Monoclonal antibodies with neutralizing activity against adenovirus type 5 and uses thereof

Abstract

The present invention belongs to the field of virus detection technology, and specifically relates to monoclonal antibodies with neutralizing activity against adenovirus type 5 and their uses. The monoclonal antibody 8B9 of the present invention can specifically recognize and neutralize adenovirus type 5, and the amino acid sequences of the heavy and light chain variable regions and their complementarity determining regions of the monoclonal antibody 8B9 are provided. The present invention also provides an in vitro neutralization activity assessment model based on HEK-293A cells. At a concentration as low as 3.3 μg / mL, the titer of the neutralizing activity against the virus stock solution after 6000-fold dilution is 7×10 4 TCID 50 / mL of virus has close to 100% neutralizing activity. After further humanization, it is expected to be developed into a neutralizing antibody drug for the treatment of diseases such as severe pneumonia caused by HADV-5 infection, filling the current lack of effective treatment options in this field and possessing significant scientific research value and clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of virus detection, and in particular relates to a monoclonal antibody with neutralizing activity against type 5 adenovirus and uses thereof. Background Art

[0002] Adenoviruses are non-enveloped, icosahedral, double-stranded DNA viruses that commonly invade multiple organs, including the respiratory, digestive, urinary, and conjunctival systems. Adenoviruses are prevalent year-round in the human population and can occur in all age groups. Outbreaks of adenovirus infection have occurred in numerous countries and regions worldwide. Currently, there is no effective treatment, posing a significant threat to public health, particularly in children and immunocompromised individuals. Adenoviruses that have been discovered to infect humans are divided into seven subgroups, A and B. Over 100 serotypes have been identified, including HADV-5, HADV6, and HADV41. Type 5 belongs to subgroup C and primarily causes respiratory infections. Adenoviruses can also infect a variety of animals, including canine adenovirus (CAV) and avian adenovirus. The risk of cross-species transmission poses a significant challenge to public safety.

[0003] The adenovirus genome is approximately 36 kb long, with an inverted terminal repeat (ITR) at each end, flanked by viral packaging signals. The genome contains four early transcription elements (E1, E2, E3, and E4) that perform regulatory functions, as well as a gene encoding structural proteins. E3 is non-essential for replication, and its deletion significantly increases the insertion capacity of exogenous genes. Adenovirus is a widely used biotechnology tool in gene therapy and vaccine development. The principle is to use replication-defective adenoviruses as vectors to deliver target genes into human cells to stimulate immune responses or repair genetic defects. In gene therapy, recombinant adenovirus vectors lacking E1 / E3 and E2 / E4 have been developed, capable of packaging exogenous genes up to 8-12 kb. Currently, adenovirus vectors are mostly based on adenovirus type 5 (Ad5) and adenovirus type 2 (Ad2), and a variety of genotypes have been developed. Due to their efficient transduction capacity, broad tissue tropism, and mature production processes, adenovirus vectors continue to attract attention in gene therapy, vaccine development, and tumor immunotherapy. For example, in the field of new coronavirus vaccines, many vaccines have been launched on the market, including CanSino's Ad5-nCoV, Johnson & Johnson's Ad26.COV2.S, and Oxford / AstraZeneca's ChAdOx1, which have verified the safety and effectiveness of adenovirus vectors.

[0004] Adenovirus type 5 (Ad5) plays a crucial role as a widely used gene therapy and vaccine vector. However, Ad5 itself possesses strong immunogenicity, and pre-existing immunity against it is prevalent in the population, potentially compromising vaccine efficacy and posing safety risks. Currently, there is a lack of highly effective and specific neutralizing antibodies against Ad5, which not only limits the development of vaccine immunological assessment tools but also hinders the development of therapeutic strategies for Ad5-associated diseases. Although some studies have reported on anti-advanced antibodies, most are polyclonal or polymorphically cross-reactive, with poor specificity and limited neutralizing capacity. Therefore, there is an urgent need to obtain monoclonal antibodies with high affinity and strong neutralizing activity against Ad5 for use in vaccine development, vector optimization evaluation, and potential passive immunotherapy. Summary of the Invention

[0005] Given the lack of highly effective, specific neutralizing monoclonal antibodies against adenovirus type 5 in the prior art, and the widespread existence of existing antibodies with strong cross-reactivity, low neutralization efficiency, and difficulty in standardized application, the present invention provides a monoclonal antibody with potent neutralizing activity against adenovirus type 5, thereby resolving the key challenge of the prior art in obtaining highly specific, high-affinity neutralizing antibodies against adenovirus type 5. This antibody can be used for immunological evaluation and vector optimization research of adenovirus vector vaccines, and possesses good neutralizing activity. After further humanization, it is expected to be developed into a neutralizing antibody drug for the treatment of diseases such as severe pneumonia caused by adenovirus type 5 infection, filling the current lack of effective treatment options in this field and possessing important scientific research value and clinical application prospects.

[0006] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0007] The present invention provides a monoclonal antibody having neutralizing activity against adenovirus type 5, wherein the monoclonal antibody is 8B9, and the monoclonal antibody 8B9 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the monoclonal antibody 8B9 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively;

[0008] The light chain variable region of the monoclonal antibody 8B9 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8B9 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8B9 is shown in SEQ ID NO.14.

[0010] In some embodiments, adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 8B9 is shown in SEQ ID NO.17.

[0012] In some embodiments, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 8B9 is shown in SEQ ID NO.18.

[0013] The present invention provides a use of the monoclonal antibody having neutralizing activity against adenovirus type 5 in the preparation of a medicament for preventing or treating adenovirus type 5 infection.

[0014] The present invention provides a use of the above-mentioned monoclonal antibody having neutralizing activity against type 5 adenovirus in the preparation of diagnostic reagents, test strips and kits for detecting type 5 adenovirus infection.

[0015] The present invention provides a use of the monoclonal antibody having neutralizing activity against adenovirus type 5 in studying the biological characteristics of adenovirus type 5.

[0016] The present invention provides a use of the monoclonal antibody having neutralizing activity against adenovirus type 5 in preparing a non-disease diagnosis test model for evaluating the in vitro neutralizing activity of the monoclonal antibody.

[0017] In some embodiments, the experimental model is established based on HEK-293A cells to detect the infection and replication process of adenovirus type 5 in cells and to evaluate the neutralizing effect of monoclonal antibodies.

[0018] The monoclonal antibody 8B9 provided by the present invention is highly specific and reacts only to adenovirus type 5. The antibody demonstrated excellent neutralization ability in in vitro neutralization tests and was able to effectively inhibit viral replication in HEK-293A cells even under high viral load conditions. Experimental results showed that when the concentration of monoclonal antibody 8B9 was 3.3 μg / mL, the titer of the virus stock solution after 6000-fold dilution was 7×10 4 TCID 50 The antibody has nearly 100% neutralizing activity against 100% of the virus per mL, demonstrating extremely high neutralizing activity and stability. The present invention also provides an in vitro neutralization activity assessment model based on the antibody. This model uses HEK-293A cells as an infection system and combines cytopathic effect (CPE) observation with double-antibody sandwich ELISA virus quantitative detection. This model can efficiently, stably, and reproducibly assess the neutralizing effect of the antibody, making it suitable for research in areas such as vaccine development, vector optimization, and neutralizing antibody drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0020] Figure 1 The test results of neutralizing monoclonal antibodies inhibiting viral replication (#1-8);

[0021] Figure 2 The results of the neutralizing monoclonal antibody test on the inhibition of viral replication (9-16);

[0022] Figure 3 The results of the neutralizing monoclonal antibody test on the inhibition of viral replication (#17-25);

[0023] Figure 4 This is a comparison of the neutralizing effects of monoclonal antibody 8B9 and monoclonal antibody 7C9;

[0024] Figure 5 This is a graph showing the neutralization effect of monoclonal antibody 8B9 on a 6000-fold diluted virus;

[0025] Figure 6 This is the test result of the monoclonal antibody 8B9's inhibition of viral replication;

[0026] Figure 7 This is a diagram showing the neutralization effect of monoclonal antibody 8B9. DETAILED DESCRIPTION

[0027] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0028] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0029] Example 1

[0030] 1. Mouse immunization

[0031] First dilute HADV-5 to 1x10 8 4-6-week-old female Balb / C mice were immunized with VP / ml at a dose of 200 μl / mouse. For the first immunization, HADV-5 was mixed and emulsified with an equal volume of Freund's complete adjuvant and administered subcutaneously at multiple sites. Subsequent booster immunizations were administered two weeks apart using the same dose of HADV-5 mixed with an equal volume of Freund's incomplete adjuvant. Serum was collected after the third immunization for titer determination. Mice with the highest titer were intraperitoneally challenged with HADV-5 Hexon protein (Abcam, ab123995) at a dose of 20 μg / mouse. Three days later, spleen cells were harvested for fusion. HADV-5 was obtained by isolation and culture.

[0032] 2. Screening of hybridoma cell lines

[0033] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and cultured in HAT medium for selection. When the fused cells reached half the bottom of the well, clones positive for HADV-5 and HADV-5 Hexon proteins were screened by indirect ELISA. Positive cells were cloned to monoclonal status by limiting dilution, and the cell lines were expanded and cryopreserved.

[0034] 3. Screening of positive clones by indirect ELISA:

[0035] HADV-5 Hexon protein and diluted purified HADV-5 virus (1 x 10 12 VP / ml), (coating buffer carbonate buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, fixed to 1L pure water), coating concentration was 1μg / mL and 500-fold dilution, respectively, incubated at 4℃ overnight; 1% BSA blocking, 150μL per well, blocked at 37℃ for 2 hours, washed once with washing solution, patted dry; added 50μL hybridoma cell culture supernatant, reacted at 37℃ for 30min. The liquid in the wells was discarded, and the plates were washed four times with PBST (0.01 M PBS, 0.1% Tween 20, pH 7.4). After patting dry, 50 μL / well of 5000-fold diluted HRP-labeled goat anti-mouse secondary antibody (Beijing Solebao, SE131) was added. The reaction was carried out at 37°C for 30 min. The plates were washed four more times, patted dry, and 50 μL / well of TMB color development solution (single-component color development solution, Beijing Meikewande Biological, 1001) was added for color development at room temperature for 10 min. Finally, 50 μL of TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction, and the OD was measured using a microplate reader. 450 The positive cell lines that reacted with both HADV-5 Hexon and HADV-5 virus were selected for subsequent experiments.

[0036] 4. Preparation of Monoclonal Antibody Ascites

[0037] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Female Balb / C mice (100 cells) were pretreated with Freund's incomplete adjuvant. About 10 days later, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle.

[0038] 5. Affinity chromatography purification of monoclonal antibodies

[0039] Ascites was centrifuged at 12,000 rpm for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4) and filtered through a 0.22 μm filter. The filtered sample was pumped through a Protein L purification column equilibrated with binding buffer at a flow rate of 1 ml / min using a peristaltic pump. The column was then connected to a protein purifier and washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled out. The column was then eluted with elution buffer (0.1 M glycine, pH 2.7). The eluted peak was collected and adjusted to neutral with 1 M Tris-HCl, pH 9. The sample was placed in a dialysis bag (MW: 8,000-14,000) and dialyzed against 20 mM PBS, pH 7.4, at 2-8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody. The protein concentration of the purified monoclonal antibody was determined using an ultra-micro spectrophotometer at a wavelength of 280 nm and the antibody was aliquoted for storage.

[0040] 6. Identification of Monoclonal Antibodies

[0041] Referring to the above-mentioned indirect ELISA method, the purified viruses of HADV-2, HADV-3, HADV-6, and HADV-7 were diluted to the same concentration as HADV-5 (the purified viruses were all at 1 x 10 12 VP / ml -2 x 10 12 VP / ml) for coating, and Hep2 cell supernatant was coated as a control antigen. Referring to the above-mentioned ELISA method, the purified monoclonal antibody was diluted to a concentration of 1 μg / ml, and its reactivity with the above-mentioned five types of adenovirus and Hep-2 cells was tested. The monoclonal antibody strain that specifically reacted only with HADV-5 was screened for subsequent neutralization tests.

[0042] Example 2

[0043] The replication-deficient recombinant adenovirus type 5 originates from Beijing Wujiahe Gene Technology Co., Ltd., and the human embryonic kidney HEK-293A cells are maintained by the company. While HADV can normally infect Hep-2 cells, replication-deficient recombinant adenovirus type 5 (rADV) typically lacks the E1 gene and cannot replicate in ordinary cells, only in cell lines specifically expressing the E1 protein. 293A cells provide the E1 protein required for replication of the E1-deficient recombinant adenovirus, enabling efficient infection and replication.

[0044] 1. Virus TCID 50 Titer determination

[0045] 1) HEK-293 cells were grown in cell growth medium until 80-90% confluency was achieved. After digestion with 0.25% EDTA-trypsin, single-cell suspension was prepared and the cells were counted.

[0046] 2) Cell suspension was prepared by adding 2% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin double antibody solution to DMEM basal medium, with a cell density of 1-2 x 10 5 Prepare cell suspensions suitable for viral infection, use low-serum medium (to reduce interference), and ensure good cell viability.

[0047] 3) Place 100 μl of cell suspension into each well of two 96-well cell culture plates.

[0048] 4) Take the virus to be tested and dilute it with virus diluent from 10 -3 Start with a 10-fold serial dilution to make 10 -4 , 10 -5 …………10 -11 , 10 -12 (1 ml of each dilution will be used for subsequent inoculation).

[0049] 5) Add 100 μl of virus-free culture medium (same as above: DMEM + 2% fetal bovine serum + 1% penicillin-streptomycin) to each well in columns 11 and 12 of the 96-well plate where the cells were plated as a cell control.

[0050] 6) Add 100 μl of serially diluted virus sample to each well in columns 1-10 of the 96-well plate where the cells have been plated, with 8 wells in each column representing one virus dilution.

[0051] 7) After plating, place two 96-well plates in a 37°C, 5% CO2 incubator.

[0052] 8) Observe the cytopathic effect (CPE) under the microscope every day. On the 10th day, observe and record the number of CPE-positive wells at each dilution and calculate the TCID 50 .

[0053] For 100 μl sample, the titer is T = 10 1+d（s-0.5） .

[0054] d = log10 稀释度 =1 (for a 10-fold dilution).

[0055] s = sum of positive ratios (starting from the first 10-fold dilution).

[0056] According to the titration results, the virus titer was 4.2×10 8 TCID 50 / mL. This indicates that the virus has a high infectious activity, ensuring effective interaction between the virus and antibodies in the experiment, improving detection sensitivity and the reliability of the results. When evaluating neutralizing antibodies, high virus titers help to more accurately reflect the neutralizing ability of antibodies, especially when faced with high viral loads. They can better simulate the real infection environment and thus screen for truly effective neutralizing antibodies.

[0057] 2. Virus neutralization test

[0058] The cytopathic effect (CPE) can be directly observed, facilitating the initial screening of monoclonal antibody strains that can inhibit viral cytopathic effect. At the same time, the virus content in the culture supernatant can be determined using the established double-antibody sandwich assay to compare the neutralization effects of different antibodies. The specific steps of the neutralization test are as follows:

[0059] 1) Preparation of 293A cells: 293A cells were cultured to the logarithmic growth phase in advance, digested with trypsin, and then dispersed. 96-well cell culture plates were plated with DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin medium at a density of 1-2 × 10 5 / ml, and culture in a 37°C, 5% CO2 incubator until the cells adhere to the wall and are ready for use (plating is performed according to experimental needs).

[0060] 2) Prepare the virus to be tested: dilute the recombinant adenovirus in blank DMEM medium at different times for later use.

[0061] 3) Incubation of monoclonal antibodies with viruses: Neutralizing monoclonal antibodies must be sterile. Take 30ul of the virus to be tested and 30ul of diluted monoclonal antibodies (100ug / ml) in a sterilized EP tube, mix thoroughly, and incubate at 37°C for 1h.

[0062] 4) Infection: The supernatant of the adhered cells was discarded, and the virus-monoclonal antibody mixture was added to each well at 20 μl / well. Subsequently, 200 μL of blank DMEM + 1% penicillin-streptomycin medium was added to each well.

[0063] 5) Control settings: In virus neutralization tests, to ensure the accuracy and comparability of experimental results, each test must have three sets of controls: cell control, monoclonal antibody control, and virus control. Among them, 200uL blank culture medium (DMEM + 1% penicillin-streptomycin) was added to each well of the cell control well, without adding virus and antibody, to evaluate the normal growth status of cells under virus-free infection conditions; the monoclonal antibody control used an equal concentration of irrelevant monoclonal antibody (monoclonal antibody against enterovirus EVD68, with a concentration of 100 μg / mL). The operation method was the same as that of the monoclonal antibody to be tested: 30μL of diluted virus (select the multiple concentration corresponding to step 2) was mixed with 30μL of irrelevant control monoclonal antibody, incubated at 37℃ for 1 hour, and then 20μL of the mixture was added to the well plate, and 200uL of blank DMEM + 1% penicillin-streptomycin medium was added to eliminate the influence of nonspecific antibodies on virus replication; virus control wells were only added with virus without adding antibody, that is, 30μL of diluted virus was incubated with 30uL DMEM blank medium, and 20μL of it was added to the well plate and 200uL of culture medium was added to reflect the infection and replication level of the virus in the absence of antibody interference. Through the reasonable setting of the above three groups of controls, the neutralizing effect of the tested monoclonal antibody on adenovirus type 5 can be effectively verified, and non-specific interference factors in the experiment can be eliminated.

[0064] 6) Place the culture plate in a 37°C, 5% CO2 incubator and observe for CPE daily. Measure the viral content in the culture supernatant at 24, 48, 72, 96, 120, and 144 hours after infection.

[0065] 3. Double Antibody Sandwich ELISA Detection

[0066] HRP labeling of monoclonal antibody 4A6:

[0067] Specifically, dilute the labeled antibody to a final concentration of 2 mg / mL in carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6). Dissolve 2 mg HRP in 0.5 mL ultrapure water and mix thoroughly with 0.5 mL of 0.06 M sodium periodate solution. Add 1 mg of the diluted antibody solution to the HRP tube and pipette to mix thoroughly. Incubate at room temperature for 1 hour, mixing regularly during the incubation period. Terminate the labeling reaction by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 minutes. Finally, dialyze the labeled antibody overnight against 0.01 M PBS, pH 7.4. Add glycerol at a 1:1 ratio and store in aliquots at -20°C.

[0068] Test steps:

[0069] Step 1) Coating with Antibodies

[0070] Monoclonal antibody 8B9 was diluted with coating buffer at a concentration of 1 μg / mL, 50 μL was added to each well of the ELISA plate, and incubated at 4°C overnight.

[0071] Step 2) Closure

[0072] After incubation overnight, discard the coating solution and add 150 μL of 1-2% BSA blocking solution to each well and incubate at 37°C for 2 hours.

[0073] Step 3) Add sample

[0074] After discarding the blocking solution and patting dry, add 50 μL of the cell culture supernatant to be tested to each well and incubate at 37°C for 35 minutes.

[0075] Step 4) Wash

[0076] Wash the plate 4 times with PBST, patting dry thoroughly each time.

[0077] Step 5) Add HRP-labeled antibody

[0078] 4A6 monoclonal antibody-HRP labeled antibody was diluted 4000 times with PBS, 50 μL was added to each well, and incubated at 37°C for 35 minutes.

[0079] Step 5) Wash again

[0080] Wash the plate 4 times with PBST, patting dry thoroughly each time.

[0081] Step 6) Color development and reaction termination

[0082] Add 50 μL of TMB color development solution to each well, develop color at room temperature for 10 minutes, and then add 50 μL of TMB stop solution to each well to terminate the color development reaction.

[0083] Step 7) Read the OD value

[0084] The absorbance (OD value) of each well was read using a microplate reader at a wavelength of 450 nm.

[0085] 4. Neutralization efficiency calculation and results

[0086] Viruses and monoclonal antibodies are co-incubated and then infected with sensitive 293 cells. If the virus is neutralized, the cells will show no or minimal lesions. Observing CPE can determine differences in neutralization efficacy, offering advantages such as ease of use and intuitive results. Furthermore, combining ELISA with viral content in the supernatant can more accurately determine the neutralization efficiency of the monoclonal antibody against the virus, facilitating standardized and digitized analysis of experimental results.

[0087] The formula for calculating the neutralization efficiency of monoclonal antibodies is:

[0088] Neutralization efficiency (100%) = (OD of virus control) 450 -Detection hole OD 450 ) / (Virus control OD 450 -Cell control OD 450 ) × 100%. In the virus neutralization test, the monoclonal antibody-virus infection well in step 4 corresponds to the detection well OD 450 Data, cell control wells in step 5 correspond to cell control OD 450 Data, virus control wells correspond to virus control OD 450 data.

[0089] If the monoclonal antibody has a neutralizing effect on the virus, the virus replicates slowly or not at all, and the amount of adenovirus that can be detected in the culture supernatant is small. 450 The value is low, and the above formula can be used to determine the neutralization efficiency of monoclonal antibodies against viruses under certain concentration conditions.

[0090] See attached Figure 1 , Attachment Figure 2 and attached Figure 3 The 25 monoclonal antibodies screened were compared with 10 -4 The diluted virus mixture was incubated and infected with 293A cells. The virus replication after infection was continuously monitored. Monoclonal antibody control (enterovirus EVD-68 monoclonal antibody), cell control and virus control were also set up. The better neutralizing monoclonal antibody can effectively inhibit or delay the replication of the virus. The cells will show lesions later or the degree of lesions is not obvious. Further detection shows that the virus replicates slowly or not at all, and the detection reading is low. The test results show that the OD value of the cell control group is 2.377 / 3.03 during the whole experiment. 450 The value remained at a low level with almost no change, which indicated that the 293A cells not infected by the virus did not produce virus-related antigens or products under normal culture conditions, so the OD 450 The value is very low and stable. The virus control group begins to show obvious OD 24 hours after infection. 450 This indicates that in cells infected with virus but not treated with monoclonal antibodies, the virus can replicate effectively and produce a large amount of virus-related substances, resulting in an increase in OD 450 The monoclonal antibody control group used an unrelated monoclonal antibody against enterovirus EVD-68, and its OD 450The trend of the values ​​was similar to that of the virus control group, indicating that the unrelated monoclonal antibodies had no specific neutralizing effect on the target virus, allowing the virus to replicate in cells. Monoclonal antibodies 1# (8B9) and 12# (7C9) significantly inhibited lesions, with no viral replication detected 144 hours after infection. This indicates that the two monoclonal antibodies, 8B9 and 7C9, have a very strong neutralizing effect on the target virus and can effectively prevent viral replication in cells for a long time.

[0091] 5. Determination of neutralizing monoclonal antibodies

[0092] The monoclonal antibodies with good neutralizing effect were selected and subjected to high titer virus infection neutralization test. The recombinant adenovirus was diluted 10 times and 100 times for neutralization test. The cell pathological changes were observed under a microscope. The virus titers after dilution were 4.2×10 7 TCID 50 / mL and 4.2×10 6 TCID 50 / mL. Due to the high virus titer, the concentration of neutralizing antibodies was appropriately increased during the experiment. See the attached Figure 4 The results showed that on the third day after infection, the negative control antibody (monoclonal antibody against enterovirus EVD-68) failed to neutralize the virus, allowing cells to become infected and develop pathological changes. The cell control group, which did not receive any treatment, showed normal cell growth and no pathological changes. Monoclonal antibody 8B9, at a concentration of 850 μg / ml, significantly inhibited infection with 10- and 100-fold diluted virus, with significantly less cytopathic effects compared to the virus control, demonstrating a more pronounced neutralizing effect than monoclonal antibody 7C9, confirming monoclonal antibody 8B9 as the optimal neutralizing monoclonal antibody.

[0093] The neutralizing effect of monoclonal antibody 7C9 is not as good as that of monoclonal antibody 8B9, therefore, the corresponding sequence is not given in the present invention.

[0094] 6. Evaluation of the neutralization efficiency of monoclonal antibody 8B9

[0095] See also Figure 5-7 , monoclonal antibody 8B9 was effective against 6000-fold diluted virus (titer of 7×10 4 TCID 50 / mL) in vitro neutralization test results of 293A sensitive cell line showed that: on the 5th day after infection, the cells in the test wells with monoclonal antibody at a concentration of 7ug / ml still had no CPE, and the virus content detected by ELISA was extremely low, indicating that the virus was unable to effectively replicate after being neutralized by monoclonal antibody. Through the calculation of neutralization efficiency, monoclonal antibody at a concentration of 3.3ug / ml or above had a titer of 7×10 4 TCID 50 / mL virus has a neutralization efficiency close to 100%. Figure 7 ctrl is the virus self-control, that is, the natural replication of the virus in the cell without adding any antibodies.

[0096] 7. Gene sequence

[0097] Monoclonal antibody variable region gene cloning and sequencing.

[0098] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0099] The specific sequence is as follows:

[0100] The amino acids of the heavy chain complementary determining region of monoclonal antibody 8B9 are as follows: CDR-H1 (SEQ ID NO.1): SYYMH; CDR-H2 (SEQ ID NO.2): YLNPVNDSTKYMEKFKG; CDR-H3 (SEQ ID NO.3): AIYESYYGY.

[0101] The amino acids of the light chain complementary determining regions of monoclonal antibody 8B9 are as follows: CDR-L1 (SEQ ID NO.4): KASKRVSISGYSYMH; CDR-L2 (SEQ ID NO.5): LASNLLG; CDR-L3 (SEQ ID NO.6): QHGRLLPST.

[0102] The amino acids of the heavy chain complementary determining regions of monoclonal antibody 4A6 are as follows: CDR-H1 (SEQ ID NO. 7): GSVFK; CDR-H2 (SEQ ID NO. 8): ELSPVIDSTSVLERAQQ; CDR-H3 (SEQ ID NO. 9): MLKSVISV.

[0103] The amino acids of the light chain complementary determining regions of monoclonal antibody 4A6 are as follows: CDR-L1 (SEQ ID NO.10): FARKSVLLSRVSYHI; CDR-L2 (SEQ ID NO.11): ETSVVG; CDR-L3 (SEQ ID NO.12): MHSGYLPST.

[0104] The amino acid sequence of the heavy chain variable region of monoclonal antibody 8B9 (SEQ ID NO. 13): EVKLEESGPELVKPGASVKMSCKASGYTFTSYYMHWVKQKPGQGLEWIGYLNPVNDSTKYMEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARAIYESYYGYWGQGTTLTVSA.

[0105] The amino acid sequence of the light chain variable region of monoclonal antibody 8B9 (SEQ ID NO. 14): DIQMNQSPASLAVSLGQRATISCKASKRVSISGYSYMHWYQQKPGQPPKLLIYLASNLLGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHGRLLPSTFGGGTKLELKRTV.

[0106] The amino acid sequence of the heavy chain variable region of 4A6 (SEQ ID NO. 15): EVMLAESGPELVKPGASVKMSCKASGYTFTGSVFKWVKQKPGQGLEWIGELSPVIDSTSVLERAQQKATLTSDKSSSTAYMELSSLTSEDSAVYYCARMLKSVISVWGQGTTLTVSS.

[0107] The amino acid sequence of the light chain variable region of monoclonal antibody 4A6 (SEQ ID NO. 16): EIVLTQSPASLAVSLGQRATISCFARKSVLLSRVSYHIWYQQKPGQPPKLLIYETSVVGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMHSGYLPSTFGGGTKLEIKRTV.

[0108] Nucleotide sequence of the heavy chain variable region of monoclonal antibody 8B9 (SEQ ID NO.17): GAGGTGAAGCTGGAGGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCTACCTGAACCCCGTGAACGACAGCACCAAGTACATGGAGAAGTTCAAGGGCAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGCCATCTACGAGAGCTACTACGGCTACTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC。

[0109] Nucleotide sequence of the light chain variable region of monoclonal antibody 8B9 is as follows (SEQ ID NO.18): GACATCCAGATGAACCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAAGGCCAGCAAGAGGGTGAGCATCAGCGGCTACAGCTACATGCACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACCTGGCCAGCAACCTGCTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCCAGCACGGCAGGCTGCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0110] Nucleotide sequence of the heavy chain variable region of monoclonal antibody 4A6 (SEQ ID NO.19): GAGGTGATGCTGCCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCAGCGTGTTCAAGTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGCTGAGCCCCGTGATCGACAGCA CCAGCGTGCTGGAGAGGGCCCAGCAGAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGATGCTGAAGAGCGTGATCAGCGTGTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC.

[0111] Nucleotide sequence of the variable region of the light chain of monoclonal antibody 4A6 (SEQ ID NO.20): GAGATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCTTCGCCAGGAAGAGCGTGCTGCTGAGCAGGGTGAGCTACCACATCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCCAAGCTGCTGATCTACGAGACCA GCGTGGTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCATGCACAGCGGCTACCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0112] The present invention provides a highly effective neutralizing monoclonal antibody, 8B9, against adenovirus type 5. It possesses high affinity and strong neutralizing activity, effectively inhibiting adenovirus type 5 infection and replication. This antibody was obtained through hybridoma screening, and its variable region gene sequencing and neutralization efficacy evaluation have been completed. It demonstrates broad application prospects in vaccine development, vector optimization, and potential passive immunotherapy. This achievement fills the current lack of specific neutralizing monoclonal antibodies against adenovirus type 5, providing important tools and technical support for the research, prevention, and treatment of adenovirus-related diseases.

[0113] So far, the various embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0114] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody having neutralizing activity against adenovirus type 5, characterized in that: The monoclonal antibody is 8B9, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region of the monoclonal antibody 8B9 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 8B9 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

2. The monoclonal antibody having neutralizing activity against adenovirus type 5 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8B9 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8B9 is shown in SEQ ID NO.

14.

3. The monoclonal antibody having neutralizing activity against adenovirus type 5 according to claim 2, characterized in that The adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

4. The monoclonal antibody having neutralizing activity against adenovirus type 5 according to claim 2, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8B9 is shown in SEQ ID NO.

17.

5. The monoclonal antibody having neutralizing activity against adenovirus type 5 according to claim 2, characterized in that The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8B9 is shown in SEQ ID NO.

18.

6. Use of the monoclonal antibody having neutralizing activity against adenovirus type 5 according to any one of claims 1 to 2 in the preparation of a medicament for preventing or treating adenovirus type 5 infection.

7. Use of the monoclonal antibody having neutralizing activity against adenovirus type 5 according to any one of claims 1 to 2 in the preparation of diagnostic reagents, test strips and kits for detecting adenovirus type 5 infection.

8. Use of the monoclonal antibody having neutralizing activity against adenovirus type 5 according to any one of claims 1 to 2 in preparing a non-disease diagnosis test model for evaluating the in vitro neutralizing activity of the monoclonal antibody.

9. The use according to claim 8, characterized in that The experimental model is established based on HEK-293A cells and is used to detect the infection and replication process of adenovirus type 5 in cells and to evaluate the neutralizing effect of the monoclonal antibody.

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