Use of an influenza virus universal and coronavirus combined polypeptide and vaccine thereof

Abstract

The application discloses an influenza virus universal and coronavirus combined polypeptide and application of a vaccine thereof, and belongs to the technical field of vaccine preparation. The vaccine is an artificial synthetic vaccine, has the characteristics of easy preparation, small toxicity, low cost, high titer, broad spectrum and conservation, and solves the complicated process of obtaining a vaccine by amplification transformation in the prior art, and improves the preparation efficiency and application efficiency. A combined polypeptide vaccine component having immunoprotection for multiple subtypes of influenza viruses and coronaviruses is designed for influenza viruses and novel coronaviruses, and Montanide ISA51 is used as an adjuvant. After mice immunized with the vaccine are infected with influenza viruses by nose dropping, it is shown that the vaccine immunization has a protective effect on mice adapted strains of influenza A H1N1 and H7N9 viruses, the combined vaccine composed of the screened six polypeptides has a protective effect on multiple influenza viruses, and has the potential to resist coronaviruses.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccine preparation, and particularly relates to an influenza virus universal and coronavirus combined polypeptide and application of a vaccine thereof. BACKGROUND

[0002] The influenza virus outbreak is one of the most influential popular diseases on human production and life so far. The three influenza pandemics that broke out in the last century were caused by influenza viruses, which caused great harm and loss to human health and economy. Different influenza virus subtypes are divided based on the sequences of hemagglutinin HA and neuraminidase, and the main influenza virus subtypes currently spreading in the population are influenza A H1N1, H3N2 and influenza B virus.

[0003] The most effective method to prevent influenza is vaccination. The currently marketed vaccines against influenza viruses mainly include whole virus inactivated vaccine, split vaccine and subunit vaccine, each of which contains 3-4 kinds of influenza inactivated viruses or antigen components of influenza A1 subtype, influenza A3 subtype and influenza B (two systems), but influenza viruses are widely spread and complex and variable, vaccines need to be constantly updated, and the prediction accuracy of WHO is limited.

[0004] Traditional vaccines are prepared by inactivating or attenuating pathogens, and have problems such as biological hazard and genetic variation leading to loss of original vaccine efficacy. Compared with traditional attenuated vaccines and inactivated vaccines and other several new types of vaccines, synthetic peptide vaccines are safe, non-toxic and stable, and have few serious complications and nosocomial infection problems due to their small and simple molecular structure.

[0005] Influenza viruses and coronaviruses are both variable RNA viruses, and as the viruses mutate, the existing vaccines will not be able to play a good protective role. In addition, influenza viruses have multiple subtypes that can infect humans, and the development of universal vaccines has been a key problem to overcome. The current influenza vaccine and coronavirus vaccine are mainly obtained by amplifying the gene through PCR, transforming the gene into E. coli through a plasmid, obtaining an engineering bacterium, and finally obtaining a recombinant protein. The operation process is complicated and time-consuming. SUMMARY

[0006] The purpose of the present application is to provide an influenza virus universal and coronavirus combined polypeptide vaccine, which is an artificial synthetic vaccine, has the characteristics of easy preparation, small toxicity, low cost, high titer, broad spectrum and conservation, solves the complicated process of obtaining the vaccine by amplification and transformation in the prior art, and improves the preparation efficiency and application efficiency.

[0007] The present application is realized by the following technical solutions:

[0008] An influenza virus universal and coronavirus combined polypeptide, comprising 6 polypeptide sequences, the specific amino acid sequences are shown as SEQ ID NO. 1-SEQ ID NO. 6.

[0009] An application of an influenza virus universal and coronavirus combined polypeptide in preparing an influenza virus universal and coronavirus combined polypeptide vaccine.

[0010] An influenza virus universal and coronavirus combined polypeptide vaccine, comprising the influenza virus universal and coronavirus combined polypeptide.

[0011] As preferably, the vaccine comprises an adjuvant.

[0012] As preferably, the adjuvant is Montanide ISA51.

[0013] An application of a combined polypeptide in preparing a medicine for preventing influenza virus universal and coronavirus.

[0014] An application of a combined polypeptide vaccine in preparing a medicine for preventing influenza virus universal and coronavirus.

[0015] Compared with the prior art, the present application has at least the following technical effects:

[0016] The present application provides an influenza virus universal and coronavirus combined polypeptide and a vaccine thereof, the vaccine is an artificial synthetic vaccine, has the characteristics of easy preparation, small toxicity, low cost, high titer, broad spectrum and conservation, solves the complicated process of obtaining the vaccine by amplification transformation in the prior art, and improves the preparation efficiency and application efficiency. A combined polypeptide vaccine component having immunoprotection for multiple subtypes of influenza virus and coronavirus is designed for influenza virus and novel coronavirus, and Montanide ISA51 is used as an adjuvant. After the vaccine immunized mice are infected with influenza virus by nose dropping, it is shown that the vaccine immunization has a protective effect on the vaccine mice adapted strains of influenza virus A H1N1 and H7N9, the combined vaccine composed of the screened 6 polypeptides has a protective effect on multiple influenza viruses, and has the potential to resist coronavirus. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A flow chart for example screening and vaccine preparation;

[0018] Figure 2 A schematic diagram for polypeptide-coated ELISA detection of serum IgG level of influenza virus immunized mice;

[0019] Figure 3 A schematic diagram for polypeptide-coated ELISA detection of serum IgG level of coronavirus immunized mice;

[0020] Figure 4 Schematic diagram of detecting serum anti-IgG concentration of immunized mice by polypeptide coated ELISA for example;

[0021] Figure 5 Schematic diagram of proportion of IFN-γ / CD4 double positive cells in spleen lymphocytes after single polypeptide stimulation for example;

[0022] Figure 6 Schematic diagram of the effect of polypeptide immunized mice on body weight change and mortality rate of H1N1 influenza virus infection for example;

[0023] Figure 7 Schematic diagram of the effect of polypeptide immunized mice on body weight change and mortality rate of H7N9 influenza virus infection for example;

[0024] Figure 8 Schematic diagram of the effect of polypeptide immunized mice on lung tissue viral load of H1N1 virus infected mice for example;

[0025] Figure 9 Schematic diagram of the effect of polypeptide immunized mice on lung tissue viral load of H7N9 virus infected mice for example;

[0026] Figure 10 Schematic diagram of the level of serum coronavirus specific neutralizing antibody of polypeptide immunized mice for example. DETAILED DESCRIPTION

[0027] The embodiments of the present application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0028] Example 1:

[0029] An influenza virus universal and coronavirus combined polypeptide vaccine is based on the GeneBank and GISAID influenza virus and coronavirus sequence database and the published therapeutic monoclonal antibody literature. Six polypeptide sequences are screened out by combined omics technology comparison and screening, and a combined polypeptide vaccine is constructed, which includes two polypeptides from influenza virus NP protein, two polypeptides from influenza virus M protein and two polypeptides from novel coronavirus S protein, all of which are conservative antigen sites. The six polypeptide sequences are as follows:

[0030] SEQ ID NO. 1: Flu-M1: DLEALMEWLKTRPILSPLTKGILGFVFTLTVP

[0031] SEQ ID NO. 2: Flu-M2: IIGILHLILWILDRLFFKCIYRLF

[0032] SEQ ID NO. 3: Flu-NPA: DLIFLARSALILRGSVAHKSC

[0033] SEQ ID NO. 4: Flu-NPB: PGIADIEDLTLLARSMVVVR

[0034] SEQ ID NO. 5:

[0035] Cov-RBD1: FTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN

[0036] SEQ ID NO. 6:

[0037] Cov-RBD2: QAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

[0038] 1. The screening and vaccine preparation process is shown as follows: Figure 1

[0039] Influenza and coronavirus protein amino acid sequences were downloaded from Genebank and aligned, combined with published literature on influenza and coronavirus related broad-spectrum monoclonal antibodies, and potential monoclonal antibody highly affinity epitope regions were selected for influenza virus NP protein and M protein, and coronavirus S protein highly conserved region. The polypeptides with a length of 24-32 amino acids were intercepted. The polypeptides contained CD4, CTL T cell epitopes and stimulation of interferon-γ sites for mice and humans through bioinformatics analysis of epitope regions, and six high-quality polypeptide sequences were selected.

[0040] Each protein contains two polypeptides. After the sequences are synthesized by a biological company, they are dissolved in 0.01M PBS solution and mixed into three concentration systems containing 250 μg / ml, 500 μg / ml and 750 μg / ml of each polypeptide, respectively. Equal volume of Montanide ISA51 adjuvant is added, and the adjuvant and polypeptide solution are emulsified with a three-way syringe to form a vaccine.

[0041] According to the 200 μL dose, 6-8W C57 / 6J mice with a body weight of about 20g are immunized, and the immunization is enhanced once every 14 days, a total of two times. Before each immunization, the mice are taken a small amount of blood, and the serum is separated for antibody detection.

[0042] ​A combined polypeptide vaccine component with immunoprotection for multiple subtypes of influenza virus and coronavirus was designed for influenza virus and novel coronavirus, and Montanide ISA51 was used as an adjuvant for intramuscular injection of mice, a total of 3 times of immunization, with an interval of 14 days, and blood was taken from the eye socket before each vaccination for IgG antibody detection.

[0043] 2. Immunization test of the influenza virus universal and coronavirus combined polypeptide vaccine

[0044] 2.1 Combined polypeptide vaccine titer detection: The immunization effect of the combined polypeptide vaccine was evaluated using serological test

[0045] Among them, 25 μg, 50 μg and 75 μg are the immunization polypeptide doses, and day 15, day 30 and day 45 are the days after immunization. The blank group is the non-immunized vaccine group.

[0046] Figure 2 Schematic diagram of polypeptide-coated ELISA for detecting serum IgG levels of influenza virus (FLU) immunized mice; Figure 3 Schematic diagram of polypeptide-coated ELISA for detecting serum IgG levels of coronavirus immunized mice.

[0047] As shown in Figure 2 and 3 The results show that the mice after immunization can produce specific high-level antibodies against influenza virus polypeptides and coronavirus polypeptides.

[0048] As shown in Figure 4 , the schematic diagram of polypeptide-coated ELISA for detecting serum IgG concentration of immunized mice. The results show that the polypeptide-coated ELISA detects the serum IgG concentration of immunized mice, and the mice are immunized with 25 μg, 50 μg and 75 μg polypeptides for three times, and the specific antibody level shows a significant gradient increase trend between the three immunizations, indicating that the selected polypeptide has good immunogenicity.

[0049] As shown in Figure 5 , the schematic diagram of the proportion of IFN-γ / CD4 double positive cells after stimulation of spleen lymphocytes by single polypeptide. Among them, the experimental group is the mice immunized with 50 μg polypeptide vaccine for 45 days after immunization, and the blank group is the non-immunized vaccine group.

[0050] After collecting the spleen lymphocytes of immunized mice, 6 polypeptides were used for in vitro stimulation. The results show that 6 polypeptides can stimulate high levels of interferon gamma, suggesting that 6 polypeptides have good cellular immunogenicity.

[0051] 2.2 Evaluation of the immunoprotection effect of the combined polypeptide vaccine:

[0052] The immune group is a group of mice immunized with 50 μg of polypeptide vaccine and infected with virus 45 days after immunization.

[0053] The control group is a group of mice infected with virus without vaccination.

[0054] As shown in Figure 6 , the effect of polypeptide immunized mice on the weight change and mortality rate of H1N1 influenza virus infection is shown in the figure.

[0055] As shown in Figure 7 , the effect of polypeptide immunized mice on the weight change and mortality rate of H7N9 influenza virus infection is shown in the figure.

[0056] It is shown that the vaccine immunization has a protective effect on both A / California / 07 / 2009 (H1N1) and A / Anhui / 01 / 2013 (H7N9) (2+6rg-PR8) influenza virus vaccine mouse adapted strains by nasal infection of influenza virus in vaccine immunized mice.

[0057] As shown in Figure 8 , the effect of polypeptide immunized mice on the viral load in lung tissue of H1N1 virus infected mice is shown in the figure.

[0058] As shown in Figure 9 , the effect of polypeptide immunized mice on the viral load in lung tissue of H7N9 virus infected mice is shown in the figure.

[0059] Figure 8 and Figure 9 The results show that the combined polypeptide vaccine can effectively inhibit the viral load in lung tissue of infected mice as time increases.

[0060] As shown in Figure 10 , the level of coronavirus specific neutralizing antibody in serum of polypeptide immunized mice is shown in the figure. The immune group is a group of mice immunized with 50 μg of polypeptide vaccine and infected with virus 45 days after immunization; the blank group is a group of mice infected with virus without vaccination. The detection of specific neutralizing antibodies against S protein of new coronavirus in vitro shows that the vaccine immunization can produce high level of specific neutralizing antibodies, which has the potential as a candidate for new coronavirus vaccine.

[0061] In summary, the combined vaccine composed of the screened 6 polypeptides has a protective effect on multiple influenza viruses and has the potential against coronavirus.

[0062] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A universal influenza virus and coronavirus combined polypeptide, characterized in that, It includes 6 polypeptide sequences, the specific amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.6; The influenza virus in question is influenza A / Anhui / 01 / 2013(H7N9)(2+6rg-PR8); The coronavirus mentioned is the novel coronavirus.

2. The application of the universal influenza virus and coronavirus combined polypeptide as described in claim 1 in the preparation of a universal influenza virus and coronavirus combined polypeptide vaccine, characterized in that, The influenza virus in question is influenza A / Anhui / 01 / 2013(H7N9)(2+6rg-PR8); The coronavirus mentioned is the novel coronavirus.

3. A universal influenza virus and coronavirus combined polypeptide vaccine, characterized in that, The invention comprises the universal influenza virus and coronavirus combined polypeptide of claim 1, characterized in that the influenza virus is influenza A / Anhui / 01 / 2013(H7N9)(2+6rg-PR8); The coronavirus mentioned is the novel coronavirus.

4. The universal influenza virus and coronavirus combined polypeptide vaccine according to claim 3, characterized in that, The vaccine includes an adjuvant.

5. The universal influenza virus and coronavirus combined polypeptide vaccine according to claim 4, characterized in that, The adjuvant is Montanide ISA51.

6. The use of the combined polypeptide as described in claim 1 in the preparation of a medicament for the prevention of influenza virus and coronavirus, characterized in that, The influenza virus in question is influenza A / Anhui / 01 / 2013(H7N9)(2+6rg-PR8); The coronavirus mentioned is the novel coronavirus.

7. The use of a combined polypeptide vaccine as described in any one of claims 3-5 in the preparation of a medicament for the prevention of influenza virus and coronavirus, characterized in that, The influenza virus in question is influenza A / Anhui / 01 / 2013(H7N9)(2+6rg-PR8); The coronavirus mentioned is the novel coronavirus.

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