Antibodies against staphylococcus aureus enterotoxin b and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to an antibody against staphylococcus aureus enterotoxin B and application thereof. The heavy chain variable region of the antibody provided in the present application comprises CDR sequences as shown in SEQ ID NO. 1-3, and the light chain variable region comprises CDR sequences as shown in SEQ ID NO. 9-11. The antibody has high affinity and can specifically bind to staphylococcus aureus SEB, can block the combination of SEB and MHC II / TCR, significantly inhibit cytokine storm, and exhibit dose-dependent protection effect in the MRSA systemic infection model, and can be used for treating, preventing or diagnosing staphylococcus aureus infection, and provides a non-antibiotic therapy solution for SEB poisoning and drug-resistant staphylococcus aureus infection, and has important clinical and public health value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a monoclonal antibody targeting staphylococcus aureus enterotoxin B and a preparation method and application thereof. BACKGROUND

[0002] Staphylococcus aureus is a gram-positive pathogenic bacterium, and the enterotoxin B (SEB) secreted by the staphylococcus aureus belongs to the superantigen family, which can directly combine with MHC class II molecules and TCR Vβ region, activate a large number of T cells and release cytokines such as IFN-γ and TNF-α, resulting in high fever, multiple organ failure and even death. SEB has high stability and strong toxicity (inhalation lethal dose is only 0.02 μg / kg), and is listed as a B-level biological warfare agent.

[0003] For the current SEB infection without approved vaccine or specific drug treatment, researchers have proposed various treatment strategies, including small molecule therapeutic agents, SEB vaccines and SEB targeting monoclonal antibodies. Among them, the monoclonal antibody can quickly play a role, and is complementary to antibiotics and vaccines, which is an ideal choice for emergency exposure after infection intervention, especially for immunodeficient patients who cannot be normally vaccinated. Such antibodies provide a new possibility for relieving and treating toxic shock syndrome and staphylococcus aureus infection caused by SEB. The passive immunization strategy based on antibody drugs shows great development potential in the treatment of MRSA infection, and is expected to become a new type of "non-antibiotic" immunoprevention means, providing an important direction for controlling drug resistance development. SUMMARY

[0004] Therefore, the present application aims to provide an antibody against staphylococcus aureus enterotoxin B; the second purpose of the present application is to provide a nucleotide sequence encoding the antibody; the third purpose of the present application is to provide a vector containing the nucleotide sequence; the fourth purpose of the present application is to provide a host cell containing the vector; the fifth purpose of the present application is to provide the application of the antibody in the preparation of a reagent or a drug specifically binding to staphylococcus aureus enterotoxin B or free enterotoxin B; and the sixth purpose of the present application is to provide the application of the antibody in the preparation of a drug for treating, preventing or diagnosing staphylococcus aureus infection.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides an antibody against staphylococcus aureus enterotoxin B, and the CDR1, CDR2 and CDR3 of the heavy chain have, in turn:

[0007] (1) the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; or

[0008] (2) the amino acid sequence shown in (1) is obtained by substituting, deleting or adding one or more residues, and has the same or similar function as (1); or

[0009] (3) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence shown in (1) or (2);

[0010] the CDR1, CDR2 and CDR3 of the light chain have, in sequence:

[0011] (4) the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; or

[0012] (5) the amino acid sequence shown in (4) is obtained by substituting, deleting or adding one or more residues, and has the same or similar function as (4); or

[0013] (6) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence shown in (4) or (5);

[0014] the number of the plurality is 2, 3, 4 or 5.

[0015] In some embodiments of the present application, the FR1, FR2, FR3 and FR4 of the heavy chain variable region of the above-mentioned antibody have, in sequence:

[0016] (7) the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; or

[0017] (8) the amino acid sequence shown in (7) is obtained by substituting, deleting or adding one or more residues, and has the same or similar function as (7); or

[0018] (9) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence shown in (7) or (8);

[0019] the FR1, FR2, FR3 and FR4 of the light chain variable region have, in sequence:

[0020] (10) an amino acid sequence as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; or

[0021] (11) an amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence as shown in (10), and having the same or similar function as that of (10); or

[0022] (12) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence as shown in (10) or (11);

[0023] The number is 2, 3, 4 or 5.

[0024] In some embodiments of the present application, the amino acid sequence of the heavy chain variable region of the above-mentioned antibody is:

[0025] (13) an amino acid sequence as shown in SEQ ID NO: 8; or

[0026] (14) an amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence as shown in (13), and having the same or similar function as that of (13); or

[0027] (15) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence as shown in (13) or (14);

[0028] The amino acid sequence of the light chain variable region is:

[0029] (16) an amino acid sequence as shown in SEQ ID NO: 16; or

[0030] (17) an amino acid sequence obtained by substituting, deleting or adding one or more residues to the amino acid sequence as shown in (16), and having the same or similar function as that of (16); or

[0031] (18) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence as shown in (16) or (17);

[0032] The number is 2, 3, 4 or 5.

[0033] The present application also provides a nucleic acid molecule having a nucleotide sequence encoding the above-mentioned antibody.

[0034] In some embodiments of the present application, the nucleic acid molecule has:

[0035] (19) the nucleotide sequence as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32; or

[0036] (20) the nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the nucleotide sequence as shown in (19), and having the same or similar function as (19); or

[0037] (21) the nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence as shown in (19) or (20);

[0038] The number is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0039] The present application also provides an expression vector having the nucleotide sequence as shown in any one of SEQ ID NO: 17-32.

[0040] The present application also provides a host cell having:

[0041] (i) the nucleic acid molecule; or

[0042] (ii) the expression vector.

[0043] The present application also provides the use of the antibody, the nucleic acid molecule, the expression vector or the host cell in the preparation of a biological product targeting Staphylococcus aureus enterotoxin B.

[0044] The present application also provides a biological product comprising the antibody, the nucleic acid molecule, the expression vector or the host cell.

[0045] In some embodiments of the present application, the biological product includes a prophylactic biological product, a therapeutic biological product or a diagnostic biological product.

[0046] In some specific embodiments of the present invention, the biological product used above or the biological product described above is a preventive biological product, a therapeutic biological product, or a diagnostic biological product;

[0047] The preventive biological products mentioned are products that prevent disease by establishing active immunization, and can be vaccines or toxoids.

[0048] The therapeutic biological products are products for treating or controlling diseases, and may be neutralizing antibody drugs, monoclonal antibody drugs, or cell therapy products;

[0049] The diagnostic biological products are products for diagnosing diseases or monitoring conditions, and may be detection or diagnostic reagents, detection or diagnostic kits, or detection or diagnostic devices. In some examples, they may also be reagents, kits, or devices for indirectly detecting Staphylococcus aureus.

[0050] The present invention also provides a method for neutralizing Staphylococcus aureus enterotoxin B, comprising neutralizing the above-mentioned antibody, nucleic acid molecule, expression vector or host cell through a series of steps.

[0051] To address the abnormal immune activation induced by SEB toxin, this invention provides a fully human monoclonal antibody, SEB-15, which exhibits high affinity, specifically binding to SEB, neutralizing its toxicity, and blocking its interaction with host MHC class II molecules and T-cell receptors, thereby inhibiting the release of pro-inflammatory cytokines. In vitro experiments have confirmed that SEB-15 effectively neutralizes the superantigen effect of SEB and significantly reduces SEB-induced T-cell activation. In vivo studies show that this antibody effectively prevents methicillin-resistant Staphylococcus aureus (MRSA) infection, with a protection rate exceeding 70%, demonstrating dose-dependent protective effects. Even with a relatively small dose, effective protection is provided, and complete protection is achieved even when administered one hour after exposure. Compared to vaccine strategies, which are still in the exploratory stage, SEB-15-mediated passive immunization is rapid in onset and provides clear protection, making it more suitable for post-exposure emergency treatment and biodefense scenarios. It possesses significant translational potential and clinical application value, and will become an important technical solution in the research field of "non-antibiotic" treatment of methicillin-resistant Staphylococcus aureus infection and control of drug resistance development. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0053] Figure 1 The results of SDS-PAGE analysis of wtSEB and mSEB proteins are shown, where "M" represents the protein molecular weight standard.

[0054] Figure 2The results of screening of Fab library, wherein a, Fab (Kappa light chain) library, b, Fab (Lambda light chain), and "M" is a nucleic acid molecular weight marker;

[0055] Figure 3 The results of expression and purification of fully human monoclonal antibody SEB-15, wherein "M" is a protein molecular weight marker;

[0056] Figure 4 The results of detection of binding activity of fully human monoclonal antibody SEB-15, wherein a, the results of ELISA detection of binding activity of fully human monoclonal antibody SEB-15 to wtSEB, b, the results of ELISA detection of binding activity of fully human monoclonal antibody SEB-15 to mSEB;

[0057] Figure 5 The results of epitope type identification of fully human monoclonal antibody SEB-15, wherein "M" is a protein molecular weight marker;

[0058] Figure 6 The inhibitory effect of fully human monoclonal antibody SEB-15 on the superantigen activity of wtSEB;

[0059] Figure 7 The results of evaluation experiment of MRSA systemic infection model. DETAILED DESCRIPTION

[0060] The present application discloses an antibody against Staphylococcus aureus enterotoxin B, and those skilled in the art can refer to the content herein and appropriately improve the process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0061] It should be understood that the expression "one or more of" includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0062] The use of the terms "including", "has", or "contains", including their grammatical synonyms, should generally be understood as open and non-limiting, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

[0063] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0064] The use of any and all examples, or exemplary language herein, for example, merely to better illuminate the application, does not pose a limitation on the scope of the application. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0065] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are only chosen to encompass the more precise values in the specification. Any numerical range, unless expressly indicated otherwise, are intended to encompass all values from the lower value and to the upper value of the range. Unless otherwise indicated, all ranges include all the sub-ranges of the same endpoints. In this disclosure and during the course of proceedings in a Court in a litigation, the term "about" can be construed as meaning approximately, in the sense of not meaning an exact number.

[0066] In some implementations, the application also encompasses the use of the antibody in diagnostic reagents, pharmaceutical compositions, and genetically engineered versions of the antibody, including in some examples, humanized antibodies, Fab fragments, mRNA encoded antibodies, and the like.

[0067] Unless otherwise indicated, the materials, reagents, consumables and apparatuses involved in the present application are all commercially available and can be purchased from the market.

[0068] The application will be further described in conjunction with the following examples.

[0069] Example 1: Expression and purification of wild-type Staphylococcus aureus enterotoxin B (wtSEB) and mutant Staphylococcus aureus enterotoxin B (mSEB)

[0070] 1. Expression and purification of wild-type Staphylococcus aureus enterotoxin B (wtSEB)

[0071] The wtSEB coding sequence was obtained by PCR amplification using the SEB gene (SACOL0907) in the genome of Staphylococcus aureus MRSA COL strain (accession number NC_002951.2) as a template. After sequencing verification, the sequence was subjected to E. coli preference codon optimization, and the whole gene synthesis was completed by Shanghai Jeery Biotechnology Co., Ltd. The optimized gene was cloned into the pET-22b(+) expression vector to construct the recombinant plasmid pET-wtSEB, which was transformed into E. coli BL21 (DE3) competent cells. After resistance screening, a high-efficiency expression strain was obtained. The engineering bacteria were inoculated in LB liquid medium containing ampicillin and cultured at 37°C overnight. When the OD 600 was 0.6-0.8, IPTG was added for expression induction at 25°C. After centrifugal collection and ultrasonic disruption of the bacterial cells, the supernatant was sequentially captured by an SP HP column, and sequentially purified by a Phenyl HP column and a Q HP column to remove aggregates, host residual impurities, endotoxins and other impurities. Finally, high-purity wtSEB protein was obtained. As shown in FIG. 2, SDS-PAGE detection showed that the purity reached 99.3%. Figure 1

[0072] 2. Expression and purification of mutant Staphylococcus aureus enterotoxin B (mSEB)

[0073] To eliminate the superantigen toxicity of SEB, the key residues L45, Y89 and Y94 combined with MHC II molecules were selected for site-directed mutagenesis (L45R, Y89A, Y94A). The wild-type SEB gene was used as a template, and the QuickChange II XL site-directed mutagenesis kit was used to introduce the above mutations to obtain the non-toxic mutant mSEB gene. After codon optimization and whole sequence synthesis of the gene, it was cloned into the pET-22b(+) vector to construct the recombinant plasmid pET-mSEB, which was transformed into the BL21 (DE3) strain. The fermentation, induction expression and purification process were basically the same as those of wtSEB. As shown in FIG. 3, the final mSEB protein had a purity of 99.5% detected by SDS-PAGE. Figure 1

[0074] Example 2: Isolation of PBMC cells

[0075] ​​The recombinant Staphylococcus aureus vaccine (Escherichia coli) developed by Chengdu Oulinn Biotechnology Co., Ltd. and the Chinese People's Liberation Army Army Medical University was approved by the State Drug Administration on June 18, 2015 as a new drug for biological products for prevention (approval number: 2015L01247), and was granted to carry out phase I, II and III clinical trials. The clinical progress of the project is as follows: phase Ia was completed from May 2016 to September 2017, a total of 174 healthy subjects were included; phase Ib was completed from May 2019 to December 2019, a total of 144 healthy subjects were included; phase II was completed from October 2018 to July 2021, for 348 high-risk groups of hospital infection of bone trauma; phase III was started in June 2022, and about 6000 patients with closed fractures undergoing elective surgery were started in 55 clinical centers in June 2022.

[0076] The present application is implemented by Chongqing Yuanlun Biotechnology Co., Ltd. Based on the protective antigen SEB contained in the vaccine, the ethical review committee of Jiangsu Provincial Center for Disease Control and Prevention approved the liquid nitrogen frozen peripheral blood lymphocyte samples in accordance with the ethical requirements from the Ia phase clinical trial center, which were used for the experiments related to the present application.

[0077] Example 3: Establishment of human-specific anti-Staphylococcus aureus antibody library

[0078] 1. cDNA synthesis

[0079] Total RNA was extracted from the frozen PBMC sample, and Oligo(dT) primers were used for reverse transcription reaction to synthesize the first strand of cDNA.

[0080] 2. Fab library construction

[0081] Using cDNA as a template, human immunoglobulin-specific primers were used to amplify light chain (κ and λ) and heavy chain Fd segment genes. The light chain PCR product was digested with SacI and XbaI and then cloned into the pComb3XSS vector to construct the light chain library. Then the heavy chain Fd segment was digested with XhoI and SpeI and inserted into the vector to complete the construction of the Fab library. The ligation product was transformed into TG1 competent cells, spread on ampicillin LB plates, and incubated at 37°C overnight to calculate the library capacity. Randomly selected clones were subjected to PCR verification, the positive rate was more than 80%, and the total library capacity was greater than 1×10 8 .

[0082] 3. Library quality identification

[0083] Ten monoclonal antibodies were picked and subjected to PCR according to the following procedure: 3 minutes of pre-denaturation at 94°C; 30 seconds of denaturation at 94°C, 30 seconds of annealing at 55°C, 1 minute of extension at 72°C, 30 cycles; 10 minutes of extension at 72°C. 5 μL of the product was subjected to 2% agarose gel electrophoresis analysis, and the results are shown in Figure 2 .

[0084] Example 4: Screening of human-specific anti-S. aureus Fab antibody library

[0085] 1. Preparation of phage and determination of titer

[0086] The TG1 bacteria were activated to OD 600 = 0.5, the phage was gradient diluted and then incubated with the bacteria, and then plated on 2xYT plates to calculate the plaque forming units (PFU / mL).

[0087] 2. Enrichment of ScFv library

[0088] After co-infection of ScFv bacteria with M13K07 helper phage (MOI = 20), the culture was expanded, and SEB-coupled magnetic beads were used for negative screening. The acid elution was immediately neutralized.

[0089] 3. Identification and sequencing of positive clones

[0090] IPTG was used to induce ScFv expression, and the bacterial lysate was subjected to ELISA screening (OD 450 ≥ 2.5 times the negative control was positive), and the positive clones were sequenced.

[0091] Example 5: Cloning, expression and purification of SEB-15 fully human antibody

[0092] 1. Construction and verification of vector

[0093] Bioinformatics analysis was performed on the antibody sequences obtained by screening to confirm the integrity of the open reading frame. The synthesized heavy chain and light chain complete sequences were connected to the pcDNA3.4 vector by enzyme digestion and ligation, respectively, to construct a full antibody expression plasmid, which was transformed into DH5α competent cells. The correctness was verified by sequencing and multiple sequence alignment.

[0094] 2. Antibody expression and purification

[0095] The recombinant plasmid was transfected into 293F cells, and after 120 hours of culture, the supernatant was collected and purified by Protein A affinity chromatography. SDS-PAGE showed that the molecular weight of the antibody was about 160 kDa, with a heavy chain of 50 kDa and a light chain of 25 kDa, as shown in Figure 3 . The antibody was named SEB-15, and its sequence is shown in Table 1.

[0096] Table 1: SEB-15 antibody sequence

[0097]

[0098]

[0099] Example 6: Detection of binding activity of SEB-15 monoclonal antibody to Staphylococcus aureus enterotoxin B (SEB)

[0100] 1. Experimental method

[0101] The wtSEB and mSEB proteins (2 μg / mL) were used as antigens to coat 96-well plates (100 μL / well) at 4°C overnight, and then blocked at room temperature for 2 hours. The SEB-15 antibody was diluted from 100 μg / mL in PBST (100 μL / well, triplicate), and positive controls (vaccine serum 1:2000), negative controls (negative serum 1:100 and irrelevant antibody IgG1 0.5 μg / mL), and blank controls (PBST) were set up. The antibody-antigen binding was performed at 37°C for 1 hour. After washing with PBST for 3 times, HRP-labeled secondary antibody (Goat-Anti-Human-IgG-Fab-HRP) was added at a dilution of 1:5000 (100 μL / well) and incubated at 37°C for 1 hour. After washing again, TMB substrate was added for color development for 5-10 minutes (37°C, dark). The reaction was terminated with 2 M H2SO4, and the OD value was measured at 450 / 650 nm. The positive threshold was determined as 3 times the average value of the negative control IgG1, and the EC 50 value was calculated. This method significantly improves the sensitivity through enzyme-labeled secondary antibody signal amplification, and uses strict washing steps (PBST containing 0.05% Tween-20) and blocking optimization (3% BSA or 5% skim milk) to reduce non-specific background, which is suitable for high-throughput detection of antibody binding activity.

[0102] 2. Experimental results

[0103] The binding activity of SEB-15 to wtSEB and mSEB was determined by ELISA. The antigen was coated on the enzyme-labeled plate at 2 μg / mL, and the HRP-labeled goat anti-human Fab was used as the secondary antibody after adding gradient-diluted antibody. The OD 450 value was determined by TMB color development. The results are shown in Figure 4 , and the EC 50 values were calculated as 0.02307 μg / mL (wtSEB) and 0.01376 μg / mL (mSEB), respectively, as shown in Table 2.

[0104] Table 2: Binding activity results of full human SEB monoclonal antibody SEB-15 to mSEB and wtSEB

[0105]

[0106] Example 7: Epitope type determination of SEB-15 monoclonal antibody

[0107] 1. Experimental method

[0108] The 0.8 μg mSEB and wtSEB protein samples were treated with reducing Loading Buffer (containing β-mercaptoethanol) and denatured in boiling water bath for 5 minutes, then subjected to SDS-PAGE electrophoresis at 140 V constant voltage for 1-2 hours (bromophenol blue indicator to run out of gel termination). Subsequently, the proteins were transferred to PVDF membrane by semi-dry transfer instrument (23 V, 20 minutes), blocked with 5% skim milk powder / TBST for 1 hour (room temperature shaker), washed with TBST 3 times (5 minutes / time), incubated with 3 μg / mL SEB-15 antibody at 37°C for 1 h, washed with TBST 3 times (5 minutes / time), then incubated with 1:7500 diluted anti-human IgG-AP secondary antibody (prepared with 1% skim milk powder / TBST) at room temperature for 1 hour, washed again, and then added AP color developing solution under dark to observe the bands. The reaction was terminated with water after color development. Key steps include: reducing denaturation to ensure protein depolymerization, semi-dry transfer to optimize the retention rate of macromolecular weight proteins (such as mSEB / wtSEB), alkaline phosphatase (AP) color developing system to improve detection sensitivity, and TBST washing throughout to reduce non-specific binding.

[0109] 2. Experimental results

[0110] As shown in Figure 5 , denatured wtSEB and mSEB proteins were separated by SDS-PAGE and transferred to the membrane, then incubated with SEB-15 antibody, and detected by alkaline phosphatase color developing system. The results showed that specific bands appeared at 28 kDa, indicating that it was a linear epitope.

[0111] Example 8: Inhibition of wtSEB superantigen activity by anti-SEB antibody SEB-15

[0112] 1. Experimental method

[0113] Purification of porcine spleen lymphocytes and evaluation of the immunomodulatory effect of SEB-15 antibody on wtSEB antigen: Fresh porcine spleen was mechanically disrupted after disinfection with 75% alcohol and Hank's solution, and a single cell suspension was obtained by centrifugation at 300 g for 5 minutes. The lymphocyte layer was further purified by density gradient centrifugation (lymphocyte separation medium, specific gravity 1.077, centrifugation at 400 g for 20 minutes), and the cells were washed (centrifugation at 250 g for 10 minutes) to adjust the cell density to 5×10 6 cells / mL in RPMI 1640 culture medium containing 10% fetal bovine serum. The cells were inoculated into a 96-well plate (5×10 5cells / well) for 40 hours at 37°C with 5% CO2, during which wtSEB antigen (10 pg / mL) was pre-incubated with SEB-15 antibody at a ratio of 1:50 / 1:100 for 1 hour to form an immune complex. Positive control (untreated wtSEB), negative control (mSEB and medium blank), irrelevant antibody group (mHla-pcAb), and SEB-15 antibody pretreatment group (3 replicates) were set up. After adding the samples, incubate for 2 hours with shaking, then wash and add detection antibody, streptavidin-HRP (signal amplification), TMB substrate for color development for 20 minutes in the dark to stop the reaction. Quantify the IFN-g concentration by standard curve and analyze the inhibitory effect of the antibody on SEB stimulation. Key steps include: density gradient centrifugation to optimize lymphocyte purity (to avoid red blood cell contamination), antigen-antibody pre-incubation (to simulate in vivo immune complex formation), and HRP-streptavidin system to enhance detection sensitivity.

[0114] 2. Experimental results

[0115] The experimental results are shown in Figure 6 The wild-type Staphylococcus aureus enterotoxin B (wtSEB) treatment group induced high levels of IFN-g (about 1500 pg / mL) in pig spleen mononuclear cells, significantly higher than the mutant SEB (mSEB) and the medium control group (RPMI 1640), confirming the superantigen activity of SEB. When wtSEB was co-incubated with 1 mg mHla-pcAb (irrelevant control), the level of IFN-g secretion was not significantly different from the wtSEB treatment group alone, indicating that mHla-pcAb could not neutralize the superantigen activity of wtSEB; however, after wtSEB was co-incubated with 1 mg SEB-15, the level of IFN-g decreased to that of the negative control group (mSEB and RPMI 1640), indicating that SEB-15 could completely block the immune stimulation of wtSEB and neutralize the superantigen activity of wtSEB. Notably, the lack of effect of mHla-pcAb on wtSEB activity is in sharp contrast to the effective neutralization by SEB-15, demonstrating the high specificity of SEB-15 for inhibiting the superantigen activity of wtSEB. These data indicate that the SEB-15 antibody can specifically inhibit wtSEB-induced IFN-g secretion, providing important evidence for the development of specific immunomodulators targeting SEB.

[0116] Example 9: Establishment and protective evaluation of MRSA systemic infection model

[0117] Thirty female BALB / c mice, each weighing 17-20 g, were divided into three groups of 10 mice each. Each mouse was injected intravenously with 3.4 x 10 8MRSA COL bacterial liquid of CFUs, injection volume 100 μL. 2 hours later, group 1 intravenous injection 100 μg SEB-15 monoclonal antibody, group 2 intravenous injection 200 μg SEB-15 monoclonal antibody, group 3 intravenous injection of equal volume of normal saline. The survival time of mice was observed for 7 consecutive days and the survival rate was calculated.

[0118] Results as Figure 7 The survival rate of the normal saline control group mice decreased rapidly after infection, and sharply decreased after 50 hours, and all died around 120 hours (survival rate 0%), indicating that MRSA COL infection was highly lethal. The survival rate of the 100 μg SEB-15 monoclonal antibody treatment group mice decreased after 50 hours, but eventually stabilized at 40% (4 / 10 survived), significantly higher than the control group (P<0.05). The survival rate of the 200 μg SEB-15 monoclonal antibody treatment group mice decreased rapidly after 50 hours, but eventually stabilized at 70% (7 / 10 survived), significantly better than the normal saline group (P<0.01) and the 100 μg dose group (P<0.05). The full human anti-SEB antibody SEB-15 has a dose-dependent protective effect on MRSA COL systemic infection, and the protective effect of the 200 μg dose group is better than that of the 100 μg dose group, significantly improving the survival rate of mice. The results show that SEB-15 may neutralize the superantigen activity of Staphylococcus aureus enterotoxin B (SEB) to reduce the immunopathological damage caused by infection, thereby improving the survival outcome of MRSA infected mice, and providing experimental evidence for the clinical conversion of anti-SEB antibodies.

[0119] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An antibody directed against Staphylococcus aureus enterotoxin B, characterized in that, The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain are shown in SEQ ID NO: 1~3 in order, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO: 9~11 in order; The amino acid sequences of FR1, FR2, FR3 and FR4 of the heavy chain variable region are shown in SEQ ID NO: 4~7 in order; The amino acid sequences of FR1, FR2, FR3 and FR4 of the light chain variable region are shown in SEQ ID NO: 12~15 in order.

2. A nucleic acid molecule, characterized in that, The nucleotide sequence having the antibody of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence having any one of SEQ ID NO: 17~32.

4. An expression vector, characterized in that, The nucleic acid molecule of claim 2 or 3.

5. A host cell characterized in that, Having: (i) the nucleic acid molecule of claim 2 or 3; or (ii) the expression vector of claim 4.

6. Use of the antibody of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 4 or the host cell of claim 5 in the preparation of a biological product for preventing, treating or diagnosing Staphylococcus aureus infection.

7. A biological product comprising a prophylactic biological product, a therapeutic biological product or a diagnostic biological product, characterized in that, The antibody of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of claim 4 or the host cell of claim 5.

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