ACE2 Inhibition Assay for Evaluation of Vaccine Immunogenicity

Abstract

Assays for measuring the inhibition of the binding between the SARS-CoV-2 spike (S) glycoprotein and hACE2 are disclosed herein. Methods of using this assay to evaluate the effectiveness of COVID-19 vaccines are also provided herein. A novel biomarker of COVID-19 vaccine immunogenicity, inhibition of SARS-CoV-2 spike (S) protein binding to the hACE2 receptor, is provided herein. This assay provides a rapid high-throughput option for evaluating vaccine immunogenicity. Together with other clinical biomarkers, this can provide valuable insights into the correlations of immune escape and protection and enable vaccine development against emerging COVID-19 variants.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority based on U.S. Provisional Patent Application No. 63 / 392,390, filed on July 26, 2022; U.S. Provisional Patent Application No. 63 / 405,653, filed on September 12, 2022; and U.S. Provisional Patent Application No. 63 / 428,991, filed on November 30, 2022. Each of these applications is hereby incorporated by reference in its entirety for all purposes.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (NOVV - 102_03WO_SeqList_ST26.xml; size: 174,555 bytes; created on July 26, 2023) are hereby incorporated by reference in their entirety.

[0003] Field The present disclosure generally relates to a method for identifying whether a biological sample (e.g., serum, blood, plasma) contains an antibody that inhibits the interaction between human angiotensin - converting enzyme 2 (hACE2) and the SARS - CoV - 2 S glycoprotein.

Background Art

[0004] Background Immunogenic biomarkers may ultimately prove to be correlates of protection and are extremely important for vaccine evaluation. Some of the current biomarkers used to evaluate vaccine immunogenicity of COVID-19 vaccines are anti-spike (S) or anti-receptor binding domain (RBD) immunoglobulin G (IgG) antibodies, neutralizing antibody responses, and levels of activated T cells. Each of these markers has limitations. Neither anti-S IgG nor anti-RBD IgG shows the neutralizing function of the antibody. Neutralizing antibodies measured by micro-neutralization assays can block infection but may not be specific or homologous to a particular target. Higher numbers of CD4+ / CD8+ T cells specific for SARS-CoV-2 epitopes may be associated with a reduced severity of infection, but they are more difficult to measure and their role in COVID-19 vaccine immunogenicity remains unclear. Furthermore, emerging variants of SARS-CoV-2, such as Omicron, show differences in reliability as correlates of protection. There is a need for improved assays to measure vaccine immunogenicity while overcoming the limitations of current biomarkers.

Summary of the Invention

Means for Solving the Problems

[0005] Summary of the Invention A new biomarker for COVID-19 vaccine immunogenicity, inhibition of SARS-CoV-2 spike (S) protein binding to the hACE2 receptor, is provided herein. This assay provides a rapid high-throughput option for evaluating vaccine immunogenicity. Together with other clinical biomarkers, this can provide valuable insights into immune escape and correlates of protection and enable vaccine development against emerging COVID-19 variants.

[0006] An assay for measuring inhibition of hACE2 binding as a biomarker of COVID-19 vaccine immunogenicity is also provided herein.

[0007] A method for determining whether a biological sample contains an antibody that inhibits the binding of the SARS-CoV-2 S glycoprotein to human angiotensin-converting enzyme 2 (hACE2), comprising: (a) measuring the binding of the SARS-CoV-2 S glycoprotein to hACE2 after exposure to the biological sample by: (i) exposing a surface coated with the SARS-CoV-2 S glycoprotein to the biological sample; (ii) exposing the surface to hACE2; and (iii) detecting the hACE2 bound to the surface; (b) measuring the binding of the SARS-CoV-2 S glycoprotein to hACE2 in the absence of the biological sample of (a) by: (i) exposing a surface coated with the SARS-CoV-2 S glycoprotein to hACE2; and (ii) detecting the hACE2 bound to the surface; and (c) comparing the binding of the SARS-CoV-2 S glycoprotein to hACE2 in (a) and (b), wherein when the amount of hACE2 bound in (a) is lower than the amount of hACE2 bound in (b), the biological sample contains an antibody that inhibits the binding of the SARS-CoV-2 S glycoprotein to hACE2. In embodiments, 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 to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments, the SARS-CoV-2 S glycoprotein has an inactivated furin cleavage site. In embodiments, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 68). In embodiments, 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 an embodiment, 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 to any one of the polypeptides of SEQ ID NO: 3, 5-13, 15, 17-19, 21, and 23-66. In an embodiment, 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 to any one of the polypeptides of SEQ ID NOs: 3-13, 20, and 22-66. In an embodiment, 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 to any one of the polypeptides of SEQ ID NO: 3, 5-13, and 23-66. In an embodiment, the SARS-CoV-2 S glycoprotein is derived from a SARS-CoV-2 virus or a variant of SARS-CoV-2.In an embodiment, the variant of SARS-CoV-2 is a B.1.1.7 SARS-CoV-2 strain; a B.1.351 SARS-CoV-2 strain; a P.1 SARS-CoV-2 strain; a Cal.20C SARS-CoV-2 strain; a B.1.617.2 SARS-CoV-2 strain; a B.1.525 SARS-CoV-2 strain; a B.1.526 SARS-CoV-2 strain; a B.1.617.1 SARS-CoV-2 strain; a C.37 SARS-CoV-2 strain; a B.1.621 SARS-CoV-2 strain; or a B.1.1.529 SARS-CoV-2 strain. In an embodiment, 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 to a SARS-CoV-2 S glycoprotein derived from a SARS-CoV-2 Omicron variant selected from the group consisting of BA.1, BA.2.12.1, BA.2, BA.3, BA.4, BA.5, XBB.1.5, XBB.2.3 and XBB.1.16. In an embodiment, hACE2 is bound to a tag. In an embodiment, the tag is a His tag. In an embodiment, the biological sample is serum, plasma or blood from a patient who had COVID-19 previously. In an embodiment, the biological sample is serum, plasma or blood from a patient to whom an immunogenic composition against the SARS-CoV-2 virus or its variant has been administered. In an embodiment, the SARS-CoV-2 S glycoprotein comprises a transmembrane domain.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0018] DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" can refer to one protein or a mixture of such proteins, and reference to "the method" includes reference to equivalent steps and / or methods known to those of ordinary skill in the art, and the like.

[0019] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise modifies or alters the immune response induced against the immunogen. Modification of the immune response can include an increase or expansion in the specificity of either or both of the antibody and cellular immune responses.

[0020] As used herein, the term "about" or "approximately", when preceding a numerical value, indicates a range of that value plus or minus 10%. For example, "about 100" includes 90 and 110.

[0021] As used herein, the terms "immunogen", "antigen", and "epitope" refer to substances such as proteins that include glycoproteins and peptides capable of eliciting an immune response.

[0022] As used herein, an "immunogenic composition" is a composition that contains an antigen, and administration of the composition to a subject results in the development of a humoral and / or cellular immune response against the antigen in the subject.

[0023] As used herein, the terms "treating", "treatment" and "treatment thereof" refer to an approach for obtaining a beneficial or desired result, e.g., a clinical result. For the purposes of the present disclosure, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; alleviating the symptoms of an infection or disease, or reducing its incidence; or a combination thereof.

[0024] "Prevention", as used herein, is used interchangeably with "prophylaxis" and can mean the complete prevention of an infection or disease, or the prevention of the onset of symptoms thereof; a delay in the onset of an infection or disease or its symptoms; or a decrease in the severity of an infection or disease or its symptoms that subsequently develop.

[0025] As used herein, the term "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. The effective dose or effective amount can be determined, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA) or micro-neutralization assay, for example, by measuring the amount of neutralizing secreted antibodies and / or neutralizing serum antibodies.

[0026] As used herein, the term "vaccine" refers to an immunogenic composition, e.g., an immunogen derived from a pathogen that is used to induce an immune response against the pathogen that provides a protective immunity (e.g., an immunity that protects a subject against infection by the pathogen and / or reduces the severity of a disease or condition caused by infection by the pathogen). The protective immune response can include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" can also refer to a suspension or solution of an immunogen that is administered to a subject to generate a protective immunity.

[0027] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject can be an adult, a teenager, a child (2 to 14 years old), an infant (birth to 2 years old) or a neonate (up to 2 months old). In certain embodiments, the subject is up to 4 months old or up to 6 months old. In an embodiment, an adult is an elderly person about 65 years old or older or about 60 years old or older. In an embodiment, the subject is a pregnant female or a female intending to become pregnant. In other embodiments, the subject is not a human; for example, a non-human primate; for example, a baboon, a chimpanzee, a gorilla or a macaque. In certain embodiments, the subject can be a pet, such as a dog or a cat.

[0028] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the United States Federal or State government or listed in the United States Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia with respect to use in mammals, more specifically humans. These compositions can be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0029] As used herein, the term "modification", when referring to a CoV S polypeptide, refers to a mutation, deletion or addition of one amino acid of the CoV S polypeptide. The position of the modification within the CoV S polypeptide can be determined based on aligning the polypeptide sequence to SEQ ID NO: 1 (CoV S polypeptide containing a signal peptide) or SEQ ID NO: 2 (mature CoV S polypeptide lacking a signal peptide).

[0030] The term "variant" of SARS-CoV-2, which is used interchangeably with "heterologous SARS-CoV-2 strain" in this specification, refers to a SARS-CoV-2 virus comprising a CoV S polypeptide having one or more modifications as compared to the SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. For example, a SARS-CoV-2 variant can have at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 modifications as compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. For example, a SARS-CoV-2 variant can have at least one and up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, up to 26, up to 27, up to 28, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35 modifications, up to 40 modifications, up to 45 modifications, up to 50 modifications, up to 55 modifications, up to 60 modifications, up to 65 modifications, up to 70 modifications, up to 75 modifications, up to 80 modifications, up to 85 modifications, up to 90 modifications, up to 95 modifications, or up to 100 modifications as compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.In an embodiment, the SARS-CoV-2 variant can have from about 2 to about 35 modifications, from about 5 to about 10 modifications, from about 5 to about 20 modifications, from about 10 to about 20 modifications, from about 15 to about 25 modifications, from about 20 to about 30 modifications, from about 20 to about 40 modifications, from about 25 to about 45 modifications, from about 25 to about 100 modifications, from about 25 to about 45 modifications, from about 35 to about 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0031] In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 70% and about 99.9% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 70% and about 99.5% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 90% and about 99.9% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 90% and about 99.8% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 95% and about 99.9% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 95% and about 99.8% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity between about 95% and about 99% to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.In an embodiment, the heterologous SARS-CoV-2 strains have a PANGO lineage selected from the group consisting of B.1.1.529; BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621 or B.1.621.1. The following document describes the Pango lineage names and is hereby incorporated by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0032] In embodiments, the heterologous SARS-CoV-2 strain has the World Health Organization label of Omicron. In embodiments, the heterologous SARS-CoV-2 strain having the World Health Organization label of Omicron has at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strain having the World Health Organization label of Omicron has 35-55, 35-65, 35-75, 35-85, 35-95 or 35-105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In embodiments, the modifications are T6I, T6R, A14S, A54V, V70A, T82I, G129D, H133Q, K134E, W139R, E143G, F144L, Q170E, I197V, L199I, V200E, V200G, G239V, G244S, G326D, G326H, R333T, L355I, S358F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, V432P, G433S, L439R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F477S, Q480R, G483S, Q485R, N488Y, Y492H, T534K, T591I, D601G, G626V, H642Y, N645S, N666K, P668H, S691L, N751K, D783Y, N843K, Q941H, N956K, L968F, D1186N, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215, and combinations thereof selected from the group consisting of.

[0033] In embodiments, the variant CoV S polypeptide is (i) A54V, T82I, G129D, L199I, G326D, S358L, S360P, S362F, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215; (ii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (iii) T6R, A14S, T82I, G129D, E143G, L199I, G326D, S358L, S360P, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215; (iv) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, K404N, N427K, L439Q, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, S691L, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (v) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (vi) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, D601G, H642Y, N645S, N666K, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (vii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, G626V, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (viii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (ix) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (x) T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, N447K, S464N, T465K, E471A, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (xi) T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447K, S464N, T465K, E471A, F473S, Q485R, N488Y, Y492H, T591I, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, D1186N, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (xii) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N645S, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xiii) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xiv) T6I, A14S, V70A, G129D, H133Q, Q170E, V200E, G239V, G326H, R333T, L355I, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, and deletion of amino acid 131; (xv) T6I, A14S, G129D, H133Q, Q170E, V200E, G326H, R333T, L355I, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131; (xvi) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xvii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; and (xviii) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131; (xix) deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131, N488Y, A557D, D601G, P668H or P668R, T703I, S969A and D1105H; (xx) D67A, K404N, E471K, N488Y, D601G and A688V; (xxi) D67A, D202G, L229H, K404N, E471K, N488Y, D601G and A688V; (xxii) D67A, D202G, deletion of one, two or three amino acids among amino acids 228 - 230, K404N, E471K, N488Y, D601G and A688V; (xxiii) D67A, L229H, R233I, N488Y, K404N, E471K, D601G and A688V; (xxiv) L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I and V1163F; (xxv) W139C and L439; (xxvi) deletion of amino acid 144, deletion of amino acid 145, T6R, E143G, L439R, T465K, D601G, P668R and D937N; (xxvii) deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, L439R, T465K, D601G, P668R and D937N; (xxviii) Deletion of amino acid 144, deletion of amino acid 145, T6R, T82I, G129D, Y132H, E143G, A209V, K404N L439R, T465K, D601G, P668R and D937N; (xxix) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R and D937N; (xxx) Deletion of amino acid 144, deletion of amino acid 145, T6R, W51H, H53W, G129D, E143G, D200V, L201R, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R and D937N; (xxxi) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, K404N, L439R, T465K, E471Q, D601G, P668R and D937N; (xxxii) Q39R, A54V, E471K; D601G, Q664H, F875L, and deletion of 1, 2, 3, or 4 of amino acids 56, 57, 131, 132; (xxxiii) T82I, D240G, E471K, D601G and A688V; (xxxiv) L439R, E471Q, D601G, P668R and Q1058H; (xxxv) G62V, T63I, R233N, L439Q, F477S, D601G, T846N, and deletion of 1, 2, 3, 4, 5, or 6 of amino acids 234 - 240; (xxxvi) T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H and D937N; and (xxxvii) G129D, G326D, S360P, S362F, K404N, N427K, T465K, E471A or E471K, Q480K or Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H and N953K comprising a combination of modifications selected from the group consisting of; The amino acids of the CoV S protein are numbered relative to the polypeptide having the sequence of SEQ ID NO: 2.

[0034] As used herein, the term "NVX-CoV2373" refers to a vaccine composition comprising the BV2373 spike glycoprotein (SEQ ID NO: 3) and fraction A and fraction C iscom matrix (e.g., MATRIX-M™). Method for evaluating the immunogenicity of a vaccine composition against SARS-CoV-2

[0035] A method for determining whether a biological sample contains an antibody that inhibits the binding of the SARS-CoV-2 S glycoprotein to human angiotensin-converting enzyme 2 (hACE2), comprising: (a) determining the binding of the SARS-CoV-2 S glycoprotein to hACE2 after exposure to the biological sample by: (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 hACE2; and (iv) detecting the hACE2 bound to the surface; (b) determining the binding of the SARS-CoV-2 S glycoprotein to hACE2 in the absence of the biological sample of (a) by: (i) providing a surface coated with the SARS-CoV-2 S glycoprotein; (ii) exposing the surface to hACE2; and (iii) detecting the hACE2 bound to the surface; and (c) comparing the binding of the SARS-CoV-2 S glycoprotein to hACE2 in (a) and (b), wherein when the amount of hACE2 bound in (a) is lower than the amount of hACE2 bound in (b), the biological sample contains an antibody that inhibits the binding of the SARS-CoV-2 S glycoprotein to hACE2. In embodiments, step a(ii) is performed before step a(iii). In embodiments, step a(iii) is performed before step a(ii).

[0036] In embodiments, these methods further comprise the step of washing the solid surface. In embodiments, these methods comprise the step of contacting the plate with a blocking buffer. The blocking buffer is a solution that removes the possibility of non-specific binding to the plate.

[0037] In embodiments, these methods can be utilized to detect antibody isotypes selected from the group consisting of IgA, IgG, IgM, IgD, and IgE. SARS-CoV-2 S glycoprotein for use in the method

[0038] In an embodiment, suitable SARS-CoV-2 S glycoproteins for use in the method include SARS-CoV-2 S glycoproteins associated with any one of the following Protein Data Bank (PDB) IDs: 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, 6YZ5, 6YZ7, 6Z2M, 6Z43, 6Z97, 6ZB4, 6ZB5, 6ZBP, 6ZCZ, 6ZDG, 6ZDH, 6ZER, 6ZFO, 6ZGE, 6ZGG, 6ZGH, 6ZGI, 6ZH9, 6ZHD, 6ZLR, 6ZOW, 6ZOX, 6ZOY, 6ZOZ, 6ZP0, 6ZP1, 6ZP2, 6ZP5, 6ZP7, 6ZWV, 6ZXN, 7A25, 7A29, 7A4N, 7A5R, 7A5S, 7A91, 7A92, 7A93, 7A94, 7A95, 7A96, 7A97, 7A98, 7AD1, 7AKD, 7B0B, 7B14, 7B17, 7B18, 7B27, 7B3O, 7B62, 7BEH, 7BEI, 7BEJ, 7BEK, 7BEL, 7BEM, 7BEN, 7BEO, 7BEP, 7BH9, 7BNM, 7BNN, 7BNO, 7BNV, 7BWJ, 7BYR, 7BZ5, 7C01, 7C2L, 7C53, 7C8D, 7C8J, 7C8V, 7C8W, 7CAB, 7CAC, 7CAH, 7CAI, 7CAK, 7CAN, 7CDI, 7CDJ, 7CH4, 7CH5, 7CHB, 7CHC, 7CHE, 7CHF, 7CHH, 7CHO, 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, 7KJ47KJ5, 7KKK, 7KKL, 7KL9, 7KLG, 7KLH, 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, 7NKT7NLL, 7NP1, 7NS6, 7NT9, 7NTA, 7NTC, 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, 7SN3, 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, 7SY87T01, 7T3M, 7T67, 7T72, 7T7B, 7T9J, 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, 7TLT, 7TLY, 7TM0, 7TN0, 7TNW, 7TO4, 7TOU, 7TOV, 7TOW, 7TOX, 7TOY, 7TOZ, 7TP0, 7TP1, 7TP2, 7TP3, 7TP4, 7TP7, 7TP8, 7TP9, 7TPA, 7TPC, 7TPE, 7TPF, 7TPH, 7TPK, 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, 7UM7WCH, 7WCK, 7WCP, 7WCR, 7WCU, 7WCZ, 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, 7XTZ, 7XU0, 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, 8CSJ, 8CWI, 8CWK, 8CWU, 8CWV, 8CXN, 8CXQ, 8CY6, 8CY7, 8CY9, 8CYA, 8CYB, 8CYC, 8CYD, 8CYJ, 8CZI, 8D0Z, 8D36, 8D47, 8D48, 8D55, 8D56, 8D5A, 8D6Z, 8D8Q, 8D8R, 8DAD, 8DAO, 8DCC, 8DCE, 8DF5, 8DGU, 8DI5, 8DLI, 8DLJ, 8DLK, 8DLL, 8DLM, 8DLN, 8DLO, 8DLP, 8DLQ, 8DLR, 8DLS, 8DLT, 8DLU, 8DLV, 8DLW, 8DLX, 8DLY, 8DLZ, 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, 8EL28ELH, 8ELJ, 8ELO, 8ELP, 8ELQ, 8EOO, 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. The amino acid sequence of the SARS-CoV-2 S glycoprotein associated with each entry can be accessed by downloading the FASTA file associated with the PDB ID at www.rcsb.org.,

[0039] In embodiments, the SARS-CoV-2 S glycoprotein is a wild-type SARS-CoV-2 S glycoprotein or a SARS-CoV-2 S glycoprotein derived from a SARS-CoV-2 variant thereof. In embodiments, the variant of SARS-CoV-2 is the B.1.1.7 SARS-CoV-2 strain; the B.1.351 SARS-CoV-2 strain; the P.1 SARS-CoV-2 strain; the Cal.20C SARS-CoV-2 strain; the B.1.617.2 SARS-CoV-2 strain; the B.1.525 SARS-CoV-2 strain; the B.1.526 SARS-CoV-2 strain; the B.1.617.1 SARS-CoV-2 strain; the C.37 SARS-CoV-2 strain; the B.1.621 SARS-CoV-2 strain; or the B.1.1.529 SARS-CoV-2 strain. In embodiments, the SARS-CoV-2 S glycoprotein includes a transmembrane domain.

[0040] The wild-type SARS-CoV-2 S glycoprotein contains a furin cleavage site RRAR (SEQ ID NO: 67) at positions 669-672 of the SARS-CoV-2 S glycoprotein of 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 NOs: 68-97. In an embodiment, the amino acid sequence of the inactive furin cleavage site is GG. In an embodiment, non-limiting examples of SARS-CoV-2 S glycoproteins having a GG inactive furin cleavage site include the glycoproteins of SEQ ID NOs: 26-28 and 30.

[0041] In an embodiment, the amino acid sequence of the inactive furin cleavage site is QQAQ (SEQ ID NO: 68). In an embodiment, non-limiting examples of SARS-CoV-2 S glycoproteins having a QQAQ (SEQ ID NO: 68) inactive furin cleavage site include the glycoproteins of SEQ ID NOs: 3, 5-13, 15, 17-19, 21, 23-25, 29 and 31-66.

[0042] In an embodiment, one or more of the amino acids constituting the native furin cleavage site are mutated to any natural amino acid. In an embodiment, one or more of the amino acids constituting the native furin cleavage site are deleted.

[0043] In an embodiment, one or more of the amino acids constituting the native furin cleavage site are mutated to glutamine. In an embodiment, one, two, three or four amino acids can be mutated to glutamine. In an embodiment, one of the arginines constituting the native furin cleavage site is mutated to glutamine. In an embodiment, two of the arginines constituting the native furin cleavage site are mutated to glutamine. In an embodiment, three of the arginines constituting the native furin cleavage site are mutated to glutamine.

[0044] In an embodiment, one or more of the amino acids constituting the native furin cleavage site are mutated to alanine. In an embodiment, one, two, three, or four amino acids may be mutated to alanine. In an embodiment, one of the arginines constituting the native furin cleavage site is mutated to alanine. In an embodiment, two of the arginines constituting the native furin cleavage site are mutated to alanine. In an embodiment, three of the arginines constituting the native furin cleavage site are mutated to alanine.

[0045] In an embodiment, one or more of the amino acids constituting the native furin cleavage site are mutated to glycine. In an embodiment, one, two, three, or four amino acids may be mutated to glycine. In an embodiment, one of the arginines of the native furin cleavage site is mutated to glycine. In an embodiment, two of the arginines constituting the native furin cleavage site are mutated to glycine. In an embodiment, three of the arginines constituting the native furin cleavage site are mutated to glycine.

[0046] In an embodiment, one or more of the amino acids constituting the native furin cleavage site are mutated to asparagine. For example, one, two, three, or four amino acids may be mutated to asparagine. In an embodiment, one of the arginines constituting the native furin cleavage site is mutated to asparagine. In an embodiment, two of the arginines constituting the native furin cleavage site are mutated to asparagine. In an embodiment, three of the arginines constituting the native furin cleavage site are mutated to asparagine.

[0047] In an embodiment, the active furin cleavage site (SEQ ID NO: 67) of the SARS-CoV-2 S glycoprotein described herein is replaced with the inactivated furin cleavage site in the following table. Inactivated furin cleavage site [Table A]

[0048] In an 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 an embodiment, Lys-973 is mutated to any natural amino acid. In an embodiment, Lys-973 is mutated to proline. In an embodiment, Lys-973 is mutated to glycine.

[0049] In an 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 an embodiment, Val-974 is mutated to any natural amino acid as compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In an embodiment, Val-974 is mutated to proline. In an embodiment, Val-974 is mutated to glycine as compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2.

[0050] In an embodiment, the SARS-CoV-2 S glycoprotein contains mutations at Lys-973 and Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In an embodiment, Lys-973 and Val-974 are mutated to any natural amino acid as compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In an embodiment, Lys-973 and Val-974 are mutated to proline as compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. Non-limiting examples of the SARS-CoV-2 S glycoprotein in which amino acids Lys-973 and Val-974 are mutated to proline include the glycoproteins of SEQ ID NOs: 3-13, 20, and 22-60.

[0051] In an embodiment, 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 to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0052] In an embodiment, 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 to any one polypeptide of SEQ ID NOs: 3-66.

[0053] Providing a surface coated with the SARS-CoV-2 S glycoprotein In an embodiment, the method described herein requires providing a surface coated with the SARS-CoV-2 S glycoprotein. In an embodiment, the surface is a chip or a microplate. In an embodiment, the microplate comprises polystyrene. In an embodiment, the microplate is a 96-well or 384-well polystyrene plate. In an embodiment, the surface is a microplate filled with a solution.

[0054] Exposing the surface to a biological sample In an embodiment, the surface is exposed to a biological sample. In an embodiment, the surface is exposed to the biological sample for 10 minutes to about 72 hours. In an embodiment, the surface is exposed to the biological sample for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, or about 72 hours, all values and ranges therebetween being included. In an embodiment, the surface is exposed to the biological sample for about 1 hour or 2 hours. In an embodiment, the surface is exposed to the biological sample at a temperature of 2 - 8°C or 8 - 37°C. In an embodiment, the surface is exposed to the biological sample at a temperature of 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C or about 37°C.

[0055] In an embodiment, the biological sample is saliva, nasopharyngeal swab, sputum, saliva, urine, fecal sample, cerebrospinal fluid, synovial fluid, serum, blood or plasma. In an embodiment, the biological sample is from a patient who previously had COVID-19. In an embodiment, the biological sample is from a patient to whom an immunogenic composition against the SARS-CoV-2 virus or its variant has been administered. Expose the surface to hACE2

[0056] In an embodiment, hACE2 comprises a polypeptide having 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 to the polypeptide of SEQ ID NO: 98 or 99.

[0057] In embodiments, hACE2 is conjugated to a tag. In embodiments, hACE2 is covalently conjugated to a tag. In embodiments, hACE2 is non-covalently conjugated to a tag. In some aspects, the tag is useful for the detection of hACE2 binding. In embodiments, the tag is a His tag. In embodiments, the tag contains an epitope. For example, the tag can be a polyglutamic acid tag, a FLAG-tag, an HA-tag, a polyHis-tag (having about 5 to 10 histidines) (SEQ ID NO: 100), a hexahistidine tag (SEQ ID NO: 101), a 7×-His-tag (having 7 histidines) (SEQ ID NO: 102), an 8×-His-tag (having 8 histidines) (SEQ ID NO: 103), 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 embodiments, 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 embodiments, the protease cleavage site is the HRV3C protease cleavage site.

[0058] In an embodiment, the surface is exposed to hACE2 for 10 minutes to about 72 hours. In an embodiment, the surface is exposed to hACE2 for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, or about 72 hours, with all values and ranges therebetween included.

[0059] Detect hACE2 bound to the surface In embodiments, these methods include the step of detecting hACE2 bound to a surface. In embodiments, hACE2 is detected by contacting the surface with an antibody that binds to hACE2. In embodiments, the surface is contacted with the antibody that binds to hACE2 for from about 10 minutes to about 72 hours. In embodiments, the surface is contacted with the antibody that binds to hACE2 for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, or about 72 hours, all values and ranges therebetween being included.

[0060] In an embodiment, the antibody is an anti-polyhistidine tag antibody. In an embodiment, the antibody is tagged with horseradish peroxidase. In an embodiment, the antibody is tagged with alkaline phosphatase. In an embodiment, the step of detecting hACE2 comprises: (i) contacting the surface with an antibody that binds to hACE2, wherein the antibody is tagged with horseradish peroxidase; and (ii) contacting the surface with a 3,3’,5,5’-tetramethylbenzidine substrate. In an embodiment, the step of detecting comprises determining the absorbance of the surface. In an embodiment, the step of detecting comprises determining the absorbance of the surface at a wavelength of from 400 nm to about 650 nm. In an embodiment, the step of detecting comprises determining the absorbance of the surface at a wavelength 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, wherein all values and ranges therebetween are included. In an embodiment, the step of detecting comprises determining the absorbance of the surface at a wavelength of about 450 nm.

[0061] In embodiments, the methods used herein can be used to evaluate the immunogenicity of compositions and vaccine compositions against SARS-CoV-2. In embodiments, the immunogenic and vaccine compositions target the SARS-CoV-2 virus or heterologous SARS-CoV-2 strains. In embodiments, the immunogenic or vaccine composition comprises the SARS-CoV-2 S glycoprotein, or a nucleic acid (e.g., mRNA) encoding the SARS-CoV-2 S glycoprotein. In embodiments, the immunogenic or vaccine composition comprises a viral vector that expresses the SARS-CoV-2 S glycoprotein. In embodiments, the immunogenic or vaccine composition comprises an SARS-CoV-2 S glycoprotein having a sequence that is 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% identical to SEQ ID NO: 1. In embodiments, the SARS-CoV-2 S glycoprotein comprises a sequence that is 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% identical to SEQ ID NO: 2. In embodiments, the SARS-CoV-2 S glycoprotein comprises a sequence that is at least 90%, 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 to SEQ ID NO: 3. The amino acid sequences of SEQ ID NOs: 1-3 are in the following table.

Table B-1

Table B-2

Table B-3

[0062] In embodiments, the immunogenic composition or vaccine composition comprises an adjuvant. Exemplary adjuvants are described below.

[0063] Aluminum-based adjuvant In embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to alum. For example, the nanoparticles may be bound to alum at least 80%, at least 85%, at least 90% or at least 95%. Often, the nanoparticles are 92% - 97% bound to alum in the composition. The amount of alum is present per dose and is typically in the range of about 400 μg to about 1250 μg. For example, alum can be present in an amount per dose 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. Typically, alum is present at about 400 μg per dose of 120 μg of protein nanoparticles.

[0064] Saponin adjuvant Adjuvants containing saponin can also be combined with the immunogens disclosed herein. Saponin is a glycoside derived from the bark of the Quillaja saponaria Molina tree. Typically, saponin is prepared using a multi-step purification process that yields multiple fractions. As used herein, the term "saponin fraction derived from Quillaja saponaria Molina" is used generically to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

[0065] Saponin fraction Several approaches are suitable for producing saponin fractions. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows. The lipophilic fraction from Quil A, a crude aqueous Quillaja saponaria Molina extract, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semi-preparative HPLC by elution using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is the fraction eluted at approximately 39% acetonitrile or corresponds to such a fraction. The fraction referred to herein as "Fraction B" or "QH-B" is the fraction eluted at approximately 47% acetonitrile or corresponds to such a fraction. The fraction referred to herein as "Fraction C" or "QH-C" is the fraction eluted at approximately 49% acetonitrile or corresponds to such a fraction. Further information regarding the purification of the fractions can be found in U.S. Patent No. 5,057,540. When prepared as described herein, Fractions A, B, and C of Quillaja saponaria Molina each represent a group or family of chemically closely related molecules with definable properties. The chromatographic conditions under which they are obtained are such that the batch-to-batch reproducibility with respect to the elution profile and biological activity is highly consistent.

[0066] Other saponin fractions are described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (lsconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). 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 of EP 0 3632 279 B2, in particular QA-7, QA-17, QA-18 and QA-21, can be used. These are obtained as described in EP 0 3632 279 B2, in particular in Examples 1 on pages 6 as well as 8 and 9.

[0067] The saponin fractions described herein and used to form an adjuvant are often substantially pure fractions; that is, the fractions are substantially free from contamination by other materials. In certain embodiments, a substantially pure saponin fraction can contain up to 40 wt%, up to 30 wt%, up to 25 wt%, up to 20 wt%, up to 15 wt%, up to 10 wt%, up to 7 wt%, up to 5 wt%, up to 2 wt%, up to 1 wt%, up to 0.5 wt% or up to 0.1 wt% of other compounds, such as other saponins or other adjuvant materials.

[0068] ISCOM structure The saponin fraction can be administered in the form of cage-like particles called ISCOM (Immune Stimulating COMplex). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546 B1. In certain embodiments, the transport and / or passenger antigen can be used as described in EP 9600647-3 (PCT / SE97 / 00289).

[0069] Matrix adjuvant In embodiments, the ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, 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 activity) substances, but does not necessarily contain glycosides, and can be produced as described in EP0436620B1, which is hereby incorporated by reference in its entirety.

[0070] In other aspects, the ISCOM is an ISCOM complex. The 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 by surfactant treatment so that a portion of the antigen is incorporated into the particle. In contrast, 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.

[0071] According to one embodiment, the saponin fraction incorporated into an ISCOM matrix complex or an ISCOM complex, or at least one additional adjuvant also incorporated into or mixed with an ISCOM or an ISCOM matrix complex, is fraction A, fraction B or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any purified subfraction, for example, selected from QA 1-21.

[0072] In certain embodiments, each ISCOM particle can contain at least two saponin fractions. Any combination of weight percentages of different saponin fractions can be used. Any combination of weight percentages of any two fractions can be used. For example, the particles can each contain any weight percentage of fraction A and any weight percentage of another saponin fraction, for example, a crude saponin fraction or fraction C. Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle can contain 0.1-99.9 wt%, 5-95 wt%, 10-90 wt%, 15-85 wt%, 20-80 wt%, 25-75 wt%, 30-70 wt%, 35-65 wt%, 40-60 wt%, 45-55 wt%, 40-60 wt%, or 50 wt% of one saponin fraction, for example, fraction A, and the balance up to 100% in each case of another saponin, for example, any crude fraction or any other fraction, for example, fraction C. The weights are calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in U.S. Patent Application Publication No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0073] In certain embodiments, the ISCOM matrix or ISCOM complex comprises from 5 to 99% by weight of one fraction, such as fraction A, and up to 100% by weight of another fraction, such as a crude saponin fraction or fraction C. The weights are calculated as the total weight of the saponin fractions.

[0074] In another embodiment, the ISCOM matrix or ISCOM complex comprises from 40 to 99% by weight of one fraction, such as fraction A, and from 1 to 60% by weight of another fraction, such as a crude saponin fraction or fraction C. The weights are calculated as the total weight of the saponin fractions.

[0075] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises from 70 to 95% by weight of one fraction, such as fraction A, and from 30 to 5% by weight of another fraction, such as a crude saponin fraction or fraction C. The weights are calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA 1 - 21.

[0076] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles can each be formed using only one saponin fraction. The compositions disclosed herein can contain a plurality of particles, each particle containing only one saponin fraction. That is, a particular composition can contain one or more different types of ISCOM - matrix complex particles and / or one or more different types of ISCOM complex particles, each individual particle containing one saponin fraction from Quillaja saponaria Molina, and the saponin fraction in one complex is different from the saponin fraction in other complex particles.

[0077] In certain embodiments, one type of saponin fraction or crude saponin fraction can be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction can be incorporated into another ISCOM matrix complex or particle. The composition or vaccine can include at least two types of complexes or particles, with each type having one type of saponin incorporated into physically distinct particles.

[0078] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles in which one saponin fraction of Quillaja saponaria Molina and another saponin fraction of Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles can be used.

[0079] ISCOM matrix or ISCOM complex particles, each having one saponin fraction, can be present in the composition in any combination of weight percentages. In certain embodiments, the composition can contain an ISCOM matrix or complex containing from 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 to 60 wt%, or 50 wt% of a first saponin fraction, with the remainder being constituted by an ISCOM matrix or complex containing a different saponin fraction. In an embodiment, the remainder is one or more ISCOM matrices or complexes, with each matrix or complex particle containing only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles can contain more than one saponin fraction.

[0080] In certain compositions, 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.

[0081] In embodiments, of the total weight of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant, fraction A of Quillaja Saponaria Molina is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% by weight, and fraction C of Quillaja Saponaria Molina makes up the remainder.

[0082] Preferred compositions comprise a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C, wherein the ISCOM matrix of fraction A constitutes about 70% by weight of the total saponin adjuvant and the ISCOM matrix of fraction C constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the ISCOM matrix of fraction A constitutes about 85% by weight of the total saponin adjuvant and the ISCOM matrix of fraction C constitutes about 15% by weight of the total saponin adjuvant. In another preferred composition, the ISCOM matrix of fraction A constitutes about 92% by weight of the total saponin adjuvant and the ISCOM matrix of fraction C constitutes about 8% by weight of the total saponin adjuvant. Thus, in certain compositions, the ISCOM matrix of fraction A is present in the range of about 70% to about 85% of the total amount by weight of the saponin adjuvant in the composition, and the ISCOM matrix of fraction C is present in the range of about 15% to about 30%. In certain compositions, the ISCOM matrix of fraction A is present in the range of about 70% to about 92% of the total amount by weight of the saponin adjuvant in the composition, and the ISCOM matrix of fraction C is present in the range of about 8% to about 30%. In embodiments, of the total weight of the ISCOM matrix of fraction A and the ISCOM of fraction C in the adjuvant, the ISCOM matrix of fraction A occupies 50 - 96% by weight respectively, and the ISCOM matrix of fraction C occupies the remainder. In a particularly preferred composition herein referred to as MATRIX-M™, the ISCOM matrix of fraction A is present at about 85% and the ISCOM matrix of fraction C is present at about 15% of the total amount by weight of the saponin adjuvant in the composition. MATRIX-M™ may be referred to interchangeably with Matrix-M1.

[0083] Exemplary QS-7 and QS-21 fractions, their production, and their use are described in U.S. Patent Nos. 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.

[0084] In embodiments, other adjuvants can be used additionally, or alternatively. The inclusion of 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, is contemplated to be within the scope of the present disclosure. Other adjuvants include complete Freund's adjuvant (a non-specific stimulant of the immune response containing killed 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 monophosphoryl lipid A (MPL), MF-59, RIBI containing three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / TWEEN® polysorbate 80 emulsion. In embodiments, the adjuvant can be paucilamellar lipid vesicles; for example, NOVASOMES®. NOVASOMES® are paucilamellar non-lipid vesicles in the range of about 100 nm to about 500 nm. These contain BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® have been shown to be effective adjuvants (see U.S. Patent Nos. 5,629,021, 6,387,373, and 4,911,928).

[0085] (Example 1) SARS-CoV-2 Receptor (ACE2) Inhibition Assay: A Rapid High-Throughput Assay Useful for Vaccine Immunogenicity Assessment The inventors have discovered a rapid high-throughput option for assessing vaccine immunogenicity. This assay is a cost-effective and rapid, BSL2-based assay. This assay enables the assessment of vaccine immunogenicity against emerging SARS-CoV-2 variants. This assay can replace the live virus neutralization assay.

[0086] Introduction: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus variants evade protective immunity generated by currently available vaccines, highlighting the need for better clinical immunogenicity biomarkers.

[0087] Methods: One novel biomarker is the inhibition of the interaction between hACE2 and the SARS-CoV-2 S glycoprotein. To measure this biomarker, the inventors developed and validated an enzyme-linked immunosorbent assay (ELISA) assay to measure the inhibition between the SARS-CoV-2 S glycoprotein and hACE2. This assay was validated for accuracy, specificity, and linearity.

[0088] ELISA assay: A 96-well format enzyme-linked immunosorbent assay (ELISA)-based assay was developed to evaluate the inhibition of binding of the prototype / Wuhan strain or variants (Delta, Omicron BA.1 / BA.5) SARS-CoV-2 S-protein (full-length rS protein) to the receptor hACE2 by human sera from clinical trials of the NVX-CoV2373 vaccine. The following SARS-CoV-2 S glycoproteins were evaluated: the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2 (prototype, Wuhan); the SARS-CoV-2 S glycoprotein from the SARS-CoV-2 Delta strain; the SARS-CoV-2 S glycoprotein from the Omicron BA.1 strain; and the SARS-CoV-2 S glycoprotein from the Omicron BA.5 strain. Figure 1 shows a schematic of the assay. A 96-well assay plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated overnight at 2 - 8°C with SARS-CoV-2 rS-protein (produced by Novavax, Inc., Gaithersburg, MD, USA) using standard plate-coating methods. This was then washed with phosphate-buffered saline with Tween® 20 (PBST) and blocked with blocking buffer (Thermo Fisher Scientific) for 1 hour. Diluted serum samples were then added to the plate, followed by washing with PBST and addition of polyhistidine-tagged hACE2. After unbound hACE2 was removed by washing with PBST, the plate was incubated with an anti-polyhistidine-tagged / horse radish peroxidase secondary antibody at room temperature (RT) for 1 hour, then washed with PBST, and then bound hACE2 was detected by incubating with 3,3’,5,5’-tetramethylbenzidine substrate (Thermo Fisher Scientific) for 30 minutes. Since the amount of bound hACE2 that gives a signal is inversely proportional to the amount of hACE2 binding inhibitor in the serum, quantification of the positive 3,3’,5,5’-tetramethylbenzidine reaction signal indicates the level of inhibition of hACE2 binding by serum constituents.The 50% inhibitory titer was calculated using a 4-parameter logistic fit and compared to the hACE2 control.

[0089] Samples: The following samples were evaluated: (i) human serum samples collected in 2018 before the COVID-19 pandemic (n = 13); serum samples collected during the COVID-19 pandemic (n = 26); serum samples from patients who recovered from SARS-CoV-2 infection (convalescent serum, n = 24), or from patients vaccinated with the NVX-CoV2373 vaccine; serum samples from individuals vaccinated with the influenza vaccine; and quality control (QC) samples containing pooled human serum samples that were either positive or negative in the hACE2 binding inhibition assay. The negative control was pre-pandemic serum that was negative for hACE2 binding inhibition in this assay. The QC samples were tested in duplicate on the first plate of each run.

[0090] Verification assay - Precision: Twenty samples were tested twice in assay runs (a total of 6 runs by 2 analysts over 3 days), with each sample tested in duplicate, and the geometric mean titer (GMT) of the duplicate values was considered the inhibitory titer. Precision was then estimated by calculating the percent geometric coefficient of variation (%GCV) based on a variance components analysis using the sample as a fixed effect and the analyst and day as random effects.The target precision was such that at least 80% of the samples had %GCV ≤ 20% and samples at the lower limit of quantification (LLoQ) had %GCV ≤ 25%. The %GCV was calculated using the equation in Figure 2 based on a variance components analysis using the sample as a fixed effect and the analyst and day as random effects. The equation for the overall %GCV is found in Figure 3. The target precision was set such that at least 80% of the samples had %GCV ≤ 20% and samples at the lower limit of quantification (LLOQ) had %GCV ≤ 25%.

[0091] Verification assay - Specificity: The hACE2 binding inhibition-positive serum samples were incubated at RT for approximately 1 hour with the rS protein prior to testing (5 samples). The control used to set the baseline was the same samples incubated with assay buffer only. The unrelated non-specific protein group used the same samples but were incubated with respiratory syncytial virus (RSV) F protein or Ebola glycoprotein (GP) produced using the same recombinant protein platform as for the rS protein. The samples were then tested in this assay and the inhibitory titers were compared to the % reduction calculated as shown in Figure 4.

[0092] Verification assay - Selectivity: Nineteen samples (expected to be negative for SARS-CoV-2 antibodies) collected prior to the COVID-19 pandemic and below the LLoQ were tested for hACE2 binding inhibitory titers. Some samples were also tested for influenza hemagglutination inhibition (HAI) titers to evaluate whether varying levels of HAI titers interfered with the detection of hACE2 binding inhibitory titers.

[0093] Verification assay - Linearity: Two hACE2 binding inhibition-positive samples were tested in this assay undiluted or in a 1:2 dilution series (5 assays by different analysts), the accuracy and precision of the titers were calculated at each dilution point, and linear regression was performed for the observed GMT vs. the predicted GMT. The predicted titer at each dilution was calculated from the overall GMT from all runs of the most dilute sample divided by the dilution factor, and the observed GMT was the overall GMT from all runs. The % relative bias at each dilution point was calculated as shown in Figure 5.

[0094] Verification assay - Sensitivity: The lowest (LLoQ) titer value determined accurately and with high precision was evaluated for two samples in the linearity analysis. The LLoQ for the assay was set at 10 based on previous data accumulated using convalescent sera from COVID-19 cases; the LLoQ was confirmed based on the acceptable GCV obtained for samples and dilutions, with data from both the accuracy and dilution linearity experiments revealed here, and the result was <15.

[0095] Verification assay - Incubation time robustness: The assay was performed for each step using the upper and lower limits of the incubation time, and the results were then compared with six runs (reference conditions) performed for the accuracy analysis. The following time conditions (lower / upper limits) were used: plate coating (14 / 72 hours, reference 18 - 20 hours), plate blocking (60 / 90 minutes, reference 60 minutes), sample incubation (55 / 62 or 65 minutes, reference 60 minutes), hACE2 incubation (55 / 65 minutes, reference 60 minutes), secondary antibody (55 / 65 minutes, reference 60 minutes), TMB incubation (25 / 35 minutes, reference 30 minutes). The goal was that ≥80% of the samples should have values within ±20% of the reference (80 - 120% of the reference value). % Recovery and % Difference were calculated as shown in Figure 6.

[0096] Verification Assay - Sample Stability: Sample stability - Samples were stored at various temperatures (6 hours or 24 hours at RT [n = 16], 7 days or 14 days at 2 - 8°C [n = 15], 9 months or 24 months at -80 ± 10°C [n = 16]), then tested in this assay, and the results were compared to freshly thawed samples (RT / refrigerated) or original accuracy results (freezer). Samples were thawed at RT and then placed in a 24 ± 2°C incubator for 6 hours or 24 hours. Samples stored at -80°C were aliquots of the original samples from the accuracy assay. Samples were also tested after undergoing 7 or 8 freeze / thaw cycles (1 hour at RT followed by refreezing), and the results were compared to aliquots of the same samples that had undergone only 1 freeze / thaw cycle. % Recovery was calculated for all samples as shown in Figure 6. Since clinical trial samples were not available at the time of the test, only convalescent sera were used.

[0097] Verification Assay - Matrix Effect: For hemolysis analysis, hemolyzed human serum was spiked into 5 samples and 1 negative control sample to create 50% hemolyzed samples or 25% hemolyzed samples (indicating severe hemolysis). These samples were then tested in this assay, percent recovery was calculated as shown in Figure 6, and compared to normal samples without hemolysis. For lipemia analysis, serum with high levels of triglycerides was spiked into 6 samples to create samples with final triglyceride concentrations of 500 or 250 mg / dL (normal level, <150 mg / dL). These samples were then tested in this assay, percent recovery was calculated as shown in Figure 6, and compared to normal samples without lipemia.

[0098] Variant assay: The assay verification methods and protocols for variants (Delta, Omicron BA.1 / BA.5) followed a similar experimental, quantitative, and assay verification plan as for the prototype / Wuhan strain, but the S protein coated on the plate was replaced with a protein reflecting each variant sequence. Assay accuracy, dilution linearity, assay specificity, selectivity, LLoQ / upper limit of quantification level (ULoQ), and assay robustness (coating time) were evaluated for each variant. Matrix interference, assay robustness (incubation time), and sample stability were evaluated as part of the validation process for the original prototype / Wuhan strain assay.

[0099] Correlation analysis: For each sample, detection of anti-rS IgG antibody and microneutralizing antibody was performed using previously described methods, and the results were then compared to the hACE2 binding inhibition assay titers, and linear regression analysis was performed using GraphPad Prism software (San Diego, CA; version 9.3.1). For some serum samples, the hACE2 binding inhibition assay results for the prototype and Omicron BA.1 were observed at the limit of detection (LOD) level, most likely due to low levels of hACE2 binding inhibition. To avoid the influence of these samples on the correlation, correlation analysis was performed with and without including the hACE2 binding inhibition results at the LOD. Since there was no significant difference in the strength of the correlation, a plot of hACE2 binding inhibition vs anti-rS IgG levels for the prototype and Omicron BA.1 excluding the data points at the LOD is shown in the text.

[0100] Results: Inter-assay and intra-assay accuracy was <20% GCV for all 20 convalescent serum samples available at the time of assay development. The overall assay accuracy for 90% (18 / 20) of the samples was <20% GCV, meeting the acceptance criteria (Table 1). Subsequently, 21 serum samples from vaccinated individuals also showed <20% GCV and exhibited a wide range of titers (data not shown).

Table 1-1

Table 1-2

[0101] The assay specificity met the acceptance criteria. All samples tested showed at least a 74.1% reduction in hACE2 binding inhibitory titer after pre - incubation of serum with SARS - CoV - 2 rS - protein (a ≧50% reduction is required for acceptance). These samples also showed less than a 20% change in inhibitory titer against irrelevant proteins when incubated with RSV F - protein or Ebola GP (Table 2A). Since all samples collected before the pandemic showed negative (<LLoQ) results for hACE2 binding inhibition, the assay selectivity met the acceptance criteria. Paired pre / post (Day 0 / 21) samples from participants showing a strong response to influenza immunization showed that detection was not affected by large vaccine - induced changes in HAI titer (Table 2B).

Table 2A

Table 2B

[0102] The linearity of the assay was successfully demonstrated with an R 2 value of 0.999 for both of the two individual samples tested (Figure 7). Ten titers were assigned to the LLoQ based on the lowest titer value (predicted titers of 8.3 or 13.9) accurately and precisely detected for the two samples. The ULoQ was determined to be at least 2540.1 based on the highest hACE2 binding inhibitory titer from the linearity analysis and on clinical samples available at the time of validation of this assay. If additional samples from clinical trials become available, accumulation of sera with titers greater than the current ULoQ is expected. At regular time intervals, for any such sera, assay accuracy and dilution linearity will be retested and the ULoQ updated. Table 3A shows the appropriate parameters relevant to the chart in Figure 2.

Table 3A

[0103] The presence of free hemoglobin (Table 3B) or a lipemic matrix (Table 3C) had a minimal impact on the assay.

Table 3B

Table 3C

[0104] When sample stability was tested at room temperature, refrigeration (2 - 8°C) and freezing (-80°C), these samples were stable for up to 8 freeze - thaw cycles and up to 24 months in freezer storage (Figure 8).

[0105] Assay robustness for incubation time was demonstrated for the prototype strain as 95% of samples at the lower limit of incubation time and 85% of samples at the upper limit of incubation time had hACE2 binding inhibitory GMTs between 80 - 120% of the reference condition value (Tables 4 + 5).

Table 4-1

Table 4-2

Table 5-1

Table 5-2

[0106] When the serum incubation step was tested at the upper limit of 65 minutes, only 7 out of 20 samples (35%) had a recovery of 80 - 120%, but when retested at 62 minutes, 85% of the samples were within the acceptable range of 80 - 120%. The original assay was developed using the prototype (Wuhan) strain, but the controls (different controls for each strain) functioned equally well even when the assay was modified for the Delta and Omicron BA.1 strains (Figures 9A - 9C). Similar results for the validation parameters were seen for the Delta and Omicron BA.1 strains.

[0107] The results from the hACE2 assay were for the prototype / Wuhan strain (Pearson's r = 0.846, R 2= 0.7157, P < 0.0001) and Omicron BA.1 variant (Pearson's r = 0.8626, R 2 = 0.7442, P < 0.0001), showed a significant correlation with anti-rS IgG titers. Due to the correlation without data points at the LOD value for the hACE2 binding inhibition assay, for the prototype / Wuhan strain (Pearson's r = 0.789, R 2 = 0.6223, P < 0.0001) and Omicron BA.1 variant (Pearson's r = 0.8445, R 2 = 0.7113, P < 0.0001), a similar significant correlation with anti-rS IgG titers was demonstrated. hACE2 binding inhibition for the Omicron BA.5 variant also showed a similar significant correlation with anti-rS IgG assay data (Pearson's r = 0.7464, R 2 = 0.5571, P < 0.0006). Results from the hACE2 assay also showed a significant correlation with neutralizing antibody titers for the prototype / Wuhan strain (Pearson's r = 0.9148, R 2 = 0.8368, P < 0.0001) and Omicron BA.1 variant (Pearson's r = 0.8639, R 2 = 0.7464, P < 0.0001). This assay functioned well for multiple SARS-CoV-2 variants (e.g., Delta and Omicron BA.1 / BA.5 variants) (Figure 10A - G).

[0108] Conclusion: The results of the present inventors suggest that the ACE2 binding inhibition assay provides a rapid high-throughput option without concerns of biological containment for evaluating vaccine immunogenicity, which correlates with micro-neutralization and can detect serological differences between variants with higher sensitivity than simple anti-spike IgG binding. Together with other clinical biomarkers, the ACE2 inhibition assay can provide valuable insights into the correlates of protection (CoP) and further enable vaccine development against emerging COVID-19 variants.

[0109] Furthermore, due to the limited throughput and turnaround time of assays related to Biosafety Level 3 (BSL-3) procedures, hACE2 binding inhibition assays conducted in BSL-2 laboratories can provide important value. This procedural difference results in lower assay costs for hACE2 binding assays. Since the hACE2 binding inhibition assay is an in vitro assay (no need to culture cells), in contrast to the infectious SARS-CoV-2 microneutralization assay, this can also simplify the assay procedure and provide faster assay data for clinical trial samples. The robust correlation between hACE2 binding inhibition and microneutralization assays using infectious SARS-CoV-2 assays (performed in BSL-3) for both the prototype and Omicron BA.1 variant demonstrates the utility of hACE2 binding inhibition as a surrogate for BSL-3 based infectious SARS-CoV-2 neutralization assays.

[0110] Numbered embodiments of the present disclosure Notwithstanding the appended claims, the present disclosure presents the following numbered embodiments: 2. A method for measuring the ability of a biological sample to inhibit the binding of the SARS-CoV-2 S glycoprotein to human angiotensin-converting enzyme 2 (hACE2), comprising: (a) measuring the binding of the SARS-CoV-2 S glycoprotein to hACE2 after exposure to the biological sample, (i) coating a surface (e.g., a plate) with the SARS-CoV-2 S glycoprotein; (ii) exposing the surface to the biological sample (e.g., human serum); (iii) exposing the surface to hACE2; and (iv) detecting the hACE2 bound to the surface by: (b) measuring the binding of the SARS-CoV-2 S glycoprotein to hACE2 in the absence of the biological sample of (a); and Step of comparing the binding of SARS-CoV-2 S glycoprotein to hACE2 in (c), (a) and (b). A method comprising, when the amount of bound hACE2 in (a) is lower than the amount of bound hACE2 in (b), the biological sample inhibits the binding of SARS-CoV-2 S glycoprotein. 2. The method according to embodiment 1, wherein the biological sample comprises serum from a patient administered with a COVID-19 vaccine. 3. The method according to embodiment 2, wherein the COVID-19 vaccine is (i) a nucleic acid encoding the SARS-CoV-2 S glycoprotein or (ii) comprises the SARS-CoV-2 S glycoprotein. 4. The method according to any one of embodiments 1 to 3, wherein the SARS-CoV-2 S glycoprotein comprises a transmembrane domain.

[0111] Incorporation by reference All references, papers, publications, patents, patent publications and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. However, any reference to any reference, paper, publication, patent, patent publication and patent application cited herein is not an admission or any form of suggestion that they constitute prior art effective in any country in the world or a part of common general knowledge and should not be construed as such. The following patent documents are hereby incorporated by reference in their entirety herein: International Publication No. 2021 / 154812; International Publication No. 2022 / 203963; International Publication No. 2023 / 102448.

Claims

**Claim 1** A method for determining whether a biological sample contains an antibody that inhibits the binding of SARS-CoV-2 S glycoprotein to human angiotensin-converting enzyme 2 (hACE2), comprising: (a) determining the binding of SARS-CoV-2 S glycoprotein to hACE2 after exposure to the biological sample by: (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 hACE2; and (iv) detecting the hACE2 bound to the surface; (b) determining the binding of SARS-CoV-2 S glycoprotein to hACE2 in the absence of the biological sample of (a) by: (i) providing a surface coated with the SARS-CoV-2 S glycoprotein; (ii) exposing the surface to hACE2; and (iii) detecting the hACE2 bound to the surface; and (c) comparing the binding of the SARS-CoV-2 S glycoprotein to hACE2 in (a) and (b), wherein when the amount of bound hACE2 in (a) is lower than the amount of bound hACE2 in (b), the biological sample contains an antibody that inhibits the binding of the SARS-CoV-2 S glycoprotein to hACE2. **Claim 2** The method of claim 1, 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 to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3. **Claim 3** The method according to any one of claims 1 to 2, wherein the SARS-CoV-2 S glycoprotein has an inactivated furin cleavage site. **Claim 4** ​ ​ ​ The method according to claim 3, wherein the SARS-CoV-2 S glycoprotein has an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 68).

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

2.

6. The method according to any one of claims 1 to 3, 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 to any one polypeptide of SEQ ID NOs: 3, 5 to 13, 15, 17 to 19, 21, and 23 to 66.

7. The method according to any one of claims 1 to 2 and 5, 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 to any one polypeptide of SEQ ID NOs: 3 to 13, 20, and 22 to 66.

8. The method according to any one of claims 1 to 5, 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 to any one polypeptide of SEQ ID NO: 3, 5-13, and 23-66.

9. The method according to any one of claims 1 to 8, wherein the SARS-CoV-2 S glycoprotein is derived from a SARS-CoV-2 virus or a variant of SARS-CoV-2.

10. The method according to claim 9, wherein the variant of SARS-CoV-2 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.

11. The method according to any one of claims 1 to 10, 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 to a SARS-CoV-2 S glycoprotein derived from a SARS-CoV-2 Omicron variant selected from the group consisting of BA.1, BA.2.12.1, BA.2, BA.3, BA.4, BA.5, XBB.1.5, XBB.2.3, and XBB.1.

16.

12. The method according to any one of claims 1 to 11, wherein the hACE2 is conjugated to a tag. **Claim 13** The method according to claim 12, wherein the tag is a His tag. **Claim 14** The method according to any one of claims 1 to 13, wherein the biological sample is serum, plasma, blood, saliva, a nasopharyngeal swab or mucus. **Claim 15** The method according to any one of claims 1 to 14, wherein the biological sample is derived from a patient who previously had COVID-19. **Claim 16** The method according to any one of claims 1 to 14, wherein the biological sample is derived from a patient who has been administered an immunogenic composition against the SARS-CoV-2 virus or a variant thereof. **Claim 17** The method according to any one of claims 1 to 16, wherein the SARS-CoV-2 S glycoprotein comprises a transmembrane domain.

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