Monoclonal ascites antibody to kdo glycoprotein conjugates and uses thereof

Abstract

The application provides a monoclonal ascites antibody for Kdo glycoprotein conjugate and an application thereof, and a Kdo monoclonal hybridoma cell strain with a preservation number of CCTCC NO: C2022211. The Kdo monoclonal hybridoma cell strain disclosed by the application can generate the monoclonal ascites antibody for the Kdo glycoprotein conjugate, and has different recognition and combination abilities for drug-resistant bacteria with Kdo existing in different parts of bacterial surfaces, such as Staphylococcus aureus and Escherichia coli, and has potential application in development of an anti-drug-resistant bacteria vaccine and a diagnostic tool.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a monoclonal ascites antibody against Kdo glycoprotein conjugate and its application. BACKGROUND

[0002] In the face of the challenge of antibiotics, bacteria can quickly evolve drug resistance, and even evolve into superbugs with multiple drug resistance. Especially Staphylococcus aureus, one of the serious drug-resistant pathogens with high infection rate, can cause serious and even fatal diseases such as osteomyelitis, pneumonia, meningitis and toxic shock syndrome. With the spread of drug-resistant bacteria worldwide and the continuous rise of infection cases, humans may face the danger of having no antibiotics available. The currently marketed vaccines for Neisseria meningitidis, Haemophilus influenzae type b, Streptococcus pneumoniae, Salmonella typhi and the like have a narrow antibacterial range, and the methods for preventing and diagnosing drug-resistant bacteria still have certain limitations. Therefore, it is necessary to develop vaccines and diagnostic tools for different drug-resistant bacteria to provide humans with more tools to fight drug-resistant bacterial infections.

[0003] 3-deoxy-D-manno-2-octulosonic acid (Kdo) belongs to a specific highly conserved structure present on the surface of drug-resistant bacterial cell walls, which is usually involved in maintaining the integrity of the cell membrane and can stimulate the immune system to produce specific antibodies, and has excellent immunogenicity. It has important potential application value for the development of vaccines and diagnostic tools for drug-resistant bacterial infections.

[0004] In order to further explore the application of Kdo derivatives in anti-drug-resistant bacterial vaccines and bacterial detection and diagnosis, it is necessary to couple them with appropriate carrier proteins to form glycoprotein conjugates, induce T cell-dependent immune responses, and effectively enhance the immunogenicity of carbohydrate substances. This is an important strategy for developing carbohydrate vaccines. Compared with the reported antibacterial glycoprotein conjugate vaccines, the Kdo glycoprotein conjugate with simple structure and clear configuration used in the present application can induce a large amount of antibodies in vivo at a small dose. The monoclonal ascites antibody prepared by monoclonal technology shows different recognition and binding abilities to drug-resistant bacteria such as Staphylococcus aureus and Escherichia coli, in which Kdo exists in different parts of the bacterial surface, and has potential application in the development of anti-drug-resistant bacterial vaccines and diagnostic tools. SUMMARY

[0005] The first object of the present application is to provide a Kdo monoclonal hybridoma cell strain capable of producing a monoclonal ascites antibody against Kdo glycoprotein conjugate.

[0006] The second object of the present application is to provide a monoclonal ascites antibody against Kdo glycoprotein conjugate, which can be used as a diagnostic tool for drug-resistant bacteria containing Kdo structure and has potential application in the development of antibacterial vaccine.

[0007] The third object of the present application is to provide the application of Kdo monoclonal hybridoma cell strain.

[0008] To achieve the first object of the present application, the present application provides a Kdo monoclonal hybridoma cell strain, which has a preservation number of CCTCC NO: C2022211.

[0009] The hybridoma cell strain is named Kdo monoclonal hybridoma cell strain 3N11, which is preserved in China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, and has a preservation date of September 18, 2022 and a preservation number of CCTCC NO: C2022211.

[0010] To achieve the second object of the present application, the present application provides a monoclonal ascites antibody against Kdo glycoprotein conjugate, which is secreted by the Kdo monoclonal hybridoma cell strain.

[0011] To achieve the third object of the present application, the present application provides the application of the Kdo monoclonal hybridoma cell strain in the preparation of Staphylococcus aureus or Escherichia coli antibacterial vaccine.

[0012] Further, the application of the Kdo monoclonal hybridoma cell strain in the preparation of drug-resistant bacteria detection and diagnostic reagents is also provided, wherein the drug-resistant bacteria refer to Staphylococcus aureus or Escherichia coli.

[0013] In a preferred embodiment of the present application, the structure of Kdo glycoprotein conjugate is shown in CRM197-1 or HSA-1:

[0014]

[0015] In the following examples, an excess of chemical cross-linking agent, succinimidyl glutarate, reacts with the amino group derived from the C-2 position of Kdo monosaccharide to form an amide bond, which is converted into the corresponding activated monoester. After removing the organic solvent by centrifugal extraction method, the Kdo glycoprotein conjugate is obtained by covalent coupling with carrier protein CRM197 or HSA, and protein concentration and purification are performed using ultrafiltration centrifugation method. The average number of monosaccharides coupled to each carrier protein is analyzed and calculated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-4800plus).

[0016] In the preferred embodiment of the present application, the Kdo glycoprotein conjugate is mixed with Freund's adjuvant (FA) to form an emulsion, and female C57BL / 6J mice are immunized subcutaneously in the groin according to the immunization schedule of 0-14-28 days. The mixed emulsion induces more cross-reactive antibodies, and the orbital blood is collected 7 days after the last immunization to obtain antisera. The results of enzyme-linked immunosorbent assay show that the IgG antibody titer level in the mouse serum is significantly improved, and the immune response is strong. In particular, the IgG1, IgG2b, and IgG3 antibody titer levels are also significantly increased, proving that the glycoprotein conjugate can induce a strong T cell-dependent protective immune response, and can be used as a potential antigen for the development of an anti-drug resistant bacteria vaccine.

[0017] In the preferred embodiment, the Kdo glycoprotein conjugate is mixed with Freund's adjuvant (FA) to form an emulsion, and female BALB / c mice are immunized subcutaneously at multiple points according to the immunization schedule of 0-14-28-42 days. The orbital blood is collected 7 days after the last immunization to select mice with an antisera titer of 8K dilution. According to the principle that B lymphocytes can produce antibodies and tumor cells can be passaged in vitro indefinitely, the mouse spleen and lymph tissue are extracted and fused with myeloma SP2 / 0 cells in the logarithmic growth phase and pretreated with 8-AG to form hybridoma cells that can both proliferate indefinitely and produce antibodies. Multiple cell subcloning is performed until a cell strain with a Kdo antigen recognition rate of 100% is obtained. The selected cell strain is continuously injected intraperitoneally into 10-week-old BALB / c mice, and abdominal fluid is collected one week later to obtain a large amount of ascites monoclonal antibody against Kdo.

[0018] In the preferred embodiment, several inactivated drug-resistant bacteria with or without Kdo structure are first incubated with monoclonal ascites antibody. Based on AF488 goat anti-mouse IgG antibody (green) staining labeling, about 10% of the bacteria can be extracted for fluorescence analysis by flow cytometry, and the binding between the inactivated bacteria and the ascites antibody can be observed using a confocal laser microscope (LeicaTCS SP8). The experimental results show that when Kdo exists in different positions on the surface of bacteria, the binding results of bacteria and monoclonal ascites antibody are significantly different. Therefore, the large amount of prepared monoclonal antibody can be used as a diagnostic tool for drug-resistant bacteria containing Kdo structure, and has potential application in the development of antibacterial vaccines.

[0019] The present application has the advantage that the present application provides a Kdo monoclonal hybridoma cell strain that can produce monoclonal ascites antibody against Kdo glycoprotein conjugate, and exhibits different recognition and binding abilities for drug-resistant bacteria with Kdo existing in different positions on the surface of bacteria, such as Staphylococcus aureus and Escherichia coli. The present application has potential application in the development of anti-drug resistant bacteria vaccines and diagnostic tools. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1Aand Figure 1B Figure 2 shows the characterization results of the glycoprotein conjugate CRM197-1 obtained in Example 1 of the present application.

[0021] Figure 2 Figure 3 shows the immune response results of the glycoprotein conjugate CRM197-1 in C57BL / 6J mice according to the present application.

[0022] Figure 3 Figure 4 shows the IgG antibody titers of the six ascites 3N11, 4G9, 4P20, 2E6, 3N14 and 2G20 for ELISA analysis.

[0023] Figure 4 Figure 5 shows the binding of ascites monoclonal antibodies 3N11, 2E6, 2G20 to inactivated bacteria for immunofluorescence analysis, wherein (4A, 4C, 4E, 4G, 4I, 4K) laser confocal microscope observation of AF488 fluorescence labeling results of inactivated bacteria; (4B, 4D, 4F, 4H, 4J, 4L) flow cytometry analysis of the binding of ascites 3N11, 2E6, 2G20 to inactivated bacteria. Note: inactivated bacteria: inactivated Streptococcus pneumoniae-19F (ATCC49619 / BNCC238812), Staphylococcus aureus (ATCC6538 / BNCC186335), Escherichia coli-K12 (ATCC25404 / BNCC313406), Escherichia coli-DH5α, Providencia alcalifacien-O36 (ATCC9886 / BNCC137352), Helicobacter pylori (NCTC 11637); (a) bacteria without ascites treatment, (b) bacteria treated with ascites 3N11, (c) bacteria treated with ascites 2E6, (d) bacteria treated with ascites 2G20; the associated panels a’-c’ show bright field images, the associated panels a”-c” show AF488 fluorescence field and bright field superimposed images, and the length of the white bar is 10 μm. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described in detail below in conjunction with the specific embodiments. It should be understood that the following specific embodiments are only used to help those skilled in the art to understand and realize the present application, and are not a limitation on the present application.

[0025] Example 1. Kdo glycoprotein conjugate

[0026] In this embodiment, a Kdo glycoprotein conjugate is provided, the Kdo monosaccharide derivative used is provided by a colleague of the research group of the present application, and can also be synthesized according to the method disclosed in Gold (I)-catalyzed Synthesis of β-Kdo Glycosides Using Kdo ortho-Hexynylbenzoate as Donor. Xuemeng Mi, Qixin Lou, Wenjing Fan, Liqin Zhuang, You Yang*. Carbohydr. Res. 2017, 448, 161-165. The glycoprotein conjugate is connected by a linker, bis (N-hydroxysuccinimide) glutarate (DSG), between the Kdo derivative and the carrier protein CRM197, and has the structure of structural formula CRM197-1:

[0027]

[0028] In this embodiment, the Kdo derivative and the linker bis (N-hydroxysuccinimide) glutarate are coupled by the activated ester method. Then, the activated Kdo derivative is connected to the carrier protein CRM197 to obtain the glycoprotein conjugate CRM197-1.

[0029] In this embodiment, the synthesis route of the glycoprotein conjugate CRM197-1 is as follows:

[0030]

[0031] The preparation method of the glycoprotein conjugate CRM197-1 is specifically as follows:

[0032] To a solution of bis-succinimidyl glutarate (15 mg, 46 μmol) and triethylamine (10 μL, 0.07 mmol) in DMSO (200 μL) was added a solution of Kdo derivative 1 (1.5 mg, 4.6 μmol) in DMSO (80 μL). After stirring at room temperature for 2 h, the reaction was diluted with PBS (100 mM, pH = 7.4, 640 μL) and extracted with 5 mL chloroform. The aqueous phase was centrifuged (2 min, 1800 g) and the supernatant was transferred to a 1.5 mL EP tube and centrifuged (1 min, 14500 g) until no phase separation was observed. The supernatant was mixed with 1 mg of CRM197 (17.3 nmol, Creative BioMart) in 1 mL of PBS (100 mM, pH = 7.4) and stirred at room temperature for 18 h. After completion of the reaction, the mixture was transferred to an ultrafiltration tube (30 kDa MWCO, Millipore) and centrifuged three times (4000 rpm, 15 min, 4°C) with ultrapure water and PBS, respectively, to obtain the glycoprotein conjugate CRM197-1.

[0033] The average molecular weight of the glycoprotein conjugate CRM197-1 was determined by 10% SDS-PAGE (120 V, 90 min) and MALDI-TOF-MS, and the results are shown in Figure 1A and Figure 1B The results show that the average number of Kdo derivatives attached to each CRM197 protein in the glycoprotein conjugate CRM197-1 is about 9.2.

[0034] Example 2. Immune activity test of the glycoprotein conjugate CRM197-1

[0035] In this example, the immune activity test results of the glycoprotein conjugate CRM197-1 obtained in Example 1 are provided. The immune results by enzyme-linked immunosorbent assay show that the glycoprotein conjugate CRM197-1 can induce a strong immune response in the body. High titers of IgGl, IgG2b and IgG3 antibodies, especially IgGl and IgG3 antibodies, are produced, indicating that the CRM197-1 glycoconjugate can induce a stronger T cell-dependent protective immune response, which is very ideal for a prophylactic vaccine.

[0036] In this example, the immune activity test of the glycoprotein conjugate CRM197-1 specifically includes the following specific steps.

[0037] 1. Immunization of C57BL / 6J mice with the glycoprotein conjugate CRM197-1

[0038] C57BL / 6J mice (4-6 per group) were immunized subcutaneously with CRM197-1 glycoconjugate antigen. The mice in the treatment group were injected with 0.4 μg of the glycoprotein conjugate CRM197-1 on day 0, and 0.8 μg of the glycoprotein conjugate CRM197-1 on days 14 and 28. Each mouse was injected with 100 μL of the emulsion of the glycoprotein conjugate CRM197-1 mixed with Freund's adjuvant 1:1 (V / V). The control group was injected with PBS or PBS plus adjuvant, and the treatment group was injected with the glycoconjugate antigen or the glycoconjugate plus adjuvant. The primary immunization was with Freund's complete adjuvant, and the booster immunization was with Freund's incomplete adjuvant. The mice were bled from the orbital vein on days 0, 14, 21 and 35, and the serum was extracted.

[0039] 2. ELISA of the polyclonal serum of C57BL / 6J mice after immunization

[0040] The ELISA was performed using a high-binding 96-well plate (Corning). HSA-1 (10 μg / mL) was dissolved in carbonate buffer (0.05 M, pH = 9.6) and incubated at 4°C for 20 hours to coat the plate. The next day, the plate was washed with PBS-T (PBS containing 0.1% Tween-20) for 3 times, and blocked with 2% BSA-PBS at 37°C for 1 hour. After washing with PBS-T for 3 times, the antiserum was diluted with 1% BSA-PBS, and incubated at 37°C for 2 hours. After washing with PBS-T for 3 times, HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG2b and IgG3 secondary antibodies were added, and incubated at 37°C for 1 hour in the dark. After washing with PBS-T for 3 times, TMB substrate was added and incubated at 37°C for 20 minutes to develop color. The reaction was stopped by adding 2% sulfuric acid, and the absorbance at 450 nm was measured using a Synergy2 multifunctional microplate reader to obtain the detection results as shown in Table 1. Figure 2

[0041] The results showed that the IgG antibody titer in the serum of the mice was significantly increased after 35 days of immunization, and the immune response was strong. The IgG1, IgG2b and IgG3 antibody titers were also significantly increased after immunization. The glycoprotein conjugate can induce a strong T cell-dependent protective immune response in the body.

[0042] Example 3. Preparation and binding ability evaluation of ascites monoclonal antibody based on Kdo glycoprotein conjugate

[0043] 1. Preparation of ascites monoclonal antibody:

[0044] ​Female BALB / c mice were immunized subcutaneously with the glycoprotein conjugate CRM197-1 mixed with Freund's adjuvant (FA) to form an emulsion, according to a four-time routine immunization program of 0-14-28-42 days, and the post-immune serum was extracted one week after the last immunization, and the antibody titer of the serum was determined by ELISA. The mice with the antibody titer reaching 1:8K dilution were selected for cell fusion. The mouse spleen and lymphocytes were extracted and fused with SP2 / 0 mouse myeloma cells in the logarithmic growth phase to form hybridoma cells, and the hybridoma cells were subcloned multiple times. The hybridoma cells were controlled by indirect ELISA, the HSA-1 glycoprotein conjugate was used as an antigen capture reagent, and the supernatant of the cells was diluted by 1:3.125K / 6.25K / 12.5K / 25K / 50K / 100K to obtain six kinds of hybridoma cells with a titer of 1:100K or more (Table 1), which is considered to have a 100% recognition rate of Kdo. The selected hybridoma cells were continuously cultured, and ascitic monoclonal antibodies were obtained by in vivo preparation. The hybridoma cells in the logarithmic growth phase were injected intraperitoneally into 10-week-old BALB / c female mice pretreated with Freund's incomplete adjuvant, and the ascites was collected one week later. After centrifugation to remove red blood cells and oil, a large amount of ascitic monoclonal antibodies against the CRM197-1 glycoprotein conjugate were obtained.

[0045] Table 1 Absorbance values of supernatants of six hybridoma cells specifically recognizing Kdo

[0046]

[0047] 2. Binding ability of ascitic monoclonal antibodies to antigens

[0048] The HSA-1 glycoprotein conjugate was used as an antigen capture reagent, the PBS-treated mouse serum was used as a negative control, and the enzyme-labeled IgG antibody was used as a research object. The titers of the six ascitic monoclonal antibodies were evaluated by ELISA, and the titers were calculated.

[0049] The ascitic ELISA results are shown in the green curve representing the ascitic monoclonal antibody, the blue curve representing the serum antibody on day 35, and the orange-red curve representing the PBS blank control. It was found that the curves of five ascitic monoclonal antibodies were higher than the blue curve representing the serum antibody, indicating that the ascitic monoclonal antibodies were more easily recognized than the serum antibodies. According to the rule that P / N≥2.1 is a positive result, the titers of the six ascitic monoclonal antibodies were calculated, and it was found that the titers of ascitic 3N11, 4G9 and 4P20 were about 1:6400000, the titers of ascitic 2E6 and 3N14 were about 1:3200000, and the titer of ascitic 2G20 was about 1:80000. Figure 3). From each class of titer, one ascites (3N11, 2E6, 2G20) was selected to perform immunofluorescence analysis again to observe the difference in the ability of ascites of different titers to bind to bacteria.

[0050] 3. The ability of ascites monoclonal antibodies to bind to inactivated bacteria

[0051] A gram-positive bacterium Streptococcus pneumoniae-19F without Kdo structure and a bacterium containing Kdo structure were selected as controls. Several inactivated drug-resistant bacteria were first incubated with ascites, and goat anti-mouse IgG antibody (green) based on AF488 was used for staining and labeling, about 10% of the bacteria were extracted for fluorescence analysis by flow cytometry, and the remaining bacteria were observed for fluorescence binding between ascites antibodies using a laser confocal microscope. The excitation wavelength (494 nm) and emission wavelength (513 nm) of AF488 fluorescent dye are basically the same as those of FITC fluorescent dye, and the fluorescence of AF488 dye fades more slowly and has better fluorescence effect, so AF488 is selected as the fluorescent dye for this immunofluorescence experiment.

[0052] The results show that the three ascites do not have fluorescence signals with Streptococcus pneumoniae-19F without Kdo structure, indicating that no binding occurs between them Figure 4 A, 4B). In the immunofluorescence experiment, the three ascites can produce significant fluorescence with Staphylococcus aureus Figure 4 C), and the highest peak of the corresponding curve of the three ascites in the flow cytometry data is significantly shifted from the highest peak of the gray shadow curve of the blank group Figure 4 D), further indicating that the ascites can bind to Staphylococcus aureus. Similarly, it can be observed that the binding of the three ascites to Escherichia coli-K12 is also very obvious Figure 4 E, 4F), the binding ability of Alcaligenes faecalis is second Figure 4 I, 4J), and the binding of Escherichia coli-DH5a and Helicobacter pylori is weaker Figure 4 G, 4H, 4K, 4L).

[0053] In summary, the ascites monoclonal antibodies can more obviously observe the differences in binding to different bacteria, and the closer the Kdo structure is to the outer layer of the bacteria, the easier it is to be recognized by the ascites monoclonal antibodies. And the binding ability of ascites 3N11 to several bacteria is the strongest, which can be considered to continue to be purified to prepare a medical diagnostic tool or reagent for assisting in the detection of drug-resistant bacteria. The 3N11 hybridoma cell is preserved in the China Center for Type Culture Collection, the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is September 18, 2022. The strain is named Kdo monoclonal hybridoma cell strain 3N11, and the preservation number is CCTCC NO: C2022211.

[0054] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A Kdo monoclonal hybridoma cell strain, with the preservation number of CCTCC NO: C2022211.

2. A monoclonal ascites antibody directed against a Kdo glycoconjugate, characterized in that, A Kdo monoclonal hybridoma cell strain of claim 1 secretes.

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