Monoclonal antibody for resisting staphylococcus aureus iron ion regulatory surface determinant B protein and application thereof

Abstract

The invention discloses a monoclonal antibody for resisting staphylococcus aureus iron ion regulatory surface determinant B protein and application of the monoclonal antibody. Amino acid of a structural domain recognized by the monoclonal antibody is shown as SEQ ID NO.1. The monoclonal antibody is generated by culturing a host cell of an expression vector, the generated monoclonal antibody is secreted into supernate, and the supernate is secreted into the staphylococcus aureus iron ion regulatory surface determinant B protein to obtain the staphylococcus aureus iron ion regulatory surface determinant B. The staphylococcus aureus iron ion regulatory surface determinant B protein. Purifying through a chromatography technology; the obtained antibody can be used for treating septicopyemia caused by staphylococcus aureus / MRSA infection, and has important significance in preventing and treating staphylococcus aureus infection.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, in particular to a fully humanized monoclonal antibody against Staphylococcus aureus iron ion regulating surface determinant cluster B protein, and also to the application of the antibody. Background Art

[0002] Staphylococcus aureus (abbreviated as S. aureus) is an important pathogen that causes hospital and community infections. It is highly pathogenic. The mortality rate of sepsis caused by S. aureus infection is as high as 20%, and it is multidrug-resistant and pan-drug-resistant. In recent years, antibiotic resistance has made S. aureus infection face a severe situation of "no cure", and the emergence of methicillin-resistant Staphylococcus aureus (MRSA) has exacerbated the dilemma of clinical treatment. In a study published in the journal "Lancet" in 2022, a statistical analysis of mortality rates associated with drug-resistant bacterial infections worldwide was conducted. The results showed that S. aureus infection ranked first, directly causing more than 1 million deaths each year, of which more than 500,000 deaths were caused by sepsis caused by infection. WHO identified MRSA as one of the 12 "super bacteria" that pose a deadly threat to humans, and called on countries to introduce policies to "highly prioritize" the development of new drugs. Therefore, there is an urgent need for effective means to deal with S. aureus infection and prevent antibiotic resistance.

[0003] Studies have shown that antibody-based immune strategies are an effective means of treating S. aureus infections and an important alternative therapy to prevent antibiotic resistance. S. aureus has many virulence factors and complex pathogenic factors. Iron Surface Determinant B (IsdB) is a cell wall anchor protein widely expressed by S. aureus. It is well conserved and plays an important role in the bacterial uptake and metabolism of the nutrient heme iron and in maintaining normal bacterial growth. Antibody therapy targeting IsdB showed significant protective effects in a mouse model of S. aureus infection.

[0004] Therefore, based on the above background, it is necessary to develop a fully human monoclonal antibody against the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a monoclonal antibody against the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus; a second object of the present invention is to provide the use of the monoclonal antibody against the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus in the preparation of a reagent that specifically binds to the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus; a third object of the present invention is to provide the use of the monoclonal antibody against the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus in the preparation of a drug for treating or preventing Staphylococcus aureus infection.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A monoclonal antibody against Staphylococcus aureus iron ion regulation surface determinant cluster B protein, wherein the domain recognized by the monoclonal antibody at least includes the NEAT2 domain at positions 341-458 of the Staphylococcus aureus iron ion regulation surface determinant cluster B protein, and its amino acids are shown in SEQ ID NO.1.

[0008] KMTDLQDTKYVVYESVENNESMMDAFVKHPIKTGMLNGKKYMVMETTNDDYWKDFMV

[0009] EGQRVRTISKDAKNNTRTIIFPYVEGKTLYDAIVKVHVKTIDYDGQYHV RIVDKEAFTKAN

[0010] Preferably, the monoclonal antibody of the present invention comprises a heavy chain and a light chain, the heavy chain comprises variable regions CDR1, CDR2 and CDR3, and the amino acid sequences thereof are as shown in SEQ ID NO.2 (GGSLNKYY), SEQ ID NO.3 (IQDSGRT), and SEQ ID NO.4 (ARDRVDSPYSNSWSRFDY); the light chain comprises variable regions CDR1, CDR2, and CDR3, and the amino acid sequences thereof are as shown in SEQ ID NO.5 (NLGSKS), DDD, and SEQ ID NO.6 (QVWDSTSDVVV).

[0011] Preferably, the constant region of the monoclonal antibody includes any one of human IgM, IgA or IgA constant regions.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.7; and the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.8.

[0013] SEQ ID NO.7:

[0014] QVQLQESGPGLVKPSETLSLTCTVSGGSLNKYYWTWIRQSPNNGLEWIGYIQDSGRTDSNP

[0015] SLKSRVSISLDTSTNQISLKLTSVAAADTAIYYCARDRVDSPYSNSWSRFDYWGQGKLVTV

[0016] SS

[0017] SEQ ID NO.8:

[0018] QSVLTQPPSVPVAPGRSAVITCGGHNLGSKSVHWYQQKPGQAPVLVVYDDDDRPSGIPAR

[0019] FSGSTSERTATLTISRVEAGDEADYYCQVWDSTSDVVVFGGGTKLAVL

[0020] 2. Use of the monoclonal antibody against the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus in the preparation of a reagent that specifically binds to the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus.

[0021] 3. Use of the monoclonal antibody against the iron ion regulating surface determinant cluster B protein of Staphylococcus aureus in the preparation of a drug for treating or preventing Staphylococcus aureus infection.

[0022] The beneficial effects of the present invention are as follows: the present invention provides an antibody against the Staphylococcus aureus IsdB NEAT2 domain protein, the antibody is produced by culturing a host cell expressing a vector to generate a monoclonal antibody, the generated monoclonal antibody is secreted into a supernatant, and is purified by chromatography technology; the obtained antibody can treat sepsis caused by Staphylococcus aureus / MRSA infection, and is of great significance in preventing and treating Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0024] Figure 1 This is the SDS-PAGE image of the purified protein of NEAT2 domain of IsdB of Staphylococcus aureus;

[0025] Figure 2 Purification and SDS-PAGE analysis of fully human antibodies against Staphylococcus aureus IsdB NEAT2;

[0026] Figure 3ELISA was used to detect the specific binding of fully human monoclonal antibodies to IsdB NEAT2 protein;

[0027] Figure 4 To measure antigen-antibody affinity by biofilm interferometry (BLI);

[0028] Figure 5 To determine the epitope type of the fully human antibody against Staphylococcus aureus IsdB NEAT2;

[0029] Figure 6 Different concentrations of antibodies inhibited the proliferation of MRSA252 bacteria;

[0030] Figure 7 Established a lethal model for MRSA252 and USA300 challenge;

[0031] Figure 8 The antibody has a preventive and protective effect in the MRSA252 and USA300 lethal challenge models;

[0032] Fig. 9 To evaluate the protective effect of the antibody in the MRSA252 sublethal challenge model;

[0033] Fig.10 To evaluate the protective effect of this antibody in the USA300 sublethal challenge model. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] Example 1. Expression and purification of Staphylococcus aureus IsdB NEAT2 domain protein

[0036] The IsdB NEAT2 gene was amplified by PCR from genomic DNA of Staphylococcus aureus strain. After DNA sequencing, it was expressed in Escherichia coli, cultured overnight at 37°C in LB medium containing ampicillin, and the cells were harvested by centrifugation. The IsdB NEAT2 protein was purified using GST affinity filler. The amino acid sequence of the IsdB NEAT2 protein is shown in SEQ ID NO.1. SDS-PAGE analysis of the IsdB NEAT2 domain protein is shown in Figure 1 shown.

[0037] Example 2: Cloning of anti-Staphylococcus aureus IsdB NEAT2 antibody and expression of fully human antibody

[0038] HEK293F cells were cultured in a 1L cell culture flask and the concentration was adjusted to 1×10 6 / ml, placed in a constant temperature incubator at 37°C, 5% CO2, 120rpm.

[0039] Sequences encoding the heavy chain (SEQ ID NO.9) and the light chain (SEQ ID NO.10) were constructed into the pcDNA3.1 vector, respectively, and connected for 24 hours at a 16°C thermostat. The connection system is shown in Table 1:

[0040]

[0041] The ligated plasmids were transferred into DH5α competent cells, single clones were selected, and after identification, they were preserved for subsequent plasmid extraction and use.

[0042] The heavy chain and light chain plasmids constructed above were mixed at a mass ratio of 1:1, and co-transfected with transfection reagent PEI into HEK293 cells. HEK293 basal medium (OPM) was supplemented 4 hours after transfection, and cultured in a 37°C, 5% CO2 incubator for 96 hours. The transfection supernatant was collected by centrifugation at 3000×g for 30 minutes, purified by protein A affinity chromatography (IgG Protein A beads), concentrated by ultrafiltration tube, detected by NanoDrop, and the expression and purification of the antibody were tested by SDS-PAGE ( Figure 2 ). The results showed that the purified antibody had high purity.

[0043] Example 3. Determination of expressed antibody binding activity

[0044] The recombinantly expressed IsdB NEAT2 protein was used as the antigen to coat the ELISA plate, 100 μL per well, coated overnight at 4°C, and blocked with blocking solution at 37°C for 2 hours. 100 μL Overlapping PCR transfection expression antibody (primary antibody) stock solution was added. The positive control was the positive plasma sample stock solution (1:50 dilution) and the purified antibody (1:1000 dilution), 100 μL per well. The negative control was the negative plasma sample stock solution (1:50 dilution) and the negative control irrelevant antibody IgG1 0.5 μg / mL, 100 μL per well, blank plus 100 μL blocking solution for 3 replicates, incubated at 37°C for 1 hour. 200 μL PBST buffer was added to each well to wash the plate, and Goat-Anti-IgG-Fab-HRP (secondary antibody) diluted 1:5000 in PBST was incubated at 37°C for 40 minutes. Wash the plate with PBST buffer as above, add 100 μL of TMB to each well, incubate at 37°C in the dark for 8 min, immediately terminate with 50 μL of 2M H2SO4, and detect OD value at 450 nm. Calculate the mean of negative control irrelevant antibody IgG1, calculate the threshold value (3 times the mean value), and the value greater than the threshold value is considered positive antibody.

[0045] Experiments show that the fully human monoclonal antibody of the present invention can specifically bind to the IsdB NEAT2 protein ( Figure 3 ).

[0046] Example 4: Determination of antigen-antibody affinity using biofilm interferometry (BLI)

[0047] Biomembrane interferometry was used to measure the affinity of the antibody to the IsdB NEAT2 protein. 50 nM of the monoclonal antibody obtained by expression and purification was immobilized on the AHC sensor. The IsdB NEAT2 protein was gradient diluted to different final concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM) and added to the sample plate in sequence. The program was set as: "Baseline detection-Loading detection-Baseline detection-Binding detection-Dissociation detection-Probe regeneration". The equilibrium dissociation constant (KD) was calculated. Figure 4 The results showed that the KD of the antibody and IsdB NEAT2 was 9.724×10 -9 M.

[0048] The results showed that the antibody of the present invention had good affinity to the IsdB NEAT2 antigen.

[0049] Example 5. Determination of Antibody Epitope Type

[0050] Add 2 μL of reduced loading buffer (containing β-mercaptoethanol) to 1 μg of IsdB NEAT2 protein sample (8 μL), and cook in a 100°C constant temperature metal bath for 5 minutes. Use for SDS-PAGE loading analysis, adjust the voltage to 120V, and the electrophoresis time to 1-2 hours. Stop the electrophoresis when bromophenol blue just runs out. Take out the electrophoresis gel and then rinse with water.

[0051] The PVDF membrane activated by methanol was transferred with a transfer instrument (3 layers of filter paper, PVDF membrane, electrophoresis gel, 3 layers of filter paper), and the voltage was adjusted to 60V for 25 minutes. After the transfer, the PVDF membrane was removed and washed once with PBST, and then the PVDF membrane was blocked in 5% skim milk powder at 4°C for 2 hours. The PVDF membrane was removed and washed 3 times with PBST for 5 minutes each time, and then the purified antibody was diluted to 1μg / mL with PBST and incubated at 4°C for 1 hour. The PVDF membrane was washed 3 times with PBST solution, and then anti-human HRP-labeled IgG was added at 1:5000 and incubated on a horizontal shaker at room temperature for 40 minutes. The PVDF membrane was washed 3 times with PBST solution, and the PVDF membrane was placed in a clean dish. About 1mL of exposure color solution was added to each membrane, and photos were taken for observation. Figure 5It was shown that it was able to bind to the denatured IsdB NEAT2, thus judging that the antibody epitope described in the present invention was a linear epitope.

[0052] Example 6: The antibody inhibits the proliferation of Staphylococcus aureus

[0053] After activating and culturing the MRSA252 strain of Staphylococcus aureus, the OD was adjusted to 1.0, and 90 μL of bacterial solution and 10 μL of antibodies of different concentrations (final concentrations were 3.734, 0.951, and 0.414 μg / μL, respectively) and saline as a control were added to a 96-well plate. The plates were incubated in a 37°C incubator for 16 hours, and the number of bacterial colonization was detected. Figure 6 As shown, the antibody can significantly inhibit the proliferation of MRSA252 strain in vitro, and is dose-dependent with the antibody concentration.

[0054] Example 7: Establishment of Staphylococcus aureus lethal model

[0055] The concentration of MRSA252 was adjusted to 9.0×10 9 CFU / mL, 6.0×10 9 CFU / mL, 4.0×10 9 CFU / mL, 2.0×10 9 CFU / mL and 1.0×10 9 CFU / mL. Adjust the concentration of USA300 to 1.5×10 9 CFU / mL, 1.0×10 9 CFU / mL, 0.8×10 9 CFU / mL, 0.5×10 9 CFU / mL. Mouse challenge: Take female Balb / C mice aged 6-8 weeks and divide them into cages according to their weight. Inject the above concentrations of MRSA252 and USA300 into tail veins at a volume of 100 μL / mouse. After the challenge, record the survival of the mice every 24 hours and continue to observe for 7 days. Figure 7 As shown, the results showed that the dose was 6.0×10 8 CFU / MRSA252 and 1.0×10 8 The mortality rate of mice challenged with USA300 CFU / mouse through the tail vein can reach over 90%.

[0056] Example 8: Evaluation of Antibody Preventive Protection Effect

[0057] In the MRSA252 lethal model, 20 mice were divided into 2 groups, 10 mice in each group. The first group was injected with 100 μg of antibody, 100 μL; the second group was injected with sterile PBS, 100 μL. After 24 hours of administration, 6.0×10 8The mice were challenged with MRSA252 at a concentration of 100 CFU / mouse via the tail vein. The mice were observed for 7 days and their survival was recorded.

[0058] In the USA300 lethal model, 20 mice were divided into 2 groups, 10 mice in each group. The first group was injected with 100 μg of antibody, 100 μL; the second group was injected with sterile PBS, 100 μL; 24 hours after administration, 1.0×10 8 The mice were challenged with USA300 at a concentration of 100 CFU / mouse via the tail vein. The mice were observed for 7 days and their survival was recorded.

[0059] like Figure 8 As shown, the results indicate that the antibody of the present invention can effectively prevent sepsis caused by Staphylococcus aureus infection.

[0060] In the MRSA252 sublethal challenge model, 20 mice were divided into 2 groups, 10 mice in each group. The first group was injected with 100 μg of antibody, 100 μL; the second group was injected with sterile PBS, 100 μL; 24 hours after administration, 1.0×10 8 The mice were challenged with MRSA252 at a concentration of 100 CFU / mouse via the tail vein. 24 hours after the challenge, the organs of the mice were removed to detect bacterial colonization.

[0061] In the USA300 sublethal challenge model, 20 mice were divided into 2 groups, 10 mice in each group. The first group was injected with 100 μg of antibody, 100 μL; the second group was injected with sterile PBS, 100 μL; 24 hours after administration, 0.5×10 8 CFU / mouse USA300 was challenged via the tail vein of mice. 24 hours after challenge, the organs of mice were removed and the bacterial colonization was detected.

[0062] like Fig. 9 As shown, in the MRSA252 sublethal challenge model, the antibody can significantly reduce the bacterial colonization in the liver, spleen, and kidney, but the protective effect in the lung tissue is not obvious.

[0063] like Fig.10 As shown, in the USA300 sublethal challenge model, the antibody can significantly reduce the bacterial colonization in the liver, spleen, lungs, and kidneys, and protect tissues against MRSA252 infection.

[0064] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A fully human monoclonal antibody against Staphylococcus aureus iron ion regulation surface determinant cluster B protein, characterized in that: The structural domain recognized by the monoclonal antibody at least includes the NEAT2 structural domain at positions 341-458 of the iron ion regulation surface determinant cluster B protein of Staphylococcus aureus, and its amino acids are shown in SEQ ID NO.

1.

2. The fully human monoclonal antibody against Staphylococcus aureus iron ion regulation surface determinant cluster B protein according to claim 1, characterized in that: The monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises variable regions CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; the light chain comprises variable regions CDR1, CDR2 and CDR3, and the amino acid sequences thereof are shown in SEQ ID NO.5, DDD and SEQ ID NO.

6.

3. The fully human monoclonal antibody against Staphylococcus aureus iron ion regulating surface determinant cluster B protein according to claim 1, characterized in that: The constant region of the monoclonal antibody includes any one of human IgM, IgA or IgA constant regions.

4. The fully human monoclonal antibody against Staphylococcus aureus iron ion regulating surface determinant cluster B protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO.

8.

5. Use of the fully human monoclonal antibody against the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus according to any one of claims 1 to 4 in the preparation of a reagent that specifically binds to the iron ion-regulated surface determinant cluster B protein of Staphylococcus aureus.

6. Use of the fully human monoclonal antibody against the iron ion regulating surface determinant cluster B protein of Staphylococcus aureus according to any one of claims 1 to 4 in the preparation of a drug for treating or preventing Staphylococcus aureus infection.

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