Broad-spectrum influenza vaccine antigen fragments, recombinant probiotics and their applications

Abstract

The present invention discloses a broad-spectrum influenza vaccine antigen fragment, recombinant probiotics and applications. The present invention utilizes the high conservation of the M2e sequence in different subtypes of influenza viruses and is expected to develop into a broad-spectrum influenza vaccine with cross-protection efficacy. The probiotic Escherichia coli EcN is used to express and secrete the 5M2e antigen. The vaccine preparation process does not involve live viruses. Compared with the traditional chicken embryo influenza vaccine preparation method, the operation is safer and simpler, and it is suitable for rapid large-scale production. The vaccine of the present invention can be immunized by nasal drops. Compared with the commonly used intramuscular injection immunization route, it is safer and simpler to use, and is closer to the natural invasion route of influenza viruses into the host, which is conducive to inducing mucosal immune responses.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to a broad-spectrum influenza vaccine antigen fragment, a recombinant probiotic and applications thereof. Background Art

[0002] Influenza, an acute respiratory infectious disease caused by the influenza virus, poses a serious threat to human health. In recent years, influenza viruses have caused approximately 1 billion cases annually worldwide, of which 3-5 million cases develop into severe cases, ultimately resulting in an estimated 290,000-650,000 deaths. Furthermore, the global COVID-19 pandemic has not yet been completely eradicated, and the peak influenza seasons of spring and autumn may see overlapping epidemics with respiratory infections like COVID-19. The prevention and control of influenza remains a major public health challenge, both now and in the future.

[0003] Currently, vaccination is the most effective way to prevent influenza. The influenza vaccines in use are mainly inactivated, attenuated or recombinant trivalent and quadrivalent influenza vaccines, which improve the body's ability to resist influenza virus infection by inducing the production of specific antibodies against the main influenza virus antigens HA or NA. Due to the continuous antigenic drift of HA and NA of influenza viruses and the recombination between different influenza virus subtypes, the vaccine needs to be updated every year. During seasonal influenza epidemics or influenza outbreaks, it is difficult to produce sufficient vaccines in a timely manner. In addition, the cross-species transmission of influenza viruses from animal hosts, such as avian influenza viruses H5N1 and H7N9 and swine influenza viruses H1N1, makes the development of a broad-spectrum influenza vaccine with broad protective efficacy urgent.

[0004] Current research on developing broad-spectrum influenza vaccines is primarily focused on two conserved epitopes of influenza A viruses: the extracellular functional domain of matrix protein 2 (M2e) and the conserved hemagglutinin stalk. M2 is a nonglycosylated homotetrameric transmembrane protein that functions as a proton channel in influenza A and B viruses. In influenza A viruses, the M2 protein consists of three functional regions: an extracellular N-terminal region (M2e, amino acid residues 1–23), an intermediate transmembrane helix, and an intracellular C-terminal region. Compared to HA and NA, M2e is the region where the M2 antigenic determinants are concentrated. This region is highly conserved among influenza A viruses from diverse host species, including humans, avian, and swine, making it a potential antigenic target for the development of broad-spectrum influenza vaccines.

[0005] Developing a broad-spectrum influenza vaccine that effectively protects against different influenza virus subtypes and generates diverse immune responses remains a major challenge. The probiotic Escherichia coli Nissle 1917 (EcN), a highly biosafe probiotic that acts as a "living factory" for biomacromolecules, has demonstrated excellent potential as a novel mucosal immune carrier in previous studies. EcN, isolated over a century ago, boasts well-characterized genetics, ease of genetic manipulation, ease of culture expansion, and a short growth cycle, and has been in clinical use for decades. The presence of multiple microbial-associated pattern molecules and curli pili on its surface potently promotes its persistent colonization and adhesion to the mucosa. Furthermore, EcN can nonspecifically stimulate the innate immune system, enhancing the host immune system's ability to fight viral infections. Studies have shown that using EcN as a mucosal delivery vehicle to express pollen factors can prevent multiple allergic reactions through nasal immunization. Summary of the Invention

[0006] The purpose of the present invention is to achieve effective expression of influenza virus antigens in prokaryotes, provide an antigen fragment design for a broad-spectrum influenza vaccine, and construct and apply a recombinant probiotic strain, thereby achieving effective exogenous secretion of influenza virus antigens by the recombinant probiotic strain and effective protection against influenza virus infection in mice, and developing a new broad-spectrum influenza vaccine.

[0007] To achieve the above object, the present invention provides an antigenic fragment of a broad-spectrum influenza vaccine, wherein the antigenic fragment is composed of a 5M2e antigen and a secretory system located thereafter; or the antigenic fragment is composed of a 5M2e antigen and a signal peptide located thereafter;

[0008] The 5M2e antigen is obtained by connecting the M2e antigen sequences of H1N1, H2N2, H3N2, H5N1 and H7N9 subtype influenza viruses in random order;

[0009] The secretion system is used to secrete the antigen fragment outside the cell;

[0010] The signal peptide is used to guide the antigen fragment to the periplasmic space for secretory expression or directly secrete expression outside the cell.

[0011] The present invention provides a concatenation and optimization strategy for the major conserved immunogen M2e of influenza viruses: amino acid conservation analysis is performed on M2e sequences from H1N1, H2N2, H3N2, H5N1, and H7N9 subtype influenza viruses, site modifications are performed, and 5M2e antigen sequences are formed through codon optimization and concatenation. The amino acid sequences of M2e from different subtypes of influenza viruses are connected using liners (GGGGS) (3-10). The amino acid sequence encoding the 5M2e antigen is shown in SEQ ID NO. 1.

[0012] Preferably, the secretion system is a prokaryotic secretion system, optionally a type III secretion system or a type VI secretion system.

[0013] More preferably, the secretion system is the HlyABD secretion system. The efficient expression and exogenous secretion of the 5M2e antigen in EcN is achieved through the type I secretion system HlyABD secretion system of Escherichia coli, wherein the amino acid sequence of HlyA in the HlyABD secretion system is preferably as shown in SEQ ID No. 4, and the amino acid sequence of HlyBD is preferably as shown in SEQ ID No. 5.

[0014] Preferably, the signal peptide is selected from PeIB, PhoA, OmpA, OmpC, OmpF, InPNC, YebF.

[0015] The present invention also discloses a nucleic acid sequence encoding the antigen fragment of the broad-spectrum influenza vaccine, comprising a promoter as shown in SEQ ID No. 2 and a strong terminator as shown in SEQ ID No. 3.

[0016] The present invention also discloses a recombinant probiotic strain integrated with the antigen fragment of the broad-spectrum influenza vaccine.

[0017] Recombinant probiotic strains are based on the transformation of probiotics in the present invention, and the selection of probiotics is determined according to the probiotic colonization situation in the subject population. Commonly used probiotics can be Escherichia coli (Escherichia coli), Lactococcus lactis (Lactococcus lactis), Lactobacillus casei (Lactobacillus casei), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Bifidobacterium bifidum (Bifidobacterium bifidum), Bacillus coagulans (Bacillus coagulans), Streptococcus thermophilus (Streptococcus thermophilus) etc. In the present invention, preferred probiotic Escherichia coli Nissle 1917 (EcN).

[0018] The present invention also provides a recombinant Escherichia coli for a broad-spectrum influenza vaccine, which has a deposit number of CCTCC NO. M20232245 and is named probiotic EcN-5M2e. It was deposited at the China Center for Type Culture Collection (Wuhan, China) on November 16, 2023, and its deposit number is CCTCC NO. M20232245. The recombinant Escherichia coli is EcN carrying the influenza virus antigen 5M2e and the HlyABD secretion system, and uses an artificially optimized promoter P1 (SEQ ID No. 2) and a strong terminator rrnBT (SEQ ID No. 3) to initiate and terminate transcription, respectively.

[0019] The present invention also provides the use of the above-mentioned recombinant Escherichia coli in the preparation of a broad-spectrum influenza vaccine, wherein the effective dose of the preparation is 1×10 8 CFU count. Three rounds of immunization were performed, with each round of nasal immunization lasting two consecutive days. Combination vaccines prepared using the EcN-5M2e recombinant probiotic strain of the present invention in combination with other influenza virus vaccine active ingredients were also effective. The broad-spectrum influenza vaccine of the present invention has high biosafety, is easy to mass-produce, and has a long effective period, allowing for large-scale stockpiling. Immunization of mice with the EcN-5M2e broad-spectrum influenza vaccine of the present invention not only induced a strong humoral immune response, but also effectively activated the mouse lung mucosal immune response and fully protected the immunized mice against high-lethal doses of the virus, effectively preventing influenza A H1N1 and H3N2 viruses. Combination vaccines prepared using the EcN-5M2e recombinant probiotic strain of the present invention with other influenza vaccine active ingredients also effectively protected against lethal doses of other influenza virus subtypes.

[0020] The present invention also provides the use of the above-mentioned recombinant Escherichia coli in the preparation of a broad-spectrum influenza vaccine nasal spray, wherein the effective dose of the preparation is 1×10 8 CFU bacterial count; the nasal spray is selected from aerosol, spray and powder spray.

[0021] In summary, the present invention is directed to the design of a broad-spectrum influenza vaccine, utilizing probiotic Escherichia coli to effectively present an optimized 5M2e antigen. The broad-spectrum vaccine prepared from this recombinant probiotic strain, EcN-5M2e, is easy to produce on a large scale, has a long effective period, and can be stockpiled in large quantities. This overcomes the shortcomings of traditional influenza vaccine development and production technology routes, thereby achieving timely and effective prevention and control of epidemics caused by various influenza viruses.

[0022] The beneficial effects of the present invention are:

[0023] 1) The present invention utilizes probiotic Escherichia coli EcN to express and secrete the 5M2e antigen. The vaccine preparation process does not involve live viruses. Compared with the traditional chicken embryo influenza vaccine preparation method, the operation is safer and simpler, and is suitable for rapid large-scale production.

[0024] 2) Using probiotic E. coli E. coli (EcN) as an antigen delivery system offers high biosafety, and its flagella and lipopolysaccharide components act as adjuvants, significantly enhancing the immunogenicity of the M2e antigen. The use of recombinant live bacteria vaccines eliminates the need for additional adjuvants, simplifying vaccine preparation.

[0025] 3) The vaccine of the present invention is administered via intranasal drops, which is safer and more convenient than the commonly used intramuscular injection route. It is also closer to the natural invasion pathway of influenza viruses and is more conducive to inducing mucosal immune responses.

[0026] 4) The present invention utilizes the high conservation of M2e sequences among different subtypes of influenza viruses and is expected to be developed into a broad-spectrum influenza vaccine with cross-protective efficacy.

[0027] 5) Intranasal immunization of BALB / c mice with EcN-5M2e induced the production of M2e-specific IgG and IgA antibodies. The recombinant probiotic strain EcN-5M2e protected immunized mice against lethal infection with both homotypic and heterotypic influenza viruses. Probiotic expression and delivery of conserved viral antigenic peptides provides a novel strategy for the development of novel vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram for the construction of 5M2e-HlyABD.

[0029] Figure 2 Western blot identification of exogenously secreted 5M2e antigen by the recombinant probiotic strain EcN-5M2e.

[0030] Figure 3 This is a statistical chart showing the changes in M2e-specific IgG antibody titers in immune serum.

[0031] Figure 4 This is a statistical chart showing the changes in M2e-specific IgA antibody titers in immune lung wash fluid.

[0032] Figure 5 This is a test chart of the binding ability of serum IgG antibodies after three immunizations to synthetic peptides of M2e of different subtypes of influenza viruses.

[0033] Figure 6 This is a statistical chart of the weight changes of mice after infection with A / Puerto Rico / 8 / 34 (H1N1) influenza virus.

[0034] Figure 7 This is a statistical graph showing the survival rate of mice after infection with A / Puerto Rico / 8 / 34 (H1N1) influenza virus.

[0035] Figure 8This is a statistical chart of the weight changes of mice after infection with A / Hong Kong / 4801 / 14 (H3N2) influenza virus.

[0036] Figure 9 This is a statistical chart of mouse survival rate after infection with A / Hong Kong / 4801 / 14 (H3N2) influenza virus. DETAILED DESCRIPTION

[0037] The present invention is described in detail below in conjunction with the embodiments and drawings. The following embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.

[0038] The reagents, antibodies, enzymes, culture media, antibiotics, and other chemical materials used in the following examples are all commercially available. Some commonly used biomaterials, such as competent cells and vectors, are also commercially available. Some synthetic biomaterials, such as synthetic peptides of influenza virus M2e antigen, which require artificial synthesis, were commissioned to synthesis companies.

[0039] Example 1: Preparation method of EcN-5M2e recombinant probiotic strain

[0040] The steps are:

[0041] Design of 5M2e antigen fragment: The M2e sequences from H1N1, H2N2, H3N2, H5N1 and H7N9 subtype influenza viruses were compared and analyzed. The sites were modified, and the 5M2e antigen fragment was formed through codon optimization and concatenation. The GGGGS linker was introduced in the middle. It was then synthesized through gene recombination technology. The correctness of the 5M2e antigen fragment was confirmed by PCR and sequencing methods. The construction diagram is shown below. Figure 1 Among them, the bold sequence of the 5M2e antigen fragment is the GGGGS linker, and the amino acid sequence of the 5M2e antigen fragment is shown in SEQ ID No. 1.

[0042] Construction of EcN-5M2e recombinant probiotic strain: In this embodiment, the pKT-HlyABD-5M2e expression plasmid was constructed, using pKT100 (Hu, Y., et al. (2011). "Cra negatively regulates acid survival in Yersiniapseudotuberculosis." FEMS Microbiol Lett 317(2):190-195.) as the starting plasmid, replacing the Cm promoter with the artificially modified constitutive promoter P1 (SEQ ID No. 2), and introducing the influenza virus antigen 5M2e encoding gene into the type I secretion system HlyABD of Escherichia coli. After being inserted into the constitutive promoter P1, the recombinant vector pKT-5M2e-HlyABD was obtained. Escherichia coli DH5a was transformed with CaCl2, and a single clone was picked to extract the plasmid, and the sequence and insertion position were verified to be correct by sequencing. After obtaining the recombinant plasmid, it was electroporated into EcN using an Eppendorf Electroporator 2510 to obtain the recombinant probiotic strain EcN-5M2e. The construction diagram is shown in the figure. Figure 1 As shown. The corresponding sequences involved above are as follows:

[0043] The amino acid sequence of HlyA in the HlyABD type I secretion system is shown in SEQ ID No.4.

[0044] The amino acid sequence of HlyBD is shown in SEQ ID No.5.

[0045] The nucleotide sequence of the artificially modified constitutive promoter P1 is shown in SEQ.ID.NO:2.

[0046] The nucleotide sequence of terminator rrnB T1 is shown in SEQ.ID.NO:3.

[0047] Example 2: Western blot detection of exogenous expression and secretion of 5M2e antigen by recombinant probiotic strains. The steps are as follows:

[0048] First, the recombinant probiotic strain EcN-5M2e was propagated and transferred to 30 ml of LB medium at a ratio of 1:100. The culture was cultured until the OD600 reached 1.0. Subsequently, the culture was centrifuged at 4000 g for 30 minutes at 4°C, and the supernatant was collected. The supernatant was quickly filtered through a 0.22 μm filter, and 27 ml of the supernatant was filtered. TCA (3 ml) was added at a ratio of 9:1, and the supernatant was precipitated overnight at 4°C to precipitate the protein. The supernatant of the overnight TCA precipitation was centrifuged at 8000 g for 30 minutes at 4°C, the waste liquid was removed, and the precipitated protein was collected. The protein was then dissolved in 500 μl of 2% SDS and transferred to a 2 ml EP tube. The protein was then precipitated by adding 1.5 ml of pre-chilled acetone at 4°C for 6-8 hours. After the cold acetone precipitation is completed, centrifuge at 4°C and 12000rpm for 30 minutes, then invert the EP tube on absorbent paper and dry it in a fume hood for 5 minutes to remove the acetone, and collect the 5M2e extracellular protein secreted into the culture medium by the recombinant probiotic strain. Then add a certain volume of 2xloading buffer to the strain supernatant protein, heat it at 95°C for 10 minutes, and complete the sample preparation. The sample was separated by 12% SDS-PAGE, transferred to the membrane, and blocked with 5% skim milk. Anti-M2e monoclonal antibody 14C2 was added and incubated at 4°C overnight. HRP-labeled goat anti-mouse IgG antibody was incubated at room temperature for 1 hour and then chemiluminescence ECL color development was performed. The corresponding 5M2e antigen-specific band can be detected in the culture supernatant of the EcN-5M2e recombinant probiotic strain, with a size between 25-35kDa, while it cannot be detected in the culture supernatant of the EcN-Vector control strain ( Figure 2 shown).

[0049] Example 3: Mouse immunization and ELISA detection of mouse serum IgG antibody levels and lung lavage IgA antibody levels

[0050] The steps are:

[0051] BALB / c mice aged 6 to 8 weeks were randomly divided into three groups: PBS group, EcN-Vector control group, and EcN-5M2e antigen group. Mice were immunized via intranasal route for a total of three rounds, with a two-week interval between each round of immunization, and each round of immunization lasting 2 days. 1×10 per mouse in the EcN-Vector control group and EcN-5M2e antigen group. 8 CFU / 10 μL, and mice in the PBS group were treated with 10 μL. Serum was collected from mice 1, 3, and 5 weeks after immunization to detect IgG antibody levels, and bronchoalveolar lavage fluid was collected from mice 6 weeks after immunization to detect mucosal IgA antibody levels.

[0052] The ELISA method was used to detect M2e-specific antibodies. The antibody detection results showed that after three immunizations, no or low IgG antibodies were detected in the serum of mice in the PBS control group and the EcN-Vector control group, while the EcN-5M2e antigen group induced the production of a large amount of M2e-specific IgG antibodies ( Figure 3 ), demonstrating that the EcN-5M2e antigen group induced a strong humoral immune response. Similarly, M2e-specific IgA antibodies in undiluted lung lavage fluid were measured by ELISA. M2e-specific IgA antibodies were detected in the lung lavage fluid of mice in the EcN-5M2e-immunized group. This result demonstrates that the EcN-5M2e recombinant probiotic strain can induce an M2e-specific mucosal immune response in mice when immunized intranasally. Figure 4 shown).

[0053] Example 4: Determination of cross-binding ability of IgG antibodies in serum with synthetic peptides of different influenza virus subtypes M2e

[0054] The steps are:

[0055] Seven days after the three immunizations with the EcN-5M2e recombinant probiotic strain, the serum of the immune mice was collected, and the ELISA method was used to determine the cross-binding ability of the IgG antibodies in the serum with the M2e polypeptides of different subtypes of influenza viruses. Among them, the synthetic peptides of the M2e of H2N2, H3N2, H5N1 and H7N9 subtype influenza viruses are contained in the 5M2e antigen fragment, and the synthetic peptides of the M2e of H6N6 and H9N2 subtype influenza viruses are not included in the 5M2e antigen fragment. Bovine serum albumin BSA was used as a negative control. The experimental results showed that the IgG antibodies in the serum of mice immunized with the EcN-5M2e recombinant probiotic strain had different degrees of binding ability with the above six different subtypes of influenza virus M2e synthetic peptides, and had no binding ability with the negative control BSA ( Figure 5 This also suggests that the EcN-5M2e recombinant probiotic strain will induce cross-protection against different subtypes of influenza virus after immunization.

[0056] Example 5: Verification of the protective effect of EcN-5M2e influenza vaccine against influenza virus infection in mice

[0057] The steps are:

[0058] Influenza virus infection experiments were performed in an ABSL-2 laboratory. Two weeks after the third round of immunization, the immunized mice were anesthetized by intraperitoneal injection of 300 μl of Avertin and then infected with 20 μl of 10×LD 50The mice were infected with either A / Puerto Rico / 8 / 34(H1N1) (PR / 8) or A / Hong Kong / 4801 / 14(H3N2) (H3N2) influenza virus at a dose of 1:1. The weight changes and survival rates of the mice were observed for two consecutive weeks. Mice were considered dead if their weight loss exceeded 30%.

[0059] After infection with A / Puerto Rico / 8 / 34(H1N1) virus, the body weight of mice in the EcN-5M2e immunization group decreased slightly and then began to recover ( Figure 6 The final survival rate was 100% ( Figure 7 As shown in the figure, all PBS mice died 9 days after virus infection, indicating that the EcN-5M2e recombinant probiotic strain protected mice from lethal infection with the H1N1 influenza virus. After infection with the A / HongKong / 4801 / 14 (H3N2) virus, the weight of mice in the EcN-5M2e immunization group began to recover on the eighth day of infection ( Figure 8 The final survival rate was 100% ( Figure 9 The weight of mice in the other immunized groups continued to decrease, and all PBS mice died 10 days after virus infection, indicating that the EcN-5M2e recombinant probiotic strain protected mice against lethal infection with the H3N2 influenza virus.

Claims

1. An antigen fragment of a broad-spectrum influenza vaccine, characterized in that: The antigen fragment consists of the 5M2e antigen and the secretion system located thereafter; or the antigen fragment consists of the 5M2e antigen and the signal peptide located thereafter; The 5M2e antigen is obtained by concatenating the M2e antigen sequences of H1N1, H2N2, H3N2, H5N1 and H7N9 subtype influenza viruses, and its amino acid sequence is shown in SEQ ID No. 1; The secretion system is used to secrete the antigen fragment outside the cell; The signal peptide is used to guide the antigen fragment to the periplasmic space for secretory expression or directly secrete expression outside the cell.

2. The antigenic fragment of the broad-spectrum influenza vaccine according to claim 1, characterized in that: The secretion system is the HlyABD secretion system.

3. The antigenic fragment of the broad-spectrum influenza vaccine according to claim 1, characterized in that: The signal peptide is selected from PeIB, PhoA, OmpA, OmpC, OmpF, InPNC or YebF.

4. A nucleic acid encoding an antigenic fragment of the broad-spectrum influenza vaccine according to claim 1, characterized in that: The nucleic acid further comprises a promoter as shown in SEQ ID No.

2.

5. The nucleic acid encoding the antigenic fragment of the broad-spectrum influenza vaccine according to claim 1 according to claim 4, characterized in that: The nucleic acid further comprises a strong terminator as shown in SEQ ID No.

3.

6. A recombinant probiotic strain incorporating antigenic fragments of the broad-spectrum influenza vaccine according to claim 1.

7. The recombinant probiotic strain according to claim 6, characterized in that The recombinant probiotic strain is selected from probiotic Escherichia coli Nissle 1917, Lactobacillus plantarum, Bacillus coagulans, Bifidobacterium, Lactobacillus paracasei, Lactococcus lactis, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus bulgaricus or Streptococcus thermophilus.

8. A recombinant Escherichia coli for a broad-spectrum influenza vaccine, whose deposit number is CCTCC NO. M 20232245.

9. Use of the recombinant Escherichia coli according to claim 8 in the preparation of a broad-spectrum influenza vaccine, wherein the effective dose of the preparation is 1 x 10 8 CFU bacterial count; the influenza vaccine is a nasal spray, and the nasal spray is selected from aerosol, spray and powder spray.

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