Application of Brucella OMP16 monoclonal antibody in the preparation of products for inhibiting Brucella

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a Brucella OMP16 monoclonal antibody in the preparation of a product for inhibiting Brucella, wherein the heavy chain variable region amino acid sequence of the Brucella OMP16 monoclonal antibody is shown in SEQ ID NO.1, and the light chain variable region amino acid sequence is shown in SEQ ID NO.2. Through in vivo and in vitro tests, the present invention verifies the potential of the OMP16 monoclonal antibody as a therapeutic antibody in the treatment of brucellosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of a Brucella OMP16 monoclonal antibody in the preparation of a product for inhibiting Brucella. Background Art

[0002] Brucella is a Gram-negative, facultative intracellular parasite that can infect humans, livestock and a variety of wild animals, causing an important zoonotic infectious disease - brucellosis. The disease is widely prevalent around the world, with about 500,000 new infections each year, posing a huge threat to human health. After humans are infected with Brucella, the initial symptoms are mainly uncertain fever and flu-like symptoms. If they are not treated promptly and effectively, they will turn into chronic infections, manifested as arthritis, orchitis, spleen and liver enlargement and other systemic debilitating symptoms. At present, the treatment of brucellosis usually adopts antibiotic combination therapy, such as rifampicin, doxycycline, tetracycline, streptomycin, gentamicin, etc. However, due to the unique survival mode of Brucella, the effectiveness of antibiotics is reduced, resulting in long medication time, high risk of adverse side effects, and easy recurrence. Therefore, the treatment of brucellosis is still mainly aimed at alleviating and shortening the symptom period, reducing complications, recurrence and chronic infection. In addition, since the treatment of brucellosis usually requires the use of antibiotics for several months, some Brucella strains have developed varying degrees of resistance to multiple antibiotics, which poses great risks and challenges to the treatment of brucellosis.

[0003] As a biomolecular drug, antibody drugs can not only bind to antigens to cause pathogen neutralization or active site closure, but also play an immunotherapeutic role by inducing antibody-mediated complement-dependent killing activity, macrophage opsonized phagocytosis and killing activity, neutrophil extracellular traps and other immunological effects. They have been widely studied in the treatment of various diseases such as cancer, autoimmune diseases, and infectious diseases, and are one of the ideal drugs for the treatment of brucellosis. Brucella outer membrane protein 16 (OMP16) is a structural protein of about 16kDa on the cell membrane of Brucella. It is highly conserved among various species of Brucella and can act as a pathogen-associated molecular pattern (PAMP) to interact with the TLR4 receptor and regulate the host immune response. In addition, OMP16 contains a conserved peptidoglycan-associated lipoprotein (PAL) domain, which plays an important role in maintaining the integrity of the bacterial outer membrane and preventing the adsorption of organic molecules. Studies have found that omp16 is a lethal gene of Brucella. Direct knockout affects the normal survival of Brucella. Conditional knockout destroys the integrity of Brucella cell membranes and reduces its in vitro stress and survival ability. OMP16 is also considered an important protective antigen. After immunizing mice, it produces an immune protection level equivalent to that of Brucella live attenuated vaccines. It has been widely studied in subunit vaccines, vector vaccines, and nucleic acid vaccines.

[0004] Previously, the team of the present invention prepared an IgM anti-Brucella OMP16 monoclonal antibody named B7 through mouse hybridoma cell technology. Existing studies have shown that IgM antibodies are pentamers formed by 5 Ab monomers linked by disulfide bonds and J chains, containing ten antigen recognition sites, which can expose more complement binding sites after binding to antigens, and have the strongest ability to activate the complement system among all types of antibodies. In addition, the cell receptor that binds to the Fc region of IgM antibodies in the host is FcμR, which is mainly expressed in human B cells, NK cells, T cells and mouse B cells, so the production of its immunomodulatory effect is greatly dependent on the activation of the complement system. However, it is not yet known whether the Brucella OMP16 monoclonal antibody has an inhibitory killing effect on Brucella. Summary of the invention

[0005] In view of the above technical problems, the present invention provides an application of a Brucella OMP16 monoclonal antibody in the preparation of a product for inhibiting Brucella.

[0006] The specific technical solutions provided by the present invention are as follows:

[0007] In a first aspect, the present invention provides an application of a Brucella OMP16 monoclonal antibody in the preparation of a product for inhibiting Brucella, wherein the heavy chain variable region amino acid sequence of the Brucella OMP16 monoclonal antibody is shown in SEQ ID NO.1, and the light chain variable region amino acid sequence is shown in SEQ ID NO.2.

[0008] Preferably, the Brucella OMP16 monoclonal antibody is used to prepare a product for improving the immune protection of a Brucella infected population.

[0009] Preferably, the Brucella OMP16 monoclonal antibody is used to prepare a product for reducing spleen or liver damage caused by Brucella infection.

[0010] Preferably, the Brucella OMP16 monoclonal antibody is used to prepare a product for preventing or treating brucellosis.

[0011] Preferably, the product is a medicine or a vaccine.

[0012] Preferably, the vaccine comprises a vaccine adjuvant or auxiliary material. Vaccine adjuvants include but are not limited to aluminum hydroxide, mineral oil, Tween-80, Freund's incomplete adjuvant, synthetic polyinosinic acid, lipopolysaccharide, lipoylglycerol, squalane, etc. The auxiliary material may be selected from preservatives, and the preservatives include but are not limited to sodium thiothioate, EDTA, thimerosal, 2-phenoxyethanol, nitroform, etc.

[0013] Preferably, the drug includes a pharmaceutically acceptable excipient or carrier. The excipient or carrier is specifically selected according to the dosage form of the drug. The drug can be in the form of an injection or an oral preparation, and the selected excipients include fillers, dispersants, emulsifiers, disintegrants, binders, preservatives, buffers, glidants, lubricants, solubilizers, coating agents, pH regulators, antioxidants, etc.

[0014] Exemplary fillers include mannitol, lactose, sucrose, dextrose, maltodextrin, sorbitol, xylitol, sodium lactose phosphate, kaolin, sodium chloride, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, calcium carbonate, sodium carbonate, cellulose, microcrystalline cellulose, silicified microcrystalline cellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, powdered sugar, starch, and the like.

[0015] Exemplary dispersants include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, agar, bentonite, cellulose, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone, sodium starch glycolate, carboxymethyl cellulose, cross-linked carboxymethyl cellulose, methyl cellulose, pregelatinized starch, microcrystalline starch, calcium carboxymethyl cellulose, magnesium aluminum silicate, sodium lauryl sulfate, and the like.

[0016] Exemplary disintegrants include crosslinked sodium carboxymethylcellulose, sodium alginate, calcium alginate, alginic acid, starch, pregelatinized starch, sodium starch glycolate, soybean polysaccharides, guar gum, ion exchange resins, crosslinked polyvidone, cellulose and its derivatives, calcium carboxymethylcellulose, sodium carboxymethylcellulose, and the like.

[0017] Exemplary binders include starch, gelatin, sugars, gum arabic, sodium alginate, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, alginates, silicic acid, and the like.

[0018] Exemplary preservatives include alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, sodium sulfite, tocopheryl acetate, cetrimonium bromide, butylated hydroxyanisole, ethylenediamine, sodium lauryl sulfate, sodium lauryl ether sulfate, sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, and the like.

[0019] Exemplary solubilizing agents include ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, glycerol, cyclodextrin, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, docusate sodium, poloxamers, sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, and the like.

[0020] Preferably, the Brucella is Brucella bovis A19.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention first verifies the ability of IgM type OMP16 monoclonal antibody B7 to agglutinate bovine Brucella A19, then detects the antibody-mediated complement-dependent lysis, complement-mediated macrophage opsonophagocytosis and killing effect exerted by activating the complement system, and finally establishes a mouse infection model to further evaluate the immunoprotective property of the antibody. In short, the present invention verifies the immunoprotective effect of IgM type OMP16 monoclonal antibody in vivo and in vitro, respectively, and evaluates its potential as a therapeutic antibody in the treatment of brucellosis. In addition, the previous research on the protectiveness of Brucella OMP16 antigen was mainly based on the humoral and cellular immunity levels induced after OMP16 antigen immunization and the attack and poison protection test research of the antigen, and the immunoprotective effect of OMP16 antibody self-mediated is rarely reported, therefore, the present invention further proves the immunoprotective property of OMP16 from the antibody perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The binding and agglutination of monoclonal antibody B7 with Brucella; A. Verify the binding of monoclonal antibody B7 with Brucella in macrophages through immunofluorescence test; B. Verify the binding of monoclonal antibody B7 with Brucella through iELISA test; C. Verify whether monoclonal antibody B7 can agglutinate Brucella through agglutination test.

[0024] Figure 2 It is the complement-mediated lysis effect of the complement system activated by the binding of monoclonal antibody B7 to Brucella; A, lysis of Brucella after incubation with monoclonal antibody B7 and addition of 10% guinea pig serum; B and D, deposition of complement C3 on the surface of Brucella after incubation with monoclonal antibody B7 and addition of 10% guinea pig serum; C and E, deposition of complement C5 on the surface of Brucella after incubation with monoclonal antibody B7 and addition of 10% guinea pig serum.

[0025] Figure 3 It is the opsonization, phagocytosis and killing effect mediated by monoclonal antibody B7; A. The number of Brucella phagocytosed by macrophages after being conditioned by monoclonal antibody B7 and adding 10% guinea pig serum; B. Immunofluorescence test to verify the number of Brucella phagocytosed by macrophages after being conditioned by monoclonal antibody B7 and adding 10% guinea pig serum; C. Detection of the number of live bacteria in macrophages 48 hours after infection, expressed as a killing index.

[0026] Figure 4 The immunoprotective effect of monoclonal antibody B7 in the Brucella mouse infection model; A, experimental flow chart; B, the effect of intraperitoneal injection of monoclonal antibody B7 on the body weight of infected mice; C, the effect of intraperitoneal injection of monoclonal antibody B7 on the spleen index of infected mice; D, the effect of intraperitoneal injection of monoclonal antibody B7 on the bacterial load in the spleen of infected mice; E, fluorescent staining to verify the effect of intraperitoneal injection of monoclonal antibody B7 on the bacterial load in the spleen of infected mice.

[0027] Figure 5 The spleen tissue sections of mice in each group reflect the effect of intraperitoneal injection of monoclonal antibody B7 on the pathological changes of the spleen of mice caused by Brucella infection.

[0028] Figure 6 The liver tissue sections of mice in each group reflect the effect of intraperitoneal injection of monoclonal antibody B7 on the pathological changes in the liver of mice caused by Brucella infection. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0030] 1. Materials

[0031] 1.1. Strains, cells and culture conditions

[0032] Brucella bovis A19 was obtained from the Shaanxi Provincial Veterinary Drug Supervision Institute. The strain was cultured in TSB medium at 37°C with shaking or statically cultured on TSA plates at 37°C. RAW264.7 macrophages were stored in the laboratory of the present invention team in high-glucose DMEM cell culture medium supplemented with 10% fetal bovine serum and cultured at 37°C, 5% CO2. Six-week-old female BALB / c mice were purchased from Chengdu Dashuo Biological Co., Ltd. and raised in an environment that meets biosafety requirements. They were given sufficient feed and drinking water to maintain circadian rhythms. The entire experiment was approved and supervised by the Experimental Animal Management and Ethical Review Executive Committee of Northwest A&F University.

[0033] 2. Indirect ELISA

[0034] In order to obtain inactivated Brucella with good antigenicity, the culture of Brucella bovis A19 in the logarithmic growth phase was centrifuged, the cells were collected, resuspended in PBS solution containing 0.7% formaldehyde, and placed at 37°C for 72 hours. A portion of the culture was spread on a TSA plate and cultured at 37°C for 72 hours to check for live bacteria. The inactivated culture was heated to 1×10 9 CFU / mL concentration dilution, 100μL per well, added to 96-well microplate, overnight coated with antigen, washed 3 times with PBS solution (PBST) containing 0.05% Tween 20 on the next day, added PBST containing 5% skim milk powder, 37℃, blocked for 2h. Then washed 3 times with PBST solution, added 100μl PBS solution containing 2μg monoclonal antibody B7, incubated at 37℃ for 1h, and set PBS solution as negative control; after washing 3 times with PBST, added 100μL PBS containing HRP-labeled goat anti-mouse IgG antibody (1:5000), incubated at 37℃ for 1h, washed 3 times with PBST solution, added 100μL TMB colorimetric solution to each well, incubated at 37℃ for 30min, finally added 50μL 2M H2SO4 solution to terminate the reaction, and read at absorbance 450nm using an enzyme reader.

[0035] 3. Immunofluorescence

[0036] RAW264.7 cells were cultured at 2×10 5100 μg / mL of gentamicin were added to the 24-well plate containing the cell slide, and the plate was placed in a 37°C, 5% CO2 cell culture incubator for overnight culture. Brucella bovis A19 was then added at a multiplicity of infection (MOI) of 100, and the plate was placed in a 37°C, 5% CO2 incubator for 4 h. The culture medium was discarded, the plate was washed 3 times with sterile PBS, and then a cell culture medium containing 50 μg / mL gentamicin was added and cultured for 1 h to kill the bacteria adhering to the cell membrane. The culture medium was discarded, the plate was washed 3 times with sterile PBS, a 4% paraformaldehyde solution was added, and the plate was allowed to stand at room temperature for 30 min. After washing 3 times with PBS, a 0.2% TritonX-100 solution was added, and the plate was allowed to stand at room temperature for 20 min. The cells were washed three times with PBS, and 100 μL of PBS solution containing 2 μg of monoclonal antibody B7 was added to each well, and incubated overnight at 4°C. At the same time, PBS solution was set as a negative control. After washing three times with PBS, AlexaFluor488-labeled goat anti-mouse IgG antibody (1:500) was added and incubated at room temperature in the dark for 1 h. The cells were washed three times with PBS, and then the cell slides were removed and inverted on a slide with anti-fluorescence quencher (containing DAPI) added, and the photos were scanned using a laser confocal microscope.

[0037] For the macrophage phagocytosis test, the cell membrane of RAW264.7 macrophages was labeled with red fluorescence using the Dil cell membrane labeling kit in advance, and the cells were infected with Brucella bovis A19 at an MOI of 10, and subsequent experiments were carried out according to the above steps.

[0038] Monoclonal antibody B7 is a Brucella OMP16 monoclonal antibody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.2.

[0039] SEQ ID NO.1:

[0040] EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISY

[0041] SGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARYDYDGDWY

[0042] FDVWGAGTTVTVSS

[0043] SEQ ID NO.2:

[0044] ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTL

[0045] RPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK

[0046] Note: The black bold font is the CDR area

[0047] 4. Agglutination test

[0048] 1×10 5 CFU bovine Brucella A19 was resuspended in sterile PBS solution, and 20 μg monoclonal antibody B7 was added and incubated at 37°C for 1 hour. At the same time, 20 μg IgM isotype control or an equal volume of sterile PBS solution was added to the bacterial solution as an isotype or negative control. Then, an appropriate amount of bacterial solution was added to the slide, and after heat fixation, it was stained with methylene blue stain and observed under a 1000x optical microscope.

[0049] 5. Complement killing test

[0050] In order to test whether the monoclonal antibody B7 can kill Brucella by activating the complement system, we injected 1×10 3 CFU bovine Brucella A19 was resuspended in sterile PBS solution, 40 μg monoclonal antibody B7 (incubated at 56°C for 30 min to inactivate complement) was added and incubated at 37°C for 30 min. IgM isotype control antibody or sterile PBS solution was set as isotype control or negative control. Guinea pig serum at a final concentration of 10% was added as a complement source, incubated at 37°C for 2 h. Subsequently, the mixture was spread on a TSA plate, cultured at 37°C for 72 h, and the number of colonies was recorded.

[0051] 6. Complement deposition test

[0052] To detect the deposition of membrane attack complex (MAC) on the bacterial surface, complement C3 and C5 were detected by flow cytometry. Briefly, 1×10 8CFU bovine Brucella A19 was resuspended in PBS solution containing 1% BSA, 40 μg monoclonal antibody B7 was added and incubated at 37°C for 30 min. At the same time, IgM isotype control antibody or sterile PBS solution was set as isotype control or negative control, guinea pig serum with a final concentration of 10% was added as a complement source, and the mixture was incubated at 37°C for 2 h. Then centrifuge at 3000r / min for 5min, add 4% paraformaldehyde solution at room temperature for 30min, centrifuge, discard the supernatant, add PBS solution containing 1% BSA to resuspend, centrifuge and discard the supernatant once, add anti-mouse complement C3 antibody (1:100) or anti-mouse complement C5 antibody (1:100), incubate at 4℃ overnight, wash twice with PBS solution containing 1% BSA, add Alexa Fluor 488-labeled goat anti-mouse IgG antibody (1:200), incubate at room temperature for 2h, wash twice with PBS solution, and resuspend in 200μL PBS solution, and analyze using flow cytometer. The integrated geometric mean fluorescence is measured as the product of the percentage of gated events that pass the fluorescence threshold and the average fluorescence of events that pass the threshold.

[0053] 7. Macrophage opsonization and killing assay

[0054] RAW264.7 cells were cultured at 2×10 5 The cells were added to a 24-well cell culture plate at a concentration of 100 / well and cultured in a 37°C, 5% CO2 cell culture incubator overnight. Subsequently, Brucella bovis A19 pre-conditioned with 40 μg monoclonal antibody B7 for 30 min was added at an infection multiplicity MOI = 10. At the same time, an IgM isotype control antibody or a sterile PBS solution was set as an isotype or negative control. After adding 10% guinea pig serum, the cells were cultured in a 37°C, 5% CO2 incubator for 4 h. The culture medium was discarded, the cells were washed 3 times with sterile PBS, and then cell culture medium containing 50 μg / mL gentamicin was added and cultured for 1 h. After washing 3 times with sterile PBS, 0.5% TritonX-100 was added and the cells were cultured at 37°C for 10 min. Subsequently, the cells were serially diluted in multiples, spread on TSA plates, and CFU was calculated.

[0055] In order to detect the opsonization and killing effect of macrophages on Brucella, we conducted a macrophage bactericidal test. The cell conditioning and infection steps are as shown above. After culturing in a cell culture medium containing 50μg / mL gentamicin for 1h, the cells were washed 3 times with sterile PBS and a cell culture medium containing 25μg / mL gentamicin was added. This time was considered to be the 0th hour after infection. Then, the culture medium was discarded 48h after infection, and after washing 3 times with sterile PBS, 0.5% TritonX-100 was added and placed at 37°C for 10min. Then, the cells were diluted in multiples and spread on TSA plates to calculate CFU. The killing ability of macrophages is expressed by the killing index, which is the macrophage killing index = (the number of bacteria phagocytosed by macrophages - the number of bacteria surviving at 48h) / the number of bacteria phagocytosed by macrophages × 100 (%).

[0056] 8. Mouse immune protection test

[0057] Six-week-old female BALB / c mice were raised for one week before the formal experiment to reduce the experimental error caused by environmental stress. Brucella bovis A19 in the logarithmic growth period was taken and washed three times with sterile PBS. 7 The concentration of CFU / mouse was suspended in 0.2 ml sterile PBS, and mice were infected by intraperitoneal injection with monoclonal antibody B7 (80 μg / mouse). This was the 0th day after infection. The weight of mice was measured on the 1st, 3rd, 7th, 14th and 21st days after infection. After anesthesia with anhydrous ether, the mice were killed by dislocation of the neck, and the spleens of mice were removed and weighed. Then a part was placed in 4% paraformaldehyde fixative, and pathological sections were made after fixation, embedding, HE staining or fluorescent staining, and images were collected under 100x and 400x digital slice scanners. Another part was weighed, placed in sterile PBS, ground to a homogenate state, and spread on TSA plates after serial dilution to calculate the bacterial load in the spleen.

[0058] 9. Results

[0059] 9.1. Monoclonal antibody B7 can bind and agglutinate Brucella

[0060] To test the immunoprotective properties of monoclonal antibody B7, we used Brucella bovis A19 as a model strain and first tested whether monoclonal antibody B7 could bind to Brucella bovis A19. We used monoclonal antibody B7 as the primary antibody in an immunofluorescence assay to test its binding to Brucella in infected cells.

[0061] The results are as follows Figure 1 As shown in A. Monoclonal antibody B7 binds significantly to Brucella bovis A19 in the cell (green fluorescent part). Through iELISA test, we further proved that monoclonal antibody B7 can effectively bind to Brucella bovis A19 ( Figure 1 B). In addition, due to its special pentamer structure, IgM can better agglutinate related pathogens, limit the spread of pathogens in the host, and facilitate the host cells to eliminate pathogens. Therefore, we further tested whether this mAb can agglutinate Brucella. The results are as follows: Figure 1 As shown in Figure C, the Brucella incubated with monoclonal antibody B7 underwent significant agglutination and showed a clustered distribution under the microscope, while the Brucella incubated with IgM isotype control antibody and PBS still showed a scattered distribution of single bacteria under the microscope. The above results show that monoclonal antibody B7 can effectively bind and agglutinate Brucella.

[0062] 9.2 Monoclonal antibody B7 exerts complement-dependent lysis by activating the formation of membrane attack complex (MAC)

[0063] The complement system is an important component of the host immune system. After activating the complement system cascade reaction through the classical pathway, alternative pathway and lectin pathway, it can exert a series of immune activities such as immune killing and conditioning. Among them, the cascade reaction of antigen-antibody binding to form an immune complex to recruit and activate complement is the classic activation pathway of the complement system. Among all antibody subtypes, IgM has the strongest ability to activate the complement system. Therefore, we first tested whether the monoclonal antibody B7 can effectively activate the complement system after binding to Brucella and exert complement-mediated lytic activity.

[0064] The results are as follows Figure 2 As shown in A, after adding 10% guinea pig serum as a complement source, the colony count of the reaction system of Brucella opsonized with monoclonal antibody B7 was significantly lower than that of the PBS and IgM isotype control antibody opsonized groups (P < 0.01). Studies have shown that after the antigen-antibody complex is formed, complement C1 is first recruited to form the C1 complex, followed by cleavage of C2 and C4 to form C3 convertase, C3 convertase cleaves C3 and C4b2a to further form C5 convertase, C5 convertase cleaves C5 and C6, C7, C8 and C9 to form a membrane attack complex (Membrane Attack Complex, MAC) deposited on the bacterial surface to form hydrophilic pores, destroying the balance of osmotic pressure inside and outside the bacterial cells and causing cell lysis. In this process, the formation of C3 convertase is considered to be the central step of the complement cascade reaction, and complement C5 is a marker of MAC formation. Therefore, we used flow cytometry to detect the deposition level of complement C3 and C5 on the surface of Brucella. The results showed that the surface deposition of complement C3 and C5 (integrated geometric mean fluorescence intensity) of Brucella opsonized with monoclonal antibody B7 was significantly higher than that of the two control groups (p<0.001)( Figure 2B~E), which shows that monoclonal antibody B7 forms an antigen-antibody complex after binding to Brucella, and further activates complement C3 and C5 through cascade reactions, forming a membrane attack complex that is deposited on the surface of Brucella, causing Brucella to dissolve.

[0065] 9.3 Monoclonal antibody B7 promotes macrophage phagocytosis and killing of Brucella through opsonization

[0066] When the complement system is activated, in addition to exerting direct bacteriolytic effects, it can also enhance the phagocytic and killing abilities of macrophages through complement receptors (CR) on the surface of macrophages. Therefore, we further tested the opsonophagocytic and killing effects of B7 mAb mediated by complement.

[0067] The results are as follows Figure 3 As shown in A, the number of Brucella phagocytosis by macrophages after conditioning with monoclonal antibody B7 and adding 10% guinea pig serum was significantly higher than that in the IgM isotype control, PBS and inactivated complement groups (P < 0.01). Immunofluorescence assay also further verified this result ( Figure 3 B), which indicates that monoclonal antibody B7 may enhance the phagocytic ability of macrophages through complement receptors after binding to Brucella.

[0068] Brucella, as a facultative intracellular parasite, can survive and proliferate in a variety of mammalian cells, including professional or non-professional phagocytes, epithelial cells and fibroblasts, and macrophages are its main host cells. Therefore, in order to verify whether the above test results show that monoclonal antibody B7 enhances the phagocytosis of macrophages by activating the complement system, or whether Brucella enhances its ability to infect macrophages through this pathway, we detected the number of live bacteria in macrophages 48 hours after infection. The results are as follows: Figure 3 As shown in Figure C, after being conditioned by monoclonal antibody B7 and activated by the complement system, the killing ability of macrophages against Brucella was significantly enhanced (p < 0.001). The above results show that after being conditioned by monoclonal antibody B7 and the complement system, the phagocytosis and killing ability of mouse macrophages against Brucella was significantly enhanced.

[0069] 9.4 Monoclonal antibody B7 exhibits effective immune protection in the Brucella mouse infection model

[0070] In in vitro experiments, we have verified that monoclonal antibody B7 can exert complement lysis and macrophage anti-Brucella activity by activating the complement system. To further verify the immunoprotective effect of the antibody, we selected BALB / C mice to establish a Brucella mouse infection model and intraperitoneally injected monoclonal antibody B7 and Brucella 1 day before infection to detect the immunoprotective effect of the antibody ( Figure 4 A).

[0071] The results showed that the body weight of mice injected with monoclonal antibody B7 was significantly higher than that of mice in the IgM isotype control group and PBS group on days 1, 3, 7, 14 and 21 after infection (p < 0.05 or p < 0.01). Figure 4 B), the spleen index of mice in the monoclonal antibody B7 group had no significant difference with that of the IgM isotype control group and PBS group on the first day after infection (p>0.05), and was significantly higher than that of the IgM isotype control group and PBS group on the third day after infection, while it was significantly lower than that of the two control groups on the 7th, 14th and 21st days after infection (p<0.05; p<0.01; p<0.001). Moreover, after infection, the spleen index of mice in the monoclonal antibody B7 group tended to be more stable than that of the other two groups ( Figure 4 C). After testing the bacterial load in the spleen of mice at different times after infection, we found that except for the 1st day after infection, there was no significant difference in the bacterial load in the spleen of the three groups of mice (p>0.05), the bacterial load in the spleen of the monoclonal antibody B7 group mice was significantly lower than that of the IgM isotype control group and PBS group mice on the 3rd, 7th, 14th and 21st days after infection (p<0.05, p<0.01 or p<0.001) ( Figure 4 D). Subsequently, after fluorescent staining of Brucella in the spleen of mice, it was found that the number of Brucella in the spleen of mice in the monoclonal antibody B7 group was significantly lower than that in the IgM isotype control group and PBS group (green fluorescence), which was consistent with the results of the bacterial load in the spleen of mice ( Figure 4 E). In conclusion, the above results indicate that intraperitoneal injection of monoclonal antibody B7 can significantly reduce the effect of Brucella on the body weight and spleen index of mice and the number of Brucella in the spleen of mice.

[0072] 9.5 Monoclonal antibody B7 reduces spleen and liver damage caused by Brucella infection

[0073] To further evaluate the effect of injection of monoclonal antibody B7 on pathological changes in the spleen and liver caused by Brucella infection in mice, we observed the pathological damage of the spleen and liver of mice through pathological tissue sections. Figure 5As shown in the figure, in general, the capsule of the spleen tissue was intact, the capsule and splenic trabecular fibrous connective tissue did not proliferate, and the boundary between the white pulp and the red pulp was clear. However, after infection with Brucella, the three groups of mice all showed varying degrees of white pulp atrophy, decreased lymphocyte counts, and extramedullary hematopoiesis, that is, increased red blood cells and granulocytes. Among them, on the 7th day after infection, the spleen lesions of mice were the most serious. The mice in the PBS and IgM isotype control groups showed obvious white pulp atrophy, decreased lymphocyte counts, and extramedullary hematopoiesis. The mice in the antibody group also showed a certain degree of extramedullary hematopoiesis. On the 14th and 21st days after infection, the degree of spleen lesions in the three groups of mice was alleviated. In addition, on the 7th, 14th, and 21st days after infection, the degree of lesions in the antibody group mice was significantly lower than that in the PBS and IgM control group mice.

[0074] During the experiment, we found that the pathological sections of mouse liver showed obvious necrotic lesions on the 7th, 14th and 21st days after infection. Figure 6 As shown in the figure, on the 7th day after Brucella infection, the liver tissue lesions were the most severe, with necrosis, inflammatory cell infiltration and hemorrhage. The degree of lesions in the antibody-injected mice was significantly lower than that in the control group. On the 14th day, the liver lesions in the mice gradually alleviated, and the degree of lesions in the antibody-injected mice was still significantly lower than that in the control group. On the 21st day, only slight lesions were found in the livers of the three groups of mice, and there was no significant difference in the degree of damage. The above results show that after the mice were intraperitoneally injected with monoclonal antibody B7, the pathological damage of the spleen and liver was significantly reduced, and the pathological damage caused by Brucella infection in mice could be significantly reduced.

[0075] Brucellosis is a global bacterial infectious disease with endemic characteristics. Humans are mainly infected through direct contact with sick animals and their secretions or eating meat and dairy products that are not strictly sterilized. Currently, there is no licensed human brucellosis vaccine for the prevention of the disease, nor is there an effective treatment for the treatment of brucellosis. Once the disease occurs, patients need to receive antibiotic treatment for weeks or months. Despite this, relapses and treatment failures still occur. Here, we report an IgM monoclonal antibody against Brucella OMP16. The immunoprotective effect of this antibody was evaluated by complement killing test, macrophage phagocytosis and killing test and mouse protection test. Our study showed that the anti-Brucella OMP16 monoclonal antibody can bind and agglutinate Brucella A19, can directly kill Brucella by activating complement C3 and C5 to form a membrane attack complex (MAC) on the surface of Brucella, and can enhance the phagocytic and killing ability of macrophages against Brucella by activating the complement system. Finally, the antibody was further confirmed to have good immunoprotective activity in mouse experiments.

[0076] The Fab and Fc segments of antibodies are the main functional regions of antibodies and have been shown to play a role in multiple pathogenic steps of bacterial infection. In the early stage of bacterial invasion of the host, the inhibitory adhesion mediated by antibody Fab can be achieved by binding to pathogen-specific sites, followed by agglutination reactions, causing multiple bacteria to aggregate to form aggregates, preventing further spread of bacteria in the host and facilitating the clearance of bacteria by phagocytes in the host. Therefore, we first tested whether B7 monoclonal antibody could agglutinate Brucella. Our results showed that after binding to Brucella, B7 monoclonal antibody further agglutinated and formed obvious aggregates, indicating that the antibody can play a certain immune protective role through agglutination reactions. Previous studies have shown that after antibody Fab binds to antigens, it will cause changes in the antibody structure, expose the complement binding sites in the CH3 region of the antibody, recruit and activate complement components, and cause a series of cascade reactions, exerting complement-mediated lysis, complement-dependent cellular phagocytosis, and complement-dependent cytotoxicity. Therefore, we further tested whether the antibody can exert complement-dependent macrophage phagocytosis and killing by activating the complement system. The results showed that the antibody can activate complement C3 and C5 to form a membrane attack complex (MAC) and deposit on the surface of Brucella, exerting a direct bacteriolytic effect; it can regulate macrophages through the complement system to enhance the phagocytic and killing ability of Brucella.

[0077] In order to further study whether this antibody also has a good immune protection effect in vivo, we established a Brucella mouse infection model to further verify the immune protection effect of this antibody in mice. Our study showed that after Brucella infection in mice, the weight of mice in the antibody treatment group was significantly higher than that of mice in the control group, and the spleen index was significantly lower than that of mice in the control group except for the 1st and 3rd days, which indicated that the injection of antibodies significantly reduced the impact of Brucella infection on the physiological condition of mice. Further spleen bacterial load detection and pathological damage assessment also showed that after the injection of antibodies, not only the Brucella load in the spleen of mice was significantly reduced, but also the pathological damage of the spleen and liver was greatly reduced, which further proved the immune protection effect of this antibody.

[0078] In summary, the present invention proves that IgM anti-Brucella OMP16 monoclonal antibody can agglutinate Brucella, exert complement-mediated bacteriolysis by activating the complement system, enhance macrophage phagocytosis and killing activity by antibody opsonization, and this protective effect is confirmed in mice. The present invention study proves that Brucella OMP16 monoclonal antibody has significant immune protection effect, and can be further developed and utilized as a potential therapeutic antibody. At the same time, from the perspective of antibodies, it is further proved that Brucella OMP16 is a good immune protection antigen, which can be used as an effective immune target for the study of related vaccines.

[0079] The above embodiments are only preferred implementation cases of the present invention, and are not intended to limit the implementation scope of the present invention. All technical solutions within the scope of the present invention belong to the protection scope of the present invention.

Claims

1. Use of a Brucella OMP16 monoclonal antibody in the preparation of a product for inhibiting Brucella, characterized in that: The amino acid sequence of the heavy chain variable region of the Brucella OMP16 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2. The Brucella OMP16 monoclonal antibody is an IgM antibody, and the Brucella is Brucella bovis A19.

2. The use according to claim 1, characterized in that: The Brucella OMP16 monoclonal antibody is used for preparing a product for improving the immune protection of a Brucella infected population.

3. The use according to claim 1, characterized in that: The Brucella OMP16 monoclonal antibody is used to prepare a product for reducing spleen or liver damage caused by Brucella infection.

4. The use according to claim 1, characterized in that: The Brucella OMP16 monoclonal antibody is used for preparing products for preventing or treating brucellosis.

5. The use according to any one of claims 1 to 4, characterized in that: The product is a medicine or a vaccine.

6. The use according to claim 5, characterized in that: The vaccine includes a vaccine adjuvant or excipient.

7. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients or carriers.

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