Chimpanzee adenoviral vector-based vaccine liquid formulation and preparation method

Abstract

The present invention relates to a vaccine formulation. An effective ingredient is a recombinant chimpanzee adenovirus expressing an antigen protein, and adjuvants are ethanol and glycerol. The present formulation has no animal-derived ingredient and has high safety. The formulation can maintain good stability of a chimpanzee adenoviral vector vaccine liquid formulation. The vaccine formulation can be stably stored for more than 12 months at 2-8°C, the abnormal toxicity test of the vaccine formulation satisfies the standards, and the vaccine formulation is safe to use. The vaccine formulation can be used as a liquid injection and an atomized inhalation formulation at the same time.

Description

A chimpanzee adenovirus vector vaccine liquid preparation and preparation method Technical Field

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

[0002] Viral vectors are defective viruses whose wild-type viral genomes have been genetically modified to reduce or eliminate their pathogenicity while retaining high transfection efficiency. These viruses can then be used to introduce target genes into cells for expression through biological infection. Due to difficulties in preparation, limited target gene storage capacity, and concerns about immunogenicity and biosafety, only a few viruses can be engineered into vectors for gene therapy. Based on the type of viral genome, they can be categorized as DNA viral vectors (adenovirus, adeno-associated virus, and herpes simplex virus), RNA viral vectors (retroviral and lentiviral), and chimeric viral vectors. While adenovirus offers many advantages as a vaccine virus, this approach to vaccine development suffers from a significant drawback. Because adenovirus is so common, approximately one-third of the population develops immunity to it by adulthood. These individuals' immune systems then destroy the vaccine rather than producing antibodies against the virus carrying the inserted gene.

[0003] Given that most people are much less likely to contract microbial infections from chimpanzees, chimpanzee adenovirus could be used as a vaccine vector. Chimpanzee adenovirus vector vaccines, as viral vector vaccines themselves, are unstable in liquid form. Changes in storage conditions and prolonged storage can affect the vaccine's effectiveness. Therefore, ensuring vaccine stability is a core technical challenge in vaccine manufacturing. Compared to human adenovirus, chimpanzee adenovirus is even more challenging to control in terms of stability.

[0004] To further enhance the immunogenicity of vaccines, extensive research has been conducted on vaccine administration methods. Studies have found that inhalation administration can further stimulate the body's mucosal immunity, allowing the drug to directly reach the respiratory tract through aerosol, covering the entire respiratory mucosa. This is an important method for preventing and treating respiratory diseases and an alternative to oral and injection administration. This aerosol administration method allows for the delivery of vaccines through the respiratory tract. Aerosol inhalation vaccines offer advantages such as low dosage, a painless vaccination process, minimal side effects, and a wide range of applications. However, the low dosage of inhalation vaccines poses new challenges in maintaining the active ingredient in the inhalation formulation. Furthermore, inhalation formulations differ from traditional injection formulations in that the aerosol rate, aerosol concentration, aerosol particle size, and particle size distribution all affect the formulation's effectiveness, placing higher demands on the formulation. In actual R&D and production, developing a vaccine that can be used as both an injection and an aerosol inhalation formulation could significantly reduce enterprise costs and clinical timelines, accelerating vaccine launch. The present invention represents a further improvement on the aforementioned existing research technologies. Therefore, a vaccine preparation with high safety, good stability and few side effects is developed, and the vaccine preparation can be prepared as a liquid injection preparation and an aerosol inhalation preparation at the same time.

[0005] Summary of the Invention

[0006] One object of the present invention is to provide a chimpanzee adenovirus vector vaccine preparation with high safety and good stability.

[0007] Another object of the present invention is to provide a chimpanzee adenovirus vector vaccine formulation. The formulation can be a liquid injection formulation or an aerosol inhalation formulation, comprising an active ingredient and excipients; the active ingredient is a recombinant chimpanzee adenovirus expressing an antigenic protein, and the excipients include a protective agent, which includes ethanol and glycerol.

[0008] In one embodiment, the vaccine formulation further comprises a carbohydrate compound or a sugar alcohol, including: sucrose, trehalose, lactose, glycine and / or mannitol, preferably: sucrose and mannitol.

[0009] Preferably, the volume percentage (v / v%) of ethanol in the preparation is 0.1-10%. More preferably, the volume percentage (v / v%) of ethanol in the preparation is 0.1-2%, 0.1-1%, preferably 0.5%.

[0010] Preferably, the concentration of glycerol in the preparation is 0.5-10 mg / ml. More preferably, the concentration of glycerol in the preparation is 0.5-5 mg / ml, 0.5-2.5 mg / ml, 0.5-1 mg / ml, or 0.5 mg / ml.

[0011] Preferably, the sucrose content in the auxiliary material is 25-100 mg / ml; preferably 25-75 mg / ml, 25-75 mg / ml, 25 mg / ml, and the mannitol content in the auxiliary material is 25-100 mg / ml, preferably 50 mg / ml;

[0012] Preferably, the optimal ratio of sucrose and mannitol in the auxiliary material is 1:2-1:3; more preferably 1:2;

[0013] Preferably, in one embodiment, sucrose is 25 mg / ml and mannitol is 50 mg / ml;

[0014] In one embodiment, sucrose is 25 mg / ml and mannitol is 75 mg / ml.

[0015] Preferably, the auxiliary material further comprises one or more of a chelating agent, a buffer, a surfactant, and a salt.

[0016] Preferably, the excipient further comprises a chelating agent; preferably, the concentration of the chelate is 0.01-1 mM; preferably, the chelating agent is selected from one or more of EDTA-Na2, EDTA-Na, sodium citrate, and citric acid, more preferably, EDTA-Na2.

[0017] Preferably, the vaccine excipient also includes a surfactant. Preferably, the surfactant is one or more selected from non-ionic surfactants, anionic surfactants, zwitterionic surfactants and; more preferably, it is one or more selected from sodium lauryl sulfate, polyvinyl alcohol, sodium lauryl sulfate, Tween 80, Span 80, Span 20, Tween 20; preferably, the surfactant concentration is 0.01-1 mg / ml.

[0018] Preferably, the vaccine adjuvant further comprises salt, and the salt is one or more of sodium chloride, magnesium chloride, and calcium chloride. Preferably, the concentration of the salt is 30-70 mM.

[0019] In a specific embodiment of the present invention, the excipients in the vaccine preparation include buffer, sucrose, mannitol, sodium chloride, magnesium chloride, Tween-80, and EDTA-Na2, and the protective agent includes one or more of ethanol, glycerol, or propylene glycol.

[0020] Preferably, the pH of the vaccine preparation is 5.8-6.6, preferably 6-6.6; 6.2-6.6; 6.4-6.6; wherein 6.6 is the best;

[0021] Preferably, the excipient further comprises a buffer selected from an amino acid buffer, a phosphate buffer or HEPES. More preferably, the buffer concentration is 1-20 mM, more preferably an amino acid buffer, more preferably a histidine buffer. Preferably, the buffer concentration is 2-15 mM, for example, the buffer concentration is 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mM.

[0022] Preferably, the chimpanzee virus is selected from AdC3, AdC6, AdC63, AdC68, Y25, preferably Y25.

[0023] Preferably, the antigen is selected from the group consisting of SARS-CoV, SARS-CoV-2, Ebola virus, hepatitis B virus, hepatitis C virus, dengue virus, herpes zoster virus, rabies virus, and human immunodeficiency virus, preferably varicella-zoster virus.

[0024] Varicella-zoster virus (VZV), also known as human herpesvirus type 3, is a ubiquitous alphaherpesvirus, a double-stranded DNA virus. VZV infects humans in two ways: primary infection with varicella (varicella) and recurrent infection with zoster (zoster). VZV naturally infects only humans, lacking an animal reservoir, and its primary targets are T lymphocytes, epithelial cells, and ganglia. Initial infection causes varicella, and after recovery, the virus remains latent within ganglia. With aging, cell-mediated immunity gradually declines, or in immunocompromised patients, the virus can relapse, causing herpes zoster. Herpes zoster can be complicated by chronic pain (postherpetic neuralgia, PHN) and other serious neurological and ocular disorders (e.g., meningoencephalitis, myelitis, cranial nerve palsies, vasculopathy, keratitis, and retinopathy), as well as a variety of visceral and gastrointestinal diseases, including ulcers, hepatitis, and pancreatitis. Vaccination with the herpes zoster vaccine is an effective means of preventing the disease.

[0025] As a preference, the content of the recombinant viral vector is 1×10 9 ~1ⅹ10 12 VP / ml.

[0026] The preparation can be an injection preparation or an aerosol inhalation preparation.

[0027] In one embodiment, the pH of the vaccine formulation is 5.8-6.8, preferably 6.0-6.8; 6.2-6.8; 6.4-6.8; wherein 6.6 is optimal.

[0028] In one embodiment, the buffer system of the vaccine formulation is His.

[0029] The present invention relates to a method for preparing a vaccine liquid preparation, comprising the following steps:

[0030] 1) Purification of recombinant adenovirus antigen stock solution;

[0031] 2) Prepare vaccine excipients in proportion;

[0032] 3) Preparation of semi-finished products;

[0033] 4) Pack the semi-finished products into controlled bottles.

[0034] Preferably, the purification step includes: the virus harvest liquid is subjected to operations such as lysis, centrifugation, and filtration 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 subjected to ultrafiltration liquid exchange, and finally sterile filtration to obtain the vaccine stock solution.

[0035] The beneficial effects of the present invention are:

[0036] Vaccine excipients refer to the protective agents, excipients, and additives used in the formulation of vaccine products. They are essential components of vaccine preparations and play a vital role in their stability. A suitable formulation plays a key role in enhancing the biological activity of vaccine products, developing new dosage forms and varieties, and meeting the requirements for preventive use. This preparation contains no animal-derived ingredients and is highly safe.

[0037] The formulation of the present invention can maintain good stability in the chimpanzee adenovirus vector vaccine liquid formulation. It effectively prevents antigen aggregation, can be stably stored at 2-8°C for more than 12 months, and has passed abnormal toxicity tests, making it safe to use. The vaccine formulation of the present invention can be used as both a liquid formulation and an aerosol inhalation formulation, eliminating the need to adjust the formulation due to dosage form changes, maximizing cost savings and reducing time, allowing for simultaneous clinical trials for both injection and inhalation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1. IFU stability test results of different stabilizer systems at 37°C

[0039] Figure 2. IFU stability test results of different sugar / sugar alcohol systems at 37°C

[0040] Figure 3. IFU recovery test results after atomization under different pH conditions

[0041] Figure 4. IFU stability test results under different pH conditions DETAILED DESCRIPTION

[0042] 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.

[0043] Example 1 Preparation of model vaccine

[0044] Varicella-zoster gE protein was selected as the model antigen.

[0045] Preparation of recombinant varicella-zoster virus: Recombinant varicella-zoster virus is generated by modifying chimpanzee adenovirus Y25. First, two homology arms LF1 and LF2 at the left end of the Y25 genome and one homology arm RF at the right end are inserted into a bacterial artificial chromosome (BAC) system to construct the plasmid pBACe3.6. This plasmid and the Y25 genome are then co-transformed into Escherichia coli. Through homologous recombination, the viral genome integrates into the plasmid and the E1 region is deleted. Correct recombinant clones are screened for chloramphenicol resistance and restriction enzyme digestion. Using primers E3delLH and E3delRH, the E3 region is replaced with galactokinase (GalK). Finally, two GalK residues are inserted to replace the E4 region open reading frames ORF4, 6, and 7. The GalK gene is then replaced with the E4 region of human adenovirus type 5, resulting in a replication-defective chimpanzee adenovirus vector. The vector has a unique AsiSI restriction site in the E1 deletion region and is inserted with a modified attR1 attR2 Gateway assembly (Invitrogen). Using this assembly, the varicella-zoster virus gE protein gene was inserted into its genome to construct a replication-defective chimpanzee adenovirus vector that can express varicella-zoster virus gE protein, which is denoted as recombinant varicella-zoster virus.

[0046] Preparation method of recombinant varicella-zoster vaccine stock solution: HEK293 cells are infected with recombinant varicella-zoster virus, and the virus harvest liquid is obtained by amplification. The virus harvest liquid is subjected to lysis, centrifugation, filtration and other operations 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 by ultrafiltration, and finally the vaccine stock solution is obtained by sterile filtration.

[0047] Example 2 Screening of Stabilizer Systems for Replication-Deficient Chimpanzee Adenovirus Vector Preparations

[0048] The effect of different stabilizers on the stability of the preparation was studied by setting up different stabilizer systems and storing them at 37°C. Samples were taken at 0, 2, 4, 7, 10, and 14 days to detect the viral infection titer.

[0049] Virus infection titer detection; detection using adenovirus detection kit;

[0050] Table 1. Screening of different stabilizer systems

[0051] Experimental results: The IFU stability test results of different stabilizer systems at 37°C are shown in Figure 1. The combination of ethanol and glycerol is better.

[0052] Example 3 Screening of Sugar and Sugar Alcohol Systems for Replication-Deficient Chimpanzee Adenovirus Vector Preparations

[0053] An appropriate amount of recombinant herpes zoster vaccine antigen stock solution; the purified recombinant herpes zoster vaccine antigen stock solution is prepared into a semi-finished product according to the following formula;

[0054] Table 2. Screening of different sugar / sugar alcohol systems Note: The other excipients are sodium chloride 50mM, magnesium chloride 2mM, Tween-80 0.1mg / ml, EDTA-Na2 0.1mM, and the rest is water for injection.

[0055] Long-term stability test method: Place the vaccine at 37°C and take samples for viral infection titer detection at 2, 4, 7, 10, and 14 days. Use an adenovirus detection kit for detection;

[0056] The experimental results are shown in Figure 2. The optimal ratio of sucrose and mannitol in the auxiliary materials is 1:2-1:3; more preferably 1:2

[0057] Example 4 Screening of pH Systems for Replication-Deficient Chimpanzee Adenovirus Vector Preparations

[0058] Based on the experimental results of Examples 2 and 3, the effect of pH on the atomization of the formulation system was further verified.

[0059] The effect of different pH values ​​on the atomized preparation was tested. The sample preparation method is as follows:

[0060] Table 3. Screening of different pH systems Note: The other excipients are sodium chloride 50mM, magnesium chloride 2mM, Tween-80 0.1mg / ml, EDTA-Na2 0.1mM, and the rest is water for injection.

[0061] The virus harvest liquid is subjected to lysis, centrifugation, filtration and other operations to obtain a clarified liquid, which is then subjected to two-step chromatography steps of ion exchange chromatography and composite mode chromatography to obtain a purified virus liquid. The liquid is then exchanged into buffer solutions of different pH values ​​through ultrafiltration, and finally sterile filtration is used to obtain vaccine stock solutions of different pH values ​​for studying the effects of different pH values ​​on virus stability.

[0062] The IFU recovery rate after nebulization was tested under different pH conditions (IFU recovery rate = IFU in nebulized sample / IFU in unnebulized sample × 100%). Samples numbered 1-10 in the table above were nebulized, 1 ml each time, and the nebulized liquid was collected for IFU testing. Three samples were collected for each number. The results are shown in Figure 3. The highest IFU recovery rate after nebulization was achieved at a pH of 6.4-6.8.

[0063] The stability of the vaccine liquid preparation was tested under different pH environments. The results are shown in Figure 4. The optimal pH range of the liquid preparation is 5.8-6.6.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0065] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

[0066] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0068] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

Claims

1. A vaccine preparation, characterized in that The vaccine is a liquid preparation, which contains an active ingredient and excipients; the active ingredient is a recombinant chimpanzee adenovirus expressing an antigen protein, the excipients include a protective agent, and the protective agent includes ethanol and glycerol. The pH value of the liquid preparation is 6.0-6.

8. Preferably, the vaccine liquid preparation is an injection preparation or an aerosol inhalation preparation.

2. The vaccine preparation according to claim 1, characterized in that The volume percentage (v / v%) of the ethanol in the preparation is 0.1-10%.

3. The vaccine preparation according to any one of claims 1 to 2, characterized in that The concentration of glycerol in the formulation is 0.5-10 mg / ml.

4. The vaccine preparation according to any one of claims 1 to 3, characterized in that The auxiliary materials also include sucrose and mannitol.

5. The vaccine preparation according to any one of claim 4, characterized in that The sucrose content is 25-100 mg / ml, preferably 25 mg / ml, and the mannitol content in the auxiliary material is 25-100 mg / ml, preferably 50 mg / ml.

6. The vaccine preparation according to any one of claims 1 to 5, characterized in that The auxiliary material further comprises a buffer, and the buffer is selected from an amino acid buffer, a phosphate buffer or a HEPES buffer. More preferably, the buffer concentration is 1-20 mM, preferably, it is an amino acid buffer, and more preferably, it is a histidine buffer.

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

8. The vaccine preparation according to any one of claims 1 to 7, characterized in that The vaccine adjuvant also includes salt, which is one or more of sodium chloride, magnesium chloride, and calcium chloride. Preferably, the concentration of the salt is 30mM-70mM.

9. The vaccine preparation according to any one of claims 1 to 8, characterized in that The chimpanzee virus is selected from AdC3, AdC6, AdC63, AdC68, Y25, preferably Y25.

10. The vaccine preparation according to any one of claims 1 to 9, characterized in that The antigen is selected from the group consisting of: SARS-CoV, SARS-CoV-2, Ebola virus, hepatitis B virus, hepatitis C virus, dengue virus, herpes zoster virus, rabies virus, human immunodeficiency virus, preferably varicella-zoster virus.

11. The vaccine preparation according to any one of claims 1 to 10, characterized in that The content of the recombinant chimpanzee adenovirus is 1×10 9 ~1ⅹ10 12 VP / ml.

12. The vaccine preparation according to any one of claims 1 to 11, characterized in that The pH value of the liquid preparation is 6.4-6.

6.

13. A method for preparing a vaccine liquid preparation according to any one of claims 1 to 12, characterized in that: The following steps are involved: 1) Purifying the recombinant adenovirus antigen stock solution; 2) Prepare vaccine excipients in proportion; 3) Preparation of semi-finished products; 4) Pack the semi-finished products into controlled bottles.

14. The method for preparing the vaccine liquid preparation according to claim 13, characterized in that: The purification step includes: the virus harvest liquid is lysed, centrifuged and filtered to obtain a clarified liquid, then subjected to two-step chromatography of ion exchange chromatography and composite mode chromatography to obtain a purified virus liquid, then subjected to ultrafiltration to exchange the liquid, and finally subjected to sterile filtration to obtain a vaccine stock solution.

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