Monoclonal antibody with neutralizing activity on type-5 adenovirus and application thereof

Abstract

The invention belongs to the technical field of virus detection, and particularly relates to a monoclonal antibody with neutralizing activity on type-5 adenovirus and application of the monoclonal antibody. The monoclonal antibody 8B9 disclosed by the invention can specifically recognize and neutralize type 5 adenovirus, and provides amino acid sequences of variable regions of a heavy chain and a light chain of the monoclonal antibody 8B9 and complementary determining regions of the amino acid sequences. The invention further provides an in-vitro neutralization activity evaluation model based on the HEK-293A cell, and the model has the neutralization activity close to 100% for viruses with the titer of 7 * 10 < 4 > TCID50 / mL obtained after a virus stock solution is diluted by 6000 times under the concentration as low as 3.3 mu g / mL. After further humanized transformation, the antibody is expected to be developed into a neutralizing antibody drug for treating severe pneumonia and other diseases caused by HADV-5 infection, the blank of lack of effective treatment means in the field at present is filled, and the antibody has important scientific research value and clinical application prospect.

Description

Technical Field

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

[0002] Adenovirus is an icosahedral double-stranded DNA virus without an envelope, which often invades multiple organs such as the respiratory system, digestive system, urinary system, and conjunctiva. Adenovirus is prevalent throughout the year in the population and can occur in any age group. Outbreaks of adenovirus infection have occurred in many countries and regions around the world. Currently, there is no specific treatment method, posing a great threat to public health, especially children and immunocompromised populations. Among the adenoviruses that have been found to infect humans, they can be divided into 7 subgroups A-G, and more than 100 serotypes have been identified so far (such as HADV-5, HADV6, HADV41, etc.). Among them, type 5 belongs to subgroup C and mainly causes respiratory infections. In addition, many animals can also be infected with adenoviruses such as canine adenovirus (CAV) and avian adenovirus. The risk of cross-species transmission of the virus also poses a significant challenge to public safety.

[0003] The adenovirus genome is about 36 kb in length, with an inverted terminal repeat region (ITR) at each end. The viral packaging signal is located inside the ITR. Four early transcription units (E1, E2, E3, E4) responsible for regulatory functions are distributed on the genome, as well as a gene responsible for encoding structural proteins. Among them, E3 is a non-essential region for replication, and its deletion can greatly increase the insertion capacity of foreign genes. As a biotechnology tool widely used in gene therapy and vaccine research and development, the principle of adenovirus is to use a replication-defective adenovirus as a vector to deliver the target gene to human cells to stimulate an immune response or repair gene defects. In the field of gene therapy, recombinant adenovirus vectors lacking E1 / E3 and E2 / E4 have been developed, which can package foreign genes up to 8-12 kb. Currently, most adenovirus vectors are based on adenovirus type 5 (Ad5) and adenovirus type 2 (Ad2), and multiple genotypes have been developed. Due to its high transduction ability, broad tissue tropism, and mature preparation process, adenovirus vectors have continued to receive attention in the fields of gene therapy, vaccine development, and tumor immunotherapy. For example, in the field of COVID-19 vaccines, several vaccines have been launched, such as Ad5-nCoV developed by CanSino Biologics, Ad26.COV2.S developed by Johnson & Johnson, and ChAdOx1 developed by Oxford / AstraZeneca, which have verified the safety and effectiveness of adenovirus vectors.

[0004] Adenovirus type 5 (Ad5) has played an important role in widely used gene therapy and vaccine vectors. However, adenovirus type 5 itself has strong immunogenicity, and pre-existing immunity to it is widespread in the population, which may affect the vaccine effect and pose safety hazards. Currently, there is a lack of highly efficient and specific neutralizing antibodies against adenovirus type 5, which not only restricts the development of vaccine immunological evaluation methods but also hinders the development of treatment strategies for adenovirus type 5-related diseases. Although there have been some research reports on anti-adenovirus antibodies, most of them are polyclonal antibodies or multi-type cross-reactive antibodies, with poor specificity and limited neutralizing ability. Therefore, there is an urgent need to obtain a monoclonal antibody with high affinity and strong neutralizing activity against adenovirus type 5 for vaccine development research, vector optimization evaluation, and potential passive immunotherapy. Summary of the Invention

[0005] In view of the lack of highly efficient and specific neutralizing monoclonal antibodies against adenovirus type 5 in the prior art, and the existing antibodies generally have problems such as 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, thus solving the key problem of the inability to obtain highly specific and high-affinity anti-adenovirus type 5 neutralizing antibodies in the prior art. This antibody can be used for the immunological evaluation of adenovirus vector vaccines and vector optimization research, and at the same time has good neutralizing activity. After further humanization, it is expected to be developed into a neutralizing antibody drug for the treatment of severe pneumonia and other diseases caused by adenovirus type 5 infection, filling the gap in the lack of effective treatment means in this field and having important scientific research value and clinical application prospects.

[0006] To achieve the above object, the main technical solutions adopted by the present invention include: The present invention provides a monoclonal antibody with neutralizing activity against adenovirus type 5. The monoclonal antibody is 8B9. The monoclonal antibody 8B9 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region of the monoclonal antibody 8B9 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are respectively shown as SEQ ID NO.1 - SEQ ID NO.3; 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 respectively shown as SEQ ID NO.4 - SEQ ID NO.6.

[0007] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8B9 is shown as SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8B9 is shown as SEQ ID NO.14.

[0008] In some embodiments, adenovirus type 5 includes HADV-5 and replication-deficient recombinant adenovirus type 5.

[0009] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8B9 is as shown in SEQ ID NO. 17.

[0010] In some embodiments, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8B9 is as shown in SEQ ID NO. 18.

[0011] The present invention provides the use of the above-mentioned monoclonal antibody having neutralizing activity against adenovirus type 5 in the preparation of a medicament for preventing or treating adenovirus type 5 infection.

[0012] The present invention provides the use of the above-mentioned monoclonal antibody having neutralizing activity against adenovirus type 5 in the preparation of diagnostic reagents, test strips and kits for detecting adenovirus type 5 infection.

[0013] The present invention provides the use of the above-mentioned monoclonal antibody having neutralizing activity against adenovirus type 5 in the study of the biological characteristics of adenovirus type 5.

[0014] The present invention provides the use of the above-mentioned monoclonal antibody having neutralizing activity against adenovirus type 5 in the preparation of a test model for non-diagnostic purposes of evaluating the in vitro neutralizing activity of monoclonal antibodies.

[0015] In some embodiments, the test 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 neutralization effect of monoclonal antibodies.

[0016] The monoclonal antibody 8B9 provided by the present invention has high specificity and only reacts with adenovirus type 5. This antibody shows excellent neutralizing ability in in vitro neutralization tests and can effectively inhibit the replication of the virus in HEK-293A cells even under high virus load conditions. Experimental results show that when the concentration of the monoclonal antibody 8B9 is 3.3 μg / mL, it has nearly 100% neutralizing activity against the virus with a titer of 7×10 4 TCID 50 / mL. It shows extremely high neutralizing activity and stability. The present invention also provides an in vitro neutralizing activity evaluation model established based on this antibody. This model uses HEK-293A cells as the infection system, combines the observation of cytopathic effect (CPE) and the quantitative detection of virus by double antibody sandwich ELISA, and can efficiently, stably and reproducibly evaluate the neutralization effect of antibodies, and is applicable to research fields such as vaccine development, vector optimization and neutralizing antibody drug screening. Brief Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0018] Figure 1 Detection results of the inhibition of virus replication by neutralizing monoclonal antibodies (1 - 8#); Figure 2 Detection results of the inhibition of virus replication by neutralizing monoclonal antibodies (9 - 16#); Figure 3 Detection results of the inhibition of virus replication by neutralizing monoclonal antibodies (17 - 25#); Figure 4 Comparison chart of the neutralization effects of monoclonal antibody 8B9 and monoclonal antibody 7C9; Figure 5 Neutralization effect diagram of monoclonal antibody 8B9 on the virus diluted 6000 times; Figure 6 Detection results of the inhibition of virus replication by monoclonal antibody 8B9; Figure 7 Neutralization effect diagram of monoclonal antibody 8B9. Specific embodiments

[0019] The following further describes the embodiments of the present application in detail in conjunction with the accompanying drawings and examples. The detailed description and accompanying drawings of the following examples are used to exemplarily illustrate the principle of the present application, but cannot be used to limit the scope of the present application. The present application can be implemented in many different forms, not limited to the specific embodiments disclosed in the text, but including all technical solutions falling within the scope of the claims.

[0020] These embodiments of the present application are provided to make the present application thorough and complete, and to fully express the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, the components of materials, numerical expressions and values described in these embodiments should be interpreted as merely exemplary, rather than as limitations.

[0021] Example 1 1. Mouse immunization First, dilute with HADV - 5 to 1x10 8Female Balb / C mice aged 4 - 6 weeks were immunized with a dose of 200 μl / mouse of VP / ml. At the first immunization, it was mixed and emulsified with an equal volume of Freund's complete adjuvant and immunized subcutaneously at multiple points. For the subsequent second and third booster immunizations, the same dose of HADV-5 was mixed and emulsified with an equal volume of Freund's incomplete adjuvant, with an interval of 2 weeks each time. After the third immunization, sera were collected to measure the titer. Mice with higher titers were boosted intraperitoneally with 20 μg / mouse of HADV-5 Hexon protein (Abcam, ab123995). Three days later, spleen cells were taken for fusion. HADV-5 is a whole virus obtained by isolation and culture.

[0022] 2. Screening of hybridoma cell lines After fusing all the spleen cells of immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, they were placed in HAT medium for screening culture. When the fused cells grew to half of the bottom of the well, positive clones of HADV-5 and HADV-5 Hexon protein were screened by indirect ELISA. The positive cells were cloned to the monoclonal state by limiting dilution method, and then the cell lines were expanded and cryopreserved.

[0023] 3. Screening of positive clones by indirect ELISA: HADV-5 Hexon protein and diluted purified HADV-5 virus (1 x 10 12 VP / ml) were added to the microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), and the coating concentrations were 1 μg / mL and 500-fold dilution respectively, and incubated overnight at 4°C; blocked with 1% BSA, 150 μL per well, blocked at 37°C for 2 hours, washed the plate once with the washing solution, and patted dry; added 50 μL of the culture supernatant of hybridoma cells, reacted at 37°C for 30 min. Discarded the liquid in the well, washed the plate 4 times with PBST washing solution (0.01 M PBS, 0.1% Tween 20, pH 7.4), patted dry and then added 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solarbio, SE131) diluted 5000-fold, reacted at 37°C for 30 min, washed the plate 4 times again, patted dry and then added 50 μL / well of TMB chromogenic solution (single-component chromogenic solution, Beijing MedKovan Biotech, 1001) for color development at room temperature for 10 min, and finally added 50 μL of TMB stop solution (acidic, Beijing MedKovan Biotech, 1001SA) to terminate the reaction, and measured the OD 450 nm value with an enzyme-linked immunosorbent assay reader. Positive cell lines that reacted with both HADV-5 Hexon and HADV-5 virus were selected for subsequent experiments.

[0024] 4. Preparation of monoclonal antibody ascites After the selected monoclonal cell lines were expanded in culture, 0.2 mL (containing 2.5×10 6 cells) of female Balb / C mice pretreated with Freund's incomplete adjuvant were injected intraperitoneally. Approximately 10 days later, when the abdomen of the mice was significantly swollen, ascites was collected using a sterile syringe needle.

[0025] 5. Affinity Chromatography Purification of Monoclonal Antibodies The ascites was centrifuged at 12,000 r / min for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4), filtered through a 0.22-μm filter, and the filtered sample was pumped into a Protein L purification column equilibrated with binding buffer at a flow rate of 1 ml / min using a peristaltic pump. The protein purification instrument was connected, and it was washed with 5 - 10 column volumes of binding buffer until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1 M glycine, pH 2.7). The elution peak was collected, and the collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, loaded into a dialysis bag (MW: 8000 - 14000), and dialyzed in 20 mM PBS, pH 7.4 solution at 2 - 8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12,000 r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The protein concentration of the purified monoclonal antibody was measured at a wavelength of 280 nm using an ultra-micro spectrophotometer and stored in aliquots.

[0026] 6. Identification of Monoclonal Antibodies Referring to the above indirect ELISA method, purified viruses of 4 types, namely HADV-2, HADV-3, HADV-6, and HADV-7, were respectively 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. At the same time, the supernatant of Hep2 cells was coated as a control antigen. Specifically referring to the aforementioned ELISA method, the purified monoclonal antibody was diluted at a concentration of 1 μg / ml to detect its reactivity with the above five types of adenoviruses and Hep-2 cells, and a monoclonal antibody strain that specifically reacted only with HADV-5 was screened for subsequent neutralization tests.

[0027] Example 2 The replication-deficient recombinant adenovirus type 5 is derived from Beijing Wujiahe Gene Technology Co., Ltd., and the human embryonic kidney cell line HEK-293A is preserved by our company. Under normal circumstances, the HADV virus can infect Hep-2 cells, but the replication-deficient recombinant adenovirus type 5 (rADV) usually lacks the E1 gene and cannot replicate in normal cells. It can only replicate in cell lines that specifically express the E1 protein. The 293A cells can provide the E1 protein required for the replication of recombinant adenoviruses lacking the E1 gene, so they can be effectively infected and replicated.

[0028] 1. Virus TCID 50 Titer determination 1) HEK-293 cells are cultured with cell growth medium until they reach 80-90% confluence. After digestion with 0.25% EDTA-trypsin, a single-cell suspension is prepared and cell counting is performed.

[0029] 2) A cell suspension is prepared by adding 2% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin solution to DMEM basal medium, with a cell density of 1-2 x 10 5 / ml. Prepare a cell suspension suitable for virus infection, use low-serum medium (to reduce interference), and ensure good cell viability.

[0030] 3) Pipette 100 μl of the cell suspension into each well, and seed two 96-well cell culture plates simultaneously.

[0031] 4) Take the virus to be tested and perform 10-fold serial dilutions starting from 10 -3 to make it 10 -4 、10 -5 ………10 -11 、10 -12 (each dilution is 1 ml for subsequent inoculation).

[0032] 5) Add 100 μl of virus-free medium (the same as above: DMEM + 2% fetal bovine serum + 1% penicillin-streptomycin) to each well in columns 11 and 12 of the seeded 96-well plate as cell controls.

[0033] 6) Add 100 μl of the serially diluted virus samples to each well in columns 1-10 of the seeded 96-well plate, with 8 wells in each column for one virus dilution.

[0034] 7) After seeding the two 96-well plates, incubate them at 37°C and 5% CO2.

[0035] 8) Observe the cytopathic effect (CPE) under the microscope every day. On the 10th day, observe and record the number of CPE-positive wells for each dilution, and calculate the TCID 50 .

[0036] For a 100 μl sample, the titer T = 10 1+d（s-0.5） .

[0037] d = log10 稀释度 = 1 (for a 10-fold dilution).

[0038] s = sum of positive ratios (starting from the first 10-fold dilution).

[0039] According to the titration results, the virus titer was measured to be: 4.2×10 8 TCID 50 / mL. This indicates that the virus has high infectivity, ensuring effective interaction between the virus and antibodies in the experiment, improving the detection sensitivity and the reliability of the results. When conducting neutralizing antibody evaluation, a high virus titer helps to more accurately reflect the neutralizing ability of antibodies. Especially when facing a high virus load, it can better simulate the real infection environment, thus screening out truly efficient neutralizing antibodies.

[0040] 2. Virus neutralization test The cytopathic effect (CPE) can be directly observed, facilitating the primary screening of monoclonal antibody strains that can inhibit the cytopathic effect of the virus. At the same time, the virus content in the culture supernatant is measured using the established double-antibody sandwich detection method to compare the neutralizing effects of different antibodies. The specific steps of the neutralization test are as follows: 1) Prepare 293A cells: Cultivate 293A cells in advance until the logarithmic growth phase, digest them with trypsin and disperse them. Then, use DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin medium to seed a 96-well cell culture plate at a density of 1 - 2×10 5 / ml, and place it in an incubator at 37°C and 5% CO2 for culture. After the cells adhere, set aside for use (seed the plate according to the experimental needs).

[0041] 2) Prepare the virus to be tested: Dilute the recombinant adenovirus with blank DMEM medium at different multiples for standby.

[0042] 3) Incubate the monoclonal antibody with the virus: The neutralizing monoclonal antibody must be sterily treated. Take 30 μl of the virus to be tested and 30 μl of the diluted monoclonal antibody (100 μg / ml), and mix them thoroughly in a sterilized EP tube. Incubate at 37°C for 1 h.

[0043] 4) Infection: Aspirate the culture supernatant of the adherent cells, and then add 20 μl / well of the virus-monoclonal antibody mixture to each well. Subsequently, add 200 μL of blank DMEM + 1% penicillin-streptomycin medium to each well.

[0044] 5) Control settings: In the virus neutralization test, to ensure the accuracy and comparability of experimental results, three groups of controls, namely cell control, monoclonal antibody control, and virus control, should be set up for each test. Among them, 200 μL of blank culture medium (DMEM + 1% penicillin-streptomycin) is added to each well of the cell control wells, without adding virus and antibody, to evaluate the normal growth state of cells under virus-free infection conditions; for the monoclonal antibody control, an equal concentration of irrelevant monoclonal antibody (a monoclonal antibody against enterovirus EVD68, with a concentration of 100 μg / mL) is used, and its operation method is the same as that of the monoclonal antibody to be tested: 30 μL of the diluted virus (select the corresponding multiple concentration in step 2) is mixed with 30 μL of the irrelevant control monoclonal antibody, incubated at 37 °C for 1 hour, then 20 μL of the mixed solution is taken and added to the well plate, and 200 μL of blank DMEM + 1% penicillin-streptomycin medium is supplemented to exclude the influence of non-specific antibodies on virus replication; for the virus control wells, only virus is added without adding antibody, that is, 30 μL of diluted virus is incubated with 30 μL of blank DMEM medium, then 20 μL of it is taken and added to the well plate and 200 μL of medium is supplemented to reflect the infection and replication level of the virus without antibody interference. Through the reasonable setting of the above three groups of controls, the neutralizing effect of the monoclonal antibody to be tested on adenovirus type 5 can be effectively verified, and non-specific interference factors in the experiment can be excluded.

[0045] 6) Place the culture plate in an incubator at 37 °C and 5% CO2, observe CPE every day, and measure the virus content in the culture supernatant at 24 / 48 / 72 / 96 / 120 / 144 hours after infection.

[0046] 3. Detection of double antibody sandwich ELISA HRP labeling of monoclonal antibody 4A6: Specifically: Dilute the antibody to be labeled with carbonate coupling buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6) to a final concentration of 2 mg / mL. Dissolve 2 mg of HRP in 0.5 mL of ultrapure water, mix it well with 0.5 mL of 0.06 M sodium periodate solution, then add the diluted 1 mg antibody solution to the supporting tube containing HRP, and pipette and mix well. Incubate at room temperature for 1 h, and mix regularly during the incubation period. Add 50 μL of 5 mg / ml sodium borohydride and mix for 15 min to terminate the labeling reaction. Finally, dialyze the labeled antibody overnight in a buffer of 0.01 M PBS, pH 7.4, add glycerol in a volume ratio of 1:1, and store in aliquots at -20 °C.

[0047] Test procedure: Step 1) Coating antibody Dilute the monoclonal antibody 8B9 to a concentration of 1 μg / mL with coating buffer, add 50 μL to each well of the ELISA plate, and incubate overnight at 4 °C.

[0048] Step 2) Blocking After incubating overnight, discard the coating solution, add 150 μL of 1 - 2% BSA blocking solution to each well, and incubate at 37 °C for 2 hours.

[0049] Step 3) Sample addition After discarding the blocking solution and patting dry, add 50 μL of the cell culture supernatant to be tested to each well; incubate at 37 °C for 35 minutes.

[0050] Step 4) Washing Wash the plate 4 times with PBST washing solution, and pat dry thoroughly each time.

[0051] Step 5) Add HRP-labeled antibody Dilute the 4A6 monoclonal antibody-HRP labeled antibody 4000-fold with PBS, add 50 μL to each well, and incubate at 37 °C for 35 minutes.

[0052] Step 5) Wash again Wash the plate 4 times with PBST washing solution, and pat dry thoroughly each time.

[0053] Step 6) Color development and termination reaction 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 termination solution to each well to terminate the color development reaction.

[0054] Step 7) Read OD value Use an ELISA reader to read the absorbance (OD value) of each well at a wavelength of 450 nm.

[0055] 4. Neutralization efficiency calculation and results The virus and the monoclonal antibody are co-incubated and then used to infect sensitive 293 cells. If the virus is neutralized, the cells will not show cytopathic effects or will show weak cytopathic effects. The neutralization effect can be judged by observing CPE, which has the advantages of simple operation and intuitive results. At the same time, combining the ELISA method to detect the virus content in the supernatant can more accurately determine the neutralization efficiency of the monoclonal antibody against the virus, which is helpful for the standardization and data analysis of experimental results.

[0056] Formula for calculating the neutralization efficiency of monoclonal antibody: Neutralization efficiency (100%) = (OD of virus control 450 - OD of test well 450 ) / (OD of virus control 450 - OD of cell control 450) × 100%. In the virus neutralization test, the OD of the detection well corresponding to the monoclonal antibody-virus infection well in step 4 450 data, the OD of the cell control well corresponding to the cell control in step 5 450 data, the OD of the virus control well corresponding to the virus control 450 data.

[0057] If the monoclonal antibody has a neutralizing effect on the virus, the virus replication is slow or does not occur, and less adenovirus can be detected in the culture supernatant, then the OD of the detection well 450 value is low, and the neutralization efficiency of the monoclonal antibody against the virus under a certain concentration condition can be judged by the above formula.

[0058] See Appendix Figure 1 and Appendix Figure 2 and Appendix Figure 3 For the 25 monoclonal antibodies screened, after being mixed and incubated with the virus diluted 10 -4 times and then infecting 293A cells, continuously monitor the replication of the virus after infection, and at the same time set up a monoclonal antibody control (enterovirus EVD-68 monoclonal antibody), a cell control and a virus control. The better neutralizing monoclonal antibody can effectively inhibit or delay the replication of the virus, and the cytopathic effect appears later or is not obvious. Further, it is detected that the virus replication is slow or does not occur, and the detection reading value is low. The test results show that: the OD of the cell control group 450 value remains at a low level and hardly changes throughout the experiment, which indicates that the uninfected 293A cells do not produce virus-related antigens or products under normal culture conditions. Therefore, the OD 450 value is very low and stable. The OD of the virus control group 450 value began to increase significantly 24 hours after infection, which indicates that in the cells only infected with the virus but not treated with the monoclonal antibody, the virus can effectively replicate and produce a large amount of virus-related substances, resulting in a significant increase in the OD 450 value. The monoclonal antibody control group used an irrelevant monoclonal antibody against enterovirus EVD-68, and the change trend of its OD 450 value is similar to that of the virus control group, which indicates that the irrelevant monoclonal antibody has no specific neutralizing effect on the target virus. Therefore, the virus can still replicate in the cells. Monoclonal antibody 1# (monoclonal antibody 8B9) and 12# (monoclonal antibody 7C9) significantly inhibited the cytopathic effect, and no virus replication was detected even 144 hours after infection, indicating that these two monoclonal antibodies, 8B9 and 7C9, have a very strong neutralizing effect on the target virus and can effectively prevent the virus from replicating in cells for a long time.

[0059] 5. Determination of neutralizing monoclonal antibodies For the selected monoclonal antibody with better neutralization effect, an infection neutralization test of high-titer virus was carried out. The recombinant adenovirus was diluted 10-fold and 100-fold respectively to test the neutralization effect. The cytopathic effect was 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 antibody added was appropriately increased during the experiment. See Appendix Figure 4 . The results showed that on the third day after infection, the negative control antibody (enterovirus EVD-68 monoclonal antibody) could not neutralize the virus, and the cells were infected and showed cytopathic effect. The cell control group was not treated with anything, and the cells grew normally without cytopathic effect. Monoclonal antibody 8B9 had obvious inhibition on the virus infection diluted 10-fold and 100-fold at a concentration of 850 μg / ml. Compared with the virus control, the cytopathic effect of the cells was significantly weaker, showing a more obvious neutralization effect than monoclonal antibody 7C9. Monoclonal antibody 8B9 was determined as the optimal neutralizing monoclonal antibody.

[0060] The neutralization effect of monoclonal antibody 7C9 was not as good as that of monoclonal antibody 8B9. Therefore, the corresponding sequence was not given in the present invention.

[0061] 6. Evaluation of the neutralization efficiency of monoclonal antibody 8B9 See Figures 5 - 7 . The results of the in vitro neutralization test of monoclonal antibody 8B9 against a 6000-fold diluted virus (titer 7×10 4 TCID 50 / mL) in the 293A sensitive cell line showed that: on the 5th day after infection, there was still no CPE in the test wells of the monoclonal antibody at a concentration of 7 μg / ml, and the virus content detected by ELISA was extremely low, indicating that the virus could not replicate effectively after being neutralized by the monoclonal antibody. Through the calculation of the neutralization efficiency, the monoclonal antibody had a neutralization efficiency close to 100% against the virus with a titer of 7×10 4 TCID 50 / mL at a concentration above 3.3 μg / ml. Among them, Figure 7 the ctrl was the virus self-control, that is, the natural replication of the virus in cells without adding any antibody.

[0062] 7. Gene sequence Cloning and sequencing of the variable region gene of monoclonal antibody.

[0063] Total RNA of hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription with RandomPrimers; universal primers for the variable regions of mouse antibodies were designed, and the VH and VL genes were amplified by 2 rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the 3rd round of PCR. After gel purification of the PCR products, they were ligated into the pUC19 vector, transformed into TOP10 strains, and single colonies were picked for sequencing after culturing at 37°C for 14 h to obtain the gene sequences of the heavy and light chains of the monoclonal antibody.

[0064] The specific sequences are as follows: Among them, the amino acids of the heavy chain complementarity-determining regions 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.

[0065] The amino acids of the light chain complementarity-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.

[0066] The amino acids of the heavy chain complementarity-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.

[0067] The amino acids of the light chain complementarity-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.

[0068] The amino acid sequence of the heavy chain variable region of monoclonal antibody 8B9 (SEQ ID NO.13): EVKLEESGPELVKPGASVKMSCKASGYTFTSYYMHWVKQKPGQGLEWIGYLNPVNDSTKYMEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARAIYESYYGYWGQGTTLTVSA.

[0069] Amino acid sequence of the light chain variable region of monoclonal antibody 8B9 (SEQ ID NO.14): DIQMNQSPASLAVSLGQRATISCKASKRVSISGYSYMHWYQQKPGQPPKLLIYLASNLLGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHGRLLPSTFGGGTKLELKRTV.

[0070] Amino acid sequence of the heavy chain variable region of 4A6 (SEQ ID NO.15): EVMLAESGPELVKPGASVKMSCKASGYTFTGSVFKWVKQKPGQGLEWIGELSPVIDSTSVLERAQQKATLTSDKSSSTAYMELSSLTSEDSAVYYCARMLKSVISVWGQGTTLTVSS.

[0071] Amino acid sequence of the light chain variable region of monoclonal antibody 4A6 (SEQ ID NO.16): EIVLTQSPASLAVSLGQRATISCFARKSVLLSRVSYHIWYQQKPGQPPKLLIYETSVVGGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCMHSGYLPSTFGGGTKLEIKRTV.

[0072] Nucleotide sequence of the heavy chain variable region of monoclonal antibody 8B9 (SEQ ID NO.17): GAGGTGAAGCTGGAGGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCTACCTGAACCCCGTGAACGACAGCACCAAGTACATGGAGAAGTTCAAGGGCAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGGCCATCTACGAGAGCTACTACGGCTACTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC.

[0073] The nucleotide sequence of the light chain variable region of monoclonal antibody 8B9 is as follows (SEQ ID NO.18): GACATCCAGATGAACCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAAGGCCAGCAAGAGGGTGAGCATCAGCGGCTACAGCTACATGCACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACCTGGCCAGCAACCTGCTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCCAGCACGGCAGGCTGCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0074] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 4A6 (SEQ ID NO.19): GAGGTGATGCTGGCCGAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCGGCAGCGTGTTCAAGTGGGTGAAGCAGAAGCCCGGCCAGGGCCTGGAGTGGATCGGCGAGCTGAGCCCCGTGATCGACAGCACCAGCGTGCTGGAGAGGGCCCAGCAGAAGGCCACCCTGACCAGCGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGATGCTGAAGAGCGTGATCAGCGTGTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

[0075] Nucleotide sequence of the light chain variable region of monoclonal antibody 4A6 (SEQ ID NO.20): GAGATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCTTCGCCAGGAAGAGCGTGCTGCTGAGCAGGGTGAGCTACCACATCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACGAGACCAGCGTGGTGGGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCATGCACAGCGGCTACCTGCCCAGCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0076] The present invention provides a highly efficient neutralizing monoclonal antibody 8B9 against adenovirus type 5, which has high affinity and strong neutralizing activity and can effectively inhibit the infection and replication of adenovirus type 5. This antibody was obtained by screening through the hybridoma technology, and the variable region gene sequencing and neutralization effect evaluation were completed, showing broad application prospects in vaccine development, vector optimization and potential passive immunotherapy. This achievement fills the blank of the current lack of specific neutralizing monoclonal antibodies against adenovirus type 5 and provides important tools and technical support for the research and prevention and treatment of adenovirus-related diseases.

[0077] So far, the 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. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0078] Although some specific embodiments of the present application have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration and not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or partial technical features can be equivalently replaced without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody with neutralizing activity against adenovirus type 5, characterized in that, The monoclonal antibody is 8B9. The monoclonal antibody 8B9 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 respectively as shown in SEQ ID NO.1-SEQ ID NO.3; The light chain variable region of the monoclonal antibody 8B9 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are respectively as shown in SEQ ID NO.4-SEQ ID NO.

6.

2. The monoclonal antibody with 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 as shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 8B9 is as 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, wherein, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8B9 is as shown in SEQ ID NO.

17.

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

18.

6. Use of a monoclonal antibody having neutralizing activity against adenovirus type 5 as described in any one of claims 1-2 in the preparation of a drug for preventing or treating adenovirus type 5 infection.

7. Use of a monoclonal antibody having neutralizing activity against adenovirus type 5 as described in any one of claims 1-2 in the preparation of a diagnostic reagent, test strip and kit for detecting adenovirus type 5 infection.

8. Use of a monoclonal antibody having neutralizing activity against adenovirus type 5 as described in any one of claims 1-2 in the study of the biological characteristics of adenovirus type 5.

9. Use of a monoclonal antibody having neutralizing activity against adenovirus type 5 as described in any one of claims 1-2 in the preparation of a test model for non-diagnostic purposes for evaluating the in vitro neutralizing activity of the monoclonal antibody.

10. The use according to claim 9, characterized in that, The test model is established based on HEK-293A cells, and is used for detecting the infection and replication process of adenovirus type 5 in cells and evaluating the neutralizing effect of the monoclonal antibody.

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