Construction method and application of adenovirus infected animal model

Abstract

The invention relates to a construction method and application of an adenovirus infected animal model, and the method comprises the following steps: transplanting lung cancer cells or laryngeal cancer cells to subcutaneous or lung tumor formation of an animal, and constructing a transplanted tumor animal model; and infecting the transplanted tumor animal model with an adenovirus, and constructing the adenovirus infected animal model. The method is high in modeling success rate, small in individual difference, simple and rapid to operate and low in cost, can support infection and efficient replication of adenovirus in an animal body, and is used as an in-vivo animal evaluation model of an antiviral drug.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a method for constructing an adenovirus-infected animal model and its application. Background Technology

[0002] Human adenoviruses (HAdVs) belong to the family Adenoviridae, genus Adenovirus of mammals. They are non-enveloped, double-stranded DNA viruses, classified into 7 groups (AG) and over 100 different serotypes. Different HAdV types exhibit varying tissue tropism and can cause different diseases. For example, group B (HAdV-3, -7, -14, and -55) are highly associated with acute respiratory distress syndrome (ARDS) and lead to higher morbidity and mortality. In immunocompromised patients, adenoviruses can cause multi-organ infections, including pharyngoconjunctival fever, pneumonia, epidemic keratoconjunctivitis, gastroenteritis, hepatitis, cystitis, and encephalitis, which can be life-threatening in severe cases. HAdV can also cause latent infection. The mortality rate of untreated severe HAdV pneumonia or disseminated disease can exceed 50%.

[0003] To date, animal models used to study the pathogenesis and infection of adenoviruses include mouse models, transgenic mouse models, and non-human primate models. However, because viral replication relies entirely on host cell machinery and is species-specific, adenoviruses cannot replicate effectively in mice or other animals. The lack of suitable animal models to support adenovirus infection and replication has greatly limited research on adenovirus infection, antiviral drugs, and vaccines. Summary of the Invention

[0004] Based on this, one embodiment of this application needs to provide a method for constructing an animal model of adenovirus infection, which can solve the problem that human adenovirus cannot effectively infect and replicate in animals, and can be used for adenovirus infection mechanism research, antiviral drug screening and evaluation, vaccine screening and evaluation of active and passive immunization of vaccines, etc.

[0005] The technical solutions include the following:

[0006] The method for constructing an animal model of adenovirus infection includes the following steps:

[0007] Transplanting lung cancer cells or laryngeal cancer cells subcutaneously or into the lungs of animals to create tumors, thus constructing animal models of xenograft tumors; and,

[0008] Adenovirus-infected animal models were constructed by infecting the transplanted tumor animal model with adenovirus.

[0009] In one embodiment, the lung cancer cells or laryngeal cancer cells are transplanted into the subcutaneous tissue or lungs of an animal to form tumors, including by subcutaneous injection or nasal instillation.

[0010] In one embodiment, the lung cancer cells or laryngeal cancer cells include A549 cells, and / or laryngeal cancer cells include Hep-2 cells.

[0011] In one embodiment, the animal comprises an immunodeficient mouse. Optionally, the immunodeficient mouse comprises a female NCG mouse.

[0012] In one embodiment, the step of transplanting lung cancer cells or laryngeal cancer cells into the subcutaneous tumor of an animal via subcutaneous injection includes:

[0013] A cell suspension of the lung cancer cells or laryngeal cancer cells was mixed with matrix gel at a volume ratio of 1:(0.8-1.2) to prepare a mixture; wherein the density of the lung cancer cells or laryngeal cancer cells in the cell suspension was 5×10⁻⁶. 7 Cells / mL~2×10 8 cells / mL; and,

[0014] The injection volume of the mixture into the animals was 90 μL to 110 μL. Tumors formed 7 to 21 days after injection.

[0015] In one embodiment, the step of transplanting lung cancer cells or laryngeal cancer cells into the lungs of an animal via nasal instillation to form a tumor includes:

[0016] After intranasal instillation of a cell suspension of the aforementioned lung cancer cells or laryngeal cancer cells into animals, tumors formed after 7 to 21 days of feeding; wherein the density of the lung cancer cells or laryngeal cancer cells in the cell suspension was 3.0 × 10⁻⁶. 7 cells / mL ~ 5.0 × 10⁻⁶ 7 The dose per mL is 90 μL to 110 μL; or,

[0017] Animals were intranasally instilled with a mixture of lung cancer cells or laryngeal cancer cells and matrix gel, and then fed for 7 to 21 days to induce tumor formation; wherein the cell density of the lung cancer cells or laryngeal cancer cells in the mixture was 3.0 × 10⁻⁶. 7 cells / mL ~ 5.0 × 10⁻⁶ 7 The dose per mL is 90 μL to 110 μL.

[0018] In one embodiment, the matrix gel comprises a Matrix-Gel matrix gel with a protein concentration of 16 mg / mL to 26 mg / mL.

[0019] In one embodiment, the infection includes intratumoral injection, intravenous injection, or nasal instillation; optionally, the infection dose is (4-6)*102. 6 TCID50 / (90μL~100μL).

[0020] In one embodiment, the adenovirus includes one or more of HAdV-3, HAdV-7, and HAdV-55.

[0021] In one embodiment, the adenovirus is a replicating recombinant human adenovirus, wherein the E3 region of the replicating recombinant adenovirus is deleted and a reporter gene is inserted. Optionally, the reporter gene includes an enhanced green fluorescent protein reporter gene and a luciferase reporter gene.

[0022] The animal model constructed by the method can be applied in any of the following ways:

[0023] (1) Application in screening or evaluating adenovirus vaccines;

[0024] (2) Its use in screening or evaluating anti-adenovirus drugs; and

[0025] (3) Application in the study of adenovirus infection mechanism.

[0026] Compared with traditional technologies, this application has the following advantages:

[0027] The method for constructing an adenovirus-infected animal model provided in this application has a high success rate, small individual differences, is simple and fast to operate, and has low cost. It can support adenovirus infection and efficient replication in animals and can be used as an in vivo animal evaluation model for antiviral drugs. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 The viral bands of adenovirus Adv7L-1 and Adv7L-2 after CsCl density gradient centrifugation;

[0030] Figure 2 The graph shows the linear relationship between the luminescence value and the viral dilution after Ad7L infection of A549 cells. The left graph represents Ad7L-1, and the right graph represents Ad7L-2.

[0031] Figure 3 Changes in mouse body weight after injection of A549 cells;

[0032] Figure 4The change in tumor size in mice after injection of A549 cells;

[0033] Figure 5 This is a schematic diagram of the experimental procedure for a mouse model infected with rAd7E and Ad7L.

[0034] Figure 6 In vivo imaging of a mouse model of recombinant human adenovirus type 7-infected xenograft tumor after infection with Ad7L virus; Note: Tail vein injection, IV, intratumoral injection, IT.

[0035] Figure 7 This image shows organ imaging of a mouse model of recombinant human adenovirus type 7-infected xenograft tumor after infection with Ad7L virus. No visible luminescent signal was observed in the tumors of mice in the PBS control group, while obvious luminescent signals were observed in the tumors of mice in the Ad7L tail vein and intratumoral injection groups. Note: In the image, no luminescent signal was observed in the right tumor of one mouse in the Ad7L tail vein injection group (left image in the group), and two tumors grew in the right axilla of one mouse in the Ad7L intratumoral injection group (left image in the group).

[0036] Figure 8 This is an in vivo imaging image of a mouse model of recombinant human adenovirus type 7-infected xenograft tumor after infection with rAd7E virus; Note: Tail vein injection, iv, intratumoral injection, it; Due to the short excitation wavelength required for enhanced green fluorescent protein (EGFP), poor penetration ability, and susceptibility to signal interference, only background-level signal values ​​can be observed in the in vivo fluorescence imaging of the experimental and control groups.

[0037] Figure 9 This is an organ imaging image of a mouse model of recombinant human adenovirus type 7-infected xenograft tumors after infection with rAd7E virus; Note: No fluorescence signal was observed in the tumor imaging of mice in the PBS control group, while obvious fluorescence signal was observed in mice in the rAd7E experimental group; Two mice in the rAd7E intratumoral injection group developed two tumors in their right axilla.

[0038] Figure 10 Viral load in organs of tumorigenic mice after infection with Ad7L (left) and rAd7E (right);

[0039] Figure 11 Changes in mouse body weight after injection of A549 cells;

[0040] Figure 12 The change in tumor size in mice after injection of A549 cells;

[0041] Figure 13 In vivo imaging of mice in a xenograft mouse model for evaluating the Ad7L neutralizing antibody 3F11;

[0042] Figure 14Organ imaging of mice used to evaluate the Ad7L neutralizing antibody 3F11 in a xenograft mouse model;

[0043] Figure 15 The viral load in tumor cells was measured in the Ad7L+3F11 group, the Ad7L virus group, and the PBS control group.

[0044] Figure 16 The viral load in tumors of the Ad55E+3F11 group, the Ad55E virus group, and the PBS control group was measured; high and low viral loads were detected in the Ad55E group (10... 7 -10 8 (copies / mL), no virus was detected in the Ad55E+3F11 group and the PBS control group;

[0045] Figure 17 To detect viral load in the lungs of the Ad7E-A5E4 group, the Ad7E virus group, and the PBS control group using qPCR;

[0046] Figure 18 To detect viral load in tumors of the Ad7E-A5E4 group, the Ad7E virus group, and the PBS control group using qPCR;

[0047] Figure 19 To detect viral load in the lungs of the Ad7E-A5E4 group, the Ad7E virus group, and the PBS control group using the IFU method;

[0048] Figure 20 To detect viral load in tumors of the Ad7E-A5E4 group, the Ad7E virus group, and the PBS control group using the IFU method;

[0049] Figure 21 HE sections of lungs from mice with A549-RFP tumor formation aided by Matricene (left image) / without Matricene (middle image);

[0050] Figure 22 DAPI staining, RFP fluorescence, ADV-IF fluorescence, and three-color superimposed imaging images of A549-RFP tumor-forming mice in the lungs;

[0051] Figure 23 Imaging of the lungs in A549-RFP mice infected with Ad7L for 9 days after intranasal administration of chemiluminescence in vivo and lung organs;

[0052] Figure 24 To detect viral load in tumors after Ad7L nasal inoculation following Matrix-assisted tumorigenesis experimental group, non-Matrix-assisted tumorigenesis experimental group, PBS nasal inoculation control group, and qPCR method. Detailed Implementation

[0053] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0055] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0056] In this article, TCID 50 The half-maximal dose of infection (MCD), also known as the 50% tissue culture infection dose, refers to the amount of virus required to induce cytopathic effect (CPE) in the wells of a culture plate or test tube. It is used to characterize the viral titer.

[0057] In this article, VPs stands for Viral particles.

[0058] Due to the lack of humanized receptors for adenoviruses and the absence of species specificity, adenoviruses cannot effectively replicate and multiply in some animals. Although some may cause pathological damage, higher viral doses are required, and they cannot well mimic the clinical symptoms and certain pathological features of human adenovirus infection.

[0059] Based on this, one embodiment of this application provides a method for constructing an adenovirus-infected animal model.

[0060] This includes the following steps S1 to S2:

[0061] Step S1: Transplant lung cancer cells or laryngeal cancer cells into the subcutaneous tissue or lungs of animals via subcutaneous injection or nasal drip to form tumors and construct an animal model of transplanted tumors.

[0062] In one specific example, the animal includes an immunodeficient mouse. Preferably, the immunodeficient mouse is a female NCG mouse.

[0063] In one specific example, lung cancer cells or laryngeal cancer cells include A549 cells, and / or laryngeal cancer cells include Hep-2 cells.

[0064] In a specific example, the subcutaneous tumorigenesis of lung cancer cells or laryngeal cancer cells in an animal via subcutaneous injection includes steps (1) to (2):

[0065] (1) Prepare a mixture by mixing a suspension of lung cancer cells or laryngeal cancer cells with matrix gel at a volume ratio of 1:(0.8-1.2).

[0066] In a specific example, in step (1), the cell suspension of lung cancer cells or laryngeal cancer cells is mixed with matrix gel at a volume ratio of 1:0.8, 1:1 or 1:1.2.

[0067] In one specific example, in step (1), the cell density of lung cancer cells or laryngeal cancer cells in the cell suspension is 5 × 10⁻⁶. 7 Cells / mL~2×10 8 cells / mL, selectable value is 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, 1×10 8 cells / mL or 2×10 8 per mL.

[0068] In a specific example, in step (1), the matrix gel assists in tumor formation. The matrix gel includes a high-concentration Matrix-Gel matrix gel with a protein concentration of 16 mg / mL to 26 mg / mL. Preferably, the matrix gel is a high-concentration, low-growth-factor Matrix-Gel matrix gel.

[0069] (2) The injection volume of the mixture into the animal is 90μL to 110μL. After injection, the animal is fed for 7 to 21 days and tumors form.

[0070] In a specific example, in step (2), the injection volume of the mixture into the animal is 90 μL to 110 μL, with selectable values ​​of 90 μL, 95 μL, 100 μL, 105 μL or 110 μL.

[0071] In one specific example, in step (2), the baby is fed for 7, 8, 9, 10, 11, 12, 13, 15, 18 or 21 days after the injection.

[0072] In one specific example, after tumor formation, the mouse's body weight and tumor size (volume) were observed.

[0073] This application uses subcutaneous inoculation of xenografts in immunodeficient mice, followed by intratumoral or intravenous viral infection. This method can be used for in vivo evaluation of antiviral drugs, which can be administered intravenously / intraperitoneally or orally. Compared to cell models, this method is an in vivo model where the drug is metabolized in vivo.

[0074] In a specific example, transplanting lung cancer cells or laryngeal cancer cells into the lungs of animals via nasal instillation includes two methods: Method 1 and Method 2.

[0075] Option 1: After intranasal instillation of a mixture of lung cancer cells or laryngeal cancer cells and matrix gel into the animals, they were fed for 7–21 days to induce tumor formation; the cell density of lung cancer cells or laryngeal cancer cells in the mixture was 3.0 × 10⁻⁶. 7 cells / mL ~ 5.0 × 10⁻⁶ 7 The dose per mL is 90 μL to 110 μL.

[0076] In a specific example, in Scheme 1, the role of matrix gel is to assist tumor formation. The matrix gel includes high-concentration matrix-Gel matrix gel with a protein concentration of 16 mg / mL to 26 mg / mL.

[0077] In a specific example, in Scheme 1, the cell density of lung cancer cells or laryngeal cancer cells in the mixture is 3.0 × 10⁻⁶. 7 cells / mL ~ 5.0 × 10⁻⁶ 7 cells / mL, with an optional value of 3.0 × 10⁻⁶. 7 cells / mL, 4.0×10 7 cells / mL or 5.0 × 10⁻⁶ 7 per mL.

[0078] Option 2: After intranasal instillation of a suspension of lung cancer cells or laryngeal cancer cells into the animals, they were fed for 7–21 days until tumor formation occurred. The cell density of the lung cancer cells or laryngeal cancer cells in the cell suspension was 3.0 × 10⁻⁶. 7 cells / mL ~ 5.0 × 10⁻⁶ 7 cells / mL, with an optional value of 3.0 × 10⁻⁶. 7 cells / mL, 4.0×10 7 cells / mL or 5.0 × 10⁻⁶ 7 The dose per mL is 90 μL to 110 μL.

[0079] Option 2 has a higher success rate in transplanting lung cancer cells or laryngeal cancer cells into the lungs of animals to form tumors, and the viral replication efficiency is high after infection with adenovirus.

[0080] This application employs methods such as nasal instillation / lung injection to inoculate lung cancer cells or laryngeal cancer cells to establish an animal model of lung tumor formation. Adenovirus is then administered via nasal instillation. Antiviral drugs are administered in a manner mimicking human administration, including intravenous / intraperitoneal, nasal instillation, inhalation, and oral administration, to evaluate the antiviral drugs in vivo. This approach better simulates natural viral infection and drug administration methods.

[0081] Step S2: Construct an adenovirus-infected animal model by infecting a xenograft tumor animal model.

[0082] In a specific example, infection can occur via intratumoral injection, intravenous injection, or nasal instillation.

[0083] In a specific example, the infection dose is (4–6) * 10-1 6 TCID 50 / (90μL~100μL), selectable value is 4*10 6 TCID 50 / 100μL, 5*10 6 TCID 50 / 100μL or 6*10 6 TCID 50 / 100μL. The infectious dose of the virus used in this application for modeling is low, and it can also replicate efficiently in the model.

[0084] In one specific example, the adenovirus includes one or more of HAdV-3, HAdV-7, and HAdV-55.

[0085] In one specific example, the adenovirus is a replicating recombinant adenovirus with its E3 region deleted and a reporter gene inserted. Optionally, the reporter gene includes an enhanced green fluorescent protein reporter gene and a luciferase reporter gene.

[0086] In a specific example, on one or more days from day 3, 4, 5, 6, 7, 8, 9, and 10 post-infection, the viral replication and distribution in animal models or organs were observed using in vivo imaging, and / or by quantitative PCR, IFU, TCID... 50 Detect viral load or viral titer.

[0087] In vivo imaging technology is used to detect the susceptibility of animal models obtained by the method described in this application to infection. The method of calculating the amount of viral fluorescence in the animal models obtained in this application using in vivo imaging shows a high correlation with the traditional method of detecting viral load using quantitative PCR, thus serving as an effective method for real-time and intuitive observation of viral distribution in animal models.

[0088] This application also provides, in one embodiment, the application of the animal model constructed by the above-described method in screening or evaluating adenovirus vaccines. For example, in the evaluation of vaccine attenuation.

[0089] An embodiment of this application also provides the application of the animal model constructed by the above construction method in screening or evaluating anti-adenovirus drugs.

[0090] An embodiment of this application also provides a method for screening or evaluating anti-adenovirus drugs, the method comprising steps S10 to S30:

[0091] Step S10: Constructing an animal model of xenograft tumor;

[0092] Step S20: Inject anti-adenovirus drugs one day before and one day after infection with adenovirus in the xenograft animal model; and,

[0093] Step S30: Infect the xenograft animal model with adenovirus.

[0094] In a specific example, in step S20, the dose of the antiviral drug injected is (40~60) μg / 100μL each time.

[0095] In a specific example, the method also includes observing the infection, replication, and distribution of adenovirus in animal models or organs by in vivo imaging 3–10 days after adenovirus infection, and / or by quantitative PCR, IFU, TCID. 50 Detect viral load or viral titer.

[0096] In a specific example, the steps of constructing the xenograft animal model in step S10 and infecting the xenograft animal model with adenovirus in step S30 are the same as described above.

[0097] An embodiment of this application also provides a method for screening or evaluating adenovirus vaccines, the method comprising steps S100 to S200:

[0098] Step S100, constructing the xenograft animal model; and,

[0099] Step S200: Infect the transplanted tumor animal model with adenovirus vaccine and adenovirus, respectively.

[0100] In a specific example, this also includes observing the infection replication and distribution of the adenovirus vaccine in animal models or organs 3–10 days after adenovirus infection using in vivo imaging, and / or using quantitative PCR, IFU, TCID... 50 Detect viral load or viral titer.

[0101] In a specific example, the dosage and steps for infecting the transplanted tumor animal model with adenovirus vaccine and adenovirus, respectively, are the same as the steps for infecting the transplanted tumor animal model with adenovirus described above.

[0102] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0103] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0104] Example 1: Construction of an adenovirus-infected xenograft mouse model

[0105] I. Preparation of recombinant human adenovirus type 7 expressing firefly luciferase

[0106] Based on the whole genome sequence of human adenovirus type 7 strain CQ1198 (GenBank No. JX625134), a replicative human adenovirus type 7 vector rAd7E (Ad7E) with an E3 region deletion and an insertion of an enhanced green fluorescent protein (EGFP) expression cassette was prepared (see Qiong Zhang, et al. Higher affinities of fibers with cell receptors increase the infection capacity and virulence of human adenovirus type 7 and type 55 compared to type 3. Microbiology Spectrum. January 2024;12 (1):e01090-23. DOI: 10.1128 / spectrum.01090-23). ​​Using the same method as Ad7E, a replicative human adenovirus type 7 vector rAd7Luc (Ad7L) with an E3 region deletion and an insertion of a firefly luciferase expression cassette was prepared. Ad7L was cultured in large quantities in two batches using A549 and then purified by CsCl density gradient centrifugation. Figure 1 This data will be used for further in vivo animal experiments. The viral titer of Ad7L-1 is 1.925*10⁻⁶. 12 VPs / mL, 10 9 TCID50 / mL; Viral titer of Ad7L-2: 2.255*10 12 VPs / mL, 10 9.23 TCID 50 / mL.

[0107] Luciferase assay: Adenovirus Adv7L-1 and Adv7L-2 were serially diluted 5-fold, and the result was taken as a 5-fold dilution. -3 5 -4 5 -5 5 -6 Viral fluid, with 2×10 4 A549 cells were added to 96-well plates along with control wells. After culturing for 48 hours, the supernatant was discarded. Following the instructions for the TransDetect® Single-Luciferase (Firefly) Reporter Assay Kit, 50 μL of cell lysis buffer was added to each well, and the cells were lysed at room temperature for 10 min. 20 μL of the lysis product was collected, and 50 μL of luciferase reaction substrate was added. The fluorescence value was detected using a multi-mode microplate reader. The fluorescence value of the uninfected control cells was only 8836, while the lowest value for Ad7L cells was 108471 (5...). -6 When diluted, the value exceeded the control by more than 12 times; moreover, the infection dose of Ad7L was positively correlated with the fluorescence intensity, proving that the preparation of Ad7L was successful. Figure 2 ).

[0108] II. Establishment of a mouse model of xenograft infection with recombinant human adenovirus type 7

[0109] 1. Subcutaneous tumor formation of lung cancer cells A549:

[0110] Subcutaneous tumorigenesis experiments were conducted using 4-8 week old female NCG mice, with 8 × 10⁸ tumor cells inoculated. 6 Each / each.

[0111] a. Place the Matrix-Gel™ matrix gel (high concentration) (C0382 / C0383 or C0386 / C0387) on ice and thaw overnight at 4°C. Mix the matrix gel with a pre-cooled pipette or tip until homogeneous. Note: High-concentration, low-factor matrix gel promotes faster tumor formation.

[0112] b. Cell Collection: Suspended cells were collected directly as a cell suspension. Adherent cells were digested with trypsin, and the cell suspension was collected afterward. The collected cell suspension was centrifuged at 300×g for 5 minutes and washed twice with PBS. The cells were resuspended in PBS and counted, and the cell density was adjusted to 5×10⁻⁶ cells / mL. 7 ~2×10 8 Cells / mL. Place cells on ice until ready to use. Note: Collected cells should be inoculated within 2 hours.

[0113] c. Mix the cell suspension with the matrix gel at a 1:1 volume ratio. At this point, the cell density will be 2.5 × 10⁻⁶. 7 ~1×10 8 Cells / mL. Note: The mixture of cell suspension and matrix gel should be placed on ice to slow down cell apoptosis and prevent matrix gel from solidifying.

[0114] d. Hair was removed from the injection site of the mice using an experimental animal electric hair shaver (FS600), and then the area was disinfected with an alcohol swab.

[0115] e. Using a 1mL syringe without a needle, draw the cell suspension and matrix gel mixture into the syringe, then attach the needle. Note: Before drawing the mixture, you can gently pipette the cells to mix them.

[0116] f. Administer a subcutaneous injection, typically 100 µl per animal. Note: The injection process should be kept as short as possible.

[0117] g. After feeding the mice for approximately 7 days following the injection, a more noticeable tumor can be observed. Measure the size of the tumor using calipers and calculate its volume.

[0118] Tumor volume (mm 3 ) = length (mm) × width (mm) × width (mm) ×1 / 2.

[0119] Changes in body weight and tumor size after subcutaneous and axillary inoculation of A549 cells in mice are as follows: Figure 3 and Figure 4 As shown.

[0120] 2. Recombinant human adenovirus type 7 infection:

[0121] Twenty-one days after tumor formation, visible tumors appeared in the axilla of mice. Recombinant human adenovirus type 7 (rAd7E) and Ad7L were inoculated via both intratumoral and intravenous injection routes. Experimental groups are shown in Table 1, and the experimental flowchart is shown below. Figure 5 As shown.

[0122] Table 1 Experimental groupings and virus inoculation methods in the rAd7E and Ad7L-infected tumorigenic mouse model

[0123]

[0124] III. In vivo and organ imaging of a mouse model of xenograft tumor infection with recombinant human adenovirus type 7.

[0125] a. Within 10-30 minutes after intraperitoneal injection of 150 μl of firefly luciferase luminescent substrate (VivoGlo™ Luciferin, Promega, 15 mg / mL) into the experimental group (Ad7L-injected) and control mice, in vivo imaging was performed using a Tanon ABL-X6 small animal in vivo imaging system in chemiluminescence mode. At the experimental endpoint, blood was collected from the mice after in vivo imaging, and they were euthanized. Lung and tumor organs were harvested for imaging. Organ imaging should be completed within 30 minutes after the injection of the firefly luciferase luminescent substrate to prevent signal attenuation.

[0126] b. Mice in the experimental group and control group injected with rAd7E were subjected to in vivo imaging using a Tanon ABL-X6 small animal in vivo imaging system in fluorescence mode. At the end of the experiment, blood was collected from the mice, they were euthanized, and lung and tumor organs were harvested for imaging.

[0127] c. After grinding the collected lung and tumor organs, adenovirus genomic DNA was extracted using a viral genomic DNA / RNA extraction kit (Tiangen, DP315), and the virus was quantified by qPCR.

[0128] In vivo and organ imaging results on day 10 after Ad7L infection of tumor-forming mice are as follows: Figure 6 and Figure 7 As shown. In vivo and organ imaging results on day 10 after rAd7E infection of tumor-forming mice are as follows. Figure 8 and Figure 9 As shown. The viral load in the lungs and tumor organs of mice infected with Ad7L and rAd7E on day 10 is as follows. Figure 10 As shown.

[0129] Example 2: In vivo evaluation of the human adenovirus type 7 / 55 neutralizing antibody 3F11 using a xenograft mouse model.

[0130] The inventors previously prepared a humanized monoclonal antibody, 3F11, which can broadly neutralize Ad7 and Ad55 (XinguiTian, ​​Ye Fan, Zhenwei Liu, Ling Zhang, Jiayi Liao, Zhichao Zhou, Xiao Li, Tiantian Liu, Wenkuan Liu, Hongling Qiu and Rong Zhou. Broadly neutralizing monoclonal antibodies against human adenovirus types 55, 14p, 7, and 11 generated with recombinant type 11 fiber knob. Emerg Microbes Infect, 2018;7(1):206. doi: 10.1038 / s41426-018-0197-8.). This murine antibody was then humanized to obtain a humanized antibody with high neutralizing activity. This study attempts to evaluate its antiviral activity using a xenograft mouse model.

[0131] 1. Experimental Methods

[0132] a. Following the method for establishing the adenovirus-infected xenograft mouse model in Example 1, mice were injected with A549 cells to induce tumor formation. The number of tumor cells inoculated was 3.5 × 10⁻⁶. 6 Each mouse was individually identified as a tumor-forming mouse. Tumor-forming mice were grouped according to Table 2.

[0133] b. Mice were injected intraperitoneally with antibodies one day before and one day after inoculation with the virus, at a dose of 50 μg / 100 μl each time.

[0134] c. Each mouse was injected via tail vein with adenovirus Ad7L / Ad55E at a dose of 5 x 10⁻⁶ mg / L. 6 TCID 50 / 100μl.

[0135] d. In vivo imaging was performed on mice on day 3 and day 5 after viral inoculation.

[0136] e. On day 5 after mice were inoculated with the virus, in vivo imaging was performed, blood was collected, the mice were euthanized, and tumors were harvested for tumor organ imaging.

[0137] f. Tumor organs and mouse lung tissue were ground, and adenovirus genomic DNA was extracted using a viral genomic DNA / RNA extraction kit (Tiangen, DP315). The virus was quantified by qPCR.

[0138] Table 2. Experimental grouping of xenograft mouse models for evaluating human adenovirus neutralizing antibodies

[0139]

[0140] 2. Results:

[0141] There were no significant differences in body weight and tumor size among the groups of mice. Figure 11 and Figure 12 In the Ad7L virus group, obvious luminescent signals were observed in the tumors and tails of mice, and the signal value increased on day 5 compared to day 3 after virus injection; the signal value in the Ad7L+3F11 antibody group was significantly lower than that in mice injected with Ad7L virus only, and the signal value did not increase on day 5 compared to day 3; no visible signal value was observed in the PBS control group mice. Figure 13 ).

[0142] Imaging of the tumor samples showed that the tumors in the Ad7L virus group mice exhibited significant luminescent signal values, while the tumors in the Ad7L+3F11 antibody group mice showed no significant fluorescence signal, and the PBS control group mice showed no visible signal values. Figure 14 ); Quantification of the virus within the tumor showed that the viral load was significantly lower in the Ad7L+3F11 antibody group compared to the Ad7L virus group ( ). Figure 15 The results showed that the 3F11 antibody significantly inhibited the replication of Ad7L in the tumor.

[0143] The neutralizing effect of 3F11 on Ad55E was also investigated using the same experimental method. Ad55E is a replicative recombinant human adenovirus type 55 vector Ad55E with an EGFP expression cassette deleted from the E3 region and inserted (Zhang Q, Zhou Z, Fan Y, Liu T, Guo Y, Li X, Liu W, Zhou L, Yang Y, Mo C, Chen Y, Liao X, Zhou R, Ding Z, Tian X. 2024. Higher affinities of fibers with cell receptors increase the infection capacity and virulence of human adenovirus type 7 and type 55 compared to type 3. Microbiol Spectr 12:e01090-23. https: / / doi.org / 10.1128 / spectrum.01090-23). ​​No virus was detected in the tumor in the Ad55E+3F11 group, while high and low viral loads were detected in the Ad55E group (10). 7-10 8 Genome copies / mL Figure 16 The results showed that the 3F11 antibody significantly inhibited the replication of Ad55E within the tumor.

[0144] These results indicate that the A549 xenograft mouse model can be used for the evaluation or screening of antiviral drugs against human adenovirus types 7 and 55.

[0145] Example 3: Evaluation of attenuated adenovirus vaccine using a xenograft mouse model

[0146] The inventors previously prepared an adenovirus vaccine candidate strain, Ad7E-A5E4 (CN202311576004.X), using genetic engineering technology. Cell experiments showed that its ability to infect cells was significantly reduced compared to Ad7E. This study attempts to evaluate its virulence using a xenograft mouse model.

[0147] 1. Experimental Methods:

[0148] a. Following the method for establishing the adenovirus-infected xenograft mouse model in Example 2, mice were injected with A549 cells to induce tumor formation. The number of tumor cells inoculated was 3.5 × 10⁻⁶. 6 Each mouse was individually identified as a tumor-forming mouse. Tumor-forming mice were grouped according to Table 3.

[0149] b. Each mouse was injected intravenously with adenovirus Ad7E-A5E4 / Ad7E at a dose of 5 x 10⁻⁶. 6 TCID 50 / 100μl.

[0150] c. On day 5 after mice were inoculated with the virus, in vivo imaging was performed, blood was collected, the mice were sacrificed, and lung and tumor tissues were taken.

[0151] d. Tumor organs and mouse lung tissue were ground, and adenovirus genomic DNA was extracted using a viral genomic DNA / RNA extraction kit (Tiangen, DP315). The virus was quantified by qPCR.

[0152] Table 3. Experimental Grouping for Attenuated Human Adenovirus Vaccine

[0153]

[0154] 2. Results

[0155] The experimental results show that, for the qPCR assay to determine viral genome copy number, the viral load in the lungs and tumors of mice infected with Ad7E-A5E4 and rAd7E groups was comparable. Figures 17-18); however, for the IFU assay to determine infectious live viruses, the viral load in the hind lungs of Ad7E-A5E4 and Ad7E mice was comparable ( Figure 19 ), while the Ad7E-A5E4 infection group in the tumor was significantly lower than that in the Ad7E infection group ( Figure 20 ).

[0156] Example 4 Evaluation of the NCG mouse-A549-RFP cell lung xenograft-AdV infection model

[0157] I. Lung cancer cells A549-RFP instilled via nasal drops to induce lung tumor formation

[0158] Lung tumorigenesis experiments were conducted using 4-8 week old female NCG mice, with 4 × 10⁴ tumor cells inoculated. 6 Each / each

[0159] a. Cell Collection: Suspended cells were collected directly as a cell suspension. Adherent cells were digested with trypsin and then the cell suspension was collected. The collected cell suspension was centrifuged at 300×g for 5 minutes, washed twice with PBS, and the cells were resuspended in PBS and counted. Two intranasal injection methods were used: the first method used matrix gel to assist tumor formation, adjusting the cell density to 6.0×10⁻⁶. 7 Cells / mL (cell density 6.0 × 10⁶) 7 -1.0×10 8 (The concentration can be within the range of cells / mL), and it is mixed 1:1 with matrix gel (C0382 / C0383 or C0386 / C0387) to obtain a cell concentration of 3.0 × 10⁻⁶ cells / mL. 7 Cells / mL (cell density 3.0 × 10⁶) 7 -5.0×10 7 (The range of cells / mL is acceptable); the second method is to directly dilute the cells to a concentration of 3.0 × 10⁻⁶. 7 Cells / mL. Place cells on ice until ready to use. Note: Collected cells should be inoculated within 2 hours.

[0160] b. Mice were anesthetized with isoflurane and then injected intranasally with the prepared cell suspension containing or without matrix gel, typically 100 µl per mouse.

[0161] c. Mice treated with A549-RFP cells via intranasal instillation were fed for another 10 days, and some mice were sacrificed for lung and tumor tissue collection. The remaining mice were then intranasally infected with Ad7L adenovirus at a dose of 5 x 10⁻⁶ cells / mL. 6 TCID 50 / 100μl.

[0162] d. Nine days after nasal infection with Ad7L adenovirus, chemiluminescence in vivo imaging and tumor organ imaging were performed, and samples were collected from the lungs and tumors.

[0163] e. The lung and tumor tissue samples are sent for pathological sectioning.

[0164] f. Tumor organs and mouse lung tissue infected with Ad7L adenovirus were ground up, and adenovirus genomic DNA was extracted using a viral genomic DNA / RNA extraction kit (Tiangen, DP315). The virus was quantified by qPCR.

[0165] Depend on Figure 21 The HE slices of the lungs showed that the lungs of A549-RFP tumor-forming mice with and without matrix gel assisted (Figure 1-left) and without matrix gel assisted (Figure 2-middle) exhibited densely growing clusters of tumor cells (indicated by arrows), while the lungs of mice in the nasal PBS control group showed no corresponding characteristics (Figure 3-right).

[0166] Figure 22 DAPI staining showed that all nucleated cells and RFP fluorescence indicated the location of A549-RFP, while the green fluorescence of ADV-IF indicated the location of the virus. It can be seen that ADV virus, when administered via nasal drops, can only infect A549 cells and cannot infect mouse lung cells.

[0167] Figure 23 The experimental results in the upper right figure show that after mice with lung tumors caused by nasal drops of A549-RFP cells were infected with Ad7L virus, the in vivo imaging instrument may not have been sensitive enough, and no luminescent signal was observed in the in vivo imaging. However, luminescent signal was observed in the virus group in the lung organ imaging, while no luminescent signal was observed in the PBS control group.

[0168] Depend on Figure 24 It can be seen that in 2 out of 4 mice that underwent Matrigel-assisted genome sequencing, the Ad7L virus genome (10) could be detected. 5 -10 10 Genome copies / mL), 2 mice were not detected, indicating that A549-RFP tumors successfully formed in the lungs of 2 mice, while the other 2 mice failed to form tumors; Ad7L virus genome (10) was detected in all 3 mice that underwent direct intranasal tumor formation with A549-RFP. 5 -10 10 The number of *genome copies / mL* indicated successful tumor formation; no *genome* was detected in the PBS control group.

[0169] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing an animal model of adenovirus infection, characterized by, comprising the following steps: transplanting lung cancer cells or laryngeal cancer cells to the subcutaneous or lung of an animal to form a tumor, to construct a transplanted tumor animal model; and infecting the transplanted tumor animal model with adenovirus to construct an adenovirus infection animal model; wherein the lung cancer cells or laryngeal cancer cells are transplanted to the subcutaneous or lung of an animal by subcutaneous injection or nasal instillation.

2. The construction method of claim 1, wherein, The lung cancer cells include A549 cells, and / or the laryngeal cancer cells include Hep-2 cells.

3. The construction method according to claim 1 or 2, characterized in that, The animal includes an immunodeficient mouse. Optionally, the immunodeficient mouse includes an NCG mouse.

4. The construction method of claim 2, wherein, The step of transplanting lung cancer cells or laryngeal cancer cells to the subcutaneous of an animal by subcutaneous injection includes: The cell suspension of the lung cancer cells or the laryngeal cancer cells is mixed with Matrigel according to a volume ratio of 1:(0.8-1.2) to prepare a mixed solution; wherein the density of the lung cancer cells or the laryngeal cancer cells in the cell suspension is 5×10 7 6 / mL-2×10 8 6 / mL; and, injecting the mixed solution into the animal at an injection volume of 90 μL to 110 μL, and feeding the animal for 7 days to 21 days after injection to form a tumor.

5. The construction method of claim 2, wherein, The step of transplanting lung cancer cells or laryngeal cancer cells to the lung of an animal by nasal instillation includes: After intranasal instillation of a cell suspension of the aforementioned lung cancer cells or laryngeal cancer cells into animals, tumors formed after 7 to 21 days of feeding; wherein the density of the lung cancer cells or laryngeal cancer cells in the cell suspension was 3.0 × 10⁻⁶. 7 cells / mL ~ 5.0 × 10⁻⁶ 7 The dose per mL is 90 μL to 110 μL; or, The mixture of the cell suspension of the lung cancer cells or the laryngeal cancer cells and the Matrigel is instilled into the nasal cavity of the animals, and the animals are bred for 7-21 days to form tumors; wherein the cell density of the lung cancer cells or the laryngeal cancer cells in the mixture is 3.0×10 7 / mL-5.0×10 7 / mL, and the instillation dose is 90-110 μL.

6. The construction method according to claim 4 or 5, characterized in that, The Matrigel includes Matrix-Gel Matrigel, and the protein concentration of the Matrix-Gel Matrigel is 16 mg / mL to 26 mg / mL.

7. The construction method according to any one of claims 1-2 and 4-5, wherein, The infection includes intratumoral injection, intravenous injection, or nasal instillation; Optionally, the infectious dose is (4-6)*10 6 TCID 50 / (90 μL-100 μL).

8. The construction method according to any one of claims 1-2 and 4-5, wherein, The adenovirus includes one or more of HAdV-3, HAdV-7, and HAdV-55.

9. The construction method according to claim 8, characterized in that, The adenovirus is a replication-type recombinant human adenovirus, and the E3 region of the replication-type recombinant adenovirus is deleted and inserted with a reporter gene; Optionally, the reporter gene includes an enhanced green fluorescent protein reporter gene and a luciferase reporter gene.

10. The animal model constructed by the construction method of any one of claims 1-9 for use in any one of: (1) screening of adenovirus vaccines; (2) screening of anti-adenovirus drugs; and (3) adenovirus infection mechanism research.

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