WO2023109740A1 - Inhalation administration delivery system of recombinant adenovirus vector vaccine - Google Patents
Inhalation administration delivery system of recombinant adenovirus vector vaccine Download PDFInfo
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- WO2023109740A1 WO2023109740A1 PCT/CN2022/138331 CN2022138331W WO2023109740A1 WO 2023109740 A1 WO2023109740 A1 WO 2023109740A1 CN 2022138331 W CN2022138331 W CN 2022138331W WO 2023109740 A1 WO2023109740 A1 WO 2023109740A1
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- adenovirus
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- adenovirus vector
- virus
- albumin
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Definitions
- the invention relates to the technical field of vaccines, in particular to a recombinant adenovirus vector vaccine inhalation delivery system for solving adenovirus pre-existing immunity.
- Adenoviruses are non-enveloped double-stranded DNA viruses, and 53 serotypes of human adenoviruses have been identified.
- Adenoviruses consist of an outer capsid protein and an inner core protein. Core proteins include: protein V, protein VII and protein X, which are combined with the viral genome.
- the terminal protein TP is covalently bound to the 5' end of the viral DNA; surrounding the viral core is the viral capsid, which is composed of seven proteins (II, III, IIIa, IV, VI, VIII and IX) through non-covalent
- Adenovirus has a non-enveloped spherical structure, and its virus particles are often arranged in a lattice in the infected cell nucleus.
- Each virus particle contains a 36kb linear double-stranded DNA, with a 100-600bp reverse end at each end Repeated sequence (inverted terminal re-peat, ITR), the inner side of ITR is the viral packaging signal, which is the cis-acting element required for viral packaging.
- the genome contains early expressed E1-E4 genes related to adenovirus replication and late expressed L1-L5 genes related to adenovirus particle assembly.
- the linear double-stranded DNA and the core protein form a core with a diameter of 60-65nm, which is wrapped in the capsid.
- the capsid is icosahedrally symmetrical and consists of 252 capsomers with a diameter of 8-10 nm.
- the capsomers are arranged on a triangular surface, with 6 on each side, of which 240 are hexons (non-apex capsomers), and the other 12 are hexons.
- One is the base of the penton (apex capsomer).
- Each hexon is a homotrimer of a hexon protein.
- the hexon molecule of the trimer has a triangular spire and a pentahedral base.
- the tower region consists of 4 rings, namely loop1, loop2, and loop3. , loop4, the base includes two regions P1 and P2.
- the current problem with adenovirus vector vaccines is the existence of human body’s pre-existing immunity to adenoviruses.
- type 5 adenoviruses are more common in the environment, and it is easy for the population to generate an immune response against adenoviruses, which will It will reduce the neutralizing antibody level of the adenovirus vector vaccine in the human body.
- the ways to solve the pre-existing immunity of adenovirus mainly include chemical methods and genetic engineering methods.
- the chemical method mainly wraps the adenovirus with PEG to shield its antigenic epitope, thereby escaping the immune function of the host, but it is difficult to obtain high-titer virus by this method.
- the method of genetic engineering is to modify the adenovirus, such as constructing a chimera-modified adenovirus capsid, or constructing a chimeric adenovirus.
- adenovirus-based vaccines with other rare adenovirus serotypes, such as from humans or non-humans (Chen H, Xiang Z Q, Li Y, et al.
- the recombinant novel coronavirus vaccine (type 5 adenovirus vector) developed by CanSino Biological Co., Ltd. was proposed by the National Health and Health Commission, and the National Medical Products Administration organized demonstrations and agreed to include it as a booster injection for emergency treatment. use.
- adenovirus including albumin-binding moiety can obtain Albumin shield that enables the virus to evade neutralizing antibodies and prolong its retention in the blood after systemic administration.
- Albumin is widely used as a drug carrier in the hot research and development field, thanks to its long half-life in plasma, and the non-covalent binding with the loaded drug will not cause the drug complex to be quickly cleared in the blood.
- the current technical route is administered by injection. Due to the presence of specific CD4+ and CD8+ T cells that have been infected with type 5 adenovirus, it still has an adverse effect on the potency of the albumin-bound recombinant adenovirus vaccine.
- the invention relates to an adenovirus vector vaccine preparation, which contains recombinant adenovirus and auxiliary components; the vector backbone genome sequence of the recombinant adenovirus includes a sequence encoding albumin binding domain; the auxiliary components include albumin.
- the vaccine can effectively escape the pre-existing immunity to the adenovirus vector in the body.
- a specific dose of human albumin is added to the formulation of the vaccine preparation for inhalation to ensure that a defense barrier effectively evading the adenovirus neutralizing antibody has been formed before administration.
- an inhalation vaccine based on the Ad-ABD platform it is difficult to form an effective protective shell against neutralizing antibodies after the vaccine is delivered to the target organ.
- an adenovirus vector vaccine with an albumin-binding domain enters the body, it may be quickly recognized and cleared by specific neutralizing antibodies before binding to albumin in the body; There is mutual competition in the binding force, resulting in the inability of albumin to fully bind to the viral vector and thus fail to form a complete and effective pre-existing immune barrier.
- the hexon is the most abundant protein in the capsid of adenovirus and is also considered to be the main target of neutralizing antibodies.
- Loop 1-L1 and loop 2-L2 of the hexon protein are located outside the capsomer structure of adenovirus.
- Loop 1 of the hexon protein has six HVRs, (HVR1-HVR6).
- HVRs hypervariable regions
- L2 contains the 7th hypervariable region (HVR7), containing specific epitopes.
- the sequence encoding the albumin binding domain is inserted in the coding region of HVR-1 of the hexon protein, the albumin binding domain being located on the outer surface of the hexon protein.
- the albumin binding domain is selected from the albumin binding domain of streptococcal protein G or its functionally equivalent variants, preferably, the albumin binding domain is the albumin binding domain 3 of streptococcal protein G.
- sequence of the albumin binding domain 3 of streptococcal protein G is SEQ ID NO:1.
- sequence of SEQ ID NO:1 is as follows:
- the inserted sequence encoding albumin binding is the binding domain included in the fusion protein after the D150 amino acid of the hexon protein.
- the sequence of the hinge region between the hexon protein and the albumin binding domain is called the linker sequence, and its main function is to provide space between the two elements so that the albumin binding domain (ABD) part does not interfere with the hexon affect the secondary structure of body proteins. Because the linker can usually maintain the length of the three-dimensional structure of the element, and make the elements independent.
- the N-terminal and/or C-terminal of the albumin binding domain is connected to the hexon protein via a linker sequence.
- the linker sequence includes the sequence GSGS, as shown in SEQ ID NO:2.
- the adenovirus is a human adenovirus.
- the human adenovirus is selected from one or more of human adenoviruses from serotype 1 to serotype 57. More preferably, the human adenovirus is selected from the group consisting of human adenoviruses of serotype 1 to serotype 57; more preferably, the human adenovirus is a human adenovirus of serotype 5, 26 or 35.
- the albumin added in the auxiliary component is one or more of human serum albumin or recombinant human serum albumin or bovine serum albumin.
- Albumin binds to the outer surface capsid region of the adenoviral hexon protein.
- the molar ratio of albumin to adenovirus vector is 1-10 5 :1; preferably, 1-240:1.
- the vaccine is a mucosal administration preparation or an injection preparation
- the mucosal administration preparation is selected from: liquid dosage form, solid dosage form, semi-solid dosage form or gas dosage form; more preferably, the mucosal administration preparation
- the pharmaceutical preparations are nasal drops, aerosols, sprays, powder sprays, powders, liquid preparations, freeze-dried preparations, gels, microspheres, liposomes, films, and suspensions;
- formulations for mucosal administration are inhalation liquid formulations, inhalation aerosols and inhalation sprays.
- the mucous membrane includes nasal mucous membrane, oral mucosa or pulmonary mucous membrane.
- auxiliary components also include pharmaceutically acceptable excipients.
- pharmaceutically acceptable auxiliary materials include, but are not limited to: one or more of buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives and inactivators.
- the buffering agent includes but is not limited to one or more of HEPES, HIS, TRIS, PB, succinic acid, citric acid;
- the protective agent includes but is not limited to gelatin, ethanol, diethylaminotetraacetic acid (EDTA), one or more of disodium diethylamine tetraacetate (EDTA-2Na) and magnesium chloride;
- the stabilizer includes but not limited to one or more of sucrose, mannitol, fucose and maltose
- the surfactant includes, but is not limited to, one or more of Tween, Span, and glycerin;
- the osmotic pressure regulator includes, but is not limited to, sodium chloride or omitted.
- the auxiliary material components include mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, glycerin, preferably, mannitol 10-150mg/ml, sucrose 10-150mg/ml, sodium chloride 10-150mM, magnesium chloride 1-10mM, HEPES 1-15mM, polysorbate 80 0.001%-0.5% by weight, glycerin 0.05-2% by weight.
- the preparation components include: sucrose 10-50mg/ml, mannitol 15-75mg/ml, sodium chloride 40-60mM, glycerin 0.5-5mg/ml, magnesium chloride 1-5mM, Tween 80 0.05-0.5mg/ml ml, HEPES 1-5mM, HAS content 0.1%-1% by weight percentage.
- the dosage form is an atomized inhalation agent
- the vaccine is atomized by an atomized drug delivery device to form particles below 10 ⁇ m.
- the content of the albumin in the vaccine is 0.001%-50% by weight, preferably 0.005% or 0.01% or 0.05% or 0.1% or 0.2% or 0.3% or 0.4% or 0.5% or 0.6% or 0.7% or 0.8% or 0.9% or 1% or 2% or 3% or 4% or 5% or 6% or 7% or 8% or 9% or 10% or 15% or 20% or 25 % or 30% or 35% or 40% or 45% or 50%.
- the adenovirus vector also contains viral or bacterial antigenic proteins.
- viral or bacterial antigenic proteins examples include HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, respiratory syncytial virus (RSV), herpes zoster One or more of virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and Plasmodium falciparum, Mycobacterium tuberculosis.
- HIV HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, respiratory syncytial virus (RSV), herpes zoster One or more of virus (VZV),
- the selected packaging cells are HEK293 cells.
- the invention provides a preparation method of an adenovirus vector vaccine preparation, the preparation steps comprising:
- S1 introduces the albumin binding domain (ABD) of Streptococcus protein G into the HVR region of the adenovirus vector backbone hexon gene to construct an adenovirus-ABD vector;
- ABSD albumin binding domain
- S2 introduces the viral or bacterial antigen protein gene sequence into the modified adenovirus-ABD vector to construct an Ad-ABD vector vaccine;
- S3 adds albumin to the preparation as an auxiliary component.
- the selected packaging cells are HEK293 cells.
- the invention provides a drug delivery system for an adenovirus vector vaccine preparation.
- the drug delivery system is atomized inhalation drug delivery, and the atomized drug delivery device is a jet atomization drug delivery device or an ultrasonic atomization drug delivery device or a vibratory atomization drug delivery device. drug delivery device.
- the atomized drug delivery device includes an atomizer and a component for collecting atomized vaccine particles.
- the invention provides an application of an adenovirus vector vaccine preparation in preparing vaccines for preventing infectious diseases.
- adenoviruses carry viral or bacterial disease antigens.
- the viral or bacterial antigens contained therein are HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, Respiratory syncytial virus (RSV), herpes zoster virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and Plasmodium falciparum, Mycobacterium tuberculosis or one of Various.
- the present invention provides a method of preventing infectious diseases, said method comprising administering an adenovirus vector vaccine to a subject.
- the subject may be human or mammal or cells, tissues or organs thereof.
- the present invention has been verified by experiments to provide an original vaccine inhalation drug delivery system, so that the vaccine can be administered in the form of atomized inhalation, which can activate the protective immune response including mucosal immunity, and at the same time solve the pre-existing immunity of rAd. Verified that it can effectively reduce the impact of Ad pre-existing immunity on vaccine immunogenicity
- the present invention adds albumin as a compounding component to the preparation to better combine with the adenovirus containing the albumin binding domain to form an albumin-coated capsid to evade pre-existing immunity.
- albumin as a compounding component to the preparation to better combine with the adenovirus containing the albumin binding domain to form an albumin-coated capsid to evade pre-existing immunity.
- inhalation administration it also solves the problem that the single administration of ABD-modified adenovirus cannot effectively stimulate humoral immunity and cellular immune response, and can simultaneously activate mucosal immunity to obtain higher titers.
- Figure 1 shows a schematic diagram of the molecular design of the recombinant Ad-ABD vector
- Figure 4 14 days antigen type strain binding antibody neutralization titer (log10)
- Figure 5 28 days antigen type strain binding antibody neutralization titer (log10)
- Figure 6 42 days antigen type strain binding antibody neutralization titer (log10)
- Adenovirus vector vaccine refers to a vaccine made by using adenovirus as a carrier, recombining the target antigen gene into the adenovirus genome, and using a recombinant adenovirus capable of expressing the antigen gene.
- Embodiment 1 Preparation of Ad-ABD adenovirus
- the recombinant new coronavirus vaccine with replication-deficient adenovirus as the carrier was selected as the evaluation model:
- the target antigen of the recombinant new coronavirus vaccine is the S protein of the new coronavirus strain (Genebank number: NC_045512.2);
- Backbone plasmid The schematic diagram of the molecular design of the recombinant Ad-ABD vector is shown in Figure 1: using the existing technology, the albumin binding domain (SEQ ID NO: 1) with two linkers (sequence: GSGS) at the side end is inserted into the adenovirus six In the adjacent HVR1 (after D150 amino acid);
- step (2) transfecting the shuttle plasmid vector described in step (1) together with the backbone plasmid into the host cell HEK293;
- step (3) cultivating the host cell HEK293 described in step (2);
- step (6) Purifying the culture product in step (5) to obtain Ad capable of expressing the target gene.
- HEK293 cells were cultured in culture medium and passaged.
- MOI multiplicity of infection
- the multiplicity of infection (MOI) of inoculated virus is 1, 5 and 10 respectively, culture at 37°C, 5% CO 2 , 125rpm, The harvesting time is 42h-48h, and the adenovirus seed harvesting liquid is obtained.
- the Ad5-ABD virus harvest liquid and supernatant amplified by HEK293 cells were detected with the Ad-FITC fluorescent antibody method to detect the titer IFU. The results are shown in Table 1.
- Embodiment 2 ELISA detects the combination of Ad-ABD adenovirus and albumin
- Example 3 In vitro neutralization experiment of Ad-ABD adenovirus evading pre-existing immunity - qualitative experiment
- Virus sample and antibody dilution Dilute Ad-ABD stock solution and NCVA stock solution to virus titer with DMEM complete culture (DMEM+10%FBS+1%P/S) and DMEM complete medium+1mg/ml HSA respectively It is 1.5 ⁇ 10 6 IFU/ml.
- the Ad5 rabbit polyclonal antibody (sourced from CanSino Biological Co., Ltd.) was diluted with DMEM complete medium, and the antibody dilutions were original times, 10 times and 100 times respectively.
- Ad rabbit polyclonal antibody 2 Ad-ABD stock solution+1mg/ml HSA+10 -1
- Ad rabbit polyclonal antibody 3 Ad-ABD stock solution+1mg/ml HSA+10 -2
- Ad rabbit polyclonal antibody 4 Ad-ABD stock solution + 10 0
- Ad rabbit polyclonal antibody 5 Ad-ABD stock solution + 10 -1
- Ad rabbit polyclonal antibody 8 NCVA stock solution+1mg/ml HSA+10 -1
- Ad rabbit polyclonal antibody 9 NCVA stock solution+1mg/ml HSA+10 -2
- Ad rabbit polyclonal antibody 12 NCVA stock solution + 10 -2
- HEK293 cells were diluted with DMEM complete medium to a cell density of 3 ⁇ 10 5 cells/ml. After the virus sample and the diluted Ad rabbit polyclonal antibody were incubated at 37°C for 1 hour, 100 ⁇ L HEK293cells/well was added to a 96-well plate, mixed evenly, and incubated at 37°C, 5% CO 2 for 4-5 hours. Wash the plate 3 times with PBS,
- Ad-FITC fluorescent antibody was diluted with 1% BSA/1 ⁇ PBS, the dilution ratio was 1:500; after washing the plate, 50 ⁇ L/well of the diluted fluorescent antibody was added to the 96-well plate, and incubated for 1 hour at room temperature in the dark Afterwards, the plate was washed twice with PBS.
- Ad or Ad-ABD virus vaccine stock solution was incubated with serially diluted anti-Ad antibodies in the presence of HSA, and then infected HEK293 cells.
- the neutralization degree of Ad-ABD was lower than that of Ad carrier, indicating that ABD modification and adding albumin in the preparation can escape pre-immunization.
- mice/group 6-8 weeks old female BALB/c mice, 8 mice/group.
- NCVA group the recombinant new coronavirus vaccine (Ad) without ABD transformation prepared in Example 1;
- Ad-ABD+HSA group human serum albumin (HSA) solution was added to the stock solution of the new coronavirus vaccine of Ad-ABD prepared in Example 1, and the final concentration of HSA was 1 mg/ml;
- Ad-ABD+BSA group Add bovine serum albumin (BSA) solution to the stock solution of the new coronavirus vaccine of Ad-ABD prepared in Example 1, and the final concentration of BSA is 1mg/ml;
- BSA bovine serum albumin
- Ad-ABD group the stock solution of the new coronavirus vaccine of Ad-ABD prepared in Example 1.
- the way of mucosal inoculation use the inhalation exposure tower to immunize by atomization inhalation, and the immunization dose is 5 ⁇ 10 9 VP per mouse.
- the second immunization was carried out 28 days after the first immunization, and the rats were killed 14 days after the second immunization to evaluate the level of cellular immunity and the titer of total S antibody.
- the test results are shown in Figure 6;
- the results of the experiment showed that two doses were used for immunization in this experiment, and the level of total antibody specific to S protein was detected 14 days after the first immunization.
- the results showed that the level of antibody induced by the Ad-ABD carrier was lower than that of the non-modified ABD after administration by inhalation. Ad.
- Ad-ABD+HSA and Ad-ABD+BSA in the experimental group were significantly higher than those in the other groups, that is, the technical scheme of pre-adding albumin in the preparation and combining Ad-ABD for co-inhalation can make the evasion of pre-existing immunity
- the positive impact has increased significantly.
- the reason may be that the lung organs are different from the in vitro and injection environments.
- the present invention fundamentally solves the druggability problem of the Ad-ABD mucosal administration preparation evading pre-existing immunity by adding albumin to the preparation, that is, through in vitro prefabricated combination and re-inhalation administration.
- Example 5 In vivo immunogenicity evaluation - cellular immune response.
- mice/group 6-8 weeks old female BALB/c mice, 8 mice/group.
- TNF ⁇ , IFN ⁇ , IL2, and IL5 were detected by flow cytometry, Anti-TNF ⁇ -FITC antibody, Anti-IFN ⁇ -PE, mo ⁇ se antibody, Anti-IL2-APC, mo ⁇ se antibody, Anti-IL5-AF700 antibody (Table 7- 14).
- the experimental results show that: through inhalation (mucosal administration), mucosal immunization, NCVA group, Ad-ABD+HSA group, Ad-ABD+BSA group, Ad-ABD group vectors can induce the production of S-IgG in mouse serum Antibody titer. Both can stimulate humoral immunity and cellular immunity in mice, mainly CD8+ T cells, but the level of cellular immunity in the Ad-ABD group was significantly lower than that of the control NCVA.
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Abstract
Disclosed is an inhalation administration delivery system of a recombinant adenovirus vector vaccine. In the delivery system, an adenovirus vector is reformed and matched with a specific preparation prescription, and after being administered by means of inhalation, the vaccine can reach the lungs by means of nasal inhalation or mouth inhalation, so that a protective immune response including mucosal immunity is generated, the effective utilization rate of the vaccine is increased, the pre-existing immunity of an adenovirus is solved, and an effect of the vaccine is improved.
Description
本发明涉及疫苗技术领域,具体涉及一种解决腺病毒预存免疫的重组腺病毒载体疫苗吸入给药递送系统。The invention relates to the technical field of vaccines, in particular to a recombinant adenovirus vector vaccine inhalation delivery system for solving adenovirus pre-existing immunity.
腺病毒属于无包膜的双链DNA病毒,人类的腺病毒目前已鉴定出53个血清型。腺病毒由外部的衣壳蛋白和内部的核心蛋白组成。核心蛋白包括:蛋白Ⅴ、蛋白Ⅶ以及蛋白Ⅹ,它们与病毒基因组结合。末端蛋白TP与病毒DNA的5'端共价结合;围绕着病毒核心的是病毒的衣壳,它是由7种蛋白(Ⅱ、Ⅲ、Ⅲa、Ⅳ、Ⅵ、Ⅷ和Ⅸ)通过非共价相互作用形成的二十面体对称结构,其中240个三聚六邻体(蛋白Ⅱ)是该20面体的主要组成蛋白。Adenoviruses are non-enveloped double-stranded DNA viruses, and 53 serotypes of human adenoviruses have been identified. Adenoviruses consist of an outer capsid protein and an inner core protein. Core proteins include: protein V, protein VII and protein X, which are combined with the viral genome. The terminal protein TP is covalently bound to the 5' end of the viral DNA; surrounding the viral core is the viral capsid, which is composed of seven proteins (II, III, IIIa, IV, VI, VIII and IX) through non-covalent The icosahedral symmetric structure formed by interaction, in which 240 trimeric hexons (protein II) is the main constituent protein of the icosahedron.
腺病毒呈无囊膜的球形结构,其病毒粒子在感染的细胞核内常呈晶格状排列,每个病毒颗粒包含一个36kb的线性双链DNA,两端各有一个100~600bp的反向末端重复序列(inverted terminal re-peat,ITR),ITR的内侧为病毒包装信号,是病毒包装所需要的顺式作用元件。基因组包含早期表达的与腺病毒复制相关的E1~E4基因和晚期表达的与腺病毒颗粒组装相关的L1~L5基因。线状双股DNA与核心蛋白形成直径为60~65nm的髓芯,被包裹于衣壳内。衣壳呈二十面体对称,由252个直径8~10nm的壳粒组成,壳粒排列在三角形的面上,每边6个,其中240个为六邻体(非顶点壳粒),另12个为五邻体基底(顶点壳粒)。每个六邻体是六邻体蛋白的同源三聚体,三聚体的六邻体分子有一个三角形的塔尖和五面体的基底,塔区由4个环构成即loop1、loop2、loop3、loop4,基底包含两个区域P1、P2区。Adenovirus has a non-enveloped spherical structure, and its virus particles are often arranged in a lattice in the infected cell nucleus. Each virus particle contains a 36kb linear double-stranded DNA, with a 100-600bp reverse end at each end Repeated sequence (inverted terminal re-peat, ITR), the inner side of ITR is the viral packaging signal, which is the cis-acting element required for viral packaging. The genome contains early expressed E1-E4 genes related to adenovirus replication and late expressed L1-L5 genes related to adenovirus particle assembly. The linear double-stranded DNA and the core protein form a core with a diameter of 60-65nm, which is wrapped in the capsid. The capsid is icosahedrally symmetrical and consists of 252 capsomers with a diameter of 8-10 nm. The capsomers are arranged on a triangular surface, with 6 on each side, of which 240 are hexons (non-apex capsomers), and the other 12 are hexons. One is the base of the penton (apex capsomer). Each hexon is a homotrimer of a hexon protein. The hexon molecule of the trimer has a triangular spire and a pentahedral base. The tower region consists of 4 rings, namely loop1, loop2, and loop3. , loop4, the base includes two regions P1 and P2.
腺病毒载体疫苗目前存在的问题就是人体对腺病毒预存免疫(pre-existing immunity)的存在,例如,5型腺病毒在环境中较为常见,人群中容易产生抗腺病毒的免疫反应,而这将会降低腺病毒载体疫苗在人体的中和抗体水平。目前解决腺病毒预存免疫方式主要包括化学法和基因工程方法。化学法主要通过用PEG包裹腺病毒,屏蔽其抗原表位,从而逃逸宿主的免疫作用,但此类方法难以获得高滴度的病毒。The current problem with adenovirus vector vaccines is the existence of human body’s pre-existing immunity to adenoviruses. For example, type 5 adenoviruses are more common in the environment, and it is easy for the population to generate an immune response against adenoviruses, which will It will reduce the neutralizing antibody level of the adenovirus vector vaccine in the human body. At present, the ways to solve the pre-existing immunity of adenovirus mainly include chemical methods and genetic engineering methods. The chemical method mainly wraps the adenovirus with PEG to shield its antigenic epitope, thereby escaping the immune function of the host, but it is difficult to obtain high-titer virus by this method.
基因工程的方法是对腺病毒进行修饰,例如构建嵌合体修饰的腺病毒衣壳、构建嵌合型的腺病毒。将基于腺病毒的疫苗完全替换为其他稀有的,例如来自人类或非人类的腺病毒血清型(Chen H,Xiang Z Q,Li Y,et al.Adenovirus-based vaccines:comparison of vectors from three species of adenoviridae[J].Virol,2010,84(20):10522-10532),但是外源基因引发的特异性抗体反应强度均显著低于由5型腺病毒载体携带所激发的免疫反应。The method of genetic engineering is to modify the adenovirus, such as constructing a chimera-modified adenovirus capsid, or constructing a chimeric adenovirus. Complete replacement of adenovirus-based vaccines with other rare adenovirus serotypes, such as from humans or non-humans (Chen H, Xiang Z Q, Li Y, et al. Adenovirus-based vaccines: comparison of vectors from three species of adenoviridae[J]. Virol, 2010,84(20):10522-10532), but the intensity of specific antibody responses triggered by exogenous genes was significantly lower than that induced by type 5 adenovirus vectors.
2022年9月4日,康希诺生物股份公司研发的吸入用重组新型冠状病毒疫苗(5型腺病毒载体),经国家卫生健康委提出建议,国家药品监督管理局组织论证同意作为加强针纳入紧急使用。On September 4, 2022, the recombinant novel coronavirus vaccine (type 5 adenovirus vector) developed by CanSino Biological Co., Ltd. was proposed by the National Health and Health Commission, and the National Medical Products Administration organized demonstrations and agreed to include it as a booster injection for emergency treatment. use.
现有技术(WO2015166082A1,发明名称:包括白蛋白结合部分的腺病毒)在衣壳外表面上,尤其在腺病毒六邻体蛋白的外表面上经白蛋白结合部分进行基因修饰的腺病毒能够获得白蛋白保护罩,使病毒在全身给药后能够逃避中和抗体并延长其在血液中的保留时间。白蛋白作为药物载体在热门研发领域被广泛使用,得益于它在血浆中有较长半衰期,并且与被载药物间的非共价结合不会使药物复合物在血液中被快速清除掉。但是目前该技术路线通过注射给药,由于已感染5型腺病毒存在的特异的CD4+和CD8+T细胞,对于白蛋白结合重组腺病毒疫苗效价的发挥仍有不利的影响。Prior art (WO2015166082A1, title of invention: adenovirus including albumin-binding moiety) can obtain Albumin shield that enables the virus to evade neutralizing antibodies and prolong its retention in the blood after systemic administration. Albumin is widely used as a drug carrier in the hot research and development field, thanks to its long half-life in plasma, and the non-covalent binding with the loaded drug will not cause the drug complex to be quickly cleared in the blood. However, the current technical route is administered by injection. Due to the presence of specific CD4+ and CD8+ T cells that have been infected with type 5 adenovirus, it still has an adverse effect on the potency of the albumin-bound recombinant adenovirus vaccine.
发明内容Contents of the invention
本发明涉及一种腺病毒载体疫苗制剂,含有重组腺病毒和辅助组分;所述重组腺病毒的载体骨架基因组序列中包括编码白蛋白结合域的序列;所述辅助组分包括白蛋白。该疫苗能有效逃逸体内对腺病毒载体的预存免疫。The invention relates to an adenovirus vector vaccine preparation, which contains recombinant adenovirus and auxiliary components; the vector backbone genome sequence of the recombinant adenovirus includes a sequence encoding albumin binding domain; the auxiliary components include albumin. The vaccine can effectively escape the pre-existing immunity to the adenovirus vector in the body.
本发明在疫苗递送之前,添加特定剂量的人源白蛋白到吸入用疫苗制剂配方中以保证在给药前已形成有效逃避腺病毒中和抗体的防御屏障。In the present invention, before the delivery of the vaccine, a specific dose of human albumin is added to the formulation of the vaccine preparation for inhalation to ensure that a defense barrier effectively evading the adenovirus neutralizing antibody has been formed before administration.
作为以Ad-ABD为平台的吸入用疫苗,在疫苗递送到靶向器官后不易形成有效的抗中和抗体的保护壳。此外,即使带有白蛋白结合域的腺病毒载体疫苗进入体内也可能在结合体内白蛋白之前就快速被特异性中和抗体识别并清除;或者特异性中和抗体和白蛋白在与腺病毒载体结合力上存在相互竞争,导致白蛋白不能充分结合病毒载体继而不能形成完整有效的预存免疫屏障。As an inhalation vaccine based on the Ad-ABD platform, it is difficult to form an effective protective shell against neutralizing antibodies after the vaccine is delivered to the target organ. In addition, even if an adenovirus vector vaccine with an albumin-binding domain enters the body, it may be quickly recognized and cleared by specific neutralizing antibodies before binding to albumin in the body; There is mutual competition in the binding force, resulting in the inability of albumin to fully bind to the viral vector and thus fail to form a complete and effective pre-existing immune barrier.
六邻体是腺病毒衣壳中含量最高的蛋白,也被认为是中和抗体的主要靶点。六邻体蛋白的环1-L1以及环2-L2位于腺病毒壳粒结构外侧。六邻体蛋白的环1有6个HVR,(HVR1-HVR6)。在六邻体蛋白三聚体结构的塔尖区域中有7个高变区(HVR),即,L2含有第7高变区(HVR7),包含特异性表位。The hexon is the most abundant protein in the capsid of adenovirus and is also considered to be the main target of neutralizing antibodies. Loop 1-L1 and loop 2-L2 of the hexon protein are located outside the capsomer structure of adenovirus. Loop 1 of the hexon protein has six HVRs, (HVR1-HVR6). There are 7 hypervariable regions (HVRs) in the spire region of the hexon protein trimer structure, ie, L2 contains the 7th hypervariable region (HVR7), containing specific epitopes.
所述编码白蛋白结合域的序列插入在六邻体蛋白的HVR-1的编码区中,所述白蛋白结合域位于所述六邻体蛋白的外表面上。The sequence encoding the albumin binding domain is inserted in the coding region of HVR-1 of the hexon protein, the albumin binding domain being located on the outer surface of the hexon protein.
具体地,所述白蛋白结合域选自链球菌蛋白G的白蛋白结合域或其的功能等同变体,优选地,所述白蛋白结合域为链球菌蛋白G的白蛋白结合结构域3。Specifically, the albumin binding domain is selected from the albumin binding domain of streptococcal protein G or its functionally equivalent variants, preferably, the albumin binding domain is the albumin binding domain 3 of streptococcal protein G.
更优选地,所述链球菌蛋白G的白蛋白结合结构域3的序列为SEQ ID NO:1。More preferably, the sequence of the albumin binding domain 3 of streptococcal protein G is SEQ ID NO:1.
SEQ ID NO:1序列如下:The sequence of SEQ ID NO:1 is as follows:
Cys Glu Trp Asp Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His ValCys Glu Trp Asp Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His Val
所述插入的编码白蛋白结合的序列是融合蛋白包括在六邻体蛋白的D150氨基酸后的结合域。The inserted sequence encoding albumin binding is the binding domain included in the fusion protein after the D150 amino acid of the hexon protein.
在六邻体蛋白以及白蛋白结合域间的铰链区的序列称之为连接子序列,主要作用是为两个元件之间提供空间,以使白蛋白结合域(ABD)部分不会对六邻体蛋白的二级结构产生影响。由于连接子通常可保持元件的三位结构长度,并且使元件各自独立。The sequence of the hinge region between the hexon protein and the albumin binding domain is called the linker sequence, and its main function is to provide space between the two elements so that the albumin binding domain (ABD) part does not interfere with the hexon affect the secondary structure of body proteins. Because the linker can usually maintain the length of the three-dimensional structure of the element, and make the elements independent.
所述白蛋白结合域的N-末端和/或C-末端通过连接子序列与所述六邻体蛋白相连。The N-terminal and/or C-terminal of the albumin binding domain is connected to the hexon protein via a linker sequence.
所述连接子序列包括序列GSGS,如SEQ ID NO:2所示。The linker sequence includes the sequence GSGS, as shown in SEQ ID NO:2.
具体地,所述腺病毒为人腺病毒。Specifically, the adenovirus is a human adenovirus.
优选地,人腺病毒选自血清1型~血清57型的人腺病毒中的一种或多种。更优选地,人腺病毒选自由血清1型~血清57型的人腺病毒组成的组;更优选地,所述人腺病毒为血清5型、26型或35型人腺病毒。Preferably, the human adenovirus is selected from one or more of human adenoviruses from serotype 1 to serotype 57. More preferably, the human adenovirus is selected from the group consisting of human adenoviruses of serotype 1 to serotype 57; more preferably, the human adenovirus is a human adenovirus of serotype 5, 26 or 35.
具体地,所述辅助组分中添加的白蛋白为人源血清白蛋白或重组的人源 血清白蛋白或牛血清白蛋白中的一种或多种。白蛋白可与腺病毒六邻体蛋白的外表面衣壳区域结合。Specifically, the albumin added in the auxiliary component is one or more of human serum albumin or recombinant human serum albumin or bovine serum albumin. Albumin binds to the outer surface capsid region of the adenoviral hexon protein.
具体地,白蛋白与腺病毒载体的摩尔比例为1-10
5:1;优选地,1-240:1。
Specifically, the molar ratio of albumin to adenovirus vector is 1-10 5 :1; preferably, 1-240:1.
具体地,所述疫苗为黏膜给药制剂或注射给药制剂,优选地,所述黏膜给药制剂选自:液体剂型、固体剂型、半固体剂型或者气体剂型;更优选地,所述黏膜给药制剂为滴鼻剂、气雾剂、喷雾剂、粉雾剂、粉末剂、液体制剂、冻干制剂、凝胶剂、微球剂、脂质体、膜剂、混悬剂;Specifically, the vaccine is a mucosal administration preparation or an injection preparation, preferably, the mucosal administration preparation is selected from: liquid dosage form, solid dosage form, semi-solid dosage form or gas dosage form; more preferably, the mucosal administration preparation The pharmaceutical preparations are nasal drops, aerosols, sprays, powder sprays, powders, liquid preparations, freeze-dried preparations, gels, microspheres, liposomes, films, and suspensions;
进一步地,所述黏膜给药制剂为吸入液体制剂、吸入气雾剂和吸入喷雾剂。Further, the formulations for mucosal administration are inhalation liquid formulations, inhalation aerosols and inhalation sprays.
更优选地,为鼻吸入或经口吸入;更优选地,所述黏膜包括鼻黏膜、口腔黏膜或肺粘膜。More preferably, it is nasal inhalation or oral inhalation; more preferably, the mucous membrane includes nasal mucous membrane, oral mucosa or pulmonary mucous membrane.
具体地,所述辅助组分中还包含药学上可接受的辅料。所述药学上可接受的辅料包括但不限于:缓冲剂、保护剂、稳定剂、表面活性剂、渗透压调节剂、佐剂、防腐剂和灭活剂等中的一种或多种。Specifically, the auxiliary components also include pharmaceutically acceptable excipients. The pharmaceutically acceptable auxiliary materials include, but are not limited to: one or more of buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives and inactivators.
具体地,所述缓冲剂包括但不限于HEPES、HIS、TRIS、PB、琥珀酸、柠檬酸中的一种或多种;所述保护剂包括但不限于明胶、乙醇、二乙氨四乙酸(EDTA)、二乙氨四乙酸二钠(EDTA-2Na)和氯化镁中的一种或者多种;所述稳定剂包括但不限于蔗糖、甘露醇、岩藻糖和麦芽糖中的一种或多种;所述表面活性剂包括但不限于吐温、司盘、甘油中的一种或者多种;所述渗透压调节剂包括但不限于氯化钠或省去。Specifically, the buffering agent includes but is not limited to one or more of HEPES, HIS, TRIS, PB, succinic acid, citric acid; the protective agent includes but is not limited to gelatin, ethanol, diethylaminotetraacetic acid ( EDTA), one or more of disodium diethylamine tetraacetate (EDTA-2Na) and magnesium chloride; the stabilizer includes but not limited to one or more of sucrose, mannitol, fucose and maltose The surfactant includes, but is not limited to, one or more of Tween, Span, and glycerin; the osmotic pressure regulator includes, but is not limited to, sodium chloride or omitted.
具体地,所述辅料组分包括甘露醇,蔗糖,氯化钠,氯化镁,HEPES,聚山梨酯80,甘油,优选地,甘露醇10-150mg/ml,蔗糖10-150mg/ml,氯化钠10-150mM,氯化镁1-10mM,HEPES 1-15mM,以重量百分比计聚山梨酯80 0.001%-0.5%,以重量百分比计甘油0.05-2%。Specifically, the auxiliary material components include mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, glycerin, preferably, mannitol 10-150mg/ml, sucrose 10-150mg/ml, sodium chloride 10-150mM, magnesium chloride 1-10mM, HEPES 1-15mM, polysorbate 80 0.001%-0.5% by weight, glycerin 0.05-2% by weight.
具体地,制剂组分包含:蔗糖10-50mg/ml、甘露醇15-75mg/ml、氯化钠40-60mM、甘油0.5-5mg/ml、氯化镁1-5mM、吐温80 0.05-0.5mg/ml、HEPES 1-5mM、以重量百分比计HAS含量0.1%-1%。Specifically, the preparation components include: sucrose 10-50mg/ml, mannitol 15-75mg/ml, sodium chloride 40-60mM, glycerin 0.5-5mg/ml, magnesium chloride 1-5mM, Tween 80 0.05-0.5mg/ml ml, HEPES 1-5mM, HAS content 0.1%-1% by weight percentage.
具体地,剂型为雾化吸入剂,所述疫苗经雾化给药装置雾化后形成10μm 以下的颗粒。Specifically, the dosage form is an atomized inhalation agent, and the vaccine is atomized by an atomized drug delivery device to form particles below 10 μm.
具体地,所述白蛋白在疫苗中的含量以重量百分比计为0.001%-50%,优选地,为0.005%或0.01%或0.05%或0.1%或0.2%或0.3%或0.4%或0.5%或0.6%或0.7%或0.8%或0.9%或1%或2%或3%或4%或5%或6%或7%或8%或9%或10%或15%或20%或25%或30%或35%或40%或45%或50%。Specifically, the content of the albumin in the vaccine is 0.001%-50% by weight, preferably 0.005% or 0.01% or 0.05% or 0.1% or 0.2% or 0.3% or 0.4% or 0.5% or 0.6% or 0.7% or 0.8% or 0.9% or 1% or 2% or 3% or 4% or 5% or 6% or 7% or 8% or 9% or 10% or 15% or 20% or 25 % or 30% or 35% or 40% or 45% or 50%.
具体地,所述腺病毒的载体中还包含病毒或细菌抗原蛋白。例如HIV、狂犬病病毒、登革热病毒、埃博拉病毒、冠状病毒、人乳头瘤病毒、丙型肝炎病毒、乙型肝炎病毒、轮状病毒、麻疹病毒、呼吸道合胞病毒(RSV)、带状疱疹病毒(VZV)、巨细胞病毒、2型单纯疱疹病毒、爱泼斯坦巴尔病毒、流感病毒、克氏锥虫和恶性疟原虫、结核杆菌的一种或多种。Specifically, the adenovirus vector also contains viral or bacterial antigenic proteins. Examples include HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, respiratory syncytial virus (RSV), herpes zoster One or more of virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and Plasmodium falciparum, Mycobacterium tuberculosis.
具体地,选择的包装细胞为HEK293细胞。Specifically, the selected packaging cells are HEK293 cells.
本发明提供一种腺病毒载体疫苗制剂的制备方法,制备步骤包括:The invention provides a preparation method of an adenovirus vector vaccine preparation, the preparation steps comprising:
S1将链球菌(Streptococcus)蛋白G的白蛋白结合域(ABD)引入到腺病毒载体骨架hexon基因的HVR区,构建腺病毒-ABD载体;S1 introduces the albumin binding domain (ABD) of Streptococcus protein G into the HVR region of the adenovirus vector backbone hexon gene to construct an adenovirus-ABD vector;
S2将病毒或细菌抗原蛋白基因序列引入到改造的腺病毒-ABD载体中,构建为Ad-ABD载体疫苗;S2 introduces the viral or bacterial antigen protein gene sequence into the modified adenovirus-ABD vector to construct an Ad-ABD vector vaccine;
S3在制剂中加入白蛋白作为辅助组分。S3 adds albumin to the preparation as an auxiliary component.
具体地,选择的包装细胞为HEK293细胞。Specifically, the selected packaging cells are HEK293 cells.
本发明提供一种腺病毒载体疫苗制剂的给药系统,给药系统为雾化吸入给药,所述雾化给药装置为射流雾化给药装置或超声雾化给药装置或振动雾化给药装置。The invention provides a drug delivery system for an adenovirus vector vaccine preparation. The drug delivery system is atomized inhalation drug delivery, and the atomized drug delivery device is a jet atomization drug delivery device or an ultrasonic atomization drug delivery device or a vibratory atomization drug delivery device. drug delivery device.
优选地,所述雾化给药装置包括雾化器和雾化后的疫苗颗粒收集部件。Preferably, the atomized drug delivery device includes an atomizer and a component for collecting atomized vaccine particles.
本发明提供一种腺病毒载体疫苗制剂在制备用于预防传染性疾病的疫苗中的用途。具体地,腺病毒包载了病毒或细菌病抗原。The invention provides an application of an adenovirus vector vaccine preparation in preparing vaccines for preventing infectious diseases. In particular, adenoviruses carry viral or bacterial disease antigens.
具体地,其中包载的病毒或细菌抗原为HIV、狂犬病病毒、登革热病毒、埃博拉病毒、冠状病毒、人乳头瘤病毒、丙型肝炎病毒、乙型肝炎病毒、轮状病毒、麻疹病毒、呼吸道合胞病毒(RSV)、带状疱疹病毒(VZV)、巨细胞病毒、2型单纯疱疹病毒、爱泼斯坦巴尔病毒、流感病毒、克氏锥虫和恶性疟原虫、结核杆菌的一种或多种。Specifically, the viral or bacterial antigens contained therein are HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, Respiratory syncytial virus (RSV), herpes zoster virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and Plasmodium falciparum, Mycobacterium tuberculosis or one of Various.
本发明提供一种预防传染性疾病的方法,所述的方法包括向受试者施加腺病毒载体疫苗。The present invention provides a method of preventing infectious diseases, said method comprising administering an adenovirus vector vaccine to a subject.
所述的受试者可以为人或哺乳动物或其细胞、组织或器官。The subject may be human or mammal or cells, tissues or organs thereof.
本发明的有益效果包括:The beneficial effects of the present invention include:
本发明经过实验验证独创性的提供一种疫苗吸入给药系统,使得疫苗以雾化吸入的方式给药,可激活包括黏膜免疫在内的保护性免疫反应,同时解决rAd的预存免疫,经实验验证,能够有效降低Ad预存免疫对疫苗免疫原性的影响The present invention has been verified by experiments to provide an original vaccine inhalation drug delivery system, so that the vaccine can be administered in the form of atomized inhalation, which can activate the protective immune response including mucosal immunity, and at the same time solve the pre-existing immunity of rAd. Verified that it can effectively reduce the impact of Ad pre-existing immunity on vaccine immunogenicity
本发明为了配合对于腺病毒的改造,在制剂中加入配合成分白蛋白,与含有白蛋白结合域的腺病毒更好地结合形成白蛋白包覆的衣壳,以逃避预存免疫。通过吸入给药,同时解决了单独施用ABD改造腺病毒无法有效刺激体液免疫和细胞免疫免疫反应的问题,可同时激活黏膜免疫以获得较高滴度。In order to cooperate with the transformation of adenovirus, the present invention adds albumin as a compounding component to the preparation to better combine with the adenovirus containing the albumin binding domain to form an albumin-coated capsid to evade pre-existing immunity. Through inhalation administration, it also solves the problem that the single administration of ABD-modified adenovirus cannot effectively stimulate humoral immunity and cellular immune response, and can simultaneously activate mucosal immunity to obtain higher titers.
图1所示为重组Ad-ABD载体的分子设计示意图;Figure 1 shows a schematic diagram of the molecular design of the recombinant Ad-ABD vector;
图2Ad和Ad-ABD与HSA和BSA结合-ELISA结果(注*无对应柱状图即未检测到);Figure 2Ad and Ad-ABD combined with HSA and BSA-ELISA results (note * no corresponding histogram is not detected);
图3体外中和实验-定量实验IFU计算结果;Fig. 3 in vitro neutralization experiment-quantitative experiment IFU calculation result;
图4 14天抗原型株结合抗体中和滴度(1og10);Figure 4 14 days antigen type strain binding antibody neutralization titer (log10);
图5 28天抗原型株结合抗体中和滴度(1og10);Figure 5 28 days antigen type strain binding antibody neutralization titer (log10);
图6 42天抗原型株结合抗体中和滴度(1og10);Figure 6 42 days antigen type strain binding antibody neutralization titer (log10);
图7抗S-IgG抗体滴度;Fig. 7 anti-S-IgG antibody titer;
图8 CD4+T cell细胞免疫结果;Figure 8 CD4+ T cell immune results;
图9 CD8+T cell细胞免疫结果。Figure 9 CD8+ T cell immune results.
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.
“腺病毒载体疫苗”是指以腺病毒作为载体,将目的抗原基因重组到腺病毒基因组中,使用能表达该抗原基因的重组腺病毒制成的疫苗。下面将结合 本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。"Adenovirus vector vaccine" refers to a vaccine made by using adenovirus as a carrier, recombining the target antigen gene into the adenovirus genome, and using a recombinant adenovirus capable of expressing the antigen gene. 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 described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
实施例1:Ad-ABD腺病毒的制备Embodiment 1: Preparation of Ad-ABD adenovirus
选择复制缺陷腺病毒为载体的重组新型冠状病毒疫苗作为评价模型:The recombinant new coronavirus vaccine with replication-deficient adenovirus as the carrier was selected as the evaluation model:
重组新型冠状病毒疫苗的目标抗原为新型冠状病毒毒株(Genebank编号:NC_045512.2)的S蛋白;The target antigen of the recombinant new coronavirus vaccine is the S protein of the new coronavirus strain (Genebank number: NC_045512.2);
(1)构建含有编码新型冠状病毒S蛋白的多核苷酸的穿梭质粒载体;(1) Construct a shuttle plasmid vector containing a polynucleotide encoding the novel coronavirus S protein;
骨架质粒:重组Ad-ABD载体的分子设计示意图参见图1:利用现有技术,将侧端具有两个接头(序列:GSGS)的白蛋白结合域(SEQ ID NO:1)插入到腺病毒六邻体的HVR1中(D150氨基酸后);Backbone plasmid: The schematic diagram of the molecular design of the recombinant Ad-ABD vector is shown in Figure 1: using the existing technology, the albumin binding domain (SEQ ID NO: 1) with two linkers (sequence: GSGS) at the side end is inserted into the adenovirus six In the adjacent HVR1 (after D150 amino acid);
(2)将步骤(1)所述穿梭质粒载体与骨架质粒一起转染入宿主细胞HEK293;(2) transfecting the shuttle plasmid vector described in step (1) together with the backbone plasmid into the host cell HEK293;
(3)培养步骤(2)所述宿主细胞HEK293;(3) cultivating the host cell HEK293 described in step (2);
(4)收获从步骤(3)所述细胞中释放的人复制缺陷重组腺病毒(5型腺病毒载体);(4) Harvest the human replication-deficient recombinant adenovirus (type 5 adenovirus vector) released from the cells described in step (3);
(5)对步聚(4)中的重组腺病毒进行扩大培养;(5) Expanding and culturing the recombinant adenovirus in step (4);
(6)对步聚(5)中的培养产物进行纯化得到能表达目的基因的Ad。(6) Purifying the culture product in step (5) to obtain Ad capable of expressing the target gene.
将HEK293细胞在培养基中培养,传代。当HEK293细胞密度>1.5×10
6cells/ml时,用培养基稀释进行病毒扩增,接毒病毒感染复数(MOI)分别为1,5和10,37℃,5%CO
2,125rpm培养,收获时间42h-48h,得到腺病毒毒种收获液。HEK293细胞所扩增的Ad5-ABD病毒收获液和上清用Ad-FITC荧光抗体法检测滴度IFU。结果如表1所示。
HEK293 cells were cultured in culture medium and passaged. When HEK293 cell density > 1.5×10 6 cells/ml, dilute with culture medium for virus amplification, the multiplicity of infection (MOI) of inoculated virus is 1, 5 and 10 respectively, culture at 37°C, 5% CO 2 , 125rpm, The harvesting time is 42h-48h, and the adenovirus seed harvesting liquid is obtained. The Ad5-ABD virus harvest liquid and supernatant amplified by HEK293 cells were detected with the Ad-FITC fluorescent antibody method to detect the titer IFU. The results are shown in Table 1.
表1 Ad-ABD腺病毒制备病毒滴度检测结果Table 1 Virus titer detection results of Ad-ABD adenovirus preparation
将上述腺病毒毒种Ad-ABD收获液在HEK 293SF-3F6细胞密度0.8-1.2×10
6cells/ml,MOI=3接毒,进行扩增,48h后收获。
The adenovirus seed Ad-ABD harvest liquid was inoculated in HEK 293SF-3F6 cells at a density of 0.8-1.2×10 6 cells/ml, MOI=3, amplified, and harvested after 48 hours.
实施例2:ELISA检测Ad-ABD腺病毒与白蛋白的结合Embodiment 2: ELISA detects the combination of Ad-ABD adenovirus and albumin
(1)包被:用包被液稀释牛血清白蛋白(BSA)和人血清白蛋白(HSA)至2mg/ml,加到96孔酶标板中,2-8℃过夜包被。洗涤,加入封闭液。(1) Coating: Dilute bovine serum albumin (BSA) and human serum albumin (HSA) to 2 mg/ml with coating solution, add to 96-well microtiter plate, and coat overnight at 2-8°C. Wash and add blocking solution.
(2)加样:待实施例1中的Ad-ABD原液,NCVA原液分别原倍,10倍和100倍稀释,加入到酶标板;对照孔加PBS。37℃孵育1h后洗板。(2) Sample addition: the Ad-ABD stock solution in Example 1 and the NCVA stock solution were respectively original times, 10 times and 100 times diluted, and added to the microtiter plate; PBS was added to the control well. After incubation at 37°C for 1 h, the plate was washed.
(3)孵育:用1%BSA/PBST 1:1000稀释腺病毒单抗(invitrogen),100μL/孔,37℃孵育1h后洗板;用1%BSA/PBST 1:1000稀释羊抗小鼠IgG(Proteintech),37℃孵育30min后洗板。(3) Incubation: Dilute adenovirus monoclonal antibody (invitrogen) with 1% BSA/PBST 1:1000, 100 μL/well, wash the plate after incubating at 37°C for 1 hour; dilute goat anti-mouse IgG with 1% BSA/PBST 1:1000 (Proteintech), wash the plate after incubating at 37°C for 30min.
(4)测定:显色液室温回温后,A液和B液1:1混合,加入酶标板,显色15min后用酶标仪读数。(4) Determination: After the chromogenic solution is warmed to room temperature, mix liquid A and liquid B at a ratio of 1:1, add to a microplate plate, and read with a microplate reader after color development for 15 minutes.
附图2结果表明,同一稀释度的Ad-ABD原液与HSA的结合能力大于Ad5原液与HSA的结合能力;随着病毒原液的稀释,与HSA的结合能力逐渐减弱。Ad-ABD腺病毒可以在体外与HSA结合,证明了Ad-ABD腺病毒载体改造以及通过添加白蛋白进行结合的可行性。Accompanying drawing 2 results show that the binding ability of Ad-ABD stock solution and HSA of the same dilution is greater than the binding ability of Ad5 stock solution and HSA; with the dilution of virus stock solution, the binding ability with HSA gradually weakens. Ad-ABD adenovirus can be combined with HSA in vitro, which proves the feasibility of Ad-ABD adenovirus vector transformation and binding by adding albumin.
实施例3:Ad-ABD腺病毒逃避预存免疫的体外中和实验-定性实验Example 3: In vitro neutralization experiment of Ad-ABD adenovirus evading pre-existing immunity - qualitative experiment
(1)病毒样品以及抗体稀释:将Ad-ABD原液和NCVA原液分别用DMEM完全培养(DMEM+10%FBS+1%P/S)和DMEM完全培养基+1mg/ml HSA稀释至病毒滴度为1.5×10
6IFU/ml。用DMEM完全培养基稀释Ad5兔多抗(来源康希诺生物股份公司),抗体稀释度分别为原倍,10倍和100倍。
(1) Virus sample and antibody dilution: Dilute Ad-ABD stock solution and NCVA stock solution to virus titer with DMEM complete culture (DMEM+10%FBS+1%P/S) and DMEM complete medium+1mg/ml HSA respectively It is 1.5×10 6 IFU/ml. The Ad5 rabbit polyclonal antibody (sourced from CanSino Biological Co., Ltd.) was diluted with DMEM complete medium, and the antibody dilutions were original times, 10 times and 100 times respectively.
(2)孵育:96孔板中加入100μL/孔病毒样品和1μL稀释后的抗体,37℃孵育1h,每个实验组做4个平行,实验分组如表2:(2) Incubation: Add 100 μL/well of virus samples and 1 μL of diluted antibody to a 96-well plate, incubate at 37°C for 1 hour, and do 4 parallel experiments in each experimental group. The experimental groups are shown in Table 2:
表2体外中和实验-定性分析分组Table 2 In Vitro Neutralization Experiment - Qualitative Analysis Grouping
编号 | 分组group | |
11 |
Ad-ABD原液+1mg/ml HSA+10
0Ad兔多抗
Ad-ABD stock solution+1mg/ml HSA+10 0 Ad rabbit |
|
22 | Ad-ABD原液+1mg/ml HSA+10 -1Ad兔多抗 Ad-ABD stock solution+1mg/ml HSA+10 -1 Ad rabbit polyclonal antibody | |
33 |
Ad-ABD原液+1mg/ml HSA+10
-2Ad兔多抗
Ad-ABD stock solution+1mg/ml HSA+10 -2 Ad rabbit |
|
44 |
Ad-ABD原液+10
0Ad兔多抗
Ad-ABD stock solution + 10 0 Ad rabbit |
|
55 |
Ad-ABD原液+10
-1Ad兔多抗
Ad-ABD stock solution + 10 -1 Ad rabbit |
|
66 |
Ad-ABD原液+10
-2Ad兔多抗
Ad-ABD stock solution + 10 -2 Ad rabbit |
|
77 |
NCVA原液+1mg/ml HSA+10
0Ad兔多抗
NCVA stock solution+1mg/ml HSA+10 0 Ad rabbit |
|
88 | NCVA原液+1mg/ml HSA+10 -1Ad兔多抗 NCVA stock solution+1mg/ml HSA+10 -1 Ad rabbit polyclonal antibody | |
99 | NCVA原液+1mg/ml HSA+10 -2Ad兔多抗 NCVA stock solution+1mg/ml HSA+10 -2 Ad rabbit polyclonal antibody |
1010 | NCVA原液+10 0Ad兔多抗 NCVA stock solution + 10 0 Ad rabbit polyclonal antibody |
1111 | NCVA原液+10 -1Ad兔多抗 NCVA stock solution + 10 -1 Ad rabbit polyclonal antibody |
1212 | NCVA原液+10 -2Ad兔多抗 NCVA stock solution + 10 -2 Ad rabbit polyclonal antibody |
(3)制备HEK293细胞悬液:将HEK293细胞用DMEM完全培养基稀释至细胞密度3×10
5cells/ml。待病毒样品和稀释好的Ad兔多抗37℃孵育1h后,100μL HEK293cells/孔加入96孔板中,混合均匀后,在37℃,5%CO
2中培养4-5h。用PBS洗板3次,
(3) Preparation of HEK293 cell suspension: HEK293 cells were diluted with DMEM complete medium to a cell density of 3×10 5 cells/ml. After the virus sample and the diluted Ad rabbit polyclonal antibody were incubated at 37°C for 1 hour, 100 μL HEK293cells/well was added to a 96-well plate, mixed evenly, and incubated at 37°C, 5% CO 2 for 4-5 hours. Wash the plate 3 times with PBS,
(4)抗体孵育:Ad-FITC荧光抗体用1%BSA/1×PBS稀释,稀释比例1:500;洗板后向96孔板中加入50μL/孔稀释好的荧光抗体,室温避光孵育1h后,PBS洗板2次。(4) Antibody incubation: Ad-FITC fluorescent antibody was diluted with 1% BSA/1×PBS, the dilution ratio was 1:500; after washing the plate, 50 μL/well of the diluted fluorescent antibody was added to the 96-well plate, and incubated for 1 hour at room temperature in the dark Afterwards, the plate was washed twice with PBS.
(5)显色:在荧光显微镜下观察细胞。(5) Color development: observe the cells under a fluorescent microscope.
从图3可以看出将Ad或Ad-ABD病毒疫苗原液在HSA存在下与连续稀释的抗Ad抗体一起孵育,然后感染HEK293细胞。Ad-ABD的中和程度低于Ad载体,表明ABD修饰同时在制剂中添加白蛋白可以逃避预先免疫。It can be seen from Figure 3 that the Ad or Ad-ABD virus vaccine stock solution was incubated with serially diluted anti-Ad antibodies in the presence of HSA, and then infected HEK293 cells. The neutralization degree of Ad-ABD was lower than that of Ad carrier, indicating that ABD modification and adding albumin in the preparation can escape pre-immunization.
实施例4体内免疫原性评价-抗体水平测试Example 4 In vivo immunogenicity evaluation-antibody level test
实验对象:6-8周龄雌性BALB/c小鼠,8只/组。Subjects: 6-8 weeks old female BALB/c mice, 8 mice/group.
预存免疫的建立:取解冻针对腺病毒的中和滴度为3000的豚鼠血,每只小鼠腹腔注射50ul。24h后,检测血清的Ad5型腺病毒载体的中和抗体,所有小鼠的中和抗体滴度在200-1000之间。Establishment of pre-existing immunity: take thawed guinea pig blood with a neutralizing titer of 3000 against adenovirus, and inject 50ul intraperitoneally into each mouse. After 24 hours, the neutralizing antibody of Ad5 adenovirus vector in serum was detected, and the neutralizing antibody titer of all mice was between 200-1000.
NCVA组:实施例1制备的未经ABD改造的重组新型冠状病毒疫苗(Ad);NCVA group: the recombinant new coronavirus vaccine (Ad) without ABD transformation prepared in Example 1;
Ad-ABD+HSA组:实施例1制备的Ad-ABD的新冠病毒疫苗原液中加入人血清白蛋白(HSA)溶液,HSA终浓度为1mg/ml;Ad-ABD+HSA group: human serum albumin (HSA) solution was added to the stock solution of the new coronavirus vaccine of Ad-ABD prepared in Example 1, and the final concentration of HSA was 1 mg/ml;
Ad-ABD+BSA组:实施例1制备的Ad-ABD的新冠病毒疫苗原液中加入牛血清白蛋白(BSA)溶液,BSA终浓度为1mg/ml;Ad-ABD+BSA group: Add bovine serum albumin (BSA) solution to the stock solution of the new coronavirus vaccine of Ad-ABD prepared in Example 1, and the final concentration of BSA is 1mg/ml;
Ad-ABD组:实施例1制备的Ad-ABD的新冠病毒疫苗原液。Ad-ABD group: the stock solution of the new coronavirus vaccine of Ad-ABD prepared in Example 1.
粘膜接种的方式:利用吸入式暴露塔通过雾化吸入免疫的方式进行免疫,免疫剂量为每只5×10
9VP。
The way of mucosal inoculation: use the inhalation exposure tower to immunize by atomization inhalation, and the immunization dose is 5×10 9 VP per mouse.
雾化吸入免疫后14天和28天后,分别眼眶取血,检测血清样本中针对新型冠状病毒S株中和滴度,检测结果如表3-5及图4-5所示;14 days and 28 days after nebulization inhalation immunization, blood was taken from the orbits respectively, and the neutralization titer against the new coronavirus S strain in the serum samples was tested. The test results are shown in Table 3-5 and Figure 4-5;
表3 14天结合抗体中和滴度(1og10)Table 3 14 days binding antibody neutralization titer (log10)
组别 | 鼠1Rat 1 |
鼠2 |
鼠3Rat 3 |
鼠4 |
鼠5 |
鼠6 |
鼠7 |
鼠8 |
|
NCVANCVA | 5.515.51 | 5.195.19 | 5.815.81 | 5.745.74 | 5.515.51 | 5.525.52 | 5.205.20 | 5.015.01 | |
Ad-ABD+BSAAd-ABD+BSA | 5.865.86 | 5.725.72 | 5.725.72 | 5.665.66 | 5.995.99 | 5.395.39 | 5.875.87 | 6.106.10 | |
Ad-ABD+HSAAd-ABD+HSA | 5.895.89 | 5.815.81 | 5.895.89 | 6.516.51 | 5.815.81 | 6.116.11 | 6.326.32 | 6.116.11 | |
Ad-ABDAd-ABD | 4.024.02 | 4.604.60 | 4.004.00 | 5.205.20 | 4.604.60 | 4.604.60 | 4.604.60 | 4.904.90 |
表4 28天结合抗体中和滴度(1og10)Table 4 28 days binding antibody neutralizing titer (log10)
组别 | 鼠1Rat 1 |
鼠2 |
鼠3Rat 3 |
鼠4 |
鼠5 |
鼠6 |
鼠7 |
鼠8 |
|
NCVANCVA | 3.903.90 | 3.823.82 | 4.204.20 | 3.993.99 | 4.464.46 | 4.584.58 | 4.244.24 | 3.903.90 | |
Ad-ABD+BSAAd-ABD+BSA | 4.904.90 | 4.174.17 | 4.834.83 | 4.094.09 | 5.155.15 | 4.904.90 | 4.224.22 | 4.804.80 | |
Ad-ABD+HSAAd-ABD+HSA | 5.225.22 | 5.115.11 | 4.814.81 | 4.204.20 | 4.814.81 | 4.934.93 | 5.755.75 | 4.974.97 | |
Ad-ABDAd-ABD | 3.603.60 | 4.204.20 | 3.903.90 | 3.813.81 | 4.814.81 | 3.763.76 | 3.543.54 | 3.303.30 |
表5 42天结合抗体中和滴度(1og10)Table 5 42 days binding antibody neutralizing titer (log10)
组别 | 鼠1Rat 1 |
鼠2 |
鼠3Rat 3 |
鼠4 |
鼠5 |
鼠6 |
鼠7 |
鼠8 |
|
NCVANCVA | 4.604.60 | 4.214.21 | 5.125.12 | 5.245.24 | 5.175.17 | 4.904.90 | 4.894.89 | 4.334.33 | |
Ad-ABD+BSAAd-ABD+BSA | 5.525.52 | 5.975.97 | 5.815.81 | 5.515.51 | 5.875.87 | 6.116.11 | 5.985.98 | 5.585.58 | |
Ad-ABD+HSAAd-ABD+HSA | 5.985.98 | 5.995.99 | 5.815.81 | 5.745.74 | 5.905.90 | 6.216.21 | 6.016.01 | 6.116.11 | |
Ad-ABDAd-ABD | 4.074.07 | 4.604.60 | 4.344.34 | 5.205.20 | 4.024.02 | 4.584.58 | 4.134.13 | 3.933.93 |
第一次免疫28天后进行第二次免疫,第二次免疫14天后杀鼠评估细胞免疫水平及S总抗体效价,检测结果如图6所示;The second immunization was carried out 28 days after the first immunization, and the rats were killed 14 days after the second immunization to evaluate the level of cellular immunity and the titer of total S antibody. The test results are shown in Figure 6;
实验结果表明:本次实验采用2种剂量进行免疫,一免14天后,检测S蛋白特异性总抗体水平,结果显示,通过吸入方式给药,Ad-ABD载体激发抗体水平低于未经ABD改造的Ad。The results of the experiment showed that two doses were used for immunization in this experiment, and the level of total antibody specific to S protein was detected 14 days after the first immunization. The results showed that the level of antibody induced by the Ad-ABD carrier was lower than that of the non-modified ABD after administration by inhalation. Ad.
可能原因为:基于特定的吸入给药方式,经过ABD改造的Ad虽然对体内预存腺病毒抗体敏感性降低,但是同时也导致了其感染宿主效率的降低。而逃避预存免疫的积极作用无法抵消免疫刺激能力降低的消极影响。实验组Ad-ABD+HSA和Ad-ABD+BSA抗体水平显著高于其他组别,即,在制剂中预先添加白蛋白再结合Ad-ABD共同吸入给药的技术方案,可使逃避预存免疫的积极影响大幅提升。究其原因,可能为,肺部器官与体外及注射环境不同,在利用吸入给药递送Ad时,肺部白蛋白与含ABD结构域的Ad结合存在一定困难,因此并不能达到理想的逃避预存免疫目的,甚至效果不及未改造的Ad,因此不能成药(黏膜给药)。而本发明通过在制剂中添加白蛋白,即,通过在体外预制结合的方式再吸入给药,在根本上解决了Ad-ABD黏膜给药制剂逃避预存免疫的成药性问题。The possible reason is: based on the specific inhalation administration method, although the ABD-modified Ad is less sensitive to the pre-existing adenovirus antibody in the body, it also leads to a decrease in the efficiency of its infection of the host. The positive effect of evading pre-existing immunity cannot offset the negative effect of reduced immune stimulation. The antibody levels of Ad-ABD+HSA and Ad-ABD+BSA in the experimental group were significantly higher than those in the other groups, that is, the technical scheme of pre-adding albumin in the preparation and combining Ad-ABD for co-inhalation can make the evasion of pre-existing immunity The positive impact has increased significantly. The reason may be that the lung organs are different from the in vitro and injection environments. When Ad is delivered by inhalation, it is difficult to combine the albumin in the lungs with the Ad containing the ABD domain, so the ideal escape from pre-existing cells cannot be achieved. For immunization purposes, the effect is even lower than that of the unmodified Ad, so it cannot be made into a drug (mucosal administration). However, the present invention fundamentally solves the druggability problem of the Ad-ABD mucosal administration preparation evading pre-existing immunity by adding albumin to the preparation, that is, through in vitro prefabricated combination and re-inhalation administration.
实施例5体内免疫原性评价-细胞免疫反应。Example 5 In vivo immunogenicity evaluation - cellular immune response.
实验对象:6-8周龄雌性BALB/c小鼠,8只/组。Subjects: 6-8 weeks old female BALB/c mice, 8 mice/group.
实验方法:采用与实施例4相同的实验方法。Experimental method: The same experimental method as in Example 4 was adopted.
检测:在免疫后21天,取眼眶血,做S-IgG抗体检测,结果如表6所示;杀鼠取脾细胞,做抗原特异性CD4和CD8 T细胞免疫应答。Detection: 21 days after immunization, the orbital blood was taken for S-IgG antibody detection, the results are shown in Table 6; the mice were killed to obtain splenocytes for antigen-specific CD4 and CD8 T cell immune responses.
表6 S-IgG抗体滴度检测结果Table 6 S-IgG antibody titer detection results
TNFα、IFNγ、IL2、IL5采用流式细胞术,Anti-TNFα-FITC抗体、Anti-IFNγ-PE,moμse抗体、Anti-IL2-APC,moμse抗体、Anti-IL5-AF700抗体进行检测(表7-14)。TNFα, IFNγ, IL2, and IL5 were detected by flow cytometry, Anti-TNFα-FITC antibody, Anti-IFNγ-PE, moμse antibody, Anti-IL2-APC, moμse antibody, Anti-IL5-AF700 antibody (Table 7- 14).
表7 CD4+T细胞TNFα免疫反应检测结果Table 7 CD4+ T cell TNFα immune response detection results
表8 CD4+T细胞IFNγ免疫反应检测结果Table 8 CD4+ T cell IFNγ immune response detection results
表9 CD4+T细胞IL2免疫反应检测结果Table 9 CD4+T cell IL2 immune response detection results
表10 CD4+T细胞IL5免疫反应检测结果Table 10 CD4+T cell IL5 immune response detection results
表11 CD8+T细胞TNFα免疫反应检测结果Table 11 CD8+T cell TNFα immune response detection results
表12 CD8+T细胞IFNγ免疫反应检测结果Table 12 CD8+ T cell IFNγ immune response detection results
表13 CD8+T细胞IL2免疫反应检测结果Table 13 Detection results of CD8+T cell IL2 immune response
表14 CD8+T细胞IL5免疫反应检测结果Table 14 CD8+ T cell IL5 immune response detection results
实验结果如图7-9所示。The experimental results are shown in Figure 7-9.
实验结果表明:通过吸入方式(黏膜给药),进行黏膜免疫,NCVA组、Ad-ABD+HSA组、Ad-ABD+BSA组、Ad-ABD组载体均能诱导小鼠血清中产生S-IgG抗体滴度。均能够在小鼠中激发体液免疫与细胞免疫,主要以 CD8+T细胞为主,但Ad-ABD组其细胞免疫水平显著低于对照NCVA。而在Ad-ABD制剂中预先添加白蛋白再结合Ad-ABD共同吸入的技术方案,对于CD4+T细胞、CD8+T细胞均能够诱导小鼠产生的针对COVID-19 S蛋白THFα、IFNγ、IL2应答水平高于NCVA组及Ad-ABD组。结果趋势与“体内免疫原性评价-抗体水平测试”实验结果吻合。The experimental results show that: through inhalation (mucosal administration), mucosal immunization, NCVA group, Ad-ABD+HSA group, Ad-ABD+BSA group, Ad-ABD group vectors can induce the production of S-IgG in mouse serum Antibody titer. Both can stimulate humoral immunity and cellular immunity in mice, mainly CD8+ T cells, but the level of cellular immunity in the Ad-ABD group was significantly lower than that of the control NCVA. However, the technical scheme of pre-adding albumin in the Ad-ABD preparation combined with Ad-ABD co-inhalation can induce CD4+ T cells and CD8+ T cells to produce anti-COVID-19 S protein THFα, IFNγ, IL2 The response level was higher than that of NCVA group and Ad-ABD group. The result trend is consistent with the experimental results of "in vivo immunogenicity evaluation-antibody level test".
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。The aforementioned embodiments and methods described in the present invention may vary based on the ability, experience and preference of those skilled in the art.
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。In the present invention, only listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.
Claims (18)
- 一种腺病毒载体疫苗制剂,其特征在于,含有重组腺病毒和辅助组分;所述重组腺病毒的载体骨架基因组序列中包括编码白蛋白结合域的序列;所述辅助组分包括白蛋白。An adenovirus vector vaccine preparation is characterized in that it contains a recombinant adenovirus and an auxiliary component; the vector backbone genome sequence of the recombinant adenovirus includes a sequence encoding an albumin binding domain; and the auxiliary component includes albumin.
- 根据权利要求1所述的腺病毒载体疫苗制剂,其特征在于,所述腺病毒为人腺病毒;优选的,人腺病毒选自血清1型~血清57型的人腺病毒中的一种或多种;更优选的,所述人腺病毒为血清5型、26型或35型人腺病毒。The adenovirus vector vaccine preparation according to claim 1, wherein the adenovirus is a human adenovirus; preferably, the human adenovirus is selected from one or more of human adenoviruses from serotype 1 to serotype 57 species; more preferably, the human adenovirus is serotype 5, 26 or 35 human adenovirus.
- 根据权利要求1-2任一所述的腺病毒载体疫苗制剂,其特征在于,所述辅助组分中添加的白蛋白为人源血清白蛋白、重组的人源血清白蛋白、牛血清白蛋白中的一种或多种。According to the adenovirus vector vaccine preparation described in any one of claims 1-2, it is characterized in that the albumin added in the auxiliary component is human serum albumin, recombinant human serum albumin, and bovine serum albumin. one or more of.
- 根据权利要求1-3任一所述的腺病毒载体疫苗制剂,其特征在于,白蛋白与腺病毒载体的摩尔比例为1-10 5:1;优选地,1-240:1。 The adenovirus vector vaccine preparation according to any one of claims 1-3, characterized in that the molar ratio of albumin to adenovirus vector is 1-10 5 :1; preferably, 1-240:1.
- 根据权利要求1-4任一所述的腺病毒载体疫苗制剂,其特征在于,所述编码白蛋白结合域的序列插入在六邻体蛋白的高变区1(HVR1)的编码区中,所述白蛋白结合域位于所述六邻体蛋白的外表面上。The adenovirus vector vaccine preparation according to any one of claims 1-4, wherein the sequence encoding the albumin binding domain is inserted into the coding region of the hypervariable region 1 (HVR1) of the hexon protein, so The albumin binding domain is located on the outer surface of the hexon protein.
- 根据权利要求1-5任一所述的腺病毒载体疫苗制剂,其特征在于,所述白蛋白结合域为链球菌蛋白G的白蛋白结合域或其的功能等同变体,优选地,所述白蛋白结合域为链球菌蛋白G的白蛋白结合结构域3。The adenovirus vector vaccine formulation according to any one of claims 1-5, wherein the albumin binding domain is an albumin binding domain of streptococcal protein G or a functionally equivalent variant thereof, preferably, the The albumin binding domain is albumin binding domain 3 of streptococcal protein G.
- 根据权利要求1-6任一所述的腺病毒载体疫苗制剂,其特征在于,所述疫苗为黏膜给药制剂或注射给药制剂,优选的,所述黏膜给药制剂选自:液体剂型、固体剂型、半固体剂型或者气体剂型;更优选的,所述黏膜给药制剂为滴鼻剂、气雾剂、喷雾剂、粉雾剂、粉末剂、液体制剂、冻干制剂、凝胶剂、微球剂、脂质体、膜剂、混悬剂;进一步优选地,所述黏膜给药制剂为吸入液体制剂、吸入气雾剂和吸入喷雾剂;更优选地,为鼻吸入或经口吸入;更优选地,所述黏膜包括鼻黏膜、口腔黏膜或肺粘膜。The adenovirus vector vaccine preparation according to any one of claims 1-6, wherein the vaccine is a preparation for mucosal administration or an preparation for injection, preferably, the preparation for mucosal administration is selected from: liquid dosage form, solid dosage form, semi-solid dosage form or gas dosage form; more preferably, the described mucosal administration preparation is nasal drop, aerosol, spray, powder mist, powder, liquid preparation, lyophilized preparation, gel, Microspheres, liposomes, films, suspensions; more preferably, the formulations for mucosal administration are inhalation liquid formulations, inhalation aerosols and inhalation sprays; more preferably, nasal inhalation or oral inhalation ; More preferably, the mucosa comprises nasal mucosa, oral mucosa or pulmonary mucosa.
- 根据权利要求1-7任一所述的腺病毒载体疫苗制剂,其特征在于,所述辅助组分中还包含药学上可接受的辅料;优选地,所述药学上可接受的辅料包括但不限于:缓冲剂、保护剂、稳定剂、表面活性剂、渗透压调节剂、佐剂、防腐剂和灭活剂等中的一种或多种。According to the adenovirus vector vaccine preparation according to any one of claims 1-7, it is characterized in that, the auxiliary components also include pharmaceutically acceptable adjuvants; preferably, the pharmaceutically acceptable adjuvants include but not Limited to: one or more of buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives and inactivators.
- 根据权利要求1-8任一所述的腺病毒载体疫苗制剂,其特征在于,所述缓冲剂包括但不限于HEPES、HIS、TRIS、PB、琥珀酸、柠檬酸中的一种或多种;所述保护剂包括但不限于明胶、乙醇、二乙氨四乙酸(EDTA)、二乙氨四乙酸二钠(EDTA-2Na)和氯化镁中的一种或者多种;所述稳定剂包括但不限于蔗糖、甘露醇、岩藻糖和麦芽糖中的一种或多种;所述表面活性剂包括但不限于吐温、司盘、甘油中的一种或者多种;所述渗透压调节剂包括但不限于氯化钠或省去。The adenovirus vector vaccine formulation according to any one of claims 1-8, wherein the buffering agent includes but is not limited to one or more of HEPES, HIS, TRIS, PB, succinic acid, and citric acid; Described protective agent includes but not limited to one or more in gelatin, ethanol, diethylaminotetraacetic acid (EDTA), disodium diethylaminotetraacetic acid (EDTA-2Na) and magnesium chloride; Described stabilizer includes but not limited to Limited to one or more of sucrose, mannitol, fucose and maltose; the surfactant includes but not limited to one or more of Tween, Span, glycerin; the osmotic pressure regulator includes But not limited to sodium chloride or omitted.
- 根据权利要求1-9任一所述的腺病毒载体疫苗制剂,其特征在于,所述辅料组分包括甘露醇,蔗糖,氯化钠,氯化镁,HEPES,聚山梨酯80,甘油,优选地,甘露醇10-150mg/ml,蔗糖10-150mg/ml,氯化钠10-150mM,氯化镁1-10mM,HEPES 1-15mM,以重量百分比计聚山梨酯80 0.001%-0.5%,以重量百分比计甘油0.05-2%。The adenovirus vector vaccine formulation according to any one of claims 1-9, wherein the adjuvant components include mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, glycerin, preferably, Mannitol 10-150mg/ml, sucrose 10-150mg/ml, sodium chloride 10-150mM, magnesium chloride 1-10mM, HEPES 1-15mM, by weight percentage polysorbate 80 0.001%-0.5%, by weight percentage Glycerin 0.05-2%.
- 根据权利要求1-10任一所述的腺病毒载体疫苗制剂,其特征在于,制剂组分包含:蔗糖10-50mg/ml、甘露醇15-75mg/ml、氯化钠40-60mM、甘油0.5-5mg/ml、氯化镁1-5mM、吐温80 0.05-0.5mg/ml、HEPES 1-5mM、以重量百分比计HAS含量0.1%-1%。The adenovirus vector vaccine preparation according to any one of claims 1-10, characterized in that the preparation components include: sucrose 10-50 mg/ml, mannitol 15-75 mg/ml, sodium chloride 40-60 mM, glycerin 0.5 -5mg/ml, magnesium chloride 1-5mM, Tween 80 0.05-0.5mg/ml, HEPES 1-5mM, HAS content 0.1%-1% by weight percentage.
- 根据权利要求1-11任一所述的腺病毒载体疫苗制剂,其特征在于,剂型为雾化吸入剂,所述疫苗经雾化给药装置雾化后形成10μm以下的颗粒。The adenovirus vector vaccine preparation according to any one of claims 1-11, characterized in that the dosage form is an atomized inhalation, and the vaccine is atomized by an atomized drug delivery device to form particles with a diameter of less than 10 μm.
- 根据权利要求1-12任一所述的腺病毒载体疫苗制剂,其特征在于,所述白蛋白在疫苗中的含量以重量百分比计为0.001%-50%,优选地为0.005%或0.01%或0.05%或0.1%或0.2%或0.3%或0.4%或0.5%或0.6%或0.7%或0.8%或0.9%或1%或2%或3%或4%或5%或6%或7%或8%或9%或10%或15%或20%或25%或30%或35%或40%或45%或50%。The adenovirus vector vaccine preparation according to any one of claims 1-12, characterized in that the content of the albumin in the vaccine is 0.001%-50% by weight, preferably 0.005% or 0.01% or 0.05% or 0.1% or 0.2% or 0.3% or 0.4% or 0.5% or 0.6% or 0.7% or 0.8% or 0.9% or 1% or 2% or 3% or 4% or 5% or 6% or 7% Or 8% or 9% or 10% or 15% or 20% or 25% or 30% or 35% or 40% or 45% or 50%.
- 一种权利要求1-13任一所述的腺病毒载体疫苗制剂的制备方法,其特征在于,制备步骤包括:A preparation method of the adenovirus vector vaccine preparation described in any one of claims 1-13, characterized in that the preparation step comprises:S1将链球菌(Streptococcus)蛋白G的白蛋白结合域(ABD)引入到腺病毒载体骨架hexon基因的HVR区,构建腺病毒-ABD载体;S1 introduces the albumin binding domain (ABD) of Streptococcus protein G into the HVR region of the adenovirus vector backbone hexon gene to construct an adenovirus-ABD vector;S2将病毒或细菌抗原蛋白基因序列引入到改造的腺病毒-ABD载体中,构建为Ad-ABD载体疫苗;S2 introduces the viral or bacterial antigen protein gene sequence into the modified adenovirus-ABD vector to construct an Ad-ABD vector vaccine;S3在制剂中加入白蛋白作为辅助组分。S3 adds albumin to the preparation as an auxiliary component.
- 根据权利要求14所述的腺病毒载体疫苗制剂的制备方法,其特征在于,选择的包装细胞为HEK293细胞。The preparation method of the adenovirus vector vaccine preparation according to claim 14, characterized in that the selected packaging cells are HEK293 cells.
- 根据权利要求14或15所述的制备方法,其特征在于,病毒或细菌抗原为HIV、狂犬病病毒、登革热病毒、埃博拉病毒、冠状病毒、人乳头瘤病毒、丙型肝炎病毒、乙型肝炎病毒、轮状病毒、麻疹病毒、呼吸道合胞病毒(RSV)、带状疱疹病毒(VZV)、巨细胞病毒、2型单纯疱疹病毒、爱泼斯坦巴尔病毒、流感病毒、克氏锥虫和恶性疟原虫、结核杆菌的一种或多种。The preparation method according to claim 14 or 15, wherein the virus or bacterial antigen is HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B Viruses, rotavirus, measles virus, respiratory syncytial virus (RSV), herpes zoster virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and malignant One or more of Plasmodium and Mycobacterium tuberculosis.
- 一种权利要求1-13任一所述的腺病毒载体疫苗制剂的给药系统,其特征在于,给药系统为雾化吸入给药,所述雾化给药装置为射流雾化给药装置或超声雾化给药装置或振动雾化给药装置,优选的,所述雾化给药装置包括雾化器和雾化后的疫苗颗粒收集部件。A drug delivery system for the adenovirus vector vaccine preparation described in any one of claims 1-13, characterized in that the drug delivery system is atomized inhalation drug delivery, and the atomized drug delivery device is a jet atomized drug delivery device Or an ultrasonic atomization drug delivery device or a vibratory atomization drug delivery device, preferably, the atomized drug delivery device includes an atomizer and an atomized vaccine particle collection component.
- 一种权利要求1-13任一所述的腺病毒载体疫苗制剂在制备用于预防传染性疾病的疫苗中的用途。A use of the adenovirus vector vaccine preparation according to any one of claims 1-13 in the preparation of vaccines for preventing infectious diseases.
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CN106471125A (en) * | 2014-04-30 | 2017-03-01 | 贝尔韦什生物医学研究学会 | Adenoviruss including albumin binding moieties |
CN112451811A (en) * | 2014-10-13 | 2021-03-09 | 欧米伽生命科学公司 | Atomizer and use thereof |
WO2021194183A1 (en) * | 2020-03-25 | 2021-09-30 | ㈜큐리진 | Immunoevasive anti-tumor adenovirus |
CN113750227A (en) * | 2020-06-01 | 2021-12-07 | 康希诺生物股份公司 | SARS-CoV-2 vaccine |
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CN106471125A (en) * | 2014-04-30 | 2017-03-01 | 贝尔韦什生物医学研究学会 | Adenoviruss including albumin binding moieties |
CN112451811A (en) * | 2014-10-13 | 2021-03-09 | 欧米伽生命科学公司 | Atomizer and use thereof |
WO2021194183A1 (en) * | 2020-03-25 | 2021-09-30 | ㈜큐리진 | Immunoevasive anti-tumor adenovirus |
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