WO2023025031A1 - Épitope neutralisant la protéine s du virus sars-cov-2 destiné à être utilisé dans le test d'anticorps de vaccin ou de neutralisation - Google Patents

Épitope neutralisant la protéine s du virus sars-cov-2 destiné à être utilisé dans le test d'anticorps de vaccin ou de neutralisation Download PDF

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WO2023025031A1
WO2023025031A1 PCT/CN2022/113307 CN2022113307W WO2023025031A1 WO 2023025031 A1 WO2023025031 A1 WO 2023025031A1 CN 2022113307 W CN2022113307 W CN 2022113307W WO 2023025031 A1 WO2023025031 A1 WO 2023025031A1
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protein
fragment
epitope
polypeptide
seq
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PCT/CN2022/113307
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Chinese (zh)
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姜毅楠
秦丽丽
陈宜顶
苗景赟
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北京百普赛斯生物科技股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus

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  • This application relates to the field of biotechnology, in particular, to a neutralizing epitope of the S protein of the new coronavirus, and its application in the preparation of vaccines and detection kits for neutralizing antibodies.
  • Novel coronavirus SARS-CoV-2 (2019-nCoV) is a virus discovered in 2019 that can cause viral pneumonia or lung infection in humans, leading to an unprecedented public health crisis, as of August 16, 2021, global infections The number of people has exceeded 200 million, and more than 4 million people have died. After the failure of various drug trials, preventive vaccines have become the main tool to prevent the spread of the virus and ensure the health of the people through "herd immunity”.
  • New vaccine technologies include recombinant protein technology and nucleic acid technology, which focus on the key proteins of viral pathogenesis rather than the whole virus. This type of technology greatly reduces the difficulty of vaccine development and production, and at the same time increases the proportion of key anti-virus protective antibodies, that is, "neutralizing antibodies” in total antibodies.
  • the infection process of the new coronavirus is started by the mutual recognition of the spike glycoprotein (SPIKE protein, S protein) on its surface and the angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell, so almost all the new coronavirus vaccines currently use
  • S protein is the target, and the expression of the S protein induces the human immune system to produce protective antibodies that can bind to the new coronavirus, thereby achieving the goal of preventing infection. If there is a significant mutation on the virus S protein, it may lead to reduced vaccine protection, that is, "immune escape".
  • VOC coronavirus variants of concern
  • the latest real-world study released by the Israeli Ministry of Health found that the Pfizer vaccine’s protection against symptomatic new coronavirus infection was reduced to 64%.
  • the Pfizer/BioNTech mRNA vaccine was only 39% protective against infection with symptomatic COVID-19 caused by the Delta mutant strain in Israel.
  • One purpose of this application is to seek a method or kit suitable for the detection of novel coronavirus neutralizing antibody titers
  • Another purpose of this application is to seek a method suitable for the preparation of a novel coronavirus vaccine.
  • the application first provides a neutralizing epitope selected from any one of (i)-(vi) S protein RBD of the new coronavirus:
  • the present application also provides a polypeptide or protein comprising the above epitope.
  • the sequence of the epitope (i) is SEQ ID No.1 or SEQ ID No.4; the sequence of the epitope (ii) is SEQ ID No.1 or SEQ ID No.4; the sequence where the epitope (iii) is located is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4; where the epitope (iv) is The sequence is SEQ ID No.3; the sequence where the epitope (v) is located is SEQ ID No.1 or SEQ ID No.4; the sequence where the epitope (vi) is located is SEQ ID No.3 or SEQ ID No.4; or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Amino acid sequences with 99% sequence identity.
  • the present application also provides an application of the above-mentioned neutralizing epitope, or polypeptide or protein in detection/screening/purification/preparation of neutralizing antibodies.
  • the present application also provides an application of the above-mentioned neutralizing epitope, polypeptide or protein in the preparation of a neutralizing antibody detection/screening/purification kit.
  • the present application also provides an application of the above-mentioned neutralizing epitope, polypeptide or protein in preparing a vaccine.
  • test kit which comprises:
  • S protein or fragment thereof encoded by SARS-CoV-2 which comprises an epitope selected from any one of (i)-(vi) S protein RBD of the new coronavirus:
  • it also includes:
  • the detection substance for detecting the interaction between the S protein or its fragments of (1) and the ACE2 or its fragments of (2); in some preferred modes, the spike protein of (1) or its fragment, or (2) ACE2 protein or its fragment is coupled with the detection substance.
  • the spike protein of (1) or a fragment thereof is coupled to the detection substance, and the ACE2 of (2) or a fragment thereof is immobilized on a solid support;
  • ACE2 or its fragment of (2) is coupled to the detection substance, and the spike protein or its fragment of (1) is immobilized on a solid support.
  • the detection substance is labeled with fluorescent labels, luminescence labels, immunodetectable labels, radiation labels, chemical labels, nucleic acid labels or polypeptide labels;
  • the present application also provides a method for detecting neutralizing antibody titers in vivo or in vitro in a subject, which includes:
  • the neutralizing epitope of the present application contains the amino acid core sequence of the new coronavirus and the amino acid sequences on both sides of the core sequence. Incorporation of relevant neutralizing epitope sequences can specifically induce neutralizing antibodies in vaccinated populations.
  • FIG. 1 Structure-based antigen clustering of SARS-CoV-2 RBD neutralizing antibodies.
  • Figure 4 Schematic representation of the surface representation model of six neutralizing antibodies bound to the RBD.
  • FIG. 1 Schematic diagram of the conserved amino acid positions of the SARS-CoV-2 RBD.
  • FIG. 7 Sequence comparison of SARS-CoV-2 wild type and VOC variants B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, B.1.526 and C.37 right. Residues involved in direct interaction with ACE2 are marked with balls.
  • FIG. 1 Heat map of monoclonal antibody epitopes from 3-dose vaccinated subject patients.
  • the terms “comprising”, “comprising”, “having”, “containing” or “involving” are inclusive or open-ended and do not exclude other unrecited elements or method steps .
  • the term “consisting of” is considered as a preferred embodiment of the term “comprising”. If in the following a certain group is defined as comprising at least a certain number of embodiments, this is also to be understood as revealing a group which preferably consists only of these embodiments.
  • This application provides a main antigenic epitope, that is, the neutralizing epitope of the S protein of the new coronavirus SARS-CoV-2.
  • This application provides the location of the main neutralizing epitope that is important for the design of an effective vaccine and the detection of neutralizing antibodies, which contains the amino acid core sequence of the new coronavirus and the amino acid sequences on both sides of the core sequence, these sequences include the new coronavirus Neutralizing epitope on the S protein RBD.
  • Neutralizing antibodies can be specifically induced in vaccinated populations by incorporating relevant neutralizing epitope sequences in various types of vaccines.
  • Vaccines containing the neutralizing epitopes provided herein are prepared so as to induce measurable neutralizing antibodies.
  • sequence of the S protein RBD located at a sequence corresponding to approximately amino acid positions 340-510 found in wild-type 2019-nCoV contains a neutralizing epitope.
  • the neutralizing epitope is selected from any one of the following (i)-(vi):
  • the amino acid sequence located corresponding to about positions 400 to 510 more specifically constitutes a broadly reactive neutralizing site.
  • Other embodiments of the neutralization sites provided in this application can be found among the various novel coronavirus VOC isolates known or to be discovered.
  • Teen working in the field of molecular biology can easily compare the sequence containing the neutralization site provided in this application with the amino acid sequence of the S protein RBD of another VOC.
  • the Spike protein or fragment thereof may comprise at least 70%, 75%, 80%, 85% of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 Amino acid sequences having %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the present application provides the above-mentioned neutralizing epitope, or the application of the polypeptide/protein in the preparation of antibodies. It can be understood that any preparation method or use of antibodies or antigen-binding fragments, etc., based on the epitopes, polypeptides or proteins of the present application, using conventional experiments in the technical field, etc., all belong to the scope of rights of the present application, because the core of its technology is all An epitope, polypeptide or protein derived from the present application.
  • the antibody preparation methods described in this application include but are not limited to: for example, 1) hybridoma technology based on mice/rabbits, etc.; 2) antibody screening technology based on phage antibody display library; 3) construction of antibodies for immune antibody phage display library Screening technology, etc.; 3) Single B cell sequencing and antibody cloning, these specific preparation methods are not limited to the invention.
  • antibodies described herein can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimed antibodies, Conjugated antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers Amers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions, heteroconjugated antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, pro and bodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, Synthetic antibodies and antigen-binding fragments of any of the above.
  • an antigen-binding fragment or antibody fragment as used herein refers to any molecule comprising an antigen-binding fragment (eg, a CDR) of the antibody from which the molecule is derived.
  • Antigen binding molecules may include complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies and multispecific antibodies formed from antigen binding molecules.
  • the antigen-binding molecule binds to the S protein of the new coronavirus.
  • the antigen-binding molecule has neutralizing activity, and can inhibit the binding of the S protein of the new coronavirus to the receptor.
  • the present application provides the use of the above-mentioned neutralizing epitope, polypeptide or protein in the preparation of a vaccine or vaccine composition.
  • the vaccine can be a therapeutic vaccine or a preventive vaccine, and the types of vaccines can include but are not limited to: live attenuated vaccines, inactivated vaccines, antitoxins, subunit vaccines (containing polypeptide vaccines), vector vaccines, nucleic acid vaccines (mRNA vaccine and DNA vaccine) and so on.
  • the above-mentioned epitope, polypeptide or protein can be prepared into a corresponding vaccine composition through traditional vaccine preparation methods; Vaccine or mRNA vaccine etc.
  • These vaccine compositions can be used to immunize animals to elicit highly specific immune responses against the novel coronavirus.
  • the result of immunization will be down-regulation of the function of SARS-CoV-2 in the immunized animals.
  • the animal is a mammal, including but not limited to, rodents, such as mice, rats, rabbits, horses, dogs, cats, cattle, sheep, primates, etc.; the animal of the present application is especially directed at primates, For example, monkeys, great apes and humans etc.
  • the application provides a kit or composition comprising:
  • SARS-CoV-2 which comprises an epitope of the new coronavirus S protein selected from any one of (i)-(vi):
  • kits and compositions may also comprise a solid support.
  • the solid support can be any solid support on which the polypeptide can be easily immobilized (eg, by adsorption or conjugation) and which is suitable for analysis of antibody-containing samples (eg, blood-derived samples according to the present application, such as serum samples). Suitable solid supports for use in such kits and compositions are known in the art.
  • a solid support can include polystyrene, polypropylene, polycarbonate, cycloolefin, glass, or quartz.
  • a solid support can be a microtiter (or "well") plate or a microarray plate.
  • the solid support can be beads, such as magnetic beads.
  • polypeptide or protein according to the present application can be immobilized (or "coated") on the solid support of the present application by methods known in the art.
  • Polypeptides can be immobilized to a solid support either covalently or non-covalently.
  • the system used to detect the interaction can be any suitable system.
  • the system can use an antibody that can specifically bind to a polypeptide complex formed by a polypeptide or fragment encoded by the new coronavirus or a polypeptide or fragment that specifically binds to the polypeptide or fragment encoded by the new coronavirus, or can use antibodies that reflect the polypeptide or fragments of the new coronavirus.
  • a reporter protein that interacts with polypeptides or fragments that specifically bind to novel coronavirus-encoded polypeptides or fragments.
  • a system for detecting an interaction may employ a detection substance.
  • SARS-CoV-2 S1 and RBD polypeptides were coupled with horseradish peroxidase (HRP). After washing to remove unbound SARS-CoV-2 S1 and RBD polypeptides, horseradish peroxidase activity levels indicate the amount of S1/RBD bound to immobilized ACE2.
  • HRP horseradish peroxidase
  • the polypeptide encoded by the new coronavirus or a fragment thereof such as the spike protein or a fragment thereof encoded by the new coronavirus
  • the polypeptide specifically combined with the polypeptide or fragment encoded by the new coronavirus Polypeptides or fragments for example, ACE2 proteins or fragments thereof that specifically bind to the spike protein or fragments thereof encoded by the novel coronavirus
  • polypeptides or Substance coupling for detecting interactions between fragments.
  • the detection substance may for example be a detectable group such as a fluorescent label, a luminescent label, an immunodetectable label, a radioactive label, a chemical label, a nucleic acid label or a polypeptide label.
  • a detection substance can be a moiety that has a detectable activity, eg, enzymatic activity towards a specific substrate.
  • detection substances having detectable activity include, for example, horseradish peroxidase (HRP) and luciferase groups.
  • a polypeptide encoded by a new coronavirus or a fragment thereof for example, a spike protein or a fragment thereof (RBD) encoded by a new coronavirus
  • a kit or composition of the present application for analysis of a sample, wherein
  • the amount or concentration of a polypeptide encoded by a new coronavirus or a fragment thereof (such as a spike protein or a fragment thereof (such as RBD) encoded by a new coronavirus is (a) lower than the neutralization of a new coronavirus in a sample analyzed using the kit or composition
  • the amount/concentration of the antibody, and/or (b) in the absence of the new coronavirus neutralizing antibody in the sample it is sufficient to produce a polypeptide that reflects the new coronavirus encoding or its fragment (such as the new coronavirus encoding the spike protein or its fragment ( For example, a detectable signal of interaction between RBD)) and a polypeptide or fragment that
  • kits and compositions can be determined/referenced, for example, by determining or known samples containing neutralizing antibodies to novel coronavirus It is put into use after the amount/concentration of the new coronavirus neutralizing antibody in it.
  • the amount/concentration of suitable polypeptides or fragments may be (in molar ratio) lower than or equal to the average (e.g., arithmetic mean) amount/concentration.
  • the serial dilution of the sample to be tested can be used to determine the appropriate amount of a novel coronavirus-encoded polypeptide (such as spike protein) or a fragment thereof.
  • compositions and kits are generated by, for example, determining/referring to the absence of SARS-CoV-2 neutralizing antibodies in the sample Required for detectable signals reflecting interactions between novel coronavirus-encoded polypeptides or fragments thereof (such as novel coronavirus-encoded spike proteins or fragments thereof) and polypeptides (such as ACE2) or fragments thereof that specifically bind novel coronavirus-encoded polypeptides or fragments Put into use after the minimum amount/concentration.
  • novel coronavirus-encoded polypeptides or fragments thereof such as novel coronavirus-encoded spike proteins or fragments thereof
  • polypeptides such as ACE2
  • the kit of the present application can be used to detect the presence of an antibody that reduces or inhibits the binding of a novel coronavirus-encoded polypeptide or fragment thereof to a polypeptide or fragment that specifically binds a novel coronavirus-encoded polypeptide or fragment.
  • antibodies may be referred to as neutralizing antibodies.
  • the kit of the present application can be applied to a method for detecting the presence of antibodies to the new coronavirus in a sample. It should be appreciated that this method can be used to detect the presence of antibodies against the spike protein or fragments thereof encoded by 2019-nCoV employed in the kit/composition.
  • the assays presented herein employ the S1 subunit of the SARS-CoV-2 Spike protein or the RBD of the SARS-CoV-2 Spike protein and thus can be used to detect Antibodies to the SARS-CoV-2 spike protein RBD.
  • the presence of antibodies to the novel coronavirus in a test sample can indicate that the subject in which the sample was located is or has previously been infected with the new coronavirus.
  • the detection of the presence of antibodies to the new coronavirus in a sample can indicate the immune response, especially the humoral immune response, of the subject before the sample to the current or previous infection with the new coronavirus.
  • the present application provides methods for determining whether a subject is or has been infected by a novel coronavirus, and determining whether a subject is or has elicited an immune response (eg, a humoral immune response) to a novel coronavirus.
  • an immune response eg, a humoral immune response
  • the method may generally comprise analyzing a sample to determine whether, relative to the level of interaction observed under suitable negative control conditions, whether the content of the sample reduces or inhibits a novel coronavirus-encoded polypeptide (e.g., spike protein) or a fragment thereof with a specific The level of interaction between polypeptides or fragments (such as ACE2) that are sexually bound to novel coronavirus-encoded polypeptides or fragments thereof.
  • a novel coronavirus-encoded polypeptide e.g., spike protein
  • a fragment thereof with a specific The level of interaction between polypeptides or fragments (such as ACE2) that are sexually bound to novel coronavirus-encoded polypeptides or fragments thereof.
  • Appropriate negative control conditions can, for example, be employed, known to lack the ability to reduce or inhibit the interaction between a novel coronavirus-encoded polypeptide (such as spike protein) or a fragment thereof and a polypeptide or fragment (such as ACE2) that specifically binds to a new coronavirus-encoded polypeptide or a fragment thereof
  • a control sample can be from a subject known to be uninfected, such as a subject known not to be infected by the novel coronavirus.
  • Confirmation of reduced or suppressed interaction levels indicates the presence of neutralizing antibodies to novel coronavirus-encoded polypeptides or fragments thereof (such as spike proteins or fragments thereof) in the sample.
  • a "reduced” or “inhibited” interaction can be compared to a novel coronavirus-encoded polypeptide (e.g., spike protein) or fragment thereof that specifically binds to a novel coronavirus-encoded polypeptide or fragment thereof observed under negative control conditions.
  • a novel coronavirus-encoded polypeptide e.g., spike protein
  • fragment thereof that specifically binds to a novel coronavirus-encoded polypeptide or fragment thereof observed under negative control conditions.
  • 1-fold lower interaction level of peptides or fragments such as ⁇ 0.99-fold, ⁇ 0.95-fold, ⁇ 0.9-fold, ⁇ 0.85-fold, ⁇ 0.8-fold, ⁇ 0.75-fold, ⁇ 0.7-fold, ⁇ 0.65-fold, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times, ⁇ 0.05 times, or ⁇ 0.01 times
  • the "reduced” or “inhibited” interaction can be defined as the expression of a novel coronavirus-encoded polypeptide (such as a spike protein) or a fragment thereof and a polypeptide or fragment that specifically binds a new coronavirus-encoded polypeptide or a fragment thereof observed under negative control conditions. (eg, ACE2) expressed as a percentage of inhibition of the interaction.
  • a novel coronavirus-encoded polypeptide such as a spike protein
  • ACE2 eg., ACE2
  • “reduced” or “inhibited” interaction may refer to the specific binding of a new coronavirus-encoded polypeptide (such as spike protein) or a fragment thereof to a new coronavirus-encoded polypeptide or a fragment thereof observed under negative control conditions.
  • the percentage of inhibition of the interaction of the polypeptide or fragment is higher than 0%, such as higher than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 %, or 100% inhibition percentage.
  • an aspect of the present application may include:
  • a sample obtained from a subject with (i) a novel coronavirus-encoded polypeptide (e.g., spike protein) or a fragment thereof, and (ii) a polypeptide that specifically binds to a novel coronavirus-encoded polypeptide or fragment (e.g., ACE2) or a fragment thereof ,as well as
  • a novel coronavirus-encoded polypeptide e.g., spike protein
  • a polypeptide that specifically binds to a novel coronavirus-encoded polypeptide or fragment e.g., ACE2
  • an aspect of the present application may include:
  • step (b) contacting the mixture formed in step (a) with (ii) a polypeptide (eg, ACE2) or a fragment thereof that specifically binds to a novel coronavirus-encoded polypeptide or fragment, and
  • a polypeptide eg, ACE2
  • a fragment thereof that specifically binds to a novel coronavirus-encoded polypeptide or fragment
  • an aspect of the present application may include:
  • a sample obtained from the subject with (i) a polypeptide (eg, ACE2) or a fragment thereof that specifically binds to a polypeptide or fragment encoded by a novel coronavirus,
  • a polypeptide eg, ACE2
  • a fragment thereof that specifically binds to a polypeptide or fragment encoded by a novel coronavirus
  • step (b) contacting the mixture formed in step (a) with (ii) a novel coronavirus-encoded polypeptide (e.g., spike protein) or a fragment thereof, and
  • a novel coronavirus-encoded polypeptide e.g., spike protein
  • the application may include contacting a sample with a composition comprising a novel coronavirus-encoded polypeptide (such as spike protein) or a fragment thereof (RBD), wherein the amount or concentration of the polypeptide or fragment thereof (a) is in molar ratio It is lower than or equal to the amount/concentration of neutralizing antibodies in the sample, and/or (b) is sufficient to generate polypeptides or fragments reflecting (i) and (ii) polypeptides in the absence of new coronavirus neutralizing antibodies in the samples or detectable signals of interactions between fragments.
  • a novel coronavirus-encoded polypeptide such as spike protein
  • RBD fragment thereof
  • this aspect of the application can include determining the presence or absence of SARS-CoV-2 neutralizing antibodies in a sample. In some embodiments, this aspect of the application can include determining the amount and/or concentration of SARS-CoV-2 neutralizing antibodies in a sample.
  • this aspect of the application may also include one or more of the following:
  • a sample such as a blood sample
  • a blood-based sample such as a serum sample
  • a sample obtained from blood eg, a serum sample
  • a composition or kit of the present application administering a sample obtained from blood (eg, a serum sample) to a composition or kit of the present application;
  • this aspect of the application further includes determining the level (eg, percentage) of inhibition of the interaction between the polypeptide or fragment of (1) and the polypeptide or fragment of (2).
  • this aspect of the present application may also include comparing the observed inhibition level (eg percentage) of the interaction between the polypeptide or fragment of (1) and the polypeptide or fragment of (2) with Including the reference threshold of the new coronavirus neutralizing antibody for comparison. In some embodiments, this aspect of the application also includes determining, based on the comparison, whether the sample contains, or does not contain, neutralizing antibodies to SARS-CoV-2 (ie, determining the presence or absence of neutralizing antibodies to SARS-CoV-2 in the sample).
  • the subject can be a patient with COVID-19, a patient who has recovered from COVID-19, or an individual who has been vaccinated against COVID-19.
  • the protective efficacy of the vaccine can be judged by testing the obtained neutralizing antibody titer.
  • PBMCs peripheral blood mononuclear cells
  • CD19 microspheres (Miltenyi Biotec). CD19+ B lymphocytes were then incubated sequentially with human Fc fragment (BD Biosciences), anti-CD20-PECy7 (BD Biosciences), S-ECD-PE, and S-ECD-APC. Single memory B cells (CD20-PECy7+S-ECD-PE+S-ECD-APC+) were then sorted into 96-well plates using a FACSAria II (BD Biosciences) and used for antibody cloning. The amplified PCR products of immunoglobulin heavy chain and ⁇ / ⁇ light chain Fab regions were subjected to electrophoresis and Sanger sequencing. Their nucleotide sequences were analyzed by IMGT/V-QUEST and IgBlast, and the V(D)J gene fragment and CDR3 sequence of each antibody were determined.
  • Fab fragments were processed using the Pierce FAB prep kit (Thermo Scientific). Briefly, the sample is first applied to a desalting column to remove salt. After centrifugation, the flowthrough is collected and incubated with papain-attached beads to cleave Fab fragments from whole antibodies. The mixture is then transferred to a protein A affinity column, which specifically binds the Fc fragment of the antibody. After centrifugation, Fab fragments were obtained and dialyzed into phosphate buffered saline (PBS) (ThermoFisher).
  • PBS phosphate buffered saline
  • S protein-Fab Purified S protein (purchased from AcroBiosystems) was mixed with neutralizing antibody Fab fragments and incubated at a molar ratio of 1:1.5 (S protein-Fab) to obtain an S-Fab complex.
  • a 3-microliter aliquot of each complex was deposited on a glow-discharged porous carbon-coated gold grid (C-flat, 300 mesh, 1.2/1.3, Protochips In.) and adsorbed at 100% relative humidity. Dry for 7 seconds, then immerse in liquid ethane using a Vitrobot (FEI).
  • Cryo-EM datasets were collected using a Titan Krios microscope (FEI) at 300 kV.
  • Antigen block I K417, S477, F486, N501;
  • Antigen block II K417, L455, Y489, T478, E484, F486, Q493, N501;
  • Antigen block III L452, Q493, S494, E484, F486, F490;
  • Antigen block IV R346, N440, K444, G446;
  • Antigen block V K417, S477, F486, N501;
  • Antigen block VI Y369, F377, K378, S383;
  • Fab fragments of six representative antibodies, RBD are shown in gray. Schematic illustrating the antigenic blocks targeted by six representative antibodies. Dashed lines indicate overlap between two adjacent antigen patches.
  • residues with large side chains such as F486, Y489, Q493, L455, F456, etc. (the top 5 hottest, each amino acid residue has 96, 96, 81, 73, and 70 antibody residues) were significantly affected by circulating SARS -CoV-2 strains show a very low frequency of mutations.
  • Embodiment 7 Pseudovirus neutralization experiment
  • Plasma samples collected from convalescent and vaccinated volunteers were first inactivated at 56 °C for 0.5 h.
  • Inactivated serum samples or purified mAbs were serially diluted with cell culture medium from 1:4 or 50,000 ng/mL in two steps and mixed with virus suspension containing 100 TCID50 and incubated at 36.5°C for 2 hours. Afterwards, the mixture was added to a 96-well plate seeded with confluent Vero cells, and cultured for another 5 days in an incubator at 36.5° C., 5% CO 2 .
  • the cytopathic effect (CPE) of each well was observed and recorded microscopically by three different individuals and then used to calculate neutralization titers by the Reed-Muench method.
  • CPE cytopathic effect
  • antibodies that bind to six types of epitopes on RBD have shown good neutralization capabilities against different VOCs of the new crown.
  • XGv031 which binds to class III epitopes
  • XGv016, which binds to class IV epitopes and XGv042 also exhibit excellent broad-spectrum neutralization capabilities.

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Abstract

L'invention concerne un épitope de neutralisation de la protéine S du virus du SARS-CoV-2 destiné à être utilisé dans un vaccin ou un test d'anticorps neutralisant, et un kit correspondant de celui-ci.
PCT/CN2022/113307 2021-08-23 2022-08-18 Épitope neutralisant la protéine s du virus sars-cov-2 destiné à être utilisé dans le test d'anticorps de vaccin ou de neutralisation WO2023025031A1 (fr)

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CN202110971560.1 2021-08-23

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