WO2023142999A1 - 细菌外膜囊泡来源的核酸纳米疫苗及其应用 - Google Patents

细菌外膜囊泡来源的核酸纳米疫苗及其应用 Download PDF

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WO2023142999A1
WO2023142999A1 PCT/CN2023/071288 CN2023071288W WO2023142999A1 WO 2023142999 A1 WO2023142999 A1 WO 2023142999A1 CN 2023071288 W CN2023071288 W CN 2023071288W WO 2023142999 A1 WO2023142999 A1 WO 2023142999A1
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nucleic acid
vesicle
omv
present application
mrna
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PCT/CN2023/071288
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English (en)
French (fr)
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聂广军
赵潇
李瑶
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国家纳米科学中心
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the application relates to the field of biomedicine, in particular to a nano-vaccine for bacterial outer membrane vesicles transporting mRNA antigens and its application.
  • Tumor vaccines are considered to be one of the most potential tumor immunotherapies, in which mRNA is not only simple, fast, but also inexpensive. More importantly, unlike most tumor drugs, the mRNA will be automatically degraded after functioning, and will not cause other toxicity or side effects to the human body.
  • mRNA vaccines must rely on effective transport carriers to enter cells, and the ability to enter antigen-presenting cells is mRNA Prerequisites for vaccines to be effective.
  • the mainstream mRNA transport carrier in vivo is the lipid nano-delivery system, but this carrier needs to be re-synthesized and entrapped for each antigen, which is time-consuming and labor-intensive.
  • the successful activation of adaptive immunity also requires the assistance of innate immunity, which requires the doping of immune adjuvants in the vaccine, which further increases the complexity of the synthesis of nanocarriers.
  • the application provides a modified vesicle and its preparation method and application.
  • This application mainly uses bacterial outer membrane vesicles (OMV) as a carrier to creatively fuse and express nucleic acid binding molecules on the surface of OMV, so as to realize the binding and delivery of nucleic acid of interest (such as exogenous mRNA antigen) on the surface of OMV.
  • OMV outer membrane vesicles
  • the present application provides a vaccine of complex and heterogeneous antigens suitable for clinical delivery.
  • the modified vesicle of the present application can be used as a nano-vaccine of a novel OMV-mRNA antigen complex, and has one or more of the following characteristics: (1) Stable, including the stability of the vesicle carrier, the stability of the loaded nucleic acid, and the stability of the nucleic acid and vesicle
  • the entire vaccine system is stable, (2) easy to store or transport, (3) capable of stimulating a strong innate immune response, (4) capable of effectively delivering nucleic acids to antigen-presenting cells and being effectively presented on the cell surface, (5) capable of eliciting Strong antigen-specific immune response, (6) able to generate antigen-specific immune memory, providing long-term tumor prevention effect, (7) able to flexibly deliver various nucleic acid antigens and effectively elicit antigen-specific anti-tumor immunity, these characteristics show that The nano-vaccine of the present application has great potential in the development and application of clinical vaccines in the future.
  • OMV can be a nano-sized bilayer proteoliposome, which can be a natural vesicle released by budding and secreting from the outer membrane of bacteria during growth, or it can be an engineered bacterial source of vesicles.
  • OMV can deliver molecules of interest, such as protein molecules, lipid substances or nucleic acid molecules, into host cells and carry immunogenic components into the cytoplasm.
  • the nanometer size for example, 1-1000nm, another example, 20-100nm
  • surface natural structure of OMV itself can promote the efficient uptake of OMV by antigen-presenting cells (for example, DC cells) and stimulate the natural immune system, Thereby promoting the processing and presentation of antigens, that is, the immune adjuvant effect.
  • the present application provides a vesicle, the outer surface of which comprises nucleic acid.
  • the present application provides a vesicle, the vesicle comprises a nucleic acid binding molecule capable of displaying the bound nucleic acid on the outer surface of the vesicle.
  • the present application provides a fusion protein, the fusion protein comprises a nucleic acid binding molecule and a membrane protein, and the nucleic acid binding molecule can display the bound nucleic acid on the outer surface of the vesicle.
  • the present application provides a nucleic acid encoding the fusion protein of the present application.
  • the present application provides a vector comprising the nucleic acid of the present application, and optional non-coding regions and/or antigen structure optimization regions.
  • the present application provides a cell comprising the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, and/or the vector of the present application.
  • the present application provides a composition comprising the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, and/or the cell of the present application, and an optional carrier.
  • the present application provides a kit comprising the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, and/or the composition of the present application.
  • the present application provides a method for displaying and/or expressing exogenous nucleic acid, comprising administering the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, and the combination of the present application Objects, and/or kits of the present application.
  • the present application provides a method for affecting immune response and/or inhibiting tumor growth, comprising administering the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the carrier of the present application, the cell of the present application, the Compositions, and/or kits of the present application.
  • the present application provides a vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, the composition of the present application, and/or the kit of the present application in the preparation of reagents
  • said agent is used for preventing and/or treating disease and/or disease.
  • Fig. 1 A is the transmission electron microscope picture of OMV universal vaccine carrier OMV-L in PBS solution
  • Fig. 1 B is the dynamic light scattering particle size analysis of OMV universal vaccine carrier OMV-L in PBS solution
  • Fig. 1 C is the application's Schematic diagram of the OMV-based mRNA delivery system.
  • FIG 2A-2B shows that the universal vaccine vector OMV uses the anchor protein ClyA to successfully construct and express the protein immunoblotting band of L7Ae
  • Figure 2C shows that the universal vaccine vector OMV uses the anchor protein INP to successfully construct and express the protein immunoblot of L7Ae Bands.
  • Figure 3 shows the quantitative diagram of OMV universal vaccine carrier transporting EGFP mRNA at 293T, the quantitative diagram of flow cytometry and the immunofluorescence diagram in BMDCs; 3A is the quantitative diagram of flow cytometry of 293T, and 3B is BMDCs 3C is the immunofluorescence confocal image.
  • 4A in Figure 4 shows the OD quantification results of luciferase signal in HEK293-derived TLR/NOD reporter cells treated with OMV-LL
  • 4B shows the heat of inflammatory cytokines secreted by BMDCs after incubation with OMV-LL picture.
  • 5A in Figure 5 is the antigen presentation efficiency diagram of OVA mRNA transported by the OMV universal vaccine carrier in BMDCs, and 5B is the expression level diagram of MHCI-OVA at different times; the expression level of MHCI-OVA molecules was detected by flow cytometry.
  • Figure 6 shows the detection of DC maturation and MHCI-OVA antigen presentation levels stimulated by OVA mRNA transported by OMV universal vaccine carrier in vivo, where 6A is the quantitative expression of lymph node DC co-stimulatory molecules CD80&CD86 after nano-vaccine injection, and 6B is Flow cytometry results of DC expressing MHCI-OVA complex.
  • 6A is the quantitative expression of lymph node DC co-stimulatory molecules CD80&CD86 after nano-vaccine injection
  • 6B is Flow cytometry results of DC expressing MHCI-OVA complex.
  • the above molecular level detection is carried out by cell flow cytometry.
  • Figure 7 shows the ability of CD3 + CD4 + , CD3 + CD8 + T cells to secrete IFN- ⁇ in the splenocytes extracted after three injections of immunization with OVA mRNA delivered by the OMV universal vaccine vector after stimulation with OVA polypeptide antigen , where Fig. 7A is the flow cytometry diagram of IFN- ⁇ secreted by CD3 + CD8 + T cells, Fig. 7B is the flow cytometry diagram of IFN- ⁇ secreted by CD3 + CD4 + T cells, and Fig. 7C shows the flow cytometry diagram using CFSE staining and flow cytometry The proliferation of splenocytes re-stimulated with OVA peptide antigen was analyzed by technique.
  • Figure 7D shows the antigen-specific killing ability of splenocytes on B16-OVA cells.
  • Figure 8 shows the evaluation of the anti-tumor effect of OMV nano-vaccine in melanoma-bearing lung metastasis
  • 8A is a schematic diagram of the evaluation procedure for the OMV-based mRNA vaccine to inhibit lung metastasis
  • 8B is a schematic diagram of the evaluation procedure from a tumor carrying B16-OVA on the 20th day.
  • Representative images of lung metastases collected from metastatic mice 8C is a quantitative statistical map of metastases
  • 8D is the rate of metastasis inhibition calculated based on the number of tumor nodules in 8C
  • 8E is the metastatic B16- Survival curves of OVA tumor mice.
  • Figure 9 shows the evaluation of the immune memory effect produced by the OMV nano-vaccine in C57/BL6 mice, wherein 9A is the program of the long-term immune memory program caused by the OMV-based mRNA vaccine, and 9B is the immune memory T cell ( CD3 + CD8 + CD44 + CD62L - ), 9C is the quantitative measurement and analysis of immune memory T cells, 9D is the image of the resected lung on day 80, and 9E is the number of lung metastatic nodules.
  • 9A is the program of the long-term immune memory program caused by the OMV-based mRNA vaccine
  • 9B is the immune memory T cell ( CD3 + CD8 + CD44 + CD62L - )
  • 9C is the quantitative measurement and analysis of immune memory T cells
  • 9D is the image of the resected lung on day 80
  • 9E is the number of lung metastatic nodules.
  • Figure 10 shows the tumor suppression effects of OMV nano-vaccine in the mouse subcutaneous colon cancer (MC38) model, wherein 10A is the MC38 tumor growth curve; 10B is the tumor weight of the MC38 tumor excised from each group of mice; 10C is the statistics of tumor inhibition rate calculated according to tumor weight.
  • 10A is the MC38 tumor growth curve
  • 10B is the tumor weight of the MC38 tumor excised from each group of mice
  • 10C is the statistics of tumor inhibition rate calculated according to tumor weight.
  • Figure 11 shows the changes of tumor-infiltrating immune cells after the OMV nanovaccine was implemented in the mouse subcutaneous colon cancer (MC38) model
  • 11A is CD3 + T cells
  • 11B is CD3 + CD8 + T cells
  • 11C is CD3 + CD4 + T cells
  • 11D is CD3 + CD4 + Foxp3 + Tregs.
  • FIG. 12 shows is, specific antibody level in serum, wherein 12A is OMV-L-mRNA HZV nano vaccine adopts intramuscular injection to immunize mice, 12B is OMV-L-mRNA HZV nano vaccine immunized mice by nasal route, 12C is OMV-L-OMV-L-mRNA HBeAg nano-vaccine is used to immunize mice by intramuscular injection, and 12D is OMV-L-mRNA HBeAg nano-vaccine to immunize mice by nasal route.
  • Figure 13 shows that OMV nano vaccines display nucleic acid expression on the inner and outer surfaces.
  • nucleic acid binding molecule generally refers to a molecule having the ability to bind nucleic acid.
  • the nucleic acid binding molecule can specifically bind nucleic acid.
  • the nucleic acid binding molecule may comprise a region that specifically recognizes a nucleic acid.
  • nucleic acid binding molecules can recognize box C/D sequences.
  • vesicle generally refers to a sac-like structure with a membrane structure.
  • the periphery of the vesicle is only a lipid bilayer molecular membrane with one layer.
  • Vesicles of the present application include vesicles of any diameter. The diameter of the vesicles can be characterized by transmission electron microscopy.
  • herpes virus antigen generally refers to a protein derived from herpes virus.
  • a nucleic acid encoding a protein of a herpes virus can be displayed by the method of the present application.
  • hepatitis B e antigen generally refers to a protein derived from hepatitis B virus.
  • the nucleic acid encoding the protein of hepatitis B e antigen can be displayed by the method of the present application.
  • Adpgk generally refers to ADP Dependent Glucokinase.
  • the Uniprot accession number of Adpgk can be Q9BRR6.
  • Adpgk of the present application may cover its unprocessed form, any processed form, its variant or substances comprising its functionally active fragments.
  • ClyA generally refers to Cytolysin A.
  • the Uniprot accession number of ClyA may be Q68S90.
  • the ClyA of the present application may cover its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • the term "INP" generally refers to Ice nucleation protein.
  • the Uniprot accession number for INP may be P06620.
  • the INP of the present application may encompass its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • the term "L7Ae” generally refers to an RNA binding protein.
  • An example Uniprot accession number could be D0KRE2.
  • the L7Ae of the present application may encompass its unprocessed form, any processed form, its variants or substances comprising its functionally active fragments.
  • the present application provides a modified vesicle, the surface of which comprises a nucleic acid binding molecule capable of binding nucleic acid and displaying the bound nucleic acid on the outer surface of the vesicle.
  • the present application provides a modified vesicle, the surface of which comprises nucleic acid, and the nucleic acid is displayed on the outer surface of the vesicle by a nucleic acid binding molecule.
  • the vesicles of the present application can be derived from the membrane structure of cells, for example, can be derived from the membrane structure of bacteria.
  • the vesicles described herein may be derived from the membrane structures of Gram-negative bacteria and/or Gram-positive bacteria.
  • the vesicles described herein may comprise membrane structures derived from Enterobacteriaceae and/or Neibacteriaceae.
  • the vesicles described herein can be derived from the membrane structures of Salmonella, E. coli, and/or meningococci.
  • the vesicles described herein can be derived from Gram-negative bacteria, attenuated Salmonella, Staphylococcus, Lactobacillus, Bacillus, Pseudomonas, or Bifidobacterium pseudolongum.
  • the vesicles described herein may be derived from bacterial outer membrane vesicles (OMVs).
  • OMVs are rich in pathogen-associated molecular patterns (PAMPs), which can strongly stimulate the innate immune system to promote antigen presentation and T cell activation, making them ideal vaccine carriers.
  • PAMPs pathogen-associated molecular patterns
  • the OMVs can be secreted by Gram-negative bacteria, have intrinsic nanometer dimensions and bacterial components, can be efficiently recognized and taken up by dendritic cells (DCs), and can be engineered as described in this application so that they can be delivered nucleic acid.
  • DCs dendritic cells
  • the cells and/or bacteria of the present application can express foreign protein plasmids by adding inducers, the inducers include tetracycline, doxycycline or in vivo metabolites such as dopamine, tea polyphenols, etc., preferably the induction of pET28a plasmids
  • inducers include tetracycline, doxycycline or in vivo metabolites such as dopamine, tea polyphenols, etc., preferably the induction of pET28a plasmids
  • the agent is IPTG.
  • the vesicles of the present application are detected by transmission electron microscopy, and the average diameter of the vesicles may be about 28 nm.
  • the vesicles of the present application can be secreted by host bacteria in a growing state and extracted by ultracentrifugation or the like.
  • the vesicles of the present application can be modified to comprise nucleic acid binding molecules.
  • the nucleic acid binding molecule can be fused to a membrane protein of the vesicle.
  • the membrane protein may be a membrane protein naturally present on the surface of the vesicle, or a heterologous membrane protein expressed on the surface of the vesicle after engineering modification, as long as the protein can be stably expressed on the surface of the vesicle.
  • the membrane protein may be selected from the group consisting of Cytolysin A (ClyA), Ice Nucleation Protein (INP), OmpA and Hbp.
  • the nucleic acid binding molecule can be indirectly expressed on the surface of the vesicle by fusing to a membrane protein.
  • the nucleic acid binding molecule can also be directly expressed on the surface of the vesicle in the form of a transmembrane protein.
  • the membrane protein and the nucleic acid binding molecule may be directly or indirectly linked.
  • the C-terminus of the membrane protein and the N-terminus of the nucleic acid binding molecule can be directly or indirectly connected.
  • the connection manner can be adjusted as required.
  • the linking manner is adjusted so that the nucleic acid binding molecule is expressed on the outer surface of the vesicle membrane.
  • the nucleic acid binding molecule can bind the nucleic acid of interest, so that the bound nucleic acid is displayed on the surface of the vesicle.
  • a nucleic acid binding molecule may be a protein capable of binding a specific nucleic acid sequence or a specific nucleic acid fragment, which may be referred to herein as a binding sequence.
  • Common nucleic acid binding molecules in the prior art such as RNA binding proteins, including iron regulatory protein (IRP) or U1A protein from eukaryotes, and L7Ae, MS2 coat protein or target of RNAIII activation protein (TRAP) from prokaryotes, all Can be used in the vesicles of the present application.
  • L7Ae was used as a nucleic acid binding molecule.
  • L7Ae can be combined with box C/D.
  • the box C/D sequence adopts a standard k-turn conformation, which can be specifically recognized by L7Ae protein, thereby stabilizing the stem-loop and forming a stable L7Ae-k-turn complex.
  • the box C/D sequence can be as shown in SEQ ID NO:1.
  • the corresponding binding sequence can be selected to label the target nucleic acid to be bound.
  • nucleic acid binding molecules are fused to surface proteins of OMV.
  • the nucleic acid binding molecule is L7Ae, fused to the C-terminus of OMV surface protein ClyA.
  • the nucleic acid can be loaded on the outer surface of the vesicle described in the present application through the interaction between the binding sequence and the nucleic acid binding molecule.
  • the nucleic acid is displayed on the surface of the OMV through the interaction between the binding sequence box C/D and L7Ae fused to the surface of the OMV.
  • the nucleic acid may comprise exogenous nucleic acid.
  • the exogenous nucleic acid may encode a protein of interest, may also be a non-coding nucleic acid, or any other nucleic acid that needs to be delivered.
  • the nucleic acid may comprise RNA.
  • the nucleic acid may comprise RNA encoding tumor-associated antigens and/or infectious disease-associated antigens.
  • the tumor-associated antigen may comprise OVA and/or carcinoembryonic antigen Adpgk (ADP Dependent Glucokinase).
  • the nucleic acid sequence of OVA can be as shown in SEQ ID NO:6.
  • the nucleic acid sequence of Adpgk ADP Dependent Glucokinase
  • the antigen may include OVA, carcinoembryonic antigen CEA, neoantigen Adpgk, tumor-associated antigen MUC1, hepatitis B e antigen, etc., and any combination of the above.
  • the infectious disease-associated antigen may comprise hepatitis B e antigen and/or herpes virus antigen.
  • the nucleic acid sequence of hepatitis B e antigen can be as shown in SEQ ID NO:10.
  • the nucleic acid sequence of herpes virus capsid antigen can be as shown in SEQ ID NO:9.
  • it can contain more than 99%, more than 98%, more than 97%, more than 96%, more than 95%, more than 90%, more than 80%, more than 70%, more than 60%, more than 50% of the nucleic acid sequence of the present application homologous nucleic acid sequences.
  • the nucleic acid may comprise a binding sequence and a nucleic acid molecule encoding an antigen.
  • the binding sequence is linked to the nucleic acid molecule encoding the antigen and does not affect the expression of the nucleic acid molecule encoding the antigen.
  • the binding sequence may be located in the non-coding region of the nucleic acid encoding the antigen.
  • the binding sequence may be located at the 3'UTR of the nucleic acid encoding the antigen.
  • the nucleic acid may further comprise an antigen structure optimization region.
  • the antigen structure optimization region of the present application can be used to improve antigen translation efficiency and/or improve MHCI presentation efficiency.
  • the antigen structure optimization region of the present application includes MITD (MHC class I trafficking signal) sequence, for example, the sequence of MITD can be shown as SEQ ID NO:4.
  • the optimized region of the antigen structure of the present application includes the sequence of Ub, for example, the sequence of Ub can be as shown in SEQ ID NO:5.
  • the nucleic acid of the present application is RNA, and its sequence may sequentially include 5' non-coding region-antigen structure optimization region-antigen coding region-3' non-coding region-RNA binding protein binding sequence.
  • the antigen coding region is the mRNA region encoding the final antigen protein.
  • the 5' and 3' non-coding regions can be used to stabilize the mRNA structure and improve translation efficiency.
  • the sequence of the 5' non-coding region can be as shown in SEQ ID NO: 2.
  • the sequence of the 3' non-coding region can be as shown in SEQ ID NO: 3.
  • the RNA binding protein binding sequence is a sequence that can specifically bind to the nucleic acid binding molecule expressed on the surface of OMV. When L7Ae is selected as the nucleic acid binding molecule, box C/D can be selected as the binding sequence to mark the nucleic acid encoding the antigen.
  • the outer surface of the vesicle can be engineered to obtain properties of interest (e.g., fusion with a protein of interest) to make the nucleic acid more stable, to allow for easier presentation of the nucleic acid by antigen-presenting cells, to render the entire vesicle
  • properties of interest e.g., fusion with a protein of interest
  • the vesicle system can more easily enter the target cells or make the immune response stronger.
  • listerialysin O (LLO) can be fused to the surface protein of vesicles to enhance OMV escape and mRNA translation in vivo.
  • the present application also provides a fusion protein, which may comprise a nucleic acid binding molecule and a membrane protein, and the nucleic acid protein can display its bound nucleic acid on the outer surface of the vesicle.
  • the membrane protein may comprise cytolysin A (ClyA) and/or ice nucleation protein (INP), as well as the above-mentioned functionally active fragments.
  • ClyA cytolysin A
  • INP ice nucleation protein
  • the nucleic acid binding molecule can recognize the box C/D sequence.
  • the nucleic acid binding molecule may comprise L7Ae or a functionally active fragment thereof.
  • the nucleic acid binding molecules of the present application may also comprise IRP and U1A derived from eukaryotes, and L7Ae, MS2 and TRAP derived from prokaryotes.
  • the membrane protein and the nucleic acid binding molecule can be linked directly or indirectly.
  • the C-terminus of the membrane protein and the N-terminus of the nucleic acid binding molecule can be directly or indirectly linked.
  • the connection manner can be adjusted as required.
  • the linking manner is adjusted so that the nucleic acid binding molecule is expressed on the outer surface of the vesicle membrane.
  • the present application also provides a nucleic acid encoding the fusion protein of the present application.
  • the present application also provides a vector, which may contain the nucleic acid of the present application, and optional non-coding regions and/or antigen structure optimization regions.
  • the vectors of the present application may contain multiple expression cassettes in a tandem structure.
  • the present application also provides a cell, which may contain the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, and/or the vector of the present application.
  • the present application also provides a composition, which may comprise the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, and/or the cell of the present application, and an optional carrier.
  • the present application also provides a kit, which may comprise the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application, and/or the composition of the present application.
  • the present application provides a vaccine, in particular, a universal nucleic acid nano-vaccine.
  • the engineered vesicle of the present application can be used as a nucleic acid vaccine carrier to deliver the nucleic acid antigen of interest.
  • the present application provides a method for displaying and/or expressing exogenous nucleic acid, which may comprise administering the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the vector of the present application, the cell of the present application , the composition of the present application, and/or the kit of the present application.
  • the fusion protein of the present application can be expressed on the outer surface of OMV.
  • the RNA bound by the fusion protein of the present application can be expressed as the target protein in dendritic cells.
  • the present application provides a method for affecting immune response and/or inhibiting tumor growth, which may comprise administering the vesicle of the present application, the fusion protein of the present application, the nucleic acid of the present application, the carrier of the present application, the Cells, compositions of the present application, and/or kits of the present application.
  • the method may be a method for non-therapeutic and/or non-diagnostic purposes.
  • the effect of influencing the immune response includes activating the immune response
  • the effect of activating the immune response may include activating dendritic cells, making dendritic cells (such as CD11c positive cells) mature (such as increasing the proportion of CD80 positive CD86 positive cells), making immune
  • the cells secrete cytokines eg, T cells secrete IFN- ⁇
  • increase the proportion of effector T cells in tumor-infiltrating lymphocytes and/or activate immune memory responses (eg, increase the proportion of CD3 + CD4 + CD44 + CD62L- cells).
  • inhibiting tumor growth in the present application may include inhibiting tumor metastasis (eg, lung metastasis of tumor), and/or inhibiting tumor growth volume.
  • a vesicle of the present application, a fusion protein of the present application, a nucleic acid of the present application, a carrier of the present application, a cell of the present application, a composition of the present application, a vaccine of the present application and/or a kit of the present application are prepared in the reagent
  • said agent is used for preventing and/or treating disease and/or disease.
  • diseases including but not limited to melanoma, colon cancer, breast cancer, human papilloma, hepatitis B, herpes, etc.
  • the disease and/or condition may comprise tumors and/or infectious diseases.
  • the disease and/or condition may comprise solid tumors.
  • the disease and/or condition may comprise melanoma and/or colorectal tumor.
  • the diseases and/or conditions may include herpes virus and/or hepatitis B virus infection-related infectious diseases.
  • the diseases and/or conditions may comprise tumors and/or infectious diseases.
  • the disease and/or condition may comprise a solid tumor.
  • the disease and/or condition may comprise melanoma and/or colorectal tumors.
  • the disease and/or condition may comprise an infection associated with herpes virus and/or hepatitis B virus infection.
  • the diseases and/or conditions may comprise tumors and/or infectious diseases.
  • the disease and/or condition may comprise a solid tumor.
  • the disease and/or condition may comprise melanoma and/or colorectal tumors.
  • the disease and/or condition may comprise an infection associated with herpes virus and/or hepatitis B virus infection.
  • the present application provides a vaccine platform capable of flexibly and rapidly displaying OMVs of mRNA tumor antigens.
  • the archaeal protein L7Ae is fused and expressed at the C-terminus of the OMV surface protein ClyA, and the box C/D sequence is modified in the 3' non-coding region of the mRNA tumor antigen synthesized in vitro.
  • OMV delivers the mRNA antigen into antigen-presenting cells and translates it into epitope information to form a complex with MHCI and present it on the cell surface.
  • nucleic acid encoding an antigen-optimized region such as MITD
  • introduction of nucleic acid encoding an antigen-optimized region (such as MITD) into mRNA increases the expression of antigen-presenting molecules; the natural components of OMV activate multiple pathways to stimulate DC cell maturation and cytokine release, which promote successful recruitment of T cells.
  • This application is based on the tumor vaccine platform of OMV that can be combined with mRNA, which can significantly activate the host immune response, quickly display tumor antigens, and finally efficiently induce the anti-tumor immune effect mediated by antigen-specific T cells.
  • the OMV has a natural immune adjuvant effect, which can enhance the immune effect of the vaccine; the surface of the OMV is embedded with an RNA binding protein (preferably archaebacteria ribosomal protein L7Ae), which can bind to the sequence containing the RNA binding protein (preferably with L7Ae matching box C/D sequence) mRNA rapidly binds and displays on the surface of OMV.
  • the mRNA consists of a 5' non-coding region-antigen structure optimization region-antigen region-3' non-coding region-RNA binding protein binding sequence, wherein the non-coding region and the antigen structure optimization region can promote the translation and translation of mRNA antigens in cells MHCI presentation efficiency.
  • this nano-vaccine based on the OMV-mRNA antigen complex achieves the simultaneous and efficient delivery of natural adjuvants and mRNA antigens, and can efficiently activate antigen-specific immune responses, showing strong application potential in the field of tumor vaccines.
  • a combined tumor nanovaccine co-transported by bacterial outer membrane vesicles and antigenic nucleic acids is formed by the anchoring combination of outer membrane vesicles secreted by Escherichia coli and antigen mRNA, and the antigen mRNA can be Antigen-presenting cells take it up and translate it into an antigen with epitope information.
  • an example of preparing the vesicle is as follows:
  • the bacterial outer membrane vesicle anchor protein is digested with a restriction endonuclease and then integrated into a prokaryotic expression plasmid through genetic engineering technology to construct a prokaryotic expression plasmid containing a membrane-localized protein and a functional protein.
  • the 3' end of the membrane anchor sequence of the recombinant plasmid is inserted into the foreign protein sequence L7Ae and the HA gene sequence to form a recombinant plasmid for fusion expression of "membrane localization protein-mRNA binding protein". It is also possible to insert the foreign protein Listeriatolysin O, using the Flag sequence as a tag.
  • the recombinant plasmid is transformed into Escherichia coli by heat shock at 42°C, induced by the inducer IPTG, and the exogenous protein is correctly translated and folded into a soluble fusion protein after low temperature induction, and the localized protein sequence is pulled to the bacteria by the membrane traction protein adventitia surface.
  • the extraction method of bacterial outer membrane vesicles is gradient ultracentrifugation, specifically:
  • Centrifuge the bacteria liquid that was induced to express overnight at low temperature collect the culture supernatant, filter the supernatant through a 0.45 ⁇ m sterile filter membrane to remove residual bacterial fragments, concentrate the filtrate through an ultrafiltration tube with a molecular weight cut-off of 100 kDa, and filter the concentrated filtrate again 0.22 ⁇ m sterile filter membrane, collect the filtrate for ultracentrifugation, resuspend and wash the precipitate with phosphate buffer saline (PBS), and finally ultracentrifuge again. Resuspend the pelleted OMV in PBS or mRNA binding reaction solution and store at -20°C.
  • PBS phosphate buffer saline
  • the presence of the foreign protein is detected in the bacterial outer membrane vesicle (OMV) derived from Escherichia coli after the recombinant expression, and it is proved that it is on the surface of the OMV.
  • OMV bacterial outer membrane vesicle
  • the tumor mRNA antigen is synthesized by in vitro transcription of a recombinant plasmid, and the nucleic acid transport ability of OMV is verified by using green fluorescent protein (EGFP), and finally the tumor model antigen OVA nucleic acid sequence is used to detect its induction of antigen-specific T cell immunity responsiveness and anti-tumor ability.
  • EGFP green fluorescent protein
  • the EGFP mRNA antigen successfully binds to the surface of the OMV, and the level of protein translation after its transport is verified in vitro on 293T cells.
  • the mass ratio of the modified OMV vector (calculated by protein mass) to the mRNA load can be 20:1 to 2:1.
  • the mass ratio of OMV vector (calculated by protein mass) to mRNA payload can be 10:3.
  • the EGFP mRNA was combined and transported by OMV, and after co-incubating with BMDCs for 24 hours, the expression of green fluorescent protein was found in flow cytometry, which further proved the success of OMV transporting mRNA in BMDCs.
  • the nano-vaccine OMV-L-mRNA OVA promotes the maturation level of antigen-presenting cells in lymph node tissue after immunizing mice by subcutaneous administration.
  • the nano-vaccine OMV-L-mRNA OVA after immunizing the mice three times, the mice have the ability to activate immune T cells against the OVA antigen.
  • the mouse spleen at the immune end point is extracted, ground to obtain a T cell suspension, and the T cell suspension is stimulated with OVA 257-264 (SIINFEKL, SEQ ID NO: 11) polypeptide antigen fragments again, and the T cell suspension is stimulated by IFN- ⁇
  • OVA 257-264 SIINFEKL, SEQ ID NO: 11
  • the nano-vaccine OMV-L-mRNA OVA exhibits a significant tumor-suppressing effect in C57/BL6 mice bearing melanoma lung metastases.
  • the nano-vaccine OMV-L-mRNA OVA exhibits long-lasting immune memory and long-lasting tumor prevention ability in C57/BL6 mice.
  • the nanovaccine OMV-L-mRNA ADPGK exhibits a significant tumor-suppressing effect in C57/BL6 mice in a subcutaneous colon cancer (MC38) model.
  • the nano-vaccine OMV-L-mRNA HZV (the amino acid sequence of the HZV antigen is shown in SEQ ID NO: 9) or OMV-L-mRNA HBeAg (the amino acid sequence of HBeAg is shown in SEQ ID NO: 10) can activate the body Antigen-specific immune response, including cellular immunity and humoral immunity.
  • the nano-vaccine platform does not need additional adjuvants, and can cause effective T cell antigen recognition immune response, greatly To enhance the tumor-specific killing ability, a new mRNA nano-vaccine delivery platform was constructed.
  • the OMV tumor nano-vaccine described in this application can be prepared by existing conventional genetic engineering means and ultracentrifugation technology.
  • the OMV tumor nanovaccine combined with bacterial outer membrane vesicles and nucleic acid antigens described in this application is a novel vaccine platform capable of flexibly and rapidly displaying OMV of mRNA tumor antigens.
  • the bacterial outer membrane vesicle (OMV) in this application has good genetic engineering operability and natural adjuvant immune activation.
  • mRNA-OMV has higher biological stability and improves its protein translation and expression level in cells; at the same time, the obtained OMV tumor nanovaccine has a stronger immune response, Compared with single mRNA, it showed a stronger ability to induce the maturation of antigen-presenting cells, and caused higher specific T cell cytotoxic killing effect and anti-tumor effect.
  • the OMV tumor nanovaccine can provide a feasible method and technology for constructing a new type of mRNA vaccine for tumor immunotherapy.
  • the solvents of the aqueous solutions used in the following examples are sterile ultrapure aqueous solutions.
  • reagents used in the following examples are analytical reagents.
  • PBS solutions used in the following examples are all 1 ⁇ PBS solutions.
  • mRNA was synthesized by IVT using the HiScribe T7ARCA mRNA kit.
  • the template for IVT was the PCR product of pST1374 plasmid using Q5 high fidelity 2 ⁇ master mix (M0492, NEB, USA). Template contains T7 promoter.
  • the IVT reaction was then performed following the standard protocol, replacing CTP and UTP with 5-methyl-CTP and pseudo-UTP, respectively.
  • mRNA was 3′-poly(A)-tailed using poly(A) polymerase (M0276L, NEB, USA), and mRNA purification was achieved by LiCl precipitation according to the manufacturer’s instructions.
  • BMDC bone marrow-derived dendritic cells
  • BMDCs were stained with anti-mouse CD11c-FITC, anti-mouse CD80-PE, or anti-mouse CD86-PE antibodies for flow cytometry.
  • BMDCs were incubated with OVA 257-264 peptide (2 ⁇ g mL ⁇ 1 ) or OMV-LL-mRNA OVA (2 ⁇ g mL ⁇ 1 ) for 0, 6, 12, 24, 36 and 48 hours.
  • OVA antigen presentation efficacy was assessed by flow cytometry assessment of MHCI-OVA expression.
  • OMVs refers to the carrier of OMV surface membrane protein ClyA without further fusion of other molecules
  • OMV-L refers to the vector fused with L7Ae on the membrane protein ClyA on the surface of OMV, that is, the OMV expressing ClyA-L7Ae;
  • OMV-LLO refers to the carrier fused with LLO on the membrane protein ClyA on the surface of OMV, that is, the OMV expressing ClyA-LLO;
  • OMV-LL refers to the vector fused with L7Ae and LLO on the membrane protein ClyA on the surface of OMV, that is, the OMV that expresses ClyA-L7Ae and ClyA-LLO at the same time;
  • OMV-L-mRNA antigen refers to the vaccine in which the mRNA antigen is fused to the membrane protein ClyA on the surface of the OMV through the nucleic acid binding molecule L7Ae, that is, the OMV expressing ClyA-L7Ae, and the antigen mRNA passes through the box C/D between L7Ae Interactions are loaded on the surface of the OMV.
  • OMV-LL-mRNA antigen refers to the vaccine in which the mRNA antigen is fused to the membrane protein ClyA on the surface of the OMV through the nucleic acid binding molecule L7Ae, and the membrane protein ClyA is fused with LLO, that is, the OMV expressing both ClyA-L7Ae and Cly-LLO , and the antigen mRNA is loaded on the surface of OMV through the interaction between box C/D and L7Ae.
  • a nano-vaccine vector OMV is prepared from plasmid pET28a-ClyA-L7Ae or pET28a-ClyA in Escherichia coli BL21 (DE3), induced, fermented, and ultracentrifuged.
  • the obtained nano-vaccine vectors are called OMV-L and OMVs respectively.
  • the specific extraction steps are as follows:
  • the morphology and particle size of the collected OMV vaccine universal carrier were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS); 10 ⁇ L of the PBS solution sample of OMV universal vaccine carrier was dropped on the surface after the discharge treatment. On a carbon-coated copper grid, let stand for 5 minutes, filter paper to dry the solution, take 5 ⁇ L of 2% uranyl acetate (centrifuge at 4000 rpm for 5 minutes before use, remove the staining solution that is not completely dissolved) and stain for 60 seconds, filter paper to dry the stain Liquid, observe the sample with a transmission electron microscope (TEM, HT7700, Hitachi Corporation), as shown in Figure 1, Figure 1B is the electron microscope morphology of the universal carrier of OMV vaccine, showing a uniformly distributed spherical structure, Figure 1C is the general carrier of OMV Particle size, its size is 28.1nm.
  • TEM transmission electron microscopy
  • DLS dynamic light scattering
  • the purpose of this example is to verify the successful expression of the OMV universal vaccine vector fusion protein.
  • a nano-vaccine vector OMV prepared by transforming, inducing, fermenting and ultracentrifuging Escherichia coli BL21 (DE3) from pET28a-ClyA-L7Ae, pET28a-ClyA, pET-ClyA-L7Ae-3HA-ClyA-LLO-3Flag, obtained
  • the nano-vaccine vectors are called OMV-L, OMVs and OMV-LL respectively, and the specific steps are referred to in Example 1; wherein, the HA tag (HAYPYDVPDYA, SEQ ID NO: HAYPYDVPDYA, SEQ ID NO: 13)
  • a Flag tag was coupled to LLO for Western blot verification. Through the Western blot experiment, the label antibody was used to indicate the position of the target protein band to verify that L7Ae was successfully constructed on the OMV universal vaccine vector.
  • PVDF polyvinylidene fluoride
  • the protein band of ClyA-L7Ae-3HA is about 48kDa position, indicating L7Ae was successfully expressed on OMV-L and OMV-LL vectors, and the protein band of ClyA-LLO-3Flag was at the 75kDa position, indicating that Listerialysin O (LLO) was successfully expressed on OMV-LL.
  • L7Ae was not detected in native OMVs or when the inducer isopropyl- ⁇ -d-thiogalactoside (IPTG) was not expressed (Fig. 2A, lane 3).
  • the purpose of this example is to explore the versatility of different surface proteins for OMV vaccine vectors.
  • a kind of nano-vaccine carrier OMV is prepared by pET28a-INP-L7Ae, pET28a-INP in Escherichia coli BL21 (DE3), induced, fermented, and prepared by ultracentrifugation, and the nano-vaccine carrier obtained is respectively called OMV-L (here OMV-L refers to the vector in which L7Ae is fused to the membrane protein INP) and OMVs (here OMV refers to the carrier in which the membrane protein INP is not fused to L7Ae); among them, the RNA anchor protein (i.e.
  • L7Ae Conjugate HA tag (HAYPYDVPDYA, SEQ ID NO: 13) for protein immunoblotting verification. Further, through protein immunoblotting experiments, use the tag antibody to indicate the position of the target protein band to verify that L7Ae was successfully used in the OMV universal vaccine Build on the carrier.
  • PVDF polyvinylidene fluoride
  • the purpose of this example is to verify that the OMV universal vaccine vector can successfully transport mRNA for expression in cells.
  • the OMV nucleic acid nano-vaccine was co-incubated with the human kidney epithelial cell line 293T for 24 hours, and the expression of EGFP was detected by flow cytometry.
  • EGFP was highly translated and expressed in 293T cells; at the same time, the OMV universal vaccine vector delivered EGFP mRNA (OMV-L-mRNA EGFP ) in the expression level of dendritic cells (BMDCs) induced to differentiate from the main immune cells bone marrow, the results are shown in Figure 3B, 3C, OMV-L-mRNA EGFP green fluorescent protein expression is the strongest, can effectively transport mRNA, It is then translated into a protein by the cell.
  • the vaccine OMV+mRNA EGFP
  • no EGFP protein expression was detected on 293T
  • the purpose of this example is to verify that the OMV universal vaccine vector can activate the innate immune response.
  • the ClyA-L7Ae-3HA-ClyA-LLO-3Flag gene was cloned into an expression plasmid vector, transformed into Escherichia coli (strain BL21(DE3)), and the transformed bacteria were inoculated into 300mL LB broth at 37°C to grow An OD600 of 0.6 to 0.8 is achieved. Add IPTG to a final concentration of 1 ⁇ 10 -3 m to induce fusion protein expression. After overnight incubation, the sterile supernatant was collected by centrifugation, concentrated and filtered to isolate OMV.
  • Treatment of HEK293-derived TLR recipient cells with OMV-LL showed that OMV-LL significantly activated TLR2, TLR4, and TLR5, and slightly activated TLR9 (Fig. 4A); meanwhile, OMV-LL stimulated BMDCs to secrete pro-inflammatory cytokines, such as IFN - ⁇ , IL-1 ⁇ , TNF- ⁇ and IL-12p70 (Fig. 4B).
  • cytokines such as IFN - ⁇ , IL-1 ⁇ , TNF- ⁇ and IL-12p70
  • the purpose of this example is to verify that the OMV universal vaccine vector can transport the tumor antigen OVA mRNA and express it in immune cells.
  • the MHCI tracking domain (MITD) or ubiquitin (Ub) was fused to the mRNA antigen fragment to obtain MITD-containing mRNA OVA (MITD-OVA) and Ub-containing mRNA OVA (Ub-OVA).
  • MITD-OVA MITD-containing mRNA OVA
  • Ub-OVA Ub-containing mRNA OVA
  • the 3'UTR of the mRNA antigen fragment is inserted into the box C/D sequence, and the mass ratio of the universal vaccine carrier to the mRNA antigen fragment is 10:3.
  • the OMV nucleic acid nano-vaccine was co-incubated with BMDCs for 24 hours. After the nano-vaccine co-incubation was detected by flow cytometry, the BMDCs expressed the antigen-presenting molecule MHCI-OVA, as shown in Figure 5.
  • Figure 5A shows that there is basically no expression of mRNA in the absence of vector assistance, but two OMV universal vaccines (OMV-L-mRNA OVA and OMV-LL-mRNA OVA ) significantly increased the expression of MHCI-OVA molecules in BMDCs, and fusion Both vaccines containing Ub and mRNA antigens fused with MITD were effective in antigen presentation.
  • Figure 5B is a comparison of the OVA 257-264 (SIINFEKL, SEQ ID NO: 11) polypeptide group as a control, comparing the changes in the molecular level of MHCI-OVA on BMDCs after different incubation times.
  • the results show that after OMV-LL-mRNA OVA acts on BMDC cells , the duration of high-level expression of MHCI-OVA was longer than that of BMDC treated with only OVA polypeptide. It shows that the modified OMV vector of the present application significantly improves the expression and presentation of antigens on DC cells.
  • the purpose of this example is to verify the immune response level of DCs induced by the OMV universal vaccine vector transporting tumor antigen OVA mRNA.
  • the mRNA antigen fragment is OVA mRNA with 3'UTR inserted into box C/D, and the mass ratio of the universal vaccine carrier to mRNA is 10:3; the mixture of natural OMV and mRNA OMV is OMVs+mRNA OVA .
  • the OVA mRNA, OMVs+mRNA OVA and OMV-L-mRNA OVA vaccines were immunized subcutaneously at the base of the tail of C57/BL6 healthy mice, each mouse was injected with 5 ⁇ g of mRNA, and the left and right inguinal lymph nodes were extracted and prepared by grinding with a sieve After forming a single cell suspension and incubating with different flow cytometry antibodies, the maturation and antigen presentation of DCs in lymph nodes were detected by flow cytometry.
  • Figure 6A is the flow cytometric statistical results of DCs maturation after OMV nano-vaccination, showing that under the action of OMV universal vaccine vector, the expression of CD80 and CD86, the marker molecules of dendritic cell DCs maturation, was observed Significantly up-regulated, indicating an increase in the maturation level of DCs;
  • Figure 6B shows that the proportion of CD11c + MHCI-OVA + cells in the OMV-L-mRNA OVA vaccine group was significantly increased, indicating that OMV vector-mediated mRNA delivery successfully made MHCI-OVA molecules in DCs The expression on it provides conditions for the subsequent activation of T cell immune killing.
  • the purpose of this example is to verify the T cell immune response level induced by the OMV universal vaccine vector transporting tumor antigen OVA mRNA.
  • OMV nanovaccine OMV-L-mRNA OVA or OMV-LL-mRNA OVA was administered to healthy C57BL/6 mice for three subcutaneous immunization courses, and each mouse was injected with a dose of 5 ⁇ g of mRNA, and the intervals were respectively the 0th, 5th, and 10th days, and the small mice were dissected after 15 days.
  • CFSE 5,6-carboxyfluorescein diacetate succinimidyl ester
  • OMV nanovaccine OMV-L-mRNA OVA and OMV-LL-mRNA OVA can effectively activate CD3 + CD4 + , CD3 + CD8 + T cells and promote the secretion of IFN- ⁇ .
  • Fig. 7A is a flow cytometry quantitative diagram of IFN- ⁇ secreted by CD3 + CD8 + T cells
  • Fig. 7B is a flow cytometric quantitative diagram of IFN- ⁇ secreted by CD3 + CD4 + T cells.
  • FIG. 7C shows that the splenocytes of mice immunized with OMV-L-mRNA OVA and OMV-LL-mRNA OVA vaccines were re-stimulated with OVA antigen peptides, and the proliferation of splenocytes was significantly stronger than that of the control group, mRNA or mRNA and natural OMV mixed group.
  • the above results indicated a strong T cell response.
  • Cytotoxicity of splenocytes to ovalbumin-expressing B16 (B16-OVA) and MC38 cells was next assessed.
  • splenocytes in OMV-L-mRNA OVA and OMV-LL-mRNA OVA groups had strong cytotoxicity to B16-OVA cells and could kill a large number of target cells.
  • the purpose of this example is to evaluate the ability of OMV nano-vaccine to inhibit lung metastasis of melanoma.
  • the inhibitory ability of the OMV nano-vaccine prepared by the aforementioned method on the lung metastasis model of melanoma was established by injecting B16-OVA cells into the tail vein of C57BL/6 mice, and the anti-tumor effect was evaluated by the number of lung metastases, metastasis inhibition rate and mouse survival rate.
  • G1 PBS control group
  • G2 OMV-LL vector
  • G3 OMV-LL-mRNA EGFP vaccine
  • G4 simple mixture of OMV vector and OVA mRNA
  • G5 OMV-L-mRNA OVA vaccine
  • G6 OMV- LL-mRNA OVA vaccine.
  • mice were given three subcutaneous injections of nano-vaccine immunization courses, each mouse was injected with a dose of 5 ⁇ g of mRNA, and the intervals were 0, 5, and 10 days, respectively. That is, day 0 was taken as the injection of B16-OVA, and three immunizations were carried out on days 3, 8, and 13. On the 20th day, lung tissue was extracted, the number of lung tumor metastases was counted, and the tumor lung metastasis inhibitory effect of the vaccine was evaluated.
  • both G5 and G6 OVA vaccine groups could inhibit lung tumor metastasis and prolong the survival time of tumor mice.
  • the OMV nano-vaccine showed an excellent tumor suppressive effect.
  • Figure 8B is a map of lung metastases
  • Figure 8C is a quantitative statistical map of lung metastases.
  • the inhibition rate of OMV-L-mRNA OVA (G5) and OMV-LL-mRNA OVA (G6) two OVA vaccine groups was significantly higher than that of OMV-LL vector control group (G2), and that of OMV-LL-mRNA EGFP (G3) EGFR vaccine group and a simple mixed control group of OMV and OVA mRNA (G4) (Fig. 8D).
  • the mice in the G5 and G6 groups still survived after 35 days or even 40 days, while the mice in the other groups died within 26-32 days (Fig. 8E).
  • the purpose of this example is to explore the long-term immune memory and tumor prevention ability of the OMV nano-vaccine on the body.
  • mice were subcutaneously immunized with the OMV nano-vaccine prepared in the foregoing example, and each mouse was injected with a dose of 5 ⁇ g of mRNA, and subcutaneously immunized three times at intervals of 0, 5, and 10 days, respectively.
  • a part of mouse spleen tissue was taken and prepared into a single cell suspension, and the number of memory T cells (CD3 + CD4 + CD44 + CD62L - ) in the spleen cells was detected by flow cytometry to evaluate the long-term immune memory effect of the vaccine.
  • Another part of the mice was taken and inoculated with B16-OVA tumor cells in the tail vein. After another 20 days, the lung tissue was dissected, and the number of lung tumor metastases was counted to evaluate the tumor prevention ability of the vaccine.
  • the purpose of this example is to explore the ability of OMV nanocarriers to load different mRNAs to form vaccines and treat different tumors, that is, the ability to inhibit the growth of colorectal tumors (MC38).
  • G1 PBS control group
  • G2 OMV-LL-mRNA EGFR
  • G3 poly(I:C)+ADPGK polypeptide, a mixture of adjuvant poly(I:C) and ADPGK polypeptide (50ug+5ug per mouse) , as a positive control
  • G4 OMV+mRNA ADPGK
  • G5 OMV-LL-mRNA OMV (5ug per mouse).
  • the anti-tumor effect of the OMV nano-vaccine on a subcutaneous tumor model of colorectal cancer was explored. Specifically, 6 days after C57BL/6 mice were subcutaneously inoculated with MC38 tumor cells, the tumors formed, and the mice were inoculated with nano-vaccine, once every 5 days, a total of three times, and each mouse was injected with a dose of 5 ⁇ g of mRNA; every 2 days Tumor growth was monitored with a vernier caliper, and tumor tissue was extracted and weighed 23 days after tumor inoculation.
  • OMV-LL-mRNA ADPGK inhibited the best.
  • the OMV-LL-mRNA ADPGK group also had the lowest tumor volume and weight at the end of treatment compared to the other groups (Fig. 10A, 10B).
  • the tumor inhibition rates of OMV-LL-mRNA EGFP , poly(I:C)+ADPGK , OMV+mRNA ADPGK , and OMV-LL-mRNA ADPGK groups were 61.8%, 80.7%, 76.6%, and 93.6%, respectively (Fig. 10C).
  • the purpose of this example is to explore the analysis of tumor immune cell infiltration of colorectal tumor (MC38) by OMV nano-vaccine (OMV-L-mRNA ADPGK ).
  • the MC38 tumor tissue after the vaccine treatment of the nano-vaccine in Example 10 is analyzed for immune cell infiltration, and then the anti-tumor mechanism of the nano-vaccine is explored; flow cytometry is used to analyze the concentration of T cells in the mouse MC38 tumor tissue. Infiltration condition.
  • Figure 11A is a diagram of the population change of CD3 + T cells infiltrating into the tumor
  • Figure 11B is a diagram of the change in the number of CD3 + CD8 + T cells infiltrating into the tumor
  • Figure 11C is a diagram of the number of CD3 + CD4 T cells infiltrating into the tumor
  • Figure 11D is a map of the group changes of Treg cells (CD3 + CD4 + CD25 + ) infiltrating the tumor; it can be seen that the universal carrier of nano vaccines can effectively induce CD3 + , CD3 + after carrying the antigen ADPGK
  • the infiltration of + CD4 + and CD3 + CD8 + T cells in the tumor tissue, while the immunosuppressive cells (Treg) did not show a tendency to increase, indicating the good immunity of the nano-vaccine.
  • the purpose of this example is to explore the versatility of OMV nano-vaccine for different nucleic acid vaccines.
  • OMV-L-mRNA HZV OMV nano vaccine
  • the OMV universal vaccine carrier is OMV
  • the mRNA antigen fragment is box C/D-HZV
  • the mass ratio of the universal vaccine carrier to mRNA is 10:3;
  • mice BALB/c healthy mice were immunized with OMV-L-mRNA intramuscularly or intranasally. The first three immunizations were separated by two weeks, and a booster immunization was carried out one month after the three immunizations. There were four immunizations in total. Serum samples from mice at 0, 2, 4, 6, and 10w were collected, and the antibody concentration in serum at different time points was detected by ELISA method.
  • Figure 12A and Figure 12B are the antibody concentrations in serum at different time points after OMV-L-mRNA HZV is immunized by intramuscular injection or intranasal route
  • Figure 12C and Figure 12D are OMV-L-mRNA HBeAg Antibody concentrations in serum at different time points after mice were immunized by intramuscular injection or intranasal drip. The results showed that whether the mice were immunized by intramuscular injection or intranasal drip, with the increase of the number of immunizations, the antibody level in the serum was on the rise. The level is higher than before.
  • This example studies the expression effect of the OMV nano-vaccine and the stimulation of the immune response.
  • the results show that the OMV nano-vaccine of the present application is versatile for different nucleic acid vaccines.
  • the purpose of this example is to explore the effect of nucleic acid display on the inner and outer surfaces of OMV nano-vaccine.
  • the anti-tumor nano-vaccine of the present application well expands the in vivo delivery system of mRNA, promotes the development of expandable personalized tumor vaccines, and has good application prospects. Due to space limitations, this article only cites some of the most convincing examples.
  • the present application illustrates the process method of the present application through the above examples, but the present application is not limited to the above process steps, that is, it does not mean that the application must rely on the above process steps to be implemented.
  • Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of the raw materials selected in the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

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Abstract

一种细菌外膜囊泡输运mRNA抗原的纳米疫苗及其应用，具体提供一种在囊泡外表面展示核酸的技术。

Description

细菌外膜囊泡来源的核酸纳米疫苗及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种细菌外膜囊泡输运mRNA抗原的纳米疫苗及其应用。
背景技术
肿瘤疫苗被认为是最具潜力的肿瘤免疫疗法之一，其中mRNA不仅简单快速，且经济低廉。更为重要的是，与大部分肿瘤药物不同，行使功能后的mRNA便会自动降解，不会对人体产生其他的毒害或副作用。
然而，目前该领域仍然存在着较大的发展瓶颈。由于其分子量大和负电荷高，裸露的mRNA直接进入体内时极易被降解，这是优势同时也是劣势，mRNA疫苗必须依赖于有效的输运载体才能进入细胞，而且能够进入抗原递呈细胞是mRNA疫苗有效的前提。目前主流的mRNA体内输运载体是脂质纳米递送系统，但这种载体针对每一种抗原都要重新合成并包载，过程费时费力。另外，成功的适应性免疫激活还需要天然免疫的辅助，这就要求在疫苗中掺杂免疫佐剂，进一步加大了纳米载体的合成复杂程度。
与此同时，天然药物载体递送平台发展迅速，其中具有代表性的例子之一是细胞膜衍生的囊泡。由于这些囊泡天然参与生物分子的细胞间交换，其作为新型药物输送载体具有巨大潜力，已有人将细胞外囊泡改造以携带一些编码或非编码核酸，但目前仍然存在很多挑战，例如核酸载量不高、不稳定、递送的核酸无法翻译为蛋白等。
因此，本领域急需一种能够提高核酸递送效率或抗原呈递效果、从而有效激活免疫的系统。
发明内容
本申请提供了一种改造的囊泡及其制备方法和应用。本申请主要以细菌外膜囊泡(OMV)为载体，创造性地在OMV表面融合表达核酸结合分子，实现将感兴趣的核酸(例如外源mRNA抗原)结合在OMV表面并进行递送。本申请提供了一种适用于临床递送的复杂和异质性抗原的疫苗。本申请的改造的囊泡可作为一种新型OMV-mRNA抗原复合物的纳米疫苗，具有以下一个或多个特点：(1)稳定，包括囊泡载体稳定、载荷核酸稳定，以及核酸与囊泡整个疫苗体系稳定，(2)易于贮存或运输，(3)能够刺激产生强烈的先天免疫反应，(4)能够有 效递送核酸至抗原呈递细胞，并被有效呈递在细胞表面，(5)能够引发强烈的抗原特异性免疫反应，(6)能够产生抗原特异性免疫记忆，提供长期的肿瘤预防效果，(7)能够灵活递送各种核酸抗原并有效引发抗原特异性抗肿瘤免疫能力，这些特点显示本申请的纳米疫苗在未来临床疫苗开发应用中具有巨大的潜力。
在本申请中，OMV可以是一种纳米尺寸下的双层蛋白脂质体，其可以是由细菌在生长过程中自外膜出芽分泌释放的天然囊泡，也可以是工程化改造的细菌来源的囊泡。在本申请中，OMV可以将感兴趣的分子，例如蛋白分子、脂类物质或核酸分子传递进入宿主细胞并且携带具有免疫原性的成分进入细胞质中。在本申请中，OMV本身的纳米尺寸(例如，1-1000nm，又例如，20-100nm)和表面天然结构可以促进抗原呈递细胞(例如，DC细胞)对OMV的高效摄取、刺激天然免疫系统，从而促进抗原的处理和递呈，即免疫佐剂效应。
本申请提供了一种囊泡，所述囊泡的外表面包含核酸。
本申请提供了一种囊泡，所述囊泡包含核酸结合分子，所述核酸结合分子能够使其结合的核酸展示在所述囊泡的外表面。
本申请提供了一种融合蛋白，所述融合蛋白包含核酸结合分子和膜蛋白，所述核酸结合分子能够使其结合的核酸展示在所述囊泡的外表面。
本申请提供了一种核酸，所述核酸编码本申请的融合蛋白。
本申请提供了一种载体，包含本申请的核酸，以及任选的非编码区和/或抗原结构优化区。
本申请提供了一种细胞，包含本申请的囊泡、本申请的融合蛋白、本申请的核酸、和/或本申请的载体。
本申请提供了一种组合物，包含本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、和/或本申请的细胞，以及任选的载剂。
本申请提供了一种试剂盒，包含本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、和/或本申请的组合物。
本申请提供了一种展示和/或表达外源核酸的方法，包含施用本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、和/或本申请的试剂盒。
本申请提供了一种影响免疫反应和/或抑制肿瘤生长的方法，包含施用本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、和/或本申请的试剂盒。
本申请提供了一种本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、和/或本申请的试剂盒在制备试剂中的用途，所述试剂用于预防和/或治疗疾病和/或病症。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1中图1A为OMV通用疫苗载体OMV-L在PBS溶液中的透射电镜图片；图1B为OMV通用疫苗载体OMV-L在PBS溶液中的动态光散射粒径分析，图1C为本申请的基于OMV的mRNA递送系统示意图。
图2A-2B显示的是，通用疫苗载体OMV利用锚定蛋白ClyA使L7Ae成功构建表达的蛋白免疫印记条带；图2C为通用疫苗载体OMV利用锚定蛋白INP使L7Ae成功构建表达的蛋白免疫印记条带。
图3显示的是，OMV通用疫苗载体输运EGFP mRNA在293T的流式检测定量图，BMDCs中的流式检测定量图和免疫荧光图；其中3A为293T的流式检测定量图，3B为BMDCs中的流式检测定量图，3C为免疫荧光共聚焦图。
图4中4A显示的是用OMV-LL处理的HEK293衍生的TLR/NOD报告细胞中的荧光素酶信号OD定量结果，4B显示的是BMDCs与OMV-LL孵育后分泌的炎性细胞因子的热图。
图5中5A为OMV通用疫苗载体输运OVA mRNA在BMDCs中的抗原递呈效率图，5B为不同时间下MHCI-OVA的表达水平图；MHCI-OVA分子表达水平由细胞流式计数仪检测。
图6显示的是，OMV通用疫苗载体输运OVA mRNA在体内刺激DC成熟和MHCI-OVA抗原呈递水平的检测，其中6A为纳米疫苗注射后，淋巴结DC共刺激分子CD80&CD86的表达定量图，6B为DC表达MHCI-OVA复合物的流式定量结果图，以上分子水平检测由细胞流式细胞术实施。
图7显示的是，OMV通用疫苗载体输运OVA mRNA在体内三针免疫后，提取的脾细胞在OVA多肽抗原刺激后，CD3 +CD4 +，CD3 +CD8 +T细胞分泌IFN-γ能力的检测，其中图7A为CD3 +CD8 +T细胞分泌IFN-γ流式定量图，图7B为CD3 +CD4 +T细胞分泌IFN-γ流式定量图，图7C显示的是使用CFSE染色和流式细胞术分析用OVA肽抗原再刺激的脾细胞的增殖，图7D显示的是脾细胞对B16-OVA细胞的抗原特异性杀伤能力。
图8显示的是，OMV纳米疫苗在荷黑色素瘤肺转移中的抗肿瘤效果评价，其中8A为基于OMV的mRNA疫苗抑制肺转移的评估程序示意图，8B为第20天从携带B16-OVA肿瘤的转移性小鼠身上采集的代表性的肺转移灶图，8C为转移灶定量统计图，8D为根据8C中肿瘤结节的数量计算的转移抑制率，8E用指定疫苗制剂免疫的转移性B16-OVA肿瘤小鼠的存活曲线。
图9显示的是，OMV纳米疫苗在C57/BL6小鼠体内产生的免疫记忆效果评价，其中9A为由基于OMV的mRNA疫苗引起的长期免疫记忆的程序的方案，9B为免疫记忆性T细胞(CD3 +CD8 +CD44 +CD62L -)的流式图，9C为免疫记忆性T细胞的定量测量分析图，9D为第80天切除的肺部图像，9E为肺转移结节的数量。
图10显示的是，OMV纳米疫苗在小鼠皮下结肠癌(MC38)模型中的肿瘤抑制效果图，其中10A为MC38肿瘤生长曲线；10B为从每组小鼠中切除的MC38肿瘤的肿瘤重量；10C为根据肿瘤重量计算的肿瘤抑制率统计。
图11显示的是，OMV纳米疫苗在小鼠皮下结肠癌(MC38)模型实施后，肿瘤浸润的免疫细胞变化情况，11A为CD3 +T细胞，11B为CD3 +CD8 +T细胞，11C为CD3 +CD4 +T细胞，11D为CD3 +CD4 +Foxp3 +Tregs。
图12显示的是，血清中特异性抗体水平，其中12A为OMV-L-mRNA HZV纳米疫苗采用肌肉注射免疫小鼠，12B为OMV-L-mRNA HZV纳米疫苗经鼻途径免疫小鼠，12C为OMV-L-OMV-L-mRNA HBeAg纳米疫苗采用肌肉注射免疫小鼠，12D为OMV-L-mRNA HBeAg纳米疫苗经鼻途径免疫小鼠。
图13显示的是，OMV纳米疫苗在内外表面展示核酸的表达情况。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“核酸结合分子”通常是指具有与核酸结合能力的分子。例如，所述核酸结合分子可以特异性地结合核酸。例如，所述核酸结合分子可以包含特异性识别核酸的区域。例如，核酸结合分子可以识别box C/D序列。
在本申请中，术语“囊泡”通常是指具有膜结构的囊状构造。例如，囊泡外围只是具有一层的脂质双层分子膜。本申请的囊泡包含任意直径的囊泡。所述囊泡的直径可以通过透射电子显微镜进行表征。
在本申请中，术语“疱疹病毒抗原”通常是指来源于疱疹病毒的蛋白。例如，编码疱疹病毒的蛋白的核酸可以通过本申请的方法进行展示。
在本申请中，术语“乙肝e抗原”通常是指来源于乙肝病毒的蛋白。例如，编码乙肝e抗原的蛋白的核酸可以通过本申请的方法进行展示。
在本申请中，术语“Adpgk”通常是指ADP Dependent Glucokinase。例如Adpgk的Uniprot登录号可以为Q9BRR6。本申请的Adpgk可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。在本申请中，术语“ClyA”通常是指Cytolysin A。例如ClyA的Uniprot登录号可以为Q68S90。本申请的ClyA可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。在本申请中，术语“INP”通常是指Ice nucleation protein。例如INP的Uniprot登录号可以为P06620。本申请的INP可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。在本申请中，术语“L7Ae”通常是指一种RNA结合蛋白。例如的Uniprot登录号可以为D0KRE2。本申请的L7Ae可以涵盖其未加工形式、任何的加工形式、其变体或包含其功能活性片段的物质。
发明详述
修饰的囊泡
一方面，本申请提供了一种修饰的囊泡，其表面包含核酸结合分子，所述核酸结合分子能够结合核酸，并使所结合的核酸展示在所述囊泡的外表面。
另一方面，本申请提供了一种修饰的囊泡，其表面包含核酸，并且所述核酸通过核酸结合分子展示在囊泡的外表面。
本申请的囊泡可以来源于细胞的膜结构，例如可以来源于细菌的膜结构。本申请所述的囊泡可以来源于革兰氏阴性菌和/或革兰氏阳性菌的膜结构。本申请所述的囊泡可以包含来源于肠杆菌科和/或奈氏菌科的膜结构。本申请所述的囊泡可以来源于沙门氏菌、大肠杆 菌、和/或脑膜炎球菌的膜结构。例如，本申请所述的囊泡可以来源于革兰氏阴性菌，减毒沙门氏菌属、葡萄球菌属、乳酸杆菌属、芽孢杆菌属、假单胞菌或假长双歧杆菌。
在某些实施方式中，本申请所述囊泡可以来源于细菌外膜囊泡(OMV)。OMV具有丰富的病原体相关分子模式(PAMP)，可以强烈刺激先天免疫系统以促进抗原呈递和T细胞活化，是理想的疫苗载体。例如，所述OMV可以由革兰氏阴性菌分泌，具有内在的纳米尺寸和细菌成分，能够被树突状细胞(DC)有效识别和摄取，并可以按照本申请的描述被改造使得其能递送核酸。
例如，本申请的细胞和/或细菌可以通过添加诱导剂的方式表达外源蛋白的质粒，诱导剂包含四环素，强力霉素或者体内来源代谢产物如多巴胺，茶多酚等，优选pET28a质粒的诱导剂为IPTG。
例如，本申请的囊泡通过透射电镜检测，所述囊泡的平均直径可以为约28nm。例如，本申请的囊泡可以通过宿主细菌在生长状态下分泌，并通过超速离心法等提取获得。
本申请的囊泡可以修饰以包含核酸结合分子。例如，所述核酸结合分子可以融合至所述囊泡的膜蛋白上。所述膜蛋白可以是天然存在囊泡表面的膜蛋白，也可以是工程化改造后表达在囊泡表面的异源膜蛋白，只要该蛋白能稳定地表达在囊泡表面即可。例如，所述膜蛋白可以选自下组：溶细胞素A(ClyA)、冰核蛋白(INP)、OmpA和Hbp。所述核酸结合分子可以通过与膜蛋白融合，间接表达在囊泡表面。所述核酸结合分子也可以以跨膜蛋白的形式直接表达在囊泡表面。
如本申请的囊泡，所述膜蛋白与所述核酸结合分子可以直接或间接连接。如本申请的囊泡，所述膜蛋白的C端与所述核酸结合分子的N端可以直接或间接连接。例如，所述连接方式可以根据需要调整。例如根据使得所述核酸结合分子表达在囊泡膜的外表面，调整所述连接方式。
在本申请中，所述核酸结合分子可以结合感兴趣的核酸，使得所结合的核酸展示在所述囊泡的表面。核酸结合分子可以是能够结合特定核酸序列或特定核酸片段的蛋白，所述特定核酸序列或特定核酸片段在本申请中可以被称为结合序列。现有技术中常见的核酸结合分子例如RNA结合蛋白，包括真核生物来源的铁调节蛋白(IRP)或U1A蛋白，以及原核生物来源的L7Ae、MS2外壳蛋白或RNAIII活化蛋白靶标(TRAP)，都可以应用在本申请的囊泡中。作为本申请的一个实施例，使用L7Ae作为核酸结合分子。L7Ae可以结合box C/D。box C/D序列采用一种标准k-turn构象，能够被L7Ae蛋白特异性识别，从而稳定茎环并形成稳定的L7Ae-k-turn复合体。例如，box C/D序列可以如SEQ ID NO：1所示。根据选择的核酸结合分子的特性，可以选择相应的结合序列来标记所需要结合的目标核酸。
在本申请中，核酸结合分子融合至OMV的表面蛋白。在一个实施方式中，核酸结合分子为L7Ae，融合至OMV表面蛋白ClyA的C端。
在本申请中，所述核酸可以通过结合序列和核酸结合分子之间的相互作用装载在本申请所述的囊泡外表面。在一个实施方式中，核酸通过结合序列box C/D与融合在OMV表面的L7Ae之间的相互作用展示在OMV表面。
如本申请的囊泡，所述核酸可以包含外源核酸。所述外源核酸可以编码感兴趣的蛋白质，也可以是非编码核酸，或者其他任何需要被递送的核酸。如本申请的囊泡，所述核酸可以包含RNA。如本申请的囊泡，所述核酸可以包含编码肿瘤相关抗原和/或传染病相关抗原的RNA。如本申请的囊泡，所述肿瘤相关抗原可以包含OVA和/或癌胚抗原Adpgk(ADP Dependent Glucokinase)。例如，OVA的核酸序列可以如SEQ ID NO：6所示。例如，Adpgk(ADP Dependent Glucokinase)核酸序列可以如SEQ ID NO：7所示。如本申请的囊泡，所述抗原可以包含OVA、癌胚抗原CEA，新抗原Adpgk，肿瘤相关抗原MUC1，乙肝e抗原等，以及上述的任意组合。如本申请的囊泡，所述传染病相关抗原可以包含乙肝e抗原和/或疱疹病毒抗原。例如，乙肝e抗原的核酸序列可以如SEQ ID NO：10所示。例如，疱疹病毒衣壳抗原核酸序列可以如SEQ ID NO：9所示。例如，可以包含与本申请的核酸序列具有99％以上、98％以上、97％以上、96％以上、95％以上、90％以上、80％以上、70％以上、60％以上、50％以上的同源性的核酸序列。
在本申请中，所述核酸可以包含结合序列和编码抗原的核酸分子。所述结合序列与编码抗原的核酸分子连接，且不影响编码抗原的核酸分子的表达。在本申请中，所述结合序列可以位于编码抗原的核酸的非编码区。在本申请中，所述结合序列可以位于编码抗原的核酸的3’UTR。
在本申请中，所述核酸还可包含抗原结构优化区。例如，本申请的抗原结构优化区，可以用于提高抗原翻译效率和/或提高MHCI的呈递效率。例如，本申请的抗原结构优化区包含MITD(MHC class I trafficking signal)序列，例如MITD的序列可以如SEQ ID NO：4所示。例如，本申请的抗原结构优化区包含Ub序列，例如Ub的序列可以如SEQ ID NO：5所示。
例如，本申请的核酸为RNA，其序列可以依次包含5’非编码区-抗原结构优化区-抗原编码区-3’非编码区-RNA结合蛋白结合序列。所述抗原编码区为编码最终抗原蛋白的mRNA区域。所述5’和3’非编码区，可以用于稳定mRNA结构和提高翻译效率。例如，5’非编码区的序列可以如SEQ ID NO：2所示。例如，3’非编码区的序列可以如SEQ ID NO：3所示。RNA结合蛋白结合序列为能够与OMV表面融合表达的核酸结合分子特异性 结合的序列，当选择L7Ae作为核酸结合分子时，可以选择box C/D作为结合序列标记编码抗原的核酸。
在一个实施方式中，所述囊泡的外表面可以改造以获得感兴趣的性质(例如，融合感兴趣的蛋白)，以使得核酸更稳定、使得核酸更容易被抗原呈递细胞呈递、使得整个囊泡体系更容易进入靶细胞或使得免疫应答更强烈。例如，可以将李斯特菌溶血素O(LLO)融合至囊泡的表面蛋白，以增强OMV的体内逃逸和mRNA翻译。
蛋白、载体和细胞
本申请还提供了一种融合蛋白，所述融合蛋白可以包含核酸结合分子和膜蛋白，所述核酸蛋白能够使其结合的核酸展示在所述囊泡的外表面。
如本申请的融合蛋白，所述膜蛋白可以包含溶细胞素A(ClyA)和/或冰核蛋白(INP)，以及上述的功能活性片段。
如本申请的融合蛋白，所述核酸结合分子可以识别box C/D序列。如本申请的融合蛋白，所述核酸结合分子可以包含L7Ae或其功能活性片段。例如，本申请的核酸结合分子还可以包含真核生物来源的IRP和U1A，以及原核生物来源的L7Ae，MS2和TRAP。
如本申请的融合蛋白，所述膜蛋白与所述核酸结合分子可以直接或间接连接。
如本申请的融合蛋白，所述膜蛋白的C端与所述核酸结合分子的N端可以直接或间接连接。例如，所述连接方式可以根据需要调整。例如根据使得所述核酸结合分子表达在囊泡膜的外表面，调整所述连接方式。
本申请还提供了一种核酸，所述核酸编码本申请的融合蛋白。
本申请还提供了一种载体，可以包含本申请的核酸，以及任选的非编码区和/或抗原结构优化区。
例如，本申请的载体可以包含多个串联结构的表达盒。
本申请还提供了一种细胞，可以包含本申请的囊泡、本申请的融合蛋白、本申请的核酸、和/或本申请的载体。
本申请还提供了一种组合物，可以包含本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、和/或本申请的细胞，以及任选的载剂。
本申请还提供了一种试剂盒，可以包含本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、和/或本申请的组合物。
疫苗和方法
另一方面，本申请提供了一种疫苗，特别的，一种通用的核酸纳米疫苗。本申请的改造的囊泡可以作为核酸疫苗载体，递送感兴趣的核酸抗原。
另一方面，本申请提供了一种展示和/或表达外源核酸的方法，可以包含施用本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、和/或本申请的试剂盒。例如，本申请的融合蛋白可以表达在OMV的外表面。例如本申请融合蛋白结合的RNA可以在树突细胞中表达为目的蛋白。
另一方面，本申请提供了一种影响免疫反应和/或抑制肿瘤生长的方法，可以包含施用本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、和/或本申请的试剂盒。例如，所述方法可以是非治疗和/或非诊断目的的方法。例如，所述影响免疫反应包含激活免疫反应，例如激活免疫反应的效果可以包含激活树突细胞、使树突细胞(例如CD11c阳性细胞)成熟(例如CD80阳性CD86阳性细胞的比例提高)、使免疫细胞分泌细胞因子(例如使T细胞分泌IFN-γ)、提高肿瘤浸润淋巴细胞中效应T细胞的比例和/或激活免疫记忆反应(例如CD3 +CD4 +CD44 +CD62L -细胞比例提高)。例如，本申请的抑制肿瘤生长可以包含抑制肿瘤的转移(例如肿瘤的肺转移)、和/或抑制肿瘤增长的体积。
一种本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、本申请的疫苗和/或本申请的试剂盒在制备试剂中的用途，所述试剂用于预防和/或治疗疾病和/或病症。例如，对疾病的预防和/或治疗，包括但不限于黑色素瘤、结肠癌、乳腺癌、人乳头状瘤，以及乙肝、疱疹等。
如本申请的用途，所述疾病和/或病症可以包含肿瘤和/或传染病。
如本申请的用途，所述疾病和/或病症可以包含实体瘤。如本申请的用途，所述疾病和/或病症可以包含黑色素肿瘤和/或结直肠肿瘤。
如本申请的用途，所述疾病和/或病症可以包含疱疹病毒和/或乙肝病毒感染相关的感染病。
一种预防和/或治疗疾病和/或病症的方法，包含施用本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、本申请的疫苗和/或本申请的试剂盒。例如，所述疾病和/或病症可以包含肿瘤和/或传染病。例如，所述疾病和/或病症可以包含实体瘤。例如，所述疾病和/或病症可以包含黑色素肿瘤和/或结直肠肿瘤。例如，所述疾病和/或病症可以包含疱疹病毒和/或乙肝病毒感染相关的感染病。
一种本申请的囊泡、本申请的融合蛋白、本申请的核酸、本申请的载体、本申请的细胞、本申请的组合物、本申请的疫苗和/或本申请的试剂盒，其用于预防和/或治疗疾病和/或病症。例如，所述疾病和/或病症可以包含肿瘤和/或传染病。例如，所述疾病和/或病症可以包含实体瘤。例如，所述疾病和/或病症可以包含黑色素肿瘤和/或结直肠肿瘤。例如，所述 疾病和/或病症可以包含疱疹病毒和/或乙肝病毒感染相关的感染病。
本申请提供一种能够灵活快速展示mRNA肿瘤抗原的OMV的疫苗平台。在一个实施方案中，将古细菌蛋白L7Ae融合表达在OMV表面蛋白ClyA的C端，同时将box C/D序列修饰在体外转录合成的mRNA肿瘤抗原的3’非编码区。通过L7Ae与box C/D的高效特异性结合，OMV将mRNA抗原递送进入抗原递呈细胞并翻译成表位信息与MHCI形成复合物，呈递在细胞表面。在一个实施方案中，将编码抗原结构优化区(例如MITD)的核酸引入mRNA，提高了抗原呈递分子的表达；OMV的天然成分可激活多条通路刺激DC细胞成熟和细胞因子释放，这些都促进了T细胞的成功招募。本申请基于可结合mRNA的OMV的肿瘤疫苗平台，能够显著激活宿主免疫反应，快速展示肿瘤抗原，最终高效的引起抗原特异性T细胞介导的抗肿瘤免疫效应。
所述OMV有天然免疫佐剂作用，可增强疫苗的免疫效应；所述OMV表面嵌合有RNA结合蛋白(优选为古细菌核糖体蛋白L7Ae)，能够与含有RNA结合蛋白结合序列(优选为与L7Ae匹配的box C/D序列)的mRNA快速结合并展示在OMV表面。所述mRNA由5’非编码区-抗原结构优化区-抗原区-3’非编码区-RNA结合蛋白结合序列组成，其中非编码区和抗原结构优化区可提升mRNA抗原在细胞内的翻译和MHCI递呈效率。最终，这种基于OMV-mRNA抗原复合物的纳米疫苗实现了天然佐剂和mRNA抗原的同时高效递送，能够高效地激活抗原特异性免疫反应，在肿瘤疫苗领域展现出强大的应用潜力。
本申请采用如下技术方案：
本申请的一个实施方式中，一种细菌外膜囊泡和抗原核酸共输运的联合肿瘤纳米疫苗，由大肠杆菌分泌的外膜囊泡和抗原mRNA锚定结合形成，所述抗原mRNA能够被抗原提呈细胞摄取，并翻译成具有抗原表位信息的抗原。在本申请的设计中，制备所述囊泡的一个实例如下：
其中：
所述细菌外膜囊泡锚定蛋白通过基因工程技术，以限制性内切酶酶切后整合到原核表达质粒，构建出含有膜定位蛋白和功能蛋白的原核表达质粒。
该重组质粒的膜锚定序列的3'端插入外源蛋白序列L7Ae以及HA基因序列，以此形成“膜定位蛋白-mRNA结合蛋白”融合表达的重组质粒。也可以插入外源蛋白李斯特菌溶血素O，用Flag序列作为标记。
优选地，所述重组质粒以热激42℃转化导入大肠杆菌，通过诱导剂IPTG，低温诱导后使得外源性蛋白正确翻译折叠为可溶性融合蛋白，并随膜牵引蛋白将定位蛋白序列牵引到细 菌外膜表面。
本申请中，细菌外膜囊泡的提取方法为梯度超速离心法，具体为：
将低温诱导表达过夜的菌液离心，收集培养液上清，0.45μm无菌滤膜过滤上清去除残余菌体碎片，将滤液通过截留分子量为100kDa的超滤管浓缩，将浓缩后滤液再次过0.22μm无菌滤膜，收集滤液进行超速离心，利用磷酸盐缓冲液(PBS)重悬洗涤沉淀，最后再次超速离心。PBS或mRNA结合反应溶液再次重悬沉淀OMV，置于-20℃保存。
优选地，所述重组表达后的大肠杆菌来源细菌外膜囊泡(OMV)中检测到了外源蛋白的存在，并证明其在OMV表面。
本申请中，所述肿瘤mRNA抗原由重组质粒体外转录合成，并利用绿色荧光蛋白(EGFP)验证了OMV的核酸输运能力，最后利用肿瘤模式抗原OVA核酸序列检测其引起抗原特异性T细胞免疫应答能力及抗肿瘤能力。
优选地，所述EGFP的mRNA抗原成功结合到OMV表面，并在293T细胞上体外验证了其输运后蛋白翻译水平。
在本申请中，改造的OMV载体(以蛋白质量计)与mRNA载荷的质量比例可以为20：1至2：1。例如，OMV载体(以蛋白质量计)与mRNA载荷的质量比例可以为10：3。
所述EGFP的mRNA由OMV结合输运，经与BMDCs共孵育24h后，在流式检测中发现了绿色荧光蛋白的表达，进而证明OMV输运mRNA在BMDCs中的成功。
以载带OVA mRNA(SIINFEKL，SEQ ID NO：11)抗原纳米疫苗OMV-L-mRNA OVA为例，在与骨髓源树突状细胞(BMDCs)共孵育24h后，展示了该纳米疫苗的促进抗原提呈细胞呈递抗原效应分子的能力。
优选地，所述纳米疫苗OMV-L-mRNA OVA以皮下给药方式免疫小鼠后，促进了淋巴结组织处抗原提呈细胞的成熟水平。
所述纳米疫苗OMV-L-mRNA OVA，经过三次免疫小鼠后，小鼠产生了针对OVA抗原的免疫T细胞激活能力。
优选地，提取所述免疫终点的小鼠脾脏，研磨得到T细胞悬液，再次使用OVA 257-264(SIINFEKL，SEQ ID NO：11)多肽抗原片段刺激该T细胞悬液，并通过IFN-γ的分泌证明了T细胞的毒性杀伤效应。
优选地，所述纳米疫苗OMV-L-mRNA OVA，在荷黑色素肺转移瘤的C57/BL6小鼠中展示了显著的抑瘤效果。
优选地，所述纳米疫苗OMV-L-mRNA OVA，在C57/BL6小鼠中展示了长效的免疫记忆性 和持久的肿瘤预防能力。
优选地，所述纳米疫苗OMV-L-mRNA ADPGK(ADPGK：ASMTNMELM，SEQ ID NO：12)，在皮下结肠癌(MC38)模型中的C57/BL6小鼠中展示了显著的抑瘤效果。
优选地，纳米疫苗OMV-L-mRNA HZV(HZV抗原的氨基酸序列如SEQ ID NO：9所示)或OMV-L-mRNA HBeAg(HBeAg的氨基酸序列如SEQ ID NO：10所示)能激活机体抗原特异性的免疫反应，包括细胞免疫和体液免疫。
本申请证明了OMV输运mRNA的能力，细胞以及动物活体水平均能产生高效的mRNA输运水平，该纳米疫苗平台无需额外借助佐剂，并能引起有效的T细胞抗原识别免疫应答，大幅度的提升肿瘤特异性杀伤能力，构建了一种全新的mRNA纳米疫苗输运平台。
本申请所述OMV肿瘤纳米疫苗可由现有常规基因工程手段和超速离心技术制备得到。
与现有技术相比，本申请的有益效果可以为：
本申请所述细菌外膜囊泡和核酸抗原结合的OMV肿瘤纳米疫苗是一种能够灵活快速展示mRNA肿瘤抗原的OMV的新型疫苗平台。本申请中细菌外膜囊泡(OMV)具有良好的基因工程可操作性和天然的佐剂免疫激活作用。其次，与单纯的裸mRNA相比，mRNA-OMV有更高的生物稳定性，且提高了其在细胞内的蛋白翻译表达水平；同时所得到的OMV肿瘤纳米疫苗具有更加强烈的免疫应答作用，与单独的mRNA相比，表现出更强的诱导抗原提呈细胞的成熟，以及引起了更高的特异性T细胞毒性杀伤效应和抗肿瘤效果。该OMV肿瘤纳米疫苗可以为构建新型mRNA疫苗用于肿瘤免疫治疗提供可行的方法和技术。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的产品、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
除非特别指明，以下实施例中所用水溶液的溶剂均为无菌超纯水溶液。
除非特别指明，以下实施例中所用的试剂均为分析纯试剂。
除非特别指明，以下实施例中所用的PBS溶液均为1×PBS溶液。
10×PBS溶液的配制：NaCl 80.00g，KCl 2g，Na 2HPO 4·12H 2O 35.8g或Na 2HPO 4 14.2g，KH 2PO 4 2.7g，用超纯水定容至1000mL，调节其pH值为7.2～7.4，高压灭菌。
1×PBS溶液的配制：将10×PBS溶液用无菌超纯水稀释10倍。
mRNA合成：使用HiScribe T7ARCA mRNA试剂盒通过IVT合成mRNA。IVT的模板是使用Q5高保真2×预混液(M0492，NEB，美国)的pST1374质粒的PCR产物。模板包含T7启动子。然后按照标准方案进行IVT反应，将CTP和UTP分别替换为5-甲基-CTP和pseudo-UTP。最后，使用poly(A)聚合酶(M0276L，NEB，美国)对mRNA进行3'-poly(A)加尾，并根据制造商的说明通过LiCl沉淀实现mRNA纯化。
骨髓来源的树突状细胞(BMDC)的产生和刺激：从6-8周大的C57BL/6小鼠的胫骨和股骨中采集骨髓细胞，并在含有10％FBS、1％HEPES、0.05×10 -3mβ-巯基乙醇(β-ME)、500U mL -1mIL-4和1000U mL -1mGM-CSF的RPMI 1640培养基中培养，诱导分化为未成熟的DC。第6天，收集BMDCs用于各种实验。为了确定OMV是否可以驱动BMDC的成熟，将细胞在24孔板中培养，然后与不同的OMV制剂一起孵育12或24小时。然后收集培养上清液用于多种细胞因子分析，并用抗小鼠CD11c-FITC、抗小鼠CD80-PE或抗小鼠CD86-PE抗体对BMDCs进行染色后进行流式细胞术。对于抗原呈递测定，将BMDC用OVA 257-264肽(2μg mL -1)或OMV-LL-mRNA OVA(2μg mL -1)孵育0、6、12、24、36和48小时。通过流式细胞术评估MHCI-OVA表达来评估OVA抗原呈递功效。
除非特别指明，实施例中各OMV通用疫苗载体和纳米疫苗的代号如下：
OMVs指的是OMV表面膜蛋白ClyA没有进一步融合其他分子的载体，
OMV-L指的是OMV表面的膜蛋白ClyA上融合了L7Ae的载体，即表达ClyA-L7Ae的OMV；
OMV-LLO指的是OMV表面的膜蛋白ClyA上融合了LLO的载体，即表达ClyA-LLO的OMV；
OMV-LL指的是OMV表面的膜蛋白ClyA上融合了L7Ae和LLO的载体，即同时表达ClyA-L7Ae和ClyA-LLO的OMV；
OMV-L-mRNA 抗原指的是mRNA抗原通过核酸结合分子L7Ae融合在OMV表面的膜蛋白ClyA上得到的疫苗，即表达ClyA-L7Ae的OMV，并且抗原mRNA通过box C/D与L7Ae之间的相互作用装载在在OMV表面。
OMV-LL-mRNA 抗原指的是mRNA抗原通过核酸结合分子L7Ae融合在OMV表面的膜蛋白ClyA上，并且膜蛋白ClyA上融合了LLO的疫苗，即同时表达表达ClyA-L7Ae和Cly-LLO的OMV，并且抗原mRNA通过box C/D与L7Ae之间的相互作用装载在在OMV表面。
实施例1
一种纳米疫苗载体OMV，由质粒pET28a-ClyA-L7Ae或pET28a-ClyA于大肠杆菌BL21(DE3)转化、诱导、发酵、超速离心制备形成，得到的纳米疫苗载体分别称为OMV-L和OMVs。其中，具体提取步骤如下：
1、收集菌液，在5000g，4℃，10min去除菌体和碎片；
2、将上述收集的上清用0.45μm的滤网过滤；
3、收集滤液并利用超滤浓缩管(100kDa)，在离心力为3000g下离心5分钟，收集滤芯内未滤过液体；
4、再次将3中收集液体用0.22μm滤网过滤，收集滤液；
5、将滤液装入超离管，在离心力为150000g，4℃条件下离心3h；6、弃去上清，用PBS缓冲液重悬底部沉淀，在离心力150000g，4℃条件下再次离心3h，最后用200μL PBS重悬沉淀，获得OMV疫苗通用载体。
利用透射电镜(TEM)和动态光散射仪(DLS)对收集到的OMV疫苗通用载体进行形貌和粒径大小的表征；取10μL OMV通用疫苗载体的PBS溶液样品滴在经放电处理后表面活化的镀碳膜铜网上，静置5min，滤纸吸干溶液，取5μL 2％醋酸双氧铀(使用之前以4000rpm的转速离心5min，除去未能完全溶解的染色液)染色60s，滤纸吸干染色液，用透射电子显微镜(TEM，HT7700，日立公司)观察样品，如图1所示，图1B为OMV疫苗通用载体的电镜形貌，显示为均一分布的球形结构，图1C为OMV通用载体的粒径，其尺寸为28.1nm。
实施例2
本实施例的目的在于验证OMV通用疫苗载体融合蛋白的成功表达。
一种纳米疫苗载体OMV，由pET28a-ClyA-L7Ae，pET28a-ClyA，pET-ClyA-L7Ae-3HA-ClyA-LLO-3Flag于大肠杆菌BL21(DE3)转化、诱导、发酵、超速离心制备形成，得到的纳米疫苗载体分别称为OMV-L、OMVs和OMV-LL，具体步骤参见实施例1；其中，在RNA锚定蛋白(即核酸结合分子)L7Ae上偶联HA标签(HAYPYDVPDYA，SEQ ID NO：13)，在LLO上偶联Flag标签，用于蛋白免疫印记验证，通过蛋白免疫印记实验，利用所述标签抗体指示目的蛋白条带位置，来验证L7Ae成功在OMV通用疫苗载体上构建。
通过SDS-PAGE电泳,聚偏二氟乙烯膜(PVDF)转膜，标签抗体指示，显影液显影；如图2A-2B所示，ClyA-L7Ae-3HA的蛋白条带约在48kDa位置处，说明L7Ae成功表达在OMV-L和OMV-LL载体上，ClyA-LLO-3Flag的蛋白条带在75kDa位置处，说明李斯特菌溶 血素O(LLO)成功表达在OMV-LL上。在天然OMV中或没有表达诱导剂异丙基-β-d-硫代半乳糖苷(IPTG)时未检测到L7Ae(图2A，泳道3)。
实施例3
本实施例的目的在于探究不同表面蛋白对于OMV疫苗载体的通用性。
一种纳米疫苗载体OMV，由pET28a-INP-L7Ae，pET28a-INP于大肠杆菌BL21(DE3)转化、诱导、发酵、超速离心制备形成，得到的纳米疫苗载体分别称为OMV-L(此处的OMV-L指的是L7Ae融合在膜蛋白INP上的载体)和OMVs(此处的OMV指的是膜蛋白INP未融合L7Ae的载体)；其中，RNA锚定蛋白(即核酸结合分子)L7Ae上偶联HA标签(HAYPYDVPDYA，SEQ ID NO：13)，用于蛋白免疫印记验证，进一步地，通过蛋白免疫印记实验，利用所述标签抗体指示目的蛋白条带位置，来验证L7Ae成功在OMV通用疫苗载体上构建。
通过SDS-PAGE电泳,聚偏二氟乙烯膜(PVDF)转膜，标签抗体指示，显影液显影；如图2C所示，INP-L7Ae-3HA的蛋白条带约在32kDa位置处，说明L7Ae成功表达在OMV载体上，即将L7Ae融合至INP也可成功表达。
实施例4
本实施例的目的在于验证OMV通用疫苗载体可成功输运mRNA在细胞中表达。
将表面融合L7Ae的OMV(OMV-L7Ae，即OMV-L)和表面未融合L7Ae的OMV与mRNA抗原片段于特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中混合制备形成纳米疫苗，其中mRNA抗原片段为box C/D插入EGFR mRNA的3’UTR得到的box C/D-EGFP，两种OMV与mRNA抗原片段的质量比为10：3；
在无任何外加转染试剂条件下，OMV核酸纳米疫苗与人肾上皮细胞系293T共孵育24小时，流式细胞计数仪检测EGFP的表达情况。如图3A所示，在OMV通用疫苗载体递送下，EGFP在293T细胞中高度翻译表达；同时，利用流式细胞计数仪和荧光共聚焦显微镜检测OMV通用疫苗载体递送EGFP mRNA(OMV-L-mRNA EGFP)在主要免疫细胞骨髓来源诱导分化的树突状细胞(BMDCs)的表达水平,结果如图3B，3C所示，OMV-L-mRNA EGFP绿色荧光蛋白表达最强，可有效输运mRNA，进而被细胞翻译表达成蛋白。而表面未融合L7Ae的OMV和mRNA混合得到的疫苗(OMV+mRNA EGFP)与293T细胞孵育后，没有在293T细胞上检测到有EGFP蛋白表达。
实施例5
本实施例的目的在于验证OMV通用疫苗载体可激活先天免疫反应。
将ClyA-L7Ae-3HA-ClyA-LLO-3Flag基因克隆到表达质粒载体，转化到大肠杆菌(菌株BL21(DE3))中，将转化后的细菌接种到37℃的300mL LB肉汤中，使生长达到OD600为0.6至0.8。添加IPTG至终浓度为1×10 -3m来诱导融合蛋白表达。孵育过夜后离心收集无菌上清液，浓缩过滤分离OMV。洗涤沉淀并重悬于0.5mL DEPC处理的PBS或反应缓冲液(5×10 -3m Tris-HCl、100×10 -3m NaCl、10×10-3m MgCl 2；pH＝5.0)，得到OMV-LL。用OMV-LL处理HEK293衍生的TLR受体细胞，结果显示OMV-LL显著激活TLR2、TLR4和TLR5，并轻微激活TLR9(图4A)；同时，OMV-LL刺激BMDC分泌促炎细胞因子，例如IFN-β、IL-1β、TNF-α和IL-12p70(图4B)。以上结果证明，本申请的OMV-LL纳米载体能够刺激先天免疫反应。
实施例6
本实施例的目的在于验证OMV通用疫苗载体可输运肿瘤抗原OVA mRNA在免疫细胞中表达。
为优化抗原结构，将MHCI跟踪域(MITD)或泛素(Ub)融合至mRNA抗原片段，得到包含MITD的mRNA OVA(MITD-OVA)和包含Ub的mRNA OVA(Ub-OVA)。然后将OMV通用疫苗载体OMV-L、OMV-LL分别与mRNA抗原片段混合，特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中制备形成纳米疫苗OMV-L-mRNA OVA和OMV-LL-mRNA OVA。mRNA抗原片段的3’UTR插入box C/D序列，该通用疫苗载体与mRNA抗原片段的质量比为10：3。
在无任何外加转染试剂条件下，OMV核酸纳米疫苗与BMDCs共孵育24h,利用流式细胞计数仪检测纳米疫苗共孵育后，BMDCs表达抗原呈递分子MHCI-OVA的情况，如图5所示，图5A表明在无载体辅助下，mRNA基本没有表达，但是两种OMV通用疫苗(OMV-L-mRNA OVA和OMV-LL-mRNA OVA)均显著提高了BMDCs中MHCI-OVA分子的表达，并且融合了Ub和融合了MITD的mRNA抗原的疫苗均能有效进行抗原呈递。图5B为用OVA 257-264(SIINFEKL，SEQ ID NO：11)多肽组作对照，比较孵育不同时间后，BMDCs上MHCI-OVA分子水平变化，结果显示，OMV-LL-mRNA OVA作用BMDC细胞后，MHCI-OVA高水平表达的持续时间比仅OVA多肽处理的BMDC长。说明本申请改造的OMV载体显著提高了抗原在DC细胞上的表达和呈递。
实施例7
本实施例的目的在于验证OMV通用疫苗载体输运肿瘤抗原OVA mRNA诱导的DCs免疫应答水平。
将OMV通用疫苗载体OMV-L与OVA mRNA抗原序列于特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中制备形成mRNA疫苗OMV-L-mRNA OVA。mRNA抗原片段为3’UTR插入box C/D的OVA mRNA，该通用疫苗载体与mRNA的质量比为10：3；天然OMV与mRNA OMV混合物为OMVs+mRNA OVA。
将OVA mRNA、OMVs+mRNA OVA和OMV-L-mRNA OVA疫苗皮下免疫接种C57/BL6健康小鼠尾根位置，每只老鼠注射剂量为5μg的mRNA量，提取左右腹股沟淋巴结，并利用筛网研磨制备成单细胞悬液，孵育不同流式抗体后，流式细胞计数仪检测淋巴结中DCs的成熟和抗原递呈情况。如图6所示，图6A为OMV纳米疫苗接种后，DCs成熟的流式定量统计结果，显示OMV通用疫苗载体的作用下，均观察到树突状细胞DCs成熟的标志分子CD80和CD86的表达显著上调，说明DCs成熟水平的增加；图6B显示OMV-L-mRNA OVA疫苗组的CD11c +MHCI-OVA +细胞比例显著增加，说明OMV载体介导的mRNA递送成功地使得MHCI-OVA分子在DCs上的表达，为后续激活T细胞免疫杀伤提供条件。
实施例8
本实施例的目的在于验证OMV通用疫苗载体输运肿瘤抗原OVA mRNA诱导的T细胞免疫应答水平。
将OMV通用疫苗载体OMV-L或OMV-LL与OVA mRNA抗原序列于特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中混合制备形成OMV纳米疫苗OMV-L-mRNA OVA或OMV-LL-mRNA OVA。本实施例将该OMV纳米疫苗对健康C57BL/6小鼠上进行皮下三针免疫疗程，每只老鼠注射剂量为5μg的mRNA量，间隔时间分别为第0、5、10天，15天后解剖小鼠，提取脾脏组织制备成单细胞悬液，将获得脾细胞在抗原肽OVA 257-264(SIINFEKL)培养再刺激后，通过流式细胞术检测CD3 +CD8 +T细胞胞内细胞因子IFN-γ的含量，以评估疫苗激活抗原特异性获得性免疫的能力。使用5,6-羧基荧光素二乙酸琥珀酰亚胺酯(CFSE)染色检测细胞增殖。
如图7所示，OMV纳米疫苗OMV-L-mRNA OVA和OMV-LL-mRNA OVA可有效激活CD3 +CD4 +，CD3 +CD8 +T细胞，促使IFN-γ的分泌。图7A为CD3 +CD8 +T细胞分泌IFN-γ的流式检测定量图，图7B为CD3 +CD4 +T细胞分泌IFN-γ的流式检测定量图。图7C显示用 OMV-L-mRNA OVA和OMV-LL-mRNA OVA疫苗免疫的小鼠的脾细胞在用OVA抗原肽再刺激后，脾细胞增殖显著强于对照组、mRNA或mRNA和天然OMV的混合组。以上结果表明T细胞反应强烈。接下来还评估了脾细胞对表达卵清蛋白的B16(B16-OVA)和MC38细胞的细胞毒性。如图7D所示，OMV-L-mRNA OVA和OMV-LL-mRNA OVA组的脾细胞对B16-OVA细胞具有强烈的细胞毒性，能杀灭大量靶细胞。
实施例9
本实施例的目的在于评价OMV纳米疫苗对黑色素肿瘤肺转移的抑制能力。
利用前述方法制备的OMV纳米疫苗对黑色素肿瘤肺转移模型的抑制能力。其中，黑色素肿瘤肺转移模型由C57BL/6小鼠尾静脉注射B16-OVA细胞构建而成，抗肿瘤效果通过小鼠肺部转移灶的数量、转移抑制率和小鼠存活率来评定。其中，G1：PBS对照组，G2：OMV-LL载体，G3：OMV-LL-mRNA EGFP疫苗，G4：OMV载体与OVA mRNA的简单混合，G5：OMV-L-mRNA OVA疫苗，G6：OMV-LL-mRNA OVA疫苗。
具体为，C57BL/6小鼠尾静脉注射B16-OVA细胞3天后，分别进行纳米疫苗皮下三针免疫疗程，每只老鼠注射剂量为5μg的mRNA量，间隔时间分别为0、5、10天，即以注射B16-OVA为第0天，在第3、8、13天进行三次免疫。在第20天，提取肺组织，统计肺部肿瘤转移灶的数量，评估疫苗的肿瘤肺转移抑制效果。
如图8B-8D所示，与对照组相比，G5和G6的OVA疫苗组均能抑制肺肿瘤转移、延长肿瘤小鼠的存活时间。OMV纳米疫苗显示出优异的肿瘤抑制效应，图8B为肺部转移灶图，图8C为肺部转移灶定量统计图。OMV-L-mRNA OVA(G5)和OMV-LL-mRNA OVA(G6)两种OVA疫苗组的抑制率显著高于OMV-LL载体对照组(G2)，OMV-LL-mRNA EGFP(G3)的EGFR疫苗组和OMV与OVA mRNA的简单混合对照组(G4)(图8D)。且G5和G6组在35天甚至40天后仍有小鼠存活，而其他组的小鼠均在26-32天内死亡(图8E)。
实施例10
本实施例的目的在于探究OMV纳米疫苗对机体的长效免疫记忆和肿瘤预防能力。
利用前述实施例制备的OMV纳米疫苗皮下免疫C57BL/6小鼠，每只老鼠注射剂量为5μg的mRNA量，分别皮下免疫三针，间隔时间分别为0、5、10天。在第60天，取一部分小鼠脾脏组织，制备成单细胞悬液，流式检测脾脏细胞中记忆性T细胞(CD3 +CD4 +CD44 +CD62L -)的数量，评估疫苗的长期免疫记忆效果。取另一部分小鼠，尾静脉再接种B16-OVA肿瘤细胞，再过20天后，解剖得到肺组织，并统计肺部肿瘤转移灶的数量，评估疫苗的肿瘤预防能力。
如图9A-8E所示，和对照组相比，尽管OMVs+mRNA OVA或商业化佐剂poly(I:C)+mRNA OVA组的效应记忆性T细胞(CD3 +CD4 +CD44 +CD62L -)数量有轻微增长，但OMV-LL-mRNA OVA疫苗组的效应T细胞和中央记忆T细胞(CD3 +CD4 +CD44 +CD62L +)均显著增长(图9B-9C)。此外，OMV-LL-mRNA OVA疫苗组的肿瘤转移率最低(图9D-9E)。说明纳米疫苗接种60天后，对肿瘤仍有预防效力，具有抗原特异性的免疫记忆。
实施例11
本实施例的目的在于探究OMV纳米载体装载不同的mRNA形成疫苗，治疗不同肿瘤的能力，即对结直肠肿瘤(MC38)的生长抑制能力。
一种OMV纳米疫苗(OMV-L-mRNA ADPGK)，由OMV通用疫苗载体、ADPGK mRNA(编码的氨基酸序列为ASMTNMELM，SEQ ID NO：12)抗原片段于特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中制备形成，所述OMV通用疫苗载体为OMV、OMV-L7Ae(OMV-L)，mRNA抗原片段为3’UTR区插入box C/D的ADPGK mRNA，该通用疫苗载体与mRNA抗原片段的质量比为10：3；
G1：PBS对照组，G2：OMV-LL-mRNA EGFR，G3：poly(I:C)+ADPGK多肽，为佐剂poly(I:C)和ADPGK多肽的混合(50ug+5ug每只小鼠)，作为阳性对照，G4：OMV+mRNA ADPGK，OMV与ADPGK mRNA的简单混合，G5：OMV-LL-mRNA OMV(5ug每只小鼠)。
本实施例将该OMV纳米疫苗对结直肠癌皮下肿瘤模型进行抗肿瘤的效果探索。具体为C57BL/6小鼠皮下接种MC38肿瘤细胞6天后，肿瘤形成，分别对小鼠进行纳米疫苗的接种，每5天一次，共三次，每只老鼠注射剂量为5μg的mRNA量；每2天用游标卡尺监测肿瘤的生长情况，在肿瘤接种23天后，提取肿瘤组织称重。
如图10所示，虽然其他组在一定程度上抑制了肿瘤生长，但OMV-LL-mRNA ADPGK抑制作用最好。与其他组相比，OMV-LL-mRNA ADPGK组在治疗结束时的肿瘤体积和重量也是最低的(图10A、10B)。OMV-LL-mRNA EGFP、poly(I:C)+ADPGK、OMV+mRNA ADPGK、OMV-LL-mRNA ADPGK组肿瘤抑制率分别为61.8％、80.7％、76.6％、93.6％(图10C)。
实施例12
本实施例的目的在于探究OMV纳米疫苗(OMV-L-mRNA ADPGK)对结直肠肿瘤(MC38)的肿瘤免疫细胞浸润情况分析。
本实施例将实施例10中纳米疫苗的疫苗治疗后的MC38肿瘤组织进行免疫细胞浸润情 况分析，进而探讨纳米疫苗的抗肿瘤机制；利用流式细胞计数仪分析小鼠MC38肿瘤组织中T细胞的浸润情况。如图11所示，图11A为瘤内浸润的CD3 +T细胞的群变化图，图11B为瘤内浸润的CD3 +CD8 +T细胞的数量变化图，图11C为瘤内浸润的CD3 +CD4 +T细胞的定量统计图，图11D为瘤内浸润的Treg细胞(CD3 +CD4 +CD25 +)细胞的群变化图；可以看出纳米疫苗通用载体在携带抗原ADPGK后能够有效诱导CD3 +,CD3 +CD4 +和CD3 +CD8 +T细胞在肿瘤组织内的浸润，而免疫抑制性细胞(Treg)却没有增加的趋势，说明纳米疫苗良好的免疫能力。
实施例13
本实施例的目的在于探究OMV纳米疫苗对于不同核酸疫苗的通用性。
一种OMV纳米疫苗(OMV-L-mRNA HZV)，由OMV通用疫苗载体、HZV mRNA(SEQ ID NO：9)抗原序列于特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中制备形成，所述OMV通用疫苗载体为OMV、OMV-L7Ae(OMV-L)，mRNA抗原片段为box C/D-HZV，该通用疫苗载体与mRNA的质量比为10：3；
一种OMV纳米疫苗(OMV-L-mRNA HBeAg)，由OMV通用疫苗载体、HBeAg mRNA(SEQ ID NO：10)抗原序列于特定缓冲液(5mM Tris-HCl,100mM NaCl,10mM MgCl 2；pH＝5.0)中制备形成，所述OMV通用疫苗载体为OMV、OMV-L7Ae(OMV-L)，mRNA抗原片段为box C/D-HBeAg，该通用疫苗载体与mRNA的质量比为10：3；
将OMV-L-mRNA肌肉或滴鼻免疫接种BALB/c健康小鼠，前三次免疫分别间隔两周，在三次免疫后一个月进行一次加强免疫，共免疫四次。收集0、2、4、6、10w的小鼠血清标本，通过ELISA方法检测不同时间点血清中抗体浓度。如图12所示，图12A和图12B为OMV-L-mRNA HZV通过肌注途径或滴鼻途径免疫小鼠后不同时间点血清中抗体浓度，图12C和图12D为OMV-L-mRNA HBeAg通过肌注途径或滴鼻途径免疫小鼠后不同时间点血清中抗体浓度。结果显示，无论是通过肌注途径还是滴鼻途径免疫小鼠，随着免疫次数增加，血清中抗体水平呈上升趋势，在末次免疫间隔一个月进行加强免疫后，第10w的免疫组血清中抗体水平较之前出现更大提高。
本实施例研究该OMV纳米疫苗的表达效果以及对于免疫反应的刺激作用，结果显示，本申请的OMV纳米疫苗对于不同核酸疫苗具有通用性。
实施例14
本实施例的目的在于探究OMV纳米疫苗的内表面和外表面的核酸展示效果。
比较mRNA在OMV通用载体内部和表面的展示方式对293T细胞表达情况，如图13所示，将mRNA工程负载内部的形式，难以实现mRNA的细胞转染，而表面的展示形式则实现了mRNA的递送。结果显示，本申请的OMV纳米疫苗在外表面进行展示，具有更显著的递送效果。
本申请的抗肿瘤纳米疫苗很好地扩展了mRNA的体内递送系统，推动了可拓展个性化肿瘤疫苗的发展，具有很好的应用前景。限于篇幅，本文仅例举部分最具说服力的实施例。
本申请通过上述实施例来说明本申请的工艺方法，但本申请并不局限于上述工艺步骤，即不意味着本申请必须依赖上述工艺步骤才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请所选用原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (40)

  1. 一种囊泡，所述囊泡的外表面包含核酸。
  2. 一种囊泡，所述囊泡包含核酸结合分子，所述核酸结合分子能够结合核酸并使其结合的核酸展示在所述囊泡的外表面。
  3. 如权利要求1-2中任一项所述的囊泡，所述囊泡包含来源于细胞的膜结构。
  4. 如权利要求1-3中任一项所述的囊泡，所述囊泡包含来源于细菌的膜结构。
  5. 如权利要求1-4中任一项所述的囊泡，所述囊泡包含来源于革兰氏阴性菌和/或革兰氏阳性菌的膜结构。
  6. 如权利要求1-5中任一项所述的囊泡，所述囊泡包含来源于肠杆菌科和/或奈氏菌科的膜结构。
  7. 如权利要求1-6中任一项所述的囊泡，所述囊泡包含来源于沙门氏菌、大肠杆菌、和/或脑膜炎球菌的膜结构。
  8. 如权利要求1-7中任一项所述的囊泡，所述囊泡包含细菌外膜囊泡(OMV)。
  9. 如权利要求1-8中任一项所述的囊泡，所述囊泡包含膜蛋白。
  10. 如权利要求9所述的囊泡，所述膜蛋白包含溶细胞素A(ClyA)和/或冰核蛋白(INP)，以及上述的功能活性片段。
  11. 如权利要求1-10中任一项所述的囊泡，所述囊泡包含核酸结合分子，所述核酸结合分子包含能够与RNA特异性结合的蛋白。
  12. 如权利要求11所述的囊泡，所述核酸结合分子能够识别box C/D序列。
  13. 如权利要求11-12中任一项所述的囊泡，所述核酸结合分子包含L7Ae或其功能活性片段。
  14. 如权利要求11-13中任一项所述的囊泡，所述膜蛋白与所述核酸结合分子直接或间接连接。
  15. 如权利要求11-14中任一项所述的囊泡，所述膜蛋白的C端与所述核酸结合分子的N端直接或间接连接。
  16. 如权利要求1-15中任一项所述的囊泡，所述核酸包含外源核酸。
  17. 如权利要求1-16中任一项所述的囊泡，所述核酸包含RNA。
  18. 如权利要求1-17中任一项所述的囊泡，所述核酸编码肿瘤相关抗原和/或传染病相关抗原。
  19. 如权利要求18所述的囊泡，所述肿瘤相关抗原包含OVA、和/或ADPGK(ADP Dependent Glucokinase)。
  20. 如权利要求18所述的囊泡，所述传染病相关抗原包含乙肝e抗原和/或疱疹病毒抗原。
  21. 一种融合蛋白，所述融合蛋白包含核酸结合分子和膜蛋白，所述核酸结合分子能够结合核酸并使其结合的核酸展示在所述囊泡的外表面。
  22. 如权利要求21所述的融合蛋白，所述膜蛋白包含溶细胞素A(ClyA)和/或冰核蛋白(INP)，以及上述的功能活性片段。
  23. 如权利要求21-22中任一项所述的融合蛋白，所述核酸结合分子能够识别box C/D序列。
  24. 如权利要求21-23中任一项所述的融合蛋白，所述核酸结合分子包含L7Ae或其功能活性片段。
  25. 如权利要求21-24中任一项所述的融合蛋白，所述膜蛋白与所述核酸结合分子直接或间接连接。
  26. 如权利要求21-25中任一项所述的融合蛋白，所述膜蛋白的C端与所述核酸结合分子的N端直接或间接连接。
  27. 一种核酸，所述核酸编码权利要求21-26中任一项所述的融合蛋白。
  28. 一种载体，包含权利要求27所述的核酸，以及任选的非编码区和/或抗原结构优化区。
  29. 一种细胞，包含权利要求1-20中任一项所述的囊泡、权利要求21-26中任一项所述的融合蛋白、权利要求27所述的核酸、和/或权利要求28所述的载体。
  30. 一种组合物，包含权利要求1-20中任一项所述的囊泡、权利要求21-26中任一项所述的融合蛋白、权利要求27所述的核酸、权利要求28所述的载体、和/或权利要求29所述的细胞，以及任选的载剂。
  31. 一种疫苗，其包含权利要求1-20中任一项所述的囊泡。
  32. 如权利要求31所述的疫苗，其粒径尺寸在1-1000nm之间。
  33. 一种试剂盒，包含权利要求1-20中任一项所述的囊泡、权利要求21-26中任一项所述的融合蛋白、权利要求27所述的核酸、权利要求28所述的载体、权利要求29所述的细胞、权利要求30所述的组合物和/或权利要求31或32所述的疫苗。
  34. 一种展示和/或表达外源核酸的方法，包含施用权利要求1-20中任一项所述的囊泡、权利要求21-26中任一项所述的融合蛋白、权利要求27所述的核酸、权利要求28所述的载体、权利要求29所述的细胞、权利要求30所述的组合物、权利要求31或32所述的疫苗和/或权利要求33所述的试剂盒。
  35. 一种影响免疫反应和/或抑制肿瘤生长的方法，包含施用权利要求1-20中任一项所述的囊泡、权利要求21-26中任一项所述的融合蛋白、权利要求27所述的核酸、权利要求28所述的载体、权利要求29所述的细胞、权利要求30所述的组合物、权利要求31或32 所述的疫苗和/或权利要求33所述的试剂盒。
  36. 一种权利要求1-20中任一项所述的囊泡、权利要求21-26中任一项所述的融合蛋白、权利要求27所述的核酸、权利要求28所述的载体、权利要求29所述的细胞、权利要求30所述的组合物、权利要求31或32所述的疫苗和/或权利要求33所述的试剂盒在制备试剂中的用途，所述试剂用于预防和/或治疗疾病和/或病症。
  37. 如权利要求36所述的用途，所述疾病和/或病症包含肿瘤和/或传染病。
  38. 如权利要求36-37中任一项所述的用途，所述疾病和/或病症包含实体瘤。
  39. 如权利要求36-38中任一项所述的用途，所述疾病和/或病症包含黑色素肿瘤和/或结直肠肿瘤。
  40. 如权利要求36-39中任一项所述的用途，所述疾病和/或病症包含疱疹病毒和/或乙肝病毒感染相关的感染病。
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