Recombinant adenovirus vector vaccine formulation and preparation method therefor

Abstract

The present invention relates to an atomized inhalable vaccine formulation and a preparation method therefor. The formulation comprises an active ingredient and auxiliary materials. The active ingredient is an antigen protein-expressing recombinant human replication-defective adenovirus at a low concentration. The auxiliary materials include a buffer, a protectant, a stabilizer, a surfactant, and an osmotic pressure regulator. The atomized inhalable vaccine formulation of the present invention can retain the good stability of a human replication-defective adenovirus vector vaccine formulation.

Description

A recombinant adenovirus vector vaccine preparation and its preparation method Technical Field

[0001] The present invention belongs to the field of viral biology, and specifically relates to a recombinant adenovirus vector aerosol inhalation vaccine preparation and a preparation method thereof. Background Art

[0002] An ongoing challenge in gene therapy and vaccine research is the development of liquid viral formulations that are stable for extended periods within a specific temperature range. Adenovirus vectors are currently considered one of the leading methods for gene delivery / therapy. Adenovirus holds enormous potential in gene therapy, necessitating the development of formulations suitable for parenteral administration in humans. While live adenovirus vaccines have been developed for human use, they are administered as lyophilized formulations. The excipients used in these lyophilized formulations (gelatin, skim milk, human serum albumin, etc.) are not suitable for parenteral administration. Consequently, despite reports on the structure and characterization of adenoviruses, few studies have reported on the development of stabilized adenovirus formulations for parenteral administration in humans. Furthermore, most adenovirus formulations are lyophilized rather than liquid, presumably due to the difficulty in ensuring the stability of liquid formulations. Continuing research into adenovirus formulations has led to the development of aerosolized adenovirus inhalation formulations. Further research into adenovirus inhalation formulations led the inventors to unexpectedly discover that different concentrations of adenovirus particles require different formulation systems. Even more unexpectedly, the widely used and reported adenovirus stabilization systems are unsuitable for low-concentration adenovirus aerosolized inhalation formulations. Therefore, it is urgent to develop a formulation to improve the stability of low-concentration adenovirus aerosol inhalation preparations. Summary of the Invention

[0003] The purpose of the present invention is to provide a stable, low-virus concentration adenovirus liquid preparation. The recombinant adenovirus vector vaccine preparation of the present invention enables the vaccine preparation to maintain the stability of the preparation even under low virus concentration conditions, can effectively stimulate the body's immune response, and can be stably provided as a nebulized inhalation preparation.

[0004] The present invention provides an aerosolized inhalation vaccine formulation that can effectively stimulate the body's immune response. During research on aerosolized inhalation vaccine formulations, the present invention unexpectedly discovered that when the active ingredient of an aerosolized inhalation vaccine formulation is a low concentration of recombinant human replication-defective adenovirus particles expressing an antigen protein, the use of conventional stabilization systems can lead to insufficient formulation stability. Therefore, a low-concentration adenovirus aerosolized inhalation formulation was specifically developed.

[0005] Furthermore, the aerosol inhalation vaccine preparation comprises an active ingredient and excipients, wherein the active ingredient is a low concentration of a recombinant human replication-deficient adenovirus expressing an antigen protein, and the content of the replication-deficient adenovirus is less than 5×10 9VP / ml;

[0006] The formulations of the present invention provide stability to adenovirus at low viral concentrations and can be administered to a variety of vertebrate organisms, preferably mammals, and in particular humans. The stabilized viral formulations of the present invention are preferably compositions based on recombinant adenoviruses, which, when administered, for example, as vaccines, can provide a prophylactic advantage in previously uninfected individuals and / or a therapeutic effect by reducing viral load levels in infected individuals, thereby prolonging the asymptomatic phase of infection with a specific microorganism.

[0007] Furthermore, the excipient components of the nebulized inhalation vaccine preparation do not include ethanol;

[0008] Furthermore, in an ethanol-free system, the buffer system is preferably histidine; and the histidine concentration is greater than 2 mM.

[0009] Furthermore, when the buffer is histidine, the pH value of the vaccine preparation is preferably 6.2-6.8.

[0010] Furthermore, the excipients of the nebulized inhalation vaccine preparation also include a protective agent;

[0011] In some embodiments, the protective agent is selected from one or more of gelatin, ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetic acid (EDTA-2Na), magnesium chloride and magnesium chloride hydrate. Preferably, the protective agent is disodium ethylenediaminetetraacetic acid and magnesium chloride hexahydrate.

[0012] In some specific embodiments, the protective agent is disodium ethylenediaminetetraacetic acid (EDTA-2Na) and magnesium chloride hexahydrate, and the concentration of EDTA-2Na in the aerosol inhalation vaccine preparation is 0-1mM. Preferably, the concentration of EDTA-2Na in the preparation is 0.1mM.

[0013] In some specific embodiments, the concentration of magnesium chloride hexahydrate in the vaccine formulation is 1-10 mM. Preferably, the concentration of magnesium chloride hexahydrate in the formulation is 1-5 mM; more preferably, the concentration of magnesium chloride hexahydrate in the formulation is 2 mM.

[0014] In some embodiments, the stabilizer is selected from one or more of sucrose, lactose, maltose, trehalose, mannitol and glycerol.

[0015] Preferably, the stabilizer is sucrose, mannitol and glycerol.

[0016] In some embodiments, the concentration of glycerol in the vaccine formulation is 0.5-10 mg / ml, or the weight / volume fraction (w / v) of glycerol in the vaccine formulation is 0.05%-1%, preferably, the concentration of glycerol in the formulation is 1.5 mg / ml. Or preferably, the weight / volume fraction (w / v) of glycerol in the vaccine formulation is 0.15%.

[0017] In some embodiments, the concentration of sucrose in the vaccine formulation is 20-80 mg / ml, preferably, the concentration of sucrose in the formulation is 25 mg / ml.

[0018] In some embodiments, the concentration of mannitol in the vaccine formulation is 20-80 mg / ml, or the weight / volume fraction (w / v) of mannitol in the vaccine formulation is 2%-8%; preferably, the concentration of mannitol in the formulation is 50 mg / ml, or preferably, the weight / volume fraction (w / v) of mannitol in the vaccine formulation is 5%.

[0019] In some embodiments, the surfactant is selected from one or more of non-ionic surfactants, anionic surfactants, and zwitterionic surfactants.

[0020] Preferably, the surfactant is selected from one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, polyvinyl alcohol, Tween 80 (polysorbate 80), Tween 20 (polysorbate 20), Span 80, and Span 20.

[0021] Preferably, the surfactant is Tween 80 (polysorbate 80).

[0022] In some embodiments, the concentration of the surfactant is 0.01-1 mg / ml, or the weight / volume fraction (w / v) of the surfactant is 0.001%-0.1%; preferably, the concentration of the surfactant is 0.1 mg / ml, or preferably, the weight / volume fraction (w / v) of the surfactant is 0.01%.

[0023] In some embodiments, the osmotic pressure regulator is sodium chloride and / or calcium chloride.

[0024] Preferably, the osmotic pressure regulator is sodium chloride.

[0025] In some embodiments, the concentration of the osmotic pressure regulator is 30 mM-70 mM, preferably, the concentration of the osmotic pressure regulator is 50 mM.

[0026] In some embodiments, the human replication-defective adenovirus of the present application comprises a polynucleotide encoding a tuberculosis antigen, including one or more of a peptide or structural protein encoding a Mycobacterium tuberculosis Mtb32A, Mtb39A antigen, or Ag85A antigen; preferably, the antigen peptide or structural protein is linked by a linker.

[0027] In some embodiments, the linker is a flexible linker, and the linking peptide is a GGGGSGGGGSGGGGS linking peptide, a GGGGS linking peptide, a GlyGlyGlyGlyGlyGlyGlyGly linking peptide, a GlyGlyGlyGlyGlyGly linking peptide, a EAAAKEAAAK linking peptide, a EAAAK linking peptide, a PAPAP linking peptide, a APAPAPAPAPAPAPAP linking peptide, a KESGSVSSEQLAQFRSLD linking peptide, a AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAK linking peptide, a EAAAKA and / or a EAAAKEAAAKEAAAK linking peptide. In some embodiments, the amino acid sequence encoding the Ag85A antigen is SEQ ID NO: 1 or has at least 75% homology; preferably, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology.

[0028] In some embodiments, the antigen peptide or structural protein is a Mtb32A, Mtb39A fusion antigen (TB75K); preferably, the amino acid sequence encoding the antigen is SEQ ID NO: 2 or has at least 75% homology; preferably, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology; more preferably, wherein the linker sequence can be substituted.

[0029] In some embodiments, the Ag85A and TB75K are fused by a linker; preferably, Ag85A-linker-TB75K or TB75K-linker-Ag85A.

[0030] In some embodiments, the amino acid sequence encoding the Ag85A-linker-TB75K antigen is SEQ ID NO: 3 or has at least 75% homology; preferably, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology; more preferably, wherein the linker sequence can be substituted.

[0031] In some embodiments, the amino acid sequence encoding the TB75K-linker-Ag85A antigen is SEQ ID NO: 4 or a sequence having at least 75% homology; preferably, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology; more preferably, wherein the linker sequence can be substituted.

[0032] In some embodiments, further comprising an amino acid sequence encoding a signal peptide; preferably, the signal peptide is selected from one or more of tissue plasminogen activator (tPa) signal peptide, Ag85A wild type signal peptide, growth hormone signal peptide, recombinant human Oncostatin M (human OSM), vesicular stomatitis virus fusogenic envelope G glycoprotein (VSV-G), mouse Ig Kappa, mouse heavy chain, basement membrane protein 40 (BM40), human chymase proenzyme, human trypsinogen-2, human interleukin-2 (human IL-2), luciferase, human serum albumin (HSA), influenza hemagglutinin, human insulin, silkworm fibroin LC.

[0033] The present application also provides a vector comprising any of the above polynucleotides and a recombinant human replication-defective adenovirus comprising any of the above polynucleotides.

[0034] In some embodiments, the recombinant human replication-defective adenovirus of the present application is prepared by a method comprising the following steps:

[0035] (1) constructing a shuttle plasmid vector containing the polynucleotide encoding the antigenic peptide or structural protein;

[0036] (2) transfecting the shuttle plasmid vector of step (1) into a host cell together with a backbone plasmid;

[0037] (3) culturing the host cell of step (2);

[0038] (4) harvesting the human replication-defective recombinant adenovirus released from the cell of step (3);

[0039] (5) expanding the culture of the recombinant adenovirus of step (4);

[0040] (6) purifying the culture product of step (5).

[0041] In another aspect of the present application, a method for preparing any of the above vaccine formulations is provided, comprising the following steps: 1) purifying the recombinant adenovirus antigen stock solution; 2) proportionally configuring the vaccine adjuvant; 3) formulating the semi-finished product; 4) dispensing the semi-finished product into a tube.

[0042] In some embodiments, the purification step comprises: obtaining a clarified solution after lysing, centrifuging and filtering the virus harvest; obtaining a purified virus solution after ion exchange chromatography and complex mode chromatography; obtaining a vaccine bulk by ultrafiltration and replacement into a specified formulation; and obtaining a vaccine bulk by sterile filtration.

[0043] In another aspect of the present application, a method for preparing a recombinant adenovirus vector vaccine preparation is provided, comprising the steps of: preparing a recombinant adenovirus vector encoding a target antigen protein; and adding a pharmaceutically acceptable excipient.

[0044] Specifically, the recombinant adenovirus vector encoding the Mycobacterium tuberculosis Ag85A antigen is prepared, and optionally, a pharmaceutically acceptable excipient is added; or, the recombinant adenovirus vector encoding the Mycobacterium tuberculosis Mtb32A and Mtb39A antigens is prepared, and optionally, a pharmaceutically acceptable excipient is added; or, the recombinant adenovirus vector encoding the Mycobacterium tuberculosis Mtb32A, Mtb39A and Ag85A antigens is prepared, and optionally, a pharmaceutically acceptable excipient is added; or, the recombinant adenovirus vector encoding the Mycobacterium tuberculosis Ag85A antigen and the recombinant adenovirus vector encoding the Mycobacterium tuberculosis Mtb32A and Mtb39A antigens (TB75K) are prepared separately, mixed, and optionally, a pharmaceutically acceptable excipient is added.

[0045] Specifically, the method comprises the steps of: constructing a fusion antigen sequence; constructing a plasmid; and co-transfecting the obtained recombinant adenovirus shuttle plasmid and a backbone plasmid carrying most of the adenovirus genome for packaging.

[0046] Preferably, the Ag85A antigen sequence (SEQ ID NO. 1) is constructed using a genetic engineering method; or, the Mtb32A and Mtb39A (TB75K) fusion antigen sequence (SEQ ID NO. 2) is constructed; or, the Mtb32A and Mtb39A (TB75K) fusion antigen sequence (SEQ ID NO. 2) and the Ag85A antigen sequence (SEQ ID NO. 1) are connected using a linker.

[0047] More preferably, the TB75K-linker-Ag85A (SEQ ID NO. 4) or Ag85A-linker-TB75K (SEQ ID NO. 3) fusion antigen is constructed.

[0048] Specifically, the method further comprises the step of: adding the co-transfected original strain seed to a corresponding excipient to obtain the vaccine.

[0049] Specifically, the step of constructing the recombinant adenovirus vector encoding the Mycobacterium tuberculosis TB75K, Ag85A fusion antigen protein comprises:

[0050] (1) Construction of pDC316-TB75K-Ag85A plasmid

[0051] The synthesized TB75K-Ag85A fusion protein gene fragment was digested and the digested fragment was recovered. Meanwhile, the shuttle plasmid vector of AdMax adenovirus system was digested and the vector was recovered. The TB75K-Ag85A fragment was connected to the vector by using the method of homologous recombination, and the competent cells were transformed and plated. The single colonies were picked and inoculated by streaking, and colony PCR identification was performed. Then, the positive single colonies in the streaked plates were cultured, and the plasmid was extracted and identified by enzyme digestion. Ten positive clones identified by enzyme digestion were sequenced, and the correct vector was recorded as pDC316-TB75K-Ag85A.

[0052] (2) Recombinant adenovirus Ad5, TB75K, Ag85A virus packaging

[0053] The shuttle plasmid pDC316-TB75K-Ag85A and the backbone plasmid of AdMax adenovirus system were co-transfected to package Ad5-TB75K-Ag85A, and the obtained virus was named Ad5-105K.

[0054] The adjuvant used in the inhalation preparation of the inhalation vaccine of the present application does not produce immunosuppression on the vaccine stock solution, and has good compatibility.

[0055] After the vaccine preparation of the present application is used for immunization of the body, high levels of antigen-specific IgG, IgA and sIgA antibodies are induced, and good cellular immune response can be produced in the lungs and system. The recombinant adenovirus vector vaccine provided by the present application has good stability, safety and high efficiency.

[0056] The vaccine preparation of the present application is administered by the way of respiratory mucosa delivery, which has better compliance than injection, lower dose, and can produce triple immune effect of humoral immunity, cellular immunity and mucosal immunity. The vaccine composition provided by the present application can be used for basic immunization or booster immunization.

[0057] The vaccine of the present application can produce particles with a particle size of 3-10 μm after being atomized by a suitable device, specifically, particles with a particle size of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 μm, preferably, aerosol particles with a particle size of 5-10 μm, specifically, aerosol particles with a particle size of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 μm. The vaccine can reach the lungs after being inhaled through the nasal cavity or oral cavity, thereby producing a protective immune response to the entire respiratory tract and the lungs, enhancing the effective utilization rate of the vaccine and improving the effect of the vaccine.

[0058] Specifically, the present application also provides a preparation of a multi-component recombinant adenovirus vector vaccine formulation and its application in preventing and / or treating diseases.

[0059] Specifically, the disease is caused by SARS-CoV, SARS-CoV-2, Ebola virus, hepatitis B virus, hepatitis C virus, dengue virus, varicella-zoster virus, rabies virus, human immunodeficiency virus, varicella-zoster virus and / or Mycobacterium tuberculosis infection.

[0060] In another aspect of the present application, the use of any of the above-mentioned recombinant human replication-defective adenovirus vector vaccine formulations in the preparation of a medicament for preventing and / or treating diseases is provided.

[0061] Specifically, the disease is caused by SARS-CoV, SARS-CoV-2, Ebola virus, hepatitis B virus, hepatitis C virus, dengue virus, varicella-zoster virus, rabies virus, human immunodeficiency virus, varicella-zoster virus and / or Mycobacterium tuberculosis infection.

[0062] The beneficial effects of the present application are:

[0063] Firstly, the vaccine formulation of the present application can maintain good stability of the human replication-defective adenovirus vector vaccine formulation, especially under the condition of low virus concentration, the good stability of the formulation can still be maintained. The vaccine formulation can effectively avoid the aggregation of antigens, can be stored for a long time, and its abnormal toxicity test is qualified, safe to use.

[0064] Secondly, the vaccine formulation of the present application is suitable for administration by atomization inhalation, the vaccine formulation can also ensure the activity yield after atomization, and can produce triple immune effects of humoral immunity, cellular immunity and mucosal immunity, and can be used for basic immunization or booster immunization.

[0065] Thirdly, the vaccine preparation of the present application can target lung macrophages, and can use human adenovirus type 5 vector as a safe natural type I adjuvant to enhance the immune response of the vaccine in the lung.

[0066] Fourthly, the vaccine preparation of the present application does not affect the immunogenicity of each antigen component, and the antigen components do not interfere with each other, the auxiliary material components, the content and the pH value of the preparation are suitable, and the body can be effectively protected from infection by pathogenic bacteria or viruses and the recurrence of latent infection. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 is the Western Blot result of antigen expression verification (1: TB75K; 2: Ag85A; 3: Ag85A: TB75K = 1:1; 4: Ag85A-linker-TB75K; 5: TB75K-linker-Ag85A; 6: negative control);

[0068] Figure 2 is the change trend of LossLgIFU of different concentration preparations at 37°C for 4 weeks

[0069] Figure 3 is the change trend of 37°C accelerated stability of different buffer system preparations with an adenovirus concentration of 5×10 9 VP / ml

[0070] Figure 4 is the atomization stability of different buffer system preparations with an adenovirus concentration of 5×10 9 VP / ml

[0071] Figure 5 is the change trend of 37°C accelerated stability of different concentration histidine buffer system preparations

[0072] Figure 6 is the change trend of 37°C accelerated stability of preparations in an ethanol-histidine buffer system and a histidine system (5×10 9 vp / ml)

[0073] Figure 7 is the change trend of 37°C accelerated stability of preparations in an ethanol-histidine buffer system and a histidine system (1×10 9 vp / ml)

[0074] Figure 8 is the change trend of 37°C accelerated stability of preparations in an ethanol-histidine buffer system and a histidine system (1×10 8 vp / ml)

[0075] Figure 9 is the change trend of 37°C-4 weeks LossLgIFU accelerated stability of recombinant adenovirus vector preparations with different pH values

[0076] Figure 10 is the atomization stability of recombinant adenovirus vector preparations with different pH values

[0077] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. In the following, the technical solutions of the present application will be clearly and completely described with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0078] Example 1: Construction and packaging of recombinant adenovirus vector vaccine preparation

[0079] 1. Construction of fusion antigen strain preparation

[0080] (1) Construction of pDC316-TB75K-Ag85A plasmid

[0081] The optimized Mycobacterium tuberculosis fusion antigen sequence is shown in SEQ ID NO: 4.

[0082] The synthesized TB75K-Ag85A fusion gene fragment was digested and the digested fragment was recovered. Meanwhile, the shuttle plasmid vector of the AdMax adenovirus system was digested and the vector was recovered. The TB75K-Ag85A fragment was connected to the vector by homologous recombination, and the competent cells were transformed and plated on Amp-resistant LB plates. The next day, single colonies were picked and inoculated by streaking and colony PCR identification was performed. Then, the positive single colonies from the streaked plates were cultured, and the plasmid was extracted and digested for identification. Ten positive clones identified by enzyme digestion were sequenced, and the correct vector was designated as pDC316-TB75K-Ag85A.

[0083] (2) Packaging of recombinant adenovirus Ad5-TB75K-Ag85A strain

[0084] The shuttle plasmid pDC316-TB75K-Ag85A and the backbone plasmid of the AdMax adenovirus system were co-transfected to package Ad5-TB75K-Ag85A, and the virus obtained by packaging was named Ad5-105K.

[0085] Preparation of vaccine inhalation preparation:

[0086] 1) Preparation of recombinant tuberculosis vaccine (adenovirus vector type 5): Take the above-mentioned new recombinant tuberculosis vaccine TB75K-Ag85A stock solution (containing virus particles 5 x 1010VP), add excipients mannitol 50 mg, sodium chloride 5 mg, HEPES 1 mg, polysorbate 80 0.2 mg, glycerol 4 mg, magnesium chloride 0.2 mg, sucrose 30 mg, mix to obtain 1 ml of recombinant multi-component tuberculosis vaccine (adenovirus vector type 5) preparation. Marked as ADTB202302001.

[0087] Example 2: In vitro expression identification of recombinant adenovirus vector vaccine preparation

[0088] The recombinant adenovirus vector tuberculosis vaccine antigen expression prepared in Example 1 was detected by cell test combined with Western Blot method.

[0089] (1) Cell test

[0090] After cell plating, inoculation, virus infection of cells, lysis of cells, transfer of cell lysis supernatant to new EP tubes, obtain test sample lysis supernatant and negative control lysis supernatant. Stored in -80°C refrigerator, ready for use.

[0091] (1) Western Blot experiment to detect target gene expression

[0092] Electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody incubation.

[0093] Result analysis

[0094] 1) Test established conditions

[0095] a. The negative control sample should not have the target antigen band;

[0096] b. The target antigen band of the positive sample is clear and distinguishable and the molecular weight is accurate.

[0097] 2) Judgment criteria

[0098] TB75K antigen protein has a target band near 75 kDa, and multi-component combined antigen has a target band near 105-110 KDa, and the negative control should not have the target protein band, and the target protein expression is positive.

[0099] (2) Analysis of recombinant adenovirus tuberculosis vaccine target antigen expression results

[0100] The recombinant new tuberculosis vaccine (adenovirus vector type 5) target antigen expression should meet the above two conditions, which are positive (+) at the same time, and the result is determined to be positive. The detection results are shown in Figure 1.

[0101] Example 3: Stability of different virus concentration preparations in HEPES buffer system

[0102] The buffer is HEPES. In this system, a gradient of recombinant adenovirus concentration is set to verify the stability of the recombinant adenovirus vector preparation under different concentrations of recombinant adenovirus. The preparation contains mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol, etc. The IFU of the recombinant adenovirus preparation sample at different concentrations is detected under the condition of 37°C and HEPES buffer system to investigate the change trend of LossLgIFU.

[0103] The results are shown in Figure 2. When the adenovirus content is less than 5×10 9 When HEPES buffer is used as the system, the stability of the preparation at 37°C is significantly worse.

[0104] Example 4: Effect of different buffers on the stability of low-dose adenovirus concentration preparations and the activity recovery rate of the preparation after atomization

[0105] Different buffer systems are set to investigate the IFU changes of the recombinant adenovirus vector preparation placed for 1 week, 2 weeks, 3 weeks, and 4 weeks under different buffer systems. Histidine, PB, Tris-HCl, HEPES, histidine + Tris-HCl, and histidine + HEPES are selected. The preparation contains mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol, etc. The IFU of the recombinant adenovirus preparation sample under different buffer systems is detected under the condition of 37°C to investigate the change trend of LossLgIFU. The activity of the recombinant adenovirus preparation under different buffer systems is detected without atomization and after atomization to calculate the activity recovery rate after atomization.

[0106] The results are shown in Figures 3 and 4. When the adenovirus content is 5×10 9 When HIS buffer is used as the system, the stability of the preparation is best.

[0107] Example 5. Effect of different concentrations of histidine buffer system on the stability of low-dose adenovirus concentration preparations

[0108] The HIS buffer system is set to investigate the IFU changes of the recombinant adenovirus vector preparation placed for 1 week, 2 weeks, 3 weeks, and 4 weeks under different concentrations of histidine content and low-dose virus content. The preparation contains mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol, etc.

[0109] The results are shown in Figure 5. When the adenovirus content is 5×10 9When the concentration of the preparation is 2 mM, the stability of the preparation is poor; when the concentration of the preparation is 5-30 nM, the stability of the preparation is better.

[0110] Example 6: Effect of EtOH on the stability of the recombinant adenovirus vector preparation

[0111] The HIS buffer system was set, and the IFU changes of the recombinant adenovirus vector preparation placed at 37°C for 1 week, 2 weeks, 3 weeks and 4 weeks were investigated in the presence or absence of ethanol and different low-dose virus contents.

[0112] As shown in FIGS. 5-7, when the adenovirus content is less than 5×10 9 The stability of the preparation without ethanol is better when the buffer is the HIS system.

[0113] Example 6: Effect of pH on the stability of the recombinant adenovirus vector preparation and the yield of the aerosol activity of the preparation

[0114] The HIS buffer system was set, and the effect of pH on the stability of the recombinant adenovirus vector preparation was investigated in the absence of ethanol and different low-dose virus contents, and the IFU changes of the recombinant adenovirus vector preparation placed at 37°C for 1 week, 2 weeks, 3 weeks and 4 weeks were investigated.

[0115] As shown in FIGS. 9 and 10, when the adenovirus content is less than 5×10 9 When the buffer is the HIS system and does not contain ethanol, the stability of the preparation at 37°C and the yield of the aerosol activity of the preparation are best when the pH is between 6.2 and 6.8.

[0116] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0117] In addition, it should be noted that each specific technical feature described in the above-described specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. An aerosolized inhalation vaccine formulation, characterized in that, The vaccine formulation active ingredient is recombinant human replication-defective adenovirus type 5 expressing an antigenic protein, the formulation does not contain ethanol, the recombinant human replication-defective adenovirus type 5 content in the formulation is less than 5 x 10 9 VP / ml.

2. [Amended pursuant to Rule 26 23.06.2025] Vaccine formulation according to claim 1, characterized in that, The buffer system of the vaccine preparation is histidine.

3. The vaccine formulation of claim 4, wherein, The concentration of histidine in the vaccine preparation is greater than 2 mM.

4. [Amended pursuant to Rule 26 23.06.2025] Vaccine formulation according to any one of claims 1 to 3, characterized in that, The protective agent is selected from one or more of gelatin, ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetate (EDTA-2Na), magnesium chloride, and magnesium chloride hydrate; preferably, the protective agent is disodium ethylenediaminetetraacetate and magnesium chloride hexahydrate, the concentration of EDTA-2Na in the vaccine preparation is 0-1 mM, preferably 0.1 mM, and the concentration of magnesium chloride hexahydrate in the vaccine preparation is 1-10 mM, preferably 1-5 mM, and more preferably 2 mM.

5. The vaccine formulation according to any one of claims 1 to 4, characterized in that, The stabilizer is selected from one or more of sucrose, lactose, maltose, trehalose, mannitol, and glycerol; preferably, the stabilizer is sucrose, mannitol, and glycerol. The concentration of glycerol in the preparation is 0.5-10 mg / ml; preferably, the concentration of glycerol in the preparation is 1.5 mg / ml, the concentration of sucrose in the preparation is 20-80 mg / ml; preferably, the concentration of sucrose in the preparation is 25 mg / ml, and the concentration of mannitol in the preparation is 20-80 mg / ml; preferably, the concentration of mannitol in the preparation is 50 mg / ml.

6. The vaccine formulation according to any one of claims 1 to 6, characterized in that, The surfactant is selected from one or more of non-ionic surfactants, anionic surfactants, and zwitterionic surfactants; preferably, the surfactant is selected from one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, polyvinyl alcohol, Tween 80, Tween 20, Span 80, and Span 20; preferably, the surfactant is Tween 80, and the concentration of the surfactant is 0.01-1 mg / ml; preferably, the concentration of the surfactant is 0.1 mg / ml.

7. The vaccine formulation according to any one of claims 1 to 6, characterized in that, The pH value of the preparation is 6.2-6.

8.

8. The vaccine formulation according to any one of claims 1 to 7, characterized in that, The preparation is an aerosol inhalation preparation, and the preparation forms particles with a particle size of less than 10 μm after being atomized by an atomization administration device; preferably, 0.5-10 μm, and more preferably, 5-10 μm.

9. The vaccine formulation according to any one of claims 1 to 8, characterized in that, The antigen protein is derived from SARS-CoV, SARS-CoV-2, Ebola virus, hepatitis B virus, hepatitis C virus, dengue virus, varicella-zoster virus, rabies virus, human immunodeficiency virus, varicella-zoster virus, and / or Mycobacterium tuberculosis.

10. A method of preparing a vaccine formulation as claimed in any one of claims 1 to 9, characterised in that, The method comprises the following steps: 1) purifying a recombinant adenovirus antigen stock solution; 2) proportionally configuring vaccine adjuvants; 3) preparing a semi-finished product; 4) dispensing the semi-finished product into a tube.

11. Use of a recombinant human replication-deficient adenoviral vector vaccine formulation for the preparation of a medicament for the prevention and / or treatment of a disease, characterized in that, The vaccine preparation is as described in claims 1-11.

12. The use according to claim 12, characterized in that, The disease is caused by infection with SARS-CoV, SARS-CoV-2, Ebola virus, hepatitis B virus, hepatitis C virus, dengue virus, varicella-zoster virus, rabies virus, human immunodeficiency virus, varicella-zoster virus, and / or Mycobacterium tuberculosis.

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