DNA nano-adjuvant, preparation method and application thereof

Abstract

The application provides a DNA nano-adjuvant as well as a preparation method and application thereof. The DNA nano-adjuvant provided by the application comprises a DNA nanostructure and a MGN1703 variant, and the nano-adjuvant has good safety, stability, high cell uptake capacity and strong immune stimulation activity. The DNA nano-adjuvant provided by the application can be used for preparing a vaccine, used as a drug carrier and a drug excipient, and is expected to play an important role in the field of anti-infection immunity as a nucleic acid adjuvant. The vaccine prepared from the DNA nano-adjuvant provided by the application shows a significant prevention effect on pathogen infection, and has a very good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a DNA nanoadjuvant, its preparation method, and its application. Background Technology

[0002] Subunit vaccines are widely used in the vaccine field due to their excellent safety profile, but their immunogenicity is relatively weak, usually requiring combination with adjuvants to enhance immune protection. Traditional adjuvants, such as aluminum adjuvants, have problems such as high toxicity and slow metabolism, and primarily activate humoral immunity, with limited activation of cellular and mucosal immunity. Therefore, the development of a novel adjuvant is urgently needed. Summary of the Invention

[0003] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0004] The first aspect of the present invention provides a DNA nanoadjuvant, the DNA nanoadjuvant comprising a DNA nanostructure and an MGN1703 variant.

[0005] In this invention, adjuvants refer to drugs or immune agents administered to improve the immune response to a vaccine.

[0006] In this invention, MGN1703 is a TLR9 agonist composed of 116 nucleotides with a covalently closed dumbbell-shaped structure. All nucleotides are linked by native phosphodiester bonds and native molecules, significantly reducing toxicity. Each cyclic structure contains three unmethylated CpG motifs, enabling highly efficient immunostimulatory effects. Its cyclic structure not only improves stability but also further reduces toxicity and enhances immunostimulatory activity.

[0007] In this invention, the MGN1703 variant refers to a variant of MGN1703 whose nucleotide sequence is achieved by substitution, deletion, insertion or combination of one or more nucleotides, and which retains the functional property of activating TLR9-dependent immunostimulatory activity.

[0008] Furthermore, the MGN1703 variant nucleotide sequence has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.5.

[0009] Furthermore, the nucleotide sequence of the MGN1703 variant is shown in SEQ ID NO.5.

[0010] In this invention, DNA nanostructures are nanoscale artificial structures constructed by precisely designing base pairing rules. The core of these structures is to program the sequence of DNA strands so that they spontaneously fold or assemble into specific geometric shapes or functional structures.

[0011] Furthermore, the DNA nanostructures include, but are not limited to, cross-shaped DNA nanostructures, DNA tiles, DNA origami, DNA tubes, or DNA nanoflowers.

[0012] In this invention, DNA tiles are modular units formed by the cross-linking of multiple short-stranded DNA molecules, which can be assembled into larger two-dimensional lattices or three-dimensional frames, wherein the three-dimensional frames include, but are not limited to, trihedrons and tetrahedrons.

[0013] In this invention, DNA origami uses a long DNA chain as a scaffold and folds short DNA chains into a specific shape.

[0014] In this invention, the DNA tube is a tubular structure formed by multiple DNA double helices.

[0015] In this invention, DNA nanoflowers are formed by co-precipitation and self-assembly of DNA, inorganic molecules, and DNA enzymes through rolling circle amplification to create porous flower-like DNA-inorganic nanostructures.

[0016] Furthermore, the DNA nanostructure is selected from cross-shaped DNA nanostructures.

[0017] In this invention, the cross-shaped DNA nanostructure is designed and optimized using the NUPACK design tool.

[0018] Furthermore, the design language of NUPACK is described as follows: (((((((((((((((((((((((((((((((.....( ... (( ...

[0019] Furthermore, the cross-shaped DNA nanostructure includes four ssDNA strands, S1, S2, S3, and S4, the sequences of which are shown in SEQ ID NO.1-4.

[0020] A second aspect of the present invention provides a vaccine comprising the DNA nanoadjuvant and antigen described in the first aspect of the present invention.

[0021] In this invention, a vaccine is a biological composition that can stimulate the immune system and enable it to fight infection or disease. The vaccine includes preventative vaccines and therapeutic vaccines. Preventative vaccines are used on healthy individuals or those who have not yet been infected with a specific pathogen, activating the immune system in advance to prevent future infections or diseases. Therapeutic vaccines are used on individuals already suffering from disease, helping to control disease progression and eliminate pathogens or abnormal cells (such as cancer cells) by regulating or enhancing the immune system.

[0022] Furthermore, the vaccine is selected from preventative vaccines.

[0023] In this invention, the antigen refers to a substance that induces a specific immune response in a host animal. Types of antigens include, but are not limited to, nucleic acids, proteins, polypeptides, bacteria, fungi, viruses, or toxoids.

[0024] Based on the type of antigen, vaccines can be classified into, but are not limited to, live attenuated vaccines, inactivated vaccines, polysaccharide and polysaccharide conjugate vaccines, subunit vaccines, and nucleic acid vaccines.

[0025] In this invention, attenuated live vaccines refer to a type of live vaccine in which the structure of pathogens such as live viruses or live bacteria is changed after being treated by chemical or physical methods, resulting in a significant reduction in their toxicity while retaining their antigenicity. They generally have a long development cycle, but can induce strong immune efficacy and are of great significance for pandemics of highly lethal and highly pathogenic infectious diseases.

[0026] Inactivated vaccines are a type of vaccine in which pathogenic microorganisms are completely deactivated by physical or chemical treatments but retain their immunogenicity. Compared with live attenuated vaccines, inactivated vaccines are relatively safer but have lower immunogenicity. Considering their lower immunogenicity and immunogenicity, they are usually used together with immune adjuvants to help activate the immune system.

[0027] Subunit vaccines are composed of specific components of a pathogen that can elicit an immune response, typically including proteins and peptides. Compared to attenuated or inactivated vaccines, subunit vaccines retain only the pathogen antigens that trigger an immune response, thus effectively improving vaccine safety.

[0028] Nucleic acid vaccines are vaccines that use plasmids as a medium to inject a foreign gene sequence that encodes a specific protein into the body and express the corresponding protein antigen in the host cells, thereby inducing the host to produce a corresponding immune response and exerting the effect of a vaccine.

[0029] Furthermore, the antigen is selected from proteins.

[0030] Furthermore, the vaccine is selected from subunit vaccines.

[0031] Based on disease classification, vaccines can be divided into vaccines against tumors, vaccines against infectious diseases, and vaccines against chronic diseases.

[0032] Among these, tumor vaccines include, but are not limited to, messenger RNA tumor vaccines, viral vector tumor vaccines, and tumor peptide vaccines. Messenger RNA tumor vaccines are produced through in vitro transcription to obtain mRNA sequences encoding tumor-specific antigens or tumor-associated antigens. These sequences are then prepared as vaccines and injected into the human body, where they translate to produce antigen proteins, thereby inducing a specific immune response. Viral vector tumor vaccines utilize modified viruses with replication defects or attenuated viruses as vectors to deliver the genetic information encoding tumor antigens to host cells, prompting the expression of these antigens and activating a specific anti-tumor immune response. Tumor peptide vaccines are vaccines produced from peptides designed and chemically synthesized based on the amino acid sequences of tumor antigen epitopes.

[0033] Vaccines for infectious diseases include, but are not limited to, vaccines against diseases caused by pathogens such as bacteria and viruses. The bacteria include, but are not limited to, the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Spirochetes, and Chlamydia. Proteobacteria include, but are not limited to, Enterobacteriaceae, Vibrioceae, Pseudomonas, and Yersinia; Firmicutes include, but are not limited to, Bacillus, Staphylococcus, and Clostridium; Actinobacteria include, but are not limited to, Mycobacterium and Streptomyces; Bacteroidetes include, but are not limited to, Bacteroidetes and Prevotella; Spirochetes include, but are not limited to, Leptospira and Treponema; and Chlamydia include, but are not limited to, Chlamydia. The viruses include, but are not limited to, the following families of viruses: Retroviridae, Clonorviridae, Coronaviridae, Filoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Hepatoviridae, Herpesviridae, Flaviviridae, Baculoviridae, Poxviridae, or Picornaviridae.

[0034] Vaccines targeting chronic diseases can be used to treat chronic allergic diseases, diabetes, hypertension, obesity, Alzheimer's disease, rheumatoid arthritis, and other diseases.

[0035] Furthermore, the vaccine is selected from vaccines against infectious diseases.

[0036] In this invention, the infectious disease is a disease caused by bacteria.

[0037] Furthermore, the bacteria are selected from the phylum Proteobacteria.

[0038] Furthermore, the bacteria are selected from the genus Yersinia.

[0039] Furthermore, the Yersinia genus includes Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica.

[0040] Furthermore, the Yersinia species mentioned are selected from Yersinia pestis.

[0041] In this invention, *Yersinia pestis* is a Gram-negative bacterium and the pathogen of the deadly plague. This zoonotic disease has stable foci in the Americas, Africa, and Eurasia. Its main routes of infection include aerosol transmission, oral contamination, skin contact, and bites from wild fleas. Based on the route of infection, plague is classified into pneumonic plague, bubonic plague, and septicemic plague, with pneumonic plague being the most severe, having an incubation period of 24 hours to 4 days. Symptoms include fever, headache, weakness, and rapidly developing pneumonia. Pneumonic plague is highly contagious and has an extremely high mortality rate.

[0042] Furthermore, the plague mentioned is selected from pneumonic plague.

[0043] In this invention, the protein can be a recombinant protein or a natural protein.

[0044] Furthermore, the protein is selected from recombinant proteins.

[0045] Furthermore, the protein is a variant of the Yersinia pestis recombinant protein LcrV.

[0046] Furthermore, the mass ratio of the DNA nanoadjuvant to the antigen is 1:1 to 1:5.

[0047] Furthermore, the mass ratio of the DNA nanoadjuvant to the antigen is 1:2.

[0048] A third aspect of the present invention provides a pharmaceutical composition comprising the DNA nanoadjuvant described in the first aspect of the present invention or the vaccine described in the second aspect of the present invention.

[0049] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0050] Pharmaceutically acceptable carriers may include, for example, water, suitable oils, physiological saline, parenteral carriers (e.g., aqueous glucose, glycols, etc.), and may further include stabilizers and preservatives. Suitable stabilizers may include antioxidants (e.g., sodium bisulfite, sodium sulfite, or ascorbic acid). Suitable preservatives may include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Additionally, if necessary, depending on the method of administration and formulation, the pharmaceutical compositions according to the invention may suitably include suspending agents, dissolving adjuvants, stabilizers, isotonic agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, antioxidants, etc.

[0051] In this invention, the pharmaceutical composition may further include other adjuvants. Other adjuvants include aluminum hydroxide and aluminum phosphate, saponins such as QuilA, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions. The emulsion may be particularly based on light liquid paraffin oil; isoprene-like oils such as squalane or squalene oils resulting from the oligomerization of olefins (especially isobutylene or decene); esters of acids or alcohols containing straight-chain alkyl groups, more specifically vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, particularly isostearates. The oil is used in combination with an emulsifier to form the emulsion. The emulsifier is preferably a nonionic surfactant, particularly sorbitan, dimannitol (e.g., anhydrous mannitol oleate), glycol, polyglycerol, propylene glycol, and esters of oleic acid, isostearic acid, castor oil acid, or hydroxystearic acid (optionally ethoxylated), as well as polyoxypropylene-polyoxyethylene copolymer blocks.

[0052] The pharmaceutical compositions described in this invention can be effectively delivered through a variety of non-invasive or minimally invasive routes, including but not limited to oral, intradermal, intratracheal (e.g., nebulized inhalation), intravaginal (e.g., mucosal sustained-release gel), intranasal (e.g., spray) or transmucosal administration.

[0053] Furthermore, the drug composition is delivered intratracheally. The drug composition is formulated as an aerosol preparation, which is then atomized into inhalable particles that directly target the lungs.

[0054] Depending on the desired duration and effectiveness of treatment, the pharmaceutical compositions of the present invention can be administered once or several times, or intermittently, for example, daily for several days, weeks or months and at different doses.

[0055] The fourth aspect of the present invention provides a method for preparing the DNA nanoadjuvant described in the first aspect of the present invention, the method comprising mixing S1, S2, S3, S4 and MGN1703 variants to synthesize the DNA nanoadjuvant.

[0056] Furthermore, the synthesis method is a one-step annealing method.

[0057] Furthermore, the one-step annealing method includes stretching the ssDNA, pre-annealing, ligation, and slow cooling.

[0058] Furthermore, the ssDNA stretching is performed at 95°C.

[0059] Furthermore, the ssDNA stretching is sustained for 5 minutes.

[0060] Furthermore, the pre-annealing is carried out at 65°C.

[0061] Furthermore, the pre-annealing lasts for 2 minutes.

[0062] Furthermore, the connection is made at 62°C.

[0063] Furthermore, the connection lasts for 1 minute.

[0064] Furthermore, the slow cooling is a continuous cooling rate of 2°C / minute.

[0065] Furthermore, the slow cooling eventually brought the temperature down to 4°C.

[0066] Furthermore, the mixing is performed in TE buffer.

[0067] Furthermore, the TE buffer includes Tris, EDTA, MgCl2, and NaCl.

[0068] Furthermore, the Tris is 20 mM.

[0069] Furthermore, the EDTA is 1 mM.

[0070] Furthermore, the MgCl2 is 5 mM.

[0071] Furthermore, the NaCl concentration is 50 mM.

[0072] The fifth aspect of the present invention provides a method for preparing the vaccine described in the second aspect of the present invention, the method comprising mixing the DNA nanoadjuvant described in the first aspect of the present invention with an antigen.

[0073] The sixth aspect of the present invention provides any of the following products:

[0074] (1) A variant of MGN1703, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0075] (2) A DNA nanostructure comprising four ssDNAs, namely S1, S2, S3 and S4, wherein the sequences of S1, S2, S3 and S4 are shown in SEQ ID NO.1-4.

[0076] Furthermore, the DNA nanostructure is a cross-shaped DNA nanostructure.

[0077] The seventh aspect of the present invention provides any of the following methods:

[0078] (1) A method for promoting dendritic cell maturation, the method comprising applying the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, and the MGN1703 variant of the sixth aspect of the present invention to promote dendritic cell maturation;

[0079] (2) A method for activating the TLR9 signaling pathway, the method comprising administering the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, or the MGN1703 variant of the sixth aspect of the present invention to activate the TLR9 signaling pathway;

[0080] (3) A method for promoting cytokine secretion, the method comprising administering the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, and the MGN1703 variant of the sixth aspect of the present invention to promote cytokine secretion;

[0081] Furthermore, the cytokine is selected from IL-6.

[0082] The eighth aspect of the present invention provides any of the following applications:

[0083] (1) The application of the DNA nanoadjuvant described in the first aspect of the present invention in the preparation of vaccines;

[0084] (2) The application of the DNA nanoadjuvant described in the first aspect of the present invention as a drug carrier;

[0085] DNA nanoadjuvants can serve as drug carriers to deliver antibacterial, antiviral, or anticancer drugs, exerting their antibacterial, antiviral, or anticancer effects while enhancing immune stimulation.

[0086] (3) The application of the DNA nanoadjuvant described in the first aspect of the present invention as a pharmaceutical dressing;

[0087] DNA nanoadjuvants can be used as medicated dressings to accelerate wound healing.

[0088] (4) The application of the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, and the DNA nanostructure of the sixth aspect of the present invention in the preparation of drugs for preventing pathogen infection;

[0089] Furthermore, the pathogens include bacteria, viruses, fungi, parasites, and prions.

[0090] Furthermore, the pathogen is selected from bacteria.

[0091] Furthermore, the bacteria include Yersinia pestis.

[0092] (5) Application of MGN1703 variant in the preparation of drugs for preventing pathogen infection;

[0093] Furthermore, the MGN1703 variant nucleotide sequence has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.5.

[0094] Furthermore, the nucleotide sequence of the MGN1703 variant is shown in SEQ ID NO.5.

[0095] Furthermore, the pathogens include bacteria, viruses, fungi, parasites, and prions.

[0096] Furthermore, the pathogen is selected from bacteria.

[0097] Furthermore, the bacteria include Yersinia pestis.

[0098] (6) The use of the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, and the MGN1703 variant of the sixth aspect of the present invention in promoting dendritic cell maturation;

[0099] (7) The application of the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, and the MGN1703 variant of the sixth aspect of the present invention in activating the TLR9 signaling pathway;

[0100] (8) The use of the DNA nanoadjuvant of the first aspect of the present invention, the vaccine of the second aspect of the present invention, the pharmaceutical composition of the third aspect of the present invention, and the MGN1703 variant of the sixth aspect of the present invention in promoting cytokine secretion.

[0101] Furthermore, the cytokine is selected from IL-6.

[0102] The advantages and beneficial effects of this invention are as follows:

[0103] This invention provides a DNA nanoadjuvant, its preparation method, and its applications. The DNA nanoadjuvant provided by this invention comprises a DNA nanostructure and an MGN1703 variant. The nanoadjuvant exhibits good safety, stability, efficient cellular uptake capacity, and potent immunostimulatory activity. The DNA nanoadjuvant provided by this invention can be used in vaccine preparation, as a drug carrier, and as a pharmaceutical excipient, and is expected to play an important role as a nucleic acid adjuvant in anti-infective immunotherapy and other fields. Vaccines prepared using the DNA nanoadjuvant provided by this invention show significant preventive effects against pathogen infections and have excellent clinical application prospects. Attached Figure Description

[0104] Figure 1 The image shows a polyacrylamide gel electrophoresis diagram of AdjCRU and CrDNA prepared in Example 1 of this invention.

[0105] Figure 2 Atomic force microscopy (AFM) images and statistical particle size distribution of AdjCRU and CrDNA prepared in Example 1 of this invention.

[0106] Figure 3 In vitro biosafety evaluation of AdjCRU prepared in Example 1 of this invention.

[0107] Figure 4 This study evaluates the in vivo biocompatibility of AdjCRU prepared in Example 1 of the present invention. Scale bar: 100 μm.

[0108] Figure 5 The image shows the effect of AdjCRU prepared in Example 1 on promoting the maturation of isolated dendritic cells in mouse bone marrow.

[0109] Figure 6 The image shows the effect of AdjCRU prepared in Example 1 on activating the TLR9 and pp65 molecules of the innate immune signaling pathway.

[0110] Figure 7 The image shows the effect of AdjCRU prepared in Example 1 on promoting IL-6 secretion.

[0111] Figure 8 The image shows the effect of the AdjCRU and rV10 blend prepared in Example 1 on promoting the production of IgG antibodies in mice.

[0112] Figure 9 25×LD after immunization with AdjCRU+rV10 50 Survival curve of mice within 14 days after challenge with Yersinia pestis.

[0113] Figure 10 25×LD after immunization with AdjCRU+rV10 50 Bacterial load in mouse organs 2 days after challenge with Yersinia pestis. Detailed Implementation

[0114] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0115] Example 1: Preparation and Characterization of AdjCRU and CrDNA

[0116] The DNA sequences of cruciform DNA nanostructures (CrDNA) were designed and optimized using the NUPACK design tool. According to the design language of NUPACK, it can be described as follows: (( ... ((((((((((((((((((((............((((((((((((................................)))))))))))))+)))))))))))))))))))))))))).....) ...

[0117] Table 1 Oligonucleotide Sequences

[0118]

[0119] DNA nanoadjuvant (AdjCRU) consists of five ssDNA strands: CrDNA (S1, S2, S3, S4) and the modified nucleic acid immunostimulant MGN1703L (Loop DNA, LoDNA). CrDNA itself consists of four ssDNA strands: S1, S2, S3, and S4. The ssDNA strands constituting AdjCRU or CrDNA are mixed in TE buffer (pH 8.0, 20 mM Tris, 1 mM EDTA, 5 mM MgCl2, 50 mM NaCl). The mixture is incubated at 95°C for 5 min, 65°C for 2 min, and 62°C for 1 min, then continuously cooled to 4°C at a rate of 2°C / min to synthesize AdjCRU or CrDNA.

[0120] Polyacrylamide gel electrophoresis results showed that lanes 6 and 7 contained CrDNA and AdjCRU ( Figure 1 AFM imaging results showed that both CrDNA and AdjCRU were cross-shaped, with statistical results indicating that the size of CrDNA was approximately 35 nm and the size of AdjCRU was approximately 50 nm. Figure 2 ).

[0121] In summary, the DNA nanoadjuvant AdjCRU and the vector CrDNA were successfully prepared.

[0122] Example 2: Biosafety Validation of the AdjCRU Prepared in Example 1 of the Present Invention

[0123] The in vitro safety of AdjCRU was assessed using the CCK-8 assay on mouse bone marrow-derived dendritic cells (BMDCs). BMDC cells were seeded into 96-well plates. 4 Cells were added to each well at a concentration of 100 μL. The 96-well plates were incubated at 37 °C for 24 h. Cells were then slowly washed twice with PBS. AdjCRU was diluted with BMDC medium to 100 nM, 200 nM, 400 nM, and 800 nM, and added to the 96-well plates. Cells were incubated at 37 °C for 24 h. Cells were then slowly washed twice with PBS, and 100 μL of CCK-8 working solution (containing 10% CCK-8 stock solution and 90% culture medium) was added. After incubation at 37 °C for 4 h, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as: Cell viability = [(As-Ab) / (Ac-Ab)] × 100%. As: Absorbance of experimental wells; Ac: Absorbance of control wells; Ab: Absorbance of blank wells.

[0124] To assess the in vivo biosafety of AdjCRU, C57BL / 6J mice were used in the experiment. AdjCRU was administered via aerosol lung delivery at doses of 50 μL and 10 μg. On day 7 post-immunization, three C57BL / 6J mice were sacrificed with CO2, and their organs (heart, liver, spleen, lung, and kidney) were dissected and removed. These tissues were then fixed in 4% paraformaldehyde solution for at least 48 hours, followed by staining by Wuhan Sewell Biotechnology Co., Ltd., and finally, pathological sections were prepared. The pathological sections were observed under an optical microscope and histopathologically scored.

[0125] Experimental results showed that the in vitro safety of AdjCRU on BMDC was evaluated using the CCK-8 assay, and the survival rate of BMDC after treatment with different concentrations of AdjCRU for 24 h was greater than 80%. Figure 3 This indicates that AdjCRU has good biocompatibility at the cellular level. In vivo pathological sections showed no significant histopathological changes observed 7 days post-immunization. Figure 4 This indicates that AdjCRU has good biocompatibility in vivo.

[0126] Example 3: Determination of the effect of AdjCRU prepared in Example 1 of the present invention on activating innate immunity.

[0127] C57BL / 6J wild-type mouse BMDCs were used in four experimental groups: Mock, CrDNA (100 nM), LoDNA (400 nM), and AdjCRU (CrDNA 100 nM, LoDNA 400 nM), with three replicates in each group. The BMDCs were mixed with BMDCs and incubated at 37°C in a 5% CO2 cell culture incubator. Flow cytometry was used to assess BMDC maturity, Western blotting was used to analyze TLR3 and p65 phosphorylation levels, and ELISA was used to detect IL-6 secretion. The specific detection methods are as follows.

[0128] 1. Flow cytometry was used to detect the maturity of BMDCs.

[0129] (1) Add 1 mL of solution with a concentration of 1×10 6 The corresponding experimental group stimulants were added to BMDCs at a concentration of cells / mL and treated for 12 hours and 24 hours, respectively.

[0130] (2) Transfer the cells to a flow cytometer, centrifuge at 350×g for 5 minutes, discard the supernatant, add 2 mL of DPBS, centrifuge again at 350×g for 5 minutes, and discard the supernatant. Repeat this step twice.

[0131] (3) Antibody preparation: Prepare antibodies according to the scheme in Table 2. Set up a single staining tube for each antibody as compensation.

[0132] Table 2 BMDC Flow Cytometry Antibody Protocol

[0133]

[0134] (4) Staining

[0135] 1) Add 4.9 μL of mixed antibody to each flow cytometry tube and incubate at room temperature in the dark for 25 minutes;

[0136] 2) Add 2 mL of DPBS, centrifuge at 350×g for 5 minutes, and repeat twice;

[0137] 3) Discard the supernatant, resuspend the cells in 50 μL of DPBS, and then perform analysis using an LSL Ortessa flow cytometer.

[0138] 2. Western Blot detection of TLR9 and p65 phosphorylation expression

[0139] (1) Sample preparation: In 2 mL of solution with a concentration of 1×10 6 Add the experimental group stimulant to BMDC at a concentration of cells / mL, treat for 3 hours, discard the supernatant, and add 150 μL of cell lysis buffer. Collect cells with a cell scraper, transfer to EP tubes, centrifuge at 12000 rpm and 4 ℃ for 10 minutes, discard the supernatant, add an appropriate amount of 5× protein loading buffer, boil in a water bath for 10 minutes, cool, and store at -80 ℃.

[0140] (2) Protein gel preparation: 12.5% ​​SDS-PAGE protein gel was prepared using the PAGE gel rapid preparation kit according to the formula.

[0141] (3) Electrophoresis: Add the sample and protein marker to the gel wells, run the sample out of the stacking gel at 80 V, and then continue electrophoresis at 120 V.

[0142] (4) Transfer: Immerse the PVDF membrane in methanol solution to activate it, and then transfer it to the transfer solution for later use. Take out the protein gel, cut the gel according to the size of the target band, and place the sponge pad, filter paper, gel, PVDF membrane, filter paper and sponge pad in sequence. Close the transfer clamp, put it into the transfer tank, add the pre-cooled transfer solution and ice box, and control the transfer time according to the protein molecular weight.

[0143] (5) Sealing: After the transfer, immerse the PVDF film in a 5% skim milk powder solution and seal it in a shaker at room temperature for 1 hour.

[0144] (6) Primary antibody incubation: After blocking, the PVDF membrane is placed in a primary antibody (TLR9 ​​and p65 phosphorylation) solution diluted to 1:1000 and incubated at room temperature for 1 hour.

[0145] (7) Secondary antibody incubation: After primary antibody incubation, wash the membrane three times in 1×TBST for 5 minutes each time, and then incubate it in secondary antibody solution for 1 hour.

[0146] (8) Exposure: After incubation with the secondary antibody, wash the membrane three times in 1×TBST for 5 minutes each time. Mix equal volumes of luminescent solution A and solution B to prepare a developing solution. After draining the membrane, immerse it in the developing solution and then place it in a chemiluminescence imaging system for automatic exposure.

[0147] 3. ELISA detection of cytokine secretion

[0148] (1) Sample preparation: After stimulating the corresponding experimental groups in the prepared BMDC for 24 hours, the supernatant was aspirated, centrifuged at 2200 rpm for 2 minutes, and the supernatant was aspirated and placed at -80 ℃ for later use.

[0149] (2) ELISA detection:

[0150] 1) Reagent warming: 30 minutes before the experiment, place the reagent kit and the sample to be tested at room temperature to warm up.

[0151] 2) Prepare washing solution: Dilute 20× Washing Buffer with double-distilled water to 1×.

[0152] 3) Washing: Take out the required strips from the sealed bag after it has reached room temperature equilibrium, wash them 3 times and spin dry before use.

[0153] 4) Add standards and samples: Label 6 EP tubes and serially dilute the standards 2-fold. Use the standard / sample dilution as a blank control. Add 100 μL of standard and test sample to the reaction wells, seal the plate, and incubate at 37 ℃ for 90 minutes.

[0154] 5) Washing: Discard the liquid in the well, add 300 μL of washing solution to each well, let stand for 30 seconds and then shake dry. Repeat 5 times, and pat dry on filter paper or absorbent paper for the last time.

[0155] 6) Add biotinylated antibody: Calculate the required amount and dilute the 100× antibody concentrate to 1× working solution using detection diluent. Add 100 μL of working solution to each well, seal the membrane, and incubate at 37 ℃ for 60 minutes.

[0156] 7) Washing: Repeat step 5.

[0157] 8) Add streptavidin-enzyme conjugate: Prepare as needed. Dilute 100× concentrated enzyme conjugate to 1× working solution with enzyme conjugate dilution buffer (centrifuge before dilution). Add 100 μL of working solution to each well, seal the membrane, and incubate at 37 ℃ in the dark for 30 minutes.

[0158] 9) Washing: Repeat step 5.

[0159] 10) Add chromogenic substrate (TMB): Add 100 μL of TMB to each well and incubate at 37 °C in the dark for 15 minutes.

[0160] 11) Add stop solution: Add 50 μL of stop solution to each well.

[0161] 12) Detection results: After the reaction was terminated, the OD value was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450-630 nm and the data was analyzed.

[0162] The results showed that after BMDC stimulation with AdjCRU, the expression of cell surface maturation markers CD40, CD80, and CD86 was significantly increased. Figure 5 Meanwhile, the phosphorylation levels of key signaling pathway molecules TLR9 and p65 proteins were significantly increased. Figure 6 The secretion level of cytokine IL-6 also increased significantly, and CrDNA and LoDNA had a synergistic effect in increasing the secretion of cytokine IL-6. Figure 7 These results indicate that AdjCRU can be taken up by dendritic cells and activates the TLR9 and its downstream NF-κB signaling pathway in lysosomes, inducing the secretion of the inflammatory cytokine IL-6, thus exhibiting a significant innate immune activation effect. This process promotes the maturation of dendritic cells and the presentation of the Yersinia pestis recombinant protein LcrV variant (rV10) antigen, thereby significantly enhancing immune protection against Yersinia pestis infection.

[0163] Example 4: Determination of the immunoprotective effect of AdjCRU prepared in Example 1 of the present invention.

[0164] Four experimental groups were set up: the Mock group, the rV10 (10 μg) group, the rV10 (10 μg) + LoDNA (5 μg) group, and the rV10 (10 μg) + AdjCRU (containing 5 μg LoDNA) group. Immunization was performed via aerosol lung delivery, with immunizations every 14 days for a total of three immunizations. Fourteen days after the third immunization, 25×LD50 was administered. 50 201 strains of Yersinia pestis (500 CFU) were challenged via liquid aerosol lung delivery. Changes in antibody titers in mice after immunization were detected by ELISA, and the immunoprotective efficacy of the vaccine was evaluated by survival analysis and organ bacterial load determination.

[0165] 1. Liquid aerosol lung delivery of immunity

[0166] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 mg / kg and fixed supine on the operating table. The tracheal opening was exposed using a laryngoscope, and a handheld liquid aerosol generator head was inserted into the trachea about 2 cm. The push handle was quickly pushed to deliver the vaccine via aerosol lung delivery. The mice were immunized three times, once every 14 days.

[0167] 2. Serum antibody level detection

[0168] (1) Sample collection and preparation: Six mice were randomly selected from each immunization group, and blood was collected on days 7, 14, 21, 28, 35 and 42 after each immunization, for a total of 6 blood collections. After the collected blood was left to stand at room temperature for 2 hours, the serum was separated by centrifugation at 3000×g and 4 ℃ for 10 minutes, aliquoted and stored at -80 ℃ for later use.

[0169] (2) Coating: Dilute protein rV10 to 1 μg / mL and add 100 µL / well to a 96-well ELISA plate. Coat overnight at 4 °C.

[0170] (3) Blocking: Discard the coating solution, add 200 µL / well blocking solution, and incubate at 37 °C for 2 hours.

[0171] (4) Primary antibody incubation: The serum was serially diluted 2 times with diluent and added to the coated plate at 100 µL / well; at the same time, blank mouse serum diluted 1:10 was used as a control and incubated at 37 °C for 30 minutes.

[0172] (5) Washing: Discard the primary antibody liquid, add 300 µL of washing buffer, shake for 1 minute and then discard. Repeat 5 times.

[0173] (6) Secondary antibody incubation: Add 100 µL / well of HRP-labeled goat anti-mouse IgG and IgG1 antibody diluted 1:10000 and incubate at 37 °C for 20 minutes.

[0174] (7) Wash plate: Discard the secondary antibody liquid and repeat step (5).

[0175] (8) Color development: Add 100 µL of TMB color development solution and incubate at 37 °C in the dark for 10 minutes.

[0176] (9) Termination: Add 100 µL of termination solution to terminate the reaction.

[0177] (10) Detection: The absorbance was measured at wavelengths of 450 nm and 630 nm using an ELISA reader. A positive result was determined when the ratio of the absorbance of the sample well to that of the normal mouse serum well was greater than 2. The maximum dilution gradient corresponding to the positive result was the serum IgG antibody level.

[0178] 3. Cultivation of plague strains

[0179] (1) Activation: Take 20 μL of plague glycerol bacteria from the -80 ℃ freezer, thaw and transfer to 20 mL of BHI liquid medium, and culture at 26 ℃ and 200 rpm for 30 hours until the plateau phase to obtain the first generation bacteria.

[0180] (2) Pre-culture: The first generation bacteria were diluted 20 times and transferred to 20 mL of BHI liquid medium and cultured at 26 ℃ and 200 rpm until OD. 600 =1.0, and the second generation of bacteria is obtained.

[0181] (3) Formal culture: The second generation bacteria were diluted 100 times and transferred to 20 mL of BHI liquid medium and cultured at 26 ℃ and 200 rpm until OD. 600 =1.0, yielding the third generation of bacteria. These were then transferred to a 37 ℃ incubator for further incubation for 3 hours to bring the plague bacteria to mid-log phase.

[0182] (4) Centrifugation to collect bacteria: Take 500 μL of bacterial solution and centrifuge at 3000×g at room temperature for 10 minutes to collect the bacterial cells.

[0183] (5) Bacterial resuspension: Discard the supernatant, resuspend the bacterial cells in an equal volume of physiological saline containing 0.05% poloxamer, wash twice, and then adjust the OD of the bacterial suspension with the same solution. 600 Up to 1.0. At this point, the theoretical concentration of the bacterial culture is 2 × 10⁻⁶. 8 The concentration of CFU / mL was diluted to the required challenge concentration for subsequent experiments.

[0184] (6) Drop plate counting: The bacterial solution was serially diluted 5 times, and 10 μL of diluted bacterial solution was dropped onto a blood plate. After incubation at 26°C for 72 hours, colony counting was performed to determine the actual bacterial concentration.

[0185] 4. Liquid aerosol lung delivery of plague bacteria for challenge and observation of clinical symptoms.

[0186] On day 14 following the third immunization, administer 500 CFU (25×LD). 50 Mice in all immunized groups were challenged with Yersinia pestis strain 201 via liquid aerosol lung delivery. Survival status and weight changes were continuously monitored for 14 days post-challenge, and survival curves were plotted accordingly.

[0187] 5. Detection of bacterial load in organs after challenge with Yersinia pestis delivered by liquid aerosols via the lungs.

[0188] Forty-eight hours after challenge with Yersinia pestis via liquid aerosol lung delivery, three mice were randomly selected from each challenge group and euthanized using CO2. Whole blood was collected by removing the mice's eyeballs in a biosafety cabinet, and the lungs, spleen, and liver were dissected and placed in sterile petri dishes. Subsequently, appropriate sizes of tissue from each organ were cut and weighed, and placed into homogenization tubes containing 800 μL of sterile PBS. Homogenization was performed at 5200 rpm for 90 seconds. The homogenate was then serially diluted 5-fold with PBS, and 10 μL of each dilution was dropped onto blood agar plates. The plates were incubated upside down at 26 ℃ for 72 hours, and the colony counts for each organ at different dilutions were recorded.

[0189] The results showed that mice immunized with the AdjCRU + rV10 co-vaccine exhibited a strong humoral immune response, with their serum IgG antibody titers significantly increasing as the immunization process progressed, and being higher than those in other immunization groups. Figure 8 ). In 25×LD 50 After challenge, the protective effect of the AdjCRU + rV10 blend vaccine on mice was significantly better than that of other immunization groups. Figure 9 Furthermore, 48 hours after challenge, the bacterial load in the lungs of mice immunized with AdjCRU + rV10 was significantly reduced. Figure 10 In conclusion, AdjCRU + rV10 immunization significantly enhanced the protective ability of mice against pneumonic plague.

[0190] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A DNA nanoadjuvant, characterized in that, The DNA nanoadjuvant consists of a DNA nanostructure and an MGN1703 variant; The nucleotide sequence of the MGN1703 variant is shown in SEQ ID NO.5; The DNA nanostructure is selected from cross-shaped DNA nanostructures; The cross-shaped DNA nanostructure is composed of four ssDNA strands, S1, S2, S3, and S4, and the sequences of S1, S2, S3, and S4 are shown in SEQ ID NO.1-4, respectively.

2. A vaccine, characterized in that, The vaccine comprises the DNA nanoadjuvant and antigen as described in claim 1; The antigen is selected from proteins; The protein in question is a variant of the recombinant protein LcrV from Yersinia pestis.

3. The vaccine according to claim 2, characterized in that, The mass ratio of DNA nanoadjuvant to antigen in the vaccine is 1:1 to 1:

5.

4. The vaccine according to claim 3, characterized in that, The mass ratio of DNA nanoadjuvant to antigen in the vaccine is 1:

2.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the vaccine according to any one of claims 2-4.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition includes a pharmaceutically acceptable carrier.

7. The method for preparing the DNA nanoadjuvant according to claim 1, characterized in that, The method includes mixing S1, S2, S3, S4 and MGN1703 variants to synthesize DNA nanoadjuvants; The sequences of S1, S2, S3 and S4 are shown in SEQ ID NO.1-4, respectively; The nucleotide sequence of the MGN1703 variant is shown in SEQ ID NO.

5.

8. The preparation method according to claim 7, characterized in that, The synthesis method is a one-step annealing method.

9. A method for preparing the vaccine according to any one of claims 2-4, characterized in that, The method includes mixing the DNA nanoadjuvant of claim 1 with an antigen; The antigen is selected from proteins; The protein in question is a variant of the recombinant protein LcrV from Yersinia pestis.

10. The application of the DNA nanoadjuvant according to claim 1 in the preparation of vaccines, characterized in that, The antigen of the vaccine is a variant of the recombinant protein LcrV of Yersinia pestis.

11. The application according to claim 10, characterized in that, The DNA nanoadjuvant of claim 1 promotes dendritic cell maturation.

12. The application according to claim 10, characterized in that, The DNA nanoadjuvant of claim 1 activates the TLR9 signaling pathway.

13. The application according to claim 10, characterized in that, The DNA nanoadjuvant of claim 1 promotes cytokine secretion; The cytokine in question is IL-6.

14. The use of the vaccine according to any one of claims 2-4 and the pharmaceutical composition according to any one of claims 5-6 in the preparation of a drug for preventing pathogen infection, wherein the pathogen is Yersinia pestis.

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