WO2023025031A1 - Sars-cov-2 virus s protein neutralizing epitope for use in vaccine or neutralizing antibody testing - Google Patents

Abstract

A SARS-CoV-2 virus S protein neutralizing epitope for use in a vaccine or neutralizing antibody testing, and a corresponding kit thereof.

Description

SARS-CoV-2 virus S protein neutralizing epitope for vaccine or neutralizing antibody test

Cross References to Related Applications

This application requires a Chinese patent application with the application number 202110971560.1 and titled "Neutralization epitope of SARS-CoV-2 virus S protein for vaccine or neutralizing antibody test" submitted to the China Patent Office on August 23, 2021 priority, the entire contents of which are incorporated in this application by reference.

technical field

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.

Background technique

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 The 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". The emergence of novel coronavirus variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma, also known as B.1.1.28.1), etc., has caused a negative impact on the achievements of the anti-epidemic. Great challenge. The recently emerged B.1.617.2 (Delta), appears to be highly infectious and more resistant to neutralizing antibodies (W.F. Garcia-Beltran et al 2021).

While several types of COVID-19 vaccines are being deployed on a large scale, novel variants are responsible for reinfection after natural infection or following vaccination, respectively, observed in Brazil and the United States (E. Hacisuleyman et al 2021). Closely related to these, a general decline in immune protection against SARS-CoV-2 variants was also observed within 6–12 months after initial infection or vaccination (E.C. Sabino et al 2021). Prospects for genetic recombination and antigenic drift in recent SARS-CoV-2 variants, and heterogeneous immune protection from COVID-19 convalescence or heterogeneously weakened humoral immunity in vaccinated individuals, suggest potential risk of prolonged pandemic that could endanger the globe Human health, reduced social, economic and outdoor recreational activities. On August 10, 2021, Moderna, the National Institutes of Health (NIH) VRC and the Fred Hutch Institute jointly uploaded a preprinted article (P.B. Gilbert et al 2021) on medRxiv, announcing Moderna's COVID-19 vaccine The latest results of the COVE study (NCT04470427) analyzed the correlation between the protective power of mRNA-1273 and the immune response it induced. The study showed that the phase 3 clinical protection of mRNA-1273 was 94%. The study found that 57 days after the first vaccination (4 weeks after the second vaccination), the vaccine-induced RBD and S-specific IgG, as well as the serum neutralization titer and occurrence COVID-19 risk was inversely correlated (p=0.003-0.01). The higher the serum neutralization titer (cID50) of the 57-day vaccinated person, the lower the risk of infection in the next 100 days. If the cID50 of the vaccinated person on day 57 is undetectable, 100 and 1000, the corresponding vaccine protective powers are 50.8% (probably related to the effect of T cells), 90.7% and 96.1% respectively. On July 8, 2021, 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%. On July 23, 2021, the Pfizer/BioNTech mRNA vaccine was only 39% protective against infection with symptomatic COVID-19 caused by the Delta mutant strain in Israel.

The results of these association analyzes clearly tell us that as the vaccination time prolongs, the level of neutralizing antibodies decreases rapidly, and the protective power of the vaccine gradually decreases. The protective power of vaccines is largely based on the induction of neutralizing antibodies, so it is very feasible to predict the protective power of vaccines by neutralizing antibody titers. In countries or regions where it is difficult to enroll clinical patients for vaccines, using the neutralizing antibody titers produced by vaccines as a surrogate endpoint can speed up the marketing process of vaccines. Measuring these neutralizing antibodies in people who have been vaccinated will help understand the level of protection gained by vaccination in the population.

In this sense, it is important to know which specific sites (epitopes) on the SARS-CoV-2 virus are involved in the neutralization reaction, so as to design eliciting broad-spectrum neutralizing antibodies that are cross-reactive to multiple VOCs. Better appropriate vaccines of peptide sequences would be beneficial. On the other hand, the discovery of these epitopes can make the test of neutralizing antibody more accurate, and has the potential to develop an alternative to the current neutralizing antibody test method.

In view of this, propose this application.

Summary of the invention

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.

In order to achieve the above purpose, the present application proposes the following technical solutions.

The application first provides a neutralizing epitope selected from any one of (i)-(vi) S protein RBD of the new coronavirus:

(i) K417, S477, F486, N501;

(ii) K417, L455, Y489, T478, E484, F486, Q493, N501;

(iii) L452, Q493, S494, E484, F486, F490;

(iv) R346, N440, K444, G446;

(v) K417, S477, F486, N501;

(vi) Y369, F377, K378, S383.

The present application also provides a polypeptide or protein comprising the above epitope.

Further, in the polypeptide or protein, 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.

The application also provides a test kit, which comprises:

(1) 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:

(i) K417, S477, F486, N501;

(ii) K417, L455, Y489, T478, E484, F486, Q493, N501;

(iii) L452, Q493, S494, E484, F486, F490;

(iv) R346, N440, K444, G446;

(v) K417, S477, F486, N501;

(vi) Y369, F377, K378, S383;

In some preferred ways, it also includes:

(2) ACE2 protein or its fragment that specifically binds to the S protein or its fragment of (1).

Further, it also includes a 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.

Further, the kit, wherein:

(a) 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; or

(b) 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.

Further, wherein the detection substance is labeled with fluorescent labels, luminescence labels, immunodetectable labels, radiation labels, chemical labels, nucleic acid labels or polypeptide labels;

In some preferred forms, it is labeled with horseradish peroxidase.

The present application also provides a method for detecting neutralizing antibody titers in vivo or in vitro in a subject, which includes:

(i) Obtaining a sample (such as a blood sample) from a subject;

(ii) preparing a blood-based sample (such as a serum sample) from a subject sample;

(iii) providing the test kit described in any one of claims 6-9;

(iv) administering the kit to a sample derived from blood;

(v) incubating the sample with the polypeptide/composition for a sufficient period of time under conditions suitable for antibody:antigen complex formation;

(vi) washing to remove unbound protein;

(vii) detecting the interaction between the polypeptide or fragment of (1) and the polypeptide or fragment of (2)

Compared with the prior art, the present application has at least the following advantages:

1) 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.

2) The neutralizing epitopes, polypeptides or proteins provided by this application are suitable for the detection of new coronavirus neutralizing antibodies;

3) The neutralizing epitopes, polypeptides or proteins provided by this application are suitable for the preparation of novel coronavirus vaccines.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the description of the specific embodiments or prior art. Obviously, the accompanying drawings in the following description The drawings are some implementations of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

Figure 1. Structure-based antigen clustering of SARS-CoV-2 RBD neutralizing antibodies.

Figure 2. Overlay matrix analysis.

Figure 3. Antigenic blocks recognized by six types of neutralizing antibodies, and amino acid residues with thermal targeting frequencies.

Figure 4. Schematic representation of the surface representation model of six neutralizing antibodies bound to the RBD.

Figure 5. Immunogenicity analysis of SARS-CoV-2 RBD amino acid residues.

Figure 6. Schematic diagram of the conserved amino acid positions of the SARS-CoV-2 RBD.

Figure 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.

Figure 8. Heat map of monoclonal antibody epitopes from 3-dose vaccinated subject patients.

Figure 9. SARS-CoV pseudovirus neutralization experiment.

Figure 10. True virus neutralization experiment.

Detailed description of the invention

This application discloses a conformational epitope of the SARS-CoV-2 virus S protein used in vaccine or neutralizing antibody testing. Those skilled in the art can refer to the content of this article to realize its application. In particular, it should be pointed out that all similar substitutions and Modifications will be obvious to those skilled in the art, and they are all considered to be included in this application. The preparation method and application of the present application have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the preparation method and application herein without departing from the content, spirit and scope of the application to achieve and Apply the technology of this application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The following terms or definitions are provided only to aid in the understanding of the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

Unless otherwise defined hereinafter, all technical and scientific terms used in the detailed description of the application have the same meanings as commonly understood by those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used in this application, 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.

The use of an indefinite or definite article when referring to a noun in the singular eg "a" or "an", "the", includes a plural of that noun.

The terms "about" and "approximately" in the present application represent the range of accuracy that can be understood by those skilled in the art and still guarantee the technical effect of the mentioned feature. The term generally means ±10%, preferably ±5%, of the indicated value.

In addition, the terms first, second, third, (a), (b), (c) and the like in the specification and claims are used to distinguish similar elements and are not necessary to describe the order or time order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

The application is further explained as follows:

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. In particular, the 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. Preferably, the neutralizing epitope is selected from any one of the following (i)-(vi):

(i) K417, S477, F486, N501

(ii) K417, L455, Y489, T478, E484, F486, Q493, N501

(iii) L452, Q493, S494, E484, F486, F490,

(iv) R346, N440, K444, G446

(v) K417, S477, F486, N501

(vi) Y369, F377, K378, S383

In another aspect of the present application, 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. Anyone 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.

In some embodiments, 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.

In another aspect of the present application, 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.

While the 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. In some embodiments, the antigen-binding molecule binds to the S protein of the new coronavirus. In some embodiments, the antigen-binding molecule has neutralizing activity, and can inhibit the binding of the S protein of the new coronavirus to the receptor.

In another aspect of the present application, the present application provides the use of the above-mentioned neutralizing epitope, polypeptide or protein in the preparation of a vaccine or vaccine composition. It can be understood that any vaccine preparation based on the epitope, polypeptide or protein of the present application and its preparation and application fall within the scope of rights of the present application. 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. For example, in some aspects of the present application, 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. Preferably, 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.

In another aspect of the application, the application provides a kit or composition comprising:

(1) The S protein or fragment thereof encoded by SARS-CoV-2, which comprises an epitope of the new coronavirus S protein selected from any one of (i)-(vi):

(i) K417, S477, F486, N501

(ii) K417, L455, Y489, T478, E484, F486, Q493, N501

(iii) L452, Q493, S494, E484, F486, F490

(iv) R346, N440, K444, G446

(v) K417, S477, F486, N501

(vi) Y369, F377, K378, S383

as well as

(2) ACE2 protein or its fragment that specifically binds to the S protein or its fragment of (1).

In some embodiments according to this aspect, the 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.

In some embodiments, a solid support can include polystyrene, polypropylene, polycarbonate, cycloolefin, glass, or quartz. In some embodiments, a solid support can be a microtiter (or "well") plate or a microarray plate. In some embodiments, the solid support can be beads, such as magnetic beads.

The 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. For example, 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.

In some embodiments, a system for detecting an interaction may employ a detection substance. In the following experimental example shown by way of example, 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.

Therefore, in some embodiments according to various aspects of the present application, 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), and/or 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), and 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. In some embodiments, a detection substance can be a moiety that has a detectable activity, eg, enzymatic activity towards a specific substrate. Examples of detection substances having detectable activity include, for example, horseradish peroxidase (HRP) and luciferase groups.

In some embodiments, 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) is provided in 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 specifically binds to a polypeptide or fragment encoded by a new coronavirus (such as an ACE2 protein or a fragment thereof that specifically binds to a spike protein or a fragment thereof encoded by a new coronavirus).

Those skilled in the art can determine the appropriate amount/concentration of novel coronavirus-encoded polypeptides (such as S protein) or fragments thereof, wherein the 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. For example, in some embodiments, 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. In some embodiments, 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.

Those skilled in the art are able to determine the appropriate amount/concentration of a polypeptide encoded by SARS-CoV-2 (e.g. spike protein) or fragments thereof, wherein 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.

Specifically, 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. These antibodies may be referred to as neutralizing antibodies.

By way of further explanation, referring to the experimental example below, based on the degree of reduction of the interaction between the S1 subunit of the SARS-CoV-2 spike protein or the RBD of the SARS-CoV-2 spike protein and ACE2 immobilized on a solid support To detect the presence of antibodies that inhibit the binding of the S1 subunit of the SARS-CoV-2 spike protein and the RBD of the SARS-CoV-2 spike protein in the sample. The presence of neutralizing antibodies can be inferred from the determination of a reduction in interactions relative to a control of a sample lacking neutralizing antibodies to the SARS-CoV-2 Spike protein S1 subunit or the SARS-CoV-2 Spike protein RBD.

Therefore, 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.

By way of example, 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.

Accordingly, 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.

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.

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 comparable sample of antibody levels. For example, 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.

As used herein, 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. 1-fold lower interaction level of peptides or fragments (eg, ACE2), 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

Alternatively, 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. In this case, "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 (such as ACE2) 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.

In some embodiments, an aspect of the present application may include:

Contacting 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

- determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide or fragment of (ii).

In some embodiments, an aspect of the present application may include:

- (a) contacting a sample obtained from the subject with (i) a polypeptide encoded by a new coronavirus (for example, the spike protein) or a fragment thereof,

- (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

- (c) determining the level of interaction between the polypeptide or fragment of (i) and the polypeptide or fragment of (ii).

In some embodiments, an aspect of the present application may include:

- (a) contacting 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,

- (b) contacting the mixture formed in step (a) with (ii) a novel coronavirus-encoded polypeptide (e.g., spike protein) or a fragment thereof, and

- (c) determining the level of interaction between the polypeptide or fragment thereof of (i) and the polypeptide or fragment of (ii).

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, this aspect of the application may also include one or more of the following:

- Obtaining a sample (such as a blood sample) from a subject;

- preparation of a blood-based sample (such as a serum sample) from a blood sample from a subject;

- providing a composition or kit of the application;

administering a sample obtained from blood (eg, a serum sample) to a composition or kit of the present application;

- incubating the sample with the polypeptide/composition for a sufficient time under conditions suitable for antibody:antigen complex formation;

- aspiration of samples;

Washing to remove unbound protein;

- detecting the interaction between the polypeptide or fragment of (1) and the polypeptide or fragment of (2).

In some embodiments, 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).

In some embodiments, 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).

In some embodiments, 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.

In some embodiments, the protective efficacy of the vaccine can be judged by testing the obtained neutralizing antibody titer.

Example

The technical solutions of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Apparently, the described embodiments are some of the embodiments of the present application, not all of them. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the scope of protection of this application.

Example 1 Epitope identification and clustering

All published structures of SARS-CoV-2 neutralizing antibodies complexed with the S trimer or its subdomain (RBD) were obtained from the Protein Data Bank. The distance from the corresponding antibody on the RBD is in

All residues within are recorded as epitope residues. The binding frequency of each residue was calculated to generate an epitope heatmap. The binary binding profile for each residue (amino acid residues 319-541 of the RBD) was used to calculate the distance between pairs of sequences using an in-house python script. The clustering of the sequence distance matrix is performed by fetch in PHYLIP.

To dissect the properties of the RBD epitopes targeted by neutralizing antibodies, 171 SARS-CoV-2 RBD-targeting neutralizing antibodies with available structures were investigated. As shown in Figure 1, through the cluster analysis of the epitope structure, the antibody is mainly divided into six sites I, II, III, IV, V and VI. Neutralizing antibodies that block or do not block ACE2 binding are outlined in light pink and light yellow, respectively. Neutralizing antibodies linked or not linked to the blocking RBD are marked in grey-blue and grey-green, respectively.

Furthermore, we superimposed the structure of the RBD from these complex structures and calculated the collision area between any 2 neutralizing antibodies. Both strategies produced the same results. As shown in Figure 2, from the conflict region between the variable regions of any two Fab fragments

Overlay matrix of the 147 RBD-neutralizing antibody structures output from , showing clustering into six antibody classes.

Example 2 Single memory B cell sorting and antibody cloning

Volunteer recruitment and blood draw were approved by the Ethics Committee of Fudan University School of Basic Medicine. Study participants, four donors who received three doses of the inactivated CoronaVac vaccine and three donors who received two doses, donated blood one month after vaccination. All donors were between the ages of 23 and 52, with a male to female ratio of 3:4. After peripheral blood was collected, human peripheral blood mononuclear cells (PBMCs) were isolated, aliquoted and stored in liquid nitrogen.

Stored PBMCs were thawed and incubated with 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 κ/λ 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.

Example 3 Antibody expression

Vector construction and antibody expression were performed on selected antibodies. Briefly, all cloned human monoclonal antibodies were prepared by transient transfection of mammalian HEK293F cells using serum-free OPM-293-CD05 medium (OPM Biosciences) at 37°C, 5% CO2, and shaking at 100rpm nourish. A total of 48 cloned antibodies were expressed, named XGv01 to XGv50 (XGv37 and XGv48 were not expressed).

To generate Fab fragments, purified monoclonal antibodies 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).

Example 4 Cryo-EM sample preparation, data collection and processing

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. Movies were recorded using a K2 Summit direct detector with a defocus range between 1.5–2.7 μm (32 frames, every 0.2 s, with a total dose of

). SerialEM performs automated single particle data acquisition with a calibrated magnification of 22,500 and a final pixel size of

Based on cryo-EM data, the structures of six types of RBD neutralizing antibodies were analyzed. Antigenic blocks recognized by six classes of neutralizing antibodies (targeting frequency >30%) are depicted in Figure 3, where residues with a "hot targeting frequency" (typically greater than 65%, but greater than 85%) are in class I) Displayed in bright colors corresponding to the block they belong to. Residues involved in two (eg Y489, L452) or three (eg F486) adjacent antigenic plaques are presented in mixed colors. Representative "hot" antigenic residues are labeled.

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;

As shown in Figure 4, 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.

Example 5 The immunogenicity analysis of SARS-CoV-2 RBD amino acid residues

As shown in Figure 5, immunogenicity analysis was performed based on the SARS-CoV-2 RBD amino acid residues of 171 neutralizing antibodies. The frequency of each epitope of the six types of antibodies is counted and displayed by a histogram. Distributions of the six antibody classes are shown by fitted curves. As shown in Figure 6, eight SARS-CoV-2 strains (WT, B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, B.1.526 and C. 37) are highly conserved for the 16 most immunogenic residues. Surprisingly, none of the top 9 most immunogenic residues had a high mutation frequency. In particular, 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.

We compared related sequences of SARS-CoV-2 WT and variants B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.1, B.1.526, and C.37 Yes, as shown in Figure 7, where the amino acid residues involved in the direct interaction with ACE2 are marked with a ball. These sequences will play a role in the preparation of antibodies with broad-spectrum neutralizing activity and vaccines.

Example 6 Antibody Classification

As shown in Figure 8, we performed epitope mapping of XGv01 to XGv50 expressed in this application, and obtained antibodies with different binding sites, including those that bind to the non-RBD or NTD regions on the RBD, NTD and S1 subunits of the new coronavirus Antibody. We predict that not all antibodies have neutralizing ability, and only antibodies that bind to the neutralizing epitope in Example 4 may have higher neutralizing ability. Further, we screened the neutralizing epitope-binding antibodies of Example 4 and classified them into categories I-VI. The results obtained are shown in the following table:

Embodiment 7 Pseudovirus neutralization experiment

In vitro neutralization assays were performed using pseudoviruses, as previously described. Briefly, Huh-7 cells were seeded and then incubated with pseudovirus/antibody mixture for 12 hours. Antibodies were serially diluted 1:3 in PBS for a total of 9 dilutions with an initial concentration of 10 μg/ml. Use fresh DMEM medium instead of the mixture for further cultivation. After 24 or 48 hours, Huh-7 cells were harvested and luminescence was measured as previously described.

The experimental results are shown in Figure 9. XGv013 binding to class I epitopes, XGv014 binding to class IV and V epitopes, XGv030 binding to class V and VI epitopes, and XGv001, XGv039 and XGv049 binding to class VI epitopes inhibited pseudo The effect of virus multiplication activity is obvious.

Example 8 True virus 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.

As shown in Figure 10, antibodies that bind to six types of epitopes on RBD have shown good neutralization capabilities against different VOCs of the new crown. Among them, XGv031, which binds to class III epitopes, and XGv016, which binds to class IV epitopes, and XGv042 also exhibit excellent broad-spectrum neutralization capabilities.

The foregoing descriptions of specific exemplary embodiments of the present application have been presented for purposes of illustration and description. These descriptions are not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the application and their practical application, thereby enabling those skilled in the art to implement and utilize various exemplary embodiments of the application, as well as various Choose and change. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims (10)

1. The neutralizing epitope of the new coronavirus S protein RBD selected from any one of (i)-(vi):

   (i) K417, S477, F486, N501;

   (ii) K417, L455, Y489, T478, E484, F486, Q493, N501;

   (iii) L452, Q493, S494, E484, F486, F490;

   (iv) R346, N440, K444, G446;

   (v) K417, S477, F486, N501;

   (vi) Y369, F377, K378, S383.
2. A polypeptide or protein comprising the epitope of claim 1.
3. The polypeptide or protein according to claim 2, wherein the sequence where the epitope (i) is located is SEQ ID No.1 or SEQ ID No.4; the sequence where the epitope (ii) is located 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; the epitope ( iv) The sequence where the epitope (v) is located 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% to said sequence , an amino acid sequence with 98% or 99% sequence identity.
4. Use of the neutralizing epitope of claim 1, or the polypeptide or protein of claim 2 or 3 in detection/screening/purification/preparation of neutralizing antibodies.
5. The neutralizing epitope of claim 1, or the use of the polypeptide or protein of claim 2 or 3 in the preparation of neutralizing antibody detection/screening/purification kits; or the use in the preparation of vaccines.
6. A test kit, characterized in that the test kit comprises:

   (1) 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:

   (i) K417, S477, F486, N501;

   (ii) K417, L455, Y489, T478, E484, F486, Q493, N501;

   (iii) L452, Q493, S494, E484, F486, F490;

   (iv) R346, N440, K444, G446;

   (v) K417, S477, F486, N501;

   (vi) Y369, F377, K378, S383;

   Preferably, it also includes:

   (2) ACE2 protein or its fragment that specifically binds to the S protein or its fragment of (1).
7. The kit according to claim 6, characterized in that it also comprises a detection substance for detecting the interaction between the S protein of (1) or a fragment thereof and the ACE2 of (2) or a fragment thereof; preferably, wherein ( 1) the spike protein or its fragment, or (2) the ACE2 protein or its fragment is coupled to the detection substance.
8. The kit according to any one of claims 6-7, wherein:

   (a) 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; or

   (b) 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.
9. The kit according to any one of claims 7-8, wherein the detection substance is labeled with fluorescent labels, luminescence labels, immunodetectable labels, radiation labels, chemical labels, nucleic acid labels or polypeptide labels; Labeling with horseradish peroxidase is preferred.
10. A method for detecting neutralizing antibody titers in a subject or in vitro, characterized in that it comprises:

    (i) Obtaining a sample (such as a blood sample) from a subject;

    (ii) preparing a blood-based sample (such as a serum sample) from a subject sample;

    (iii) providing the test kit described in any one of claims 6-9;

    (iv) administering the kit to a sample derived from blood;

    (v) incubating the sample with the polypeptide/composition for a sufficient period of time under conditions suitable for antibody:antigen complex formation;

    (vi) washing to remove unbound protein;

    (vii) detecting the interaction between the polypeptide or fragment of (1) and the polypeptide or fragment of (2).

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