Preparation method of broad-spectrum vaccine

Abstract

The present invention relates to a method for preparing a broad-spectrum vaccine, comprising the following steps: combining a cross-linking agent with a pathogen; or contacting a cross-linking agent with a recombinant protein from a pathogen, and allowing them to form a new internal covalent bond of the protein; if the pathogen is used, it needs to be inactivated, and then the cross-linking agent not bound to the protein is removed, and if the recombinant protein from the pathogen is used, the cross-linking agent not bound to the protein is directly removed; the pathogen or recombinant protein modified by the cross-linking agent is separated; and the pathogen or recombinant protein modified by the cross-linking agent is combined with an adjuvant to obtain a broad-spectrum vaccine. During the maturation and hypermutation of B cells in the germinal center, the present invention allows follicular dendritic cells to present antigens with a stable original ecological structure to the B cells in the germinal center. Thereby, the B cells in the germinal center have sufficient time to repeatedly cycle mutations in the germinal center, and finally produce broad-spectrum immunoglobulin G antibodies with high affinity to the original antigen.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological medicines, and in particular relates to a method for preparing a broad-spectrum vaccine. Background Art

[0002] Influenza epidemics have brought disaster to humanity over the past two centuries. The 1918 pandemic infected about 500 million people and killed an estimated 50 to 100 million people worldwide.

[0003] In the United States, current influenza vaccines cost approximately $2.5 to $3 billion per year, and this is expected to reach approximately $5 billion by 2025. Although influenza vaccines have also been developed for influenza, due to the limitations of these vaccines, they are only about 50% effective. In this regard, the direct medical costs for treating influenza-infected people in the United States can reach approximately $10.4 billion per year, and the indirect costs are approximately $87 billion per year. The costs worldwide may be as high as 10 times these values. There is an urgent need to develop a universal and long-lasting influenza vaccine, as well as vaccines against other pathogens.

[0004] A major challenge in influenza vaccine development is that the rapid changes in influenza viruses make it difficult to find a common antigen (one that can be used in a broad-spectrum vaccine) between different influenza virus strains. For example, influenza A (A) strains have 18 H subtypes and 11 N subtypes, for a total of 18x11 different combinations. And even specific combinations, such as H1N1, can change (or mutate) rapidly over time and place. Influenza B (B) has similar problems, although not as many as influenza A. It is now clear that influenza A and B strains are the main pathogens that cause epidemics in humans each year.

[0005] The use of modern RNA and DNA sequencing technologies has led to a large number of viruses being sequenced, including influenza A and B viruses. Based on sequence comparisons, some parts of the antigens on these viruses are quite conserved and change slowly over time. For example, Dr. Peter Palese's research team found that a fragment from the hemagglutinin subunit 2 protein (HA20) is a conserved domain in most common influenza viruses, based on which it can induce the production of broadly neutralizing antibodies in mice and provide protection against influenza viruses of structurally different H3N2, H1N1, and H5N1 subtypes (Steel et al., 2010). In some cases, broadly neutralizing antibodies such as nAb12D1 were found in some patients. These results suggest that the development of a universal and long-lasting influenza vaccine has both theoretical and practical foundations. However, there are at least two problems. 1) How to effectively discover and stably preserve common antigens; 2) How to immunize these common antigens to hosts (mammals, including humans, cattle, pigs, birds, etc.).

[0006] There are three main traditional methods for preparing vaccines:

[0007] The first method is to use killed viruses (dead viruses). In theory, this method should retain all the antigens of the virus.

[0008] The second approach is to use a weakened virus as a vaccine, which should also retain the antigens.

[0009] The third method is to use recombinant proteins or peptides expressed on the surface of the virus as antigens. In theory, when the subject is infected with the virus, the subject should be exposed to all the antigens of the virus and become immune to them.

[0010] But the protection (against subsequent challenges) conferred by all three of these approaches was incomplete. Specifically, they did not lead to the production of broadly neutralizing antibodies.

[0011] Dendritic cells (DC), B cells and macrophages are called antigen presenting cells (APCs). Proteins of the immunoglobulin G (IgG) family are the most important neutralizing antibodies. According to existing theories, the production of IgG requires the coordination of T cells, B cells and follicular dendritic cells (FDC). Dendritic cells (DC) first present antigens (peptides digested from viruses within the dendritic cells) to naive T cells in the T cell zone, which will move to the B cell zone to become follicular T cells (Tfh). Follicular T helper cells (CD4 cells) interact with germinal center (GC) B cells presenting viral antigens, triggering Tfh cells to produce IL-21, which will stimulate the germinal center B cells to undergo class switching. At the same time, if there is a follicular dendritic cell presenting the antigen in its native form (protein from the virus), high-affinity IgG GB cells (through membrane-bound IgG) interact with the follicular dendritic cell, triggering the B cell to transform into a plasma cell and memory B cell. To generate broadly neutralizing antibodies, follicular dendritic cells must present antigens in native form to activated B cells in germinal centers (via Tfh).

[0012] Several factors may hamper the production of highly effective vaccines, or vaccines that suppress infectious viruses and induce broadly neutralizing antibodies:

[0013] First, due to the high mutation rate of influenza viruses, many common viral antigens disappear in new strains due to mutations.

[0014] Secondly, due to the instability of protein antigens in vivo (protein antigens are unstable both in vitro and in vivo), follicular dendritic cells may no longer be able to present the antigens in their native form when needed (the antigens have been denatured or digested in vivo).

[0015] Finally, traditional vaccine preparation processes, especially where formalin or propiolactone is used, denature the original structure of viral antigens due to the long inactivation process, which takes several days or longer. In general, it is difficult for follicular dendritic cells to present native structure antigens to B cells for further maturation and mutation to produce broad-spectrum neutralizing antibodies.

[0016] In order to overcome this obstacle, the present application discloses a new method for preparing a broad-spectrum vaccine. During the maturation and hypermutation of B cells in the germinal center, follicular dendritic cells (FDC) are allowed to present antigens with stable original ecological structures to the B cells in the germinal center. This allows the B cells in the germinal center to have sufficient time to repeatedly cycle mutations in the germinal center, and finally produce broad-spectrum immunoglobulin G antibodies with high affinity to the original antigen. At the same time, part of the B cells that produce high-affinity antibodies become plasma B cells that produce a large number of high-affinity antibodies, and part become memory B cells. In terms of T cells, in the germinal center, T cells accompany B cells through a similar maturation process. Finally, memory T cells are formed. Memory B cells and memory T cells can survive in the human body for a long time, thereby protecting humans from infection with similar types of pathogens in the future. Summary of the invention

[0017] Purpose of the invention: In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to disclose a method for preparing a broad-spectrum vaccine.

[0018] Technical solution: A method for preparing a broad-spectrum vaccine comprises the following steps:

[0019] (1) Obtaining live pathogens;

[0020] (2) diluting the live pathogen obtained in step (1) with a low-temperature buffer solution until the concentration is below 1.0 mg / ml and then proceeding to step (3);

[0021] (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent is less than 0.005%;

[0022] (4) diluting the pathogen dilution obtained in step (2) with the cross-linking agent dilution obtained in step (3) for a second time, and proceeding to step (5) when the pathogen concentration reaches 0.01 to 0.001 mg / ml;

[0023] (5) placing the pathogen solution diluted in step (4) in a cold room at 0°C to 10°C for 24 to 48 hours and then proceeding to step (6);

[0024] (6) inactivating the pathogen liquid refrigerated in step (5) to obtain an inactivated pathogen liquid;

[0025] (7) Add glycine to the inactivated pathogen solution, and then place at 37° C. for at least 2 hours before proceeding to step (8), wherein:

[0026] The molar ratio of the added glycosylamine to glutaraldehyde is 5 to 10:1;

[0027] (8) Concentrate and collect the pathogen solution treated in step (7), wash it once or twice with physiological saline or HEPES buffer, filter, concentrate and collect it, and then proceed to step (9);

[0028] (9) The pathogen solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 10-25:200-800 to obtain a broad-spectrum vaccine.

[0029] Furthermore, the pathogen in step (1) is one of a virus, a bacterium, and a fungus, preferably one of a coronavirus, an influenza virus, a retrovirus, and a rhino virus.

[0030] Furthermore, the pathogen in step (1) is influenza virus or the new coronavirus SARS-CoV-2.

[0031] Furthermore, the pathogen in step (1) has a purity of more than 95%.

[0032] Furthermore, the low temperature in the low temperature buffer in step (2) and step (3) means that the buffer temperature is lower than 10° C., and the buffer is one of the following three types:

[0033] a. Normal saline;

[0034] b. PBS buffer;

[0035] c. 50mM HEPES, 50-150mM NaCl, pH 7.0-7.2 buffer.

[0036] Furthermore, the cross-linking agent described in step (3) is one of succinaldehyde, glutaraldehyde, adipaldehyde, heptanedialdehyde, suberic acid, azelaic acid, sebacic acid, disuccinimidyl suberate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, dimethyl pimelate, dimethyl suberate, dimethyl azelaic acid, and dimethyl sebacate, preferably glutaraldehyde.

[0037] Further, step (6) comprises the following steps:

[0038] Adding an appropriate amount of a β-propiolactone aqueous solution with a mass concentration of more than 90% to the pathogen solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.01% to 0.5%;

[0039] The pathogen solution is placed at 0-10°C for at least 72 hours to obtain an inactivated pathogen solution; or

[0040] The inactivated pathogen solution is obtained by placing the pathogen solution at 18°C ​​to 37°C for at least 24 hours.

[0041] Further, step (6) comprises the following steps:

[0042] Adding an appropriate amount of a 36-37% formaldehyde solution to the pathogen solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.025-0.5%;

[0043] The pathogen solution is placed at 0-10°C for at least 50 days to obtain an inactivated pathogen solution; or

[0044] The pathogen solution was placed at 37° C. for at least 60 hours to obtain an inactivated pathogen solution.

[0045] Furthermore, step (6) can be inactivated by gamma rays, specifically comprising the following steps:

[0046] Gamma ray inactivation varies according to the size of the viral genome. For the new coronavirus SARS-CoV-2, radiation D 10 The dose is the international standard of 2.0-5.0 kGy;

[0047] For influenza A virus, radiation D is needed 10 The dose is 1.60-3.0 kGy according to the international standard;

[0048] For influenza B virus, radiation D is required 10 The dose is according to the international standard of 2.0-3.0 kGy.

[0049] Furthermore, the aluminum adjuvant in step (9) is one of aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHS), and potassium aluminum sulfate.

[0050] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0051] S1. Obtain recombinant proteins of pathogens;

[0052] S2, diluting the recombinant protein obtained in step S1 with a low-temperature buffer, and proceeding to step S3 after the concentration of the recombinant protein is below 1.0 mg / ml;

[0053] S3, diluting the cross-linking agent with a low-temperature buffer, and proceeding to step S4 after the mass concentration of the cross-linking agent is below 0.005%;

[0054] S4, diluting the recombinant protein solution obtained in step S2 with the cross-linking agent diluent obtained in step S3, and proceeding to step S5 after the concentration of the recombinant protein reaches 0.01-0.001 mg / ml;

[0055] S5, placing the recombinant protein solution treated in step S4 in a cold room at 0°C to 10°C for 24-48 hours and proceeding to step S6;

[0056] S6, adding glycine to the recombinant protein solution refrigerated in step S5, and then placing it at 37° C. for at least 2 hours, and then proceeding to S7, wherein:

[0057] The molar ratio of added glycosylamine to glutaraldehyde is 5-10:1;

[0058] S7, concentrating and collecting the recombinant protein solution treated in step S6, and washing it once or twice with physiological saline or HEPES buffer, filtering, concentrating and collecting it, and then proceeding to step S8;

[0059] S8. The recombinant protein solution treated in step S7 is mixed with an aluminum adjuvant in a mass ratio of 10-25:200-800 to obtain a broad-spectrum vaccine.

[0060] Furthermore, the recombinant protein of the pathogen in step S1 is derived from one of a virus, a bacterium, and a fungus, preferably one of a coronavirus, an influenza virus, a retrovirus, and a rhino virus.

[0061] Furthermore, the recombinant protein of the pathogen in step S1 may be derived from influenza virus or the new coronavirus SARS-CoV-2.

[0062] Furthermore, the purity of the recombinant protein of the pathogen in step S1 is above 95%.

[0063] Furthermore, the low temperature in the low temperature buffer in step S2 and step S3 means that the buffer temperature is lower than 10°C, and the buffer is one of the following three types:

[0064] a. Normal saline;

[0065] b. PBS buffer;

[0066] c. 50mM HEPES, 50-150mM NaCl, pH 7.0-7.2 buffer.

[0067] Further, the cross-linking agent described in step S3 is one of succinaldehyde, glutaraldehyde, adipaldehyde, heptanedialdehyde, suberic acid, azelaic acid, decanedial, disuccinimidyl suberate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, dimethyl pimelate, dimethyl suberate, dimethyl azelaic acid, and dimethyl sebacate, preferably glutaraldehyde.

[0068] Furthermore, the aluminum adjuvant in step S8 is one of aluminum hydroxide, aluminum phosphate, amorphous aluminum hydroxyphosphate adjuvant (AAHS), and potassium aluminum sulfate.

[0069] Disclosed herein is a method for preparing a broad-spectrum vaccine, wherein a cross-linking agent is combined with a pathogen; or

[0070] Expose cross-linking agents to recombinant proteins from pathogens and allow them to form new intra-protein covalent bonds;

[0071] Remove cross-linker that is not bound to protein;

[0072] Isolation of pathogens or recombinant proteins modified with cross-linkers;

[0073] The pathogen or recombinant protein modified by the cross-linking agent is combined with an adjuvant to obtain a broad-spectrum vaccine.

[0074] In many embodiments, the crosslinking agent is one of succinaldehyde, glutaraldehyde, adipaldehyde, heptanedialdehyde, octanedial, nonanedialdehyde, and sebacic dialdehyde.

[0075] In other embodiments, the cross-linking agent is one of bissulfosuccinimidyl suberate (BS3), dimethyl adipimidate (DMA), dimethyl suberate (DMS), dimethyl pimelic acid (DMP), dimethyl succinate, dimethyl glutarate, and dimethyl azelaic acid.

[0076] Also disclosed is a method of immunizing a subject, the method comprising injecting the subject with a pathogen or protein modified with a cross-linking agent; and

[0077] The subject is injected a second time with the pathogen or protein modified with the linker, wherein the subject is a mammal or bird, for example, a mammal or bird includes a human, a cow, a pig, a horse, a cat, a dog, a bird or a chicken.

[0078] In many embodiments, the crosslinking agent is one of succinaldehyde, glutaraldehyde, adipaldehyde, heptaldehyde, suberaldehyde, azelaic acid, sebacic acid, disuccinimidyl suberate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, dimethyl pimelate, dimethyl suberate, dimethyl azelaic acid, and dimethyl sebacate. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is a schematic diagram of the effect of the influenza A vaccine H1N1 prepared in Example 2.

[0080] Figure 2 This is a schematic diagram of the effect of the influenza A vaccine H2N3 prepared in Example 2.

[0081] Figure 3 This is a schematic diagram of the effect comparison of the influenza vaccine type B (B) prepared in Example 3.

[0082] Figure 4 Schematic diagram of the binding of RBD to 6His-tagged ACE2 after treatment with glutaraldehyde cross-linker.

[0083] Figure 5 Schematic diagram of the stability effect of the vaccine coronavirus antigen R417-RBD after treatment with glutaraldehyde in the refrigerator and at room temperature.

[0084] Figure 6 Shows the performance of the vaccine in the mouse model two months after treatment with glutaraldehyde.

[0085] Figure 7 Performance of the vaccine after three months in a mouse model following treatment with glutaraldehyde.

[0086] Figure 8 . Schematic diagram of the binding of antibodies produced by K417R mutant RBD treated with glutaraldehyde cross-linking to ACE. DETAILED DESCRIPTION

[0087] The specific embodiments of the present invention are described in detail below.

[0088] The disclosed methods and chemical reagents can be used to produce universal broad-spectrum long-acting vaccines against various pathogens (including viruses, fungi, and bacteria).

[0089] In some embodiments, the disclosed methods and chemical reagents can be used to prepare vaccines that have neutralizing capabilities against a broad spectrum of influenza, coronavirus, rhinovirus, parainfluenza, pneumonia A virus, respiratory syncytial virus, retrovirus, hepatitis C, HIV, and other viruses.

[0090] In many embodiments, the disclosed methods and chemical reagents facilitate the creation of influenza vaccines that are active against different strains.

[0091] In many embodiments, the disclosed methods and chemical reagents can be used to make coronavirus vaccines with activity against different strains, including SARS-CoV-2 that causes COVID-19. The disclosed methods and chemical reagents can also be used to prepare recombinant protein vaccines and monoclonal antibodies, for example, against various antigens (e.g., proteins).

[0092] Example 1 - Production of stable protein antigens capable of withstanding extremely harsh conditions

[0093] Applicants have analyzed and elucidated many basic mechanisms of protein folding and unfolding. It was found that the destruction of hydrogen bonds is the driving force for the destruction of the three-dimensional structure of proteins (Wang et al., 2014). It was later found that hydrogen bonds are the main driving force for the initial folding of proteins (Lee, 2017). However, the three-dimensional structure of proteins is mostly maintained by weak hydrophobic interactions (van der Waals forces). These findings show that the three-dimensional structure of proteins is fragile and therefore easily affected by environmental changes and loses its original normal structure. Minor perturbations of the protein environment may cause the denaturation of the three-dimensional structure of proteins. Formalin (formaldehyde), β-propiolactone (BPL), extreme pH values, certain solution components, vaccine adjuvants (aluminum), increased temperature, etc. can all cause changes in the original three-dimensional structure of proteins. In order to protect the fragile three-dimensional structure of proteins, others have tried to introduce internal disulfide bonds (bridges) to help maintain the original structure of proteins. However, disulfide bridges may be destroyed by the reducing environment in the body. Another popular cross-linking agent used to maintain protein structure is formalin / formaldehyde. However, formaldehyde can cause drastic denaturation of antigens. Additionally, due to the small size of formaldehyde molecules, its ability to support cross-linking is limited to those amino acid residues that are in close proximity to each other.

[0094] In order to solve the problem discussed above, applicant's cross-linking agent, comprise one of succinaldehyde, glutaraldehyde, adipaldehyde, heptanedialdehyde, suberaldehyde, azelaic acid, decanedial, disuccinimide suberate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, dimethyl pimelate, dimethyl suberate, dimethyl azelaic acid, dimethyl sebacate, they can keep the original structure of viral antigen. Glutaraldehyde is a five-carbon linear molecule with terminal active oxygen molecule. Glutaraldehyde links the side chains of lysine residues from different parts of protein (even different domains). In some embodiments, other cross-linking agents also can be used in present method (see below). The structural formula of glutaraldehyde is as follows:

[0095]

[0096] In order to test the efficiency of the cross-linking agent glutaraldehyde, the applicant used green fluorescent protein (GFP) as a test model. Specifically, the following steps are included:

[0097] (1) Add an appropriate amount of pH 7.2 buffer to dilute the GFP protein at a concentration of 1.0 mg / ml to 0.1 mg / ml. The buffer is a mixed aqueous solution of HEPES and sodium chloride, wherein the concentration of HEPES is 50 mmol / L and the concentration of sodium chloride is 50 mmol / L, pH 7.2 (physiological pH value has the best stability for the target protein or antigen);

[0098] (2) placing the protein solution / glutaraldehyde solution at room temperature for 24 hours;

[0099] (3) Add an excess amount of amino acid glycine to the protein solution / glutaraldehyde solution, with the molar ratio of glycine to glutaraldehyde being 10:1, and allow to react for 5 hours to obtain a GFP sample to be tested.

[0100] In order to test the prepared GFP sample, six equal portions of the prepared GFP sample were taken out and divided into six groups for testing. The details are shown in the following table:

[0101]

[0102] The stability of the prepared GFP samples was tested in 0.01% SDS at 80°C for about 30 minutes. The native GFP sample, without any treatment, loses its structure and fluorescence emission ability after only 30 minutes in 0.01% SDS at 80°C (see table). However, the GFP sample was treated with glutaraldehyde and left to stand for 24 hours. Then 0.1% SDS was added. The fluorescence at high temperature and in 0.1% SDS denaturant was maintained for a long time (weeks or longer). These results show that glutaraldehyde is an effective reagent for maintaining the original structure of proteins in harsh denaturing environments.

[0103] Traditionally, formalin (formaldehyde) is believed to have cross-linking ability, and therefore, may be able to protect protein from denaturation. In order to prove its ability to protect protein structure, 0.5% formaldehyde was added to the above GFP sample and left to stand for 24 hours. After about 30 minutes in 0.01% SDS at 80-100°C, the fluorescence of GFP disappeared (see table). Therefore, formaldehyde cannot protect native proteins from being stable under harsh conditions.

[0104] Example 2 - Preparation of a vaccine containing stable antigens of influenza A strains.

[0105] Next, the applicant studied the use of glutaraldehyde to treat influenza A virus to produce a vaccine with stable antigens.

[0106] The specific steps are as follows:

[0107] (1) Obtain H1N1 influenza virus, which is from Charles River (Product No.: 10100374, Influenza A / PR / 8 / 34);

[0108] (2) diluting the H1N1 influenza virus obtained in step (1) with a low-temperature buffer until the concentration is below 1.0 mg / ml and proceeding to step (3);

[0109] (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent reaches 0.005%;

[0110] (4) diluting the H1N1 influenza virus dilution obtained in step (2) with the cross-linking agent dilution obtained in step (3) for a second time, and proceeding to step (5) when the H1N1 influenza virus concentration reaches 0.01 mg / ml;

[0111] (5) placing the pathogen solution diluted in step (4) in a cold room at 0°C for 48 hours and then proceeding to step (6);

[0112] (6) inactivating the pathogen liquid refrigerated in step (5) to obtain an inactivated pathogen liquid;

[0113] (7) Add glycine to the inactivated pathogen solution, and then place at 37° C. for at least 2 hours before proceeding to step (8), wherein:

[0114] The molar ratio of the added glycosylamine to glutaraldehyde is 5:1;

[0115] (8) Concentrate and collect the pathogen solution treated in step (7), rinse once with physiological saline or HEPES buffer, filter, concentrate and collect, and then proceed to step (9);

[0116] (9) The pathogen solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 10:200 to obtain a broad-spectrum vaccine.

[0117] Furthermore, the purity of the H1N1 influenza virus in step (1) is above 95%.

[0118] Furthermore, the low temperature in the low temperature buffer in step (2) and step (3) means that the buffer temperature is lower than 10°C, and the buffer is physiological saline.

[0119] Furthermore, the cross-linking agent described in step (3) is glutaraldehyde.

[0120] Further, step (6) comprises the following steps:

[0121] Adding an appropriate amount of 90% β-propiolactone aqueous solution to the pathogen solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.1%;

[0122] The inactivated pathogen solution was obtained by placing the H1N1 influenza virus solution at 0°C for 72 hours.

[0123] In another embodiment, the H1N1 influenza virus solution is placed at 18° C. for 24 hours to obtain an inactivated pathogen solution.

[0124] In another embodiment, step (6) comprises the following steps:

[0125] Adding an appropriate amount of a 36% formaldehyde solution to the H1N1 influenza virus solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.5%;

[0126] The inactivated pathogen solution was obtained by placing the H1N1 influenza virus solution at 0°C for 50 days.

[0127] In another embodiment, the H1N1 influenza virus solution is placed at 37° C. for at least 60 hours to obtain an inactivated pathogen solution.

[0128] In another embodiment, step (6) can be inactivated by gamma ray, specifically comprising the following steps: gamma ray inactivation varies according to the size of the viral genome. For influenza A virus, radiation D 10 The dose is according to the international standard of 1.60-3.0 kGy.

[0129] Furthermore, the aluminum adjuvant in step (9) is aluminum hydroxide.

[0130] In other embodiments, the concentration of live H1N1 influenza virus may be less than 0.1 mg / ml and the concentration of glutaraldehyde may be less than or greater than 0.005%.

[0131] In other embodiments, the live H1N1 influenza virus can be diluted to less than 1.0 mg / ml, for example 0.9 mg / ml, 0.8 mg / ml, 0.7 mg / ml, 0.6 mg / ml, 0.5 mg / ml, 0.4 mg / ml, 0.3 mg / ml, 0.2 mg / ml, 0.09 mg / ml, 0.08 mg / ml, 0.07 mg / ml, 0.06 mg / ml, 0.05 mg / ml, 0.04 mg / ml, 0.03 mg / ml, 0.02 mg / ml, 0.01 mg / ml, 0.001 mg / ml, 0.0001 mg / ml.

[0132] In other embodiments, the live H1N1 influenza virus can be diluted to greater than 0.00001 mg / ml, for example, 0.0001 mg / ml, 0.001 mg / ml, 0.009 mg / ml, 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, or 0.9 mg / ml.

[0133] In many embodiments, the concentration of glutaraldehyde may be greater than 0.00001%, for example, 0.0001%, 0.0005%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%.

[0134] In many embodiments, the concentration of glutaraldehyde may be less than 0.5%, for example 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, 0.0001%.

[0135] Applicants note that 2-5% glutaraldehyde is usually used as a high-level disinfectant, which may result in precipitation of vaccine samples. The glutaraldehyde cross-linking concentration used is 0.005%, which may not be enough to kill viruses and / or other pathogens in some cases. Therefore, in some embodiments, after 24 to 48 hours of treatment with low concentration (0.005%) of glutaraldehyde at 0-10°C (cold room), formaldehyde or β-propiolactone may be added, for example, at a concentration of about 0.025-2%. This may help to completely eliminate live viruses, for example, greater than 24h, 48h, 60h, 72h, 84h, 96h, and less than 120h, 108h, 96h, 84h, 72h, 60h, 48h, 24h, 12h.

[0136] Aluminum adjuvants include one or more of the following: amorphous aluminum hydroxyphosphate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, commonly used aluminum adjuvants defined and recommended by CDC (US Centers for Disease Control). Available from Millipore Sigma (Product No. 239186-aluminum hydroxide or 255963-aluminum phosphate), or Freund's incomplete adjuvant, or Freund's complete adjuvant. Other pharmaceutically acceptable adjuvants have been approved for use.

[0137] Animal in vivo testing:

[0138] Mice were injected with 10-20ug of glutaraldehyde-treated virus sample + adjuvant in 100ul. The second injection was given two weeks after the first injection, and the third injection was given four weeks after the first injection.

[0139] Serum was collected for HAI testing (anticoagulated blood) 6-8 weeks after the first injection - or from immunized mice 2 and 4 weeks after the third injection. Here, samples were collected 60 days after injection. Samples were sent to VIRAPUR (San Diego, USA) for HAI testing.

[0140] like Figure 1 and Figure 2 It shows that the vaccine serum prepared in this example still produces inhibitory activity after about 80 to 160 times dilution (much greater than the 40-fold dilution required by the CDC standard). Specifically, about 80-160 times dilution has an inhibitory activity against H1N1 (A / Cifornia / 07 / 2009) ( Figure 1 80-fold dilution is effective against H3N2 (A / HongKong / 4801 / 2014) ( Figure 2 shown).

[0141] from Figure 1 and Figure 2 It can be seen that the present invention can improve the effect of influenza A vaccine and increase the neutralization capacity of influenza A vaccine by 8-16 times.

[0142] Interestingly, conventional killing of viruses by formaldehyde did not produce any HAI activity (see Figure 1 and Figure 2 ). These results show that our cross-linking method can effectively neutralize the 2009 California strain H1N1 virus (effective after dilution about 80-160 times) and the 2014 Hong Kong strain H3N2 virus (effective after dilution about 80 times) in a vaccine prepared from the 1934 Puerto Rico H1N1 virus. Therefore, the disclosed cross-linking method and preparation process can effectively create a vaccine with broad efficacy against a variety of viruses from different places, different times, and even different subtypes. That is, the disclosed method is effective in producing universal and long-term vaccines.

[0143] Example 3 - Preparation of Antigen-Stable Influenza B Strain Vaccine

[0144] The influenza vaccine of type B (B) strain was prepared by a method similar to that of Example 2. In addition, the vaccine was produced by a conventional method (i.e., using formaldehyde to kill the virus and prepare the vaccine) as a control. The virus of type B (B) strain was also obtained from Charles River (USA) (Product No.: 10100379, B / Lee / 40).

[0145] Two batches of virus were prepared as follows:

[0146] Preparation of the first batch of influenza B virus vaccines - The preparation method disclosed in the present invention is used to prepare a type B (B) influenza strain vaccine with stable antigens, comprising the following steps:

[0147] (1) Obtaining purified live type B virus (purchased from Charles River Company);

[0148] (2) diluting the live virus obtained in step (1) with a low-temperature buffer until the concentration is below 1.0 mg / ml and proceeding to step (3);

[0149] (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent reaches 0.005%;

[0150] (4) diluting the virus dilution obtained in step (2) with the cross-linker dilution obtained in step (3) for a second time, and proceeding to step (5) when the virus concentration reaches 0.01 mg / ml;

[0151] (5) Place the diluted virus solution in step (4) in a cold room at 10° C. for 24 hours and then proceed to step (6);

[0152] (6) inactivating the virus solution refrigerated in step (5) to obtain an inactivated virus solution;

[0153] (7) Adding glycine to the inactivated virus solution, and then placing at 37° C. for at least 2 hours before proceeding to step (8), wherein:

[0154] The molar ratio of the added glycosylamine to glutaraldehyde is 10:1;

[0155] (8) Concentrate and collect the virus solution treated in step (7), wash twice with physiological saline or HEPES buffer, filter, concentrate and collect, and then proceed to step (9);

[0156] (9) The virus solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 25:200 to obtain a broad-spectrum vaccine.

[0157] Furthermore, the virus described in step (1) has a purity of more than 95%.

[0158] Furthermore, the low temperature in the low temperature buffer in step (2) and step (3) means that the buffer temperature is lower than 10°C, and the buffer is:

[0159] Buffer of 50 mM HEPES, 50-150 mM NaCl, pH 7.0-7.2.

[0160] Furthermore, the cross-linking agent described in step (3) is glutaraldehyde.

[0161] Further, step (6) comprises the following steps:

[0162] Adding an appropriate amount of 95% β-propiolactone aqueous solution to the virus solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.5%;

[0163] The virus solution was placed at 10°C for at least 72 hours to obtain an inactivated virus solution.

[0164] In another embodiment, the virus solution is placed at 37° C. for 24 hours to obtain the inactivated virus solution.

[0165] In another embodiment, step (6) comprises the following steps:

[0166] Adding an appropriate amount of a 37% formaldehyde solution to the virus solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.5%;

[0167] The virus solution was placed at 10°C for at least 50 days to obtain an inactivated virus solution.

[0168] In another embodiment, the virus solution is placed at 37° C. for at least 60 hours to obtain the inactivated virus solution.

[0169] Furthermore, step (6) can be inactivated by gamma rays, specifically comprising the following steps:

[0170] Gamma ray inactivation varies according to the size of the viral genome. For influenza B virus, radiation D 10 The dose is according to the international standard of 2.0-3.0 kGy.

[0171] Furthermore, the aluminum adjuvant in step (9) is aluminum phosphate.

[0172] Preparation of the second batch of influenza B virus vaccine - the purified live virus was diluted to 0.1 mg / ml (10 ml in total) in 50 mM pH 7.2 HEPES buffer, 0.01-2% formaldehyde was added, and cultured at 4-6°C for more than 100 hours or 5-10 days, and quenched again by adding 10-fold molar excess of glycine, and further cultured for 2 hours.

[0173] This batch of virus was concentrated and washed. The treated virus was mixed with or without alum adjuvant. Each mouse was injected with 10-30ug of sample each time. After the first injection, the second injection was given two weeks later and the third injection was given four weeks or one month later. The serum was collected about two months after the first injection. Four groups of mice were immunized as described.

[0174] Group 1: virus treated with formaldehyde plus adjuvant (5 mice in total).

[0175] Group 2: virus treated with formaldehyde, without adjuvant (4 mice in total).

[0176] Group 3: virus treated with glutaraldehyde plus adjuvant (6 mice in total).

[0177] Group 4: virus treated with glutaraldehyde, without adjuvant (4 mice in total).

[0178] Serum was collected and tested for HAI by IITRI (Illinois Institute of Technology, Chicago, USA). Figure 3 As shown, vaccines prepared from B strain viruses had no protective effect against either influenza A (A) strain viruses H1N1 (A / Michigan / H1N1) or H3N2 (A / Singapore / H3N2) (e.g. Figure 3 However, the hepatitis B virus vaccine produced with formaldehyde, i.e., the vaccine prepared by the conventional method (Group 1) with adjuvant, did provide weak protection against a different hepatitis B strain (B / Colorado / 2017), but with an HAI score below 40 (i.e., 40-fold dilution below the standard level) (Conventional + adjuvant, Figure 3 ). This level of inhibition is not effective for protection. Without adjuvant (conventional-adjuvant, no adjuvant, group 2), the conventional vaccine performed even worse. However, the vaccine prepared with glutaraldehyde plus adjuvant had an inhibitory effect even after 80-fold dilution ( Figure 3 , HAI>>80). Similarly, when no adjuvant was used (Group 4), the inhibitory effect decreased (no adjuvant, Figure 3 , 20<HAI<40).

[0179] This data demonstrates that vaccines prepared according to the described methods can provide broad immunity. Specifically, the disclosed cross-linking method was used to produce a vaccine for a type B virus from a 1940 type B virus that was able to protect against another type B virus from Colorado in 2017. These results again demonstrate that our method is effective in producing vaccines for influenza type B strains. These vaccines can protect against a variety of type B strains from different subtypes, even strains obtained at different times and locations. Thus, the vaccines made in this way have universal, long-lasting properties. This method is versatile and can be applied to other viruses that are difficult to produce vaccines for.

[0180] like Figure 3 As described, the cross-linking method and preparation process currently disclosed can be used to make universal vaccines that are effective against various viruses and have a broad spectrum of responses to different strains. The cross-linking method and preparation process currently disclosed can be used to make universal vaccines that are effective against various viruses and have a broad spectrum of responses to variable strains and substrains from different places and times. This method should be suitable for preparing vaccines including influenza, coronaviruses (including SARS-CoV-1, SARS-CoV-2, MER, etc.), rhinoviruses, retroviruses, parainfluenza viruses, Metapneumovirus, respiratory syncytial virus, hepatitis C, HIV and other viruses. The cross-linking method and preparation process currently disclosed can be used to make vaccines against various other pathogens, such as bacteria, fungi, parasites, etc.

[0181] Immunization methods

[0182] One embodiment of the disclosed immunization method is as follows.

[0183] The treated virus was concentrated to 0.1 mg / ml using a concentration tube.

[0184] Wash pelleted virus twice with cold PBS;

[0185] The virus was resuspended in 200 μl PBS.

[0186] Add 200 μl of alum adjuvant and stir vigorously for 1-2 minutes.

[0187] The virus / adjuvant mixture was emulsified in an Eppendorf Thermomix at 1400 rpm for 5 hours at room temperature.

[0188] C57bl / 6 mice were immunized by intramuscular injection of 100 μl / μl (containing about 10-20 μg / μg virus) virus + aluminum adjuvant (first immunization).

[0189] Two weeks after the first immunization, 100 μl (about 10-20 μg / μg virus) virus + aluminum adjuvant was injected intramuscularly and immunized in the same way. (Second immunization)

[0190] Two weeks later, the animals were given a repeat intramuscular injection of 100 μl (~10-20 μg / μg virus) virus / aluminum adjuvant (third immunization).

[0191] 4-6 weeks after the third immunization, collect blood from the tail. Store the serum at -20°C for use the next day, or store at -80°C for long-term storage.

[0192] in conclusion

[0193] It has been a great challenge for people to develop universal and durable vaccines against influenza, COVID-19, HIV and other viruses. It is well known that a universal and durable vaccine should trigger the host to produce broadly neutralizing antibodies, and most of these antibodies are members of the immunoglobulin G family, usually produced by plasma B cells. Plasma B cells are produced by B cells in lymphoid germinal centers after a process of class switching and hypermutation maturation with the help of follicular dendritic cells (FDCs) and follicular T cells. One step in the process relies on FDCs to present antigens in native form throughout the process, which may last for weeks and months (2 months).

[0194] One challenge in the field of immunization is presenting antigens in their native, normal structure for such a long time. This is partly due to the instability of native antigens. In this regard, it has been discovered that adjuvants that rapidly precipitate the antigen may help preserve the native three-dimensional structure of the antigen - allowing the antigen to retain its native structure longer in the body. This is why traditional vaccines rely on adjuvants. However, for influenza, HIV, and other viruses, these traditional approaches do not work well due to the limited number of native, conserved antigens that are the same between strains. The limited number of native shared antigens is partly due to the high mutation rate of many of these viruses. Therefore, it would be very useful to be able to preserve the limited number of native shared antigens in these viruses in their native state after injection. This would increase the amount of time these native shared antigens are available to induce the complex process of B cell maturation in the biocenter.

[0195] Traditional virus inactivation and cross-linking agents, such as formaldehyde or beta-propiolactone, cannot effectively play a cross-linking role, and the addition of artificial disulfide bridges also has limited effect. On the contrary, the applicant unexpectedly found that the use of glutaraldehyde cross-linking agents and other cross-linking agents can provide more effective cross-linking for antigens, which helps to preserve the original structure of the antigen. The disclosed method can also improve the stability of GFP protein in denaturing environments, such as SDS and high temperatures. These GFP experiments show that glutaraldehyde and other cross-linking agents are effective in maintaining protein stability. And GFP is a good system to test this effective cross-linking property.

[0196] Applicants note that high concentrations of glutaraldehyde (e.g., about 2% and above) are a common disinfectant. However, such high concentrations of glutaraldehyde may result in cross-linking between molecules, which may result in precipitation of viral particles or other pathogens. This may also result in failure of internal cross-linking of protein antigens (internal cross-linking, which may help stabilize antigens). Applicants have found that using low concentrations of glutaraldehyde (0.005% or 0.5 mM or less) can solve this problem.

[0197] The applicant has found that the virus after cross-linking with low concentrations of glutaraldehyde (0.005% or 0.5 mM or lower) still has some activity. Thus, further inactivation of the virus is necessary. Traditional inactivation methods can be used, such as, adding formaldehyde or propiolactone, or gamma rays.

[0198] Applicants have shown that the disclosed cross-linking methods and preparation processes are effective for producing vaccines for both influenza A and influenza B. The disclosed cross-linking methods and preparation processes can also be used to produce effective vaccines for other virus types, such as coronaviruses, HIV, and various pathogens, such as bacteria and fungi, and recombinant protein vaccines.

[0199] Example 4 - Development of an effective COVID-19 vaccine

[0200] During the development of a universal flu vaccine, the COVID-19 pandemic (caused by SARS-CoV-2) broke out in late 2019 and spread around the world in early 2020. The SARS-COV-2 virus has now infected nearly 300 million people worldwide (COVID-19 patients) and caused an estimated nearly 5 million deaths (see worldometers.info / coronavirus). Currently, there are no specific drugs to treat COVID-19 patients. Therefore, there is an urgent need to develop vaccines to protect people from SARS-CoV-2. Many candidate vaccines are being developed and used, including several that use: 1) mRNA for the spike protein of SARS-COV-2; 2) DNA for the spike protein via an adenovirus vector; 3) inactivated virus of SARS-COV-2; and 4) recombinant protein of SARS-COV-2.

[0201] However, all of the above methods have different disadvantages, which may hinder the development of an efficient, safe, easy-to-use, conveniently transported, and reasonably priced vaccine for people around the world. Specifically, 1) mRNA as a vaccine has problems such as high production cost, low stability, and high processing requirements (such as low-temperature storage to ensure stability). In addition, most of the expressed spike proteins are also easily denatured in the host body, while only the receptor binding domain of the spike protein is relatively stable due to the four pairs of disulfide bonds, resulting in the escape of new receptor binding domain mutant strains, such as the new Delta strain. 2) Adenovirus as a vector can be destroyed in the body by the immune system of people who have been infected with the virus before, because adenovirus is a common cold virus, most people have a history of infection and have immune memory to destroy new infections. 3) Inactivated viruses also have several disadvantages-first, protein antigens, mainly the spike protein on the surface of SARS-COV-2, are highly variable and unstable, and are easily denatured by formaldehyde or Beta-proiolactone. Traditional virus killing may result in the loss of native antigens of the spike protein. Secondly, there are reports that inactivated virus vaccines may cause antibody-dependent enhancement (ADE) of infection with similar viruses, some slightly different virus strains, such as ADE found in dengue virus; 4) recombinant proteins also have similar ADE issues, as well as low stability issues. All of these shortcomings have led to the emergence of new variants of SARS-COV-2 that can escape or break through even after vaccination with all of the above vaccines. Based on these insights, we are beginning to explore a new approach to develop an effective vaccine against SARS-COV-2 and overcome all of the above shortcomings.

[0202] At present, the above disclosed cross-linking method technology and preparation process are universal for developing vaccines against all other viruses or pathogens, and are also suitable for the preparation of inactivated SARS-CoV-2 viruses. Due to the difficulty in obtaining live SARS-CoV-2 viruses, we only use the receptor binding domain of the spike protein (RBD, residue 319 to residue 541. Among them, residue 387 to residue 502, P0DTC2, as shown below) to develop an effective vaccine against SARS-COV-2. As mentioned above, this vaccine is produced by the method of cross-linking with glutaraldehyde. This method overcomes the above-mentioned shortcomings of all vaccines currently developed for the treatment of SARS-COV-2 infection.

[0203] result

[0204] As mentioned above, cross-linking with glutaraldehyde can stabilize antigens so that they maintain their original conformation in vivo for a long time after immunization. Cross-linked antigens will help the host maintain a long-term immune response, such as providing B cell class switching, B cell hypermutation affinity maturation, the production of persistent plasma B cells in germinal centers (usually producing high-affinity broadly neutralizing immunoglobulin G antibodies), memory B cells, and memory T cells. After a long immune response, the human host will form immune memory to resist future infection with the same virus or a variant version, thereby obtaining long-term protection.

[0205] To create the current vaccine, we created a K417R mutant version of the receptor binding domain. This version of the receptor binding domain is still able to bind to ACE2 after cross-linking with 0.005% glutaraldehyde. Based on the published receptor binding domain of the spike protein of SARS-COV-2 (RBD, residues 319- to residues 541, Figure 4 Only structural information between residues 387 to residue 516) and the human host receptor ACE2 (PDB ID: 6MOJ) is included.

[0206] The primary sequence alignment and rearrangement of the receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) and SARS (SARS-CoV) are as follows: * indicates the amino acid that binds to the receptor ACE2. # indicates that K417,417 lysine mutates to R417 arginine.

[0207]

[0208] It is proposed that an antibody that binds to the RBD surface and blocks ACE2 binding will protect the human host and prevent infection by SARS-CoV-2. In this regard, the RBD domain alone or the entire spike protein could be selected as candidate vaccine antigens against SARS-CoV-2.

[0209] From the structural information and sequence alignment, we found that a lysine residue (K417) is involved in the interaction between the receptor binding domain and ACE2. In order to avoid the reaction of this lysine residue with the cross-linking agent glutaraldehyde, which would lead to the loss of binding to ACE2 after cross-linking, as described above, we created a version of the receptor binding domain with a lysine to arginine (K417R) mutation, as well as a similarly mutated full-length spike protein. Both proteins were expressed in human 293T / F cells. The proteins were also expressed in Hi5 insect cells and yeast. The protein with the mutant version of the receptor binding domain (K417R) can bind to ACE2 in vitro with or without cross-linking (such as Figure 4 However, after cross-linking, the native form of the receptor binding domain no longer binds to ACE2 ( Figure 4 ). Figure 4 It can be seen that 6His-tagged ACE2 can pull out the R471 mutant RBD treated with glutaraldehyde cross-linker, but cannot pull out the RBD without mutation treated with cross-linker.

[0210] The cross-linked version of the 417R receptor binding domain is stable for long periods of time on ice or at room temperature. This is beneficial because vaccine stability is a big issue, especially when delivering vaccines to remote or underdeveloped areas around the world. Figure 5 According to our experiments, the cross-linked K417R receptor binding domain is stable after one week on ice and at room temperature ( Figure 5 However, the native form of the receptor binding domain without cross-linking is degraded after one week ( Figure 5 shown).

[0211] In some embodiments, the cross-linked antigen can be stable for an extended period of time, such as from about 1 week to about 10 weeks, from about 0°C to about 37°C.

[0212] In some embodiments, the cross-linked antigen is at greater than 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 134°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C or 39°C, and less than 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C. 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 13°C, 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, or 1°C.

[0213] In many embodiments, the stability of the disclosed cross-linked antigens is greater than 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 1 year or more, and less than about 2 years, 1 year, 50 weeks, 40 weeks, 30 weeks, 25 weeks, 20 weeks, 15 weeks, 10 weeks, 9 weeks, 8 weeks, 7 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week.

[0214] The cross-linked version of the K417R receptor binding domain produced significantly higher (approximately 3-fold) titers of IgG molecules in mice compared to IgG titers in mice immunized with the native form of the receptor binding domain. IgG titers against the receptor binding domain were determined as described above. Mice immunized with the cross-linked receptor binding domain had higher (approximately 3-fold) titers two months after immunization compared to mice immunized with the native form of the receptor binding domain, either as a single immunization or as two additional booster immunizations two weeks apart (a total of three immunizations, each with approximately 10-20ug of the receptor binding domain sample) ( Figure 6 ).

[0215] After three months, the immunoglobulin G titer remained high ( Figure 7 ). This result indicates that the cross-linked version of the K417R receptor binding domain is more effective in eliciting an immune response than the non-cross-linked version of the receptor binding domain. In many embodiments, the cross-linked antigen can increase the immunoglobulin G titer of the subject by about 3 to more times compared to the non-cross-linked antigen.

[0216] Compared with the native form of the receptor binding domain, the cross-linked version of the K417R receptor binding domain can produce a wider range of neutralizing antibodies, thereby blocking the binding of ACE2. We found that the blocking ability of mouse serum after immunization with the cross-linked K417R receptor binding domain to ACE2 is much higher than that of the native form of the receptor binding domain ( Figure 8 ). 40μg / ml of the native receptor binding domain was coated on the ELISA plate overnight and blocked with 30% FBS in PBS. Dilution buffer was also used alone for signal correction. Mouse serum was diluted 1000-fold in dilution buffer and incubated on the plate for 1 hour at room temperature. The plate was then washed three times. 50μg / ml ACE2-6his-tagged was added and incubated for 1 hour at room temperature. The plate was then washed three times. Biotinylated anti-6His*histidine tag antibody (Biorad MCA1396B) was then added and incubated for 1 hour at room temperature. The plate was then washed three times. Streptavidin-AP was then added and incubated for 1 hour at room temperature. The plate was washed five times. AP substrate was added. At different time points after substrate, the absorbance was read at 405nM. The data are expressed as the ratio of the absorbance of mouse serum divided by the absorbance of dilution buffer alone. The final bound ACE2 reading reflects the degree of blocking by the antibody. The less ACE2 detected on the ELISA plate (reading), the higher the blocking property of the antibody ( Figure 8 ).

[0217] Other cross-linking agents, such as bissulfosuccinimide (BS3), dimethyl adipate (DMA), dimethyl ferrite (DMS) and dimethyl pyrimidine (DMP), etc., are also effective in making the disclosed vaccines. As long as they can cross-link chemical groups within the protein antigen (internal cross-linking) and stabilize the protein antigen. In one embodiment, such a cross-linking agent is a bissulfosuccinimide ester (BS3) as shown below, and its structure is as follows:

[0218]

[0219] In one embodiment, such a cross-linking agent is dimethyl adipate (DMA) as shown below:

[0220]

[0221] In many embodiments, the disclosed cross-linking agents are useful in preparing the above antigens and vaccines, and can covalently link different parts of the protein while maintaining the three-dimensional structure of the protein. The ability of BS3 / DMA / DMS / DMP to cross-link receptor binding domains was analyzed. Similar to the above studies, BS3 / DMA / DMS / DMP has similar stability and effectiveness in providing receptor binding domain antigens compared to glutaraldehyde cross-linking. In addition, cross-linking agents that covalently link free amines and other groups are useful in this method. The disclosed reagents can link free amines, as well as other groups. This is the basis for cross-linking other groups from different residues in the protein selected as the antigen.

[0222] Described here are cross-linking processes for creating highly effective vaccines against a variety of targets, including SARS-COV-2 and influenza. In many embodiments, the disclosed vaccines can be prepared using a small region of the target, such as the receptor binding domain region of SARS-COV-2. This can greatly reduce the likelihood that the disclosed vaccines will suffer from antibody-dependent enhancement, as has been seen with other vaccines. Due to the small size of the receptor binding domain and the large sequence differences in the receptor binding domain region between coronaviruses, we believe that our vaccines will also overcome the antibody-dependent enhancement problem of vaccines caused by inactive virus or the entire spike protein. Interestingly, two recent reports found that more than 90% of broadly neutralizing antibodies in sera from COVID-19 patients or vaccine recipients were directed against the receptor binding domain of the spike protein (Greaney et al., 2021; Piccoli et al., 2020), indicating that the human immune system also uses the receptor binding domain as a primary antigen to generate broadly neutralizing antibodies against SARS-COV-2.

[0223] Applicants note that most cross-linking agents work through free amino groups on lysine residues, which can be involved in host and pathogen recognition. To avoid the loss of epitopes after cross-linking, intentionally replacing lysine residues with arginine in a single recombinant protein antigen can avoid this problem, as we did in the receptor binding domain of SARS-COV-2.

[0224] Example 5

[0225] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0226] (1) Obtaining live pathogens;

[0227] (2) diluting the live pathogen obtained in step (1) with a low-temperature buffer solution until the concentration is below 1.0 mg / ml and then proceeding to step (3);

[0228] (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent is less than 0.005%;

[0229] (4) diluting the pathogen dilution obtained in step (2) with the cross-linking agent dilution obtained in step (3) for a second time, and proceeding to step (5) when the pathogen concentration reaches 0.01 mg / ml;

[0230] (5) placing the pathogen solution diluted in step (4) in a cold room at 0°C for 48 hours and then proceeding to step (6);

[0231] (6) inactivating the pathogen liquid refrigerated in step (5) to obtain an inactivated pathogen liquid;

[0232] (7) Add glycine to the inactivated pathogen solution, and then place at 37° C. for at least 2 hours before proceeding to step (8), wherein:

[0233] The molar ratio of the added glycosylamine to glutaraldehyde is 5:1;

[0234] (8) Concentrate and collect the pathogen solution treated in step (7), wash with physiological saline or HEPES buffer, filter, concentrate and collect, and then proceed to step (9);

[0235] (9) The pathogen solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 10:200 to obtain a broad-spectrum vaccine.

[0236] Further, the pathogen in step (1) is a virus. In one embodiment, it is a coronavirus. In another embodiment, it is an influenza virus. In another embodiment, it is a retrovirus. In another embodiment, it is a rhino virus.

[0237] Furthermore, the pathogen in step (1) has a purity of more than 95%.

[0238] Furthermore, the low temperature in the low temperature buffer in step (2) and step (3) means that the buffer temperature is lower than 10°C, and the buffer is physiological saline.

[0239] Furthermore, the cross-linking agent described in step (3) is succinaldehyde.

[0240] Further, step (6) comprises the following steps:

[0241] Adding an appropriate amount of a β-propiolactone aqueous solution with a mass concentration of more than 90% to the pathogen solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.01%;

[0242] The pathogen solution is placed at 0°C for at least 72 hours to obtain an inactivated pathogen solution; or

[0243] The pathogen solution was inactivated by placing the pathogen solution at 18°C ​​for at least 24 hours.

[0244] Further, step (6) comprises the following steps:

[0245] Adding an appropriate amount of a 36% formaldehyde solution to the pathogen solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.025%;

[0246] The pathogen solution is placed at 0°C for at least 50 days to obtain an inactivated pathogen solution; or

[0247] The pathogen solution was placed at 37° C. for at least 60 hours to obtain an inactivated pathogen solution.

[0248] Furthermore, step (6) can be inactivated by gamma rays, specifically comprising the following steps:

[0249] Gamma ray inactivation varies according to the size of the viral genome. For the new coronavirus SARS-CoV-2, radiation D 10 The dose is the international standard of 2.0-5.0 kGy;

[0250] For influenza A virus, radiation D is needed 10 The dose is 1.60-3.0 kGy according to the international standard;

[0251] For influenza B virus, radiation D is required 10 The dose is according to the international standard of 2.0-3.0 kGy.

[0252] Furthermore, the aluminum adjuvant in step (9) is aluminum hydroxide.

[0253] Example 6

[0254] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0255] (1) Obtaining live pathogens;

[0256] (2) diluting the live pathogen obtained in step (1) with a low-temperature buffer solution until the concentration is below 1.0 mg / ml and then proceeding to step (3);

[0257] (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent is less than 0.005%;

[0258] (4) diluting the pathogen dilution obtained in step (2) with the cross-linking agent dilution obtained in step (3) for a second time, and proceeding to step (5) when the pathogen concentration reaches 0.001 mg / ml;

[0259] (5) placing the pathogen solution diluted in step (4) in a cold room at 10° C. for 24 hours and then proceeding to step (6);

[0260] (6) inactivating the pathogen liquid refrigerated in step (5) to obtain an inactivated pathogen liquid;

[0261] (7) Add glycine to the inactivated pathogen solution, and then place at 37° C. for at least 2 hours before proceeding to step (8), wherein:

[0262] The molar ratio of the added glycosylamine to glutaraldehyde is 10:1;

[0263] (8) Concentrate and collect the pathogen solution treated in step (7), wash twice with physiological saline or HEPES buffer, filter, concentrate and collect, and then proceed to step (9);

[0264] (9) The pathogen solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 25:800 to obtain a broad-spectrum vaccine.

[0265] Furthermore, the pathogen in step (1) is a bacterium.

[0266] Furthermore, the pathogen in step (1) has a purity of more than 95%.

[0267] Furthermore, the low temperature in the low temperature buffer in step (2) and step (3) refers to a buffer temperature below 10°C, and the buffer is PBS buffer.

[0268] Furthermore, the cross-linking agent described in step (3) is adipaldehyde.

[0269] Further, step (6) comprises the following steps:

[0270] Adding an appropriate amount of a β-propiolactone aqueous solution having a mass concentration of more than 90% to the pathogen solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.5%;

[0271] The pathogen solution is placed at 10°C for at least 72 hours to obtain an inactivated pathogen solution; or

[0272] The pathogen solution was inactivated by placing the pathogen solution at 37°C for at least 24 hours.

[0273] Further, step (6) comprises the following steps:

[0274] Adding an appropriate amount of a 36-37% formaldehyde solution to the pathogen solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.5%;

[0275] The pathogen solution is placed at 10°C for at least 50 days to obtain an inactivated pathogen solution; or

[0276] The pathogen solution was placed at 37° C. for at least 60 hours to obtain an inactivated pathogen solution.

[0277] Furthermore, the aluminum adjuvant in step (9) is amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHS).

[0278] Example 7

[0279] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0280] (1) Obtaining live pathogens;

[0281] (2) diluting the live pathogen obtained in step (1) with a low-temperature buffer solution until the concentration is below 1.0 mg / ml and then proceeding to step (3);

[0282] (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent is less than 0.005%;

[0283] (4) diluting the pathogen dilution obtained in step (2) with the cross-linking agent dilution obtained in step (3) for a second time, and proceeding to step (5) when the pathogen concentration reaches 0.005 mg / ml;

[0284] (5) Place the pathogen solution diluted in step (4) in a cold room at 4° C. for 36 hours and then proceed to step (6);

[0285] (6) inactivating the pathogen liquid refrigerated in step (5) to obtain an inactivated pathogen liquid;

[0286] (7) Add glycine to the inactivated pathogen solution, and then place at 37° C. for at least 2 hours before proceeding to step (8), wherein:

[0287] The molar ratio of the added glycosylamine to glutaraldehyde is 8:1;

[0288] (8) Concentrate and collect the pathogen solution treated in step (7), wash it once or twice with physiological saline or HEPES buffer, filter, concentrate and collect it, and then proceed to step (9);

[0289] (9) The pathogen solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 15:600 to obtain a broad-spectrum vaccine.

[0290] Furthermore, the pathogen in step (1) is a fungus.

[0291] Furthermore, the pathogen in step (1) has a purity of more than 95%.

[0292] Further, the low temperature in the low temperature buffer in step (2) and step (3) refers to a buffer temperature below 10°C, and the buffer is a buffer of 50mM HEPES, 50-150mM NaCl, pH 7.0. In another embodiment, the buffer is a buffer of 50mM HEPES, 50-150mM NaCl, pH 7.2. In another embodiment, the buffer is a buffer of 50mM HEPES, 50-150mM NaCl, pH 7.1.

[0293] Furthermore, the cross-linking agent in step (3) is heptanedial.

[0294] Further, step (6) comprises the following steps:

[0295] Adding an appropriate amount of a β-propiolactone aqueous solution having a mass concentration of more than 90% to the pathogen solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.1%;

[0296] The pathogen solution is placed at 4°C for at least 72 hours to obtain an inactivated pathogen solution; or

[0297] The pathogen solution was placed at 30°C for at least 24 hours to obtain an inactivated pathogen solution.

[0298] Further, step (6) comprises the following steps:

[0299] Adding an appropriate amount of a 36.5% formaldehyde solution to the pathogen solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.4%;

[0300] The pathogen solution is inactivated by placing the pathogen solution at 4°C for at least 50 days; or

[0301] The pathogen solution was placed at 30° C. for at least 60 hours to obtain an inactivated pathogen solution.

[0302] Furthermore, the aluminum adjuvant in step (9) is one of potassium aluminum sulfate.

[0303] Embodiment 8-18

[0304] The same as Example 7, the only difference is that the cross-linking agent is different

[0305] Crosslinking agent Example 8 Suberaldehyde Example 9 Azelaic acid Example 10 Secalinaldehyde Embodiment 11 Disuccinimidyl suberate Example 12 Dimethyl succinate Example 13 Dimethyl glutarate Embodiment 14 Dimethyl adipate Embodiment 15 Dimethyl pimelate Example 16 Dimethyl suberate Embodiment 17 Dimethyl Azelate Embodiment 18 Dimethyl sebacate

[0306] Embodiment 19

[0307] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0308] S1. Obtain recombinant proteins of pathogens;

[0309] S2, diluting the recombinant protein obtained in step S1 with a low-temperature buffer, and proceeding to step S3 after the concentration of the recombinant protein is below 1.0 mg / ml;

[0310] S3, diluting the cross-linking agent with a low-temperature buffer, and proceeding to step S4 after the mass concentration of the cross-linking agent is below 0.005%;

[0311] S4, diluting the recombinant protein solution obtained in step S2 with the cross-linking agent diluent obtained in step S3, and proceeding to step S5 after the concentration of the recombinant protein reaches 0.01 mg / ml;

[0312] S5, placing the recombinant protein solution treated in step S4 in a cold room at 0°C for 48 hours and then proceeding to step S6;

[0313] S6, adding glycine to the recombinant protein solution refrigerated in step S5, and then placing it at 37° C. for at least 2 hours, and then proceeding to S7, wherein:

[0314] The molar ratio of glycine to glutaraldehyde added was 5:1;

[0315] S7, concentrating and collecting the recombinant protein solution treated in step S6, and washing it once with physiological saline, filtering, concentrating and collecting it, and then proceeding to step S8;

[0316] S8. The recombinant protein solution treated in step S7 is mixed with an aluminum adjuvant at a mass ratio of 10:200 to obtain a broad-spectrum vaccine.

[0317] Further, the recombinant protein of the pathogen in step S1 is derived from a virus. In one embodiment, the recombinant protein of the pathogen in step S1 is derived from a coronavirus. In one embodiment, the recombinant protein of the pathogen in step S1 is derived from an influenza virus. In one embodiment, the recombinant protein of the pathogen in step S1 is derived from a retrovirus. In one embodiment, the recombinant protein of the pathogen in step S1 is derived from a rhino virus.

[0318] Furthermore, the purity of the recombinant protein of the pathogen in step S1 is above 95%.

[0319] Furthermore, the low temperature in the low temperature buffer in step S2 and step S3 means that the buffer temperature is lower than 10° C., and the buffer is physiological saline.

[0320] Furthermore, the cross-linking agent described in step S3 is succinaldehyde.

[0321] Furthermore, the aluminum adjuvant in step S8 is aluminum hydroxide.

[0322] Embodiment 20

[0323] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0324] S1. Obtain recombinant proteins of pathogens;

[0325] S2, diluting the recombinant protein obtained in step S1 with a low-temperature buffer, and proceeding to step S3 after the concentration of the recombinant protein is below 1.0 mg / ml;

[0326] S3, diluting the cross-linking agent with a low-temperature buffer, and proceeding to step S4 after the mass concentration of the cross-linking agent is below 0.005%;

[0327] S4, diluting the recombinant protein solution obtained in step S2 with the cross-linking agent diluent obtained in step S3, and proceeding to step S5 after the concentration of the recombinant protein reaches 0.001 mg / ml;

[0328] S5, placing the recombinant protein solution treated in step S4 in a cold room at 10°C for 24 hours and then proceeding to step S6;

[0329] S6, adding glycine to the recombinant protein solution refrigerated in step S5, and then placing it at 37° C. for at least 2 hours, and then proceeding to S7, wherein:

[0330] The molar ratio of glycine to glutaraldehyde added was 10:1;

[0331] S7, concentrating and collecting the recombinant protein solution treated in step S6, and washing it once or twice with physiological saline or HEPES buffer, filtering, concentrating and collecting it, and then proceeding to step S8;

[0332] S8. The recombinant protein solution treated in step S7 is mixed with an aluminum adjuvant in a mass ratio of 25:800 to obtain a broad-spectrum vaccine.

[0333] Furthermore, the recombinant protein of the pathogen in step S1 is derived from bacteria.

[0334] Furthermore, the purity of the recombinant protein of the pathogen in step S1 is above 95%.

[0335] Furthermore, the low temperature in the low temperature buffer in step S2 and step S3 means that the buffer temperature is lower than 10° C., and the buffer is PBS buffer.

[0336] Furthermore, the cross-linking agent in step S3 is glutaraldehyde.

[0337] Furthermore, the aluminum adjuvant in step S8 is aluminum phosphate.

[0338] Embodiment 21

[0339] The preparation method of the broad-spectrum vaccine comprises the following steps:

[0340] S1. Obtain recombinant proteins of pathogens;

[0341] S2, diluting the recombinant protein obtained in step S1 with a low-temperature buffer, and proceeding to step S3 after the concentration of the recombinant protein is below 1.0 mg / ml;

[0342] S3, diluting the cross-linking agent with a low-temperature buffer, and proceeding to step S4 after the mass concentration of the cross-linking agent is below 0.005%;

[0343] S4, diluting the recombinant protein solution obtained in step S2 with the cross-linking agent diluent obtained in step S3, and proceeding to step S5 after the concentration of the recombinant protein reaches 0.003 mg / ml;

[0344] S5, placing the recombinant protein solution treated in step S4 in a cold room at 4°C for 36 hours and then proceeding to step S6;

[0345] S6, adding glycine to the recombinant protein solution refrigerated in step S5, and then placing it at 37° C. for at least 2 hours, and then proceeding to S7, wherein:

[0346] The molar ratio of glycine to glutaraldehyde added was 8:1;

[0347] S7, concentrating and collecting the recombinant protein solution treated in step S6, and washing it once or twice with physiological saline or HEPES buffer, filtering, concentrating and collecting it, and then proceeding to step S8;

[0348] S8. The recombinant protein solution treated in step S7 is mixed with aluminum adjuvant in a mass ratio of 15:450 to obtain a broad-spectrum vaccine.

[0349] Furthermore, the recombinant protein of the pathogen in step S1 is derived from fungi.

[0350] Furthermore, the purity of the recombinant protein of the pathogen in step S1 is above 95%.

[0351] Further, the low temperature in the low temperature buffer in step S2 and step S3 means that the buffer temperature is less than 10°C, and the buffer is a buffer of 50mM HEPES, 50-150mM NaCl, pH 7.0. In another embodiment, the buffer is a buffer of 50mM HEPES, 50-150mM NaCl, pH 7.2. In another embodiment, the buffer is a buffer of 50mM HEPES, 50-150mM NaCl, pH 7.1.

[0352] Furthermore, the cross-linking agent described in step S3 is adipaldehyde.

[0353] Further, the aluminum adjuvant in step S8 is amorphous aluminum hydroxyphosphate adjuvant (AAHS). In another embodiment, the aluminum adjuvant is potassium aluminum sulfate.

[0354] Examples 22-33

[0355] It is substantially the same as Example 21, except that the cross-linking agent is different:

[0356] Crosslinking agent Embodiment 22 Heptanedial Embodiment 23 Suberaldehyde Embodiment 24 Azelaic acid Embodiment 25 Secalin Embodiment 26 Disuccinimidyl suberate Embodiment 27 Dimethyl succinate Embodiment 28 Dimethyl glutarate Embodiment 29 Dimethyl adipate Embodiment 30 Dimethyl pimelate Embodiment 31 Dimethyl suberate Embodiment 32 Dimethyl Azelate Embodiment 33 Dimethyl sebacate

[0357] The above describes the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for preparing a broad-spectrum vaccine, characterized in that: The following steps are involved: (1) Obtaining live pathogens; (2) diluting the live pathogen obtained in step (1) with a low-temperature buffer solution until the concentration is below 1.0 mg / ml and then proceeding to step (3); (3) diluting the cross-linking agent with a low-temperature buffer solution, and proceeding to step (4) when the mass concentration of the cross-linking agent reaches 0.005%; (4) diluting the pathogen dilution obtained in step (2) with the cross-linking agent dilution obtained in step (3) for a second time, and proceeding to step (5) when the pathogen concentration reaches 0.01 to 0.001 mg / ml; (5) placing the pathogen solution diluted in step (4) in a cold room at 0°C to 10°C for 24 to 48 hours and then proceeding to step (6); (6) inactivating the pathogen liquid refrigerated in step (5) to obtain an inactivated pathogen liquid; (7) Add glycine to the inactivated pathogen solution, and then place at 37° C. for at least 2 hours before proceeding to step (8), wherein: The molar ratio of the added glycosylamine to glutaraldehyde is 5 to 10:1; (8) Concentrate and collect the pathogen solution treated in step (7), wash it once or twice with physiological saline or HEPES buffer, filter, concentrate and collect it, and then proceed to step (9); (9) The pathogen solution treated in step (8) is mixed with an aluminum adjuvant at a mass ratio of 10-25:200-800 to obtain a broad-spectrum vaccine, wherein: The pathogen in step (1) is influenza virus type B (B); The low temperature in the low temperature buffer in step (2) and step (3) means that the buffer temperature is lower than 10°C; The cross-linking agent described in step (3) is glutaraldehyde; The aluminum adjuvant in step (9) is aluminum phosphate.

2. The method for preparing a broad-spectrum vaccine according to claim 1, characterized in that: The pathogen in step (1) has a purity of more than 95%.

3. The method for preparing a broad-spectrum vaccine according to claim 1, characterized in that: The low temperature buffer in step (2) and step (3) is one of the following three: a. Normal saline; b. PBS buffer; c. 50mM HEPES, 50-150mM NaCl, pH 7.0-7.2 buffer.

4. The method for preparing a broad-spectrum vaccine according to claim 1, characterized in that: Step (6) comprises the following steps: Adding an appropriate amount of a β-propiolactone aqueous solution with a mass concentration of more than 90% to the pathogen solution refrigerated in step (5), and stopping adding β-propiolactone when the mass concentration of β-propiolactone reaches 0.01% to 0.5%; The pathogen solution is placed at 0-10°C for at least 72 hours to obtain an inactivated pathogen solution; or The pathogen solution is placed at 18°C ​​to 37°C for at least 24 hours to obtain an inactivated pathogen solution.

5. The method for preparing a broad-spectrum vaccine according to claim 1, characterized in that: Step (6) comprises the following steps: Adding an appropriate amount of a 36-37% formaldehyde solution to the pathogen solution refrigerated in step (5), and stopping adding formaldehyde when the formaldehyde concentration reaches 0.025-0.5%; The pathogen solution is placed at 0-10°C for at least 50 days to obtain an inactivated pathogen solution; or The pathogen solution was placed at 37° C. for at least 60 hours to obtain an inactivated pathogen solution.

6. The method for preparing a broad-spectrum vaccine according to claim 1, characterized in that: Step (6) can be inactivated by gamma rays, and specifically comprises the following steps: For influenza B virus, radiation D is required 10 The dose is the international standard of 2.0-3.0 kGy.

7. A method for preparing a broad-spectrum vaccine, characterized in that: The following steps are involved: S1. Obtain recombinant proteins of pathogens; S2, diluting the recombinant protein obtained in step S1 with a low-temperature buffer, and proceeding to step S3 after the concentration of the recombinant protein is below 1.0 mg / ml; S3, diluting the cross-linking agent with a low-temperature buffer, and proceeding to step S4 after the mass concentration of the cross-linking agent reaches 0.005%; S4, diluting the recombinant protein solution obtained in step S2 with the cross-linking agent diluent obtained in step S3, and proceeding to step S5 after the concentration of the recombinant protein reaches 0.01-0.001 mg / ml; S5, placing the recombinant protein solution treated in step S4 in a cold room at 0°C to 10°C for 24-48 hours and proceeding to step S6; S6, adding glycine to the recombinant protein solution refrigerated in step S5, and then placing it at 37° C. for at least 2 hours, and then proceeding to S7, wherein: The molar ratio of added glycosylamine to glutaraldehyde is 5-10:1; S7, concentrating and collecting the recombinant protein solution treated in step S6, and washing it once or twice with physiological saline or HEPES buffer, filtering, concentrating and collecting it, and then proceeding to step S8; S8, mixing the recombinant protein solution treated in step S7 with an aluminum adjuvant in a mass ratio of 10-25:200-800 to obtain a broad-spectrum vaccine, wherein: The recombinant protein of the pathogen in step S1 is a recombinant protein of influenza virus type B (B); The low temperature in the low temperature buffer in step S2 and step S3 means that the buffer temperature is lower than 10°C; The cross-linking agent in step S3 is glutaraldehyde; The aluminum adjuvant in step S8 is aluminum phosphate.

8. The method for preparing a broad-spectrum vaccine according to claim 7, characterized in that: The purity of the recombinant protein of the pathogen in step S1 is above 95%.

9. The method for preparing a broad-spectrum vaccine according to claim 7, characterized in that: The low temperature buffer in step S2 and step S3 is one of the following three: a. Normal saline; b. PBS buffer; c. 50mM HEPES, 50-150mM NaCl, pH 7.0-7.2 buffer.

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