Peptides, nucleic acids, recombinant expression vectors, cells, SARS-cov-2 vaccine substance, SARS-cov-2 vaccine composition, and SARS-cov-2 immunization method

Abstract

Disclosed are peptides, nucleic acids, recombinant expression vectors, cells, a SARS-CoV-2 vaccine substance, a SARS-CoV vaccine composition, and a SARS-CoV-2 immunization method.

Description

Peptides, nucleic acids, recombinant expression vectors, cells, vaccine materials for SARS-COV-2, vaccine compositions for SARS-COV-2, and methods for immunizing against SARS-COV-2

[0001] The present invention relates to peptides, nucleic acids, recombinant expression vectors, cells, vaccine materials for SARS-CoV-2, vaccine compositions for SARS-CoV-2, and methods for immunizing against SARS-CoV-2.

[0002] The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered the global COVID-19 pandemic. Over the past two decades, three zoonotic coronaviruses have infected humans: SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. These viruses have the potential to cause severe acute respiratory distress syndrome (ARDS) in humans. These coronaviruses are known to produce four major proteins: an envelope protein, a membrane protein, a nucleocapsid protein, and a spike protein (SP). SP has been extensively utilized in vaccine development and diagnostic testing. In particular, SARS-CoV-2 is known to infect host cells by binding SP, located on the viral envelope, to the angiotensin-converting enzyme 2 (ACE2) receptor on the outer membrane of the host cell. SP consists of two protein domains (S1 and S2), and the specific interaction with the ACE2 receptor occurs through the receptor binding domain (RBD) located in the S1 domain of SP. In the present invention, Fv-antibodies having binding affinity to the RBD of SP were selected from an Fv-antibody library, and the selected Fv-antibodies were used in a single-step immunoassay for detecting SARS-CoV-2 using a switching peptide without a washing step.

[0003] Fv antibodies correspond to the VH region of immunoglobulin G (IgG) and are composed of three complementarity-determining regions (CDRs) and four framework regions (FRs). The Fv antibody library was prepared by randomizing the amino acid sequence of CDR3, which consists of eleven amino acid residues, through site-specific mutagenesis. The Fv antibody library was expressed on the outer membrane of Escherichia coli using autodisplay technology.

[0004] Autodisplay technology is an expression method that produces a target protein (Fv-antibody) on the outer membrane of E. coli through transport of the expressed target protein (Fv-antibody) through the beta-barrel of AIDA-1. Using this expression method, >10 5 High expression yield of protein / E. coli and >90% of transformed E. coli within the total E. coli population were achieved. The Fv-antibody library prepared in this way also had a high diversity of >105 clones / library. From this high expression yield Fv-antibody library, target Fv-antibodies with high affinity for the target antigen could be effectively selected without repeated biopanning processes. The Fv-antibody library was used to select Fv-antibodies with binding affinities for biotin, fluorescent dyes (fluorescein and rhodamine B), biocrystals (monosodium urate and calcium phosphate dehydrate), food allergens (mackerel, peanut, pork fat), and viral antigen (SARS-CoV-1 SP).

[0005] Fv antibodies and nanobodies have been used for the detection of target analytes using not only SPR biosensors but also other types of biosensors based on various types of transducers. For example, the detection of SARS-CoV-1 using Fv antibodies was demonstrated using impedance spectroscopy. Furthermore, the detection of vacuolar sorting receptors (VSRs) activity using nanobodies was demonstrated using a fluorescence biosensor, and a quartz crystal microbalance (QCM), a mass-sensitive transducer, was used for the detection of fibrinogen using nanobodies.

[0006] SARS-CoV-2 has been detected using various methods, including gold-standard PCR assays and lateral flow immunoassays, as summarized in the table. Recently, a single-step immunoassay based on switching peptides has been reported for the detection of viruses (influenza A and influenza B), as well as bacteria associated with foodborne illness and infection-related proteins (hepatitis B antigen). Switching peptides are synthesized from FRs located in the middle and light chains of IgG. These FRs from different chains of IgG are known to self-assemble to form the binding pocket of IgG. Since the expressed Fv antibody is composed of CDRs and FRs from the middle chain (VH region) of IgG, switching peptides from the light chain of IgG bind to the self-assembled FRs in the Fv antibody. These switching peptides (labeled with fluorescent dyes) are quantitatively released from IgG upon binding of the target antigen to the binding pocket of IgG, enabling the realization of a single-step immunoassay without a washing step. As an important prerequisite for the construction of a single-step immunoassay, the Fv-antibody must have a higher affinity constant (KD) for the target antigen compared to the switching peptide for quantitative analysis.

[0007] In previous studies, selected Fv antibodies, instead of whole IgG, were used in a single-step immunoassay for SARS-CoV-2. In the first step, Fv antibodies against the SARS-CoV-2 SP were screened from an autodisplayed Fv antibody library expressed on the outer membrane of E. coli. The selected Fv antibodies were expressed as soluble proteins, and their binding affinities (KD) to pseudovirus particles with mutated RBDs and mutated SPs on the outer membrane were measured. Finally, a single-step assay for SARS-CoV-2 based on the switching peptide was demonstrated using heat-inactivated SARS-CoV-2 (NATtrol™ reagent) as a real sample.

[0008] The present invention aims to develop an effective and safe SARS-CoV-2 vaccine to prevent infection with this virus and the resulting serious health problems. To this end, the present invention provides a unique vaccine composition that utilizes specific peptides, nucleic acids, recombinant expression vectors, and H-ferritin protein to enhance the immune response. This vaccine aims to provide broad protection, including protection against variants of SARS-CoV-2. Furthermore, this vaccine is designed to be applicable to various populations, making it a crucial tool in the global fight against SARS-CoV-2. Addressing this challenge represents a crucial step toward improving public health, preventing the spread of infectious diseases, and reducing the burden of disease associated with coronaviruses. In the present invention, vaccine development is proposed through sequential procedures of (1) selection of Fv-antibody from an auto-display Fv-antibody library, (2) antigen selection through epitope search by docking simulation, (3) expression and vaccination of antigen with H-ferritin, and (4) confirmation of neutralizing activity of the immunized antiserum against antibodies.

[0009] In one aspect, the present invention provides a peptide capable of inducing in vivo expression of an antibody against SARS-CoV-2, the peptide comprising at least one peptide sequence selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4.

[0010] [Sequence number 1]

[0011] WNRKRISGGSNGGSGGSPGGSFER

[0012] [Sequence number 2]

[0013] SGGSWNSNNLDSKVGGSP

[0014] [Sequence number 3]

[0015] RKSNLKPFERDISTEIYQ

[0016] [Sequence number 4]

[0017] KGGSGGSRGDEVRQIAPGQTGKGGSL

[0018] In another aspect, the present invention provides a nucleic acid encoding the peptide.

[0019] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0020] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0021] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0022] In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0023] In another aspect, the present invention may include a vaccine material for SARS-CoV-2, comprising a ferritin protein; and a peptide bound to the outer surface of the ferritin protein. In one embodiment, the peptide may be a peptide according to an embodiment of the present invention described above.

[0024] In one embodiment, the ferritin protein may comprise H-ferritin protein.

[0025] In another aspect, the present invention provides a vaccine composition for SARS-CoV-2 comprising the vaccine material for SARS-CoV-2.

[0026] In another aspect, the present invention provides a method for immunizing a subject against SARS-CoV-2, comprising injecting the vaccine composition for SARS-CoV-2 into a subject.

[0027] In one embodiment, the method for immunizing against SARS-CoV-2 may comprise injecting the vaccine composition for SARS-CoV-2 into a human.

[0028] In one embodiment, the SARS-CoV-2 immunization method may comprise injecting the SARS-CoV-2 vaccine composition into an animal.

[0029] The efficacy of the present invention lies in its ability to effectively prevent and manage diseases associated with SARS-CoV-2 infection, particularly by inducing a strong and sustained immune response to the virus. The vaccine composition provided by this invention utilizes H-ferritin protein to promote an enhanced immune response and provides broad protection against various variants. Furthermore, this vaccine is applicable to a wide range of populations, contributing to reducing the spread of infectious diseases and building a healthier and safer society. This effect represents a significant advancement in public health and lays a crucial foundation for preparing for similar pandemic situations in the future. By combining scientific innovation and medical advancements, this invention represents a significant step forward in supporting the global fight against the coronavirus.

[0030] Figure 1 illustrates the process of selecting clones with binding activity to SARS-CoV-2 RBD from an autodisplay Fv-antibody library.

[0031] Figure 2a shows that GFP is expressed within the cell line.

[0032] Figure 2b shows the results of quantitative analysis of flow cytometry analysis after pseudovirus infection.

[0033] Figure 3a illustrates the binding sites of four Fv antibodies.

[0034] Figure 3b shows the results of the docking simulation.

[0035] Figure 4a is a drawing showing that the binding antigen is bound to H-ferritin.

[0036] Figure 4b illustrates that expression of the antigen bound with H-ferritin was confirmed after purification with cell lysate and His-tag column.

[0037] Figure 4c shows an SEM image of the antigen bound to H-ferritin.

[0038] Figure 4d shows the size distribution of H-ferritin particles.

[0039] Figure 5a illustrates the mouse experimental process.

[0040] Figure 5b shows the results of antibody activity measurement in serum.

[0041] Figure 5c illustrates that antiserum is mixed with Fv-antibody and then processed on an RBD antigen-immobilized plate.

[0042] Figure 5d shows the results measured in which the bound antibody in the antiserum decreases as the concentration of Fv-antibody increases.

[0043] Figure 6a shows the results of the neutralizing activity of the expressed Fv-antibody measured in the range of 20 - 40% in the fluorescence image.

[0044] Figure 6b shows the quantitative results of flow cytometry analysis after pseudovirus infection.

[0045] Figure 7 shows the results of an analysis showing that CDR3 of the Fv antibody has an amino acid sequence similar to a part of ACE2.

[0046] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0047] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof. In the context of this specification, the term "about" or the like can mean about ± 1%, about ± 2%, about ± 3%, about ± 4%, about ± 5%, about ± 6%, about ± 7%, about ± 8%, about ± 9%, or about ± 10% of a numerical value described in the specification.

[0048] Additionally, the description of one aspect of the present invention may be applied identically or similarly to the same or similar configurations or terms in the description of other aspects.

[0049] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0050] A peptide according to an embodiment of the present invention is a peptide capable of inducing in vivo expression of an antibody against SARS-CoV-2, and may include one or more peptide sequences selected from the group consisting of a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, a peptide sequence of SEQ ID NO: 3, and a peptide sequence of SEQ ID NO: 4.

[0051] [Sequence number 1] WNRKRISGGSNGGSGGSPGGSFER

[0052] [Sequence number 2] SGGSWNSNNLDSKVGGSP

[0053] [Sequence number 3] RKSNLKPFERDISTEIYQ

[0054] [SEQ ID NO: 4] KGGSGGSRGDEVRQIAPGQTGKGGSL

[0055] In the context of this specification, the term "peptide" refers to a short amino acid sequence that specifically interacts with the receptor binding domain (RBD) of the SARS-CoV-2 virus. These peptides play an essential role in blocking the infectivity of the virus and stimulating an immune response. In particular, the peptide sequences disclosed herein promote the in vivo expression of antibodies against SARS-CoV-2, thereby inducing an effective immune response against the virus. These peptides are produced using various forms of recombinant expression vectors and cell systems, and are used as key components of vaccine compositions.

[0056] In the context of this specification, terms such as "antibody expression" or "immunity" refer to the process of producing and increasing antibodies through the body's defense response to a specific antigen. This process is induced by a vaccine and involves activating the immune system, enabling the body to effectively respond to pathogens such as SARS-CoV-2. In particular, the vaccine composition developed in the present invention induces a virus-specific antibody response, providing long-term immune protection. This immune response plays a crucial role in preventing infection, reducing viral transmission, and responding to public health crises such as pandemics.

[0057] Meanwhile, the nucleic acid according to an embodiment of the present invention can encode the peptide. In the context of this specification, "coding" refers to the process by which a specific DNA or RNA sequence determines the amino acid sequence of a specific protein or peptide. This refers to the process by which genetic information is translated into proteins, through which the genetic sequence can determine the structure and function of a specific protein. This gene translation mechanism plays a crucial role in biological processes, particularly in vaccine development. To produce antibodies against specific antigens, the production of peptides with precise amino acid sequences is essential, and this requires precise coding. The nucleic acid presented in the present invention comprises a coding sequence that enables the production of such specific peptides. This nucleic acid can be inserted into a recombinant expression vector and used as a template for peptide production in target cells or tissues.

[0058] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of this specification, a recombinant expression vector refers to a DNA molecule engineered for gene expression. It functions to transfer a gene into a new host cell and to translate the gene into a protein. A recombinant expression vector contains a specific gene or DNA sequence, thereby inducing the production of a specific protein in the host cell. The vector may include a promoter, an origin for gene replication, a selection marker, and other regulatory sequences for the expression of the inserted gene. These components increase the efficiency and stability of gene expression and contribute to optimizing expression for specific cell types or conditions. The use of recombinant expression vectors plays a key role in biological research and pharmaceutical development, particularly in vaccine development, enabling the effective development of preventive and therapeutic strategies for various diseases.

[0059] Meanwhile, cells according to embodiments of the present invention can be transformed with the recombinant expression vector. In the context of this specification, "transformation" refers to the process of introducing exogenous genetic material into a cell. Through this process, the cell acquires new genetic characteristics, which can lead to changes in the cell's physiological, morphological, or genetic characteristics. Transformation is widely used in gene expression, protein production, and biotechnology research and applications. These transformed cells induce the production of specific proteins or peptides through the genes encoded by the recombinant expression vector, which can be used for medical or research purposes. In particular, these cells play a crucial role in vaccine development and can contribute to the development of new treatments or diagnostic methods. This process can be used to manipulate the genetic makeup of cells to enhance the immune response to pathogens or to study specific disease states, which can significantly contribute to the advancement of life sciences.

[0060] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, and Schizosaccharomaces. It may comprise one or more cells selected from the group comprising Schizosaccharomyces sp. and Neurospora crassa.

[0061] The selection of the above cells may be based on a cell type suitable for the expression and production of a specific protein or peptide. Among various cell types, monkey kidney cell 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138 cells, baby hamster kidney (BHK) cells, MDCK cells, myeloma cell lines, HuT 78 cells, and HEK293 cells are all cells of human or animal origin, and they can be widely used for the expression and production of specific proteins. In addition, bacterial types such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus can also be used for protein production. In addition, fungal or yeast cells such as Aspergillus, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomaces, and Neurospora crassa may also be suitable for protein production. These cells can be used to purify, modify, or optimize the functional activity of proteins based on their individual characteristics. Each cell type is selected based on its production and functional properties for specific proteins or peptides, which can be crucial for efficient protein production in biomedical and diagnostic applications.

[0062] Meanwhile, a vaccine material for SARS-CoV-2 according to an embodiment of the present invention may include a vaccine material for SARS-CoV-2, including a ferritin protein; and a peptide bound to the outer surface of the ferritin protein. In one embodiment, the peptide may be a peptide according to an embodiment of the present invention described above.

[0063] In the context of this specification, the term "ferritin protein" refers to a protein structure used to stimulate an immune response. In particular, ferritin is a naturally occurring protein whose structure allows it to efficiently contain multiple replicated antigen units, making it useful for vaccine development. By binding the peptide to the ferritin protein, the complex forms an effective vaccine material that is readily recognized by the immune system and capable of inducing an antibody response. Such ferritin-based vaccine materials play a crucial role in inducing a strong and sustained immune response to the target antigen and can provide effective protection against pathogens such as SARS-CoV-2. This unique structure optimizes the safety and efficacy of the vaccine and provides flexibility to respond to a variety of immune system responses.

[0064] In one embodiment, the ferritin protein may comprise H-ferritin protein. In the context of this specification, H-ferritin protein refers to an iron-storing protein that has its own nanoparticle form and is useful for vaccine development. This protein provides an ideal platform for the effective delivery of pathogen-specific antigens. The specific structure of H-ferritin allows for the repeated presentation of multiple antigens on its surface, which helps induce a more robust and sustained immune system response. This may enhance the immune response to SARS-CoV-2, which may be particularly important in combating new variants.

[0065] A SARS-CoV-2 vaccine composition according to an embodiment of the present invention may include the above-described SARS-CoV-2 vaccine substance. The vaccine composition comprises a specific peptide bound to the H-ferritin protein, a combination essential for effectively inducing an immune response against the SARS-CoV-2 virus. The vaccine composition is designed for stability, efficacy, and optimal immune system response. The composition may also include other ingredients, such as various additives, preservatives, stabilizers, and adjuvants, which help enhance the effectiveness and extend the shelf life of the vaccine.

[0066] A method for immunizing against SARS-CoV-2 according to an embodiment of the present invention comprises injecting the vaccine composition for SARS-CoV-2 into a subject.

[0067] In one embodiment, the method for immunizing against SARS-CoV-2 may comprise injecting the vaccine composition for SARS-CoV-2 into a human.

[0068] In one embodiment, the SARS-CoV-2 immunization method may comprise injecting the SARS-CoV-2 vaccine composition into an animal.

[0069] Hereinafter, embodiments of the present invention will be described. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0070] ingredient

[0071] SARS-CoV-2 RBD (amino acid residues 319–541), ferritin H-labeled RBD antigen, and Fv antibody were custom-made by Cosmogenetech (Seoul, Korea). Fetal bovine serum (#SH-30919.03) was purchased from Cytiva. FuGENE (#E5911) was purchased from Promega (Madison, WI). Penicillin-streptomycin (Gibco, #15140122), Dulbecco's modified Eagle's medium (DMEM; Gibco #11995065), Opti-MEM (Gibco, #31985-070), and 96-well microplates (Nunc Maxisorp) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lenti-X™ HEK293T cells (#632180), Lenti-X™ SARS-CoV-2 Packaging Single Shot (#632672), and Lenti-X™ Concentrator (#631231) were purchased from Clontech (Palo Alto, CA, USA). psPAX2 (#12260) was purchased from Addgene (http: / www.addgene.org). Spike pseudotyped viral vectors (wild type, delta, Omicron BA.2, and Omicron BA.4 / BA.5) were purchased from InvivoGen (San Diego, CA, USA). HRP-conjugated anti-mouse IgG1 (ab97240), IgG2a (ab97245), IgG2b (ab97250), and IgG3 (ab97260) antibodies were purchased from Abcam (Waltham, MA, USA).

[0072] Fv-antibody library screening

[0073] The preparation of the Fv-antibody library has been described previously (Jung et al., 2021a; Jung et al., 2021b; Lee et al., 2021; Sung et al., 2022a; Sung et al., 2022b). As shown in Fig. 1, clones with binding activity to SARS-CoV-2 RBD were selected from the autodisplay Fv-antibody library through the following steps: (1) Fv antibodies with randomized CDR3 regions were expressed in the E. coli membrane using an autodisplay system. (2) GFP-labeled SARS-CoV-2 RBD (2 μM) was reacted with the Fv-antibody library (OD600 nm = 0.5, 100 μL) at 37°C for 1 h. (3) After washing with 0.01% PBST, the selected clones were sorted (n = 2,000) using a FACSCalibur™ flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). (4) The selected clones were selected after DNA sequencing of the CDR3 region. The amino acid and oligonucleotide sequences of the CDR3 region of the selected clones are summarized in Table 1. (5) The Fv-antibody expression plasmid was generated through DNA cloning.

[0074] Pseudovirus production of SARS-CoV-2 variants

[0075] Pseudoviruses were generated by co-transfection of Lenti-X™ HEK293T cells with a helper plasmid (psPAX2), a GFP expression plasmid (pLVXS-ZsGreen1-Puro), and the SARS-CoV-2 spike pseudotyped viral vector (wild type, Delta, Omicron BA.2, and Omicron BA.4 / BA.5) using FuGENE transduction reagent. Pseudovirus production was performed through the following steps: (1) Lenti-X™ HEK293T cells (3 × 106 cells) were cultured in DMEM (14 mL) supplemented with 10% FBS. (2) 10 μL of FuGENE transfer reagent and three plasmids (5 μg of psPAX2, 5 μg of pLVXS-ZsGreen1-Puro, and 5 μg of SARS-CoV-2 spike pseudotyped viral vector) were added to 1 mL of Opti-MEM and incubated at 37°C for 10 min. (3) The medium was replaced with DMEM (10 mL) containing 10% FBS. (4) The mixture from step 2 was slowly added to Lenti-X™ HEK293T cells. (5) After 72 h, the supernatant was centrifuged at 500 X g for 10 min to remove cell debris. (6) The supernatant was mixed with the Lenti-X™ concentrator and incubated at 4°C for 3 h. (7) The mixture from step 6 was centrifuged at 1,500 X g for 45 min at 4°C. (8) The pseudovirus pellet was resuspended in DMEM medium. (9) The pseudovirus was stored at -70°C. (10) The viral titer of the pseudovirus was determined using the Lenti-X™ qRT-PCR titer determination kit (#631235; Takara Bio, CA, USA) at 10 8 10 in 9 It was measured in the range of copies / mL.

[0076] Expression and purification of ferritin H

[0077] The RBD antigen-tagged ferritin H expression plasmid was custom-made by Cosmogenetech (Seoul, Korea). The amino acids of ferritin H are listed in Table 3. To express ferritin H in Escherichia coli, BL21(DE3) competitive cells were transformed with the ferritin expression plasmid. Next, the transformed E. coli were cultured in 100 mL Luria Bertani (LB) medium supplemented with 1 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) and 30 μg / mL carbenicillin and incubated at 30°C for 16 h. The E. coli were centrifuged at 2,000 × g for 2 min, and the pellet was resuspended in 30 mL of binding buffer (5 mM Tris-HCl, 0.5 M NaCl) containing 6 M urea. The E. coli in binding buffer E. coli was sonicated using an ultrasonic reactor (Vibracell VCX-130, Sonics, USA). The supernatant was then centrifuged at 25,000 × g for 10 min. Ferritin H in the supernatant was purified using a His-tag purification column (Roche, Basel, Switzerland). After washing with 3 M urea binding buffer, ferritin H was eluted with an elution buffer containing 1 M urea binding buffer and 100 mM imidazole (Spriestersbach et al., 2015). Finally, the purified ferritin H was dialyzed at 4°C for 16 h to remove imidazole and urea.

[0078] mouse immunization

[0079] Six-week-old BALB / c mice (Samtaco, Korea) were divided into five groups of five mice each. All groups received three intramuscular injections into the pectoralis muscle at two-week intervals. The control group received PBS and 50 μL Alum adjuvant, and the positive group received 100 μg of ferritin-RBD1-4 and 50 μL Alum adjuvant, respectively. Serum was collected 42 days after the initial immunization. For ELISA, SARS-CoV-2 RBD (10 μg / mL, 100 μL / well) was coated onto Maxisorp microplates for 16 h at 4°C. After washing with PBS, each well was blocked with 1 mg / mL BSA (120 μL / well) in PBS for 1 h at 37°C. Subsequently, serially diluted sera were incubated for 1 h at 37°C. After washing with PBS, HRP-conjugated anti-mouse subtype antibody (100 ng / mL, 100 μL / well) was added to the wells for 1 h at 37°C. Finally, each well was washed with 0.01% PBST, and the TMB reaction was performed using TMB reagent (#34021) for 3 min. After adding 2 M sulfuric acid (100 μL) to each well to stop the reaction, the optical density at 450 nm was measured using a Versamax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

[0080] Competition analysis between anti-RBD and immunized antibodies

[0081] A competitive assay setup was applied to detect SPs in standard samples. SARS-CoV-2 RBD (10 μg / mL, 100 μL / well) was immobilized in Maxisorp microplates at 4°C for 16 h. After washing with PBS, each well was blocked with 1 mg / mL BSA (120 μL / well) for 1 h at 37°C. Serum diluted 1000-fold was mixed with Fv antibodies at concentrations ranging from 1.3 to 1000.0 nM and then applied to the RBD-immobilized plate. After washing, HRP-labeled anti-mouse IgG antibody (250 ng / mL) was incubated for 1 h at 37°C. The TMB reaction was performed in the same manner as described above.

[0082] Pseudovirus neutralization assay of SARS-CoV-2 variants

[0083] The neutralizing activity of the four antisera was measured using an in vitro cell-based infection assay. A pseudovirus neutralization assay was performed. hACE2-293T cells (1.0 × 10 5 / well) were pre-incubated in poly-L-lysine-treated 96-well plates and incubated for 24 h. Pseudoviruses of SARS-CoV-2 variants (5 μL, 1.0 × 106 copies / mL) were pre-incubated with antiserum (100 μL) diluted 300-fold in DMEM containing 2% FBS for 30 min at 37°C. After removing the cell culture medium, the mixture of pseudovirus and antiserum was added to the cells for 48 h at 37°C. Finally, GFP and DAPI fluorescence images were observed using a microscope (model Eclipse Ts2). In the same way, the neutralizing activity of Anti-RBD was also analyzed.

[0084] result

[0085] Fv antibodies obtained from CDR3 library and epitope analysis

[0086] Four Fv antibodies were selected from an autodisplay Fv antibody library expressed on the outer membrane of E. coli. The library consists of three CDRs, CDR3 being 10 6 It has a random amino acid sequence (11 residues) with greater diversity than clonal antibodies. The Fv-antibody library was screened using a FACSCalibur™ (Becton-Dickinson, Franklin Lakes, NJ, USA) flow cytometer together with the receptor binding domain (RBD) of the spike protein (SP) of SARS-CoV-2. The screened Fv-antibodies were expressed as soluble proteins with three CDRs (Jung et al., 2023), and the binding affinities (KD) of the screened Fv-antibodies were measured to be 10-80 nM, as summarized in Table 1. As previously reported, the binding affinity of SARS-CoVs to the ACE2 receptor was measured to be 31-100 nM for SARS-CoV-1 and 4.7-10 nM for SARS-CoV-2 (Lan et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Zahradnik et al., 2021; Jung et al., 2023), and the binding affinity of the selected Fv-antibodies was considered sufficiently high compared to the interaction between the SP of SARS-CoVs and the ACE2 receptor.

[0087] Table 1 shows the results of screening CDR3 sequences with affinity for the RBD region of the SARS-CoV-2 spike protein.

[0088] Screened cloneOligonucleotide sequence (33 bp)CDR3 sequence (11 mer)Binding constant K D (nM)13-GAT ATA AGC GGA GAG AAA CAC CCC GCG GAT TTC-5 1 DISGE 5 KHPAD<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> F20-8123-GAC GGT ACA CCG AAA GGC CCT AGA AAT GAT TTC-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> DGTPK<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> GPRND<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> F13-7133-GAC CCT CCA ACT GGT ACA ATG GTA ACA GAT TTT-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> DPPTG<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> TMVTD<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> F24-8543-TAC GGA AGT CTA CAA GCA CAA GCA CCT GAT GAT-5<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> YGSLQ<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> AQAPD<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> D24-70<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] The neutralizing activity of the four expressed Fv antibodies was measured using an in vitro cell-based infection assay. When pseudovirus particles expressing SP and the indicator GFP gene on their surface were infected with a model cell line that constitutively expresses the ACE2 receptor, GFP was expressed within the cell line (as shown in Figure 2(a)). When the four Fv antibodies were treated together with pseudovirus particles bearing SP, infection was blocked, demonstrating the neutralizing activity of the Fv antibodies. Compared to infection with the positive control, VSV-G virus, the neutralizing activity of the expressed Fv antibodies was measured in the range of 20-40% in fluorescence images. Flow cytometry analysis after pseudovirus infection was quantitatively analyzed and is shown in Figure 2(b). For pseudovirus particles with expressed mutant SPs of SARS-CoV-2 variants such as Delta, Omicron BA.2, and Omicron BA.4 / 5, the neutralizing activity of the four Fv antibodies was observed to decrease as the mutation increased from the original SARS-CoV-2 type. These results indicate that the Fv antibodies can be effectively used as neutralizing antibodies in antisera generated after vaccination. The sufficiently high binding affinity of the Fv antibodies to the RBD of SARS-CoV-2 as well as the neutralizing activity of the Fv antibodies indicated that the epitope of the Fv antibody could be effective as an antigen for vaccination against the SP. In the docking simulation using the RBD of SP (Protein Database Number: 6VXX), the antigenic sites for the four Fv antibodies binding were analyzed and summarized in Table 2 with favorable docking parameters such as negative Gibbs free energy.

[0090] Anti-RBBD S443, K444, V445, P499-6.83R457, K458, S459, R466, S469, Y473, Q474-7.14K378, R403, V407, R408, Q409, A411, G413, T415, G416, K417, L455-6.7

[0091] As shown in Figure 3(a), the binding sites of the four Fv antibodies were found to be located at different locations. Furthermore, amino acids involved in the interactions between the four Fv antibodies and the RBD were found to be spatially concentrated in the 3D structure. As shown in Figure 3(b), the distances between key amino acid residues involved in the interactions between the Fv antibodies and the RBD were analyzed in docking simulations. To represent these amino acid sequences as antigens, the gaps were filled with a non-immunogenic amino acid sequence consisting of G2S, which is summarized in Table 3.

[0092] Ferritin HRBD#Amino acid sequence of RBD antigen (N -> C term)Molecular weight (kDa)5x[Antigen-(G2S)10]-Ferritin H1353WNRKRIS--5.6 Å--394N--16.4 Å--426P--7.5 Å--464FER47.9WNRKRISGGSSNGGSGGSPGGSFER2373S--7.3 Å--436WNSNNLDSKV--7.1 Å--499P44.8SGGSWNSNNLDSKVGGSP3457RKSNLKPFERDISTEIYQ47.1RKSNLKPFER DISTEIYQ4378K--17.2 Å--403RGDEVRQIAPGQTGK--6.9 Å--455L48.3KGGSGGSRGDEVRQIAPGQTGKGGSL

[0093] Ferritin-conjugated antigen challenge and neutralizing activity of antiserum

[0094] The identified epitope in the RBD of SARS-CoV-2 SP for Fv antibody binding was expressed as an antigen conjugated with the ferritin intermediate chain (H-ferritin). As shown in Figure 4(a), the conjugated antigen expressed five times with the epitope repeated and conjugated with H-ferritin (192 residues, 24.8 kDa). A non-immunogenic linker ((G2S)10, 2 kDa) was used between the repeated epitopes, resulting in a conjugated antigen with a molecular weight of approximately 45-48 kDa. The expression of the antigen conjugated with H-ferritin was confirmed after purification with cell lysate and His-tag column (see Figure 4(b)). The morphology of the antigen conjugated with H-ferritin in the SEM image was analyzed as a spherical shape with a diameter of approximately 20-30 nm, as shown in Figure 4(c). In the presence of only H-ferritin without repeated antigens, H-ferritin particles were observed as spherical particles with a diameter of less than approximately 20 nm. The size distribution of the bound antigens was measured using dynamic laser scattering (DLS). As shown in Fig. 4(d), H-ferritin particles without epitopes had a size distribution of 18.9±0.9 (4.9%) nm, while the bound antigens had size distributions of 31.9±0.8 (2.5%) nm for Ferritin-RBD1, 29.8±1.2 (4.2%) nm for Ferritin-RBD2, 27.5±0.3 (1.1%) nm for Ferritin-RBD3, and 32.3±1.2 (3.8%) nm for Ferritin-RBD4. These results demonstrated that the bound antigens were expressed together with H-ferritin and that these antigen particles had a uniform size of less than 4.9%.

[0095] The four conjugated antigens were injected into mice using alum as an adjuvant for immunization. Two weeks after the first injection of the conjugated antigens, a booster injection was administered, and the mice were terminated after another week (see Figure 5(a)). The binding affinity of the antibodies was assayed using RBD antigen-immobilized plates and 1000-fold diluted antisera. As shown in Figure 5(b), antibody activity in the serum was measured in the OD range of 2-3 at 450 nm for the four types of conjugated RBD antigens. The IgG subtype titers in serum, IgG1, IgG2a, IgG2b, and IgG3, were as follows: IgG1 4.1 × 10^5, IgG2a 7.6 × 10^3, IgG2b 3.4 × 10^4, and IgG3 2.1 × 10^3 for Ferritin-RBD1; IgG1 5.7 × 10^5, IgG2a 6.0 × 10^4, IgG2b 5.2 × 10^4, and IgG3 1.5 × 10^3 for Ferritin-RBD2; IgG1 3.2 × 10^5, IgG2a 7.6 × 10^3, IgG2b 7.6 × 10^3, and IgG3 8.3 × 10^2 for Ferritin-RBD3; and IgG1 4.9 × 10^5, IgG2a 2.8 × 10^3 for Ferritin-RBD4. 10^4, IgG2b 1.3 × 10^4, and IgG3 1.5 × 10^3 were measured. These results indicate that the antigen is assembled with ferritin, and the epitopes are arranged in a regular and repetitive manner. This arrangement is an important factor in initiating and enhancing the adaptive immune response by promoting efficient uptake by antigen-presenting cells (Bachmann and Jennings, 2010; Veggi et al., 2023).

[0096] The antibody titers of each antisera against SP were measured using a competition assay. As shown in Figure 5(c), 1000-fold diluted antisera were mixed with Fv antibodies at concentrations ranging from 1.3 to 1000.0 nM and then treated on RBD antigen-immobilized plates. As shown in Figure 5(d), the amount of antibody bound to the antisera was measured to decrease with increasing Fv antibody concentration. These results demonstrate that not only the antibodies in the antisera but also the Fv antibodies competed for binding to the RBD antigen, and that these antibodies recognized the RBD antigen as an epitope. The concentration at half maximal response for each antisera was measured as 62.1±15.2 nM for Anti-RBD1 antiserum obtained from Ferritin-RBD1, 23.8±3.0 nM for Anti-RBD2 antiserum obtained from Ferritin-RBD2, 125.4±38.2 nM for Anti-RBD3 antiserum obtained from Ferritin-RBD3, and 35.4±3.8 nM for Anti-RBD4 antiserum obtained from Ferritin-RBD4. These results indicate that the antisera (1000-fold dilution) had antibody titers ranging from 23.8 to 125.4 nM against the RBD antigen, with Anti-RBD2 having the highest antibody titer against the RBD antigen.

[0097] The neutralizing activity of the four antisera was measured using an in vitro cell-based infection assay. As described previously, pseudovirus particles expressing SP and the indicator GFP gene on their surface infected a model cell line that constitutively expressed the ACE2 receptor, and GFP was expressed within the cell line. When the four antisera were treated with the pseudovirus particles bearing SP, infection was blocked, demonstrating the neutralizing activity of the antisera. Compared to infection with the positive control, VSV-G virus, the neutralizing activity of the expressed Fv antibodies was measured in the range of 20-40% in fluorescence images (see Figure 6(a)). Flow cytometry analysis after pseudovirus infection was quantitatively analyzed (see Figure 6(b)). The relative comparison of the neutralizing activity of the antisera (300-fold dilution) was measured as 19.8% for Anti-RBD1 (23.1% for Anti-RBD Fv1, 0.5 μM), 20.7% for Anti-RBD2 (17.8% for Anti-RBD Fv2, 0.5 μM), 33.1% for Anti-RBD3 (33.4% for Anti-RBD Fv3, 0.5 μM), and 24.3% for Anti-RBD4 (22.6% for Anti-RBD Fv4, 0.5 μM). For pseudovirus particles with mutant SPs of SARS-CoV-2 variants, such as Delta, BA.2, and BA.4 / 5, the neutralizing activity of antisera was observed to decrease with increasing mutations from the original SARS-CoV-2 type (Perez-Then et al., 2022; Servellita et al., 2022). These results indicate that antisera can be effectively used as neutralizing antibodies.

[0098] discussion

[0099] A vaccine development method was proposed through the sequential steps of (1) screening of Fv antibodies from an autodisplay Fv antibody library, (2) antigen selection through epitope screening by docking simulation, (3) expression and vaccination of the antigen with H-ferritin, and (4) confirmation of neutralizing activity of the immunized antiserum. To produce high titer antiserum, the step of screening Fv antibodies with high binding affinity for the target protein from the autodisplay Fv antibody library was considered a key step. In the present invention, four Fv antibodies against the RBD of SARS-CoV-2 SP were screened, as summarized in Table 1. As the RBD binds to the ACE2 receptor, the relationship between the amino acid sequences of the screened Fv antibodies and the ACE2 receptor was investigated using BLAST analysis. As summarized in Figure 7, the CDR3 of the Fv antibodies was analyzed to have an amino acid sequence similar to a part of ACE2. The identity with the exact same amino acid sequence and the similarity with the same chemical properties such as polarity, acidity, and basicity were analyzed for the Fv antibodies and ACE2 receptor, which were 45% for Anti-RBD1, 54% for Anti-RBD2, 45% for Anti-RBD3, and 45% for Anti-RBD4. These results showed that the CDR3 of the Fv antibodies in the library did not come from random amino acid sequence selection for the RBD antigen. Therefore, it was considered that these CDR3 amino acid sequences had high similarity to the ACE2 receptor, and it was thought that high titer antisera could be obtained by using epitopes as antigens for this region.

[0100] Another key issue was the production of antisera through the application of antigens conjugated with H-ferritin. As shown in Figure 7, the CDR3 of the Fv antibody has a similar region to that of the ACE2 receptor, and the epitopes for Fv-antibody binding were distributed in different parts of the RBD, as shown in Figure 3(a). In the present invention, epitopes were searched through computer simulation, and effective antigenic sites for binding of the four Fv antibodies had to be determined using docking parameters (e.g., negative Gibbs free energy, etc.), which are summarized in Table 2. These antigenic sites were then expressed as repeat sequences in the antigens conjugated with H-ferritin. The purpose of these repeat sequences was to increase the antibody titer of the antisera. These results demonstrated that conjugated antigens with repeated epitope sequences can be used as an effective method for generally increasing antisera titer.

[0101] Antisera produced with antigens conjugated with H-ferritin were analyzed to have low binding affinity for mutant SARS-CoV-2. As the amino acid sequence variation of the RBD antigen increased compared to the original type of SP, the binding specificity statistically decreased, as shown in Figure 6(b). Therefore, high-titer antisera can be produced by screening Fv antibodies using the modified RBD antigen. By screening Fv antibodies from an autodisplay Fv library and using antigens conjugated with H-ferritin, high-titer antisera against mutant strains are expected to be effectively produced within a short preparation time.

[0102] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0103] National Research and Development Project that supported this invention

[0104] - Assignment ID (10 digits): 1465038753

[0105] - Assignment number: RS-2022-KH128357

[0106] - Ministry name: Ministry of Health and Welfare

[0107] - Project Management (Professional) Organization Name: Korea Health Industry Development Institute

[0108] - Research Project Name: Rapid Universal Vaccine Technology Development

[0109] Research Project Name: Research on a Vaccine Screening Platform Capable of Rapidly Responding to Mutant Viruses

[0110] - Contribution rate: 1 / 1

[0111] - Project implementation organization name (host organization): Yonsei University Industry-Academic Cooperation Foundation

[0112] Research period: January 1, 2023 - December 31, 2023

Claims

1. A peptide that can induce in vivo expression of antibodies against SARS-CoV-2. Comprising at least one peptide sequence selected from the group consisting of a peptide sequence of sequence number 1, a peptide sequence of sequence number 2, a peptide sequence of sequence number 3 and a peptide sequence of sequence number 4. Peptide: [Sequence number 1] WNRKRISGGSNGGSGGSPGGSFER [Sequence number 2] SGGSWNSNNLDSKVGGSP [Sequence number 3] RKSNLKPFERDISTEIYQ [Sequence number 4] KGGSGGSRGDEVRQIAPGQTGKGGSL.

2. Coding a peptide according to paragraph 1; Nucleic acid.

3. Containing a nucleic acid according to Article 2, Recombinant expression vector.

4. Transformed with a recombinant expression vector according to Article 3, cell.

5. In paragraph 4, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli and insect cells. cell.

6. In paragraph 5, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp., Aspergillus sp., Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp.) and Neurospora crassa, comprising one or more cells selected from the group consisting of cell.

7. Ferritin protein; and A peptide bound to the outer surface of the above ferritin protein; The above peptide comprises a peptide according to claim 1. Vaccine material for SARS-CoV-2.

8. In paragraph 7, The above ferritin protein comprises H-ferritin protein. Vaccine material for SARS-CoV-2.

9. Containing a vaccine material for SARS-CoV-2 according to Article 7; A vaccine composition for SARS-CoV-2.

10. Comprising injecting a SARS-CoV-2 vaccine composition according to Article 9 into a subject; Methods of SARS-CoV-2 immunization.

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