Spray of canine distemper subunit vaccine

Abstract

The invention belongs to the technical field of vaccines, and provides a canine distemper subunit vaccine spray which is formed by dissolving nanoparticles carrying key antigen epitope peptide in normal saline in the form of freeze-dried powder. Comprising nanoparticles carrying key antigen epitope peptide and a buffer solution, and the nanoparticles are dissolved in the buffer solution in the form of freeze-dried powder; the mass ratio of the nanoparticles to the buffer solution in the spray is (0.1-10): 1; the nano-particles are composed of epitope peptide of the hemagglutinin protein of the canine distemper virus and a nano-carrier. The spray type canine distemper vaccine is convenient to store and transport, meanwhile, the stability of antigens, the convenience of inoculation and the safety and effectiveness of the vaccine are guaranteed, and large-scale inoculation of the vaccine can be rapidly promoted.

Description

Technical Field

[0001] The invention relates to a dosage form of a canine distemper subunit vaccine, in particular to a spray of the canine distemper subunit vaccine. Background Art

[0002] Canine distemper virus seriously endangers the health of pet dogs and fur economic animals, and can infect seals, wild boars and non-human primates. Although there are cures, the problems of cross-infection, secondary infection and late immunosuppression still make active immunization the most effective way to prevent and control canine distemper. Currently, most canine distemper vaccines on the market are multivalent combination vaccines injected intramuscularly or subcutaneously, mainly attenuated live vaccines produced by chicken embryos or cells. The attenuated live vaccine produces virulence reversion in individuals with low immune capacity, making the vaccinated animals a source of transmission, and the spread of canine distemper virus through the respiratory tract and raw food indirectly leads to the prevalence of canine distemper virus among wild species. In addition, canine distemper virus is a negative-strand RNA virus, which is very prone to a large number of mutations. When its key pathogenic protein sites mutate, the existing vaccine cannot provide effective immune protection for the vaccine recipient. Therefore, it is urgent to develop a canine distemper vaccine spray that is effective against most canine distemper viruses, does not rely on chicken embryos and cells, and can be quickly and massively inoculated. Epitope vaccines have the potential to focus immune responses on conserved epitopes, thereby increasing the breadth of immunity. They are also simple to manufacture and can be quickly prepared in Escherichia coli, which is low-cost and more suitable for large-scale production. Hemagglutinin protein plays a primary role in receptor recognition and binding during the infection stage of canine distemper virus and participates in the virus fusion process. This feature also makes hemagglutinin protein a key target for the development of antibodies that can induce the production of neutralizing antibodies against canine distemper virus. Therefore, epitope vaccines screened based on hemagglutinin protein can be combined with nanocarriers to prepare a spray of canine distemper subunit vaccine.

[0003] The shortcomings and problems of current canine distemper vaccine technology mainly include the following:

[0004] 1. Limited scope of protection: Canine distemper virus is a negative-strand RNA virus that is prone to a large number of mutations, which limits the protective effect of the vaccine.

[0005] 2. Complex and time-consuming production: The production process of attenuated canine distemper vaccine requires culturing the virus in chicken embryos or cells and processing it to make the vaccine. This production method is complex, time-consuming and highly dependent, which is not conducive to quickly responding to virus mutations.

[0006] 3. Annual vaccination is required: The antibodies produced by the existing canine distemper vaccine can only last for one year, so it needs to be re-vaccinated intramuscularly or subcutaneously every year, which brings a heavy burden to the public health system.

[0007] 4. Challenges in the development of canine distemper vaccine: Subcutaneous and intramuscular injections are more complicated, and spray form is the best option for canine distemper vaccination. However, there is still a lack of non-toxic immunogens that can be administered in spray form. Summary of the invention

[0008] In view of the above technical problems, one of the objectives of the present invention is to provide a spray of canine distemper subunit vaccine, which comprises nanoparticles carrying key antigen epitope peptides and a buffer solution, wherein the nanoparticles are dissolved in the buffer solution in the form of lyophilized powder.

[0009] The second object of the present invention is to provide a canine distemper subunit vaccine, the immunogen of which is derived from one or more epitope peptides of the canine distemper virus hemagglutinin protein and a combination of different copy numbers.

[0010] The third object of the present invention is to provide a canine distemper subunit vaccine, wherein the immunogen is expressed in the form of fusion, cross-linking or mixing of epitope peptides and nanocarriers.

[0011] The fourth object of the present invention is to provide a spray of canine distemper subunit vaccine, wherein one or more of electrolyte salts, amino acids, pH buffers, antioxidants, glycerol and sucrose are added to the buffer.

[0012] A fifth object of the present invention is to provide a spray of canine distemper subunit vaccine, wherein the spray is a separate package or a premix of lyophilized powder and buffer.

[0013] The sixth object of the present invention is to provide a spray of canine distemper subunit vaccine, which may be free of adjuvant or contain adjuvant, and the adjuvant is a cytokine adjuvant, a toxin-based adjuvant, a liposome, a polymer, but is not limited to these adjuvants.

[0014] The seventh object of the present invention is to provide a canine distemper subunit vaccine, whose carrier is a Norovirus P particle carrier or a Ferritin carrier, but is not limited to these nanocarriers.

[0015] The eighth object of the present invention is to provide a spray of canine distemper subunit vaccine, and its immunization route is nasal spray and inhalation, but is not limited to these routes.

[0016] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0017] First, the present invention provides a spray of canine distemper subunit vaccine, which is painless and can quickly achieve large-scale animal immunization, thereby improving the convenience of existing attenuated canine distemper vaccines. The vaccine is a protein subunit vaccine, which is safe and non-toxic, and improves the applicability of existing attenuated canine distemper vaccines. The dominant epitope peptide is prepared from Escherichia coli, which is simple and rapid to produce, safe and effective to use, and has broad application prospects in the research and development of anti-canine distemper virus vaccines, antibodies, and drugs.

[0018] The present invention provides a canine distemper subunit vaccine spray, which comprises nanoparticles carrying key antigen epitope peptides and a buffer solution; the epitope peptides are antigen components, which can be prepared in Escherichia coli, can stimulate effective adaptive immune responses and improve the scope of vaccine protection; the spray is easy to vaccinate and has incomparable advantages over traditional vaccines.

[0019] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0020] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0021] The invention provides a spray of canine distemper subunit vaccine, which can be prepared based on Escherichia coli and is significantly superior to traditional vaccines in terms of production cost, production speed, safety, protection scope, vaccination route, etc.

[0022] (2) Whether the technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0023] The invention provides a canine distemper subunit vaccine spray, which can quickly complete large-scale vaccination of the vaccine and effectively control the virus transmission chain in a short time.

[0024] (3) Whether the technical solution of the present invention overcomes technical prejudice:

[0025] The technical solution overcomes the problem that the existing canine distemper vaccine cannot be produced without chicken embryos or cells; the technical solution overcomes the problem that the existing canine distemper vaccine can only be administered by subcutaneous or intramuscular injection.

[0026] Third, the canine distemper subunit vaccine spray provided by the present invention comprises the following technical features and innovations:

[0027] 1) Nanoparticles of key antigen epitope peptides: This vaccine uses nanoparticles to carry the key antigen epitope peptides of canine distemper virus. The use of nanoparticles can improve the stability of the antigen and the efficiency of antigen release.

[0028] 2) Use of multiple antigens: The antigen can be one or more epitope peptides of the canine distemper virus hemagglutinin protein, as well as combinations of different copy numbers. This can increase the immune coverage of the vaccine.

[0029] 3) Nanoparticle presentation: Epitope peptides can be fused, cross-linked or mixed with nanocarriers. This design can improve the bioavailability and immune effect of the vaccine.

[0030] 4) Composition of buffer: The buffer may contain electrolyte salts, amino acids, pH buffers, antioxidants, glycerol, sucrose, etc. These ingredients can help maintain the stability of the vaccine.

[0031] 5) Spray packaging: The spray can be packaged separately or premixed with freeze-dried powder and buffer. This design can facilitate storage and use.

[0032] 6) Use of adjuvants: The vaccine may be adjuvant-free or may contain adjuvants, such as cytokine adjuvants, toxin-based adjuvants, liposomes, polymers, etc. Adjuvants can enhance the immune effect of vaccines.

[0033] 7) Types of nanocarriers: Nanocarriers can be norovirus P particle carriers, Ferritin nanocarriers, etc., providing more options for vaccine design.

[0034] 8) Vaccine immunization route: The vaccine immunization route can be nasal spray, inhalation, etc. This design is easier to administer and can directly stimulate the immune response of the respiratory tract.

[0035] The technical features and innovations provided by the present invention bring about significant technological progress, which can improve the immune effect of the vaccine and also facilitate the storage and use of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a sequence analysis of the canine distemper virus hemagglutinin protein; A. Conservative analysis of amino acids at each position of the canine distemper virus hemagglutinin protein; B. Surface schematic diagram of the canine distemper Yanaka strain hemagglutinin protein;

[0037] Figure 2 The transmission electron microscopy observation image of the fusion protein of the epitope peptide and the Ferritin vector;

[0038] Figure 3 This is a schematic diagram of a canine distemper subunit vaccine spray;

[0039] Figure 4 The following are the mucosal immune detection images of mice immunized with canine distemper subunit vaccine spray; A. Total sIgA concentration in nasal wash fluid (NLF) and bronchoalveolar lavage fluid (BALF); B. sIgA titer of anti-hemagglutinin protein in NLF and BALF;

[0040] Figure 5 This is a humoral immune detection diagram after mice were immunized with canine distemper subunit vaccine spray; A. specific binding antibody titer in serum; B. neutralizing antibody titer in serum;

[0041] Figure 6 This is a cell-mediated immune detection image of mice immunized with canine distemper subunit vaccine spray; A. Mouse CD3 + CD4 + 、CD3 + CD8 + T cell proliferation assay; B. Peptide-specific in vivo CTL assay. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] The present invention provides a spray for measuring canine distemper subunit vaccine, comprising the following specific components:

[0044] 1. Antigenic epitope peptides: These are one or more antigenic epitope peptides extracted from the hemagglutinin protein of canine distemper virus. Their function is to simulate the virus to induce an immune response. The amino acid sequence of the epitope peptide is the 173-199th and / or 364-411th and / or 427-462th positions of the hemagglutinin protein of the Yanaka strain, including but not limited to these epitopes.

[0045] 2. Nanocarriers: These small particles are used to carry antigen epitope peptides to ensure their effective distribution in the body. Nanocarriers are Ferritin, NoV P particles, liposomes, chitosan, including but not limited to these nanocarriers.

[0046] 3. Buffer: This is the liquid used to dissolve the nanoparticles. It contains a variety of ingredients, such as electrolyte salts, amino acids, pH buffers, antioxidants, glycerol, sucrose, etc., which can help maintain the stability of the vaccine, optimize its distribution in the body, and enhance the immune response. The buffer is phosphate buffer (020mM Na2HPO4, 20mM NaH2PO4, 5% glycerol), Tris buffer (20mM Tris, 150mM NaCl, 5% sucrose), including but not limited to these buffers.

[0047] 4. Adjuvant (optional): An adjuvant is a substance used to enhance the immune response. Adjuvants include cytokine adjuvants, toxin-based adjuvants, liposomes, polymers, etc. These adjuvants can help vaccines stimulate the immune system's response more effectively. Adjuvants include interleukin-2, cholera toxin B subunit, liposomes, ferritin, chitosan, including but not limited to these adjuvants.

[0048] The above are some of the components of this vaccine. The specific components will vary depending on the specific formulation and design of the vaccine.

[0049] Application Example 1: Canine distemper subunit vaccine spray containing nanoparticles carrying a single antigen

[0050] In this embodiment, the nanoparticles are composed of a single epitope peptide of the hemagglutinin protein of canine distemper virus and a norovirus P particle carrier. First, the epitope peptide is prepared by a bioengineering method and combined with a nanocarrier to form nanoparticles. Then, these nanoparticles are dissolved in a buffer containing electrolyte salts and amino acids in the form of a lyophilized powder to obtain a spray with a mass ratio of 5:1. This spray can be packaged separately and inoculated by an inhalation immunization route when used.

[0051] Application Example 2: Nanoparticles carrying multiple antigens in canine distemper subunit vaccine spray, containing adjuvant

[0052] In this embodiment, the nanoparticles are composed of multiple epitope peptides of the canine distemper virus hemagglutinin protein (each epitope peptide has a different number of copies) and Ferritin nanocarriers. First, multiple epitope peptides are prepared by bioengineering methods and combined with nanocarriers to form nanoparticles. Then, these nanoparticles are dissolved in a buffer containing electrolyte salts, amino acids, pH buffers, antioxidants, glycerol, sucrose and liposome adjuvants in the form of lyophilized powder to obtain a spray with a mass ratio of 2:1. This spray can be pre-mixed and inoculated through a nasal spray immunization route when used.

[0053] The two embodiments provided by the present invention demonstrate how to prepare canine distemper subunit vaccine sprays with different characteristics by selecting different antigens, nanocarriers, buffer components and adjuvants, as well as selecting appropriate immunization pathways and packaging methods according to different needs.

[0054] Example 1: Antigenic Conservation Analysis

[0055] Thirty hemagglutinin protein sequences of canine distemper cases and common commercial attenuated canine distemper strains that broke out in Asia in recent years were collected from the NCBI database, and the conservation of these sequences was analyzed ( Figure 1). Although the hemagglutinin protein is not very conservative, its mutation region is relatively concentrated, which is one of the potential advantages of developing a canine distemper virus hemagglutinin protein epitope vaccine. Subsequently, the hemagglutinin protein of the Yanaka strain with a conservation rate of more than 93% was selected for vaccine design.

[0056] Example 2: Preparation of subunit vaccines

[0057] This embodiment uses the Ferritin vector, but is not limited to this vector. The genes encoding Helicobacter pylori Ferritin and the hemagglutinin epitope of the present invention are inserted into the prokaryotic expression vector pET-20b (+), but are not limited to this vector, and then transformed into the expression competent state of Escherichia coli. When the bacteria are in the logarithmic growth phase, 0.5mM IPTG is added and induced overnight at 18°C. Subsequently, after centrifugation, bacterial collection and ultrasonic disruption, the supernatant is purified by affinity chromatography and dialyzed to obtain the immunogen, which is named HAe-nanoparticle ( Figure 2 ).

[0058] Example 3: Preparation of canine distemper subunit vaccine spray

[0059] The purified HAe-nanoparticle solution is injected into the nebulizer to prepare the vaccine spray. Another method is to freeze-dry the purified HAe-nanoparticle solution and dispense it into the nebulizer, and then add a buffer to dissolve the immunogen during vaccination ( Figure 3 ).

[0060] The above-described embodiments are only for describing the preferred modes of the present invention, and are not intended to limit the scope of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications may be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0061] Example 4: Evaluation of immunogenicity of canine distemper subunit vaccine spray

[0062] Animal immunization: The immunized subjects were BALB / c female mice aged 6 to 8 weeks. The immunization was carried out three times, with an interval of two weeks between each immunization. The intranasal immunization dose was 10 μg / mouse.

[0063] Mucosal immunization: Competitive ELISA and indirect ELISA were used to detect the secretory IgA (sIgA) titers in mouse nasal wash fluid (NLF) and bronchoalveolar lavage fluid (BALF). The results showed that canine distemper subunit vaccine spray can directly stimulate the respiratory mucosa to produce a large amount of sIgA, which helps to prevent the virus from adhering to and colonizing the respiratory epithelium ( Figure 4 ).

[0064] Binding antibody: The indirect ELISA method was used to detect the binding antibody titer in the serum after immunization with canine distemper subunit vaccine spray. The results showed that canine distemper subunit vaccine spray can induce high titer anti-hemagglutinin binding antibodies ( Figure 5 .A).

[0065] Neutralizing antibody: Vero cells were passaged to 96-well plates in advance and cultured in a 37°C, 5% CO2 incubator for 24 h until a monolayer of cells was formed; 50 μL of serum was diluted two-fold in a series of gradients and then 50 μL of 100×TCID 50 The serum-virus mixture was added to Vero cells and cultured in a 37°C incubator. The half inhibitory dose (ID) was calculated by visualizing the cytopathic effect (CPE). 50 ). ID 50 It is defined as the serum dilution factor that neutralizes 50% of the virus. The results showed that canine distemper subunit vaccine spray can induce high titer neutralizing antibodies against canine distemper virus ( Figure 5 .B).

[0066] Lymphocyte detection: Mouse spleen lymphocytes were separated using red blood cell lysis buffer. The cells were stained using FITC-labeled anti-mouse CD3 antibody, PE-labeled anti-mouse CD4 antibody, and APC-labeled anti-mouse CD8a antibody. After incubation on ice in the dark for 20 minutes, centrifuge at 350×g for 5 minutes and discard the supernatant. After washing twice with cell staining buffer, the cells were suspended in cell staining buffer and immediately detected using a flow cytometer. The results showed that canine distemper subunit vaccine spray can stimulate the CD3 + CD4 + and CD3 + CD8 + T cell proliferation ( Figure 6 .A).

[0067] In vivo CTL detection: Splenic lymphocytes of blank mice were isolated using red blood cell lysis buffer. Half of the cells were incubated with the peptide for 2 hours and stained with a high concentration of CFSE (5 μM) as target cells. The other half of the cells were incubated with BSA for 2 hours and stained with a low concentration of CFSE (0.5 μM) as control cells. The same number of cells from the two groups were mixed and injected into the mice inoculated with the spray through the tail vein. Splenocytes were isolated after 10 hours, and the ratio of the two groups of cells was detected using flow cytometry. The results showed that canine distemper subunit vaccine spray can induce mice to produce peptide-specific CTLs ( Figure 6 .B).

[0068] The implementation of the present invention is significantly superior to the prior art in terms of production cost and vaccination method, and the present invention is of great significance in the rapid immune diagnosis and prevention of viral infectious diseases and the development of therapeutic vaccines. The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any person skilled in the art within the technical scope disclosed by the present invention and within the spirit and principle of the present invention shall be covered within the protection scope of the present invention.

[0069]

[0070]

Claims

1. A canine distemper subunit vaccine spray, characterized in that: The invention comprises nanoparticles carrying key antigen epitope peptides and a buffer solution, wherein the nanoparticles are dissolved in the buffer solution in the form of freeze-dried powder; the mass ratio of the nanoparticles to the buffer solution in the spray is 0.1 to 10:1; and the nanoparticles are composed of the epitope peptide of the hemagglutinin protein of canine distemper virus and a nanocarrier.

2. The spray of canine distemper subunit vaccine according to claim 1, characterized in that The antigen is one or more epitope peptides of the canine distemper virus hemagglutinin protein and a combination of different copy numbers; the amino acid sequence of the epitope peptide is the 173-199th and / or 364-411th and / or 427-462th positions of the Yanaka strain hemagglutinin protein, including but not limited to these epitopes; The amino acid sequence of positions 173-199 is SALSGARGDIFPPYRCSGATTSVGRVF Amino acid sequence from position 364 to 411 PALVSEKQEEQKNCLESACQRKSYPMCNQTSWEPFGGGQLPSYGRLTL amino acid sequence 427-462 YGPVILNGDGMDYYESPLLDSGWLTVPPKNGTVLGL 。 3. according to the spray of canine distemper subunit vaccine of claim 1, it is characterized in that, The nanoparticles are expressed in the form of fusion, cross-linking or mixing of epitope peptides and nanocarriers; the nanocarriers are ferritin, NoV P particles, liposomes, chitosan, including but not limited to these nanocarriers; the fusion expression system includes prokaryotic expression and eukaryotic expression; the cross-linking agent is dichloroanhydride, glucosamine, dithiothreose, including but not limited to these cross-linking agents.

4. The spray of canine distemper subunit vaccine according to claim 1, characterized in that One or more of electrolyte salt, amino acid, pH buffer, antioxidant, glycerol and sucrose are added to the buffer.

5. The spray of canine distemper subunit vaccine according to claim 1, characterized in that, The spray is a separate package or premix of freeze-dried powder and buffer.

6. The spray of canine distemper subunit vaccine according to claim 1, characterized in that It may be free of adjuvant or contain an adjuvant, such as a cytokine adjuvant, a toxin-based adjuvant, a liposome, a polymer, but is not limited to these adjuvants.

7. The spray of canine distemper subunit vaccine according to claim 1, characterized in that The nanocarriers are norovirus P particle carriers and Ferritin nanocarriers, but are not limited to these carriers.

8. The spray of canine distemper subunit vaccine according to claim 1, characterized in that The canine distemper subunit vaccine immunization routes are nasal spray and inhalation, but are not limited to these routes.

9. A canine distemper subunit vaccine spray, characterized in that: The invention comprises nanoparticles carrying epitope peptides of canine distemper virus hemagglutinin protein and a buffer solution. The nanoparticles are dissolved in the buffer solution in the form of freeze-dried powder. The mass ratio of the nanoparticles to the buffer solution in the spray is 0.1 to 10:

1. The nanoparticles are composed of the epitope peptide and the nanocarrier.

10. The spray of canine distemper subunit vaccine according to claim 9, characterized in that The antigen is one or more epitope peptides of canine distemper virus hemagglutinin protein and a combination of different copy numbers, the nanocarrier is a Norovirus P particle carrier or a Ferritin nanocarrier, and one or more of electrolyte salts, amino acids, pH buffers, antioxidants, glycerol, and sucrose are added to the buffer.

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