Vaccine liquid preparation based on chimpanzee adenovirus vector and preparation method

Abstract

The invention relates to a vaccine preparation, an effective component is recombinant chimpanzee adenovirus for expressing antigen protein, and auxiliary materials are ethanol and glycerol. The preparation does not contain animal-derived components and is high in safety. The preparation provided by the invention can keep good stability of the chimpanzee adenovirus vector vaccine liquid preparation. The composition can be stably stored for more than 12 months at 2-8 DEG C, and is qualified in abnormal toxicity test and safe to use; the vaccine preparation can be used as a liquid injection and an aerosol inhalation preparation at the same time.

Description

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 refer to a type of defective virus that can introduce target genes into cells for expression through biological infection pathways after the wild-type viral genome has been genetically modified to reduce or eliminate its pathogenicity while retaining the characteristics of high transfection efficiency. Due to the difficulty in preparation, the small capacity of the target gene, and the problems of immunogenicity and biosafety, only a few viruses can be transformed into the vectors required for gene therapy. According to the type of viral genome, they can be classified into DNA viral vectors (adenovirus vectors, adeno-associated virus vectors and herpes simplex virus vectors), RNA viral vectors (retroviral vectors and lentiviral vectors) and chimeric viral vectors. Although adenovirus has many advantages as a vaccine virus, this method of developing vaccines has a major flaw. Because adenovirus is so common, about one-third of the population has developed immunity to it in adulthood, and the immune systems of these people will destroy the vaccine instead of producing antibodies against the virus carrying the inserted gene.

[0003] Considering that most people are much less likely to be infected with microorganisms from chimpanzees, chimpanzee adenovirus can be used as a vaccine vector. Chimpanzee adenovirus vector vaccine is a viral vector vaccine. In liquid state, the active components contained in the vaccine are not stable. The main manifestation is that changes in storage conditions and extended storage time will affect the effectiveness of the vaccine itself. Therefore, how to ensure the stability of the vaccine is one of the most core technical issues in the vaccine production process. Compared with human adenovirus, chimpanzee adenovirus is more difficult to control its stability.

[0004] In addition, in order to further improve the immunogenicity of the vaccine, various studies have been conducted on the vaccine administration method. The study found that the administration method through atomization inhalation can further stimulate the body's mucosal immunity, allowing the drug to directly reach the respiratory tract through atomization and cover the entire respiratory mucosa; it is an important method for preventing and treating respiratory diseases, and it is also another method of administration besides oral and injection. This method of atomization administration can achieve the vaccination of the vaccine through the respiratory tract. Because the atomized inhalation vaccine has the advantages of small dosage, painless vaccination process, small side effects and wide application range. However, due to the small dosage of inhalation vaccines, new challenges are posed to the maintenance of the active ingredients of the vaccine in the inhalation dosage form. In addition, inhalation preparations are different from traditional injection preparations. The atomization rate, mist concentration, atomization particle size and particle size distribution will affect the preparation effect, and higher requirements are put forward for the preparation; in actual research and development and production, a vaccine that can be used as both an injection preparation and an atomized inhalation preparation can be developed to greatly reduce corporate costs and clinical periods, and promote the vaccine to be listed as soon as possible. The present invention is a further improvement on the above-mentioned existing research technology. 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. Summary of the invention

[0005] One object of the present invention is to provide a chimpanzee adenovirus vector vaccine preparation with high safety and good stability.

[0006] Another object of the present invention is to provide a chimpanzee adenovirus vector vaccine preparation. The preparation can be a liquid injection preparation or an aerosol inhalation preparation, and the preparation comprises an active ingredient and an excipient; the active ingredient is a recombinant chimpanzee adenovirus expressing an antigen protein, and the excipient comprises a protective agent, and the protective agent comprises ethanol and glycerol.

[0007] 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.

[0008] 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%.

[0009] 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, 0.5 mg / ml.

[0010] 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;

[0011] Preferably, the optimal ratio of sucrose and mannitol in the auxiliary material is 1:2-1:3; more preferably 1:2;

[0012] Preferably, in one embodiment, sucrose is 25 mg / ml and mannitol is 50 mg / ml;

[0013] In one embodiment, sucrose is 25 mg / ml and mannitol is 75 mg / ml.

[0014] Preferably, the auxiliary material further comprises one or more of a chelating agent, a buffer, a surfactant and a salt.

[0015] Preferably, the auxiliary material 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.

[0016] 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 dodecyl sulfate, polyvinyl alcohol, sodium dodecyl sulfate, Tween 80, Span 80, Span 20, Tween 20; preferably, the concentration of the surfactant is 0.01-1 mg / ml.

[0017] 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.

[0018] 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.

[0019] 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 optimal;

[0020] Preferably, the auxiliary material further comprises a buffer, and the buffer is 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, 15 mM.

[0021] Preferably, the chimpanzee virus is selected from AdC3, AdC6, AdC63, AdC68, Y25, preferably Y25.

[0022] 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, human immunodeficiency virus, preferably varicella-zoster virus.

[0023] Varicella-zoster virus (VZV), also known as human herpesvirus type 3, is a ubiquitous alpha herpesvirus and a double-stranded DNA virus. There are two types of VZV infection in humans, namely primary infection with varicella (Variella) and recurrent infection with herpes zoster (Zoster). VZV only infects humans in its natural state, without an animal host, and its main targets are T lymphocytes, epithelial cells and ganglia. The initial infection causes chickenpox, and after recovery, the virus remains latent in the ganglia. With age, cellular immunity gradually declines, or in immunocompromised patients, the virus recurs and causes herpes zoster. Herpes zoster can be complicated by chronic pain (postherpetic neuralgia, PHN) and other serious neurological and ocular diseases (e.g., meningoencephalitis, myelitis, cranial nerve palsy, vasculopathy, keratitis and retinopathy), as well as a variety of visceral and gastrointestinal diseases, including ulcers, hepatitis and pancreatitis. Immunization with a vaccine against shingles is an effective means of preventing the disease.

[0024] Preferably, the content of the recombinant viral vector is 1×10 9 ~1ⅹ10 12 VP / ml.

[0025] The preparation may be an injection preparation or an aerosol inhalation preparation.

[0026] 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.

[0027] In one embodiment, the buffer system of the vaccine formulation is His.

[0028] The present invention relates to a method for preparing a vaccine liquid preparation, comprising the following steps:

[0029] 1) Purifying the recombinant adenovirus antigen stock solution;

[0030] 2) Prepare vaccine excipients in proportion;

[0031] 3) Preparation of semi-finished products;

[0032] 4) Pack the semi-finished products into controlled bottles.

[0033] Preferably, the purification step includes: 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 subjected to ultrafiltration to exchange the liquid, and finally subjected to sterile filtration to obtain a vaccine stock solution.

[0034] The beneficial effects of the present invention are:

[0035] Vaccine excipients refer to the protective agents, excipients and additives used in the preparation of vaccine products. They are an indispensable and important component of vaccine preparations and play an important role in the stability of vaccine preparations. Appropriate formulations play a positive and critical role in the biological activity of vaccine products, the development of new dosage forms and new varieties, and meeting the requirements of preventive use. This preparation does not contain animal-derived ingredients and is highly safe.

[0036] The preparation of the present invention can maintain good stability of the chimpanzee adenovirus vector vaccine liquid preparation. The vaccine preparation can effectively avoid the aggregation of antigens, can be stably stored at 2-8°C for more than 12 months, and its abnormal toxicity test is qualified and safe to use. The vaccine preparation of the present invention can be used as a liquid preparation or an aerosol inhalation preparation at the same time, without the need to adjust the prescription due to dosage form changes, saving costs and reducing time to the greatest extent, and injection and inhalation clinical trials can be carried out simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 . Results of IFU stability test at 37℃ with different stabilizer systems

[0038] Figure 2 . Results of IFU stability test of different sugar / sugar alcohol systems at 37°C

[0039] Figure 3 . Results of IFU recovery after atomization under different pH conditions

[0040] Figure 4 . Results of IFU stability test under different pH conditions DETAILED DESCRIPTION

[0041] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those generally understood by those skilled in the art to which the present invention relates. The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments 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 ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0042] Example 1 Preparation of model vaccine

[0043] Varicella-zoster gE protein was selected as the model antigen.

[0044] Preparation method of recombinant varicella-zoster virus: Recombinant varicella-zoster virus is obtained by modifying chimpanzee adenovirus Y25. First, the two homologous arms LF1 and LF2 at the left end of the Y25 genome and one homologous arm RF at the right end were inserted into the bacterial artificial chromosome (BAC) system to construct the plasmid pBACe3.6. The plasmid and the Y25 genome were then co-transformed into Escherichia coli. Through homologous recombination, the viral genome was integrated into the plasmid and the E1 region gene was deleted. The correct recombinant clone was obtained by chloramphenicol resistance and restriction enzyme screening. Then, using primers E3de l LH and E3de l RH, the E3 region was replaced by galactokinase (Gal K). Finally, two Gal Ks were inserted to replace the E4 region open reading frames ORF4, 6, and 7. Subsequently, the Gal K gene was replaced by the E4 region of human adenovirus type 5 to obtain a replication-defective chimpanzee adenovirus vector. The vector has a unique AsiSI restriction site in the E1 deletion region, and a modified attR1 attR2Gateway component (Invitrogen) is inserted. This component is used to insert the varicella-zoster virus gE protein gene 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.

[0045] Preparation method of recombinant varicella-zoster vaccine stock solution: HEK293 cells are infected with recombinant varicella-zoster virus, and virus harvest solution is obtained through amplification. The virus harvest solution is subjected to lysis, centrifugation, filtration and other operations to obtain a clarified solution, which is then subjected to two-step chromatography of ion exchange chromatography and composite mode chromatography to obtain a purified virus solution, which is then subjected to ultrafiltration for liquid exchange, and finally, the vaccine stock solution is obtained through sterile filtration.

[0046] Example 2 Screening of Stabilizer Systems for Replication-Deficient Chimpanzee Adenovirus Vector Preparations

[0047] The effect of different stabilizers on the stability of the preparations was studied by setting different stabilizer systems, storing them at 37°C, and taking samples to detect the virus infection titer at 0, 2, 4, 7, 10, and 14 days.

[0048] Virus infection titer detection; detection using adenovirus detection kit;

[0049] Table 1. Screening of different stabilizer systems

[0050]

[0051] Experimental results: The results of I FU stability test under 37℃ conditions with different stabilizer systems 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

[0055]

[0056] 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.

[0057] Long-term stability test method: Place the vaccine at 37°C and take samples for virus infection titer detection at 2, 4, 7, 10, and 14 days. Use an adenovirus detection kit for detection;

[0058] The experimental results are as follows Figure 2 The optimal ratio of sucrose and mannitol in the auxiliary material is 1:2-1:3; more preferably 1:2

[0059] Example 4 Screening of pH System for Replication-Deficient Chimpanzee Adenovirus Vector Preparation

[0060] Based on the experimental results of Examples 2 and 3, the effect of pH on the formulation system after atomization was further verified.

[0061] The effect of different pH values ​​on the atomized preparation was tested. The sample preparation method is as follows:

[0062] Table 3. Screening of different pH systems

[0063]

[0064] 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.

[0065] The virus harvest fluid 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 fluid. The fluid is then exchanged into buffer solutions of different pH values ​​through ultrafiltration, and finally, aseptically filtered to obtain vaccine stock solutions of different pH values, which are used to study the effects of different pH values ​​on virus stability.

[0066] The I FU recovery rate after atomization was tested under different pH environments (I FU recovery rate = I FU of atomized samples ÷ I FU of unatomized samples × 100%). The samples No. 1-10 in the table above were atomized separately, 1 ml each time, and the atomized liquid was collected for I FU detection. Three samples were collected for each number. The results are shown in Figure 1. Figure 3 When the pH was 6.4-6.8, the I FU recovery rate after atomization was the highest.

[0067] The stability of vaccine liquid preparations under different pH conditions was tested. Figure 4 , the optimal pH range for liquid preparations is 5.8-6.6.

[0068] 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 principle of the present invention should be included in the protection scope of the present invention.

[0069] 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.

[0070] 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.

[0071] 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 principle of the present invention should be included in the protection scope of the present invention.

[0072] 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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