Monoclonal antibodies against Staphylococcus aureus iron-regulated surface determinant cluster B protein and their applications

Abstract

This invention discloses a monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein and its application. The amino acids of the domain recognized by the monoclonal antibody are shown in SEQ ID NO.1. The monoclonal antibody is generated by culturing host cells expressing the vector. The generated monoclonal antibody is secreted into the supernatant and purified by chromatography. The obtained antibody can treat sepsis caused by Staphylococcus aureus / MRSA infection and has important significance in the prevention and treatment of Staphylococcus aureus infection.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to a fully human monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein, and also to the application of the antibody. Background Technology

[0002] Staphylococcus aureus (S. aureus) is a major pathogen causing hospital and community infections. It is highly pathogenic, with sepsis caused by S. aureus infection having a mortality rate as high as 20%, and it spreads with multidrug resistance and pan-drug resistance. In recent years, antibiotic resistance has led to a severe situation where S. aureus infections face "no cure," and the emergence of methicillin-resistant Staphylococcus aureus (MRSA) has further exacerbated the clinical treatment dilemma. A study published in the *Lancet* in 2022 statistically analyzed the mortality rates associated with drug-resistant bacterial infections worldwide. The results showed that S. aureus infection ranked first, directly causing more than 1 million deaths annually, of which more than 500,000 deaths were caused by sepsis resulting from the infection. The WHO has identified MRSA as one of the 12 "superbugs" posing a deadly threat to humans and called on countries to implement policies that prioritize new drug development. Therefore, effective means to combat S. aureus infection and prevent antibiotic resistance are urgently needed.

[0003] Studies have shown that antibody-based immunization strategies are an effective treatment for Staphylococcus aureus infection and an important alternative therapy to prevent antibiotic resistance. Staphylococcus aureus has numerous virulence factors and complex pathogenic factors. Iron surface determinant B (IsdB) is a widely expressed cell wall anchor protein in Staphylococcus aureus, exhibiting good conservation. It plays a crucial role in the bacterial uptake and metabolism of heme iron, as well as in maintaining normal bacterial growth. Antibody therapy targeting IsdB has shown significant protective effects in a mouse model of Staphylococcus aureus infection.

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

[0005] In view of this, one objective of the present invention is to provide a monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein; a second objective of the present invention is to provide the application of the monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein in the preparation of reagents that specifically bind to Staphylococcus aureus iron-regulated surface determinant B protein; and a third objective of the present invention is to provide the application of the monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein in the preparation of medicaments for treating or preventing Staphylococcus aureus infection.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

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

[0008] KMTDLQDTKYVVYESVENNESMMDAFVKHPIKTGMLNGKKYMVMETTNDDYWKDFMV

[0009] EGQRVRTISKDAKNNTRTIIFPYVEGKTLYDAIVKVHVKTIDYDGQYHV RIVDKEAFTKAN

[0010] Preferably, the monoclonal antibody comprises a heavy chain and a light chain. The heavy chain comprises variable regions CDR1, CDR2, and CDR3, with amino acid sequences 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, with amino acid sequences 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 the IgA constant region.

[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. The application of the monoclonal antibody against Staphylococcus aureus iron-regulated surface-determining cluster B protein in the preparation of reagents that specifically bind to Staphylococcus aureus iron-regulated surface-determining cluster B protein.

[0021] 3. The application of the monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant cluster B protein in the preparation of drugs for the treatment or prevention of Staphylococcus aureus infection.

[0022] The beneficial effects of this invention are as follows: This invention provides an antibody against the IsdB NEAT2 domain protein of Staphylococcus aureus. This antibody is generated by culturing host cells expressing the vector to produce a monoclonal antibody. The generated monoclonal antibody is secreted into the supernatant and purified by chromatography. The obtained antibody can treat sepsis caused by Staphylococcus aureus / MRSA infection and is of great significance in the prevention and treatment of Staphylococcus aureus infection. Attached Figure Description

[0023] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

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

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

[0026] Figure 3To enable ELISA detection of the specific binding of fully human monoclonal antibody to IsdB NEAT2 protein;

[0027] Figure 4 To determine antigen-antibody affinity using biomembrane 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 were used to inhibit the proliferation of MRSA252 bacteria;

[0030] Figure 7 Establishment of lethality models for MRSA252 and USA300 virus attacks;

[0031] Figure 8 The protective efficacy of this antibody in the MRSA252 and USA300 challenge-lethal models;

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

[0033] Figure 10 To evaluate the protective effect of this antibody in the USA300 sublethal challenge model. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described 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 using genomic DNA from Staphylococcus aureus strains. After DNA sequencing confirmation, it was expressed in Escherichia coli and cultured overnight at 37°C in LB medium containing ampicillin. Cells were then harvested by centrifugation. The IsdB NEAT2 protein was purified using GST affinity chromatography. 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 below. Figure 1 As 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. 6 / ml, placed in a constant temperature incubator at 37℃, 5% CO2, 120rpm.

[0039] The sequences encoding the heavy chain (SEQ ID NO. 9) and the light chain (SEQ ID NO. 10) were constructed into the pcDNA3.1 vector and ligated at 16°C for 24 hours. The ligation 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 constructed heavy and light chain plasmids were mixed at a 1:1 mass ratio and co-transfected with PEI transfection reagent into HEK293 cells. Four hours post-transfection, HEK293 basal medium (OPM) was added, and the cells were incubated at 37°C with 5% CO2 for 96 hours. The transfection supernatant was collected by centrifugation at 3000×g for 30 minutes and purified using protein A affinity chromatography (IgG Protein A beads). The protein was concentrated using ultrafiltration tubes, and the concentration was detected by NanoDrop. Antibody expression and purification were assessed by SDS-PAGE. Figure 2 The results showed that the antibody with high purity was obtained after purification.

[0043] Example 3: Determination of antibody binding activity

[0044] ELISA plates were coated with 100 μL of recombinant IsdB NEAT2 protein as antigen per well, incubated overnight at 4°C, and blocked with blocking buffer at 37°C for 2 hours. 100 μL of overlapping PCR transfection expression antibody (primary antibody) stock solution was added. Positive controls consisted of 100 μL of positive plasma sample stock solution (1:50 dilution) and purified antibody (1:1000 dilution) per well. Negative controls consisted of 100 μL of negative plasma sample stock solution (1:50 dilution) and negative control-irrelevant antibody IgG1 0.5 μg / mL per well. Three replicates of blank were added with 100 μL of blocking buffer, and the plates were incubated at 37°C for 1 hour. The plates were washed with 200 μL of PBST buffer per well, and then incubated with Goat-Anti-IgG-Fab-HRP (secondary antibody) diluted 1:5000 with PBST 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, and immediately terminate with 50 μL of 2M H2SO4. Detect the OD value at a wavelength of 450 nm. Calculate the mean value of the negative control unrelated antibody IgG1, and calculate the threshold (3 times the mean value). Antibodies greater than the threshold are considered positive antibodies.

[0045] Experiments show that the fully human monoclonal antibody described in this invention can specifically bind to the IsdB NEAT2 protein. Figure 3 ).

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

[0047] Biomembrane interferometry was used to measure the affinity of the antibody for IsdB NEAT2 protein. A 50 nM monoclonal antibody, purified from expression, was immobilized onto an AHC sensor. IsdB NEAT2 protein was serially diluted to different final concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM) and added sequentially to the sample plate. The program was set as follows: "Baseline detection - Loading detection - Baseline detection - Binding detection - Dissociation detection - Probe regeneration". The equilibrium dissociation constant (KD) was calculated. Figure 4 As shown in the figure. The results show that the KD of this antibody with IsdB NEAT2 is 9.724 × 10⁻⁶. -9 M.

[0048] The results show that the antibody described in this invention has good affinity for the IsdB NEAT2 antigen.

[0049] Example 5: Determination of Antibody Epitope Type

[0050] Add 2 μL of reducing loading buffer (containing β-mercaptoethanol) to 1 μg IsdB NEAT2 protein sample (8 μL), and heat in a 100℃ metal bath for 5 minutes. Then load the sample onto an SDS-PAGE gel, adjusting the voltage to 120V and the electrophoresis time to 1-2 hours. Stop electrophoresis when the bromophenol blue just appears. Remove the electrophoresis gel and rinse with water.

[0051] The PVDF membrane, activated with methanol using the electrophoresis gel, was transferred using a transfer apparatus (3 layers of filter paper, PVDF membrane, electrophoresis gel, 3 layers of filter paper). The voltage was adjusted to 60V for 25 minutes. After transfer, the PVDF membrane was removed, washed once with PBST, and then blocked in 5% skim milk powder at 4°C for 2 hours. The PVDF membrane was then removed and washed three times with PBST for 5 minutes each time. The purified antibody was then diluted to 1 μg / mL with PBST and incubated at 4°C for 1 hour. The PVDF membrane was washed three times with PBST solution, and then anti-human HRP-labeled IgG was added at a 1:5000 ratio. The membrane was incubated on a horizontal shaker at room temperature for 40 minutes. The PVDF membrane was washed three times with PBST solution, placed in a clean Petri dish, and approximately 1 mL of exposure and development solution was added to each membrane for photographic observation. Figure 5The results show that it can bind to denatured IsdB NEAT2, thus indicating that the antibody epitope described in this invention is a linear epitope.

[0052] Example 6: This antibody inhibits the proliferation of Staphylococcus aureus bacteria.

[0053] After activating and culturing Staphylococcus aureus MRSA252, the OD was adjusted to 1.0. 90 μL of bacterial culture was added to each well of a 96-well plate along with 10 μL of antibodies at different concentrations (final concentrations of 3.734, 0.951, and 0.414 μg / μL) and physiological saline as controls. The plates were incubated together at 37°C for 16 hours, and the bacterial colonization was then detected. Results are as follows: Figure 6 As shown, the antibody can significantly inhibit the in vitro proliferation of MRSA252 strain in a dose-dependent manner.

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

[0055] The concentration of MRSA252 was adjusted to 9.0 × 10⁻⁶ using physiological saline. 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 × 10⁻⁶. 9 CFU / mL, 1.0×10 9 CFU / mL, 0.8×10 9 CFU / mL, 0.5×10 9 CFU / mL. Mouse challenge: Female Balb / C mice aged 6-8 weeks were separated into different cages according to their body weight. MRSA252 and USA300 at the above concentrations were injected via the tail vein, 100 μL / mouse. After challenge, the survival status of the mice was recorded every 24 hours and observed for 7 days. Figure 7 As shown, the results indicate that the dose is 6.0 × 10⁻⁶. 8 CFU / each of MRSA252 and 1.0×10 8 The mortality rate of mice challenged with the USA300 tail vein using CFU / mouse can reach over 90%.

[0056] Example 8: Evaluation of Antibody Prophylactic and Protective Effect

[0057] In the MRSA252 challenge-induced death model, the experimental groups were as follows: 20 mice were divided into two groups of 10 each. The first group was injected with 100 μg of antibody in 100 μL; the second group was injected with 100 μL of sterile PBS. 24 hours after administration, 6.0 × 10⁻⁶ ppm of PBS was administered. 8MRSA252 mice were challenged via tail vein with CFU / mouse. The mice were observed for 7 days and their survival status was recorded.

[0058] In the USA300 challenge-induced death model, the experimental groups were as follows: 20 mice were divided into two groups of 10 each. The first group was injected with 100 μg of antibody in 100 μL; the second group was injected with 100 μL of sterile PBS. 24 hours after administration, 1.0 × 10⁻⁶ ppm of PBS was administered. 8 USA300 mice were challenged via tail vein with CFU / mouse. The mice were observed for 7 days and their survival status was recorded.

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

[0060] In the MRSA252 sublethal challenge model, the experimental groups were as follows: 20 mice were divided into two groups of 10 each. The first group was injected with 100 μg of antibody in 100 μL; the second group was injected with 100 μL of sterile PBS. 24 hours after administration, 1.0 × 10⁻⁶ ppm of PBS was administered. 8 MRSA252 mice were challenged via tail vein with CFU / mouse. Twenty-four hours post-challenge, various organs were removed from the mice to assess bacterial colonization.

[0061] In the USA300 sublethal challenge model, the experimental groups were as follows: 20 mice were divided into two groups of 10 each. The first group was injected with 100 μg of antibody in 100 μL; the second group was injected with 100 μL of sterile PBS. 24 hours after administration, 0.5 × 10⁻⁶ mmol / L of PBS was administered. 8 USA300 mice were challenged via tail vein with CFU / mouse. Twenty-four hours post-challenge, organs were removed from the mice to assess bacterial colonization.

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

[0063] like Figure 10 As shown, in the USA300 sublethal challenge model, this antibody can significantly reduce the bacterial colonization in organs such as the liver, spleen, lungs, and kidneys, protecting tissues against MRSA252 infection.

[0064] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A fully human monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant cluster B protein, characterized in that: The domain recognized by the monoclonal antibody includes at least the NEAT2 domain at positions 341-458 of the Staphylococcus aureus iron-regulating surface determinant B protein, the amino acid sequence of which is shown in SEQ ID NO.1; the monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively; the light chain comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO.5, DDD and SEQ ID NO.6, respectively.

2. The fully human monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant cluster B protein according to claim 1, characterized in that: The constant region of the monoclonal antibody includes either the human IgM or IgA constant region.

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

8.

4. The use of the fully human monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein as described in any one of claims 1 to 3 in the preparation of a reagent that specifically binds to Staphylococcus aureus iron-regulated surface determinant B protein.

5. The use of the fully human monoclonal antibody against Staphylococcus aureus iron-regulated surface determinant B protein as described in any one of claims 1 to 3 in the preparation of a medicament for treating Staphylococcus aureus infection.

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