CCL4mRNA的表达，其中IL1β和IL6在3小时到最高值，IFNβ以及CCL4在6h到最高值。相对于Poloxamer凝胶(2520#)，DNA水凝胶诱导相关细胞因子产生能力更强。

Claims (9)

  1. 一种佐剂，其包含或由核酸水凝胶组成，所述核酸水凝胶包含：
    支架单元，该支架单元具备至少三个支架粘性末端，
    交联单元，该交联单元具备至少两个交联粘性末端，以及
    水性介质；
    所述支架单元和所述交联单元均由核酸以碱基互补配对的方式形成，
    所述支架单元与所述交联单元通过所述支架粘性末端与所述交联粘性末端以碱基互补配对的方式交联，从而形成三维空间网络结构，
    其中，所述支架单元或所述交联单元包含或不包含CpG序列；
    其中，所述核酸包含或不包含修饰，所述修饰包括连接基团修饰、荧光基团修饰、淬灭基团修饰、间臂修饰、核苷酸变体修饰、简并碱基修饰；
    优选地，所述连接基团修饰包括氨基修饰、羧基修饰、醛基修饰、丙烯酰胺基修饰、叠氮修饰、炔基修饰、二苯基环辛炔修饰、马来酰亚胺修饰、巯基修饰、二硫醇修饰、二茂铁修饰、生物素修饰、地高辛修饰；
    优选地，所述荧光基团修饰包括Pacific Blue、ROX、Texas red；
    优选地，所述淬灭基团修饰包括BHQ1、BHQ2；
    优选地，所述间臂修饰包括C3/C6 Spacer、Spacer 9/Spacer 18、PC Spacer/PC linker、四氢呋喃修饰；
    优选地，所述核苷酸变体修饰包括磷酸化、硫代、2-氨基嘌呤、5-溴脱氧尿嘧啶、脱氧尿嘧啶核苷、倒置dT/dG、双脱氧胞嘧啶核苷、5-甲基胞嘧啶脱氧核苷、5-羟甲基dC、N6甲基腺嘌呤核苷酸、脱氧次黄嘌呤核苷、锁核酸、5-硝基吲哚、2-甲氧基修饰、RNA碱基、2-氟修饰、2-氟RNA、2'-O-(2-甲氧基)乙基、吗啉代、桥接核苷酸、BNA、吡咯-脱氧胞嘧啶；
    优选地，所述佐剂用于疫苗；
    优选地，所述支架粘性末端或交联粘性末端的长度为4nt-150nt；
    优选地，所述支架单元由三条单链核酸以碱基互补配对的方式形成，且每一条单链核酸具有一个所述支架粘性末端；
    优选地，所述交联单元由两条单链核酸以碱基互补配对的方式形成，且每一条单链核酸具有一个所述交联粘性末端。
  2. 根据权利要求1所述的佐剂，其中所述疫苗选自mRNA疫苗、灭活疫苗、减毒疫苗和重组蛋白疫苗；
    任选地，所述疫苗是冠状病毒疫苗，所述冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV或新冠病毒(COVID-19)；
    优选地，所述冠状病毒疫苗选自mRNA-1273(Moderna,Inc.)、AZD-1222(AstraZeneca and University of Oxford)、BNT162(辉瑞和BioNTech)、CoronaVac(Sinovac)、COVILO、NVX-CoV 2372(NovoVax)、SCB-2019(赛诺菲和GSK)、ZyCoV-D(Zydus Cadila)和CoVaxin(Bharat Biotech)；
    优选地，所述疫苗是新冠病毒(COVID-19)疫苗，例如阿尔法新冠病毒疫苗、德尔塔新冠病毒疫苗、奥密克戎新冠病毒疫苗及其突变毒株的疫苗，更优选地，用于奥密克戎新冠病毒及其突变毒株的疫苗，任选地，所述奥密克戎新冠突变毒株选自BA.1、BA.2、xe、xl、BA.4、BA.5及其分支。
  3. 包括用于形成所述支架单元的核酸，用于形成所述交联单元的核酸，以及水性介质的组合，在制备根据权利要求1所述的佐剂中的用途，其中，所述支架单元或所述交联单元包含或不包含CpG序列。
  4. 一种试剂盒在制备根据权利要求1所述的佐剂的用途，其包括：用于形成所述支架单元的核酸和用于形成所述交联单元的核酸；其中，所述支架单元或所述交联单元包含或不包含CpG序列。
  5. 根据权利要求1所述的佐剂在制备用于在受试者中引起或增强免疫应答的药物中的用途。
  6. 根据权利要求1所述的佐剂，其用于在受试者中引起或增强免疫应答。
  7. 一种用于在受试者中引起或增强免疫应答的药物组合，其包含：
    (1)根据权利要求1所述的佐剂；
    (2)抗原；优选地所述抗原是冠状病毒抗原，所述冠状病毒优选COVID-19；优选地，所述抗原选自新冠病毒S蛋白或其片段、新冠病毒RBD蛋白或其二聚体等；优选地，所述新冠病毒RBD蛋白的序列与SEQ ID NO：1-3中的任一序列具有至少30％、40％、50％、60％、70％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，更优选地具有100％的同一性。
  8. 一种在受试者中引起或增强免疫应答的方法，其包含向所述受试者施用根据权利要求7所述的药物组合；任选地，将权利要求7中所述的药物组合中的佐剂和所述的抗原同时或间隔一段时间施用于受试者；任选地，间隔一段时间可以是间隔1分钟，2分钟，5分钟，10分钟，30分钟，1小时，2小时或更多或上述任一时间点之间的任一时间。
  9. 根据权利要求1所述的佐剂或权利要求7所述的组合，其用于促进免疫细胞在引流淋巴结的富集和分化、促进免疫细胞在受试者的佐剂施用部位的富集、诱导受试者的佐剂施用部位的IL-1b、CCL-2、CCL-3、CCL-4等细胞因子的表达升高、诱导骨髓来源巨噬细胞(BMDM)细胞因子的表达升高或诱导骨髓来源树突细胞(BMDC)细胞因子的表达升高。
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