Method for detecting anti-SARS-COV-2 spike immunoglobulins

Abstract

Disclosed is a method for detecting whether a biological sample (e.g., serum, blood, plasma) contains an antibody against SARS-CoV-2 S glycoprotein, the method comprising: (i) providing a surface coated with SARS-CoV-2 S glycoprotein; (ii) exposing the surface to the biological sample; (iii) exposing the surface to a secondary antibody; and (iv) detecting the secondary antibody bound to the surface; wherein if a secondary antibody is detected, the biological sample contains an antibody that binds to the SARS-CoV-2 S glycoprotein.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 581,754, filed September 11, 2023. The entire application of the above application is incorporated herein by reference.

[0002] Reference electronic sequence list The application contains a sequence list that has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The XML copy, created on September 11, 2024, is named 1450_105WO1_Sequence_Listing_09_11_2024 and has a size of 814,987 bytes. Technical Field

[0003] This disclosure generally relates to methods for identifying whether biological samples (e.g., serum, blood, plasma) contain antibodies against the SARS-CoV-2 S glycoprotein. Background Technology

[0004] The Coronavirus Disease 2019 (COVID-19) pandemic is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of variants (such as Alpha, Beta, Gamma, Delta, and multiple Omicron subvariants) has led to the sustained spread of the virus. Some SARS-CoV-2 variants (such as Omicron) possess immune evasion properties, thereby reducing the effectiveness of COVID-19 vaccines. Additional protective association values ​​(CoPs) for COVID vaccine efficacy are needed to help track immune evasion and understand the prospects for vaccine development (e.g., the development of novel vaccines based on variant S protein sequences). Validated CoPs can help infer vaccine efficacy / immunogenicity results for populations or formulations / regimens that have not been demonstrated in clinical trials. Of particular interest are CoPs on the durability and level of vaccine-driven protective effects against the original strain and variant strains.

[0005] Ultimately, biomarkers demonstrating immunogenicity associated with protective efficacy are crucial for vaccine evaluation. Specifically, the amount of anti-SARS-CoV-2 S (spike) antibodies in biological samples determines the effectiveness of the humoral response induced by the vaccine. Current assays measure total anti-SARS-CoV-2 S antibodies. Not all of these antibodies are desirable. An improved assay for evaluating vaccine-induced anti-SARS-CoV-2 S antibodies is necessary.

[0006] Human IgG has four distinct subclasses, numbered in order of relative abundance: IgG1, IgG2, IgG3, and IgG4. IgG1 and IgG3 are major contributors to the rapid IgG response to protein and membrane antigens and are active in viral neutralization, but some differences in IgG subclass responses have been identified in the context of SARS-CoV-2 infection and vaccination. In fact, the IgG3 response to SARS-CoV-2 infection has been highlighted by analysis of convalescent plasma, showing that although IgG3 accounts for 12% of total anti-spike (anti-S) protein IgG, it contributes approximately 80% of the total active SARS-CoV-2 neutralizing activity. Furthermore, IgG1, and especially IgG3, binds FcγRIIa, FcγRIIIa, and C1q, thereby supporting antibody-dependent phagocytosis (ADCP), antibody-dependent cytotoxicity (ADCC), and antibody-dependent complement deposition (ADCD).

[0007] Repeated mRNA SARS-CoV-2 vaccination has been associated with a significant increase in the proportion of immunoglobulin G4 (IgG4) in the spike-specific response and a reduction in Fc-mediated ADCP and ADCD, which may limit control of viral infection. Following repeated mRNA SARS-CoV-2 vaccination, IgG3 peaked after the second dose and steadily declined to significantly lower levels after the third and fourth doses, while IgG4 increased. IgG4 is typically low in abundance (0–5% of total IgG), but may increase slowly over time due to repeated or over-exposure to some antigens. Although repeated antigen exposure appears to be necessary, it is insufficient to induce IgG4 because prolonged expression of IL-10 on CD4 is also required. + T cell activation and expression of other anti-inflammatory cytokines. Increased IgG4 concentrations have been associated with immunosuppression and adverse clinical outcomes in COVID-19, and while generally considered anti-inflammatory, it may contribute to some autoimmune disorders and inflammatory IgG4-related diseases. Summary of the Invention

[0008] This document provides a method for determining whether a biological sample contains an antibody that binds to the SARS-CoV-2 spike (S) glycoprotein, comprising: (i) providing a surface coated with the SARS-CoV-2 S glycoprotein; (ii) exposing the surface to the biological sample; (iii) exposing the surface to a secondary antibody; and (iv) detecting the secondary antibody bound to the surface; wherein if the secondary antibody is detected, the biological sample contains an antibody that binds to the SARS-CoV-2 S glycoprotein. In an embodiment, the SARS-CoV-2 S glycoprotein is associated with SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151 ,153,156,158,174,175,176,181-184,186,188,190,195,217-228,233-236,243,255-264,273-280,283,284,287,288,291,292 and 294, The polypeptides of any one of 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236, 328, 329, 330, 331, 332, and 333 have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. In an embodiment, the SARS-CoV-2 S glycoprotein has an inactive furin protease cleavage site. In an embodiment, the inactive furin protease cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7). In this embodiment, amino acids 973 and 974 of the SARS-CoV-2 S glycoprotein are proline, compared to the wild-type SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 2. In this embodiment, the antibody binding to the SARS-CoV-2 S glycoprotein is IgG. In this embodiment, the antibody binding to the SARS-CoV-2 S glycoprotein is IgG1. In this embodiment, the antibody binding to the SARS-CoV-2 S glycoprotein is IgG2. In this embodiment, the antibody binding to the SARS-CoV-2 S glycoprotein is IgG3. In this embodiment, the antibody binding to the SARS-CoV-2 S glycoprotein is IgG4.In the implementation scheme, the secondary antibody is selected from anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, and anti-human IgG4 antibody. In the implementation scheme, the SARS-CoV-2 S glycoprotein is derived from the SARS-CoV-2 virus or a SARS-CoV-2 variant. In the implementation scheme, the SARS-CoV-2 variant is B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; or B.1.1.529 SARS-CoV-2 strain. In the implementation scheme, the SARS-CoV-2 S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SARS-CoV-2 S omicron variant selected from: BA.1, BA.2.12.1, BA.2, BA.3, BA.4, BA.5, XBB.1.5, XBB.2.3, XBB.1.16, EG.5.1, JN.1, BQ.1.1, and BF.7. In the implementation scheme, the secondary antibody is attached to a tag. In the implementation scheme, the tag is horseradish peroxidase. In this implementation, the biological sample is serum, plasma, blood, saliva, nasopharyngeal swab, or mucus. In this implementation, the biological sample is from a patient who has previously had COVID-19. In this implementation, the biological sample is from a patient who has been administered an immunogenic composition against the SARS-CoV-2 virus or a variant thereof. In this implementation, the SARS-CoV-2 S glycoprotein contains a transmembrane domain. In this implementation, evaluating the binding IgG response to the SARS-CoV-2 S glycoprotein in serum leads to the rapid development of an effective vaccine against emerging SARS-CoV-2 variants. Attached Figure Description

[0009] The invention can be more fully understood by taking into consideration the following detailed description of various embodiments of the invention in conjunction with the accompanying drawings, wherein: Figure 1 A schematic diagram of the procedure for performing anti-rS IgG assays is presented; Figures 2A-2C This shows how to detect anti-spike IgG4 ( Figure 2A ), anti-spike IgG1 ( Figure 2B ) and total anti-spike IgG ( Figure 2C A schematic diagram of the determination of ). Figure 3 The percentage of IgG1 in total IgG is shown in samples taken on day 1 (before booster) and day 29 (28 days after booster). Figure 4 The percentage of IgG4 in total IgG is shown for samples taken on day 1 (before booster) and day 29 (28 days after booster). Figure 5 The percentage of IgG4 in the total IgG in the sample on day 29 (28 days after booster) is shown. Figure 6 The percentages of IgG1 and IgG4 in the sample from day 1 (before booster) are shown. Figure 7 The percentages of IgG1 and IgG4 in the sample on day 29 (28 days after booster) are shown. Figure 8 The total anti-S glycoprotein IgG in the sample is shown; Figure 9 The total anti-S glycoprotein IgG1 in the sample is shown; Figure 10 The total anti-S glycoprotein IgG4 in the sample is shown; Figure 11 The results show that for all 27 serum samples representing all concentration ranges (low, medium, and high), the inter-assay, intra-assay, and total precision were <20% GCV. Figure 12 The assay specificity of the original strain for IgG detection is shown; Figure 13 The effects of IgG assay on the hemoglobin and lipid matrix of the original strain are shown; Figures 14A-14B The linearity of IgG assay for the original strain is shown; Figure 15 The temperature and freeze / thaw stability of the original strain for IgG assay are shown; Figures 16A-16F It shows Beta ( Figure 16B Delta Figure 16C ) and Omicron BA.1 ( Figure 16D ), BA.5 ( Figure 16E ), XBB.1.5 ( Figure 16FValidation parameters for IgG assay of variants compared with the original strain ( Figure 16A Similarity; Figures 17A-17F The correlation between IgG assay results in SARS-CoV-2 seronegative serum samples and other immunogenicity assays is shown; Figures 18A-18F The correlation between IgG assay results in SARS-CoV-2 positive serum samples and other immunogenicity assays is shown; Figure 19A The serum concentrations of IgG subclasses are shown after repeated SARS-CoV-2 vaccination; Figure 19B The alternative Fc effector functional response following repeated SARS-CoV-2 vaccination is shown.

[0010] While the invention may have various modifications and alternatives, its details have been shown by way of example in the accompanying drawings and will be described in detail. However, it should be understood that the purpose is not to limit the invention to the specific embodiments described. Rather, the purpose is to cover all modifications, equivalents, and alternatives that fall within the spirit and scope of the invention as defined by the appended claims. Detailed Implementation

[0011] definition As used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” may refer to a single protein or a mixture of such proteins, and reference to “the method” includes reference to equivalent steps and / or methods known to those skilled in the art, and so on.

[0012] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, amplifies or otherwise alters or modifies the immune response induced by that immunogen. Modification of the immune response may include enhancing or broadening the specificity of one or both of the antibody and cellular immune responses.

[0013] As used herein, the terms “about” or “approximately” preceding a numerical value indicate a range of that value plus or minus 10%. For example, “about 100” covers 90 and 110. As used herein, an “immunogenic composition” is a composition containing an antigen, wherein administration of the composition to a subject results in the development of a humoral and / or cellular immune response to the antigen in the subject.

[0014] As used herein, "subunit" compositions, such as vaccines, include one or more selected antigens, but not all antigens derived from a pathogen. Such compositions are substantially free of intact viruses or lysates of such cells or particles, and are typically prepared from at least partially purified, often substantially purified, immunogenic peptides derived from a pathogen. The antigens in the subunit compositions disclosed herein are typically recombinant-prepared, often using baculovirus systems.

[0015] As used herein, “substantially” means the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes a substantial percentage of the sample containing it. For example, in a sample, the substantially purified component constitutes 85%, preferably 85%-90%, more preferably at least 95%-99.5%, and most preferably at least 99%. If the component is substantially displaced, the amount remaining in the sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1.0%.

[0016] As used herein, the term "treatment" refers to a method for achieving a beneficial or desired outcome, such as a clinical result. For the purposes of this disclosure, a beneficial or desired outcome may include suppressing or inhibiting the onset or progression of an infection or disease; improving or reducing the development of symptoms of an infection or disease; or a combination thereof.

[0017] As used in this article, “prevention” can be used interchangeably with “prophylaxis” and can mean complete prevention of an infection or disease, or prevention of the development of symptoms of such an infection or disease; delay in the onset of an infection or disease or its symptoms; or reduction in the severity of an infection or disease or its symptoms that subsequently develop.

[0018] As used herein, “effective dose” or “effective amount” refers to an amount of immunogen sufficient to induce an immune response that reduces at least one symptom of pathogen infection. An effective dose or effective amount can be determined, for example, by measuring the amount of neutralized secreted and / or serum antibodies, such as by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0019] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, for inducing an immune response against the pathogen. An immune response may include antibody formation and / or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen administered to a subject to produce an immune response. Preferably, the vaccine induces an immune response that effectively prevents infection with SARS-CoV-2 or its variants.

[0020] As used herein, the term "subject" includes both humans and other animals. Generally, a subject is a human. For example, a subject can be an adult, adolescent, child (2 to 14 years old), infant (birth to 2 years old), or newborn (up to 2 months old). In certain respects, a subject may be up to 4 months old, or up to 6 months old. In various respects, an adult is approximately 65 years of age or older, or approximately 60 years of age or older. In some respects, a subject is a pregnant woman or a woman intending to become pregnant. In other respects, a subject is not a human; for example, a non-human primate; such as a baboon, chimpanzee, gorilla, or macaque. In some respects, a subject can be a pet, such as a dog or cat.

[0021] As used herein, the term "pharmaceutically acceptable" means approved by U.S. federal or state regulatory agencies or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeias for use in mammals, and more particularly in humans. These compositions can be used as vaccine and / or antigen compositions to induce a protective immune response in vertebrates.

[0022] As used herein, the term “NVX-CoV2373” refers to a vaccine composition comprising the BV2373 spike glycoprotein (SEQ ID NO: 87) and fractions A and C iscom matrix (e.g., MATRIX-M™).

[0023] As used herein, the term "modification" in relation to CoV S peptides refers to a mutation, deletion, or addition of one amino acid in the CoV S peptide. The location of a modification within the CoV S peptide can be determined by comparing the peptide sequence with SEQ ID NO: 1 (CoV S peptide containing a signal peptide) or SEQ ID NO: 2 (mature CoV S peptide lacking a signal peptide).

[0024] Assay for evaluating the immunogenicity of vaccine compositions against SARS-CoV-2 This disclosure provides an assay for evaluating the immunogenicity of immunogenic compositions and vaccine compositions against SARS-CoV-2. In embodiments, the assay evaluates the amounts of anti-rS protein IgG1, IgG2, IgG3, and IgG4, as well as total anti-rS IgG, in a biological sample (see Example 1). Alternative ADCP (FcγRIIa binding), alternative ADCC (FcγRIIIa binding), and ADCD (C1q binding) in the biological sample are also evaluated.

[0025] In the implementation scheme, the immunogenic composition and vaccine composition comprise a non-naturally occurring coronavirus (CoV) spike (S) polypeptide or nanoparticles containing the CoV S polypeptide.

[0026] In the implementation scheme, the CoV S polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequence to SEQ ID NO: 87. The sequence of SEQ ID NO: 87 is shown in the table below.

[0027] SEQ ID NO: 87

[0028] In some embodiments, these methods also include washing the solid surface. In others, these methods include contacting the plate with a blocking buffer. The blocking buffer is a solution that eliminates the possibility of non-specific binding to the plate.

[0029] In the implementation scheme, these methods can be used to detect antibody isotypes selected from IgA, IgG, IgM, IgD, and IgE. In the implementation scheme, these methods can be used to detect antibody isotypes selected from IgG1, IgG2, IgG3, and IgG4.

[0030] SARS-CoV-2 S glycoprotein used in these methods In the implementation scheme, a suitable SARS-CoV-2 S glycoprotein used in the methods described herein includes a SARS-CoV-2 protein database (PDB) ID associated with any of the following: S glycoprotein: 6LVN, 6LZG, 6M0J, 6M17, 6M1V, 6VSB, 6VW1, 6VXX, 6VYB, 6W41, 6WPS, 6WPT, 6X29, 6X2A, 6X2B, 6X2C, 6X45, 6X6P, 6X79, 6XC2, 6XC3, 6XC4, 6XC7, 6XCM, 6XCN, 6XDG, 6XE1, 6XEY, 6XF5, 6XF6, 6XKL, 6XKP, 6XKQ, 6XLU, 6XM0, 6XM3, 6XM4, 6XM5, 6XR8, 6XRA, 6XS6, 6YBB, 6YLA, 6YM0, 6YOR, 6 YZ5, 6YZ7, 6Z2M, 6Z43, 6Z97, 6ZB4, 6ZB5, 6ZBP, 6ZCZ, 6ZDG, 6ZDH, 6ZER, 6ZFO, 6ZGE, 6ZGG, 6ZGH, 6ZGI, 6ZH9, 6ZHD, 6ZLR, 6ZOW, 6ZOX, 6ZOY, 6 ZOZ, 6ZP0, 6ZP1, 6ZP2, 6ZP5, 6ZP7, 6ZWV, 6ZXN, 7A25, 7A29, 7A4N, 7A5R, 7A5S, 7A91, 7A92, 7A93, 7A94, 7A95, 7A96, 7A97, 7A98, 7AD1, 7AKD, 7B 0B, 7B14, 7B17, 7B18, 7B27, 7B3O, 7B62, 7BEH, 7BEI, 7BEJ, 7BEK, 7BEL, 7BEM, 7BEN, 7BEO, 7BEP, 7BH9, 7BNM, 7BNN, 7BNO, 7BNV, 7BWJ, 7BYR, 7B Z5, 7C01, 7C2L, 7C53, 7C8D, 7C8J, 7C8V, 7C8W, 7CAB, 7CAC, 7CAH, 7CAI, 7CAK, 7CAN, 7CDI, 7CDJ, 7CH4, 7CH5, 7CHB, 7CHC, 7CHE, 7CHF, 7CHH, 7CH O, 7CHP, 7CHS, 7CJF, 7CM4, 7CN9, 7COT, 7CT5, 7CWL, 7CWM, 7CWN, 7CWO, 7CWS, 7CWT, 7CWU, 7CYH, 7CYP, 7CYV, 7CZP, 7CZQ, 7CZR, 7CZS, 7CZT, 7CZU , 7CZV, 7CZW, 7CZX, 7CZY, 7CZZ, 7D00, 7D03, 7D0B, 7D0C, 7D0D, 7D2Z, 7D30, 7D4G, 7D6I, 7DCC, 7DCX, 7DD2, 7DD8, 7DDD, 7DDN, 7DEO, 7DET, 7DEU,7DF3, 7DF4, 7DHX, 7DJZ, 7DK0, 7DK2, 7DK3, 7DK4, 7DK5, 7DK6, 7DK7, 7DMU, 7DPM, 7DQA, 7DTE, 7DWX, 7DWY, 7DWZ, 7DX0, 7DX1, 7DX2, 7DX3, 7DX4, 7DX5, 7DX6 7DX7, 7DX8, 7DX9, 7DZW, 7DZX, 7DZY, 7E23, 7E39, 7E3B, 7E3C, 7E3J, 7E3K, 7E3L, 7E3O, 7E5O, 7E5R, 7E5S, 7E5Y, 7E7B, 7E7D, 7E7X, 7E7Y, 7E86, 7E88, 7E8C 7E8F, 7E8M, 7E9N, 7E9O, 7E9P, 7E9Q, 7E9T, 7EAM, 7EAN, 7EAZ, 7EB0, 7EB3, 7EB4, 7EB5, 7EDF, 7EDG, 7EDH, 7EDI, 7EDJ, 7EFP, 7EFR, 7EH5, 7EJ4, 7EJ5, 7EJL 7EJY, 7EJZ, 7EK0, 7EK6, 7EKC, 7EKE, 7EKF, 7EKG, 7EKH, 7ENF, 7ENG, 7EPX, 7EY0, 7EY4, 7EY5, 7EYA, 7EZV, 7F0X, 7F12, 7F15, 7F3Q, 7F46, 7F5H, 7F5R, 7F62 7F63, 7F6Y, 7F6Z, 7F7E, 7F7H, 7FAE, 7FAF, 7FAT, 7FAU, 7FB0, 7FB1, 7FB3, 7FB4, 7FBJ, 7FBK, 7FC5, 7FCD, 7FCE, 7FCP, 7FCQ, 7FDG, 7FDH, 7FDI, 7FDK, 7FEM 7FET, 7FG2, 7FG3, 7FG7, 7FH0, 7FJC, 7FJN, 7FJO, 7FJS, 7JJC, 7JJI, 7JJJ, 7JMO, 7JMP, 7JMW, 7JV2, 7JV4, 7JV6, 7JVA, 7JVB, 7JVC, 7JW0, 7JWB, 7JWY, 7JX3 7JZL, 7JZM, 7JZN, 7JZU, 7K43, 7K45, 7K4N, 7K8M, 7K8S, 7K8T, 7K8U, 7K8V, 7K8W, 7K8X, 7K8Y, 7K8Z, 7K90, 7K9H, 7K9I, 7K9J, 7K9K, 7K9Z, 7KDG, 7KDH, 7KDI 7KDJ, 7KDK, 7KDL, 7KE4, 7KE6, 7KE7, 7KE8, 7KE9, 7KEA, 7KEB, 7KEC, 7KFV, 7KFW, 7KFX, 7KFY, 7KGJ, 7KGK, 7KJ2, 7KJ3, 7KJ4, 7KJ5, 7KKK, 7KKL, 7KL9, 7KLG7KLH, 7KLW, 7KM5, 7KMB, 7KMG, 7KMH, 7KMI, 7KMK, 7KML, 7KMS, 7KMZ, 7KN3, 7KN4, 7KN5, 7KN6, 7KN7, 7KNB, 7KNE, 7KNH, 7KNI, 7KQE, 7KRQ, 7KRR, 7KRS, 7KS9 7KSG, 7KXJ, 7KXK, 7KZB, 7L02, 7L06, 7L09, 7L0N, 7L2C, 7L2D, 7L2E, 7L2F, 7L3N, 7L4Z, 7L56, 7L57, 7L58, 7L5B, 7L7D, 7L7E, 7L7F, 7L7K, 7LAA, 7LAB, 7LC8 7LCN, 7LD1, 7LDJ, 7LJR, 7LM8, 7LO4, 7LOP, 7LQ7, 7LQV, 7LQW, 7LRS, 7LRT, 7LS9, 7LSS, 7LWI, 7LWJ, 7LWK, 7LWL, 7LWM, 7LWN, 7LWO, 7LWP, 7LWQ, 7LWS, 7LWT 7LWU, 7LWV, 7LWW, 7LX5, 7LXW, 7LXX, 7LXY, 7LXZ, 7LY0, 7LY2, 7LY3, 7LYK, 7LYL, 7LYM, 7LYN, 7LYO, 7LYP, 7LYQ, 7M0J, 7M3I, 7M42, 7M53, 7M6D, 7M6E, 7M6F 7M6G, 7M6H, 7M6I, 7M71, 7M7B, 7M7W, 7M8J, 7M8K, 7M8S, 7M8T, 7M8U, 7MDW, 7ME7, 7MEJ, 7MF1, 7MFU, 7MJG, 7MJH, 7MJI, 7MJJ, 7MJK, 7MJL, 7MJM, 7MJN, 7MKL 7MKM, 7MLZ, 7MM0, 7MMO, 7MSQ, 7MTC, 7MTD, 7MTE, 7MW2, 7MW3, 7MW4, 7MW5, 7MW6, 7MY2, 7MY3, 7MY8, 7MZF, 7MZG, 7MZH, 7MZI, 7MZJ, 7MZK, 7MZL, 7MZM, 7MZN 7N0G, 7N0H, 7N1A, 7N1B, 7N1E, 7N1F, 7N1Q, 7N1T, 7N1U, 7N1V, 7N1W, 7N1X, 7N1Y, 7N3I, 7N4I, 7N4J, 7N4L, 7N4M, 7N5H, 7N62, 7N64, 7N6D, 7N6E, 7N8H, 7N8I 7N9A, 7N9B, 7N9C, 7N9E, 7N9T, 7NAB, 7ND3, 7ND4, 7ND5, 7ND6, 7ND7, 7ND8, 7ND9, 7NDA, 7NDB, 7NDC, 7NDD, 7NEG, 7NEH, 7NKT, 7NLL, 7NP1, 7NS6, 7NT9, 7NTA7NTC, 7NX6, 7NX7, 7NX8, 7NX9, 7NXA, 7NXB, 7NXC, 7OAN, 7OAO, 7OAP, 7OAQ, 7OAU, 7OAY, 7OD3, 7ODL, 7OLZ, 7OR9, 7ORA, 7ORB, 7OWX, 7P19, 7P3D, 7P40, 7P5G 7P5Q, 7P5S, 7P77, 7P78, 7P79, 7P7A, 7P7B, 7PBE, 7PHG, 7PQY, 7PQZ, 7PR0, 7PRY, 7PRZ, 7PS0, 7PS1, 7PS2, 7PS4, 7PS5, 7PS6, 7PS7, 7Q0A, 7Q0G, 7Q0H, 7Q0I 7Q1Z, 7Q3Q, 7Q3R, 7Q6E, 7Q9F, 7Q9G, 7Q9I, 7Q9J, 7Q9K, 7Q9M, 7Q9P, 7QEZ, 7QF0, 7QF1, 7QNW, 7QNX, 7QNY, 7QO7, 7QO9, 7QTI, 7QTJ, 7QTK, 7QUR, 7QUS, 7R0Z 7R10, 7R11, 7R12, 7R13, 7R14, 7R15, 7R16, 7R17, 7R18, 7R19, 7R1A, 7R1B, 7R40, 7R4I, 7R4Q, 7R4R, 7R6W, 7R6X, 7R7N, 7R8L, 7R8M, 7R8N, 7R8O, 7R95, 7RA8 7RAL, 7RAQ, 7RBU, 7RBV, 7RBY, 7RKU, 7RKV, 7RNJ, 7RPV, 7RQ6, 7RR0, 7RTD, 7RTR, 7RU1, 7RU2, 7RU3, 7RU4, 7RU5, 7RU8, 7RW2, 7RXD, 7RZQ, 7RZR, 7RZS, 7RZT 7RZU, 7RZV, 7S0B, 7S0C, 7S0D, 7S0E, 7S3N, 7S4S, 7S5P, 7S5Q, 7S5R, 7S6I, 7S6J, 7S6K, 7S6L, 7S83, 7SA2, 7SBK, 7SBL, 7SBO, 7SBP, 7SBQ, 7SBR, 7SBS, 7SBT 7SBU、7SC1、7SD5、7SI2、7SIS、7SIX、7SJ0、7SJS、7SKZ、7SL5、7SN0、7SN2、7S N3、7SO9、7SOA、7SOB、7SOC、7SOD、7SOE、7SOF、7SPO、7SPP、7SWN、7SWO、7SWP、 7SWW, 7SWX, 7SXR, 7SXS, 7SXT, 7SXU, 7SXV, 7SXW, 7SXX, 7SXY, 7SXZ, 7SY0, 7SY1, 7SY2, 7SY3, 7SY4, 7SY5, 7SY6, 7SY7, 7SY8, 7T01, 7T3M, 7T67, 7T72, 7T7B7T9J, 7T9K, 7T9L, 7TAS, 7TAT, 7TB4, 7TB8, 7TBF, 7TCA, 7TCC, 7TCQ, 7TEI, 7TEW, 7TEX, 7TEY, 7TEZ, 7TF0, 7TF1, 7TF2, ​​7TF3, 7TF4, 7TF5, 7TF8, 7TGE, 7TGW 7TGX、7TGY、7THE、7THK、7THT、7TIK、7TL1、7TL9、7TLA、7TLB、7TLC、7TLD、7T LT、7TLY、7TM0、7TN0、7TNW、7TO4、7TOU、7TOV、7TOW、7TOX、7TOY、7TOZ、7TP0、 7TP1、7TP2、7TP3、7TP4、7TP7、7TP8、7TP9、7TPA、7TPC、7TPE、7TPF、7TPH、7T PK、7TPL、7TPR、7TYZ、7TZ0、7U09、7U0A、7U0D、7U0E、7U0N、7U0P、7U0Q、7U0X、 7U1R, 7U2D, 7U2E, 7U8E, 7U9O, 7U9P, 7UAP, 7UAQ, 7UAR, 7UB0, 7UB5, 7UB6, 7UFK, 7UFL, 7UHC, 7UL0, 7UM2, 7UPL, 7UR1, 7URQ, 7URS, 7UZ4, 7UZ5, 7UZ6, 7UZ7 7UZ8, 7UZ9, 7UZA, 7UZB, 7UZC, 7UZD, 7V20, 7V22, 7V23, 7V24, 7V26, 7V27, 7V2A, 7V76, 7V77, 7V78, 7V79, 7V7A, 7V7D, 7V7E, 7V7F, 7V7G, 7V7H, 7V7I, 7V7J 7V7N, 7V7O, 7V7P, 7V7Q, 7V7R, 7V7S, 7V7T, 7V7U, 7V7V, 7V7Z, 7V80, 7V81, 7V82, 7V83, 7V84, 7V85, 7V86, 7V87, 7V88, 7V89, 7V8A, 7V8B, 7V8C, 7VHH, 7VHJ 7VHK, 7VHL, 7VHM, 7VHN, 7VMU, 7VNB, 7VNC, 7VND, 7VNE, 7VOA, 7VQ0, 7VRV, 7VRW, 7VX1, 7VX4, 7VX5, 7VX9, 7VXA, 7VXB, 7VXC, 7VXD, 7VXE, 7VXF, 7VXI, 7VXK 7VXM, 7VYR, 7VZT, 7W1S, 7W6U, 7W8S, 7W92, 7W94, 7W99, 7W9B, 7W9C, 7W9E, 7W9F, 7WA1, 7WB5, 7WBL, 7WBP, 7WBQ, 7WBZ, 7WCD, 7WCH, 7WCK, 7WCP, 7WCR, 7WCU7WCZ, 7WD0, 7WD1, 7WD2, 7WD7, 7WD8, 7WD9, 7WDF, 7WE7, 7WE8, 7WE9, 7WEA, 7WEB, 7WEC, 7WED, 7WEE, 7WEF, 7WEV, 7WG6, 7WG7, 7WG8, 7WG9, 7WGB, 7WGC, 7WGV 7WGX, 7WGY, 7WGZ, 7WH8, 7WHB, 7WHD, 7WHH, 7WHI, 7WHJ, 7WHK, 7WHZ, 7WJY, 7WJZ, 7WK0, 7WK2, 7WK3, 7WK4, 7WK5, 7WK6, 7WK8, 7WK9, 7WKA, 7WLC, 7WM0, 7WN2 7WNB, 7WNM, 7WO4, 7WO5, 7WO7, 7WOA, 7WOB, 7WOC, 7WOG, 7WON, 7WOP, 7WOQ, 7WOR, 7WOS, 7WOU, 7WOV, 7WOW, 7WP0, 7WP1, 7WP2, 7WP5, 7WP6, 7WP8, 7WP9, 7WPA 7WPB, 7WPC, 7WPD, 7WPE, 7WPF, 7WPH, 7WQV, 7WR8, 7WRH, 7WRI, 7WRJ, 7WRV, 7WS0, 7WS1, 7WS2, 7WS3, 7WS4, 7WS5, 7WS6, 7WS7, 7WS8, 7WS9, 7WSA, 7WSE, 7WSH 7WSK, 7WT7, 7WT8, 7WT9, 7WTF, 7WTG, 7WTH, 7WTI, 7WTJ, 7WTK, 7WUE, 7WUH, 7WVL, 7WVP, 7WVQ, 7WWI, 7WWJ, 7WWK, 7WWL, 7WWM, 7WXZ, 7WZ1, 7WZ2, 7X1M, 7X25 7X2H, 7X2K, 7X2L, 7X2M, 7X63, 7X66, 7X6A, 7X7D, 7X7E, 7X7N, 7X7T, 7X7U, 7X8W, 7X8Y, 7X8Z, 7X90, 7X91, 7X92, 7X93, 7X94, 7X95, 7X96, 7X9E, 7XA7, 7XAZ 7XB0, 7XB1, 7XBY, 7XCH, 7XCI, 7XCK, 7XCO, 7XCP, 7XCZ, 7XD2, 7XDA, 7XDB, 7XDK, 7XDL, 7XEG, 7XEI, 7XH8, 7XIC, 7XID, 7XIK, 7XIL, 7XIW, 7XIX, 7XIY, 7XIZ 7XJ6, 7XJ8, 7XJ9, 7XMX, 7XMZ, 7XNQ, 7XNR, 7XNS, 7XO4, 7XO5, 7XO6, 7XO7, 7XO8, 7XO9, 7XOA, 7XOB, 7XOC, 7XOD, 7XRP, 7XS8, 7XSA, 7XSB, 7XSC, 7XST, 7XTZ7XU0, 7XU1, 7XU2, 7XU3, 7XU4, 7XU5, 7XU6, 7XWA, 7XXL, 7Y0C, 7Y0V, 7Y1Y, 7Y1Z, 7Y42, 7Y6D, 7Y6K, 7Y6L, 7Y6N, 7Y75, 7Y76, 7Y7J, 7Y7K, 7Y8J, 7Y9N, 7Y9S 7Y9Z, 7YA0, 7YA1, 7YAD, 7YBI, 7YBJ, 7YC5, 7YCK, 7YCL, 7YCN, 7YCO, 7YD1, 7YDI, 7YDY, 7YE5, 7YE9, 7YEG, 7YH6, 7YH7, 7YHW, 7YJ3, 7YKJ, 7YOW, 7YQT, 7YQU 7YQV, 7YQW, 7YQX, 7YQY, 7YQZ, 7YR0, 7YR1, 7YR2, 7YR3, 7YTN, 7YUE, 7YV8, 7YVE, 7YVF, 7YVG, 7YVH, 7YVI, 7YVJ, 7YVK, 7YVL, 7YVM, 7YVN, 7YVO, 7YVP, 7YVU 7Z0X, 7Z0Y, 7Z1A, 7Z1B, 7Z1C, 7Z1D, 7Z1E, 7Z3Z, 7Z6V, 7Z7X, 7Z85, 7Z86, 7Z8O, 7Z9Q, 7Z9R, 7ZBU, 7ZCE, 7ZCF, 7ZDQ, 7ZF3, 7ZF4, 7ZF5, 7ZF7, 7ZF8, 7ZF9 7ZFA, 7ZFB, 7ZFC, 7ZFD, 7ZFE, 7ZJL, 7ZR2, 7ZR7, 7ZR8, 7ZR9, 7ZRC, 7ZRV, 7ZSD, 7ZSS, 7ZXU, 8A99, 8AAA, 8AQS, 8AQT, 8AQU, 8AQV, 8AQW, 8BBN, 8BBO, 8BCZ 8BE1、8BEV、8BGG、8BH5、8BON、8BSE、8BSF、8C1V、8C3V、8C8P、8CIM、8CSA、8C SJ、8CWI、8CWK、8CWU、8CWV、8CXN、8CXQ、8CY6、8CY7、8CY9、8CYA、8CYB、8CYC、 8CYD、8CYJ、8CZI、8D0Z、8D36、8D47、8D48、8D55、8D56、8D5A、8D6Z、8D8Q、8D 8R、8DAD、8DAO、8DCC、8DCE、8DF5、8DGU、8DI5、8DLI、8DLJ、8DLK、8DLL、8DLM、 8DLN、8DLO、8DLP、8DLQ、8DLR、8DLS、8DLT、8DLU、8DLV、8DLW、8DLX、8DLY、8D LZ、8DM0、8DM1、8DM2、8DM3、8DM4、8DM5、8DM6、8DM7、8DM8、8DM9、8DMA、8DNN、8DT3, 8DT8, 8DTR, 8DTT, 8DTX, 8DV1, 8DV2, 8DW2, 8DW3, 8DW9, 8DWA, 8DXS, 8DXT, 8DXU, 8DYA, 8DZH, 8DZI, 8E1G, 8EL2, 8ELH, 8ELJ, 8ELO, 8ELP, 8ELQ, 8E OO, 8EPN, 8EPP, 8EPQ, 8ERQ, 8ERR, 8F0G, 8F0H, 8FA1, 8FA2, 8FEZ, 8FU7, 8FU8, 8FU9, 8GB0, 8GB5, 8GB6, 8GB7, 8GB8, 8GJM, 8GJN, 8GOM, 8GON, 8GOU, 8GPY, 8GRY, 8GS6, 8GS9, 8GTO, 8GTP, 8GTQ, 8GX9, 8GZ5, 8GZZ, 8H00, 8H01, 8H06, 8H07, 8H08, 8H3D, 8H3E, 8H3M, 8H3N, 8H5C, 8HC2, 8HC3, 8HC4, 8HC5, 8HC6, 8HC7, 8HC8, 8HC9, 8HCA, 8HCB, 8HEB, 8HEC, 8HED, 8HHX, 8HHY, 8HHZ, 8HN6, 8HN7, 8I5H, 8I5I, 8IDN, 8IF2, 8IOS, 8IOT, 8IOU, 8IOV, 8ITU, 8J1Q, 8J26, or 8SMT. Each of these PDB IDs is incorporated herein by reference in its entirety. FASTA can be used to obtain the amino acid sequence of the SARS-CoV-2 S glycoprotein associated with each entry.

[0031] In the implementation scheme, the SARS-CoV-2 S glycoprotein is the wild-type SARS-CoV-2 S glycoprotein or a SARS-CoV-2 S glycoprotein derived from its SARS-CoV-2 variants. In the implementation scheme, the SARS-CoV-2 variants are: B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; or B.1.1.529 SARS-CoV-2 strain. In the implementation scheme, the SARS-CoV-2 S glycoprotein contains a transmembrane domain.

[0032] The wild-type SARS-CoV-2 S glycoprotein contains a furin cleavage site RRAR (SEQ ID NO: 6) at positions 669-672 of the SARS-CoV-2 S glycoprotein in SEQ ID NO: 2. In an embodiment, the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site. In an embodiment, the inactive furin cleavage site has an amino acid sequence of any one of SEQ ID NO: 7-34, 97, and 111. In an embodiment, the amino acid sequence of the inactive furin cleavage site is GG.

[0033] In the implementation scheme, the amino acid sequence of the inactive furin protease cleavage site is QQAQ (SEQ ID NO:7).

[0034] In one embodiment, one or more amino acids constituting the natural furin cleavage site are mutated to any natural amino acid. In another embodiment, one or more amino acids constituting the natural furin cleavage site are deleted.

[0035] In one embodiment, one or more amino acids constituting the natural furin cleavage site are mutated to glutamine. In another embodiment, 1, 2, 3, or 4 amino acids may be mutated to glutamine. In another embodiment, one arginine cell constituting the natural furin cleavage site is mutated to glutamine. In another embodiment, two arginine cells constituting the natural furin cleavage site are mutated to glutamine. In another embodiment, three arginine cells constituting the natural furin cleavage site are mutated to glutamine.

[0036] In one embodiment, one or more amino acids constituting the natural furin cleavage site are mutated to alanine. In another embodiment, 1, 2, 3, or 4 amino acids may be mutated to alanine. In another embodiment, one arginine cell constituting the natural furin cleavage site is mutated to alanine. In another embodiment, two arginine cells constituting the natural furin cleavage site are mutated to alanine. In another embodiment, three arginine cells constituting the natural furin cleavage site are mutated to alanine.

[0037] In one embodiment, one or more amino acids constituting the natural furin cleavage site are mutated to glycine. In another embodiment, 1, 2, 3, or 4 amino acids may be mutated to glycine. In another embodiment, one arginine residue in the natural furin cleavage site is mutated to glycine. In another embodiment, two arginine residues constituting the natural furin cleavage site are mutated to glycine. In another embodiment, three arginine residues constituting the natural furin cleavage site are mutated to glycine.

[0038] In one embodiment, one or more amino acids constituting the natural furin cleavage site are mutated to asparagine. For example, 1, 2, 3, or 4 amino acids can be mutated to asparagine. In another embodiment, one arginine cell constituting the natural furin cleavage site is mutated to asparagine. In yet another embodiment, two arginine cells constituting the natural furin cleavage site are mutated to asparagine. In yet another embodiment, three arginine cells constituting the natural furin cleavage site are mutated to asparagine.

[0039] In the implementation scheme, the active furin cleavage site (SEQ ID NO: 67) of the SARS-CoV-2 S glycoprotein described herein is replaced by the inactive furin cleavage sites listed in the table below.

[0040] Inactivation of furin cleavage site In one embodiment, the SARS-CoV-2 S glycoprotein contains a mutation at Lys-973 of the native SARS-CoV-2 S glycoprotein (SEQ ID NO: 2). In another embodiment, Lys-973 is mutated to any native amino acid. In yet another embodiment, Lys-973 is mutated to proline. In yet another embodiment, Lys-973 is mutated to glycine.

[0041] In one embodiment, the SARS-CoV-2 S glycoprotein contains a mutation at Val-974 of the native SARS-CoV-2 S glycoprotein (SEQ ID NO: 2). In another embodiment, Val-974 is mutated to any native amino acid, compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In another embodiment, Val-974 is mutated to proline. In yet another embodiment, Val-974 is mutated to glycine, compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2.

[0042] In one embodiment, the SARS-CoV-2 S contains mutations at Lys-973 and Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In another embodiment, Lys-973 and Val-974 are mutated to any native amino acid, compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In yet another embodiment, Lys-973 and Val-974 are mutated to proline, compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. An exemplary SARS-CoV-2 S glycoprotein containing proline at positions 973 and 974 is the glycoprotein of SEQ ID NO: 87, wherein the SARS-CoV-2 S glycoprotein is numbered according to SEQ ID NO: 2.

[0043] In the implementation plan, the SARS-CoV-2 S glycoprotein is associated with SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 89, 106, 110, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228. The polypeptides of any one of 233-236, 243, 255-264, 273-280, 283-284, 287-288, 291-292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236, 328, 329, 330, 331, 332, and 333 have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0044] Provide a surface coated with SARS-CoV-2 S glycoprotein In embodiments, the methods described herein require providing a surface coated with the SARS-CoV-2 S glycoprotein. Numerous examples of the SARS-CoV-2 S glycoprotein are provided herein. In embodiments, the surface is a chip or microplate. In embodiments, the microplate comprises polystyrene. In embodiments, the microplate is a 96-well or 384-well polystyrene plate. In embodiments, the surface is a microplate filled with a solution.

[0045] Expose the surface to biological samples In the implementation scheme, the surface is exposed to a biological sample. In the implementation scheme, the surface is exposed to the biological sample for approximately 10 minutes to approximately 72 hours. In the implementation scheme, the surface is exposed to the biological sample for approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 25 minutes, approximately 30 minutes, approximately 35 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, and approximately 32 hours. The timeframe is approximately 1 hour, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, or 72 hours, including any range or value within this range. In one embodiment, the surface is exposed to the biological sample for approximately 1 hour or 2 hours. In another embodiment, the surface is exposed to the biological sample for approximately 2 hours. In the implementation scheme, the surface is exposed to the biological sample at a temperature of 2-8°C or 8-37°C. In the implementation scheme, the surface is exposed to the biological sample at temperatures of 2°C, approximately 3°C, approximately 4°C, approximately 5°C, approximately 6°C, approximately 7°C, approximately 8°C, approximately 9°C, approximately 10°C, approximately 11°C, approximately 12°C, approximately 13°C, approximately 14°C, approximately 15°C, approximately 16°C, approximately 17°C, approximately 18°C, approximately 19°C, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, or approximately 37°C.

[0046] In this implementation, the biological sample is saliva, nasopharyngeal swab, sputum, urine, fecal sample, cerebrospinal fluid, synovial fluid, serum, blood, or plasma. In this implementation, the biological sample is from a patient who has previously had COVID-19. In this implementation, the biological sample is from a patient who has been given an immunogenic composition against SARS-CoV-2 virus or a variant thereof.

[0047] Expose the surface to the secondary antibody In one embodiment, the surface is exposed to a secondary antibody. In another embodiment, the secondary antibody is an anti-human IgG4 antibody. The anti-human IgG4 antibody binds to IgG4 antibodies. In yet another embodiment, the secondary antibody is an anti-human IgG1 antibody. The anti-human IgG1 antibody binds to IgG1 antibodies. In yet another embodiment, the secondary antibody is an anti-human IgG antibody. The anti-human IgG antibody binds to all IgG antibodies.

[0048] In one implementation, the secondary antibody is attached to the tag. In another implementation, the secondary antibody is covalently attached to the tag. In yet another implementation, the secondary antibody is non-covalently attached to the tag. In one implementation, the tag is a His tag. In another implementation, the tag contains an epitope. For example, the tag may be a polyglutamic acid tag, a FLAG tag, a HA tag, a polyHis tag (having approximately 5-10 histidines) (SEQ ID NO: 101), a hexahistidine tag (SEQ ID NO: 100), an 8X-His tag (having eight histidines) (SEQ ID NO: 102), a Myc tag, a glutathione S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, or an Fc tag. In another implementation, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include the HRV3C protease cleavage site, chymotrypsin, trypsin, elastase, endopeptidase, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa, granzyme B, TEV protease, and thrombin. In this embodiment, the protease cleavage site is the HRV3C protease cleavage site. In this embodiment, the tag is horseradish peroxidase (HRP).

[0049] In the implementation plan, the surface is exposed to the secondary antibody for approximately 10 minutes to approximately 72 hours. Specifically, the surface is exposed to the secondary antibody for approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, and 32 hours. The timeframe is approximately 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, or 72 hours, including any range or value within this timeframe. In this embodiment, the surface is exposed to the secondary antibody for approximately 1 hour.

[0050] Detection of anti-SARS-CoV-2 IgG1, IgG4 and total IgG bound to the surface In the implementation plan, the method includes detecting anti-SARS-CoV-2 IgG1, IgG4 and total IgG bound to the surface.

[0051] In one embodiment, detecting anti-SARS-CoV-2 IgG1 includes (i) contacting the surface with a secondary antibody that binds IgG1 antibodies, wherein the antibody is labeled with horseradish peroxidase; and (ii) contacting the surface with a 3,3',5,5'-tetramethylbenzidine substrate. In another embodiment, detecting anti-SARS-CoV-2 IgG4 includes (i) contacting the surface with a secondary antibody that binds IgG4 antibodies, wherein the antibody is labeled with horseradish peroxidase; and (ii) contacting the surface with a 3,3',5,5'-tetramethylbenzidine substrate. In yet another embodiment, detecting anti-SARS-CoV-2 IgG includes (i) contacting the surface with a secondary antibody that binds IgG antibodies, wherein the antibody is labeled with horseradish peroxidase; and (ii) contacting the surface with a 3,3',5,5'-tetramethylbenzidine substrate. In another embodiment, the detection includes determining the absorbance of the surface. In yet another embodiment, the detection includes determining the absorbance of the surface at wavelengths from 400 nm to approximately 650 nm. In this embodiment, the detection includes determining the absorbance of the surface at wavelengths of about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, or about 650 nm (inclusive of all values ​​and ranges therebetween). In this embodiment, the detection includes determining the absorbance of the surface at a wavelength of about 450 nm. In this embodiment, the surface is contacted with a 3,3',5,5'-tetramethylbenzidine substrate for about 5 minutes to about 1 hour. In the implementation scheme, the surface is brought into contact with the 3,3',5,5'-tetramethylbenzidine substrate for about 20 minutes.

[0052] In the embodiments, the methods used herein can be used to evaluate the immunogenicity of the compositions and vaccine compositions against SARS-CoV-2. In the embodiments, the immunogenic compositions and vaccine compositions target SARS-CoV-2 virus or heterologous SARS-CoV-2 strains. In the embodiments, the immunogenic compositions or vaccine compositions comprise the SARS-CoV-2 S glycoprotein or nucleic acid (e.g., mRNA) encoding the SARS-CoV-2 S glycoprotein. In the embodiments, the immunogenic compositions or vaccine compositions comprise a viral vector expressing the SARS-CoV-2 S glycoprotein. In the embodiments, the immunogenic compositions or vaccine compositions comprise at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SARS-CoV-2 S glycoprotein specified in SEQ ID NO: 87.

[0053] In some embodiments, the immunogenic composition or vaccine composition includes an adjuvant. Exemplary adjuvants are described below.

[0054] Aluminum-based adjuvants In the embodiments, the adjuvant may be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles may be at least 80%, at least 85%, at least 90%, or at least 95% bound to the alum. Often, 92% to 97% of the nanoparticles are bound to the alum in the composition. The amount of alum present in each dose is typically in the range of about 400 µg to about 1250 µg. For example, alum may be present in amounts of about 300 µg to about 900 µg, about 400 µg to about 800 µg, about 500 µg to about 700 µg, about 400 µg to about 600 µg, or about 400 µg to about 500 µg per dose. Typically, for a 120 µg dose of protein nanoparticles, alum is present in about 400 µg.

[0055] Saponin adjuvant Adjuvants containing saponins can also be combined with the immunogens disclosed herein. Saponins are derived from the soapberry tree (…). Quillaja saponaria Glycosides from the bark of *Molina*. Typically, saponins are prepared using a multi-step purification process, yielding multiple fractions. As used herein, the term "from" refers to... soapberry tree Molina's saponin fraction is generally used to describe soapberry treeSemi-purified or definitive saponin fractions or substantially pure fractions thereof.

[0056] Saponin fraction Several methods for producing saponin fractions are suitable. Fractions A, B, and C are described in US Patent 6,352,697 and can be prepared as follows: Separation of crude aqueous saponin fractions by chromatography. soapberry tree The lipophilic fraction of Molina extract Quil A was extracted and eluted with an aqueous solution of 70% acetonitrile to recover the lipophilic fraction. This lipophilic fraction was then separated by semi-preparative HPLC using an acidic aqueous solution of acetonitrile in a 25% to 60% gradient. The fraction referred to herein as “fraction A” or “QH-A” is or corresponds to the fraction eluted in approximately 39% acetonitrile. The fraction referred to herein as “fraction B” or “QH-B” is or corresponds to the fraction eluted in approximately 47% acetonitrile. The fraction referred to herein as “fraction C” or “QH-C” is or corresponds to the fraction eluted in approximately 49% acetonitrile. Further information regarding fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, soapberry tree Molina fractions A, B, and C each represent groups or families of chemically closely related molecules with definable properties. The chromatographic conditions used to obtain them result in high batch-to-batch reproducibility in terms of elution profiles and biological activity.

[0057] Other saponin fractions have been described. Fractions B3, B4, and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP03632279 B2, Q-VAC is described in (Nor-Feed, AS Denmark), and [further details omitted]. soapberry tree Molina guanylic acid. Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22, especially QA-7, QA-17, QA-18, and QA-21, can be used. They are obtained as described in EP 0 3632 279 B2, particularly in Example 1 on pages 6, 8, and 9.

[0058] The saponin fractions described herein for the formation of adjuvants are often substantially pure fractions; that is, the fractions are substantially free from contamination from other materials. In certain aspects, substantially pure saponin fractions may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds, such as other saponins or other auxiliary materials.

[0059] ISCOM Structure The saponin fraction can be administered in the form of cage-like particles called ISCOM (immunostimulatory complex). ISCOM can be prepared as described in EP0109942B1, EP0242380B1, and EP0180546 B1. In certain embodiments, transport and / or transient antigens, as described in EP 9600647-3 (PCT / SE97 / 00289), can be used.

[0060] matrix adjuvant In the implementation scheme, ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and lipids. The lipids are at least sterols, such as cholesterol. In certain aspects, the ISCOM matrix complex also contains phospholipids. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant-active) substances, not necessarily glycosides, and may be produced as described in EP0436620B1, which is incorporated herein by reference in its entirety.

[0061] In other respects, ISCOM is an ISCOM complex. An ISCOM complex contains at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex contains an antigen associated with the antigen through detergent treatment, such that a portion of the antigen is integrated into the particle. Conversely, the ISCOM matrix is ​​formulated as a mixture with the antigen, and the association between the ISCOM matrix particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0062] According to one embodiment, the saponin fraction integrated into the ISCOM matrix complex or the ISCOM complex, or at least one additional adjuvant also integrated into or mixed with the ISCOM matrix complex, is selected from... soapberry tree The grade is A, B or C. soapberry tree Semi-purified preparation, soapberry tree The purified formulation or any purified subfraction, such as QA 1-21.

[0063] In certain aspects, each ISCOM particle may contain at least two saponin fractions. Different saponin fractions in any weight percentage combination may be used. Any two fractions in any weight percentage combination may be used. For example, the particle may contain any weight percentage of fraction A and any weight percentage of another saponin fraction, such as a crude saponin fraction or fraction C. Thus, in certain aspects, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1 wt% to 99.9 wt%, 5 wt% to 95 wt%, 10 wt% to 90 wt%, 15 wt% to 85 wt%, 20 wt% to 80 wt%, 25 wt% to 75 wt%, 30 wt% to 70 wt%, 35 wt% to 65 wt%, 40 wt% to 60 wt%, 45 wt% to 55 wt%, 40 wt% to 60 wt%, or 50 wt% of one saponin fraction, such as fraction A, and in each case, the remainder reaching 100% of another saponin fraction, such as any crude fraction or any other fraction (e.g., fraction C). Weights are calculated based on the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in U.S. Publication No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0064] In a particular embodiment, the ISCOM matrix or ISCOM complex comprises 5% to 99% by weight of one fraction (e.g., fraction A) and the remainder up to 100% by weight of another fraction (e.g., crude saponin fraction or fraction C). Weights are calculated based on the total weight of the saponin fractions.

[0065] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40% to 99% by weight of one fraction (e.g., fraction A) and 1% to 60% by weight of another fraction (e.g., crude saponin fraction or fraction C). The weight is calculated based on the total weight of the saponin fractions.

[0066] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction (e.g., fraction A) and 30% to 5% by weight of another fraction (e.g., crude saponin fraction or fraction C). Weights are calculated based on the total weight of the saponin fractions. In other embodiments, from soapberry tree Molina's saponin fractions are selected from any one of QA 1-21.

[0067] In addition to particles containing a mixture of saponin fractions, both ISCOM matrix particles and ISCOM complex particles can be formed using only one saponin fraction. The compositions disclosed herein may contain multiple particle types, each containing only one saponin fraction. That is, certain compositions may contain one or more different types of ISCOM-matrix complex particles and / or one or more different types of ISCOM complex particles, wherein each individual particle contains one saponin fraction derived from… soapberry tree Molina's saponin fractions, one of the complexes has a saponin fraction that differs from that in the particles of the other complexes.

[0068] In certain respects, one type of saponin fraction or crude saponin fraction may be integrated into one ISCOM matrix complex or particle, while another type of substantially pure saponin fraction or crude saponin fraction may be integrated into another ISCOM matrix complex or particle. The composition or vaccine may comprise at least two types of complexes or particles, each type having one type of saponin integrated into physically different particles.

[0069] In the composition, ISCOM matrix complex particles and / or a mixture of ISCOM complex particles may be used, wherein a saponin fraction is present. soapberry tree Molina and another saponin fraction soapberry tree Molina is incorporated individually into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0070] ISCOM matrix or ISCOM complex particles (each having a saponin fraction) may be present in the composition in any weight percentage combination. In a particular aspect, the composition may contain 0.1 wt% to 99.9 wt%, 5 wt% to 95 wt%, 10 wt% to 90 wt%, 15 wt% to 85 wt%, 20 wt% to 80 wt%, 25 wt% to 75 wt%, 30 wt% to 70 wt%, 35 wt% to 65 wt%, 40 wt% to 60 wt%, 45 wt% to 55 wt%, 40 wt% to 60 wt%, or 50 wt% of an ISCOM matrix or complex containing a first saponin fraction, with the remainder consisting of ISCOM matrices or complexes containing different saponin fractions. In some aspects, the remainder is one or more ISCOM matrices or complexes, wherein each matrix or complex particle contains only one saponin fraction. In other aspects, the ISCOM matrix or complex particles may contain more than one saponin fraction.

[0071] In a specific composition, the only saponin fraction in the first ISCOM matrix or ISCOM complex particles is fraction A and the only saponin fraction in the second ISCOM matrix or ISCOM complex particles is fraction C.

[0072] In the implementation plan, respectively using adjuvants soapberry tree Molina's Grade A and soapberry tree The total weight of Molina's fraction C is calculated as follows: soapberry tree Molina's grade A, by weight, comprises at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, while soapberry tree Molina's fraction C accounts for the remainder.

[0073] A preferred composition comprises a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C, wherein fraction A ISCOM matrix comprises about 70% by weight of the total saponin adjuvant and fraction C ISCOM matrix comprises about 30% by weight of the total saponin adjuvant. In another preferred composition, fraction A ISCOM matrix comprises about 85% by weight of the total saponin adjuvant and fraction C ISCOM matrix comprises about 15% by weight of the total saponin adjuvant. In yet another preferred composition, fraction A ISCOM matrix comprises about 92% by weight of the total saponin adjuvant and fraction C ISCOM matrix comprises about 8% by weight of the total saponin adjuvant. Thus, in certain compositions, fraction A ISCOM matrix is ​​present in the range of about 70% to about 85% by weight of the total saponin adjuvant in the composition, and fraction C ISCOM matrix is ​​present in the range of about 15% to about 30%. In certain compositions, fraction A ISCOM matrix is ​​present in the range of about 70% to about 92% by weight of the total weight of the saponin adjuvant in the composition, and fraction C ISCOM matrix is ​​present in the range of about 8% to about 30%. In embodiments, fraction A ISCOM matrix accounts for 50% to 96% by weight of the total weight of fraction A ISCOM matrix and fraction C ISCOM matrix in the adjuvant, respectively, and fraction C ISCOM matrix accounts for the remainder. In a particularly preferred composition (referred to herein as MATRIX-M™), fraction A ISCOM matrix is ​​present in the range of about 85% by weight and fraction C ISCOM matrix is ​​present in the range of about 15% by weight of the total weight of the saponin adjuvant in the composition. MATRIX-M™ may be interchangeably referred to as matrix-M1.

[0074] Exemplary QS-7 and QS-21 fractions, their production, and their uses are described in U.S. Patents 5,057,540, 6,231,859, 6,352,697, 6,524,584, 6,846,489, 7,776,343, and 8,173,141, which are incorporated herein by reference.

[0075] In embodiments, other adjuvants may be used additionally or as alternatives. Within the scope of this disclosure, any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," which is incorporated herein by reference in its entirety for all purposes. Other adjuvants include complete Freund's adjuvant (a nonspecific immune response stimulant containing Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid A and monophospholipid A (MPL), MF-59, RIBI (containing three components extracted from bacteria), MPL, trehalose dimethicone (TDM), and cell wall skeleton (CWS) in 2% squalene / TWEEN® polysorbate 80 emulsions. In embodiments, the adjuvant may be a few-layer lipid vesicle; for example, NOVASOMES®. NOVASOMES® are few-layered nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They contain BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been proven to be an effective adjuvant (see U.S. Patents 5,629,021, 6,387,373, and 4,911,928).

[0076] Example 1: Measurement of anti-rS protein IgG1, IgG2, IgG3, and IgG4, as well as total anti-rS IgG, in the serum of patients who have received a COVID-19 vaccine or have been previously infected with SARS-CoV-2. Objective: To develop assays for anti-rS protein IgG1, IgG2, IgG3, and IgG4, as well as total anti-rS IgG, to measure anti-spike IgG levels (as a percentage of total anti-spike IgG) in serum samples from subjects repeatedly immunized with a COVID-19 vaccine. These assays allow for the identification of different IgG subtypes to guide vaccine platform selection. Alternative ADCP (FcγRIIa binding), alternative ADCC (FcγRIIIa binding), and ADCD (C1q binding) were also measured.

[0077] Samples: Samples were collected from: (1) patients who received three doses of a first vaccine containing mRNA encoding the SARS-CoV-2 spike protein before and after administration of a booster dose of a vaccine containing the SARS-CoV-2 spike glycoprotein (referred to as “heterologous 1”); (2) patients who received three doses of a second vaccine containing mRNA encoding the SARS-CoV-2 spike protein before and after administration of a booster dose of a vaccine containing the SARS-CoV-2 spike glycoprotein (referred to as “heterologous 2”); and (3) patients who received two doses of a vaccine containing the SARS-CoV-2 spike glycoprotein before and after administration of a booster dose of the same vaccine (referred to as “homologous”). The SARS-CoV-2 spike glycoprotein from the vaccine of (3) and the boosters of (1), (2) and (3) contains proline at positions 973 and 974 and an inactive primary furin cleavage site, wherein the SARS-CoV-2 S glycoprotein is polypeptide numbered according to SEQ ID NO: 2. The vaccine (3) and the boosters of (1), (2) and (3) also contain saponin adjuvants. The saponin adjuvant contains two types of iscom particles, wherein: the first iscom particle contains... soapberry tree Molina's grade A without soapberry tree Molina's fraction C; and the second iscom particle contains soapberry tree Molina's grade C without including soapberry tree Molina's fraction A. Fractions A and C account for the respective proportions of the adjuvant in the adjuvant. soapberry tree Molina's Grade A and soapberry tree Molina's fractions of C account for 85% and 15% of the total weight.

[0078] Each sample was subjected to anti-spike IgG4 assay ( Figure 2A ), Anti-spike IgG1 assay ( Figure 2B ) and total anti-spike IgG assay ( Figure 2C As described below and Figures 2A-2C As described in the description. The SARS-CoV-2 S glycoprotein (also known as "SARS-CoV-2 rS protein") used in the assay contains proline residues at positions 973 and 974 and an inactive primary furin cleavage site, wherein the SARS-CoV-2 S glycoprotein is polypeptide numbered according to SEQ ID NO: 2.

[0079] Anti-spike IgG4 assay: Assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1–2 µg / mL SARS-CoV-2 rS protein (manufactured at Novavax, Inc., Gaithersburg, MD, USA) at 2–8°C for 15–72 hours. The plates were then washed with phosphate-buffered saline (PBST) containing Tween 20 and blocked with blocking buffer (Thermo Fisher Scientific) for 1 hour. Human serum samples (reference standard, quality control, or test serum) were then added to the wells to bind anti-rS protein IgG antibodies to the rS protein coated on the plate (incubation for 2 hours). Anti-SARS-CoV-2 spike RBD IgG4 monoclonal antibody (Acro Biosystems, catalog number SPD-M402a) was used as a reference standard to measure IgG4 antibodies in the serum samples. The plates were washed again with PBST, and then mouse anti-human IgG4 secondary antibody conjugated with horseradish peroxidase (HRP; from Invitrogen) was added and incubated at room temperature for 1 hour. A final washing step was performed, followed by the addition of 3,3',5,5'-tetramethylbenzidine substrate (TMB, from Thermo Fisher Scientific). The reaction was terminated after 20 minutes with TMB stop solution (Scytek Laboratories, Logan, UT, USA). The optical density (OD) of the chromogenic signal is proportional to the amount of anti-rS IgG4 captured on the plate, providing a quantifiable measure of IgG4 concentration in the serum sample. The anti-spike IgG4 concentration was calculated by interpolating the levels against an IgG4 reference standard curve.

[0080] Anti-spike IgG1 assay: Assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1–2 µg / mL SARS-CoV-2 rS protein (manufactured at Novavax, Inc., Gaithersburg, MD, USA) at 2–8°C for 15–72 hours. The plates were then washed with phosphate-buffered saline (PBST) containing Tween 20 and blocked with blocking buffer (Thermo Fisher Scientific) for 1 hour. Human serum samples (reference standard, quality control, or test serum) were then added to the wells to bind the anti-rS protein IgG antibody to the coated S protein on the plate (incubation for 2 hours). Anti-SARS-CoV-2 spike RBD IgG1 monoclonal antibody (Acro Biosystems, catalog number SPD-SPD-M265) was used as a reference standard. The plates were washed again with PBST, and then a mouse anti-human IgG1 secondary antibody conjugated with horseradish peroxidase (HRP; from Invitrogen) was added and incubated at room temperature for 1 hour. A final washing step was performed, followed by the addition of 3,3',5,5'-tetramethylbenzidine substrate (TMB, from Thermo Fisher Scientific). The reaction was terminated after 25 minutes with TMB stop solution (Scytek Laboratories, Logan, UT, USA). The optical density (OD) of the chromogenic signal is proportional to the amount of anti-rSIgG1 captured on the plate, providing a quantifiable measure of IgG1 concentration in the serum sample. The anti-spike IgG1 concentration was calculated by interpolating the levels against an IgG1 reference standard curve.

[0081] Anti-spike IgG2 assay: Assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1–2 µg / mL SARS-CoV-2 rS protein (manufactured at Novavax, Inc., Gaithersburg, MD, USA) at 2–8°C for 15–72 hours. The plates were then washed with phosphate-buffered saline (PBST) containing Tween 20 and blocked with blocking buffer (Thermo Fisher Scientific) for 1 hour. Human serum samples (reference standard, quality control, or test serum) were then added to the wells to allow the anti-rS protein IgG antibody to bind to the S protein coated on the plate (incubation for 2 hours). Anti-SARS-CoV-2 spike RBD IgG1 monoclonal antibody (Acro Biosystems, catalog number SPD-SPD-M265) was used as a reference standard. The plates were washed again with PBST, and then mouse anti-human IgG2 secondary antibody conjugated with horseradish peroxidase (HRP; from Invitrogen) was added and incubated at room temperature for 1 hour. A final washing step was performed, followed by the addition of 3,3',5,5'-tetramethylbenzidine substrate (TMB, from Thermo Fisher Scientific). The reaction was terminated after 25 minutes with TMB stop solution (Scytek Laboratories, Logan, UT, USA). The optical density (OD) of the chromogenic signal is proportional to the amount of anti-rSIgG2 captured on the plate, providing a quantifiable measure of IgG2 concentration in the serum sample. The anti-spike IgG2 concentration was calculated by interpolating the levels against an IgG2 reference standard curve.

[0082] Anti-spike IgG3 assay: Assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1–2 µg / mL SARS-CoV-2 rS protein (manufactured at Novavax, Inc., Gaithersburg, MD, USA) at 2–8°C for 15–72 hours. The plates were then washed with phosphate-buffered saline (PBST) containing Tween 20 and blocked with blocking buffer (Thermo Fisher Scientific) for 1 hour. Human serum samples (reference standard, quality control, or test serum) were then added to the wells to allow the anti-rS protein IgG antibody to bind to the S protein coated on the plate (incubation for 2 hours). Anti-SARS-CoV-2 spike RBD IgG3 monoclonal antibody (Acro Biosystems, catalog number SPD-SPD-M265) was used as a reference standard. The plates were washed again with PBST, and then a mouse anti-human IgG3 secondary antibody conjugated with horseradish peroxidase (HRP; from Invitrogen) was added and incubated at room temperature for 1 hour. A final washing step was performed, followed by the addition of 3,3',5,5'-tetramethylbenzidine substrate (TMB, from Thermo Fisher Scientific). The reaction was terminated after 25 minutes with TMB stop solution (Scytek Laboratories, Logan, UT, USA). The optical density (OD) of the chromogenic signal is proportional to the amount of anti-rSIgG2 captured on the plate, providing a quantifiable measure of IgG3 concentration in the serum sample. The anti-spike IgG3 concentration was calculated by interpolating the levels against an IgG3 reference standard curve.

[0083] Total anti-spike IgG assay: Assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1–2 µg / mL SARS-CoV-2 rS protein (manufactured at Novavax, Inc., Gaithersburg, MD, USA) at 2–8°C for 15–72 hours. The plates were then washed with phosphate-buffered saline (PBST) containing Tween 20 and blocked with blocking buffer (Thermo Fisher Scientific) for 1 hour. Human serum samples (reference standard, quality control, or test serum) were then added to the wells to bind the anti-rS protein IgG antibody to the coated S protein on the plate (incubation for 2 hours). Anti-SARS-CoV-2 spike RBD IgG monoclonal antibody (Acro Biosystems, catalog number RAS008-02) was used as a reference standard. The plates were washed again with PBST, and then a goat anti-human IgG secondary antibody conjugated with horseradish peroxidase (HRP; from Southern Biotech, Birmingham, AL, USA) was added and incubated at room temperature for 1 hour. A final washing step was performed, followed by the addition of 3,3',5,5'-tetramethylbenzidine substrate (TMB, from Thermo Fisher Scientific). The reaction was terminated after 20 minutes with TMB stop solution (Scytek Laboratories, Logan, UT, USA). The optical density (OD) of the chromogenic signal is proportional to the amount of total anti-rS IgG captured on the plate, providing a quantifiable measure of the total IgG concentration in the serum sample. The anti-spike IgG concentration was calculated by interpolating the levels against an IgG reference standard curve.

[0084] % IgG4 calculation: Data on anti-spike IgG4, IgG1, and total IgG for individual subjects were tabulated. For each subject, anti-spike IgG4 (%) was calculated as anti-spike IgG4 / total anti-spike IgG * 100. For each subject, anti-spike IgG1 (%) was calculated as anti-spike IgG1 / total anti-spike IgG * 100.

[0085] result: Figure 3 The data shows the percentage of IgG1 in total IgG in samples taken on day 1 (before booster) and day 29 (28 days after booster). Figure 4 The figures show the percentage of IgG4 in total IgG in samples taken on day 1 (before booster) and day 29 (28 days after booster). Figure 5 The figure shows the percentage of total IgG4 in the sample taken on day 29 (28 days after booster). Figure 6The results show the levels of IgG1 and IgG4 in the sample from day 1 (before booster). Figure 7 The results show the levels of IgG1 and IgG4 in the sample taken on day 29 (28 days after booster). Figure 8 The total anti-S glycoprotein IgG in the sample is shown. Figure 9 The total anti-S glycoprotein IgG1 in the sample is shown. Figure 10 The total anti-S glycoprotein IgG4 in the sample is shown.

[0086] Table A shows the percentage of total IgG4 in each sample on day 1 (D1 in the table) and day 29 (D29 in the table). Table B shows the percentage of total IgG1 in each sample on day 1 (D1 in the table) and day 29 (D29 in the table). Table C shows the total anti-S IgG concentration in each sample on day 1 (D1 in the table) and day 29 (D29 in the table). Table D shows the total anti-S IgG1 concentration in each sample on day 1 (D1 in the table) and day 29 (D29 in the table). Table E shows the total anti-S IgG concentration in each sample on day 1 (D1 in the table) and day 29 (D29 in the table).

[0087] Table A

[0088] Table B

[0089] Table C

[0090] Table D

[0091] Table E

[0092] Total anti-S IgG and IgG1 levels were similar after three doses of homologous mRNA or NVX-CoV2373, but NVX-CoV2373 induced slightly higher levels, and a fourth dose of NVX-CoV2373 resulted in an increased response in each group. Figure 19A Compared to recipients of previous mRNA vaccines, anti-S IgG3 levels were significantly higher (>10-fold) after 3 or 4 doses of homologous NVX-CoV2373. In contrast, much higher anti-S IgG4 levels (>75-fold) were observed after repeat mRNA vaccination, but this was not observed after 3 or 4 doses of homologous NVX-CoV2373. Figure 19AThe fourth dose of NVX-CoV2373 also appeared to enhance surrogate signals for ADCP, ADCC, and ADCD activity in recipients of previous mRNA vaccines, but the effect was greater after the fourth homologous dose of NVX-CoV2373. Figure 19B ).

[0093] Data show that the NVX-CoV2373 rS protein vaccine does not appear to induce a significant increase in IgG4, nor does it attenuate the Fcγ-dependent effector response observed with the mRNA vaccine, even after multiple exposures. Instead, NVX-CoV2373 promotes a proportional increase in IgG3 (potentially the most potent SARS-CoV-2 neutralizing antibody subclass) and enhances the activity of alternative ADCP, ADCD, and ADCC.

[0094] Data showed that this assay could detect IgG4 class switching in samples from patients who received COVID-19 vaccines. Specifically, the data showed that serum samples from patients who received vaccines containing mRNA encoding the SARS-CoV-2 S glycoprotein had significantly lower levels of IgG4 compared to serum samples from patients who received vaccines containing the SARS-CoV-2 S glycoprotein.

[0095] Example 2: Measurement of serum anti-rS protein IgG against SARS-CoV-2 in original and variant strains Objective: To develop an anti-rS protein I IgG assay to quantify vaccine response and establish the correlation between such response and protective efficacy against SARS-CoV-2 variants. The accuracy, specificity, linearity, and other validation parameters of the assay, as well as its correlation with pseudovirus neutralization, wild-type virus neutralization, and hACE2 binding inhibition assays, were evaluated.

[0096] Samples: Serum samples from healthy individuals prior to the COVID-19 pandemic (collected between 2016 and 2018) were obtained from BioIVT (Westbury, NY, USA) and Valley Biomedical (Winchester, VA, USA). COVID-19 convalescent serum was obtained from Sanguine BioSciences (Waltham, MA, USA) and BioIVT. Serum samples obtained from the Novavax Clinical Trials Bank were from participants in Phase 1 to 3 trials of the COVID-19 vaccine NVX-CoV2373 (Novavax, Gaithersburg, MD, USA). Positive quality control (QC) samples (COVID-19 convalescent serum bank) with known high or low anti-rS IgG levels were used. Negative controls (NCs) were pre-COVID-19 serum that was negative for anti-rS IgG. QC samples were tested in duplicate wells on the first plate of each run. For correlation analysis, serum samples were from the Novavax Clinical Trial 2019nCoV-311 (NCT05372588). To analyze the conversion to WHO International Units, the following samples were used: high QC (HQC) / low QC (LQC) / NC samples (as described above), the internal reference standard COVID-19 convalescent serum bank, the WHO International Standard (NIBSC code 20 / 136) [10,11], and the WHO reference pool (NIBSC code 20 / 268). The reference pool contained five different pooled samples, ranging from high to low, and all were negative for antibody titers.

[0097] Assay procedure: Each sample was subjected to anti-spike IgG4 assay ( Figure 1The assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 0.80 µg / mL SARS-CoV-2 rS protein (produced at Novavax, Inc., Gaithersburg, MD, USA) at 2–8 °C for 15 to 72 hours. For assay development and initial validation, Novavax’s SARS-CoV-2 rS protein was prepared from the full-length (1273 amino acids), wild-type original SARS-CoV-2 S protein based on the Genbank gene sequence MN908947, i.e., nucleotides 21563–25384. The unstable pre-fusion protein was generated by mutating two residues in the furin cleavage site and the CH domain. The recombinant protein was expressed in Sf9 insect cells and purified by chromatography to obtain a homotrimer, which displayed the N-terminal domain and the receptor-binding domain on the top surface, as previously shown [9]. The coated plate was then washed with phosphate-buffered saline (PBST) containing Tween 20 and blocked with blocking buffer (Thermo Fisher Scientific, catalog 37542) for 1 hour. Human serum samples (reference standard, quality control, or test serum) were then added to the wells to bind the anti-rS protein IgG antibody (incubated for 2 hours). The plate was washed again with PBST, and then goat anti-human IgG secondary antibody conjugated with horseradish peroxidase (HRP) (Southern Biotech, catalog 2040-05) was added and incubated at room temperature for 1 hour. A final wash was performed, followed by the addition of 3,305,50-tetramethylbenzidine substrate (TMB, Sigma, catalog T00440-1L). After 10 minutes, the reaction was terminated with TMB stop solution (Scytek Laboratories, catalog TSB999). The optical density (OD) of the chromogenic signal is proportional to the amount of anti-rS IgG captured on the plate, providing a quantifiable measurement of the concentration of rS-specific IgG in serum samples.

[0098] Validation of Assay (Accuracy): Six distinct assay runs were conducted by three different analysts over two different days, with each run testing 27 samples twice (in duplicate). The geometric mean concentration (GMC) for each sample across the six runs was calculated. Accuracy was then estimated using component analysis of variance, with analysts and days as random effects and samples as fixed effects, by calculating the percentage geometric coefficient of variation (%GCV). Acceptance criteria for accuracy were that at least 80% of samples should have a %GCV ≤ 20%, while samples at or near the lower limit of quantitation (LLoQ) were acceptable with a %GCV ≤ 25%.

[0099] Validation assay (selective): Forty samples collected prior to the COVID-19 outbreak (and therefore expected to be negative for SARS-CoV-2 specific antibodies) were tested in an IgG assay. The concentrations in these samples were expected to be below LLoQ.

[0100] Validation assay (specificity): To confirm the specificity of anti-rS IgG detection, homologous antigen competition was assessed by incubating anti-rS IgG positive samples with varying amounts of SARS-CoV-2 rS at room temperature for 1 hour prior to testing. Controls were samples incubated only with the assay buffer. Acceptance criteria were that homologous protein incubation should reduce the detected IgG concentration by at least 50% in at least 80% of the test samples. To assess the potential for cross-reactivity with other β-coronavirus S proteins, samples were incubated with S proteins from SARS-CoV-1 and MERS-CoV. Samples were also incubated with irrelevant proteins—4 μg / mL of respiratory syncytial virus fusion protein (RSV F) and 4 HA units of influenza virus hemagglutinin (HA) (A / Kansas / 14 / 2017 virus-like particles [VLP]). These relevant and irrelevant viral proteins are similar to SARS-CoV-2 rS expressed in Sf9 insect cells and purified using conventionally parallel chromatographic methods. The target outcome was that incubation with irrelevant proteins should not reduce the detected IgG concentration (reduction of -20%) in at least 80% of the tested samples. Furthermore, in the anti-rS IgG assay, pre-COVID-19 samples from 5 participants vaccinated with RSV (Novavax clinical trial RSV-M-301, NCT02624947) and 5 participants vaccinated with influenza (Novavax clinical trial qNIV-E-301, NCT04120194) were tested. The target outcome was that vaccination with RSV or influenza using antigens produced on the same vaccine platform should not cause a detectable change in anti-rS IgG, and that anti-rS IgG levels in post-immunization samples should be lower despite a strong response to other highly immunogenic respiratory virus proteins. The percentage reduction in IgG concentration (%inhibition) was calculated for all specific results as follows: Verification assay (matrix effect): To evaluate the impact of hemolysis on the assay, 100% hemolyzed human blood (BioIVT BRH1369895) was spiked into 6 samples and the negative control to produce samples with 50% or 25% hemolysis (to represent severe hemolysis). After testing in the IgG assay, the percentage recovery was calculated (result of the hemolyzed sample divided by the result of the non-hemolyzed sample). The acceptance criteria were that the percentage recovery was between 80% and 120% of the reference value and the NC should remain < LLoQ. The same method was used to evaluate the impact of hyperlipidemia by spiking hyperlipidemic serum with a high level of triglycerides (BioIVT BRH1119533, triglycerides 1473 mg / dL) into the samples. The final concentration of triglycerides was 500 mg / dL or 250 mg / dL (normal level, < 150 mg / dL). The samples were then used in the IgG assay and the percentage recovery was calculated. The acceptable range was that the recovery was between 80% and 120% of the reference value and the NC was below LLoQ.

[0101] Verification assay (linearity): Two Phase 1 trial samples with high anti-rS IgG levels were tested in the assay undiluted and in a 1:2 dilution series (6 assay runs). The precision at each dilution point was calculated and a linear regression was performed on the observed and expected GMC. The expected EU / mL at each dilution was calculated by dividing the total GMC of all runs at the minimum dilution by the dilution factor for each dilution of each sample. The observed anti-rS protein IgG EU / mL at each dilution was the total GMC of each sample at each dilution over all runs. To evaluate the ability of the assay to return values that accurately reflect the pure sample, the % relative deviation at each dilution point was calculated as follows: Verification assay (sensitivity): In the linearity analysis, the lowest IgG level values accurately and precisely determined (as above) were evaluated for 2 linearity assessment samples. The LLoQ used for the assay was set at 200 EU / mL during assay qualification and confirmed during validation.

[0102] Verification assay (assay robustness (incubation time and plate coating time)): Incubation time robustness - The assay was performed on 18 samples using the following lower and upper limits for each incubation step: plate coating time (lower and upper limits were 15 hours and 72 hours respectively), plate blocking time (60 minutes and 90 minutes), sample incubation on the plate (110 minutes and 130 minutes), secondary antibody HRP on the plate (50 minutes and 70 minutes), TMB incubation on the plate (8 minutes and 12 minutes). The assay results were then compared to the precision analysis runs performed under reference conditions. The acceptance criteria were that ≥ 80% of the samples should have values within the range of 80% - 120% of the reference value. The percentage recovery was calculated as follows: Validation assay (sample stability): Samples were stored at different temperatures: 6 hours, 24 hours, or 48 hours at room temperature; 6 days or 7 days at 2–8°C; 29 days at -20±10°C; and 6 months or 24 months at -80±10°C. The samples were then tested in the assay, and the results were compared to those obtained under reference conditions for the precision assay run. After 3, 6, 7, or 8 freeze / thaw cycles (1 hour at room temperature, then refreeze), the samples were tested, and the results were compared to those obtained from the precision run (1 freeze / thaw cycle only). Percentage recoveries were calculated as shown above; acceptable recoveries were between 80% and 120% of the reference values.

[0103] Validation assay: This assay is also applicable to the Beta, Delta, Omicron BA.1, Omicron BA.5 and OmicronXBB.1.5 variants. Figure 1 The assay method described herein is the same, except that the original S protein used to coat the plate is replaced with a related variant sequence protein. Follow as described above. Figure 1 A similar verification process described in [the document].

[0104] Correlation analysis: Each sample was evaluated in the anti-rS IgG assay described here. The same samples were also tested in wild-type virus neutralization, pseudovirus neutralization, and hACE2 binding inhibition assays.

[0105] Results: The IgG assay was accurate, robust, linear, and specific for the SARS-CoV-2 S protein, and could be used for both the original strain and variants (tested for Beta, Delta, and Omicron BA.1 / BA.5 / XBB.1.5). For all tested variants ranging from the original virus to XBB.1.5, the results of the IgG assay were significantly correlated with pseudovirus / live wild-type virus neutralization and hACE2 binding inhibition assays.

[0106] exist Figure 11 The results show that for all 27 serum samples representing all concentration ranges (low, medium, and high), the inter-assay, intra-assay, and total precision were <20% GCV. Assay plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 0.80 µg / mL SARS-CoV-2 rS protein (manufactured at Novavax, Inc., Gaithersburg, MD, USA) at 2–8°C for 15–72 hours.

[0107] The acceptance criterion for the determination of selectivity is that the IgG level of 80% of the samples meets the requirement that it is lower than the LLoQ. Among 40 serum samples collected before the COVID-19 pandemic (collected from 2016 to 2018), the anti-rS IgG levels of 35 samples were lower than the LLoQ of 200 EU / mL (Table S1). It is known that some of these samples had high influenza hemagglutination inhibition assay titers (data not shown). Therefore, the IgG assay is not affected by the presence of antibodies against other common respiratory pathogens.

[0108] The assay specificity also meets the acceptance criteria. When 6 samples (from a phase 1 trial) were incubated with the SARS-CoV-2 rS protein, IgG detection in all 6 samples was strongly inhibited by >50%, indicating that the assay is specific for the SARS-CoV-2 rS protein (Table 2). When the same 6 samples were incubated with the rS proteins of MERS-CoV and SARS-CoV-1, the phase 1 trial sera showed much less inhibition of the IgG signal, indicating little cross-reactivity with MERS-CoV and SARS-CoV-1 in this assay. We did not test cross-reactivity with the spike proteins of HCoV-OC34 or HKU1 sequences, but the sequence homology of these proteins with the SARS-CoV-2 spike protein is significantly lower than that of SARS-CoV-1 and MERS-CoV, and has been shown to produce only minimal levels of cross-reactivity (16). Here, although there is widespread seropositivity to endemic seasonal coronaviruses, there is no significant signal >LLoQ in pre-pandemic sera, which is consistent with minimal cross-reactivity.

[0109] The same 6 samples were also incubated with unrelated proteins (RSV F protein or influenza virus-like particles [VLP]) produced on the same vaccine platform, as Figure 12 shown. In clinical trial samples, for RSV incubation, 5 out of 6 samples (83.6%) and for influenza VLP, 6 out of 6 samples (100%), the unrelated proteins did not significantly inhibit IgG detection (defined as >20%). In addition, among 5 participants vaccinated with RSV vaccine and 5 participants vaccinated with influenza vaccine, the anti-rS IgG levels (using immunogens produced on the same insect cell platform) did not change in post-vaccination samples compared to pre-vaccination samples (Table S2). One individual vaccinated with RSV vaccine showed an increase in anti-rS IgG to 2.26-fold during immunization, but the increase was from a <LLoQ value to slightly above the LLoQ value. Free hemoglobin and lipemic matrices had minimal effects on the assay, as Figure 13 shown.

[0110] The linearity of the assay was demonstrated, with an R2 value of 0.9998 for 2 test samples, as Figure 14A and Figure 14B as shown. Based on the lowest concentration values that could be accurately and precisely detected in these two samples, the LLoQ was designated as 200 EU / mL (and the precision data for samples with anti-rS levels close to 200 EU / mL Figure 11 support this choice). Based on the Phase 1 samples, the upper limit of quantification (ULoQ) was temporarily designated as at least 206,767 EU / mL. However, based on 5 serum samples from the Phase 2 and 3 trials of the COVID vaccine NVX-CoV2373, the ULoQ is currently estimated to be at least 2,904,275 EU / mL.

[0111] The stability of the samples was tested at different storage temperatures (room temperature, refrigerated [2 - 8°C], and frozen [-20°C or -80°C]). Figure 15 also shows that the samples were evaluated after multiple freeze / thaw cycles. The samples were stable after 8 freeze / thaw cycles and after 2 years of storage in an -80°C freezer, as shown by recoveries within the acceptable recovery range of 80% - 120% of the reference.

[0112] The results of the assay validation parameters for the Beta, Delta, and Omicron BA.1 / BA.5 / XBB.1.5 variants were similar to those of the original strain. As Figure 16A shown, for the variants compared to the original strain, the control samples performed equally well. For the Beta variant, for 18 out of 20 samples (90%), the GCV for inter-assay, intra-assay, and total precision was <20%. Selectivity was demonstrated as 36 out of 40 pre-COVID-19 samples (90.0%) showed results <LLoQ in the assay ( Figure 16B) Specificity was also demonstrated, as 8 out of 8 samples showed a >50% decrease in IgG detection when incubated with the homologous S protein. SARS-CoV-1 and MERS-CoV S proteins decreased IgG detection in 5 out of 8 samples and 3 out of 8 samples, respectively. RSV F protein decreased IgG detection in 3 out of 8 samples; influenza hemagglutinin did not decrease IgG detection in any samples. In 5 participants vaccinated with RSV vaccine and 5 participants vaccinated with influenza vaccine, no post-vaccination samples showed a change in anti-rS IgG detection compared to pre-vaccination. Linearity was successfully demonstrated (R2 = 0.9999 and 0.9996), the LLoQ was defined as 200 EU / mL and the ULoQ was determined to be at least 490,731 EU / mL. For the Delta variant, 19 out of 20 samples (95%) had inter-assay, intra-assay and total precision % GCV < 20%. In 40 pre-COVID-19 serum samples, 33 samples (82.5%) showed results < LLoQ in the assay. In 7 samples, when incubated with the homologous S protein (original protein, Beta and Delta proteins respectively), 6, 3 and 7 samples showed a >50% decrease in IgG detection Figure 16C ) SARS-CoV-1 and MERS-CoV S proteins decreased IgG detection in 2 out of 7 samples and 1 out of 8 samples, respectively. RSV F protein did not decrease IgG detection in any samples; influenza hemagglutinin decreased IgG detection in 1 out of 7 samples. In 5 participants vaccinated with influenza vaccine, no post-vaccination samples showed a change in anti-rSIgG compared to pre-vaccination. In 5 participants vaccinated with RSV vaccine, only 1 participant showed a change in anti-rS IgG level. Linearity was successfully demonstrated (R2 = 0.9999 and 0.9998), the LLoQ was defined as 200 EU / mL, and the ULoQ was determined to be at least 501,789 EU / mL. For Omicron BA.1, for 20 out of 21 samples (95.2%), the inter-assay, intra-assay and total precision GCV < 20%. In 40 pre-COVID-19 serum samples, 31 samples (77.5%) showed results < LLoQ. In 8 samples, when incubated with the homologous S protein, 7 to 8 samples showed a >50% decrease in IgG detection. In 6 out of 8 samples (75%), RSV F protein did not decrease IgG detection Figure 16D)。In 7 out of 8 samples (87.5%), influenza hemagglutinin did not reduce the IgG test results. Among the 5 participants vaccinated with influenza vaccine, there was no change in anti-rS IgG in the post-vaccination samples compared to before vaccination. Among the 5 participants vaccinated with RSV vaccine, only 1 participant showed a change in anti-rS IgG level. The linearity was successfully demonstrated (R2 = 0.986 and 0.966), the LLoQ was defined as 200 EU / mL, and the ULoQ was determined to be at least 391,124 EU / mL. Since the recoveries of 17 out of 21 samples (81.0%) were within 80% - 120% of the baseline value, the robustness of the plate coating time was demonstrated. For Omicron BA.5, all 21 samples had inter-assay, intra-assay, and total precision % GCV < 20%. Among the 40 pre-COVID-19 serum samples, 34 samples (85%) showed results < LLoQ. Among 8 samples, when incubated with the SARS-CoV-2 S protein, 6 to 8 samples showed a > 50% reduction in IgG test results (7 / 8 for Omicron BA.5, 6 - 7 / 8 for Omicron BA.1, and 6 / 8 for the original strain)( Figure 16E )。In 7 out of 8 samples (87.5%), RSV F protein did not reduce the IgG test results. Ebola glycoprotein did not reduce the IgG test results in any of the samples. The linearity was successfully demonstrated (R2 = 0.9988 and 0.9991). The LLoQ was defined as 200 EU / mL, and the ULoQ was determined to be at least 990,591 EU / mL. Since 17 out of 21 samples (81.0%) were within 80% - 120% of the baseline value, the robustness of the plate coating time was demonstrated. For Omicron XBB.1.5, all 21 serum samples had inter-assay, intra-assay, and total precision % GCV < 20%. Among the 40 pre-COVID-19 samples, 33 samples (82.5%) showed results < LLoQ. Among 8 test samples, when incubated with the SARS-CoV-2 S protein (original strain, Omicron XBB.1.5, or Omicron BA.5), all samples showed a > 50% reduction in IgG test results ( Figure 16FIn 7 out of 8 samples (87.5%), incubation with RSV F protein did not decrease IgG detection results, and incubation with Ebola glycoprotein did not decrease IgG detection results in any of the 8 samples. Linearity was successfully demonstrated (R² = 0.9985 and 0.9991). LLoQ was defined as 200 EU / mL, and ULoQ was determined to be at least 682,680 EU / mL. Robustness of the plate coating time was demonstrated as recoveries in all 21 test samples were within 80%–120% of baseline values.

[0113] Natural SARS-CoV-2 infection also induces anti-rS IgG as part of the body's immune response. Anti-rS IgG levels in serum from seropositive and seronegative subjects were evaluated using anti-rS IgG assays for the original, Omicron BA.1, and Omicron BA.5 strains, subsequently correlated with neutralizing antibody levels (micro-neutralization assays based on live virus or neutralization assays based on pseudovirus) and hACE2 binding inhibition assays. For seronegative samples, the IgG assay results for the original strain were compared with neutralization assays from live wild-type virus (R² = 0.73, Pearson r = 0.853, p < 0.0001). Figure 17A hACE2 binding inhibition assay (R2=0.874, Pearson r=0.935, p<0.0001) Figure 17B And the neutralization assay based on pseudoviruses (R2=0.857, Pearson r=0.926, p<0.0001) Figure 17C The results were significantly correlated with those of the OmicronBA.1 IgG assay and the results of the live wild-type virus neutralization assay (R²=0.48, Pearson r=0.695, p<0.0001). Figure 17D IgG assay results for Omicron BA5 were compared with hACE2 binding inhibition assay results (R²=0.825, Pearson r=0.906, p<0.0001). Figure 17E () and neutralization assay based on pseudovirus (R2=0.659, Pearson r=0.812, p<0.0001) Figure 17F The results were significantly correlated. Similarly, for seropositive samples, the IgG assay results of the original strain were significantly correlated with the neutralization assay from live wild-type virus (R²=0.56, Pearson r=0.746, p<0.0001). Figure 18A hACE2 binding inhibition assay (R2=0.769, Pearson r=0.877, p<0.0001) Figure 18B And the neutralization assay based on pseudoviruses (R2=0.768, Pearson r=0.876, p<0.0001) Figure 18C The results were significantly correlated with those of the Omicron BA.1 IgG assay and the results of the live wild-type virus neutralization assay (R²=0.60, Pearson r=0.778, p<0.0001). Figure 18D IgG assay results for Omicron BA5 were compared with hACE2 binding inhibition assay results (R²=0.741, Pearson r=0.861, p<0.0001). Figure 18E () and neutralization assay based on pseudovirus (R2=0.686, Pearson r=0.828, p<0.0001) Figure 18F The results were significantly correlated.

[0114] After reading this specification, various modifications, equivalent processes, and numerous methods applicable to the invention will be apparent to those skilled in the art to which this invention pertains. The claims are intended to cover such modifications and methods.

[0115] As described above, this invention is applicable to identifying whether biological samples contain antibodies against the SARS-CoV-2 S glycoprotein. Therefore, this invention should not be considered limited to the specific embodiments described above, but should be understood to cover all aspects of the invention as set forth in the appended claims.

[0116] The implementation schemes listed in this disclosure The following are specific examples of implementation schemes. <1> to <20> For illustrative purposes only and do not further limit the scope of the subject matter disclosed by the claims. These enumerated embodiments cover all combinations, sub-combinations, and multiple references (e.g., multiple dependencies) described herein.

[0117] 1. A method for determining whether a biological sample contains an antibody that binds to the SARS-CoV-2 spike (S) glycoprotein, the method comprising: (i) Provide a surface coated with SARS-CoV-2 S glycoprotein; (ii) Exposing the surface to the biological sample; (iii) Exposing the surface to the secondary antibody; and (iv) Detect the secondary antibody bound to the surface; If the secondary antibody is detected, the biological sample contains an antibody that binds to the SARS-CoV-2 S glycoprotein.

[0118] 2. The method as described in embodiment 1, wherein the SARS-CoV-2 S glycoprotein is associated with SEQ ID NO. NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283, 284, 287, 288, 291, 292 and 29 The polypeptides of any one of 4, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236, 328, 329, 330, 331, 332, and 333 have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0119] 3. The method of any one of the listed embodiments 1-2, wherein the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site.

[0120] 4. The method of embodiment 3 as listed, wherein the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0121] 5. The method of embodiment 4 as listed, wherein amino acids 973 and 974 of the SARS-CoV-2 S protein are proline, compared to the wild-type SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 2.

[0122] 6. The method of any one of the listed embodiments 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG1.

[0123] 7. The method of any one of the listed embodiments 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG2.

[0124] 8. The method of any one of the listed embodiments 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG3.

[0125] 9. The method of any one of the listed embodiments 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG4.

[0126] 10. The method of any one of the listed embodiments 1-5, wherein the secondary antibody is selected from anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody and anti-human IgG4 antibody.

[0127] 11. The method of any one of the listed embodiments 1-10, wherein the SARS-CoV-2 S glycoprotein is selected from SARS-CoV-2 virus or SARS-CoV-2 variant or subvariant of SARS-CoV-2 variant.

[0128] 12. The method as described in embodiment 11, wherein the SARS-CoV-2 variant is B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; or B.1.1.529 SARS-CoV-2 strain.

[0129] 13. The method of embodiment 11 listed herein, wherein the SARS-CoV-2 variant is a subvariant of the following strains: B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; or B.1.1.529 SARS-CoV-2 strain.

[0130] 14. The method of any one of the listed embodiments 1-13, wherein the SARS-CoV-2 S glycoprotein is associated with SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 15 1, 153, 156, 158, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283, 284, 287, 288, 291, 292 and 29 4. Any one of 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236, 328, 329, 330, 331, 332, and 333 has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0131] 15. The method of any one of the listed embodiments 1-13, wherein the SARS-CoV-2 S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SARS-CoV-2 S omicron variant selected from: BA.1, BA.2.12.1, BA.2, BA.3, BA.4, BA.5, XBB.1.5, XBB.2.3 and XBB.1.16, EG.5.1, JN.1, BQ.1.1, BF.7.

[0132] 16. The method of any one of the listed embodiments 1-15, wherein the secondary antibody is attached to the label.

[0133] 17. The method as described in embodiment 16, wherein the label is horseradish peroxidase.

[0134] 18. The method of any one of the listed embodiments 1-17, wherein the biological sample is serum, plasma, blood, saliva, nasopharyngeal swab, or mucus.

[0135] 19. The method of any one of the listed embodiments 1-18, wherein the biological sample is derived from a patient who has previously had COVID-19.

[0136] 20. The method of any one of the listed embodiments 1-18, wherein the biological sample is derived from a patient who has been administered an immunogenic composition against SARS-CoV-2 virus or a variant thereof.

[0137] 21. The method of any one of the listed embodiments 1-20, wherein the SARS-CoV-2 S glycoprotein comprises a transmembrane domain.

Claims

1. A method for determining whether a biological sample contains an antibody that binds to the SARS-CoV-2 spike (S) glycoprotein, the method comprising: (i) Provide a surface coated with SARS-CoV-2 S glycoprotein; (ii) Exposing the surface to the biological sample; (iii) Expose the surface to the secondary antibody; as well as (iv) Detect the secondary antibody bound to the surface; If the secondary antibody is detected, the biological sample contains an antibody that binds to the SARS-CoV-2 S glycoprotein.

2. The method of claim 1, wherein the SARS-CoV-2 S glycoprotein is associated with SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151 ,153,156,158,174,175,176,181-184,186,188,190,195,217-228,233-236,243,255-264,273-280,283,284,287,288,291,292 and 294, The polypeptide of any one of 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236, 328, 329, 330, 331, 332, and 333 has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

3. The method of any one of claims 1-2, wherein the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site.

4. The method of claim 3, wherein the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site, the site having the amino acid sequence QQAQ (SEQ ID NO: 7).

5. The method of any one of claims 1-4, wherein amino acids 973 and 974 of the SARS-CoV-2 S protein are proline, compared to the wild-type SARS-CoV-2 S glycoprotein having the amino acid sequence SEQ ID NO:

2.

6. The method of any one of claims 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG1.

7. The method of any one of claims 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG2.

8. The method of any one of claims 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG3.

9. The method of any one of claims 1-5, wherein the antibody binding to the SARS-CoV-2 S glycoprotein is IgG4.

10. The method according to any one of claims 1-5, wherein the secondary antibody is selected from anti-human IgG antibody, anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody and anti-human IgG4 antibody.

11. The method of any one of claims 1-10, wherein the SARS-CoV-2 S glycoprotein is selected from the SARS-CoV-2 virus or a SARS-CoV-2 variant or a subvariant of a SARS-CoV-2 variant.

12. The method of claim 11, wherein the SARS-CoV-2 variant is B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; or B.1.1.529 SARS-CoV-2 strain.

13. The method of claim 11, wherein the SARS-CoV-2 variant is a subvariant of the following strains: B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; or B.1.1.529 SARS-CoV-2 strain.

14. The method of any one of claims 1-13, wherein the SARS-CoV-2 S glycoprotein is associated with SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 15 1, 153, 156, 158, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283, 284, 287, 288, 291, 292 and 29 4. Any one of 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236, 328, 329, 330, 331, 332, and 333 has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

15. The method of any one of claims 1-13, wherein the SARS-CoV-2 S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the SARS-CoV-2 S omicron variant selected from: BA.1, BA.2.12.1, BA.2, BA.3, BA.4, BA.5, XBB.1.5, XBB.2.3 and XBB.1.16, EG.5.1, JN.1, BQ.1.1, BF.

7.

16. The method of any one of claims 1-15, wherein the secondary antibody is attached to the label.

17. The method of claim 16, wherein the label is horseradish peroxidase.

18. The method of any one of claims 1-17, wherein the biological sample is serum, plasma, blood, saliva, nasopharyngeal swab, or mucus.

19. The method of any one of claims 1-18, wherein the biological sample is derived from a patient who has previously had COVID-19.

20. The method of any one of claims 1-18, wherein the biological sample is derived from a patient who has been administered an immunogenic composition against SARS-CoV-2 virus or a variant thereof.

21. The method of any one of claims 1-20, wherein the SARS-CoV-2 S glycoprotein comprises a transmembrane domain.

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