A circular RNA encoding aaspergillus fumigatus chimeric antigen and application thereof

Abstract

The present application relates to a kind of encoding aspergillus fumigatus chimeric antigen circular RNA and its application, belong to biomedical and vaccine technology field.The present application first applies circular RNA technology to anti-aspergillus fumigatus infection, provides new strategy for fungal vaccine development.Select the non-transmembrane region of aspergillus fumigatus cell membrane protein FtrA to construct antigen, avoid the transmembrane region expression problem and immune recognition uncertainty;Fusion IgK signal peptide and Fc domain, significantly improve antigen secretion efficiency, stability and immune presentation effect.LNP delivery system mature, efficient, can effectively deliver vaccine to host cell and express antigen, the LNP@CircRNA provided by the present application, especially the LNP@CircRNA FtrA2 , can induce high level of IgG antibody and T cell immune response in mouse in vivo without using adjuvant, realize the dual activation of humoral immunity and cellular immunity.

Description

Technical Field

[0001] This invention relates to a circular RNA encoding Aspergillus fumigatus chimeric antigen and its application, belonging to the fields of biomedicine and vaccine technology. Background Technology

[0002] Invasive fungal infections (IFIs), especially invasive aspergillosis (IA) caused by Aspergillus fumigatus, pose a serious threat to immunocompromised individuals, characterized by high morbidity and mortality. Aspergillus fumigatus has been listed by the World Health Organization as a "Fungal Pathogen of Priority for Research and Action." Currently, clinical diagnosis is time-consuming, treatment options are limited, and drug resistance is increasing, thus necessitating the development of novel and effective prevention strategies.

[0003] Vaccines are one of the most effective means of preventing pathogenic microbial infections. Researchers have explored various traditional vaccine forms against Aspergillus fumigatus, including live attenuated vaccines, inactivated vaccines, recombinant protein vaccines, and polysaccharide vaccines. However, these vaccines all have significant drawbacks: live attenuated vaccines carry the risk of incomplete attenuation leading to reinfection in immunocompromised individuals, and the degree of attenuation is difficult to balance with immunogenicity, posing a risk of virulence reversion during large-scale production; inactivated vaccines destroy the native conformation of the antigen during inactivation, resulting in low immunogenicity and requiring adjuvants to enhance the immune response, which may trigger adverse reactions such as inflammatory responses; recombinant protein vaccines have a single antigenic target, making it difficult to cover the diversity of Aspergillus fumigatus antigens, making them prone to immune escape, and the recombinant proteins expressed in vitro may have conformational errors, affecting immunization efficacy; polysaccharide vaccines are T-cell-independent antigens, unable to induce durable immune memory, and polysaccharide purification is difficult, resulting in weak cross-protective ability.

[0004] In recent years, nucleic acid vaccine technology has developed rapidly, and mRNA vaccines have made initial progress in the field of antifungal treatment (such as linear mRNA vaccines against Cryptococcus neoformans, which have shown protective effects). However, linear mRNA vaccines have limitations such as poor in vivo stability, easy degradation by exonucleases, and short half-life.

[0005] Circular RNA (circRNA), as a next-generation nucleic acid vaccine platform, possesses unique technological advantages: its closed circular structure lacks free 5' and 3' ends, making it less susceptible to degradation by exonucleases, resulting in high in vivo stability and a long half-life; it achieves sustained antigen expression through a cap-independent translation mechanism, eliciting strong and durable humoral and cellular immune responses without adjuvants; it exhibits low immunogenicity, is less likely to trigger nonspecific inflammatory responses, and does not integrate into the host genome, thus offering higher safety; furthermore, its antigen sequence design is flexible, allowing for rapid adaptation to pathogen variants. However, to date, no anti-Aspergillus fumigatus vaccine based on circRNA technology has been developed. The main reasons are: limited research on in vitro circRNA synthesis systems, lacking efficient and stable preparation systems; insufficient screening of Aspergillus fumigatus protective antigens, making it difficult to identify antigen targets that can induce broad-spectrum and potent immune responses; and the in vivo delivery efficiency and targeting of circRNA still require optimization.

[0006] Based on the shortcomings of the existing technology, this invention aims to develop a circular RNA encoding Aspergillus fumigatus antigen, which will be used as the core immunogenic component to construct a novel vaccine. Through an innovative circular RNA synthesis system, precise antigen design, and efficient delivery system, this invention addresses the deficiencies of traditional vaccines and linear mRNA vaccines, providing a completely new strategy for the prevention of IA. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a circular RNA encoding Aspergillus fumigatus chimeric antigen and its applications.

[0008] The technical solution of the present invention is as follows: A chimeric antigen of Aspergillus fumigatus, the chimeric antigen comprising antigen FtrA1 or FtrA2, wherein the amino acid sequence of antigen FtrA1 is shown in SEQ ID NO.3 and the amino acid sequence of antigen FtrA2 is shown in SEQ ID NO.4.

[0009] Preferably, the chimeric antigen further includes the Fc domain of human IgG1, wherein the Fc domain of human IgG1 is linked to the C-terminus of antigen FtrA1 or FtrA2. Preferably, the chimeric antigen further includes the mouse IgK signal peptide SP, which is linked to the N-terminus of antigen FtrA1 or FtrA2. More preferably, the amino acid sequence of the Fc domain of the human IgG1 is shown in SEQ ID NO.11; More preferably, the amino acid sequence of the mouse IgK signal peptide SP is shown in SEQ ID NO.13.

[0010] In this invention, based on the non-transmembrane region of the iron ion permeability enzyme FtrA located in the cell membrane of Aspergillus fumigatus, two fusion antigens, FtrA1 and FtrA2, were designed and constructed.

[0011] A nucleotide encoding the chimeric antigen described above; Preferably, the nucleotide sequence encoding antigen FtrA1 is shown in SEQ ID NO.7; Preferably, the nucleotide sequence encoding antigen FtrA2 is shown in SEQ ID NO.8; Preferably, the nucleotide sequence encoding the Fc domain of human IgG1 is shown in SEQ ID NO.12; Preferably, the nucleotide sequence of the signal peptide SP encoding mouse IgK is shown in SEQ ID NO.14.

[0012] A circRNA comprising the aforementioned nucleotides; Preferably, the circRNA further includes nucleotides encoding IRES; More preferably, the nucleotide sequence encoding IRES is shown in SEQ ID NO.19.

[0013] A recombinant vector comprising the aforementioned nucleotides or circRNA.

[0014] A recombinant cell comprising the aforementioned nucleotides, circRNA, or recombinant vector.

[0015] The above-mentioned recombinant antigens, nucleotides, circRNAs, recombinant vectors, or recombinant cells are used in the preparation of Aspergillus fumigatus vaccines.

[0016] A vaccine comprising the above-mentioned recombinant antigen, nucleotide, circRNA, recombinant vector or recombinant cell; Preferably, the vaccine also includes lipid nanoparticles.

[0017] The above-mentioned recombinant antigens, nucleotides, circRNAs, recombinant vectors, recombinant cells, or vaccines are used in the preparation of drugs for the treatment / prevention of Aspergillus fumigatus infection.

[0018] A pharmaceutical composition comprising the above-mentioned recombinant antigen, nucleotide, circRNA, recombinant vector, recombinant cell or vaccine; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0019] Beneficial effects: (1) Innovative vaccine platform: This invention is the first to apply circular RNA technology to anti-Aspergillus fumigatus infection, providing a new strategy for fungal vaccine development.

[0020] (2) Precise antigen design and strong immunogenicity: The non-transmembrane region of the Aspergillus fumigatus cell membrane protein FtrA was selected to construct the antigen, avoiding the difficulties of transmembrane region expression and the uncertainty of immune recognition; the fusion of IgK signal peptide and Fc domain significantly improves antigen secretion efficiency, stability and immune presentation effect.

[0021] (3) Efficient and safe delivery and expression: A novel cis-acting ribozyme system is used, which has high efficiency in the synthesis of circular RNA. Its closed circular structure gives it excellent in vivo stability and a much longer half-life than linear mRNA, enabling continuous antigen expression without the need for frequent booster immunizations. The LNP delivery system is mature and efficient, and can effectively deliver the vaccine to the host cell and express the antigen.

[0022] (5) Strong dual immune response: The LNP@CircRNA provided by this invention, especially LNP@CircRNA FtrA2 Without the use of adjuvants, it can induce high levels of IgG antibodies and T cell immune responses in mice, achieving dual activation of humoral and cellular immunity.

[0023] (5) Clear protective efficacy: In a lethal Aspergillus fumigatus challenge model in immunosuppressed mice, inoculation with LNP@CircRNA FtrA2 It can significantly reduce the fungal load in the lungs, alleviate pathological damage, and increase the survival rate to 60%, demonstrating a significant protective effect.

[0024] (6) Wide range of applications: FtrA protein has homology in important pathogenic fungi of different species, suggesting that the CircRNA provided by this invention has the potential to provide cross-protection against a variety of invasive fungal infections. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the chimeric antigen structure (IgK(SP) + antigen + IgG1(Fc)). Figure 2 This is a schematic diagram of the structure and cyclization mechanism of the cyclization system; Figure 3 The HPLC chromatogram of the cyclized product is shown below. Figure 4 The image shows a 1% agarose gel electrophoresis result of four antigen circRNAs. Figure 5 A diagram showing the Sanger sequencing results for the circularized site; Figure 6 A bar graph showing the antigen expression levels of four circRNAs detected by ELISA; Figure 7 The figure shows the characterization results of LNP@CircRNA, where A represents LNP@CircRNA. FtrA1 Particle size analysis results, B represents LNP@CircRNA FtrA2 Particle size analysis results, C is the zeta potential detection result, D is the DLS detection result, E is the TEM electron microscope image; Figure 8 Flowchart of mouse immunization program experiment; Figure 9 The graph shows the in vivo IgG antibody titer results of LNP@CircRNA; Figure 10 For IFN-γ ＋ CD4 + T and IFN-γ ＋ CD8 + T cell proportion bar chart; where A represents IFN-γ ＋ CD4 + T cell proportion bar chart, B represents IFN-γ ＋ CD8 + T cell proportion bar chart; Figure 11 Microscopic image showing the binding ability of LNP@CircRNA-induced antibody to Aspergillus fumigatus; Figure 12 The figure shows the inhibition of Aspergillus fumigatus biofilm formation by LNP@CircRNA-induced antibody. Figure 13 Figure showing the effect of LNP@CircRNA-induced antibody on the phagocytic killing effect of macrophages; Figure 14 Flowchart of experiments involving lethal doses of Aspergillus fumigatus infection in mice; Figure 15 This is a survival curve for mice; Figure 16 A bar graph showing the fungal load in mouse lungs; Figure 17 Microscopic image of mouse lung tissue stained with GMS. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] Unless otherwise specified, the drugs and reagents used in the examples are common products already on the market. All contents not described in detail in the examples are based on the prior art.

[0028] Unless otherwise specified, the pH of the PBS buffer used in the examples was 7.4.

[0029] Example 1 Candidate antigen design and coding sequence optimization 1. Antigen target analysis: The full-length amino acid sequences of FtrA and FksA proteins from Aspergillus fumigatus were obtained from the NCBI database. The amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Three-dimensional structure prediction was performed using AlphaFold, and functional domain and transmembrane region analysis was performed using InterPro. FtrA was determined to be a 7-transmembrane protein and FksA to be a 17-transmembrane protein.

[0030] 2. Antigen sequence design: FtrA1: The predicted extracellular region fragment of FtrA fusion (91 aa, amino acid sequence as shown in SEQ ID NO.3); FtrA2: The predicted intracellular region fragment of FtrA fusion (121 aa, amino acid sequence as shown in SEQ ID NO.4); FksA1: The 758-1017aa region of FksA (amino acid sequence as shown in SEQ ID NO.5); FksA2: The 1018-1338aa region of FksA (amino acid sequence as shown in SEQ ID NO.6).

[0031] 3. Sequence optimization: Human codon optimization was performed on the coding genes of the above antigens to improve their expression efficiency in Expi293F cells. The optimized nucleotide sequence of the coding gene of antigen FtrA1 is shown in SEQ ID NO.7, the optimized nucleotide sequence of the coding gene of antigen FtrA2 is shown in SEQ ID NO.8, the optimized nucleotide sequence of the coding gene of antigen FksA1 is shown in SEQ ID NO.9, and the optimized nucleotide sequence of the coding gene of antigen FksA2 is shown in SEQ ID NO.10. Meanwhile, in order to improve antigen stability and presentation effect, the Fc domain of human IgG1 is linked to the C-terminus of the antigen, that is, the coding gene of the Fc domain is fused to the 3' end of the antigen coding gene. The amino acid sequence of the Fc domain is shown in SEQ ID NO.11, and the nucleotide sequence of the coding gene of the Fc domain is shown in SEQ ID NO.12. In addition, to enhance the secretion effect of the antigen, the N-terminus of the antigen is also linked to the signal peptide SP of mouse IgK, that is, the coding gene of the signal peptide SP is fused to the 5' end of the antigen coding gene. The amino acid sequence of the signal peptide SP is shown in SEQ ID NO.13, and the nucleotide sequence of the coding gene of the signal peptide SP is shown in SEQ ID NO.14. Following the optimization method described above, the final chimeric antigen structure is as follows: Figure 1 As shown.

[0032] Example 2 Preparation and purification of circular RNA The RNA cyclization system was designed according to the method described in patent document CN118086282B (application number 202410221887.0). It involves cyclization at the L9.1 position of the Anabaena type I intron. The structure of the cyclization system is as follows: first matching sequence - upstream sequence of ribozyme 9.1 - polyAC-IRES nucleotide sequence - kozak sequence - chimeric antigen nucleotide sequence - downstream sequence of polyAC-ribozyme 9.1 - second matching sequence complementary to the first matching sequence. The structure and cyclization mechanism of the cyclization system are as follows: Figure 2 As shown (taking enhanced green fluorescent protein EGFP as an example). The nucleotide sequence of the first matching sequence is shown in SEQ ID NO. 15; the nucleotide sequence of the upstream sequence of ribozyme 9.1 is shown in SEQ ID NO. 16; the nucleotide sequence of the downstream sequence of ribozyme 9.1 is shown in SEQ ID NO. 17; the nucleotide sequence of the second matching sequence is shown in SEQ ID NO. 18; the nucleotide sequence of IRES is shown in SEQ ID NO. 19; and the kozak sequence is GCCACC.

[0033] RNA was prepared and cyclized according to the conditions described in Example 1 of patent document CN118086282B to obtain a cyclized product containing CircRNA, and the cyclized product was purified.

[0034] The purification steps are as follows (using CircRNA as an example). EGFP (For example) ① Preliminary HPLC purification: A high-performance liquid chromatography (HPLC) system was used, equipped with a 4.6 × 300 mm size-limited column (5 μm particle size, 1000 Å pore size), with 150 mM sodium phosphate buffer (pH 7.0) as the mobile phase, a flow rate of 0.6 mL / min, and detection at 260 nm wavelength; the target peak corresponding to the circRNA was collected (the HPLC chromatogram of the cyclization product is shown in the figure). Figure 3 As shown, the target CircRNA peak is the latter half of the main peak, and impurities such as high molecular weight (HMW) RNA and free introns are removed.

[0035] ②RNase R enzymatic digestion and enrichment: After desalting and concentrating the HPLC collection solution, RNase R enzyme (Vazyme) was added at a ratio of 1 U / μg RNA, and incubated at 37℃ for 15 minutes to specifically degrade linear RNA and incomplete RNA with gaps, while retaining closed circular RNA.

[0036] ③ Final purification and recovery: The enzymatic hydrolysate was purified by column chromatography using the HiPure RNA Pure Micro kit to remove residual impurities such as RNase R enzyme and salt ions, obtaining high-purity circRNA. EGFP It was dissolved in RNase-free water at a concentration of 1 μg / μL. CircRNA FtrA1 CircRNA FtrA2 CircRNA FksA1 CircRNA FksA2 Purification was performed according to CircRNA. EGFP implement.

[0037] Example 3 Quality control of circRNA (based on circRNA) EGFP (For example) Agarose gel electrophoresis: CircRNA obtained in Example 2 EGFP The results of 1% agarose gel electrophoresis are as follows: Figure 4 As shown, CircRNA EGFP The band was a single band with no obvious linear RNA impurities; Sequencing validation: using CircRNA EGFP Using the pUC57 vector as a template, reverse transcription PCR (RT-PCR) was performed to amplify the circularization site fragment. The fragment was then cloned into the pUC57 vector and sequenced to confirm that the circularization site matched the design. The sequencing results of the circularization site are as follows: Figure 5 As shown; CircRNA FtrA1 CircRNA FtrA2 CircRNA FksA1 CircRNA FksA2 Quality control reference CircRNA EGFP implement; Expression validation: CircRNA was expressed separately. FtrA1 CircRNA FtrA2 CircRNA FksA1 CircRNA FksA2 (2 μg each) and PEI transfection reagent were mixed at a mass ratio of 1:4. The control group was transfected with an equal volume of Expi293F cell culture medium instead of CircRNA. (This was a gift from Academician Gao Fu's team at the Institute of Microbiology, Chinese Academy of Sciences. Other personnel skilled in the art can obtain the above biological materials from the applicant or through commercial channels to replicate the relevant experiments of this invention.) The mixture was then processed in 6-well plates at a ratio of 2 × 10⁻⁶. 6 Cells were seeded into wells after transfection and cultured for 48 h. The supernatant was then collected, and antigen expression levels were detected by ELISA. The results are as follows: Figure 6 As shown, CircRNAFksA2 CircRNA FtrA1 CircRNA FtrA2 The expression level was significantly higher than that of the control group (P < 0.05), among which, circRNA... FtrA2 The highest expression level was found in CircRNA. FtrA1 Secondly, therefore, CircRNA was selected. FtrA1 and CircRNA FtrA2 Further experiments will be conducted.

[0038] Example 4 Preparation and characterization of LNP@CircRNA 1. Preparation of LNP@CircRNA (1) Preparation of liposome (LNP) components: Ionizable lipids (iLs), DSPC, cholesterol, and Lip-PEG (polyethylene glycol-lipid) in a molar ratio of 50:10:38:2 were dissolved in ethanol to prepare an organic phase; the CircRNA prepared in Example 2 was used to prepare the organic phase. FtrA1 and CircRNA FtrA2 The aqueous phases were prepared by dissolving them separately in 100 mM citrate buffer (pH 5.0).

[0039] (2) Microfluidic mixing: Using a microfluidic device, the aqueous phase and organic phase are injected into the chip at a volume ratio of 1:3. The LNP@CircRNA complex is formed by laminar chaotic mixing, and the flow rate is controlled at 1 mL / min.

[0040] (3) Dialysis and concentration: The LNP@CircRNA complex was dialyzed in PBS buffer for 12 h using a dialysis bag (MWCO=3.5 kDa) to remove ethanol and other impurities for later use.

[0041] 2. Characterization: Particle size and PDI: Particle size analysis results are as follows Figure 7 As shown in A and B, DLS detection is as follows Figure 7 As shown in D, LNP@CircRNA FtrA1 The average particle size was 91.7 ± 3.86 nm, and the PDI was 0.097; LNP@CircRNA FtrA2 The average particle size was 97.3 ± 7.39 nm, and the PDI was 0.113, both meeting the requirements for vaccine delivery; furthermore, DLS results indicated that LNP@CircRNA FtrA1 and LNP@CircRNA FtrA2 The particle size fluctuations were relatively small within 72 hours, indicating good stability.

[0042] zeta potential: Detection results are as follows Figure 7As shown in C, the result is -3.9 to -4.5 mV, which is beneficial for in vivo stability and cellular uptake; Morphology: TEM electron microscopy, such as Figure 7 As shown in E, observations revealed LNP@CircRNA FtrA1 and LNP@CircRNA FtrA2 The particles were uniformly spherical, and their morphology did not change significantly after 72 hours. The particle size did not increase significantly, and the stability was good.

[0043] Example 5 In vivo immunogenicity assessment of LNP@CircRNA (1) Experimental animals: 6-8 week old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in an SPF-grade animal room. They were used for experiments after acclimatization to the environment for 1 week.

[0044] (2) Immunization program: Mice were randomly divided into 3 groups (n=8): LNP group, LNP@CircRNA group, and LNP@CircRNA group. FtrA1 Group, LNP@CircRNA FtrA2 Groups. Mice in each group were immunized intramuscularly with three doses, each containing 10 μg of circRNA (the LNP group received an equal volume of LNP without circRNA). The immunization interval was two weeks, administered on day 13 (after the first immunization, Prime) and day 27 (after the second immunization, Prime). st Boost), 42 days (after three free treatments, 2 nd Boost) collects mouse serum, and the experimental procedure is as follows: Figure 8 As shown.

[0045] (3) Antibody detection: IgG antibody titer was detected by ELISA. Preparation of recombinant FtrA1 and FtrA2 proteins: The encoding nucleotide sequences of FtrA1 and FtrA2 were inserted into the commercial plasmids pSmart-Ⅰ and pGEX4T-1, respectively, to construct the pSmart-Ⅰ-FtrA1 and pGEX4T-1-FtrA2 plasmids. Subsequently, the pSmart-Ⅰ-FtrA1 and pGEX4T-1-FtrA2 plasmids were introduced into BL21(DE3) strain for antigen expression and purification. During induction, IPTG was added to a final concentration of 1 mM, and induction was performed at 16°C and 100 rpm / min for 12–16 hours. The proteins were purified directly from the pretreated bacterial lysate using a His tag on an ÄKTA™ purification system (Cytiva, USA) using a Ni-NTA QZT 6FF pre-packed column (Senhui, China). The column was equilibrated with 5 column volumes of PBS buffer. Bacterial lysate was pretreated by centrifugation and filtration through a 0.22 μm filter before being loaded onto the column. The column was then washed sequentially with PBS buffer and wash buffer (PBS buffer containing 20 mM imidazole, pH 7.4) until no substances were detected in the effluent and the UV value remained stable. The flow rate during washing was set at 2 mL / min. The target protein was then eluted with elution buffer (PBS buffer containing 300 mM imidazole, pH 7.4). After concentration using a 10 kDa MWCO ultrafiltration centrifuge (Millipore, USA) at 3000 × g and 4 °C, the protein was further purified using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, USA). The purified protein was further analyzed by SDS-PAGE to obtain recombinant FtrA1 and FtrA2 proteins, which were then rapidly frozen in liquid nitrogen and stored at -80 °C.

[0046] Coating: Prepare ELISA plates with a concentration of 4 μg / mL using PBS buffer, add 100 μL to each well, and incubate overnight at 4°C. Add 250 μL of PBS buffer to each well and wash three times, then pat dry with absorbent paper.

[0047] Blocking: Add 200 μL of PBST buffer containing 5% (w / v, g / mL) skim milk powder to each well and block at 37°C for 1 h; Add 250 μL of PBS buffer to each well and wash three times, then pat dry with absorbent paper.

[0048] Primary antibody incubation: Serum samples were serially diluted starting at a ratio of 1:100, i.e., 1 μL of serum and 99 μL of PBST buffer were added to the first well, and subsequent wells were serially diluted two-fold. 100 μL of diluted serum was added to each well, and the samples were incubated at 37°C for 1 h. Add 250 μL of PBS buffer to each well and wash three times, then pat dry with absorbent paper.

[0049] Secondary antibody incubation: Add HRP (horseradish peroxidase) labeled goat anti-mouse IgG antibody (Easybio, China), dilute with PBST buffer 1:5000, add 100 μL of diluted secondary antibody to each well, and incubate at 37℃ for 1 h; Add 250 μL of PBS buffer to each well and wash three times, then pat dry with absorbent paper.

[0050] Color development: Add 100 μL of TMB (tetramethylbenzidine) solution to each well and develop color for 15 min under dark conditions. Then, add 100 μL of 2 M H2SO4 to terminate the reaction and measure the OD. 450 Value. Result as follows Figure 9 As shown, antibody levels were low after the first immunization, but significantly increased after the second and third immunizations. (LNP@CircRNA) FtrA2 The titer of IgG antibody after group 3 immunization was significantly higher than that of LNP@CircRNA. FtrA1 Group.

[0051] (4) T cell response detection: Seven days after the third immunization, mice (n=5) were sacrificed, and spleens were collected to prepare 1×10⁻⁶ T cells. 7 Single-cell suspensions of 100 cells / mL were added to each well, and the cells were incubated with recombinant FtrA1 or FtrA2 protein (final concentration 4 μg / mL) in a 37°C, 5% CO2 incubator for 12 h. Then, a blocking agent (BFA) was added to a final concentration of 1 μg / mL, and the cells were incubated for another 6 h. IFN-γ was detected by flow cytometry. ＋ CD4 + T and IFN-γ ＋ CD8 + The T cell ratio is determined as follows: Cell staining: Add Fixable Viability Dye eFluor TM 450 excluded dead cells, APC-labeled anti-mouse CD8α antibody identified CD8 + T cells, FITC-labeled anti-mouse CD4 antibody identification of CD4 + T cells, using True-Nuclear TM Buffer and PE-labeled anti-mouse IFN-γ antibody staining for intracellular factors; Flow cytometry analysis: Data were acquired using a CytoFLEX flow cytometer, and the results were analyzed using CytExpert software. Results are as follows: Figure 10 As shown, LNP@CircRNA FtrA2 Group IFN-γ ＋ CD4 +T and IFN-γ ＋ CD8 + The proportion of T cells was significantly higher than that of LNP@CircRNA. FtrA1 Group and LNP group indicate that LNP@CircRNA FtrA2 It can effectively induce antigen-specific cellular immune responses.

[0052] Example 6 Functional validation of LNP@CircRNA-induced antibodies (1) Antibody binding experiment: Aspergillus fumigatus spores were mixed at 2×10⁻⁶. 4 LNPs / well were inoculated into 96-well plates containing 100 μL of RPMI-1640 medium (Gibco, USA) and cultured at 37°C for 2 days to form hyphae. After removing the medium, the hyphae were fixed with 4% paraformaldehyde at 4°C for 1 h, followed by gentle washing three times with PBS buffer. After blocking with 5% fetal bovine serum at room temperature for 30 min, the hyphae were washed three more times with PBS buffer. LNP and LNP@CircRNA were also included. FtrA1 LNP@CircRNA FtrA2 Each group was resuspended in 100 μL of serum from mice that had undergone triple immunization, as described in Example 5 (serum diluted 1:50 with PBS buffer, i.e., 2 μL serum and 98 μL PBS buffer were added). The control group (without serum) was resuspended in 100 μL of PBS buffer and incubated at room temperature for 1 h. Then, 100 μL of Rhodamine Red-X-labeled goat anti-mouse IgG antibody (from Yisheng Biotechnology, China; antibody diluted 1:100 with PBS buffer, i.e., 1 μL antibody and 99 μL PBS buffer were added) was added, and the mixture was incubated at room temperature for 1 h. Finally, the mice were washed three times with PBS buffer and observed under a fluorescence microscope. The results are as follows: Figure 11 As shown, LNP@CircRNA FtrA1 Group and LNP@CircRNA FtrA2 The group showed significant fluorescence intensity, indicating that the serum antibody had the ability to bind to Aspergillus fumigatus hyphae. LNP@CircRNA FtrA2 The serum antibody in group A showed the strongest binding ability to Aspergillus fumigatus hyphae, and its fluorescence intensity was significantly higher than that of other groups.

[0053] (2) Biofilm inhibition experiment: Aspergillus fumigatus spores were injected at a concentration of 2×10⁻⁶. 4 LNPs and LNP@CircRNA were seeded per well into 96-well plates containing 100 μL of RPMI-1640 medium. FtrA1 LNP@CircRNA FtrA2The control group (without serum) was treated with 5 μL of heat-inactivated serum from mice after triple immunization (inactivation conditions: 56℃, 30 min), while the control group (without serum) was treated with 5 μL of PBS buffer. The plates were incubated at 37℃ for 2 days. After gently aspirating the culture medium, the plates were washed three times with PBS buffer. Then, 100 μL of methanol was added to each well, and the plates were fixed at room temperature for 15 min. After removing the methanol and allowing the plates to air dry, 100 μL of 0.1% crystal violet solution was added to each well, and the plates were stained at room temperature for 15 min. The crystal violet solution was then removed, and the plates were rinsed with double-distilled water until no more purple liquid was expelled. Finally, the crystal violet was dissolved in 95% ethanol, and the OD was measured. 595 Value. Result as follows Figure 12 As shown, LNP@CircRNA FtrA1 Group and LNP@CircRNA FtrA2 Group OD 595 The values ​​were all significantly lower than those in the LNP group, indicating that LNP@CircRNA FtrA1 LNP@CircRNA FtrA2 Inducible antibodies can effectively inhibit the formation of Aspergillus fumigatus biofilm, among which LNP@CircRNA FtrA2 The inhibitory effect was more significant in the group.

[0054] (2) Oppositional phagocytosis assay: Mouse RAW264.7 macrophages (purchased from Pronosai) cultured on RAW-specific medium (purchased from Pronosai) were activated by adding lipopolysaccharide (LPS) (Sigma-Aldrich, Germany) to a final concentration of 1 ng / mL in a 37℃, 5% CO2 incubator for 24 h. Aspergillus fumigatus spores were inoculated at a rate of 2 × 10⁻⁶. 4 LNPs / well were seeded into 96-well plates containing 90 μL of DMEM medium, followed by the addition of 10 μL of post-tertiary immunization serum and incubation at 37°C for 15 min (LNP, LNP@CircRNA). FtrA1 LNP@CircRNA FtrA2 Serum from the corresponding group in Example 5 was added to each group, while the control group (Raw264.7) received an equal volume of DMEM medium. Then, 100 μL of activated RAW264.7 macrophages (Aspergillus fumigatus spores to macrophages ratio of 1:1) were added to each well. After gently shaking the plate, it was incubated at 37°C for 2 h. The diluted solution was then inoculated onto PDA plates and incubated at 30°C for 48 h. Colony forming units (CFU) were counted, and the killing rate was calculated. Results are as follows: Figure 13 As shown, LNP@CircRNA FtrA1 Group and LNP@CircRNA FtrA2 The macrophage killing rate in the LNP group was significantly higher than that in the LNP group, indicating that LNP@CircRNA... FtrA1 LNP@CircRNA FtrA2Inducible antibodies can enhance the phagocytic and killing effects of macrophages on Aspergillus fumigatus, among which LNP@CircRNA FtrA2 Even better results.

[0055] Example 7 Protective effect of LNP@CircRNA against fatal Aspergillus fumigatus infection The experimental procedure in this embodiment is as follows: Figure 14 As shown, the specific steps and corresponding results are as follows: (1) Construction of immunosuppression model: Mice were immunized according to the method in Example 5, and LNP and LNP@CircRNA were used. FtrA1 LNP@CircRNA FtrA2 Two weeks after the third immunization, mice were given immunosuppression treatment via subcutaneous injection of hydrocortisone (125 mg / kg, once every 3 days) and intraperitoneal injection of tacrolimus (1 mg / kg, once daily) until the end of the experiment. Drinking water was supplemented with 1 mg / L tetracycline and 64 mg / L ciprofloxacin to prevent bacterial infection.

[0056] (2) Aspergillus fumigatus infection: Three days after the initial immunosuppressive treatment, mice were intranasally inoculated with 5 × 10 6 A lethal infection model was established using a suspension of Aspergillus fumigatus spores (40 μL / animal).

[0057] The control group (CK) did not undergo Aspergillus fumigatus infection, but all other procedures were the same as those in the LNP group.

[0058] (3) Survival monitoring: The survival status of mice was monitored daily after infection. Mice that lost more than 20% of their body weight were humanely euthanized and recorded as dead. The results are as follows: Figure 15 As shown, all mice in the LNP group died within 10 days after infection; LNP@CircRNA FtrA1 The final survival rate for the group was 20%; LNP@CircRNA FtrA2 The final survival rate for the group reached 60%.

[0059] (4) Detection of fungal load in the lungs: Three days after infection, some mice (n=3) were sacrificed, lung tissue was collected and weighed, and 1 mL of PBS buffer containing 0.05% (w / v, g / mL) chloramphenicol was added. After homogenization and dilution, the tissue was inoculated onto PDA plates and incubated at 30℃ for 48-72 h. CFU were then counted. The results are as follows: Figure 16 As shown, LNP@CircRNA FtrA1 Group and LNP@CircRNA FtrA2 The pulmonary fungal load in the LNP group was significantly lower than that in the LNP group (P < 0.05), and LNP@CircRNA FtrA2 Groups work better.

[0060] (5) Lung pathological analysis: Lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with GMS to observe fungal infiltration. Results are as follows: Figure 17 As shown, the LNP group had extensive fungal hyphae infiltration in the lungs, indicating a severe inflammatory response. (LNP@CircRNA) FtrA1 Group and LNP@CircRNA FtrA2 The group showed a significant reduction in pulmonary fungal infiltration and a marked decrease in inflammatory damage, and LNP@CircRNA FtrA2 Groups work better.

Claims

1. A chimeric antigen of Aspergillus fumigatus, characterized in that, The chimeric antigen includes antigen FtrA1 or FtrA2, the amino acid sequence of antigen FtrA1 is shown in SEQ ID NO.3, and the amino acid sequence of antigen FtrA2 is shown in SEQ ID NO.

4.

2. The chimeric antigen as described in claim 1, characterized in that, The chimeric antigen also includes the Fc domain of human IgG1, which is linked to the C-terminus of antigen FtrA1 or FtrA2. Preferably, the chimeric antigen further includes the mouse IgK signal peptide SP, which is linked to the N-terminus of antigen FtrA1 or FtrA2. More preferably, the amino acid sequence of the Fc domain of the human IgG1 is shown in SEQ ID NO.11; More preferably, the amino acid sequence of the mouse IgK signal peptide SP is shown in SEQ ID NO.

13.

3. A nucleotide, characterized in that, The nucleotide encodes the chimeric antigen as described in claim 1 or 2; Preferably, the nucleotide sequence encoding antigen FtrA1 is shown in SEQ ID NO.7; Preferably, the nucleotide sequence encoding antigen FtrA2 is shown in SEQ ID NO.8; Preferably, the nucleotide sequence encoding the Fc domain of human IgG1 is shown in SEQ ID NO.12; Preferably, the nucleotide sequence of the signal peptide SP encoding mouse IgK is shown in SEQ ID NO.

14.

4. A circRNA, characterized in that, The CircRNA comprises the nucleotides described in claim 3; Preferably, the circRNA further includes nucleotides encoding IRES; More preferably, the nucleotide sequence encoding IRES is shown in SEQ ID NO.

19.

5. A recombinant vector, characterized in that, The recombinant vector comprises the nucleotide of claim 3 or the circRNA of claim 4.

6. A recombinant cell, characterized in that, The recombinant cells comprise the nucleotides of claim 3, the circRNA of claim 4, or the recombinant vector of claim 5.

7. The use of the recombinant antigen of claim 1 or 2, the nucleotide of claim 3, the circRNA of claim 4, the recombinant vector of claim 5, or the recombinant cell of claim 6 in the preparation of Aspergillus fumigatus vaccine.

8. A vaccine, characterized in that, The vaccine comprises the recombinant antigen of claim 1 or 2, the nucleotide of claim 3, the circRNA of claim 4, the recombinant vector of claim 5, or the recombinant cell of claim 6; Preferably, the vaccine also includes lipid nanoparticles.

9. The use of the recombinant antigen of claim 1 or 2, the nucleotide of claim 3, the circRNA of claim 4, the recombinant vector of claim 5, the recombinant cell of claim 6, or the vaccine of claim 8 in the preparation of a medicament for the treatment / prevention of Aspergillus fumigatus infection.

10. A pharmaceutical composition comprising the recombinant antigen of claim 1 or 2, the nucleotide of claim 3, the circRNA of claim 4, the recombinant vector of claim 5, the recombinant cell of claim 6, or the vaccine of claim 8; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

Images