A recombinant adenoviral vector vaccine inhalation delivery system

Abstract

The present application relates to a kind of recombinant adenovirus vector vaccine inhalation delivery system, the delivery system is by the modification of adenovirus vector itself and cooperate specific formulation prescription, vaccine is inhaled after inhalation administration, can reach lung by nasal cavity or oral inhalation, to generate protective immune response including mucosal immunity, while solving adenovirus pre-existing immunity, enhance the effective utilization rate of vaccine, improve the effect of vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccine technology, in particular to a recombinant adenovirus vector vaccine inhalation delivery system for solving the pre-existing immunity of adenovirus. BACKGROUND

[0002] Adenovirus is a double-stranded DNA virus without envelope, and 53 serotypes of human adenovirus have been identified. Adenovirus is composed of external capsid proteins and internal core proteins. The core proteins include protein V, protein VII, and protein X, which are associated 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 a icosahedral structure formed by non-covalent interactions of seven proteins (II, III, IIIa, IV, VI, VIII, and IX), of which 240 trimeric hexamers (protein II) are the main constituent proteins of the icosahedron.

[0003] Adenovirus is a non-enveloped spherical structure, and the virions are often arranged in a crystal lattice within the nucleus of infected cells. Each virus particle contains a 36 kb linear double-stranded DNA, with a 100-600 bp inverted terminal repeat (ITR) at each end. The inner side of the ITR is the viral packaging signal, which is a cis-acting element required for viral packaging. The genome contains E1-E4 genes for early expression associated with adenovirus replication and L1-L5 genes for late expression associated with adenovirus particle assembly. Linear double-stranded DNA forms a core with a diameter of 60-65 nm with core proteins, which is wrapped in the capsid. The capsid is icosahedral symmetry, composed of 252 shell particles with a diameter of 8-10 nm, arranged on a triangular face with 6 on each side, of which 240 are hexamers (non-apex shell particles), and the other 12 are pentamer bases (apex shell particles). Each hexamer is a homotrimer of hexon proteins, and the trimeric hexon molecule has a triangular tower and a pentahedral base. The tower region consists of four loops, i.e. loop1, loop2, loop3, and loop4, and the base contains two regions P1 and P2.

[0004] The current problem of adenovirus vector vaccine is the pre-existing immunity of human body to adenovirus, for example, adenovirus type 5 is relatively common in the environment, and the human population is prone to produce an immune response against adenovirus, which will reduce the neutralizing antibody level of adenovirus vector vaccine in the human body. The current methods to solve the pre-existing immunity of adenovirus mainly include chemical method and genetic engineering method. The chemical method mainly uses PEG to wrap adenovirus to shield its antigen epitopes, so as to escape the immune effect of the host, but it is difficult to obtain high titer virus by this method.

[0005] The method of genetic engineering is to modify the adenovirus, such as constructing a chimeric modified adenovirus capsid, constructing a chimeric adenovirus. The adenovirus-based vaccine is completely replaced by other rare, such as human or non-human adenovirus serotypes (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 specific antibody response induced by the foreign gene is significantly lower than the immune response induced by the 5 type adenovirus vector.

[0006] On September 4, 2022, the inhaled recombinant novel coronavirus vaccine (5 type adenovirus vector) developed by Kangxinfu Biotechnology Co., Ltd. was recommended by the National Health Commission and approved by the National Drug Administration as a booster shot for emergency use.

[0007] The prior art (WO2015166082A1, invention name: adenovirus including albumin binding portion) can obtain an albumin protective cover on the outer surface of the capsid, especially on the outer surface of the adenovirus hexon protein, which is genetically modified by the albumin binding portion, so that the virus can escape neutralizing antibodies and prolong its retention time in the blood after systemic administration. Albumin is widely used as a drug carrier in a popular research and development field, which benefits from its long half-life in plasma and non-covalent binding between the loaded drug, which does not cause the drug complex to be quickly cleared from the blood. However, the current technical route is administered by injection, and the specific CD4+ and CD8+ T cells infected with 5 type adenovirus still have an adverse effect on the potency of the albumin-binding recombinant adenovirus vaccine. SUMMARY

[0008] The present application relates to an adenovirus vector vaccine preparation, which 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; the auxiliary component includes albumin. The vaccine can effectively escape pre-existing immunity to adenovirus vectors in the body.

[0009] The present application adds a specific dose of human albumin to the inhaled vaccine preparation formula before vaccine delivery to ensure that an effective defense barrier against neutralizing antibodies of adenovirus has been formed before administration.

[0010] As an inhaled vaccine based on Ad-ABD platform, it is not easy to form an effective anti-neutralizing antibody protective shell after the vaccine is delivered to the target organ. In addition, even if the adenovirus vector vaccine with albumin binding domain enters the body, it can be quickly recognized and cleared by specific neutralizing antibodies before binding to the albumin in the body; or there is mutual competition between specific neutralizing antibodies and albumin in binding to adenovirus vectors, resulting in insufficient binding of albumin to virus vectors and then unable to form a complete and effective pre-existing immune barrier.

[0011] Hexon is the most abundant protein in the adenovirus capsid and is also considered the main target of neutralizing antibodies. Loop 1-L1 and loop 2-L2 of hexon protein are located on the outside of the adenovirus capsid structure. Loop 1 of hexon protein has 6 HVRs, (HVR1-HVR6). There are 7 hypervariable regions (HVRs) in the tower tip region of the hexon protein trimer structure, i.e., L2 contains the 7th hypervariable region (HVR7), which contains a specific epitope.

[0012] The sequence encoding the albumin binding domain is inserted in the coding region of HVR-1 of the hexon protein, and the albumin binding domain is located on the outer surface of the hexon protein.

[0013] Specifically, the albumin binding domain is selected from the albumin binding domain of streptococcal protein G or a functionally equivalent variant thereof, preferably the albumin binding domain 3 of streptococcal protein G.

[0014] More preferably, the sequence of the albumin binding domain 3 of streptococcal protein G is SEQ ID NO: 1.

[0015] The sequence of SEQ ID NO: 1 is as follows:

[0016] Cys Glu Trp Asp Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu

[0017] Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys ThrHis Val

[0018] The inserted sequence encoding the albumin binding is a binding domain after the D150 amino acid of the hexon protein.

[0019] The sequence between the hexon protein and the hinge region of the albumin binding domain is called a linker sequence, which mainly functions to provide space between the two elements so that the albumin binding domain (ABD) part does not affect the secondary structure of the hexon protein. Since the linker usually maintains the length of the three-dimensional structure of the elements and makes the elements independent of each other.

[0020] The N-terminus and / or C-terminus of the albumin binding domain is connected to the hexon protein through a linker sequence.

[0021] The linker sequence includes the sequence GSGS, as shown in SEQ ID NO: 2.

[0022] Specifically, the adenovirus is a human adenovirus.

[0023] Preferably, the human adenovirus is selected from one or more of human adenoviruses of serotypes 1 to 57. More preferably, the human adenovirus is selected from the group consisting of human adenoviruses of serotypes 1 to 57; more preferably, the human adenovirus is a human adenovirus of serotype 5, 26 or 35.

[0024] 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 can bind to the outer surface of the capsid region of the adenovirus hexon protein.

[0025] Specifically, the molar ratio of albumin to adenovirus vector is 1-10 5 : 1; preferably, 1-240: 1.

[0026] Specifically, the vaccine is a mucosal administration preparation or an injection administration preparation, preferably the mucosal administration preparation is selected from: a liquid dosage form, a solid dosage form, a semi-solid dosage form or a gaseous dosage form; more preferably, the mucosal administration preparation is a nasal drop, an aerosol, a spray, a powder spray, a powder, a liquid preparation, a lyophilized preparation, a gel, a microsphere, a liposome, a film, a suspension;

[0027] Further, the mucosal administration preparation is an inhalation liquid preparation, an inhalation aerosol and an inhalation spray.

[0028] More preferably, it is nasal inhalation or oral inhalation; more preferably, the mucosa includes nasal mucosa, oral mucosa or lung mucosa.

[0029] Specifically, the auxiliary component further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient includes but is not limited to one or more of a buffer, a protective agent, a stabilizer, a surfactant, an osmotic pressure regulator, an adjuvant, a preservative and an inactivator, etc.

[0030] 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 one or more of gelatin, ethanol, diethylamine tetraacetic acid (EDTA), diethylamine tetraacetic acid disodium (EDTA-2Na) and magnesium chloride; the stabilizer includes but is 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, glycerol; the osmotic pressure regulator includes but is not limited to sodium chloride or is omitted.

[0031] Specifically, the adjuvant component includes mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, glycerol, preferably, mannitol 10-150 mg / ml, sucrose 10-150 mg / ml, sodium chloride 10-150 mM, magnesium chloride 1-10 mM, HEPES 1-15 mM, polysorbate 80 0.001%-0.5% by weight, glycerol 0.05-2% by weight.

[0032] Specifically, the formulation component comprises: sucrose 10-50 mg / ml, mannitol 15-75 mg / ml, sodium chloride 40-60 mM, glycerol 0.5-5 mg / ml, magnesium chloride 1-5 mM, Tween 80 0.05-0.5 mg / ml, HEPES 1-5 mM, HAS content 0.1%-1% by weight.

[0033] Specifically, the dosage form is an aerosol inhaler, and the vaccine forms particles of less than 10 μm after being atomized by an atomization administration device.

[0034] 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%.

[0035] Specifically, the adenovirus vector further comprises one or more of viral or bacterial antigen proteins, such as HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, respiratory syncytial virus (RSV), varicella-zoster virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and Plasmodium falciparum, and Mycobacterium tuberculosis.

[0036] Specifically, the selected packaging cell is HEK293 cell.

[0037] The present application provides a preparation method of an adenovirus vector vaccine preparation, the preparation steps comprising:

[0038] S1 introducing the albumin binding domain (ABD) of Streptococcus protein G into the HVR region of the hexon gene of the adenovirus vector skeleton to construct an adenovirus-ABD vector;

[0039] S2 introducing the viral or bacterial antigen protein gene sequence into the modified adenovirus-ABD vector to construct an Ad-ABD vector vaccine;

[0040] S3 adding albumin as an auxiliary component in the preparation.

[0041] Specifically, the selected packaging cell is HEK293 cell.

[0042] The present application provides an administration system of an adenovirus vector vaccine preparation, the administration system being an aerosol inhalation administration, and the aerosol administration device being a jet aerosol administration device or an ultrasonic aerosol administration device or a vibration aerosol administration device.

[0043] Preferably, the aerosol administration device comprises an atomizer and an aerosol particle collection component.

[0044] The present application provides a use of an adenovirus vector vaccine preparation in the preparation of a vaccine for preventing infectious diseases. Specifically, the adenovirus encapsulates viral or bacterial disease antigens.

[0045] Specifically, the encapsulated viral or bacterial antigens are one or more of HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, respiratory syncytial virus (RSV), varicella-zoster virus (VZV), cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma cruzi and Plasmodium falciparum, and Mycobacterium tuberculosis.

[0046] The present application provides a method for preventing infectious diseases, the method comprising administering an adenovirus vector vaccine to a subject.

[0047] The subject can be a human or a mammal or a cell, tissue or organ thereof.

[0048] The beneficial effects of the present application include:

[0049] The present application is creatively provided with a vaccine inhalation administration system through experiments, so that the vaccine is administered in the form of atomized inhalation, which can activate protective immune response including mucosal immunity, and solve the pre-existing immunity of rAd, and experiments prove that the present application can effectively reduce the influence of Ad pre-existing immunity on vaccine immunogenicity

[0050] In order to cooperate with the modification of adenovirus, the present application adds a cooperative component albumin in the preparation, and the adenovirus containing the albumin binding domain is better combined to form an albumin-coated shell to escape from pre-existing immunity. Through inhalation administration, the problem that the ABD-modified adenovirus cannot effectively stimulate humoral immune and cellular immune immune response when used alone is solved, and mucosal immunity can be activated at the same time to obtain a higher titer. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 The figure shows the schematic diagram of the molecular design of the recombinant Ad-ABD vector;

[0052] Figure 2 Ad and Ad-ABD binding with HSA and BSA-ELISA results (note * no corresponding column chart means not detected);

[0053] Figure 3 In vitro neutralization experiment-quantitative experimental IFU calculation results;

[0054] Figure 4 14-day antigenic strain binding antibody neutralization titer (log10);

[0055] Figure 5 28-day antigenic strain binding antibody neutralization titer (log10);

[0056] Figure 6 42-day antigenic strain binding antibody neutralization titer (log10);

[0057] Figure 7 Anti-S-IgG antibody titer;

[0058] Figure 8 CD4+T cell cellular immune results;

[0059] Figure 9 CD8+T cell cellular immune results. DETAILED DESCRIPTION

[0060] Unless otherwise defined, all scientific and technical terms used in the present application have the same meanings as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0061] The "adenovirus vector vaccine" refers to a vaccine prepared by using a recombinant adenovirus expressing an antigen gene, in which the adenovirus is used as a vector and the antigen gene is recombined into the adenovirus genome. The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0062] Example 1: Preparation of Ad-ABD adenovirus

[0063] The replication-deficient adenovirus vector recombinant novel coronavirus vaccine is selected as an evaluation model:

[0064] The target antigen of the recombinant novel coronavirus vaccine is the S protein of the novel coronavirus strain (Genebank No. NC_045512.2);

[0065] (1) Constructing a shuttle plasmid vector containing a polynucleotide encoding the S protein of the novel coronavirus;

[0066] The schematic diagram of the molecular design of the backbone plasmid: Ad-ABD vector is shown in Figure 1 : Using the prior art, the albumin binding domain (SEQ ID NO: 1) with two linkers (sequence: GSGS) at the side ends is inserted into the HVR1 of the adenovirus hexon (after the D150 amino acid);

[0067] (2) Transfecting the shuttle plasmid vector of step (1) into the host cell HEK293 together with the backbone plasmid;

[0068] (3) Culturing the host cell HEK293 of step (2);

[0069] (4) Harvesting the human replication-deficient recombinant adenovirus (type 5 adenovirus vector) released from the cell of step (3);

[0070] (5) Subculturing the recombinant adenovirus in step (4);

[0071] (6) Purifying the culture product in step (5) to obtain Ad expressing the target gene.

[0072] The HEK293 cells are cultured in the culture medium and subcultured. When the density of the HEK293 cells is >1.5x10 6HEK293 cells / ml, dilute with medium for virus amplification, virus infection multiplicity of infection (MOI) is 1, 5 and 10, respectively, 37℃, 5% CO2, 125rpm culture, harvest time 42h-48h, get adenovirus seed harvest. HEK293 cells amplified Ad5-ABD virus harvest and supernatant were detected by Ad-FITC fluorescent antibody method. The results are shown in Table 1.

[0073] Table 1 Ad-ABD adenovirus preparation virus titer detection results

[0074]

[0075] The above adenovirus seed Ad-ABD harvest was amplified in HEK 293SF-3F6 cells with a cell density of 0.8-1.2x10 6 cells / ml, MOI=3 infection, and harvested after 48h.

[0076] Example 2: ELISA detection of Ad-ABD adenovirus binding to albumin

[0077] (1) Coating: dilute bovine serum albumin (BSA) and human serum albumin (HSA) to 2mg / ml with coating solution, add to 96-well enzyme-labeled plate, coat overnight at 2-8℃. Wash, add blocking solution.

[0078] (2) Sample addition: dilute Ad-ABD stock solution and NCVA stock solution in Example 1 by 10-fold and 100-fold, respectively, and add to the enzyme-labeled plate; add PBS to the control wells. Incubate at 37℃ for 1h, then wash the plate.

[0079] (3) Incubation: dilute adenovirus monoclonal antibody (invitrogen) with 1% BSA / PBST 1:1000, 100μL / well, incubate at 37℃ for 1h, then wash the plate; dilute goat anti-mouse IgG (Proteintech) with 1% BSA / PBST 1:1000, incubate at 37℃ for 30min, then wash the plate.

[0080] (4) Determination: after the color developing solution is warmed at room temperature, mix A and B liquids 1:1, add to the enzyme-labeled plate, develop color for 15min, then read with an enzyme-labeled instrument.

[0081] Appendix Figure 2 The results show that the binding capacity of Ad-ABD stock solution of the same dilution to HSA is greater than that of Ad5 stock solution to HSA; with dilution of the virus stock, the binding capacity to HSA gradually weakens. Ad-ABD adenovirus can bind to HSA in vitro, proving the feasibility of Ad-ABD adenovirus vector modification and binding by adding albumin.

[0082] Example 3: In vitro neutralization experiment of Ad-ABD adenovirus to escape pre-existing immunity - qualitative experiment

[0083] (1) Virus samples and antibody dilution: Ad-ABD stock solution and NCVA stock solution were diluted with DMEM complete culture (DMEM + 10% FBS + 1% P / S) and DMEM complete culture medium + 1 mg / ml HSA, respectively, to a virus titer of 1.5 x 10 6 IFU / ml. Ad5 rabbit polyclonal antibody (from CanSino Biologies Co., Ltd.) was diluted with DMEM complete culture medium, and the antibody dilution was 1x, 10x and 100x, respectively.

[0084] (2) Incubation: 100 μL / well of virus sample and 1 μL of diluted antibody were added to the 96-well plate, incubated at 37°C for 1 h, and 4 parallel samples were prepared for each experimental group. The experimental groups are shown in Table 2:

[0085] Table 2 In vitro neutralization experiment - qualitative analysis grouping

[0086]

[0087]

[0088] (3) Preparation of HEK293 cell suspension: HEK293 cells were diluted with DMEM complete culture medium to a cell density of 3 x 10 5 cells / ml. After the virus sample and the diluted Ad rabbit polyclonal antibody were incubated at 37°C for 1 h, 100 μL of HEK293 cells / well was added to the 96-well plate, mixed well, and incubated at 37°C, 5% CO2 for 4-5 h. The plate was washed with PBS 3 times,

[0089] (4) Antibody incubation: Ad-FITC fluorescent antibody was diluted with 1% BSA / 1x PBS, and the dilution ratio was 1:500; after washing the plate, 50 μL / well of diluted fluorescent antibody was added to the 96-well plate, and incubated at room temperature for 1 h in the dark. The plate was washed with PBS 2 times.

[0090] (5) Color development: The cells were observed under a fluorescence microscope.

[0091] From Figure 3 It can be seen 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 the Ad vector, indicating that ABD modification and the addition of albumin in the preparation can escape pre-existing immunity.

[0092] Example 4 In vivo immunogenicity evaluation - antibody level test

[0093] Experimental subjects: 6-8 week old female BALB / c mice, 8 mice per group.

[0094] Pre-existing immunity: Take the guinea pig blood with a neutralizing titer of 3000 against adenovirus, and inject 50ul into the abdominal cavity of each mouse. 24 hours later, detect the neutralizing antibody of Ad5 type adenovirus vector in the serum, and the neutralizing antibody titer of all mice is between 200-1000.

[0095] NCVA group: Recombinant novel coronavirus vaccine (Ad) prepared in Example 1 without ABD modification;

[0096] Ad-ABD+HSA group: Add human serum albumin (HSA) solution to the Ad-ABD novel coronavirus vaccine stock prepared in Example 1, and the final concentration of HSA is 1mg / ml;

[0097] Ad-ABD+BSA group: Add bovine serum albumin (BSA) solution to the Ad-ABD novel coronavirus vaccine stock prepared in Example 1, and the final concentration of BSA is 1mg / ml;

[0098] Ad-ABD group: Ad-ABD novel coronavirus vaccine stock prepared in Example 1.

[0099] Mucosal inoculation method: Immunization by inhalation immunization through an inhalation exposure tower, and the immunization dose is 5x10 9 VP.

[0100] 14 days and 28 days after inhalation immunization, the orbital blood was taken, and the neutralizing titer of the serum sample against the novel coronavirus S strain was detected, and the detection results are shown in Tables 3-5 and Figure 4-5

[0101] Table 3 14-day binding antibody neutralizing titer (log10)

[0102] Group Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 Mouse 7 Mouse 8 NCVA 5.51 5.19 5.81 5.74 5.51 5.52 5.20 5.01 Ad-ABD + BSA 5.86 5.72 5.72 5.66 5.99 5.39 5.87 6.10 Ad-ABD + HSA 5.89 5.81 5.89 6.51 5.81 6.11 6.32 6.11 Ad-ABD 4.02 4.60 4.00 5.20 4.60 4.60 4.60 4.90

[0103] Table 4 28-day binding antibody neutralizing titer (log10)

[0104] Group Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 Mouse 7 Mouse 8 NCVA 3.90 3.82 4.20 3.99 4.46 4.58 4.24 3.90 Ad-ABD + BSA 4.90 4.17 4.83 4.09 5.15 4.90 4.22 4.80 Ad-ABD + HSA 5.22 5.11 4.81 4.20 4.81 4.93 5.75 4.97 Ad-ABD 3.60 4.20 3.90 3.81 4.81 3.76 3.54 3.30

[0105] Table 5 42-day binding antibody neutralizing titer (log10)

[0106] Group Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 Mouse 7 Mouse 8 NCVA 4.60 4.21 5.12 5.24 5.17 4.90 4.89 4.33 Ad-ABD + BSA 5.52 5.97 5.81 5.51 5.87 6.11 5.98 5.58 Ad-ABD + HSA 5.98 5.99 5.81 5.74 5.90 6.21 6.01 6.11 Ad-ABD 4.07 4.60 4.34 5.20 4.02 4.58 4.13 3.93

[0107] The second immunization was performed 28 days after the first immunization, and the cell immunity level and S total antibody titer were evaluated 14 days after the second immunization, and the detection results are shown in Figure 6

[0108] ​​The experimental results show that: in the experiment, two doses are used for immunization, and after 14 days of immunization, the specific total antibody level of S protein is detected, and the results show that through inhalation administration, the antibody level stimulated by the Ad-ABD carrier is lower than that of the Ad without ABD modification.

[0109] The possible reason is that: based on the specific inhalation administration method, although the ABD modified Ad has reduced sensitivity to pre-existing adenovirus antibodies in the body, it also leads to a decrease in the efficiency of infecting the host. The positive effect of escaping pre-existing immunity cannot offset the negative effect of reducing immune stimulation. The antibody level of the experimental group Ad-ABD+HSA and Ad-ABD+BSA is significantly higher than that of other groups, that is, the technical scheme of pre-adding albumin in the preparation and then inhaling the Ad-ABD together can greatly improve the positive effect of escaping pre-existing immunity. The reason is that the lung organ is different from the in-vitro and injection environment, and there is a certain difficulty in the combination of lung albumin and Ad containing ABD domain when using inhalation administration to deliver Ad, so it cannot achieve the ideal purpose of escaping pre-existing immunity, and even the effect is not as good as that of unmodified Ad, so it cannot be used for medicine (mucosal administration). The present application adds albumin in the preparation, that is, by pre-preparing the combination in vitro and then inhaling, the problem of the adhesion of the mucosal administration of the Ad-ABD to the pre-existing immunity is fundamentally solved.

[0110] Example 5 In-vivo immunogenicity evaluation-cellular immune response

[0111] Experimental subjects: 6-8 week old female BALB / c mice, 8 mice per group.

[0112] Experimental method: the same experimental method as in Example 4 is used.

[0113] Detection: 21 days after immunization, orbital blood was taken for S-IgG antibody detection, and the results are shown in Table 6; the spleen cells were taken for antigen-specific CD4 and CD8 T cell immune response.

[0114] Table 6 S-IgG antibody titer detection results

[0115]

[0116] TNFα, IFNγ, IL2, IL5 were detected by flow cytometry using Anti-TNFα-FITC antibody, Anti-IFNγ-PE, mouse antibody, Anti-IL2-APC, mouse antibody, and Anti-IL5-AF700 antibody (Tables 7-14).

[0117] Table 7 CD4+T cell TNFα immune response detection results

[0118]

[0119] Table 8 CD4+T cell IFNy immune response test results

[0120]

[0121] Table 9 CD4+T cell IL2 immune response test results

[0122]

[0123] Table 10 CD4+T cell IL5 immune response test results

[0124]

[0125] Table 11 CD8+T cell TNFa immune response test results

[0126]

[0127] Table 12 CD8+T cell IFNy immune response test results

[0128]

[0129] Table 13 CD8+T cell IL2 immune response test results

[0130]

[0131] Table 14 CD8+T cell IL5 immune response test results

[0132]

[0133] The experimental results are shown in Table 15. Figure 7-9

[0134] The experimental results show that: through the inhalation method (mucosal administration), mucosal immunization, the NCVA group, the Ad-ABD+HSA group, the Ad-ABD+BSA group, and the Ad-ABD group carriers can all induce the production of S-IgG antibody titers in the serum of mice. All can stimulate humoral immunity and cellular immunity in mice, mainly CD8+T cells, but the cellular immunity level of the Ad-ABD group is significantly lower than that of the control NCVA. And the technical scheme of pre-adding albumin in the Ad-ABD preparation and then inhaling Ad-ABD together can induce the production of CD4+T cells and CD8+T cells in mice. The THFα, IFNy, and IL2 response levels of the mice are higher than those of the NCVA group and the Ad-ABD group. The results trend is consistent with the experimental results of “in vivo immunogenicity evaluation-antibody level test”.

[0135] ​The above merely describes preferred embodiments of the present application, but is not used to limit the present application, and any modification, equivalent replacement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0136] The foregoing embodiments and methods described in the present application can vary based on the ability, experience and preference of the person skilled in the art.

[0137] The fact that the steps of the method are listed in a certain order in the present application does not constitute any limitation on the order of the steps of the method.

Claims

1. An adenovirus vector vaccine formulation, characterized in that, The vaccine 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, the sequence encoding the albumin-binding domain being inserted into the coding region of hypervariable region 1 of the hexagonal protein, the albumin-binding domain being located on the outer surface of the hexagonal protein; the albumin-binding domain is the albumin-binding domain of streptococcal protein G; the auxiliary component includes albumin; and the vaccine formulation is an inhaled vaccine formulation.

2. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The adenovirus mentioned is a human adenovirus.

3. The adenovirus vector vaccine formulation according to claim 2, characterized in that, The human adenovirus is selected from one or more of human adenoviruses of serum types 1 to 57.

4. The adenovirus vector vaccine formulation according to claim 2, characterized in that, The human adenovirus is serotype 5, 26, or 35.

5. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The albumin is one or more of human serum albumin and bovine serum albumin.

6. The adenovirus vector vaccine formulation according to claim 5, characterized in that, The human serum albumin mentioned is recombinant human serum albumin.

7. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The albumin-binding domain is albumin-binding domain 3 of streptococcal protein G.

8. The adenovirus vector vaccine formulation according to claim 7, characterized in that, The sequence of the albumin-binding domain is SEQ ID NO:

1.

9. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The adenovirus vector further includes viral or bacterial antigen protein gene sequences; the viral or bacterial antigens are one or more of HIV, rabies virus, dengue virus, Ebola virus, coronavirus, human papillomavirus, hepatitis C virus, hepatitis B virus, rotavirus, measles virus, respiratory syncytial virus, herpes zoster virus, cytomegalovirus, herpes simplex virus type 2, Epstein-Barr virus, influenza virus, Trypanosoma krusei, Plasmodium falciparum, or Mycobacterium tuberculosis.

10. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The vaccine is a mucosal delivery preparation.

11. The adenovirus vector vaccine formulation according to claim 10, characterized in that, The mucosal delivery formulation is selected from: liquid dosage form, solid dosage form, semi-solid dosage form or gas dosage form.

12. The adenovirus vector vaccine formulation according to claim 10, characterized in that, The mucosal delivery preparation is an inhaled liquid preparation, an inhaled aerosol, or an inhaled spray.

13. The adenovirus vector vaccine formulation according to claim 10, characterized in that, The mucosal delivery preparation is administered via nasal inhalation or oral inhalation.

14. The adenovirus vector vaccine formulation according to claim 10, characterized in that, The mucosa includes the nasal mucosa, oral mucosa, or lung mucosa.

15. The adenovirus vector vaccine formulation according to any one of claims 1-14, characterized in that, The auxiliary components also include pharmaceutically acceptable excipients.

16. The adenovirus vector vaccine formulation according to claim 15, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators; The buffer includes one or more of HEPES, HIS, TRIS, PB, succinic acid, and citric acid; the protective agent includes one or more of gelatin, ethanol, diethylenetriacetic acid (EDTA), disodium diethylenetriacetate (EDTA-2Na), and magnesium chloride; the stabilizer includes one or more of sucrose, mannitol, fucose, and maltose; the surfactant includes one or more of Tween, Span, and glycerol; and the osmotic pressure regulator includes sodium chloride.

17. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The auxiliary components also include mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, and glycerol.

18. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The auxiliary components also include mannitol 10-150 mg / ml, sucrose 10-150 mg / ml, sodium chloride 10-150 mM, magnesium chloride 1-10 mM, HEPES 1-15 mM, polysorbate 80 0.001%-0.5% by weight, and glycerol 0.05-2% by weight.

19. The adenovirus vector vaccine formulation according to claim 1, characterized in that, The auxiliary components include: sucrose 10-50 mg / ml, mannitol 15-75 mg / ml, sodium chloride 40-60 mM, glycerol 0.5-5 mg / ml, magnesium chloride 1-5 mM, Tween 80 0.05-0.5 mg / ml, HEPES 1-5 mM, and HSA content of 0.1%-1% by weight.

20. The adenovirus vector vaccine formulation according to any one of claims 1-14 or 16-19, characterized in that, The dosage form is an inhaled nebulizer, and the vaccine is atomized by the nebulizer to form particles smaller than 10 μm.

21. The adenovirus vector vaccine formulation according to any one of claims 1-14 or 16-19, characterized in that, The albumin content in the vaccine is 0.001%-50% by weight.

22. The adenovirus vector vaccine formulation according to claim 21, characterized in that, The albumin content in the vaccine, by weight percentage, is 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%.

23. A method for preparing an adenovirus vector vaccine formulation according to any one of claims 1-22, characterized in that, The preparation steps include: S1 introduces the sequence encoding the albumin-binding domain (ABD) of streptococcal protein G into the coding region of hypervariable region 1 of the adenovirus vector hexagonal protein to construct the adenovirus-ABD vector. S2 introduces viral or bacterial antigen protein gene sequences into a modified adenovirus-ABD vector to construct an Ad-ABD vector vaccine; S3 adds albumin as an auxiliary component to the formulation.

24. The preparation method according to claim 23, characterized in that, The viral or bacterial antigens are one or more of the following: 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, and Mycobacterium tuberculosis.

25. A drug delivery system, characterized in that, The drug delivery system comprises the adenovirus vector vaccine formulation according to any one of claims 1-22, wherein the drug delivery system is atomized inhalation drug delivery, and the atomized drug delivery device is a jet atomized drug delivery device, an ultrasonic atomized drug delivery device, or a vibration atomized drug delivery device.

26. The drug delivery system according to claim 25, characterized in that, The nebulized drug delivery device includes a nebulizer and a vaccine particle collection component after nebulization.

27. Use of an adenovirus vector vaccine formulation according to any one of claims 1-22 in the preparation of a vaccine for the prevention of infectious diseases.

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