CN116271066A

CN116271066A - Recombinant adenovirus vector vaccine inhalation administration delivery system

Info

Publication number: CN116271066A
Application number: CN202211589718.XA
Authority: CN
Inventors: 邵娟; 隋秀文; 周朝东; 曹龙龙; 苏喆; 苗伟; 吴丹; 魏搏超; 徐方; 司伟雪; 齐源远; 奚志嫒; 朱涛
Original assignee: CanSino Biologics Inc
Current assignee: CanSino Biologics Inc
Priority date: 2021-12-13
Filing date: 2022-12-12
Publication date: 2023-06-23
Also published as: WO2023109740A1

Abstract

The invention relates to a recombinant adenovirus vector vaccine inhalation administration delivery system, which is characterized in that the delivery system is used for improving adenovirus vectors and matching with a specific preparation prescription, and the vaccine can reach the lung through nasal cavity or oral inhalation after inhalation administration, so that protective immune response including mucosal immunity is generated, the effective utilization rate of the vaccine is enhanced and the effect of the vaccine is improved while the pre-existing immunity of adenovirus is solved.

Description

Recombinant adenovirus vector vaccine inhalation administration delivery system

Technical Field

The invention relates to the technical field of vaccines, in particular to a recombinant adenovirus vector vaccine inhalation administration delivery system for solving adenovirus pre-existing immunity.

Background

Adenoviruses belong to the non-enveloped double-stranded DNA viruses, and human adenoviruses have now identified 53 serotypes. Adenovirus consists of an outer capsid protein and an inner core protein. The core protein comprises: protein v, protein vii, and protein x, which bind to 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 an icosahedral symmetrical structure formed by the non-covalent interactions of 7 proteins (II, III, IIIa, IV, VI, VIII and IX), of which 240 trimeric hexons (protein II) are the main constituent proteins of the 20-face.

Adenovirus has a spherical structure without envelope, virus particles are arranged in lattice form in infected cell nucleus, each virus particle contains 36kb linear double-stranded DNA, two ends of each virus particle have 100-600 bp inverted terminal repeat sequences (inverted terminal re-peat, ITR), and the inner side of ITR is a virus packaging signal, which is a cis-acting element required by virus packaging. The genome comprises early expressed E1-E4 genes associated with adenovirus replication and late expressed L1-L5 genes associated with adenovirus particle assembly. The linear double-stranded DNA and the core protein form a marrow core with the diameter of 60-65 nm, and the marrow core is wrapped in a capsid. The capsid is icosahedral and consists of 252 capsomers with diameters of 8-10 nm, the capsomers are arranged on the triangular surface, 6 on each side, wherein 240 are hexons (non-vertex capsomers), and the other 12 are penton bases (vertex capsomers). Each hexon is a homotrimer of hexon protein, the hexon molecule of the trimer has a triangular cone tip and a pentahedral base, the cone region is composed of 4 rings, namely loop1, loop2, loop3 and loop4, and the base comprises two regions P1 and P2.

The current problem with adenovirus vector vaccines is the presence of pre-existing immunity (pre-existing immunity) to adenovirus, e.g., adenovirus type 5 is more common in the environment, and immune responses against adenovirus are readily generated in the human population, which would reduce the neutralizing antibody levels of adenovirus vector vaccines in humans. The existing method for solving the pre-existing immunity of adenovirus mainly comprises a chemical method and a genetic engineering method. Chemical methods mainly use PEG to encapsulate adenovirus and shield its epitope, thereby escaping the immune function of the host, but such methods have difficulty in obtaining high-titer viruses.

Genetic engineering methods are modification of adenovirus, e.g., construction of chimeric modified adenovirus capsids, construction of chimeric adenoviruses. Adenovirus-based vaccines are completely replaced with other rare, e.g., adenovirus serotypes from humans or non-humans (Chen H, xiang Z Q, li Y, et al adenoviruses-based vaccines: comparison of vectors from three species of adenoviridae [ J ]. Virol,2010,84 (20): 10522-10532), but the specific antibody response elicited by foreign genes is significantly lower than that elicited by type 5 adenovirus vector carrying.

Recombinant coronavirus vaccine (adenovirus type 5 vector) for inhalation developed by the biological stock company Kang Xinuo, 9, 2022, was suggested by the national health commission, and the national administration of pharmaceutical administration demonstrated consent for inclusion as an enhanced needle for emergency use.

Prior art (WO 2015166082A1, entitled adenovirus comprising an albumin binding moiety) adenoviruses genetically modified on the outer surface of the capsid, in particular on the outer surface of the adenovirus hexon protein, enable an albumin protection shield to be obtained, enabling the virus to evade neutralizing antibodies and to extend its retention time in the blood after systemic administration. Albumin is widely used as a drug carrier in the field of intensive research and development, thanks to its long half-life in plasma, and non-covalent binding with the drug to be carried does not allow rapid clearance of the drug complex in the blood. However, this current route of technology is administered by injection, and there is still a detrimental effect on the exertion of the potency of albumin-binding recombinant adenovirus vaccines due to the specific cd4+ and cd8+ T cells present in the infected adenovirus type 5.

Disclosure of Invention

The invention relates to an adenovirus vector vaccine preparation, which contains recombinant adenovirus and auxiliary components; the recombinant adenovirus vector backbone genome sequence comprises a sequence encoding an albumin binding domain; the adjunct component includes albumin. The vaccine can effectively escape the pre-existing immunity of adenovirus vectors in vivo.

The present invention adds specific doses of human albumin to the formulation of an inhalation vaccine formulation prior to vaccine delivery to ensure that a protective barrier has been formed prior to administration that effectively evades adenovirus neutralizing antibodies.

As an inhalation vaccine with Ad-ABD as a platform, it is not easy to form a protective shell of an effective anti-neutralizing antibody after the vaccine is delivered to the targeted organ. Furthermore, even an adenovirus vector vaccine with an albumin binding domain may be rapidly recognized and cleared by specific neutralizing antibodies before binding to albumin in vivo; or the specific neutralizing antibodies and albumin compete with each other for binding to the adenovirus vector, resulting in insufficient binding of albumin to the virus vector and subsequent failure to form a complete and effective pre-existing immune barrier.

Hexon is the highest protein content in the adenovirus capsid and is also considered the primary target for neutralizing antibodies. The loop 1-L1 and loop 2-L2 of the hexon protein are located outside the adenovirus capsid structure. Loop1 of hexon protein has 6 HVRs, (HVR 1-HVR 6). There are 7 hypervariable regions (HVRs) in the cone region of the hexon protein trimer structure, i.e., L2 contains the 7 th hypervariable region (HVR 7), comprising a specific epitope.

The sequence encoding an albumin binding domain is inserted in the coding region of HVR-1 of a hexon protein, the albumin binding domain being located on the outer surface of the hexon protein.

In particular, 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 is albumin binding domain 3 of streptococcal protein G.

More preferably, the albumin binding domain 3 of streptococcal protein G has the sequence of SEQ ID NO:1.

SEQ ID NO. 1 sequence 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 Val

the inserted albumin binding encoding sequence is the binding domain of the fusion protein included 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 a linker sequence and serves primarily to provide space between the two elements so that the Albumin Binding Domain (ABD) portion does not affect the secondary structure of the hexon protein. Since the connectors generally maintain the three-position structural length of the elements and allow the elements to be independent of each other.

The N-terminus and/or the C-terminus of the albumin binding domain is linked to the hexon protein by a linker sequence.

The linker sequence comprises the sequence GSGS, as shown in SEQ ID NO: 2.

In particular, the adenovirus is a human adenovirus.

Preferably, the human adenovirus is selected from one or more of the 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 serotype 5, 26 or 35 human adenovirus.

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 may bind to the outer surface capsid region of the adenovirus hexon protein.

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

specifically, the vaccine is a mucosal or injectable formulation, preferably selected from: a liquid, solid, semi-solid, or gaseous dosage form; more preferably, the mucous membrane administration preparation is nasal drops, aerosol, spray, powder fog, powder, liquid preparation, freeze-dried preparation, gel, microsphere preparation, liposome, film preparation and suspension;

further, the mucosal delivery formulation is an inhalation liquid formulation, an inhalation aerosol, and an inhalation spray.

More preferably, nasal inhalation or oral inhalation; more preferably, the mucosa comprises nasal mucosa, oral mucosa or pulmonary mucosa.

Specifically, the auxiliary component also comprises pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable excipients include, but are not limited to: one or more of buffers, protectants, stabilizers, surfactants, tonicity adjusting agents, adjuvants, preservatives, inactivating agents and the like.

In particular, 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, diethylaminotetraacetic acid (EDTA), disodium diethylaminotetraacetic acid (EDTA-2 Na), and magnesium chloride; the stabilizers include, but are 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 osmolality adjusting agent includes, but is not limited to, sodium chloride or is omitted.

Specifically, the auxiliary material components comprise mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80 and glycerin, preferably 10-150mg/ml mannitol, 10-150mg/ml sucrose, 10-150mM sodium chloride, 1-10mM magnesium chloride and 1-15mM HEPES, wherein the polysorbate 80 is 0.001% -0.5% by weight percent and the glycerin is 0.05-2% by weight percent.

Specifically, the formulation components comprise: 10-50mg/ml of sucrose, 15-75mg/ml of mannitol, 40-60mM of sodium chloride, 0.5-5mg/ml of glycerin, 1-5mM of magnesium chloride, 0.05-0.5mg/ml of Tween 80, 1-5mM of HEPES and 0.1% -1% of HAS in percentage by weight.

Specifically, the dosage form is an atomized inhalant, and the vaccine forms particles below 10 mu m after being atomized by an atomized administration device.

In particular, the albumin is present in the vaccine in a content of 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%.

Specifically, the adenovirus vector also comprises viral or bacterial antigen proteins. Such as HIV, rabies, dengue, ebola, coronavirus, human papilloma, hepatitis c, hepatitis b, rotavirus, measles, respiratory Syncytial Virus (RSV), herpes zoster (VZV), cytomegalovirus, herpes simplex virus type 2, epstein barr, influenza, trypanosoma cruzi and plasmodium falciparum, tubercle bacillus.

Specifically, the packaging cell of choice is a HEK293 cell.

The invention provides a preparation method of an adenovirus vector vaccine preparation, which comprises the following preparation steps:

s1, introducing Albumin Binding Domain (ABD) of Streptococcus (Streptococcus) protein G into HVR region of adenovirus vector skeleton hexon gene to construct adenovirus-ABD vector;

s2, introducing a virus or bacterial antigen protein gene sequence into the modified adenovirus-ABD vector to construct an Ad-ABD vector vaccine;

s3, adding albumin as an auxiliary component into the preparation.

Specifically, the packaging cell of choice is a HEK293 cell.

The invention provides a drug delivery system of an adenovirus vector vaccine preparation, which is aerosol inhalation drug delivery, and the aerosol drug delivery device is a jet aerosol drug delivery device or an ultrasonic aerosol drug delivery device or a vibration aerosol drug delivery device.

Preferably, the aerosolized drug delivery device comprises an atomizer and an atomized vaccine particle collection member.

The invention provides an application of an adenovirus vector vaccine preparation in preparing a vaccine for preventing infectious diseases. Specifically, adenoviruses harbor viral or bacterial disease antigens.

In particular, the viral or bacterial antigens entrapped therein are one or more of HIV, rabies, dengue, ebola, coronavirus, human papilloma, hepatitis c, hepatitis b, rotavirus, measles, respiratory Syncytial Virus (RSV), herpes zoster (VZV), cytomegalovirus, herpes simplex type 2, epstein barr, influenza, trypanosoma cruzi and plasmodium falciparum, tubercle bacillus.

The present invention provides a method of preventing an infectious disease comprising administering an adenovirus vector vaccine to a subject.

The subject may be a human or mammal or a cell, tissue or organ thereof.

The beneficial effects of the invention include:

the invention provides a vaccine inhalation administration system through experimental verification, so that vaccine is administered in an aerosol inhalation mode, which can activate protective immune response including mucosal immunity, and simultaneously solve the problem of preexisting immunity of rAD, and through experimental verification, the influence of the preexisting immunity of Ad on vaccine immunogenicity can be effectively reduced

In order to coordinate with the transformation of adenovirus, the invention adds the matching component albumin into the preparation, and better combines with the adenovirus containing albumin binding domain to form an albumin coated capsid so as to avoid the pre-existing immunity. By inhalation administration, the problem that the independently administered ABD modified adenovirus cannot effectively stimulate humoral immunity and cellular immunity is solved, and mucous membrane immunity can be activated simultaneously to obtain higher titer.

Drawings

FIG. 1 is a schematic diagram showing the molecular design of a recombinant Ad-ABD vector;

FIG. 2Ad and Ad-ABD binding to HSA and BSA-ELISA results (noted with no corresponding histogram, i.e., undetectable);

FIG. 3 in vitro neutralization assay-quantitative assay IFU calculation;

FIG. 4 day 14 neutralizing titer of antigen-binding antibodies (1 og 10);

FIG. 5 day 28 neutralizing titer of anti-prototype binding antibodies (1 og 10);

FIG. 6 day 42 neutralizing titer of antigen-binding antibodies (1 og 10);

FIG. 7 anti-S-IgG antibody titers;

FIG. 8CD4+T cell immunization results;

FIG. 9CD8+T cell immunization results.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

The "adenovirus vector vaccine" refers to a vaccine prepared by recombining a target antigen gene into an adenovirus genome using adenovirus as a vector, and using a recombinant adenovirus capable of expressing the antigen gene. The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: preparation of Ad-ABD adenovirus

Recombinant novel coronavirus vaccine using replication-defective adenovirus as vector was selected as evaluation model:

the target antigen of the recombinant novel coronavirus vaccine is the S protein of a novel coronavirus strain (Genebank number: NC_ 045512.2);

(1) Constructing a shuttle plasmid vector containing a polynucleotide encoding a novel coronavirus S protein;

backbone plasmid: the schematic of the molecular design of the recombinant Ad-ABD vector is shown in FIG. 1: using the prior art, an albumin binding domain (SEQ ID NO: 1) with two linkers (sequence: GSGS) at the flanking ends was inserted into HVR1 of adenovirus hexon (after D150 amino acids);

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

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

(4) Harvesting the human replication-defective recombinant adenovirus (adenovirus type 5 vector) released from the cells of step (3);

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

(6) Purifying the culture product in the step (5) to obtain the Ad capable of expressing the target gene.

HEK293 cells were cultured in medium and passaged. When HEK293 cell density is > 1.5X10 ⁶ At cells/ml, virus amplification was performed by dilution with medium, and the multiplicity of virus infection (MOI) was 1,5 and 10, 37℃and 5% CO, respectively ₂ Culturing at 125rpm, and harvesting for 42-48 h to obtain adenovirus seed harvest liquid. The titer IFU was measured by the Ad5-ABD virus harvest and supernatant from HEK293 cells using the Ad-FITC fluorescent antibody method. The results are shown in Table 1.

TABLE 1Ad-ABD adenovirus preparation Virus titre detection results

The adenovirus strain Ad-ABD harvest liquid is processed in HEK293 SF-3F6 cell density of 0.8-1.2X10 ⁶ cells/ml, moi=3, amplified, harvested 48h later.

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

(1) Coating: bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) were diluted to 2mg/ml with coating solution, added to 96-well ELISA plates and coated overnight at 2-8deg.C. Washing, and adding a sealing liquid.

(2) Sample adding: to the Ad-ABD stock solution in example 1, NCVA stock solution was diluted 10-fold and 100-fold respectively and added to the ELISA plate; control wells were added with PBS. After incubation for 1h at 37℃the plates were washed.

(3) Incubation: 1% BSA/PBST 1: adenovirus monoclonal antibody (invitrogen) at 1000 dilution, 100 μl/well, incubated at 37 ℃ for 1h and plate washed; 1% BSA/PBST 1: sheep anti-mice IgG (Proteintech) were diluted 1000, incubated at 37℃for 30min and plates were washed.

(4) And (3) measuring: after the color development liquid returns to the room temperature, the solution A and the solution B are 1:1, adding an enzyme label plate, developing color for 15min, and reading by an enzyme label instrument.

The result of the attached figure 2 shows that the binding capacity of the Ad-ABD stock solution and HSA of the same dilution is larger than that of the Ad5 stock solution and HSA; with dilution of the virus stock, the binding capacity to HSA gradually decreases. Ad-ABD adenovirus can bind to HSA in vitro, demonstrating the feasibility of Ad-ABD adenovirus vector engineering and binding by the addition of albumin.

Example 3: in vitro neutralization experiment-qualitative experiment for escape of Ad-ABD adenovirus from preexisting immunity

(1) Virus samples and antibody dilution: ad-ABD stock and NCVA stock were diluted to a virus titer of 1.5X10 with DMEM complete culture (DMEM+10% FBS+1% P/S) and DMEM complete medium+1 mg/ml HSA, respectively ⁶ IFU/ml. Ad5 rabbit polyclonal antibody (Source Kang Xinuo Biotechnology Co.) was diluted with DMEM complete medium, 10-fold and 100-fold antibody dilutions, respectively.

(2) Incubation: 100. Mu.L/well virus sample and 1. Mu.L diluted antibody were added to 96-well plates and incubated at 37℃for 1h, 4 replicates for each experimental group, and the experimental groups are shown in Table 2:

TABLE 2 in vitro neutralization assay-qualitative analysis grouping

(3) Preparation of HEK293 cell suspension: HEK293 cells were diluted to a cell density of 3X 10 with DMEM complete medium ⁵ cells/ml. After incubation of virus samples and diluted Ad rabbit polyclonal antibody for 1h at 37 ℃, 100. Mu.L HEK293 cells/well are added into a 96-well plate, and after uniform mixing, the mixture is incubated at 37 ℃ with 5% CO ₂ Culturing for 4-5h. The plate was washed 3 times with PBS,

(4) Antibody incubation: ad-FITC fluorescent antibody was diluted with 1% BSA/1 XPBS, dilution ratio 1:500; after washing the plate, 50. Mu.L/well diluted fluorescent antibody was added to the 96-well plate, and after incubation for 1h at room temperature in the dark, the plate was washed 2 times with PBS.

(5) Color development: cells were observed under a fluorescence microscope.

It can be seen from figure 3 that Ad or Ad-ABD virus vaccine stock was incubated with serial dilutions of anti-Ad antibodies in the presence of HSA, and then HEK293 cells were infected. The extent of neutralization of Ad-ABD was lower than that of Ad vectors, indicating that ABD modification with the addition of albumin in the formulation could evade pre-immunization.

Example 4 in vivo immunogenicity evaluation-antibody level test

The experimental object: female BALB/c mice of 6-8 weeks of age, 8/group.

Establishment of pre-stored immunity: guinea pig blood thawed to a neutralization titer of 3000 against adenovirus was taken and 50ul per mouse was intraperitoneally injected. After 24h, the serum was tested for neutralizing antibodies against adenovirus type 5 vectors, and the neutralizing antibody titer was between 200-1000 in all mice.

NCVA group: the recombinant novel coronavirus vaccine (Ad) prepared in example 1 without ABD modification;

ad-abd+hsa group: the novel coronavirus vaccine stock solution of Ad-ABD prepared in example 1 is added with a Human Serum Albumin (HSA) solution, and the final concentration of HSA is 1mg/ml;

Ad-ABD+BSA group: adding Bovine Serum Albumin (BSA) solution into the novel coronavirus vaccine stock solution of Ad-ABD prepared in example 1, wherein the final concentration of BSA is 1mg/ml;

Ad-ABD group: new coronavirus vaccine stock of Ad-ABD prepared in example 1.

Mode of mucosal inoculation: immunization by inhalation using inhalation type exposure tower and aerosol inhalation immunization, wherein the immunization dose is 5×10 ⁹ VP。

Blood is taken from the eyesockets 14 days and 28 days after atomization inhalation immunization, and neutralization titer of the serum sample aiming at novel coronavirus S strain is detected, and the detection results are shown in tables 3-5 and figures 4-5;

table 3 neutralizing titres of binding antibodies for 14 days (1 og 10)

Group of	Rat 1	Rat 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

Table 4 neutralizing titres of 28 days binding antibody (1 og 10)

Group of	Rat 1	Rat 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

Table 5 neutralizing titres of bound antibody for 42 days (1 og 10)

Group of	Rat 1	Rat 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

Performing a second immunization 28 days after the first immunization, and performing deratization and evaluation of the cellular immunity level and the total antibody titer of S after 14 days of the second immunization, wherein the detection result is shown in FIG. 6;

the experimental results show that: the experiment was performed at 2 doses and after 14 days of immunization, the total antibody level specific for S protein was detected, and the results showed that the level of antibody elicited by the Ad-ABD vector was lower than that elicited by the Ad without ABD modification by inhalation.

The possible reasons are: ABD engineered Ad, based on a specific inhaled administration regimen, while having reduced sensitivity to pre-existing adenovirus antibodies in vivo, also results in reduced host infection efficiency. While the positive effects of evading pre-existing immunity cannot counteract the negative effects of reduced immunostimulatory capacity. The antibody levels of the experimental groups Ad-ABD+HSA and Ad-ABD+BSA are obviously higher than those of other groups, namely, the technical scheme that albumin is added in advance in the preparation and then the albumin is combined with the Ad-ABD for co-inhalation administration can greatly improve the positive influence of escaping pre-existing immunity. As a result, it is possible that pulmonary albumin is difficult to bind to Ad containing ABD domains when Ad is delivered by inhalation administration, unlike in vitro and in an injection environment, and thus the desired escape from preexisting immunity is not achieved, and even the effect is inferior to that of unmodified Ad, and thus the drug cannot be formulated (mucosal administration). The invention fundamentally solves the problem of pharmacy of the Ad-ABD mucous membrane administration preparation for avoiding pre-existing immunity by adding albumin into the preparation, namely, by re-inhalation administration in a mode of in vitro pre-preparation combination.

Example 5 in vivo immunogenicity evaluation-cellular immune response.

The experimental object: female BALB/c mice of 6-8 weeks of age, 8/group.

The experimental method comprises the following steps: the same experimental procedure as in example 4 was used.

And (3) detection: at 21 days post immunization, orbital blood was taken and tested for S-IgG antibodies, the results are shown in Table 6; splenocytes were harvested and used as antigen-specific CD4 and CD 8T cell immune responses.

TABLE 6S-IgG antibody titre detection results

TNFα, IFNγ, IL2, IL5 were detected by flow cytometry using Anti-TNFα -FITC antibody, anti-IFNγ -PE, mo μse antibody, anti-IL2-APC, mo μse antibody, anti-IL5-AF700 antibody (tables 7-14).

TABLE 7CD4+T cell TNF alpha immune response assay results

TABLE 8CD4+T cell IFN gamma immune response detection results

TABLE 9CD4+T cell IL2 immune response test results

TABLE 10CD4+T cell IL5 immune response assay results

TABLE 11CD8+T cell TNF alpha immune response assay results

TABLE 12CD8+T cell IFN gamma immune response assay results

Table 13CD8+T cell IL2 immune response test results

TABLE 14CD8+T cell IL5 immune response assay results

The experimental results are shown in FIGS. 7-9.

The experimental results show that: mucosal immunization by inhalation (mucosal administration), NCVA group, ad-ABD+HSA group, ad-ABD+BSA group, ad-ABD group vectors induced S-IgG antibody titers in mouse serum. Can excite humoral immunity and cellular immunity in mice, mainly uses CD8+T cells, but the cellular immunity level of Ad-ABD group is obviously lower than that of control NCVA. The technical scheme of pre-adding albumin in the Ad-ABD preparation and then combining with Ad-ABD for co-inhalation can induce mice to generate the COVID-19S proteins THF alpha, IFN gamma and IL2 response level higher than NCVA group and Ad-ABD group for CD4+ T cells and CD8+ T cells. The trend of the results is consistent with the experimental results of 'in vivo immunogenicity evaluation-antibody level test'.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

1. An adenovirus vector vaccine formulation comprising a recombinant adenovirus and a helper component; the recombinant adenovirus vector backbone genome sequence comprises a sequence encoding an albumin binding domain; the adjunct component includes albumin.

2. The adenoviral vector vaccine formulation according to claim 1, wherein the adenovirus is a human adenovirus; preferably, the human adenovirus is selected from one or more of the human adenoviruses of serotypes 1 to 57; more preferably, the human adenovirus is a human adenovirus of serotype 5, 26 or 35.

3. The adenoviral vector vaccine formulation according to any one of claims 1-2, wherein the albumin added to the auxiliary component is one or more of human serum albumin, recombinant human serum albumin, bovine serum albumin.

4. An adenoviral vector vaccine preparation according to any one of claims 1-3, wherein the molar ratio of albumin to adenoviral vector is 1-10 ⁵ :1, a step of; preferably, 1-240:1.

5. the adenoviral vector vaccine formulation according to any one of claims 1-4, wherein the sequence encoding the albumin binding domain is inserted in the coding region of hypervariable region 1 (HVR 1) of the hexon protein, said albumin binding domain being located on the outer surface of the hexon protein.

6. The adenoviral vector vaccine formulation according to any one of claims 1-5, wherein the albumin binding domain is the albumin binding domain of streptococcal protein G or a functionally equivalent variant thereof, preferably the albumin binding domain is albumin binding domain 3 of streptococcal protein G.

7. The adenoviral vector vaccine formulation according to any one of claims 1-6, wherein the vaccine is a mucosal or injectable formulation, preferably the mucosal formulation is selected from the group consisting of: a liquid, solid, semi-solid, or gaseous dosage form; more preferably, the mucous membrane administration preparation is nasal drops, aerosol, spray, powder fog, powder, liquid preparation, freeze-dried preparation, gel, microsphere, liposome, film agent and suspension; further preferably, the mucosally administered formulation is an inhalation liquid formulation, an inhalation aerosol, and an inhalation spray; more preferably, nasal inhalation or oral inhalation; more preferably, the mucosa comprises nasal mucosa, oral mucosa or pulmonary mucosa.

8. The adenoviral vector vaccine formulation according to any one of claims 1-7, wherein the adjunct component further comprises pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients include, but are not limited to: one or more of buffers, protectants, stabilizers, surfactants, tonicity adjusting agents, adjuvants, preservatives, inactivating agents and the like.

9. The adenoviral vector vaccine formulation according to any one of claims 1-8, wherein the buffer comprises, 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, diethylaminotetraacetic acid (EDTA), disodium diethylaminotetraacetic acid (EDTA-2 Na), and magnesium chloride; the stabilizers include, but are 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 osmolality adjusting agent includes, but is not limited to, sodium chloride or is omitted.

10. Adenovirus vector vaccine formulation according to any one of claims 1-9, wherein the adjuvant component comprises mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, glycerol, preferably mannitol 10-150mg/ml, sucrose 10-150mg/ml, sodium chloride 10-150mM, magnesium chloride 1-10mM, HEPES 1-15mM, polysorbate 80.001% -0.5% in weight percent, glycerol 0.05-2% in weight percent.

11. The adenoviral vector vaccine formulation according to any one of claims 1-10, wherein the formulation components comprise: 10-50mg/ml of sucrose, 15-75mg/ml of mannitol, 40-60mM of sodium chloride, 0.5-5mg/ml of glycerin, 1-5mM of magnesium chloride, 0.05-0.5mg/ml of Tween 80, 1-5mM of HEPES and 0.1% -1% of HAS in percentage by weight.

12. The adenoviral vector vaccine formulation according to any one of claims 1-11, wherein the formulation is an aerosol inhalation, the vaccine forming particles below 10 μm after aerosol administration by an aerosol administration device.

13. Adenovirus vector vaccine formulation according to any one of claims 1-12, wherein the albumin is present in the vaccine in an amount of 0.001% -50%, 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% by weight.

14. A method of preparing an adenovirus vector vaccine formulation according to any one of claims 1-13, comprising the steps of:

s3, adding albumin as an auxiliary component into the preparation.

15. The method of claim 14, wherein the selected packaging cells are HEK293 cells.

16. The method of claim 14 or 15, wherein the viral or bacterial antigen is one or more of HIV, rabies, dengue, ebola, coronavirus, human papilloma, hepatitis c, hepatitis b, rotavirus, measles, respiratory Syncytial Virus (RSV), herpes zoster (VZV), cytomegalovirus, herpes simplex virus type 2, epstein barr, influenza, trypanosoma cruzi and plasmodium falciparum, tubercle bacillus.

17. A delivery system for an adenovirus vector vaccine formulation according to any one of claims 1-13, wherein the delivery system is an aerosol inhalation delivery, the aerosol delivery device being a jet aerosol delivery device or an ultrasonic aerosol delivery device or a vibratory aerosol delivery device, preferably the aerosol delivery device comprising an atomizer and an atomized vaccine particle collection means.

18. Use of an adenoviral vector vaccine formulation according to any one of claims 1-13 in the manufacture of a vaccine for preventing infectious diseases.