Mosaic swine influenza nanoparticle vaccine as well as preparation method and application thereof

Abstract

The invention provides a mosaic type swine influenza nanoparticle vaccine as well as a preparation method and application thereof, and belongs to the technical field of biological products. The fusion protein provided by the invention is formed by fusion of M2e and HA dominant epitopes derived from H1N1 and H3N2 influenza viruses and ferritin. Nanoparticles are formed based on the fusion protein through self-assembly, strong cellular immune response can be induced in vivo after mice are immunized, 100% protection is provided for H1N1 and H3N2 swine influenza viruses, and the fusion protein has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and specifically relates to a "mosaic" type swine influenza nanoparticle vaccine and a preparation method and application thereof. Background Art

[0002] Swine influenza (SI) is an acute, contact-related respiratory disease of pigs caused by the swine influenza virus (SIV). SIV infection can lead to decreased production performance in pigs and susceptibility to secondary infection with other pathogens, further compromising pig health and causing severe economic losses. Because porcine respiratory epithelial cells express both sialic acid α-2,3-galactose and α-2,6-galactose receptors, pigs are considered "mixers" of influenza viruses, promoting genetic recombination between human and avian influenza viruses, resulting in the generation of novel influenza viruses that pose a risk of infection to humans and poultry, posing a significant threat to the livestock industry and public health.

[0003] Vaccination remains the most cost-effective method for preventing and controlling influenza (SI). Although inactivated whole-virus vaccines for H1 and H3 subtype SI have been widely used for swine influenza prevention and control, they still have shortcomings. First, inactivated vaccines primarily activate humoral immunity, resulting in short-lived immunity. Second, antigenic drift and shift in influenza viruses often result in mismatches between circulating and vaccine strains, leading to reduced vaccine efficacy. Therefore, the development of a new, broad-spectrum swine influenza vaccine is urgent.

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

[0005] The present invention provides a fusion protein, which connects M2e, dominant T cell epitopes (H1H20, H1H25) derived from H1N1 influenza virus, and dominant T cell epitopes (H3H13, H3H23) derived from H3N2 influenza virus in series with ferritin to form a fusion protein, in order to effectively improve the immunogenicity and broad-spectrum protection effect of the vaccine.

[0006] The present invention provides a fusion protein, comprising M2e (H1N1-M2e) derived from an H1N1 influenza virus, M2e (H3N2-M2e) derived from an H3N2 influenza virus, a dominant T cell epitope derived from an H1N1 influenza virus, a dominant T cell epitope derived from an H3N2 influenza virus, and ferritin; 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] The present invention provides a nanoparticle assembled based on the fusion protein.

[0009] The present invention provides a swine influenza vaccine, wherein the antigen comprises the fusion protein or the nanoparticle.

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

[0011] The present invention provides use of the fusion protein or the nanoparticle in preparing a vaccine for preventing and / or controlling swine influenza virus infection.

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

[0013] The present invention provides a "mosaic" nanoparticle formed by self-assembly after fusion expression of M2e derived from H1N1 and H3N2 influenza viruses, a dominant T cell epitope derived from H1N1 influenza virus, a dominant T cell epitope derived from H3N2 influenza virus, and ferritin. This not only increases the number of epitope antigens and improves immunogenicity, but also utilizes ferritin's in vitro self-assembly properties to further enhance immunogenicity. Experiments have shown that two subcutaneous immunizations of BALB / c mice with the nanoparticles stimulated a strong cellular immune response and induced 100% protection against both H1N1 and H3N2 subtypes of SIV. The present 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the expression detection result of MH(1+3)F nanoparticles;

[0015] Figure 2 Transmission electron microscopy (TEM) observation results of MH(1+3)F nanoparticles;

[0016] Figure 3is the particle size test result of MH(1+3)F nanoparticles;

[0017] Figure 4 Flow cytometry for CD3 + CD4 + T cells and CD3 + CD8 + T cell level results;

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

[0019] Figure 6 The weight changes and survival rates of mice after immunization and challenge. DETAILED DESCRIPTION

[0020] The present invention provides a "mosaic" fusion protein, including 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 series is named MH(1+3) fusion protein; the recombinant protein obtained by fusion expression of MH(1+3) with the N-terminus of Ferritin is named MH(1+3)F fusion protein; the amino acid sequence of the MH(1+3)F fusion protein is preferably as shown in SEQ ID NO:1(SLLTEVETPTRSEWERSRSSGSSDGSGSLLTEVETPIRNGWESKSNDSSDGGGGSNNSTDTVDTILEKNVTVTHSVNLLEGGGGSKSTQTAIDGISNKVNSVIEKGGGGSNGKSSIMRSDAPIGGGGSGIFGAIAGFIENGWEGMVDGWYGGGGGSGGGGSGGG GSDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS).

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

[0022] The present invention provides a nanoparticle formed by assembling the fusion protein, which is named as MH(1+3)F nanoparticle.

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

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

[0025] In the present invention, the final concentration of the antigen is preferably 2 mg / mL, and can be 2 mg / mL. The present invention has no particular limitation on the preparation method of the swine influenza vaccine, and any preparation method of swine influenza vaccine known in the art can be used.

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

[0027] The present invention provides use of the fusion protein or the nanoparticle in preparing a vaccine for preventing and / or controlling swine influenza virus infection.

[0028] In the present invention, the swine influenza virus preferably includes an H1N1 influenza virus and / or an H3N2 influenza virus. In the embodiments of the present invention, in order to illustrate that the vaccine prepared by the present invention has a cross-protective effect against H1 subtype and H3 subtype virus infection, A / Swine / Shandong / TA27 / 2021 (Han Lebin. Isolation and Identification of Swine Influenza Viruses and Swine Influenza Serological Survey in Shandong Province from 2020 to 2021 [D]. Shandong Agricultural University, 2022.DOI:10.27277 / d.cnki.gsdnu.2022.000289.) is used as a representative of the H1N1 influenza virus; rA / PR / 8-TX98 (H3N2) is used as a representative of the H3N2 influenza virus to illustrate that the vaccine has the effect of inducing a cellular immune response in vivo. The rA / PR / 8-TX98 (H3N2) virus is based on A / Puerto Rico / 8 / 34 (A / PR / 8, GenBank: AAV41245) as a backbone virus, and 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, NA and M genes of A / PR / 8, respectively, and is named rA / PR / 8-TX98 (H3N2). The replacement method specifically comprises co-transfecting a recombinant vector plasmid carrying the HA gene, NA gene and M gene of H3N2 with a recombinant vector plasmid carrying the PB2 gene, PB1 gene, PA gene, NP gene and NS gene of the backbone virus into eukaryotic cells for virus rescue to obtain the rA / PR / 8-TX98 (H3N2) virus.

[0029] The following describes in detail a "mosaic" type swine influenza nanoparticle vaccine provided by the present invention, its preparation method and application, in conjunction with the examples. However, they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] 1. Optimize sequence synthesis

[0032] H1N1-M2e, H3N2-M2e, H1 subtype dominant T cell epitopes (H1H20 and H1H25), and H3 subtype dominant T cell epitopes (H3H13 and H3H23) were fused in series to the N-terminus of ferritin. The resulting fusion protein was named MH(1+3)F. The gene sequence of the recombinant protein was optimized using codons preferred by Escherichia coli, 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 the HindIII and XbaI restriction sites. The recombinant plasmid was transformed into the pGT-F2 competent expression strain, incubated on ice for 30 minutes, heat-shocked at 42°C for 1 minute, and then incubated on ice for another 1 minute. After that, 500 μL of antibiotic-free LB was added and the cells were incubated at 37°C and 220 rpm for 40 minutes. After incubation, the cells were centrifuged at 4000 rpm for 1 minute. The supernatant was discarded, and the precipitate was plated onto an ampicillin-resistant solid LB plate and incubated at 37°C for 12 hours.

[0035] (2) Pick a single colony from the plate and add it to 4 mL of LB liquid containing ampicillin resistance, and culture it at 37°C, 220 rpm for 10 h. Take 5 mL of the above bacterial solution and add it to 1 L of LB liquid containing ampicillin resistance, and culture it at 37°C, 220 rpm. When the OD of the bacterial culture reaches 600 When the pH value reached 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM, and the expression was induced at 18°C ​​for 18-20 h.

[0036] (3) After induction, the bacterial solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 40 mL of TBS buffer (25 mM Tris, 30 mM NaCl, pH 8.0). The resuspended cells were sonicated at 100% power for 30 min.

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

[0038] 3. Characterization of Nanoparticles

[0039] The morphology of the 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 minutes, the sample was negatively stained with 10 μL of 2% phosphotungstic acid (pH 7.0) for 5 minutes. The excess liquid was removed using 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 ZetasizerNano ZS ZEN 3600 nanoparticle size potentiometer. 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 divided into eight groups of 10 mice each, as shown in the table below. Each mouse was subcutaneously immunized with 20 μg of nanoparticles (or PBS) twice.

[0042] Table 1 Immune antigen information of each experimental group

[0043]

[0044] 5. Cellular Immunity Detection

[0045] 5.1 T lymphocyte detection

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

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

[0048] Antigen-specific splenocytes from immunized BALB / c mice were detected using mouse IFN-γ and IL-4 ELISpot kits. 14 days after the last immunization, three mice were randomly selected from each group and spleens were harvested for ELISpot analysis. Isolated spleen lymphocytes (3 × 10 cells / well) were isolated and then immunized with IFN-γ and IL-4 ELISpot kits. 5 Cells (100 cells) were added to pre-coated and activated ELISpot plates and stimulated with H1N1-M2e, H3N2-M2e, H1H20, H1H25, H3H13, and H3H23 peptides (10 μg / mL) for 24 hours. After lysing the cells with ice-cold deionized water, the plates were washed and then incubated sequentially with biotinylated antibodies, HRP-conjugated streptavidin, and 3-amino-9-ethylcarbazole (AEC) color development solution. The reaction was terminated by washing with double-distilled water, and spots were counted using an ImmunoSpot enzyme-linked immunosorbent assay (ELISpot).

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

[0050] 6.1 Seed the well-grown 293T cells at a density of 70% to 80% into a 6-well poly-lysine-coated cell culture plate;

[0051] 6.2 The PB2, PB1, PA, NP, and NS genes of 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 HA, NA, and M genes of 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 medium (OPTI-MEM) to each. Add 1 μL of each of the eight plasmids to one tube and mix thoroughly by pipetting. Add 10 μL of liposomes to the other tube and mix thoroughly by pipetting. Then mix with the OPTI-MEM containing the plasmids, for a total of 510 μL. Let stand at room temperature for 25 minutes.

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

[0054] 6.5 Incubate in a 37°C, 5% CO2 incubator for 6-8 hours, then change the medium. Aspirate the supernatant and add 2 mL of OPTI-MEM (to reduce toxicity, add TPCK trypsin at a final concentration of 0.5 μg / mL) and continue incubation for 48-72 hours.

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

[0056] 6.7 If the chicken embryo allantoic fluid has hemagglutination, the virus is successfully rescued. The hemagglutination titer is greater than 1:2. 6 , and the highest chicken embryo allantoic fluid was packaged and stored at -80℃.

[0057] 7. Mouse challenge protection test

[0058] Two weeks after the last immunization, mice were infected with A / Swine / Shandong / TA27 / 2021(H1N1) via the nasal cavity with an infection dose of 10 6 EID 50 / 50μL; rA / PR / 8-TX98(H3N2), infectious dose is 10 7.5 EID 50 Weigh mice daily, and record weight changes and survival rates up to day 14. Euthanize mice when their body weight decreases ≥ 25%.

[0059] result

[0060] 1 Protein expression and purification

[0061] The constructed plasmid was transformed into pGT-F2 competent cells, and a single clone was picked and expanded for prokaryotic expression. The supernatant of the broken cells was filtered through a 0.45 μm membrane, purified on a Ni-NTA column, and eluted with imidazole. The purified protein was analyzed by SDS-PAGE. The SDS-PAGE results showed that the target protein (such as Figure 1 The purified protein was buffer-exchanged into Tris buffer (TBS: 25 mM Tris, 150 mM NaCl, pH 8.0) and concentrated using a 100 kDa centrifugal filter.

[0062] 2 Nanoparticle characterization

[0063] This study used TEM to observe the structure of nanoparticles. The results showed that MH(1+3)F monomers can form particles of uniform size (such as Figure 2 shown).

[0064] In this study, the nanoparticle size was measured by nanometer potential instrument. The results showed that compared with Ferritin, the particle size of MH(1+3)F nanoparticles increased due to the introduction of epitopes (e.g. Figure 3 shown).

[0065] 3 Nanoparticles induce 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 in spleen lymphocytes was detected by flow cytometry. + CD4 + T cells and CD3 + CD8 + The percentage of T cells. Figure 4 As shown, CD3 + CD4 + T cells and CD3 + CD8 + The T cell level was significantly higher than that in other experimental groups.

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

[0069] Two weeks after the last immunization, the ELISpot method was used to detect the number of cytokines secreted by spleen lymphocytes after immunization of mice. Figure 5 As shown, there were a large number of IL-4 and IFN-γ secreting lymphocytes in the spleens of mice in experimental groups 7 and 8 immunized with MH(1+3)F nanoparticles.

[0070] Immunoprotective test of 4MH(1+3)F on mice

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

[0072] Two weeks after the last immunization, mice were challenged with A / Swine / Shandong / TA27 / 2021 (H1N1) and rA / PR / 8-TX98 (H3N2). The body weight and survival rate of the mice were monitored for 14 consecutive days after the challenge. The results of body weight and survival rate are shown in Figure 2. Figure 6As shown, on day 14, the weight of mice in experimental group 7 decreased by approximately 5%, and the weight of mice in experimental group 8 decreased by approximately 10%. The weight loss in both groups was less than 25%, while the weight loss of mice in the other experimental groups exceeded 25% on day 8. At the same time, all mice in experimental groups 7 and 8 survived the lethal dose of H1N1 and H3N2 virus challenge, while all mice in the other experimental groups died within 8 days after the challenge.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises M2e derived from an H1N1 influenza virus, M2e derived from an H3N2 influenza virus, a dominant epitope derived from an H1N1 influenza virus, a dominant epitope derived from an 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 ferritin is shown in SEQ ID NO:

2.

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

4. A swine flu vaccine, characterized in that The antigen comprises the fusion protein according to claim 1 or 2 or the nanoparticle according to claim 3.

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

6. Use of the fusion protein according to claim 1 or 2 or the nanoparticle according to claim 3 in the preparation of a vaccine for preventing and / or controlling swine influenza virus infection.

7. The application according to claim 6, characterized in that The swine influenza virus includes H1N1 influenza virus and / or H3N2 influenza virus.

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