WO2025218651A1 - Recombinant adenovirus vector vaccine formulation and preparation method therefor - Google Patents

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

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

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

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.

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.

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 ⁹ VP/ml;

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.

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

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

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

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

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.

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.

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.

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

Preferably, the stabilizer is sucrose, mannitol and glycerol.

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%.

In some specific 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.

In some specific 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%.

In some embodiments, the surfactant is selected from one or more of a nonionic surfactant, an anionic surfactant, and a zwitterionic surfactant.

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

Preferably, the surfactant is Tween 80 (polysorbate 80).

In some specific 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%.

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

Preferably, the osmotic pressure regulator is sodium chloride.

In some specific embodiments, the concentration of the osmotic pressure regulator is 30 mM-70 mM. Preferably, the concentration of the osmotic pressure regulator is 50 mM.

In some specific embodiments, the human replication-deficient adenovirus of the present invention comprises a polynucleotide encoding a tuberculosis antigen, including one or more peptides or structural proteins encoding Mycobacterium tuberculosis Mtb32A, Mtb39A antigen or Ag85A antigen; preferably, the antigenic peptides or structural proteins are connected by a linker.

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

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

In some specific embodiments, the Ag85A and TB75K are fused via a linker; preferably, it is Ag85A-linker-TB75K or TB75K-linker-Ag85A.

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

In some specific embodiments, the amino acid sequence encoding the TB75K-linker-Ag85A antigen is SEQ ID NO: 4 or a sequence with at least 75% homology; preferably, it has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology; more preferably, the linker sequence is replaceable.

In some specific embodiments, it further comprises 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 tumor inhibitor M (human OSM), vesicular stomatitis virus fusogenic capsid G glycoprotein (VSV-G), mouse Ig Kappa, mouse heavy chain, basement membrane protein 40 (BM40), human chymotrypsinogen, human trypsinogen-2, human interleukin-2 (human IL-2), insect luciferase, human serum albumin (HSA), influenza hemagglutinin, human insulin, and silkworm fibrin LC.

The present invention also provides a vector comprising any of the above polynucleotides and a recombinant human replication-deficient adenovirus comprising any of the above polynucleotides.

In some specific embodiments, the recombinant human replication-defective adenovirus of the present invention is prepared by the following method, which comprises the following steps:

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

(2) transfecting the shuttle plasmid vector and the backbone plasmid described in step (1) into host cells;

(3) culturing the host cells described in step (2);

(4) harvesting the human replication-deficient recombinant adenovirus released from the cells in step (3);

(5) Expanding the culture of the recombinant adenovirus in step (4);

(6) Purify the culture product in step (5).

Another aspect of the present invention provides a method for preparing any of the above-mentioned vaccine preparations, the method comprising the following steps: 1) purifying the recombinant adenovirus antigen stock solution; 2) preparing vaccine excipients in proportion; 3) preparing a semi-finished product; and 4) packaging the semi-finished product into controlled bottles.

In some specific embodiments, the purification step includes: the virus harvest liquid is lysed, centrifuged, and filtered to obtain a clarified liquid, and then subjected to two-step chromatography of ion exchange chromatography and composite mode chromatography to obtain a purified virus liquid, and then the liquid is exchanged into a specified formula by ultrafiltration, and finally the vaccine stock solution is obtained by sterile filtration.

Another aspect of the present invention provides a method for preparing a recombinant adenovirus vector vaccine preparation, comprising the following steps: preparing a recombinant adenovirus vector encoding a target antigen protein; and adding a pharmaceutically acceptable excipient.

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

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

Preferably, the Ag85A antigen sequence (SEQ ID NO.1) is constructed using genetic engineering methods; 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 connecting peptide (i.e., linker).

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

Specifically, the method further includes the following steps: adding corresponding auxiliary materials to the co-transfected and packaged original strain seeds to obtain the vaccine.

Specifically, the steps for constructing a recombinant adenovirus vector encoding the fusion antigen protein of Mycobacterium tuberculosis TB75K and Ag85A include:

(1) Construction of pDC316-TB75K-Ag85A plasmid

The synthesized TB75K and Ag85A fusion protein gene fragments were enzymatically digested and the digested fragments were recovered. Simultaneously, the shuttle plasmid vector of the AdMax adenovirus system was enzymatically digested and the vector was recovered. The TB75K-Ag85A fragment was connected to the vector using homologous recombination, transformed into competent cells, and plated. Single clones were picked for streak inoculation and colony PCR identification was performed. Subsequently, single positive clones from the streaked plates were cultured, and plasmids were extracted for enzymatic digestion identification. Ten clones identified as positive by enzymatic digestion were sequenced, and the vector identified as correct by sequencing was designated pDC316-TB75K-Ag85A.

(2) Recombinant adenovirus Ad5, TB75K, Ag85A virus seed packaging

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 packaged virus was named Ad5-105K.

The excipients used in the inhalation vaccine inhalation preparation of the present invention will not produce immunosuppression on the vaccine stock solution and have good compatibility.

The vaccine preparation of the present invention induces the production of high levels of antigen-specific IgG, IgA, and sIgA antibodies after immunization, and can generate a good cellular immune response in the lungs and system. The recombinant adenovirus vector vaccine provided by the present invention has good stability, safety, and high efficiency.

The vaccine formulation of the present invention is administered via respiratory mucosal delivery, which not only improves compliance but also provides a lower dosage than injection, and can produce a triple immune effect of humoral immunity, cellular immunity, and mucosal immunity. The vaccine composition provided by the present invention can be used for primary or booster immunization.

The vaccine of the present invention can produce particles with a particle size of 3 to 10 μm after being atomized by suitable equipment. Specifically, the particle size that can be produced includes but is not limited to particles 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, the aerosol particles have a better uniformity of 5 to 10 μm, specifically, including but not limited to aerosol particles with a better uniformity 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. It can reach the lungs by inhalation through the nasal cavity or oral cavity, thereby generating 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.

Specifically, the present invention also provides a method for preparing a multi-component recombinant adenovirus vector vaccine formulation composition and its use in preventing and/or treating diseases.

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

Another aspect of the present invention provides use of any of the above-mentioned recombinant human replication-defective adenovirus vector vaccine preparations in the preparation of drugs for preventing and/or treating diseases.

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

The beneficial effects of the present invention are:

First, the vaccine formulation of the present invention maintains excellent stability for human replication-defective adenovirus vector vaccines, particularly at low viral concentrations. This vaccine formulation effectively avoids antigen aggregation, allows for long-term storage stability, and has passed abnormal toxicity testing, demonstrating safety.

2. The vaccine preparation of the present invention is suitable for administration by aerosol inhalation. The vaccine preparation can also ensure the active yield after aerosolization, and can produce triple immune effects of humoral immunity, cellular immunity and mucosal immunity, and can be used for basic immunity or booster immunity.

3. The vaccine preparation of the present invention can target immune lung macrophages and utilize human adenovirus type 5 vector as a safe natural type I adjuvant to enhance the immune response of the vaccine in the lungs.

Fourth, the use of the vaccine preparation of the present invention will not affect the immunogenicity of each antigen component, and there will be no mutual interference of immune response between the antigens. The auxiliary material components, content and pH value of the preparation are appropriate, which can effectively protect the body from infection by pathogenic bacteria or viruses and prevent the recurrence of latent infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Western Blot results of antigen expression verification (1: TB75K; 2: Ag85A; 3: Ag85A:TB75K = 1:1; 4: Ag85A-linker-TB75K; 5: TB75K-linker-Ag85A; 6: negative control);

Figure 2 Changes in LossLgIFU of different concentrations of preparations at 37°C for 4 weeks

Figure 3 Trends in the accelerated stability of adenovirus preparations at 5×10 ⁹ VP/ml in different buffer systems at 37°C

Figure 4 Nebulization stability of adenovirus preparations in different buffer systems at a concentration of 5×10 ⁹ VP/ml

Figure 5. Trends in accelerated stability of preparations with different histidine buffer concentrations at 37°C

Figure 6. Trends in accelerated stability of formulations at 37°C in ethanol-histidine buffer and histidine systems (5×10 ⁹ vp/ml)

Figure 7. Trends in the accelerated stability of the formulations at 37°C in the ethanol-histidine buffer system and the histidine system (1×10 ⁹ vp/ml)

Figure 8. Trends in the accelerated stability of the formulations at 37°C in the ethanol-histidine buffer system and the histidine system (1×10 ⁸ vp/ml)

Figure 9. Trends in the accelerated stability of recombinant adenoviral vector preparations at different pH values at 37°C for 4 weeks after the loss of LgIFU.

Figure 10. Stability and activity yield of recombinant adenovirus vector preparations at different pH values.

Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention relates. The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

Example 1: Construction and packaging of recombinant adenovirus vector vaccine preparations

1. Construction and preparation of fusion antigen strains

(1) Construction of pDC316-TB75K-Ag85A plasmid

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

The synthesized TB75K and Ag85A fusion gene fragments were digested with enzymes, and the digested fragments were recovered. At the same time, the shuttle plasmid vector of the AdMax adenovirus system was digested with enzymes, and the vector was recovered. The TB75K-Ag85A fragment was connected to the vector using the homologous recombination method, transformed into competent cells, and coated on an Amp-resistant LB plate. The next day, a single clone was picked for streak inoculation and colony PCR identification was performed. Subsequently, a single clone of the positive clone in the streak plate was cultured, and the plasmid was extracted for enzyme digestion identification. Ten positive clones identified by enzyme digestion were taken for sequence determination, and the vector identified correctly by sequencing was recorded as pDC316-TB75K-Ag85A

(2) Packaging of recombinant adenovirus Ad5-TB75K-Ag85A virus seed

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 packaged virus was named Ad5-105K.

Preparation of vaccine inhalation formulations:

1) Preparation of recombinant tuberculosis vaccine (adenovirus type 5 vector): To the aforementioned novel recombinant tuberculosis vaccine TB75K-Ag85A stock solution (containing 5×1010 VP of viral particles), add 50 mg of mannitol, 5 mg of sodium chloride, 1 mg of HEPES, 0.2 mg of polysorbate 80, 4 mg of glycerol, 0.2 mg of magnesium chloride, and 30 mg of sucrose. Mix to obtain 1 ml of a recombinant multicomponent tuberculosis vaccine (adenovirus type 5 vector) formulation. Label: ADTB202302001.

Example 2: In vitro expression and identification of recombinant adenovirus vector vaccine preparations

The expression of target antigen of the recombinant adenovirus vector tuberculosis vaccine prepared in Example 1 was detected by cell assay combined with Western Blot method.

(1) Cell test

After plating cells, inoculating with viruses, infecting cells with viruses, and lysing cells, transfer the cell lysate supernatant to a new EP tube to obtain the test sample lysate supernatant and the negative control lysate supernatant. Store at -80°C for future use.

(1) Western Blot assay to detect target gene expression

Electrophoresis, transfer, blocking, primary antibody incubation, and secondary antibody incubation.

Result Analysis

1) Test conditions

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

b. The target antigen bands of positive samples are clearly discernible and the molecular weight is accurate.

2) Judgment criteria

The TB75K antigen protein has a target band near 75kDa, the multi-component binding antigen has a target band near 105-110kDa, and the negative control should not have the target protein band, which is determined to be positive for target protein expression.

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

The expression of the target antigen of the recombinant new tuberculosis vaccine (adenovirus type 5 vector) should meet the above two conditions and be positive (+) at the same time, and the result is considered positive. The test results are shown in Figure 1.

Example 3: Stability of preparations with different virus concentrations in a HEPES buffer system

The buffer is HEPES. In this system, a recombinant adenovirus concentration gradient is set to verify the stability of the recombinant adenovirus vector preparation at different recombinant adenovirus concentrations. The preparation contains mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol, etc. The IFU of recombinant adenovirus preparation samples with different concentrations are tested at 37°C under HEPES buffer system conditions to examine the change trend of LossLgIFU.

The results are shown in FIG2 . When the adenovirus content is lower than 5×10 ⁹ , the stability of the preparation in the 37° C. accelerated test using HEPES buffer is significantly deteriorated.

Example 4: Effects of different buffers on the stability of low-dose adenovirus concentration preparations and the yield of aerosolized active preparations

Different buffer systems were established to investigate changes in IFU (Intracellular Units) (IFU) of recombinant adenovirus vector preparations after 1, 2, 3, and 4 weeks of storage. The formulations included histidine, PB, Tris-HCl, HEPES, histidine + Tris-HCl, and histidine + HEPES. The formulations also contained mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, and ethanol. The IFU of recombinant adenovirus preparations in different buffer systems was measured at 37°C to investigate trends in Loss Lg IFU. The activity of both unnebulized and recovered samples of recombinant adenovirus preparations in different buffer systems was tested, and the nebulized activity yield was calculated.

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

Example 5. Effect of different histidine concentration buffer systems on the stability of low-dose adenovirus preparations

A HIS buffer system was established to investigate changes in IFU (Intracellular Units) of recombinant adenoviral vector preparations at different histidine concentrations and low viral loads after storage at 37°C for 1, 2, 3, and 4 weeks. The preparations contained mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, and ethanol.

The results are shown in FIG5 . When the adenovirus content was 5×10 ⁹ vp/ml, the HIS buffer was used as the system, and the concentration was 2 mM, the stability of the preparation was poor; when the concentration was 5-30 nM, the stability of the preparation was better.

Example 6: Effect of EtOH on the stability of recombinant adenovirus vector preparations

A HIS buffer system was set up to investigate the changes in IFU of recombinant adenovirus vector preparations after storage at 37°C for 1, 2, 3, and 4 weeks in the presence or absence of ethanol and at different low-dose virus contents.

The results are shown in Figures 5-7. When the adenovirus content is lower than 5×10 ⁹ and the buffer is a HIS system, the stability of the preparation without ethanol is better.

Example 6: Effect of pH on the Stability of Recombinant Adenovirus Vector Preparations and the Atomization Activity Yield of the Preparations

A HIS buffer system was set up, and the effect of pH change on the stability of recombinant adenovirus vector preparations was studied at low virus content in an ethanol-free system. The IFU changes of the recombinant adenovirus vector preparations were observed after being placed at 37°C for 1 week, 2 weeks, 3 weeks, and 4 weeks.

The results are shown in Figures 9 and 10 . When the adenovirus content is lower than 5×10 ⁹ , the buffer is a HIS system without ethanol, and the pH is between 6.2 and 6.8, the stability of the preparation in the 37°C accelerated test and the aerosolized activity yield of the preparation are optimal.

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims (12)

A vaccine preparation for aerosol inhalation, characterized in that the active ingredient of the vaccine preparation is a recombinant human replication-deficient adenovirus type 5 expressing an antigen protein, the preparation does not contain ethanol, and the content of the recombinant human replication-deficient adenovirus type 5 in the preparation is less than 5×10 ⁹ VP/ml. [Corrected 23.06.2025 in accordance with Article 26]
The vaccine preparation according to claim 1, characterized in that the buffer system of the vaccine preparation is histidine. The vaccine preparation according to claim 4, characterized in that the histidine concentration in the vaccine preparation is greater than 2 mM. [Corrected 23.06.2025 in accordance with Article 26]
The vaccine preparation 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 ethylenediaminetetraacetic acid (EDTA-2Na), magnesium chloride and magnesium chloride hydrate; preferably, the protective agent is disodium ethylenediaminetetraacetic acid and magnesium chloride hexahydrate, the concentration of EDTA-2Na in the vaccine preparation is 0-1mM, preferably 0.1mM, and the concentration of magnesium chloride hexahydrate in the vaccine preparation is 1-10mM, preferably 1-5mM, and more preferably 2mM. The vaccine preparation 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, and 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. The vaccine preparation according to any one of claims 1 to 6, characterized in that the surfactant is selected from one or more of a nonionic surfactant, an anionic surfactant and a zwitterionic surfactant; preferably, the surfactant is selected from one or more of sodium lauryl sulfate, sodium lauryl 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. The vaccine preparation according to any one of claims 1 to 6, characterized in that the pH value of the preparation is 6.2-6.8. The vaccine preparation according to any one of claims 1 to 7 is 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 aerosolized by an aerosol delivery device; preferably, 0.5 to 10 μm, more preferably, 5 to 10 μm. The vaccine preparation 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, herpes zoster virus, rabies virus, human immunodeficiency virus, varicella-zoster virus and/or Mycobacterium tuberculosis. A method for preparing a vaccine preparation according to any one of claims 1 to 9, characterized in that it comprises the following steps: 1) purifying the recombinant adenovirus antigen stock solution; 2) preparing vaccine excipients in proportion; 3) preparing a semi-finished product; and 4) packaging the semi-finished product into controlled bottles. A use of a recombinant human replication-deficient adenovirus vector vaccine preparation in the preparation of a medicament for preventing and/or treating a disease, wherein the vaccine preparation is as described in claims 1-11. 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, herpes zoster virus, rabies virus, human immunodeficiency virus, varicella-zoster virus and/or Mycobacterium tuberculosis.