CN120815063A

CN120815063A - A recombinant adenovirus vector vaccine preparation and its preparation method

Info

Publication number: CN120815063A
Application number: CN202510469343.0A
Authority: CN
Inventors: 赵晓龙; 王月然; 彭少丹; 菅慧; 王丽萍; 蒋悦颖; 司伟雪; 徐方; 朱涛
Original assignee: CanSino Biologics Inc
Current assignee: CanSino Biologics Inc
Priority date: 2024-04-15
Filing date: 2025-04-15
Publication date: 2025-10-21
Also published as: WO2025218651A1

Abstract

The invention relates to an aerosol inhalation vaccine preparation and a preparation method thereof, wherein the preparation comprises an active ingredient and auxiliary materials, the active ingredient is low-concentration recombinant human replication defective adenovirus expressing antigen protein, and the auxiliary materials comprise a buffering agent, a protective agent, a stabilizing agent, a surfactant and an osmotic pressure regulator. The aerosol inhalation vaccine preparation can keep good stability of the human replication-defective adenovirus vector vaccine preparation.

Description

Recombinant adenovirus vector vaccine preparation and preparation method thereof

Technical Field

The invention belongs to the field of virus biology, and in particular relates to a recombinant adenovirus vector aerosol inhalation vaccine preparation and a preparation method thereof.

Background

One of the continuing challenges in the field of gene therapy and vaccine research is to produce liquid viral formulations that are stable over a longer period of time at a specific temperature range. Adenovirus vectors are currently considered one of the main approaches to gene delivery/therapy. Adenoviruses have great potential in the field of gene therapy and there is therefore a need to develop formulations suitable for parenteral use in humans. Although live adenovirus vaccines have been developed for human use, administration in the form of lyophilized formulations, the excipients used in these lyophilized formulations (gelatin, skim milk, human serum albumin, etc.) are not suitable for parenteral administration routes. Thus, despite reports on the structure and characterization of adenoviruses, there are few reports on the development of stabilized formulations of adenoviruses for parenteral administration in humans. Furthermore, most adenovirus formulations are lyophilized formulations, not liquid formulations, presumably because the stability of liquid formulations is difficult to ensure. Along with the continuous research of adenovirus preparations, adenovirus aerosol inhalation preparations have been developed in the prior art, and in the further research of adenovirus inhalation preparations, the inventor unexpectedly discovers that adenovirus particles with different concentrations have different requirements on a preparation system, and more unexpectedly discovers that currently, the reported stable adenovirus system is widely used and disclosed, and is not suitable for low-concentration adenovirus aerosol inhalation preparations. For this reason, there is a need to develop a formulation to improve the stability of low concentration adenovirus aerosol inhalation formulations.

Disclosure of Invention

The invention aims to provide a stable adenovirus liquid preparation with low virus concentration, and the recombinant adenovirus vector vaccine preparation can maintain the stability of the preparation under the condition of low virus concentration, can effectively excite the immune response of organisms, and can be stably provided as an atomization inhalation preparation.

The invention provides an aerosol inhalation vaccine preparation which can effectively excite immune response of organisms. According to the invention, in the research on the aerosol inhalation vaccine preparation, the problem of insufficient preparation stability can be caused by using a conventional stabilizing system when the effective component of the aerosol inhalation vaccine preparation is low-concentration recombinant human replication defective adenovirus particles expressing antigen proteins, so that the aerosol inhalation preparation of the low-concentration adenovirus is developed in a targeted manner.

Furthermore, the aerosol inhalation vaccine preparation comprises an active ingredient and auxiliary materials, wherein the active ingredient is low-concentration recombinant human replication defective adenovirus expressing antigen proteins, and the content of the replication defective adenovirus is lower than 5x10 ⁹ VP/ml;

The formulations of the invention provide stability to adenoviruses at low viral concentrations and can be administered to a variety of vertebrate organisms, preferably mammals, especially humans. The stabilized viral formulations of the present invention are preferably recombinant adenovirus-based compositions, wherein administration as a vaccine, for example, may provide a prophylactic advantage to previously uninfected individuals and/or a therapeutic effect by reducing the level of viral load in an infected individual, thereby prolonging the asymptomatic phase of infection by a particular microorganism.

Furthermore, the adjuvant component of the aerosol inhalation vaccine preparation does not contain ethanol;

Further, in a system without ethanol, the buffer system is preferably histidine with a histidine concentration greater than 2mM.

Further, when the buffer is histidine, the pH of the vaccine formulation is preferably between 6.2 and 6.8.

Furthermore, the adjuvant of the aerosol inhalation vaccine preparation also comprises a protective agent;

In some embodiments, the protective agent is selected from one or more of gelatin, ethylenediamine tetraacetic acid (EDTA), disodium ethylenediamine tetraacetate (EDTA-2 Na), magnesium chloride, and magnesium chloride hydrate. Preferably, the protectant is disodium edetate and magnesium chloride hexahydrate.

In some embodiments, the protective agent is disodium ethylenediamine tetraacetate (EDTA-2 Na) and magnesium chloride hexahydrate, and the concentration of EDTA-2Na in the aerosol inhalation vaccine formulation is 0-1mM, preferably, the concentration of EDTA-2Na in the formulation is 0.1mM.

In some embodiments, the concentration of magnesium chloride hexahydrate in the vaccine formulation is 1-10mM, preferably the concentration of magnesium chloride hexahydrate in the formulation is 1-5mM, more preferably the concentration of magnesium chloride hexahydrate in the formulation is 2mM.

In some embodiments, the stabilizing agent 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 0.5-10mg/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 1.5mg/ml. Or preferably, the weight/volume fraction (w/v) of glycerol in the vaccine formulation is 0.15%.

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

In some embodiments, the concentration of mannitol in the vaccine formulation is 20-80mg/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 50mg/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 dodecyl sulfate, sodium dodecyl 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 embodiments, the surfactant is present in a concentration of 0.01-1mg/ml, or the surfactant has a weight/volume fraction (w/v) of 0.001% -0.1%, preferably the surfactant has a concentration of 0.1mg/ml, or preferably the surfactant has a weight/volume fraction (w/v) of 0.01%.

In some embodiments, the osmolality adjusting agent is sodium chloride and/or calcium chloride.

Preferably, the osmotic pressure regulator is sodium chloride.

In some embodiments, the osmolality adjusting agent is at a concentration of 30mM-70mM, preferably at a concentration of 50mM.

In some embodiments, the human replication defective adenovirus of the invention comprises a polynucleotide encoding a tuberculosis antigen, including one or more of a peptide or structural protein encoding a mycobacterium tuberculosis Mtb32A, mtb a antigen or an Ag85A antigen, preferably the antigenic peptide or structural protein is linked by a linker.

In some embodiments, the linker is a flexible linker, the sequence of which the linker peptide is a ggggsggggsggs linker peptide, a GGGGS linker peptide, a GLYGLYGLYGLYGLYGLYGLYGLY linker peptide, a GLYGLYGLYGLYGLYGLY linker peptide, a EAAAKEAAAK linker peptide, an EAAAK linker peptide, a PAPAP linker peptide, a APAPAPAPAPAPAPAP linker peptide, a KESGSVSSEQLAQFRSLD linker peptide, a AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAK linker peptide, a EAAAKA, and/or a EAAAKEAAAKEAAAK linker peptide. In some embodiments, the amino acid sequence encoding the Ag85A antigen is SEQ ID NO 1 or has a sequence of at least 75% homology, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology.

In some embodiments, the peptide or structural protein of the antigen is a Mtb32A, mtb A fusion antigen (abbreviated as TB 75K), preferably the amino acid sequence encoding the antigen is SEQ ID NO. 2 or has a sequence of at least 75% homology, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, more preferably wherein the linker sequence may be substituted.

In some embodiments, the Ag85A is fused to TB75K via l inker, preferably Ag85A-l inker-TB75K or TB75K-l inker-Ag85A.

In some embodiments, the amino acid sequence encoding the Ag85A-l inker-TB75K antigen is SEQ ID NO 3 or has a sequence of at least 75% homology, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, more preferably wherein the linker sequence may be substituted.

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

In some embodiments, further comprising an amino acid sequence encoding a signal peptide, preferably the signal peptide is selected from one or more of a tissue plasminogen activator (tPa) signal peptide, an Ag85A wild-type signal peptide, a growth hormone signal peptide, recombinant human Oncoinhibin M (human OSM), vesicular stomatitis virus fusion coat G glycoprotein (VSV-G), mouse Ig Kappa, mouse heavy chain, basement membrane protein 40 (BM 40), human chymotrypsinogen, human trypsinogen-2, human interleukin-2 (human IL-2), luciferase, human albumin (HSA), influenza hemagglutinin, human insulin, and silkworm fibrin LC.

The invention also provides a vector comprising any of the polynucleotides described above and a recombinant human replication defective adenovirus comprising any of the polynucleotides described above.

In some embodiments, the recombinant human replication defective adenovirus of the invention is prepared by a method comprising the steps of:

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

(2) Transfecting the shuttle plasmid vector of step (1) into a host cell along with a backbone plasmid;

(3) Culturing the host cell of step (2);

(4) Harvesting the human replication-defective recombinant adenovirus released from the cells of step (3);

(5) Performing amplification culture on the recombinant adenovirus in the step (4);

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

In another aspect of the invention, a method for preparing any of the vaccine formulations described above is provided, comprising the steps of 1) purifying a recombinant adenovirus antigen stock solution, 2) formulating vaccine adjuvants in proportion, 3) formulating a semi-finished product, and 4) packaging the semi-finished product in a vial.

In some specific embodiments, the purification step comprises the steps of obtaining clarified liquid after splitting, centrifuging and filtering virus harvest liquid, obtaining purified virus liquid after ion exchange chromatography and compound mode chromatography, changing liquid into a specified formula through ultrafiltration, and obtaining vaccine stock solution through sterile filtration.

In another aspect, the invention provides a method for preparing a recombinant adenovirus vector vaccine preparation, which comprises the steps of preparing a recombinant adenovirus vector encoding target antigen protein, and adding pharmaceutically acceptable auxiliary materials.

Specifically, preparing a recombinant adenovirus vector for encoding the mycobacterium tuberculosis Ag85A antigen, optionally adding pharmaceutically acceptable auxiliary materials, or preparing a recombinant adenovirus vector for encoding the mycobacterium tuberculosis Mtb32A and Mtb39A antigens, optionally adding pharmaceutically acceptable auxiliary materials, or preparing a recombinant adenovirus vector for encoding the mycobacterium tuberculosis Mtb32A, mtb A and Ag85A antigens, optionally adding pharmaceutically acceptable auxiliary materials, or respectively preparing a recombinant adenovirus vector for encoding the mycobacterium tuberculosis Ag85A antigen and a recombinant adenovirus vector for encoding the mycobacterium tuberculosis Mtb32A and Mtb39A antigen (TB 75K), mixing, optionally adding pharmaceutically acceptable auxiliary materials.

The method comprises the steps of 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 adenovirus genome.

Preferably, the Ag85A antigen sequence (SEQ ID NO. 1) is constructed by using a genetic engineering method, or the Mtb32A and Mtb39A (TB 75K) fusion antigen sequence (SEQ ID NO. 2) is constructed, or the fusion antigen sequence (SEQ ID NO. 2) containing the Mtb32A and Mtb39A (TB 75K) and the Ag85A antigen sequence (SEQ ID NO. 1) are linked by using a linker 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 comprises the step of adding corresponding auxiliary materials into the original strain seeds subjected to cotransfection packaging to obtain the vaccine.

Specifically, the construction steps of the recombinant adenovirus vector for encoding the mycobacterium tuberculosis TB75K, ag A fusion antigen protein comprise:

(1) Construction of pDC316-TB75K-Ag85A plasmid

And (3) enzyme cutting the synthesized TB75K, ag A fusion protein gene fragment, and recovering the enzyme cut fragment. Meanwhile, the shuttle plasmid vector of AdMax adenovirus system is subjected to enzyme digestion, and the vector is recovered. The TB75K-Ag85A fragment was ligated to the vector using homologous recombination, rendered competent, and plated. Monoclonal was picked for streaking and colony PCR identification. Then, the positive clone monoclonal in the streak plate is taken for culture, and plasmids are extracted for enzyme digestion identification. 10 positive clones were sequenced and sequenced, and the correct vector identified by sequencing was designated pDC316-TB75K-Ag85A.

(2) Recombinant adenovirus Ad5, TB75K, ag A virus seed package

Packaging of the shuttle plasmid pDC316-TB75K-Ag85A and the backbone plasmid of AdMax adenovirus system is carried out by a cotransfection method, and the packaged virus is named as Ad5-105K.

The auxiliary materials used for inhaling the vaccine inhalation preparation can not generate immunosuppression on the vaccine stock solution, and have good compatibility.

The vaccine preparation of the invention induces high level antigen specific IgG, igA, sIgA antibodies after immunization of the organism and can generate good cellular immune response in the lung and the system. The recombinant adenovirus vector vaccine provided by the invention has good stability, safety and high efficiency.

Compared with injection, the vaccine preparation of the invention is administered by using a respiratory tract mucous membrane delivery mode, has compliance, has lower administration dosage, and can generate triple immune effects of humoral immunity, cellular immunity and mucous membrane immunity. The vaccine composition provided by the invention can be used for basic immunity or booster immunity.

The vaccine of the invention can generate particles with the particle size of 3-10 mu m after being atomized by a proper device, particularly, aerosol particles with the 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 mu m, preferably aerosol particles with the particle size of 5-10 mu m, particularly, aerosol particles with the 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, which are preferably uniform. The medicine can reach the lung through nasal or oral inhalation, thereby generating protective immune response to the whole respiratory tract and the lung, enhancing the effective utilization rate of the vaccine and improving the effect of the vaccine.

In particular, the invention also provides a preparation method of the multicomponent recombinant adenovirus vector vaccine preparation composition and application thereof in preventing and/or treating diseases.

Specifically, the disease is a disease caused by 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 infection.

In another aspect, the invention provides the use of any of the recombinant human replication-defective adenovirus vector vaccine formulations described above in the manufacture of a medicament for the prevention and/or treatment of a disease.

The beneficial effects of the invention are as follows:

1. The vaccine preparation can keep good stability of the human replication-defective adenovirus vector vaccine preparation, and can keep good stability of the preparation under the condition of low virus concentration. The vaccine preparation can effectively avoid aggregation of antigens, can be stably stored for a long time, is qualified in abnormal toxicity test and is safe to use.

2. The vaccine preparation is suitable for administration by utilizing an aerosol inhalation mode, can ensure the activity yield after aerosol, can generate triple immune effects of humoral immunity, cellular immunity and mucosal immunity, and can be used for basic immunity or reinforcing immunity.

3. The vaccine preparation can target immune lung macrophages, and can enhance the immune response of the vaccine in the lung by using a human type 5 adenovirus vector as a safe natural type I adjuvant.

4. The vaccine preparation of the invention does not influence the immunogenicity of each antigen component, does not generate mutual interference of immune reaction among antigens, has proper auxiliary material components, content and pH value of the preparation, and can effectively protect organisms from infection of pathogenic bacteria or viruses and prevent recurrence of latent infection.

Drawings

FIG. 1 Western Blot results of antigen expression validation (1: TB75K;2: ag85A;3: ag85A: TB75K=1: 1;4: ag85A-linker-TB75K;5: TB75K-l inker-Ag85A;6: negative control);

FIG. 2 trend of LossLgIFU changes in different concentration formulations at 37℃for 4 weeks

FIG. 3 tendency of accelerated stability at 37℃of different buffer system formulations with adenovirus concentration of 5X 10 ⁹ VP/ml

FIG. 4 atomization stability of different buffer System formulations with adenovirus concentration of 5X 10 ⁹ VP/ml

FIG. 5 trend of accelerated stability at 37℃for formulations of histidine buffer systems of different concentrations

FIG. 6 accelerated stability towards 37℃of formulations with ethanol-histidine buffer System and histidine System (5X 10 ⁹ vp/ml)

FIG. 7 accelerated stability towards 37℃of formulations with ethanol-histidine buffer System and histidine System (1X 10 ⁹ vp/ml)

FIG. 8 accelerated stability towards 37℃of formulations with ethanol-histidine buffer System and histidine System (1X 10 ⁸ vp/ml)

FIG. 9 accelerated stability profiles of recombinant adenovirus vector formulations at different pH's at 37℃to 4 weeks LossLgIFU

FIG. 10 specific embodiment of the spray stability Activity yield of recombinant adenovirus vector formulations at different pH

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 construction packaging of recombinant adenovirus vector vaccine formulations

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.

And (3) enzyme cutting the synthesized TB75K, ag A fusion gene fragment, and recovering the enzyme cut fragment. Meanwhile, the shuttle plasmid vector of AdMax adenovirus system is subjected to enzyme digestion, and the vector is recovered. The TB75K-Ag85A fragment was ligated to the vector using homologous recombination, rendered competent, and plated on Amp-resistant LB plates. The following day the monoclonal was picked for streaking and colony PCR identification was performed simultaneously. Then, the positive clone monoclonal in the streak plate is taken for culture, and plasmids are extracted for enzyme digestion identification. Sequencing 10 positive clones, and marking the correct vector as pDC316-TB75K-Ag85A

(2) Recombinant adenovirus Ad5-TB75K-Ag85A virus seed package

Preparation of vaccine inhalation formulation:

1) Preparation of recombinant tuberculosis vaccine (type 5 adenovirus vector) by adding adjuvants such as mannitol 50mg, sodium chloride 5mg, HEPES1mg, polysorbate 80.2 mg, glycerol 4mg, magnesium chloride 0.2mg, sucrose 30mg into the novel recombinant tuberculosis vaccine TB75K-Ag85A stock solution (containing virus particle number 5×1010 VP), and mixing to obtain 1ml recombinant multicomponent tuberculosis vaccine (type 5 adenovirus vector) preparation. Labeled ADTB202302001.

Example 2 in vitro expression characterization of recombinant adenovirus vector vaccine formulations

The recombinant adenovirus vector tuberculosis vaccine prepared in example 1 was tested for target antigen expression by cell assay combined with Western Blot.

(1) Cell assay

Cell plating, virus inoculation, virus infection, cell lysis, and transfer of cell lysis supernatant to new EP tube to obtain sample lysis supernatant and negative control lysis supernatant. The refrigerator is kept at-80 ℃ for standby.

(1) Western Blot experiment to detect target gene expression

Electrophoresis, membrane transfer, sealing, primary antibody incubation and secondary antibody incubation.

Analysis of results

1) Conditions for establishment of test

A. the negative control sample should not have the antigen band of interest;

b. The target antigen band of the positive sample is clearly discernable and the molecular weight is accurate.

2) Decision criterion

The TB75K antigen protein has a target band around 75kDa, the multicomponent binding antigen has a target band around 105-110 kDa, and the negative control should not have the target protein band, and the target protein is judged to be positive in expression.

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

The recombinant novel tuberculosis vaccine (5-type adenovirus vector) can be used for expressing target antigen, and the result is judged to be positive when the two positive (+) are satisfied. The detection results are shown in FIG. 1.

Example 3 stability of formulations of different viral concentrations in HEPES buffer System

The buffer is HEPES, the concentration gradient of the recombinant adenovirus is set under the system, and the stability of the recombinant adenovirus vector preparation under different concentrations of the recombinant adenovirus is verified, wherein the preparation comprises mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol and the like. IFU of recombinant adenovirus preparation samples with different concentrations under the buffer system condition of 37 degrees C, HEPES is detected, and LossLgIFU variation trend is examined

Group of	Number of viral particles VP/ml	PH value of
			1	1.00E+08	7.5
2	1.00E+09	7.5
			3	5.00E+09	7.5
4	1.00E+10	7.5
			5	1.00E+11	7.5

As a result, as shown in FIG. 2, when the adenovirus content was less than 5X 10 ⁹, the stability of the preparation was significantly deteriorated at 37℃in the accelerated test using HEPES buffer as a system.

Example 4 Effect of different buffers on stability of formulations at Low dose adenovirus concentration and formulation aerosolization Activity yield

Different buffer systems are arranged, and IFU change conditions of the recombinant adenovirus vector preparation placed for 1 week, 2 weeks, 3 weeks and 4 weeks under the different buffer systems are examined. Selecting histidine, PB, tris-HCl, HEPES, histidine+Tris-HCl, histidine+HEPES, wherein the preparation contains mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol, etc. And detecting IFU of the recombinant adenovirus preparation sample under different buffer systems at 37 ℃ and examining LossLgIFU variation trend. And detecting the activity of the sample which is not atomized and recovered by the recombinant adenovirus preparation of different buffer systems, and calculating the atomization activity yield.

Group of	Number of viral particles VP/ml	PH value of	Buffer solution
				1	5.00E+09	7.5	HEPES
2	5.00E+09	7.5	HIS
				3	5.00E+09	7.5	Tris-HCl
4	5.00E+09	7.5	PB
				5	5.00E+09	7.5	HIS-Tris-HCl
6	5.00E+09	7.5	HIS-HEPES

As shown in FIGS. 3 and 4, when the adenovirus content was 5X 10 ⁹, the HIS buffer was used as the system, and the stability of the preparation was optimal.

Example 5 Effect of different concentration histidine buffer systems on stability of Low dose adenovirus concentration formulations

The HIS buffer system is set, and the IFU change condition of the recombinant adenovirus vector preparation at 37 ℃ for 1 week, 2 weeks, 3 weeks and 4 weeks under the conditions of different histidine content and low-dose virus content is examined. The preparation comprises mannitol, sucrose, sodium chloride, magnesium chloride, glycerol, PS-80, ethanol, etc.

Group of	Histidine concentration	PH value of
			1	2mM	7.5
2	5mM	7.5
			3	10mM	7.5
4	15mM	7.5
			5	20mM	7.5
6	30mM	7.5

As shown in FIG. 5, when the adenovirus content was 5X 10 ⁹ vp/ml, the HIS buffer was used as the system, the stability of the preparation was poor at a concentration of 2mM, and the stability of the preparation was better at a concentration of 5-30 nM.

Example 6 Effect Rate of EtOH on stability of recombinant adenovirus vector formulations

Setting HIS buffer system, examining IFU change condition of recombinant adenovirus vector preparation at 37 deg.C for 1 week, 2 weeks, 3 weeks and 4 weeks in the presence of ethanol and different low dose virus contents.

Group of	Number of viral particles VP/ml	Buffer solution	Ethanol
				1	5.00E+09	HIS	0
2	1.00E+09	HIS	0
				3	1.00E+08	HIS	0
4	5.00E+09	HIS	0.5%
				5	1.00E+09	HIS	0.5%
6	1.00E+08	HIS	0.5%

As shown in FIGS. 5-7, when the adenovirus content was less than 5X 10 ⁹ and the buffer was HIS, the stability of the ethanol-free formulation was better.

Example 6 ratio of influence of pH on stability of recombinant adenovirus vector preparation and preparation aerosolization Activity yield

The HIS buffer system is arranged, the effect of pH change on the stability of the recombinant adenovirus vector preparation is studied by low-dose virus content in the absence of ethanol, and the recombinant adenovirus vector preparation is placed at 37 ℃ for 1 week, 2 weeks, 3 weeks and 4 weeks to change IFU.

Group of	Number of viral particles VP/ml	Buffer solution	pH
				1	5.00E+09	HIS	7
2	1.00E+09	HIS	7
				3	1.00E+08	HIS	7
4	5.00E+09	HIS	6.8
				5	1.00E+09	HIS	6.8
6	1.00E+08	HIS	6.8
				7	5.00E+09	HIS	6.6
8	1.00E+09	HIS	6.6
				9	1.00E+08	HIS	6.6
	5.00E+09	HIS	6.2
					1.00E+09	HIS	6.2
	1.00E+08	HIS	6.2
				10	5.00E+09	HIS	6.0
11	1.00E+09	HIS	6.0
				12	1.00E+08	HIS	6.0

As shown in FIGS. 9 and 10, when the adenovirus content is lower than 5×10 ⁹, the buffer is HIS system and contains no ethanol, the preparation has the best stability and atomization activity yield at 37 ℃ under the pH value of 6.2-6.8.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. An aerosol inhalation vaccine preparation, characterized in that the effective component of the vaccine preparation is recombinant type 5 human replication defective adenovirus expressing antigen protein, the preparation does not contain ethanol, and the content of the recombinant type 5 human replication defective adenovirus in the preparation is lower than 5x10 ⁹ VP/ml.

2. The vaccine formulation of claim 1, wherein the buffer system of the vaccine formulation is histidine.

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

4. A vaccine formulation according to any one of claims 1-3, wherein the protective agent is selected from one or more of gelatin, ethylenediamine tetraacetic acid (EDTA), disodium ethylenediamine tetraacetic acid (EDTA-2 Na), magnesium chloride and magnesium chloride hydrate, preferably the protective agent is disodium ethylenediamine tetraacetic acid and magnesium chloride hexahydrate, the concentration of EDTA-2Na in the vaccine formulation is 0-1mM, preferably 0.1mM, and the concentration of magnesium chloride hexahydrate in the vaccine formulation is 1-10mM, preferably 1-5mM, more preferably 2mM.

5. The vaccine formulation according to any one of claims 1-4, wherein 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 glycerin in the preparation is 0.5-10mg/ml, the concentration of glycerin in the preparation is preferably 1.5mg/ml, the concentration of sucrose in the preparation is 20-80mg/ml, the concentration of sucrose in the preparation is preferably 25mg/ml, the concentration of mannitol in the preparation is 20-80mg/ml, and the concentration of mannitol in the preparation is preferably 50mg/ml.

6. The vaccine formulation according to any one of claims 1-6, wherein the surfactant is selected from one or more of a non-ionic surfactant, an anionic surfactant and a zwitterionic surfactant, preferably the surfactant is selected from one or more of sodium dodecyl sulphate, polyvinyl alcohol, tween 80, tween 20, span 80 and span 20, preferably the surfactant is tween 80, the concentration of the surfactant is 0.01-1mg/ml, preferably the concentration of the surfactant is 0.1mg/ml.

7. The vaccine formulation according to any one of claims 1-6, wherein the pH of the formulation is between 6.2 and 6.8.

8. Vaccine formulation according to any one of claims 1-7, characterized in that the formulation is an aerosol inhalation formulation, which upon aerosol administration device forms particles with a particle size below 10 μm, preferably 0.5-10 μm, more preferably 5-10 μm.

9. Vaccine formulation according to any one of claims 1-8, characterized in that the antigenic 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.

10. A method for preparing a vaccine formulation according to any one of claims 1 to 9, comprising the steps of 1) purifying a recombinant adenovirus antigen stock solution, 2) proportioning vaccine adjuvants, 3) formulating a semi-finished product, and 4) packaging the semi-finished product in a vial.

11. Use of a recombinant human replication defective adenovirus vector vaccine preparation for the preparation of a medicament for the prevention and/or treatment of a disease, characterized in that the vaccine preparation is as defined in claims 1-11.

12. Use according to claim 12, wherein the disease is caused by 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 infection.