A "mosaic" swine flu nanoparticle vaccine and methods of making and using the same

Abstract

The application provides a "mosaic" swine flu nanoparticle vaccine and a preparation method and application thereof, and belongs to the technical field of biological products. The fusion protein provided by the application comprises M2e and HA dominant epitopes from H1N1 and H3N2 influenza viruses and is fused with ferritin. Nanoparticles are formed by self-assembly based on the fusion protein, and the nanoparticles can induce strong cellular immune response in vivo by immunizing mice, and provide 100% protection against H1N1 and H3N2 swine flu viruses, and have wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a "mosaic" type swine influenza nanoparticle vaccine, its preparation method, and its application. Background Technology

[0002] Swine influenza (SI) is an acute, contagious respiratory disease in pigs caused by the swine influenza virus (SIV). SIV infection can lead to decreased production performance in pig herds and increased susceptibility to secondary infections with other pathogens, further impacting herd health and causing severe economic losses. Because pig respiratory epithelial cells can simultaneously express sialic acid α-2,3-galactose and α-2,6-galactose receptors, pigs are considered "mixing vessels" for influenza viruses. They can promote gene reassortment between human and avian influenza viruses, resulting in new influenza viruses that pose a risk of infecting humans and poultry, thus constituting a significant threat to the livestock industry and public health.

[0003] Vaccination remains the most economical and effective method for controlling swine influenza (SI). Although inactivated whole-virus vaccines against H1 and H3 subtype SI have been widely used for swine influenza control, there are still shortcomings. First, inactivated vaccines mainly activate humoral immunity, resulting in short-lasting immunity. Second, antigenic drift and antigenic shift of influenza viruses often cause mismatches between circulating strains and vaccine strains, leading to reduced vaccine efficacy. Therefore, the development of novel broad-spectrum swine influenza vaccines is particularly urgent.

[0004] In recent years, ferritin nanoparticle vaccines have become a hot topic in vaccine research. Ferritin nanoparticles are near-spherical proteins self-assembled from 24 identical subunits, exhibiting good thermal and chemical stability. Due to the oligomerization of this protein, multiple viral antigenic components can be displayed on its surface, making it a highly efficient antigen epitope delivery carrier. Currently, the ferritin nanoparticle platform has been used to display protein antigens of various viruses, such as influenza HA protein and SARS-CoV-2 spike protein, and has induced effective humoral and cellular immunity in animals. Summary of the Invention

[0005] This invention provides a fusion protein that combines dominant T-cell epitopes (H1H20, H1H25) derived from M2e and H1N1 influenza virus, and dominant T-cell epitopes (H3H13, H3H23) derived from H3N2 influenza virus with Ferritin to form a fusion protein, aiming to effectively improve the immunogenicity and broad-spectrum protective effect of the vaccine.

[0006] This invention provides a fusion protein comprising M2e (H1N1-M2e) derived from H1N1 influenza virus, M2e (H3N2-M2e) derived from H3N2 influenza virus, a dominant T cell epitope derived from H1N1 influenza virus, a dominant T cell epitope derived from H3N2 influenza virus, and Ferritin fused together; the amino acid sequence of the fusion protein is shown in SEQ ID NO:1.

[0007] Preferably, the amino acid sequence of the Ferritin is shown in SEQ ID NO:2.

[0008] This invention provides nanoparticles assembled based on the fusion protein.

[0009] This invention provides a swine influenza vaccine, wherein the antigen comprises the fusion protein or the nanoparticles.

[0010] Preferably, the concentration of the antigen is 2 mg / mL.

[0011] This invention provides the use of the fusion protein or the nanoparticles in the preparation of vaccines for the prevention and / or control of swine influenza virus infection.

[0012] Preferably, the swine influenza virus includes H1N1 influenza virus and / or H3N2 influenza virus.

[0013] This invention provides a "mosaic"-like nanoparticle, which is formed by the self-assembly of M2e epitopes derived from H1N1 and H3N2 influenza viruses, dominant T-cell epitopes derived from H1N1 influenza virus, dominant T-cell epitopes derived from H3N2 influenza virus, and Ferritin. This not only increases the number of epitope antigens and enhances immunogenicity, but also utilizes the in vitro self-assembly properties of Ferritin to further improve immunogenicity. Experiments show that two subcutaneous immunizations of BALB / c mice with the aforementioned nanoparticles stimulate a strong cellular immune response, inducing 100% immune protection against H1N1 and H3N2 subtypes of swine influenza (SIV). This invention utilizes the Ferritin platform to develop a novel swine influenza nanoparticle vaccine. This vaccine can induce cross-protective immunity against swine influenza and has the potential to become a universal vaccine. Attached Figure Description

[0014] Figure 1 The expression detection results of MH(1+3)F nanoparticles;

[0015] Figure 2 The results are from transmission electron microscopy (TEM) observations of MH(1+3)F nanoparticles.

[0016] Figure 3The particle size distribution of MH(1+3)F nanoparticles is shown.

[0017] Figure 4 For the detection of CD3 by flow cytometry + CD4 + T cells and CD3 + CD8 + T cell level results;

[0018] Figure 5 The results of ELISpot detection of IL-4 and IFN-γ levels secreted by lymphocytes;

[0019] Figure 6 The study included changes in body weight and survival rate in mice after immunization with the virus. Detailed Implementation

[0020] This invention provides a "mosaic"-like fusion protein comprising H1N1-M2e, H3N2-M2e, H1 subtype dominant T cell epitopes (H1H20 and H1H25), H3 subtype dominant T cell epitopes (H3H13 and H3H23), and Ferritin. The recombinant protein obtained by sequentially expressing H1N1-M2e, H3N2-M2e, H1 subtype dominant T cell epitopes (H1H20 and H1H25), and H3 subtype dominant T cell epitopes (H3H13 and H3H23) in tandem is named MH(1+3) fusion protein; the recombinant protein obtained by fusing MH(1+3) with the N-terminus of Ferritin is named MH(1+3)F fusion protein; the preferred amino acid sequence of the MH(1+3)F fusion protein is as shown in SEQ ID. NO:1(SLLTEVETPTRSEWERSRSSGSSDGSGSLLTEVETPIRNGWESKSNDSSDGGGGSNNSTDTVDTILEKNVTVTHSVNLLEGGGGSKSTQTAIDGISNKVNSVIEKGGGGSNGKSSIMRSDAPIGGGGSGIFGAIAGFIENGWEGMVDGWYGGGGGSGGGGSGGG GSDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS).

[0021] In this invention, the preferred amino acid sequence of Ferritin is as shown in SEQ ID NO:2(DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS). The Ferritin nanoparticles in the fusion protein possess virus-like characteristics, a highly ordered and repetitive symmetrical structure, and can be effectively presented by dendritic cells to enhance the immune response. Under in vitro conditions, Ferritin can spontaneously assemble from 24 subunits to form a hollow spherical structure, thereby achieving the polymerization and display of the antigenic epitope fused with Ferritin, and improving immunogenicity.

[0022] This invention provides a nanoparticle based on the assembly of the fusion protein, named MH(1+3)F nanoparticles.

[0023] In this invention, the nanoparticles are approximately spherical particles self-assembled from 24 identical subunits (fusion proteins), with a particle size of 30-40 nm.

[0024] This invention provides a swine influenza vaccine, wherein the antigen comprises the aforementioned nanoparticles.

[0025] In this invention, the final concentration of the antigen is preferably 2 mg / mL, but can be any concentration of 2 mg / mL. This invention does not impose any particular limitation on the preparation method of the swine influenza vaccine; any swine influenza vaccine preparation method well-known in the art can be used.

[0026] In this invention, the preferred immunization method for the swine influenza vaccine is to administer an immunization dose of MH(1+3)F nanoparticles, with each mouse receiving 20 μg of nanoparticles subcutaneously, for a total of two immunizations.

[0027] This invention provides the use of the fusion protein or the nanoparticles in the preparation of vaccines for the prevention and / or control of swine influenza virus infection.

[0028] In this invention, the swine influenza virus preferably includes H1N1 influenza virus and / or H3N2 influenza virus. In the embodiments of this invention, to illustrate that the vaccine prepared according to this invention has cross-protective effects against H1 and H3 subtype virus infections, A / Swine / Shandong / TA27 / 2021 (Han Lebin. Isolation and Identification of Swine Influenza Viruses and Serological Survey of Swine Influenza in Shandong Province, 2020-2021 [D]. Shandong Agricultural University, 2022. DOI:10.27277 / d.cnki.gsdnu.2022.000289.) is used as a representative of the H1N1 influenza virus; and rA / PR / 8-TX98(H3N2) is used as a representative of the H3N2 influenza virus, illustrating that the vaccine has the effect of inducing cellular immune responses in vivo. The rA / PR / 8-TX98(H3N2) virus is based on A / PuertoRico / 8 / 34 (A / PR / 8, GenBank: AAV41245). The HA (SEQ ID NO:3), NA (SEQ ID NO:4), and M (SEQ ID NO:5) genes of A / Swine / Texas / 4199-2 / 1998(H3N2) (GenBank; AEK70348) are used to replace the HA (SEQ ID NO:3), NA (SEQ ID NO:4), and M (SEQ ID NO:5) genes of A / PR / 8, respectively, resulting in the virus rA / PR / 8-TX98(H3N2). Specifically, the replacement method involves co-transfecting a recombinant vector plasmid carrying the HA, NA, and M genes of H3N2 with a recombinant vector plasmid carrying the PB2, PB1, PA, NP, and NS genes of the aforementioned virus backbone into eukaryotic cells for virus rescue, yielding the rA / PR / 8-TX98(H3N2) virus.

[0029] The following detailed description, in conjunction with embodiments, illustrates a "mosaic" type swine influenza nanoparticle vaccine, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of this invention.

[0030] Example 1

[0031] 1. Optimize sequence synthesis

[0032] The H1N1-M2e, H3N2-M2e, H1 subtype dominant T cell epitopes (H1H20 and H1H25), and H3 subtype dominant T cell epitopes (H3H13 and H3H23) were tandemly fused to the N-terminus of Ferritin to form a fusion protein named MH(1+3)F. The gene sequence of the recombinant protein was optimized using E. coli-preferred codons, enabling efficient and soluble expression. The gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0033] 2. Plasmid construction, protein expression and purification

[0034] (1) The synthesized gene was cloned into the pCold vector using HindIII and Xba I restriction sites. The recombinant plasmid was transformed into pGT-F2 competent expression strain, incubated on ice for 30 min, heat-shocked at 42℃ for 1 min, then incubated on ice for 1 min. 500 μL of antibiotic-free LB agar was added, and the mixture was incubated at 37℃ and 220 rpm for 40 min. After incubation, the mixture was centrifuged at 4000 rpm for 1 min. The supernatant was discarded, and the precipitate was plated onto ampicillin-resistant solid LB agar plates and incubated at 37℃ for 12 h.

[0035] (2) Pick a single colony from the plate and add it to 4 mL of ampicillin-resistant LB broth, incubate at 37°C and 220 rpm for 10 h. Add 5 mL of the above bacterial culture to 1 L of ampicillin-resistant LB broth, incubate at 37°C and 220 rpm. When the OD of the bacterial culture... 600 When the concentration reaches 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) is added to a final concentration of 0.6 mM, and expression is induced at 18°C ​​for 18–20 h.

[0036] (3) After induction, centrifuge the bacterial culture at 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial cells in 40 mL TBS buffer (25 mM Tris, 30 mM NaCl, pH 8.0). Sonicate the resuspended bacterial cells at 100% power for 30 min.

[0037] (4) After disruption, the protein was centrifuged at 9500 rpm for 10 min. The supernatant was filtered through a 0.45 μm membrane and purified using Ni-NTA packing material, followed by further purification by size exclusion chromatography (SEC). The purified protein was analyzed by SDS-PAGE. The purified protein buffer was exchanged for Tris buffer (TBS: 25 mM Tris, 150 mM NaCl, pH 8.0) and concentrated using a 100 kDa centrifuge filter. The protein concentration was determined by the BCA assay.

[0038] 3. Characterization of nanoparticles

[0039] The morphology of nanoparticles was observed using TEM. The purified protein was diluted to 0.2 mg / mL in TBS, and 10 μL of the sample was added to a carbon-coated copper grid. After adsorption for 5 min, the sample was negatively stained with 10 μL of 2% phosphotungstic acid (pH 7.0) for 5 min. Excess liquid was blotted away with filter paper, and after drying, the nanoparticle structure was observed at an accelerating voltage of 80 kV. The particle size distribution was analyzed at 25 °C using a Zetasizer Nano ZS ZEN 3600 nanoparticle size potentiometry instrument. The purified protein sample was added to a quartz cuvette and analyzed at a fixed scattering angle of 90°.

[0040] 4. Mouse Immunization

[0041] To evaluate the protective effect of MH(1+3)F nanoparticles against swine influenza virus infection in mice, 6-8 week old BALB / c mice were randomly selected and divided into 8 groups of 10 mice each, as shown in the table below. Each mouse was immunized subcutaneously with 20 μg of nanoparticles (or PBS) twice.

[0042] Table 1. Immunogen information for each experimental group

[0043]

[0044] 5. Cellular Immunoassay

[0045] 5.1 T lymphocyte detection

[0046] Fourteen days after the last immunization, splenic lymphocytes were isolated for analysis. Splenic lymphocytes from immunized mice were obtained using a mouse spleen lymphocyte isolation kit. The mouse spleen lymphocytes were resuspended in cell staining buffer, and the cell count was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells were collected at a concentration of 1000 μL and incubated on ice for 20 min with anti-mouse CD16 / 32 antibody. Then, FITC-labeled anti-mouse CD3 antibody, PE-labeled anti-mouse CD4 antibody, and APC-labeled anti-mouse CD8 antibody were added. After incubation on ice in the dark for 20 min, the cells were centrifuged at 350 g for 5 min, the supernatant was discarded, and the cells were washed twice with cell staining buffer. The cells were then resuspended in 500 μL of cell staining buffer and analyzed by flow cytometry fluorescence sorting.

[0047] 5.2 Enzyme-linked spot immunoassay (ELISpot)

[0048] Antigen-specific spleen cells from immunized BALB / c mice were detected using a mouse IFN-γ and IL-4 ELISpot kit. Fourteen days after the last immunization, three mice from each group were randomly selected, and spleens were collected for ELISpot detection. Isolated splenic lymphocytes (3 × 10⁶ cells per well) were... 5 Cells were added to pre-coated and activated ELISpot plates and stimulated for 24 h with peptides of H1N1-M2e, H3N2-M2e, H1H20, H1H25, H3H13, and H3H23 (10 μg / mL). Cells were lysed with ice-cold deionized water, the plates were washed, and then incubated sequentially with biotin-labeled antibody, HRP-conjugated streptavidin, and 3-amino-9-ethylcarbazole (AEC) chromogenic solution. The reaction was terminated by washing with double-distilled water, and spots were counted using an ImmunoSpot ELISA reader.

[0049] 6.rA / PR / 8-TX98(H3N2) virus rescue

[0050] 6.1 Seed 293T cells in good growth condition into poly-L-lysine-coated 6-well cell culture plates at a density of 70%–80%;

[0051] 6.2 The five genes PB2, PB1, PA, NP, and NS from A / Puerto Rico / 8 / 34 (A / PR / 8) were constructed into the pBD vector and named pBD-PB2, pBD-PB1, pBD-PA, pBD-NP, and pBD-NS, respectively; the three genes HA, NA, and M from A / Swine / Texas / 4199-2 / 1998 (H3N2) were constructed into the pBD vector and named pBD-HA, pBD-NA, and pBD-M, respectively; the concentration of the eight recombinant plasmids constructed above was adjusted to 0.5 μg / μL.

[0052] 6.3 Take two sterile 1.5 mL EP tubes and add 250 μL of serum-free culture medium (OPTI-MEM) to each tube. Add 1 μL of 8 plasmids to one tube and mix thoroughly by pipetting. Add 10 μL of liposomes to the other tube, mix thoroughly by pipetting, and then mix with the OPTI-MEM containing plasmids, for a total of 510 μL. Let stand at room temperature for 25 min.

[0053] 6.4 During the static incubation period, wash the 6-well plate with cells three times with PBS, add 1.5 mL of OPTI-MEM medium, and then evenly drop 510 μL of the well-mixed mixture onto the cells.

[0054] 6.5 After culturing in a 37℃, 5% CO2 incubator for 6-8 hours, change the medium, aspirate the supernatant, add 2 mL of OPTI-MEM (for weak toxicity, add TPCK trypsin to a final concentration of 0.5 μg / mL) and continue culturing for 48-72 hours;

[0055] 6.6 Collect the supernatant and cells, mix thoroughly, and inoculate into 9-10 day old SPF chicken embryos, 400 μL per embryo;

[0056] 6.7 If the chicken embryo allantoic fluid exhibits hemagglutination activity, the virus rescue is successful; the hemagglutination titer collected should be greater than 1:2. 6 Furthermore, the highest quality chicken embryo allantoic fluid is aliquoted and stored at -80℃.

[0057] 7. Mouse challenge protection test

[0058] Two weeks after the last immunization, mice were infected nasally with A / Swine / Shandong / TA27 / 2021(H1N1) at an infectious dose of 10. 6 EID 50 / 50μL; rA / PR / 8-TX98(H3N2), infectious dose is 10 7.5 EID 50 / 50μL. Weigh the mice daily and record changes in body weight and survival rate for 14 days. Euthanize the mice when their body weight drops by ≥25%.

[0059] result

[0060] 1. Protein Expression and Purification

[0061] The constructed plasmid was transformed into pGT-F2 competent cells, and single clones were selected for expansion culture for prokaryotic expression. The supernatant from the lysed bacterial cells was filtered through a 0.45 μm membrane and purified using a Ni-NTA column with imidazole elution. The purified protein was analyzed by SDS-PAGE. The SDS-PAGE results showed that the target protein (e.g., ...) was successfully purified. Figure 1 (As shown). The purified protein buffer was exchanged for Tris buffer (TBS: 25 mM Tris, 150 mM NaCl, pH 8.0), and the protein was concentrated using a 100 kDa centrifugal filter.

[0062] 2 Nanoparticle Characterization

[0063] This study used TEM to observe the structure of nanoparticles. The results show that MH(1+3)F monomers can form uniformly sized particles, similar to Ferritin monomers (e.g., ...). Figure 2 (As shown).

[0064] This study used a nanoparticle size potentiometer to determine the particle size of nanoparticles. The results showed that, compared to Ferritin, MH(1+3)F nanoparticles exhibited increased particle size due to the introduction of epitopes (e.g., ...). Figure 3 (As shown).

[0065] 3 nanoparticles induce a strong cellular immune response

[0066] 3.1 Flow cytometry detection of CD3 + CD4 + T cells and CD3 + CD8 + T cell levels

[0067] Two weeks after the last immunization, CD3 levels in spleen lymphocytes were detected by flow cytometry. + CD4 + T cells and CD3 + CD8 + The percentage of T cells. Results as follows: Figure 4 As shown, CD3 in experimental groups 7 and 8 of the immunoimmune MH(1+3)F nanoparticles + CD4 + T cells and CD3 + CD8 + The level of T cells was significantly higher than that of other experimental groups.

[0068] 3.2 ELISpot detection of lymphocyte levels secreting IL-4 and IFN-γ

[0069] Two weeks after the final immunization, the number of cytokines secreted by splenic lymphocytes in mice was measured using the ELISpot method. Results are as follows: Figure 5 As shown, the spleens of mice in experimental groups 7 and 8, which were immunized with MH(1+3)F nanoparticles, contained a large number of lymphocytes that secreted IL-4 and IFN-γ.

[0070] Immunoprotective assay of 4MH(1+3)F in mice

[0071] 4.1 Evaluation of the in vivo protective effect of MH(1+3)F

[0072] Two weeks after the final immunization, mice were challenged with A / Swine / Shandong / TA27 / 2021 (H1N1) and rA / PR / 8-TX98 (H3N2). Mouse body weight and survival rate were monitored for 14 consecutive days post-challenge. The results of body weight and survival rate are as follows: Figure 6As shown, on day 14, mice in experimental group 7 lost approximately 5% of their body weight, and mice in experimental group 8 lost approximately 10% of their body weight. Both groups experienced a weight loss of less than 25%, while mice in other experimental groups experienced a weight loss of more than 25% on day 8. Furthermore, all mice in experimental groups 7 and 8 survived after being challenged with lethal doses of H1N1 and H3N2, while all mice in the other experimental groups died within 8 days of challenge.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein is formed by fusing M2e from H1N1 influenza virus, M2e from H3N2 influenza virus, dominant epitope from H1N1 influenza virus, dominant epitope from H3N2 influenza virus, and ferritin; the amino acid sequence of the fusion protein is shown in SEQ ID NO:

1.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the ferritin is shown in SEQ ID NO:

2.

3. A nanoparticle assembled based on the fusion protein of claim 1 or 2.

4. A swine influenza vaccine, characterized in that, The antigen includes the fusion protein of claim 1 or 2 or the nanoparticle of claim 3.

5. The swine influenza vaccine according to claim 4, characterized in that, The concentration of the antigen is 2 mg / mL.

6. The use of the fusion protein of claim 1 or 2 or the nanoparticle of claim 3 in the preparation of a vaccine for the prevention and / or control of swine influenza virus infection, wherein the swine influenza virus is an H1N1 influenza virus and / or an H3N2 influenza virus.

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