Pseudovirus-based neutralization assay for evaluating vaccine immunogenicity

Abstract

Pseudoviruses expressing the SARS-CoV-2 S protein are provided herein. Assays using these pseudoviruses to evaluate the immunogenicity of biological samples against the SARS-CoV-2 virus or variants thereof are also provided herein. Methods for evaluating the immunogenicity of COVID-19 vaccines using these assays are also provided herein. Pseudoviruses encoding (i) the SARS-CoV-2 spike (S) protein; (ii) one or more of the Rev, Tat, Gag, and Pol proteins; and (iii) a reporter protein are provided herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Patent Application No. 63 / 392,397, filed on July 26, 2022, and U.S. Provisional Patent Application No. 63 / 407,371, filed on September 16, 2022. These applications are hereby incorporated by reference in their entirety for all purposes.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (NOVV - 101_02WO_SeqList_ST26.xml; size: 203,198 bytes; creation date: July 26, 2023) is hereby incorporated by reference in its entirety.

[0003] Field The present disclosure generally relates to pseudoviruses expressing the SARS - CoV - 2 S protein. The present disclosure also relates to assays using these pseudoviruses to evaluate the immunogenicity of biological samples (e.g., whether the biological sample contains neutralizing antibodies) against the SARS - CoV - 2 virus or its variants. The present disclosure also relates to methods of using these assays to evaluate the ability of COVID - 19 vaccines to stimulate an immune response against the SARS - CoV - 2 virus or its variants.

Background Art

[0004] Background of the Invention ]>The neutralizing antibody response elicited by COVID-19 vaccines plays a crucial role in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. To measure neutralizing antibodies against the SARS-CoV-2 virus, a biosafety level 3 (BSL-3) laboratory must be used. BSL-3 laboratories require more stringent laboratory safety precautions compared to BSL-2 laboratories. These include restricted / regulated laboratory access, medical surveillance and / or immunization of staff, appropriate laboratory design (including self-closing and lockable doors, continuous directional air flow, and sinks / eyewash stations), use of appropriate biosafety cabinets, and appropriate personal protective equipment (which may include respirators).

[0005] As a result of these safety precautions, BSL-3 laboratories are costly to operate and difficult to access. There is a need in the art for a novel assay that enables the measurement of SARS-CoV-2 neutralizing antibodies outside of a BSL-3 laboratory. Summary of the Invention Means for Solving the Problems

[0006] Gist of the Invention Provided herein is a pseudovirus-based neutralization assay for measuring neutralizing antibodies against the SARS-CoV-2 virus. The assay described herein can be performed in a biosafety level 2 (BSL-2) laboratory. Thus, this assay is more cost-effective than conventional assays that require the use of the SARS-CoV-2 virus.

[0007] (i) SARS-CoV-2 spike (S) glycoprotein; (ii) one or more of the Rev, Tat, Gag, and Pol proteins; and (iii) a pseudovirus encoding a reporter protein are provided herein.

[0008] A method for producing a pseudovirus, which comprises the step of transfecting a cell with (i) a plasmid encoding a lentiviral backbone and a reporter protein; (ii) one or more plasmids encoding Rev protein, Tat protein, Gag protein, Pol protein; or a combination thereof; and (iii) a plasmid expressing the SARS-CoV-2 spike (S) glycoprotein, is provided herein.

[0009] A method for measuring the ability of a biological sample to stimulate an immune response against SARS-CoV-2 virus or a variant thereof, comprising: (a) contacting the biological sample with a pseudovirus expressing the SARS-CoV-2 S glycoprotein and a reporter protein; (b) contacting a cell expressing angiotensin-converting enzyme 2 with the pseudovirus; and (c) quantifying the expression of the reporter protein is provided herein.

[0010] (i) SARS-CoV-2 spike (S) glycoprotein; (ii) one or more of Rev, Tat, Gag, and Pol proteins; and (iii) a reporter protein-encoding pseudovirus are provided herein. In embodiments, the pseudovirus encodes from 1 to about 20, 2 to about 20, 3 to about 20, 4 to about 20, 5 to about 20, 6 to about 20, 7 to about 20, 8 to about 20, 9 to about 20, about 10 to about 20, about 11 to about 20, about 12 to about 20, about 13 to about 20, about 14 to about 20, about 15 to about 20, about 16 to about 20, about 17 to about 20, about 18 to about 20, about 19 to about 20, 1 to about 10, 2 to about 10, 3 to about 10, 4 to about 10, 5 to about 10, 6 to about 10, about 7 to about 10, about 8 to about 10, 2 to 5, 3 to 5, 4 to about 8, 5 to about 8, or about 6 to about 8 species of SARS-CoV-2 S glycoproteins. In embodiments, the reporter protein is luciferase. In embodiments, the reporter protein is a fluorescent protein. In embodiments, the fluorescent protein is ZsGreen. 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 SARS-CoV-2 S glycoprotein has an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 68). In embodiments, 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.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 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 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 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, the pseudovirus comprises a Gag protein and a Pol protein. In an embodiment, the fusion protein encodes a Gag protein and a Pol protein. In an embodiment, the Gag protein and the Pol protein are derived from Moloney murine leukemia virus (MLV). In an embodiment, the Rev, Tat, Gag and Pol proteins are derived from a lentivirus.

[0011] In an embodiment, provided herein is a method for producing a pseudovirus described in this specification, the method comprising the step of transfecting a host cell with (i) a transfer plasmid encoding a reporter protein; (ii) one or more plasmids encoding Rev protein, Tat protein, Gag protein, and Pol protein; or a combination thereof; and (iii) a plasmid encoding the SARS-CoV-2 spike (S) glycoprotein. In an embodiment, the host cell is a human cell. In an embodiment, the host cell is HEK293T (also referred to as "293T") cells.

[0012] In an embodiment, a method for determining whether a biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or a variant thereof, comprising: (a) contacting the biological sample with 1 to about 20 pseudoviruses described herein; (b) contacting cells expressing angiotensin-converting enzyme 2 (ACE2) with 1 to about 20 pseudoviruses; (c) quantifying the expression of a reporter protein in the cells expressing ACE2; (d) contacting control cells expressing ACE2 with 1 to about 20 pseudoviruses, wherein the 1 to about 20 pseudoviruses have not been contacted with the biological sample; and (e) quantifying the expression of the reporter protein in the control cells expressing ACE2, wherein if the expression of the reporter protein in the cells of (c) is lower than the expression of the reporter protein in the control cells of (e), the biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or a variant thereof. A method is provided herein. In an embodiment, the method comprises contacting the biological sample and the pseudovirus for 1 hour to about 7 hours. In an embodiment, the method comprises contacting the biological sample and the pseudovirus for about 2 hours. In an embodiment, the method comprises contacting the biological sample and the pseudovirus at about 37°C. In an embodiment, the method comprises heat-inactivating the biological sample. In an embodiment, the method comprises contacting the cells with the pseudovirus for 1 day to about 7 days. In an embodiment, the method comprises contacting the cells with the pseudovirus for about 3 days. In an embodiment, the cells are selected from the group consisting of Vero E6, A549, and HEK293T cells. In an embodiment, the cells are HEK293T cells. In an embodiment, performing the method does not require a biosafety level 3 (BSL-3) laboratory. In an embodiment, the biological sample is saliva, nasopharyngeal swab, sputum, saliva, urine, fecal sample, cerebrospinal fluid, synovial fluid, serum, blood, mucosal sample, or plasma. In an embodiment, the biological sample is from a patient administered an immunogenic composition against the SARS-CoV-2 virus or a variant thereof.In an embodiment, the biological sample is from a patient who had COVID-19 previously. In an embodiment, the method includes contacting the biological sample with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 types of pseudoviruses. In an embodiment, each pseudovirus expresses a different reporter gene and a different SARS-CoV-2 S protein.

Brief Description of the Drawings

[0013]

Figure 1

[0014]

Figure 2A

Figure 2B

[0015]

Figure 3A

Figure 3B

Figure 3C

[0016]

Figure 4A

Figure 4B

[0017]

Figure 5A

Figure 5B

[0018]

Figure 6A

Figure 6B

Figure 6C

[0019]

Figure 7A

Figure 7B

[0020]

Figure 8

[0021]

Figure 9A

Figure 9B

[0022]

Figure 10A

Figure 10B

Figure 10C

Figure 10D

[0023]

Figure 11A

Figure 11B

Figure 11C

Figure 11D

[0024]

Figure 12A

Figure 12B

[0025]

Figure 13A

Figure 13B

[0026]

Figure 14A

Figure 14B

[0027] **Detailed Description of the Invention** **Definitions** As used herein and in 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, reference to "the method" includes reference to equivalent steps and / or methods known to those of ordinary skill in the art, and the like.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] As used herein, an "immunogenic composition" is a composition that includes an antigen, and administration of the composition to a subject results in the development of a humoral and / or cellular immune response to that antigen in the subject.

[0032] 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 protective immunity (e.g., 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). A 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 protective immunity.

[0033] As used herein, when the term "modification" refers to a CoV S polypeptide, it 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). (See the following sequences)

Table 1-1

Table 1-2

[0034] As used interchangeably herein with the term "heterologous SARS-CoV-2 strain", the variant of SARS-CoV-2 is compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and has 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, about 1 to about 100 modifications, about 2 to about 35 modifications, about 5 to about 10 modifications, about 5 to about 20 modifications, about 10 to about 20 modifications, about 15 to about 25 modifications, about 20 to about 30 modifications, about 20 to about 40 modifications, about 25 to about 45 modifications, about 25 to about 50 modifications, about 25 to about 55 modifications, about 30 to about 60 modifications, about 25 to about 100 modifications, about 25 to about 45 modifications or about 35 to about 100 modifications, and is a SARS-CoV-2 virus comprising a SARS-CoV-2 S glycoprotein (also referred to as "CoV S polypeptide").

[0035] 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 71%, at least 72%, at least 73%, at least 74%, at least about 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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: 1 or 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: 1 or 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: 1 or 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: 1 or 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: 1 or 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: 1 or SEQ ID NO: 2.In an embodiment, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity of between about 95% and about 99.8% to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having an identity of between about 95% and about 99% to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0100] In an embodiment, a heterologous SARS-CoV-2 strain has 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 Pango lineage names and is hereby incorporated by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0101] In embodiments, the heterologous SARS-CoV-2 strains have the World Health Organization label of Omicron. In embodiments, the heterologous SARS-CoV-2 strains having the World Health Organization label of Omicron have at least 35 modifications as compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In embodiments, the heterologous SARS-CoV-2 strains having the World Health Organization label of Omicron have 35 to 55, 35 to 65, 35 to 75, 35 to 85, 35 to 95 or 35 to 105 modifications as compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In embodiments, the modifications are selected from the group consisting of 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.

[0102] 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 from 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 one, two, three, or four 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 one, two, three, four, five, or six 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.

[0036] 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 (trademark)).

[0037] The term "pseudovirus" refers to a recombinant viral particle encoding one or more proteins from a first virus and an envelope protein from a second virus, wherein the first virus is different from the second virus. In embodiments, the protein from the first virus is selected from one or more of Gag, Pol, Rev, and Tat. In embodiments, the envelope protein is a spike protein. In embodiments, the envelope protein is a spike protein from SARS-CoV-2 S virus or a variant thereof. The following reference describes pseudoviruses and is hereby incorporated by reference in its entirety: Li et al. Rev Med Virol. 2018 Jan; 28(1): e1963.

[0038] The term "transfer plasmid" refers to a nucleic acid plasmid comprising an insertion point for a gene of interest (GOI) and nucleic acids that facilitate packaging and insertion of the GOI into the host cell genome. In embodiments, the GOI encodes a reporter protein.

[0039] Assays for evaluating the immunogenicity of vaccine compositions against SARS-CoV-2 The present disclosure provides pseudoviruses and methods of using pseudoviruses for evaluating the immunogenicity of immunogenic compositions and vaccine compositions against SARS-CoV-2 or variants thereof. Pseudoviruses are also used to determine whether a biological sample contains antibodies that neutralize SARS-CoV-2 or variants thereof.

[0040] Pseudovirus In embodiments, pseudoviruses are provided herein that encode (i) the SARS-CoV-2 spike (S) glycoprotein; and (ii) one or more of the Rev, Tat, Gag, and Pol proteins. In embodiments, the pseudovirus further encodes a reporter protein.

[0041] In embodiments, the pseudovirus encodes from 1 to 30, 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 17 to 20, 18 to 20, 19 to 20, 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 2 to 5, 3 to 5, 4 to 8, 5 to 8, or 6 to 8 species of SARS-CoV-2 S glycoproteins. In embodiments, the pseudovirus encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 species of SARS-CoV-2 S glycoproteins.

[0042] Suitable SARS-CoV-2 S glycoproteins 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, �CWO, 7CWS, 7CWT, 7CWU, 7CYH, 7CYP, 7CYV, 7CZP, 7CZQ, 7CZR, 7CZS, 7CZT, 7CZU, 7CZV, 7CZW, 7CZX, 7CZY, 7CZZ, 7D00, 7D03, 7D0B, 7D0C, 7D0D, 7D2Z, 7D30, 7D4G, 7D6I, 7DCC, 7DCX, 7DD2, 7DD8, 7DDD, 7DDN, 7DEO,7DET, 7DEU, 7DF3, 7DF4, 7DHX, 7DJZ, 7DK0, 7DK2, 7DK3, 7DK4, 7DK5, 7DK6, 7DK7, 7DMU, 7DPM, 7DQA, 7DTE, 7DWX, 7DWY, 7DWZ, 7DX0, 7DX1, 7DX2, 7DX3, 7DX4, 7DX5, 7DX6, 7DX7, 7DX8, 7DX9, 7DZW, 7DZX, 7DZY, 7E23, 7E39, 7E3B, 7E3C, 7E3J, 7E3K, 7E3L, 7E3O, 7E5O, 7E5R, 7E5S, 7E5Y, 7E7B, 7E7D, 7E7X, 7E7Y, 7E86, 7E88, 7E8C, 7E8F, 7E8M, 7E9N, 7E9O, 7E9P, 7E9Q, 7E9T, 7EAM, 7EAN, 7EAZ, 7EB0, 7EB3, 7EB4, 7EB5, 7EDF, 7EDG, 7EDH, 7EDI, 7EDJ, 7EFP, 7EFR, 7EH5, 7EJ4, 7EJ5, 7EJL, 7EJY, 7EJZ, 7EK0, 7EK6, 7EKC, 7EKE, 7EKF, 7EKG, 7EKH, 7ENF, 7ENG, 7EPX, 7EY0, 7EY4, 7EY5, 7EYA, 7EZV, 7F0X, 7F12, 7F15, 7F3Q, 7F46, 7F5H, 7F5R, 7F62, 7F63, 7F6Y, 7F6Z, 7F7E, 7F7H, 7FAE, 7FAF, 7FAT, 7FAU, 7FB0, 7FB1, 7FB3, 7FB4, 7FBJ, 7FBK, 7FC5, 7FCD, 7FCE, 7FCP, 7FCQ, 7FDG, 7FDH, 7FDI, 7FDK, 7FEM, 7FET, 7FG2, 7FG3, 7FG7, 7FH0, 7FJC, 7FJN, 7FJO, 7FJS, 7JJC, 7JJI, 7JJJ, 7JMO, 7JMP, 7JMW, 7JV2, 7JV4, 7JV6, 7JVA, 7JVB, 7JVC, 7JW0, 7JWB, 7JWY, 7JX3, 7JZL, 7JZM, 7JZN, 7JZU, 7K43, 7K45, 7K4N, 7K8M, 7K8S, 7K8T, 7K8U, 7K8V, 7K8W, 7K8X, 7K8Y, 7K8Z, 7K90, 7K9H, 7K9I, 7K9J, 7K9K, 7K9Z, 7KDG, 7KDH, 7KDI, 7KDJ, 7KDK, 7KDL, 7KE4, 7KE6, 7KE7, 7KE8, 7KE9, 7KEA, 7KEB, 7KEC, 7KFV, 7KFW, 7KFX, 7KFY, 7KGJ, 7KGK, 7KJ2, 7KJ3, 7KJ4, 7KJ5, 7KKK, 7KKL7KL9, 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, 7NKT, 7NLL, 7NP1, 7NS67NT9, 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, 7SY8, 7T01, 7T3M, 7T677T72, 7T7B, 7T9J, 7T9K, 7T9L, 7TAS, 7TAT, 7TB4, 7TB8, 7TB F, 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, 7UM2, 7UPL, 7UR1, 7URQ, 7URS, 7UZ4, 7UZ5, 7UZ6, 7UZ7, 7UZ8, 7UZ9, 7UZA, 7UZB, 7UZC, 7UZD, 7V20, 7V22, 7V23, 7V24, 7V26, 7V27, 7V2A, 7V76, 7V77, 7V78, 7V79, 7V7A, 7V7D, 7V7E, 7V7F, 7V7G, 7V7H, 7V7I, 7V7J, 7V7N, 7V7O, 7V7P, 7V7Q, 7V7R, 7V7S, 7V7T, 7V7U, 7V7V, 7V7Z, 7V80, 7V81, 7V82, 7V83, 7V84, 7V85, 7V86, 7V87, 7V88, 7V89, 7V8A, 7V8B, 7V8C, 7VHH, 7VHJ, 7VHK, 7VHL, 7VHM, 7VHN, 7VMU, 7VNB, 7VNC, 7VND, 7VNE, 7VOA, 7VQ0, 7VRV, 7VRW, 7VX1, 7VX4, 7VX5, 7VX9, 7VXA, 7VXB, 7VXC, 7VXD, 7VXE, 7VXF, 7VXI, 7VXK, 7VXM, 7VYR, 7VZT, 7W1S, 7W6U, 7W8S, 7W92, 7W94, 7W99, 7W9B, 7W9C, 7W9E, 7W9F, 7WA1, 7WB5, 7WBL, 7WBP, 7WBQ, 7WBZ, 7WCD, 7WCH, 7WCK, 7WCP7WCR, 7WCU, 7WCZ, 7WD0, 7WD1, 7WD2, 7WD7, 7WD8, 7WD9, 7WDF, 7WE7, 7WE8, 7WE9, 7WEA, 7WEB, 7WEC, 7WED, 7WEE, 7WEF, 7WEV, 7WG6, 7WG7, 7WG8, 7WG9, 7WG B, 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, 7Y7K7Y8J, 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, 8EL2, 8ELH, 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, 8SMT. The amino acid sequences of the SARS-CoV-2 S glycoprotein related to each entry can be accessed by downloading the FASTA file related to the PDB ID at www.rcsb.org. In embodiments, the pseudovirus expresses a nucleic acid encoding the SARS-CoV-2 S glycoprotein of any one of the foregoing PDB IDs.,

[0043] In an embodiment, 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 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. The wild-type SARS-CoV-2 S glycoprotein contains a furin cleavage site RRAR (SEQ ID NO: 6) at positions 669 to 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 to 97. In an embodiment, the amino acid sequence of the inactive furin cleavage site is GG. In an embodiment, non-limiting examples of the SARS-CoV-2 S glycoprotein having a GG inactive furin cleavage site include the glycoproteins of SEQ ID NOs: 26 to 28 and 30.

[0044] 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 the SARS-CoV-2 S glycoprotein having a QQAQ (SEQ ID NO: 68) inactive furin cleavage site include the glycoproteins of SEQ ID NOs: 3, 5 to 13, 15, 17 to 19, 21, 23 to 25, 29 and 31 to 66.

[0045] 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.

[0046] 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 may 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.

[0047] 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.

[0048] 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.

[0049] 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 can 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.

[0050] 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 2]

[0051] 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.

[0052] 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.

[0053] In embodiments, 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 embodiments, 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 embodiments, 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.

[0054] 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.

[0055] 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 any one polypeptide of SEQ ID NOs: 3-66.

[0056] In an embodiment, the pseudovirus encodes a Gag protein and a Pol protein. In an embodiment, the pseudovirus encodes a fusion protein that encodes a Gag protein and a Pol protein. In an embodiment, the pseudovirus encodes a Rev protein and a Tat protein. In an embodiment, the Gag protein and the Pol protein are derived from murine leukemia virus (MLV). In an embodiment, one or more of the Rev, Tat, Gag, and Pol proteins are derived from a lentivirus.

[0057] In an embodiment, the pseudovirus encodes one or more reporter proteins. In an embodiment, the pseudovirus encodes 1 to 10 reporter proteins. In an embodiment, one or more of the reporter proteins are fluorescent proteins. In an embodiment, the fluorescent proteins are green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, enhanced green fluorescent protein, and ZsGreen. In an embodiment, one or more of the reporter proteins are tagged with a fluorescent dye. In an embodiment, one or more of the reporter proteins are luciferase.

[0058] Method for producing a pseudovirus In an embodiment, provided herein is a method of producing a pseudovirus described herein, the method comprising transfecting a cell with (i) a transfer plasmid encoding a reporter protein; (ii) one or more plasmids encoding Rev protein, Tat protein, Gag protein, Pol protein; or a combination thereof; and (iii) a plasmid expressing a SARS-CoV-2 spike (S) glycoprotein. In an embodiment, the cell is a HEK293T (also referred to as "293T") cell. The SARS-CoV-2 S glycoprotein can be any SARS-CoV-2 S glycoprotein described herein. In an embodiment, the SARS-CoV-2 S glycoprotein contains mutations to prolines at amino acids 973 and 974 and is numbered according to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In an embodiment, the SARS-CoV-2 S glycoprotein contains an inactivated furin cleavage site. A number of examples of inactivated furin cleavage sites are described herein.

[0059] In an embodiment, the pseudovirus is produced in a human host cell. In an embodiment, the pseudovirus is produced in HEK293T cells (also referred to as "293T" cells).

[0060] In an embodiment, the plasmid encoding the Rev protein 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 plasmid of SEQ ID NO: 100. In an embodiment, the plasmid encoding the fusion protein encoding the Gag protein and the Pol protein 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 plasmid of SEQ ID NO: 99. In an embodiment, the plasmid encoding the Tat protein 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 plasmid of SEQ ID NO: 98.

[0061] Method for identifying neutralizing antibodies against SARS-CoV-2 or its variants In an embodiment, a method for determining whether a biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or a variant thereof, comprising: (a) contacting the biological sample with 1 to about 20 pseudoviruses described herein; (b) contacting cells expressing angiotensin-converting enzyme 2 (ACE2) with 1 to about 20 pseudoviruses; (c) quantifying the expression of a reporter gene in the cells expressing ACE2; (d) contacting control cells expressing ACE2 with 1 to about 20 pseudoviruses, wherein the 1 to about 20 pseudoviruses have not been contacted with the biological sample; and (e) quantifying the expression of the reporter gene in the control cells expressing ACE2, wherein if the expression of the reporter gene in the cells of (c) is lower than the expression of the reporter gene in the control cells of (e), the biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or a variant thereof, is provided herein.

[0062] In an embodiment, the method comprises contacting the biological sample with 1, 2, 3, 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 pseudoviruses. In an embodiment, each pseudovirus expresses a different SARS-CoV-2 S protein. In an embodiment, each pseudovirus expresses a different reporter protein.

[0063] In an embodiment, cells expressing ACE2 are produced by transfecting cells with a plasmid 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 plasmid of SEQ ID NO: 101. In an embodiment, ACE2 has an amino acid sequence 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: 102 or 103.

[0064] In an embodiment, a biological sample and a pseudovirus are incubated for 1 hour to about 24 hours, 1 hour to about 12 hours, 1 hour to about 8 hours, 1 hour to about 6 hours, 2 to about 6 hours, 2 to about 7 hours, or 2 to about 8 hours. In an embodiment, a biological sample and a pseudovirus are incubated for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 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, or about 24 hours. In an embodiment, a biological sample and a pseudovirus are incubated for 2 or 3 hours. In an embodiment, a biological sample and a pseudovirus are incubated for 1 day to about 7 days. In an embodiment, a biological sample and a pseudovirus are incubated for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In an embodiment, a biological sample and a pseudovirus are incubated for 3 days.

[0065] In an embodiment, the cells are contacted with the pseudovirus for 1 hour to about 24 hours. In an embodiment, the cells are contacted with the pseudovirus and incubated for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 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 or about 24 hours. In an embodiment, the cells are contacted with the pseudovirus for 1 day to about 7 days. In an embodiment, the cells are contacted with the pseudovirus for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In an embodiment, the cells are contacted with the pseudovirus for 3 days.

[0066] In an embodiment, the cells expressing ACE2 are human cells. In an embodiment, the cells expressing ACE2 are 293T cells. In an embodiment, the cells are A549 cells or Vero E6 cells. In an embodiment, the methods described herein do not require a biosafety level 3 (BSL-3) laboratory.

[0067] In an embodiment, the cell is selected from the group consisting of Vero E6, A549, and HEK293T cells. In an embodiment, the biological sample is serum. In an embodiment, the serum is human serum. In an embodiment, the serum is derived from an individual infected with SARS-CoV-2 or a variant thereof. In an embodiment, the serum is derived from an individual who has received an immunogenic composition or a vaccine against SARS-CoV-2 or a variant thereof. In an embodiment, before use in the methods described herein, the serum, plasma, or blood is heat inactivated. In an embodiment, heat inactivation includes incubating the serum, plasma, or blood at a temperature of about 37°C to about 90°C. In an embodiment, heat inactivation includes incubating the serum, plasma, or blood at a temperature of about 56°C. In an embodiment, heat inactivation includes incubating the serum, plasma, or blood at a temperature of about 37°C to about 90°C for about 15 minutes to about 2 hours. In an embodiment, the serum, plasma, or blood is incubated at about 37°C to about 90°C for 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 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes. In an embodiment, the serum, plasma, or blood is incubated at about 56°C for 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 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, or about 120 minutes. In an embodiment, the serum, plasma, or blood is incubated at about 37°C to about 90°C for about 30 minutes.

[0068] In an embodiment, the immunogenic composition and the vaccine composition contain a non-naturally occurring coronavirus (CoV) spike (S) polypeptide or a nanoparticle containing a CoV S polypeptide.

[0069] In an embodiment, the CoV S polypeptide 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: 1.

[0070] In an embodiment, the CoV S polypeptide 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.

[0071] In an embodiment, the CoV S polypeptide 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 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.

[0072] The sequence of SEQ ID NO: 3 is in the following table. SEQ ID NO: 3 [Table 3]

[0073] In embodiments, the immunogenic composition or vaccine composition comprises an adjuvant. Exemplary adjuvants are described throughout the present disclosure.

[0074] Aluminum-based adjuvant In embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles may be bound to the alum at least 80%, at least 85%, at least 90% or at least 95%. Often, the nanoparticles are 92% - 97% bound to the 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, the alum may 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, the alum is present at about 400 μg per 120 μg protein nanoparticle dose.

[0075] 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 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.

[0076] 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% - 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is the fraction eluted with approximately 39% acetonitrile or corresponds to such a fraction. The fraction referred to herein as "Fraction B" or "QH-B" is the fraction eluted with approximately 47% acetonitrile or corresponds to such a fraction. The fraction referred to herein as "Fraction C" or "QH-C" is the fraction eluted with 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 having 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.

[0077] 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 Example 1 on pages 6 as well as 8 and 9.

[0078] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions; that is, the fractions are substantially free from the presence of 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.

[0079] ISCOM structure The saponin fraction can be administered in the form of cage-like particles called ISCOMs (Immune Stimulating Complexes). 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).

[0080] 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.

[0081] 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 integrated 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.

[0082] According to one embodiment, the saponin fraction incorporated into an ISCOM matrix complex or an ISCOM complex, or at least one further 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 sub-fraction, for example, selected from QA 1-21.

[0083] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight % of different saponin fractions may be used. Any combination of weight % of any two fractions may be used. For example, the particles may each contain any weight % of fraction A and any weight % 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 may 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 remainder 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 US Patent Application Publication No. 2013 / 0129770, which is hereby incorporated by reference in its entirety herein.

[0084] In certain embodiments, the ISCOM matrix or ISCOM complex comprises from 5 to 99% by weight of one fraction, for example fraction A, and up to 100% by weight of another fraction, for example a crude saponin fraction or fraction C. The weights are calculated as the total weight of the saponin fractions.

[0085] In another embodiment, the ISCOM matrix or ISCOM complex comprises from 40% to 99% by weight of one fraction, for example fraction A, and from 1% to 60% by weight of another fraction, for example a crude saponin fraction or fraction C. The weights are calculated as the total weight of the saponin fractions.

[0086] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises from 70% to 95% by weight of one fraction, for example fraction A, and from 30% to 5% by weight of another fraction, for example 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 derived from Quillaja saponaria Molina is selected from any one of QA 1 - 21.

[0087] In addition to particles containing mixtures 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 derived from Quillaja saponaria Molina, and the saponin fraction in one complex is different from the saponin fraction in other complex particles.

[0088] 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. A composition or vaccine can comprise at least two types of complexes or particles, each type having one type of saponin incorporated into physically distinct particles.

[0089] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles in which one saponin fraction Quillaja saponaria Molina and another saponin fraction Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles can be used.

[0090] 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 0.1 wt% - 99.9 wt%, 5 wt% - 95 wt%, 10 wt% - 90 wt%, 15 wt% - 85 wt%, 20 wt% - 80 wt%, 25 wt% - 75 wt%, 30 wt% - 70 wt%, 35 wt% - 65 wt%, 40 wt% - 60 wt%, 45 wt% - 55 wt%, 40 - 60 wt%, or 50 wt% of a first saponin fraction, and the remainder can be 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, and each matrix or complex particle contains only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles can contain more than one saponin fraction.

[0091] 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.

[0092] 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, respectively, 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.

[0093] Preferred compositions include 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 to 96% by weight, 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% of the total amount by weight of the saponin adjuvant in the composition, and the ISCOM matrix of fraction C is present at about 15%. MATRIX-M™ may be referred to interchangeably with Matrix-M1.

[0094] 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.

[0095] 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 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).

Examples

[0096] (Example 1) Pseudovirus-based neutralization assay for SARS-CoV-2 variants: A rapid and cost-effective BSL2-based high-throughput assay useful for vaccine immunogenicity assessment Introduction: The inventors have discovered a novel pseudovirus-based neutralization assay for evaluating the immunogenicity of vaccines against SARS-CoV-2 variants. This assay is cost-effective, provides a rapid turnaround, and is a BSL-2-based assay that enables the evaluation of the immunogenicity (e.g., neutralizing antibody production) of SARS-CoV-2 vaccines against emerging SARS-CoV-2 variants. This assay serves as a surrogate for live virus neutralization assays and does not require the use of a BSL-3 laboratory.

[0097] Methods: The inventors developed a BSL-2-based pseudovirus-based neutralization assay for SARS-CoV-2 prototype and variants (Omicron BA.1 and BA.2). This assay is suitable for measuring neutralization capacity in clinical samples (serum from human subjects infected with SARS-CoV-2 or immunized with COVID-19 vaccines). Significantly, this assay can be performed in a BSL-2 laboratory rather than a BSL-3 laboratory. This assay can be used to evaluate the ability of SARS-CoV-2 vaccines to stimulate an immune response against the prototype SARS-CoV-2 virus. The prototype SARS-CoV-2 virus contains the SARS-CoV-2 S protein having the amino acid sequence of SEQ ID NO: 2. This assay can also be utilized to evaluate the ability of SARS-CoV-2 vaccines to stimulate an immune response against the prototype SARS-CoV-2 virus containing Omicron BA.1, BA.2, and BA.5 variants. This assay can further be utilized to evaluate the ability of human sera to neutralize SARS-CoV-2 and SARS-CoV-2 variants.

[0098] A plasmid encoding a lentiviral backbone that expresses a luciferase reporter under the control of a mammalian promoter, a plasmid encoding a non-surface protein for lentivirus production, and a plasmid expressing the SARS-CoV-2 spike protein (either the prototype strain or the Omicron strain) were co-transfected into cells to produce SARS-CoV-2 pseudovirus expressing the SARS-CoV-2 spike protein.

[0099] A schematic diagram of the pseudovirus neutralization assay used in this example is found in Figure 1.

[0100] SARS-CoV-2 pseudovirus was produced by transfecting HEK293T cells with (i) a plasmid encoding a lentiviral backbone that expresses a reporter protein (e.g., luciferase or ZsGreen); (ii) a lentiviral helper plasmid (e.g., a plasmid expressing Rev1b, Tat1b, Gag, and Pol); and (iii) a plasmid expressing the SARS-CoV-2 S glycoprotein. These plasmids expressed the genes of interest under the control of the cytomegalovirus (CMV) promoter. The pseudovirus was recovered from the cell culture supernatant.

[0101] The pseudovirus was then incubated with sera from patients who had recovered from SARS-CoV-2 infection or patients immunized with the SARS-CoV-2 vaccine NVX-CoV2373. As a control, the pseudovirus was incubated with cell culture medium without sera (virus only "VC" control).

[0102] The pseudovirus was used to infect cells expressing the ACE2 receptor and incubated at 37°C for 48 - 72 hours. As a negative control, the cells were infected with cell culture medium in the absence of pseudovirus. The luminescence (from luciferase) of the cells was measured. A reduction in the luciferase signal indicated neutralization of the infection.

[0103] The dose-dependence of luminescence on the pseudovirus level was determined by infecting cells expressing the ACE2 receptor with different amounts of pseudovirus and detecting luciferase luminescence (RLU) two days after infection.

[0104] The optimal cell line for the pseudovirus-based neutralization assay was determined. The following cells were evaluated: Vero-E6 (African green monkey kidney cells), A549 / ACE2-TMPRESS (human lung epithelial cells expressing ACE2 and TMPRESS), and 293T / ACE2 cells (human kidney epithelial cells expressing ACE2). The cells were infected with pseudovirus, and luminescence was measured two days after infection. The infectivity of the cell line was determined by TCID50 quantification. The suitability of the cell line for the pseudovirus neutralization assay was further confirmed by infecting 293T / ACE2 and A549 / ACE2-TMPRESS cells with Omicron BA.1 / BA.5 pseudovirus in the presence or absence of serum and then measuring luminescence. The controls were cells that were infected but not treated with serum (virus control) and cells without pseudovirus (cell control). The percent (%) inhibition / neutralization of pseudovirus infection by serum was determined compared to the virus control.

[0105] The pseudovirus-based neutralizing titer using different amounts of pseudovirus was evaluated to optimize the assay procedure.

[0106] The kinetics of the pseudovirus neutralization assay endpoint were evaluated by measuring luminescence at 48-hour and 72-hour time points and comparing the signal-to-background (S / B) ratio.

[0107] Assay quality - Precision: Quality control (QC) samples were tested by two operators (analysts) on two different days, and each duplicate sample was tested in the same assay. For the prototype / Wuhan strain, high-quality, intermediate-quality, and low-quality QC samples were used. For variants, two or three QC samples were used.

[0108] Assay quality - Linearity: One sample was serially diluted (6 times, 4-fold dilution series) and evaluated in duplicate by two different operators.

[0109] Correlation analysis using a live virus-based micro-neutralization (MN) assay: Samples were tested in a validated live virus MN assay (360biolabs) (n = 13), and then linear regression analysis was used to compare the data with data from the pseudovirus neutralization assay.

[0110] Correlation with anti-S IgG antibody and hACE2 binding inhibition - Samples were tested in a validated anti-S IgG assay (n = 15) or a validated hACE2 binding inhibition assay (n = 8), and then linear regression analysis was used to compare the data with the pseudovirus neutralization assay results.

[0111] Clinical utility: To evaluate the clinical utility of the assay, clinical serum samples from participants vaccinated with NVX-CoV2373 were tested against the prototype / Wuhan strain and variants (Omicron BA.1, BA.2, BA.5) using the pseudovirus neutralization assay. The neutralizing antibody responses against the prototype / Wuhan strain and variants in the clinical serum samples were profiled. To demonstrate the effect of the vaccine booster on the neutralizing antibody response (using PNT), sera from patients before and after administration of the booster dose of NVX-CoV2373 were evaluated in the pseudovirus neutralization assay.

[0112] Results: A pseudovirus-based neutralization assay in 96-well plate format was developed, and the dose (TCID 50Optimized in pseudovirus amount / well and kinetics (assay duration) experiments. The positive and negative controls of the assay showed a wide dynamic range. The neutralization data from the pseudovirus assay showed a robust correlation with validated anti-r spike IgG levels and ACE2 inhibitory titers (prototype). Currently, a pseudovirus assay against Omicron BA.5 is under development, and sera from patients vaccinated with NVX-CoV2373 are being evaluated in this assay.

[0113] The luminescence signal from the pseudovirus neutralization assay showed dose-dependence on the amount of pseudovirus used for infection (Figure 2A, Figure 2B). The luciferase endpoint showed a robust dynamic range and user-friendliness (ease of use and throughput).

[0114] Results - Effects of ACE2-expressing cell lines on pseudovirus infection: To evaluate the appropriateness of cell lines for pseudovirus infection, three different cell lines (Vero E6 - African green monkey kidney cell line, A549 / ACE2-TMPRESS - human lung epithelial cell line expressing ACE2 and TMPRESS, 293T-ACE2 human kidney epithelial cell line expressing ACE2) were infected with various amounts of pseudovirus expressing the SARS-CoV-2 S protein from the prototype strain (also called "prototype / Wuhan") or the S protein from SARS-CoV-2 variants (Omicron BA.1, BA.2, BA.5). The luciferase levels in the infected cells after 2 days are shown on the Y-axis. The TCID50 / mL of the pseudovirus calculated by the luciferase levels is shown in the following table. [Table 4]

[0115] Cell lines expressing ACE2 (e.g., Vero-E6, A549-ACE2-TMPRESS, and 293T-ACE2 cell lines) were differentially sensitive to infection by pseudoviruses (Figures 3A - 3C). Vero-E6 cells were less suitable for pseudovirus infection than the other cell lines evaluated, regardless of the identity of the expressed SARS-CoV-2 S protein (Figure 3C). The ability of pseudoviruses expressing SARS-CoV-2 S protein from the prototype or Wuhan strain to infect 293T-ACE2 cells and A549-ACE2-TMPRESS cells was similar (Figures 3A, 3B). However, the infectivity of the pseudovirus was higher in 293T / ACE2 cells when the pseudovirus expressed the SARS-CoV-2 S protein from the SARS-CoV-2 Omicron strain.

[0116] To compare the suitability of 293T / ACE2 and A549 / ACE2 cell lines for the pseudovirus neutralization assay (PNT), cells were infected with Omicron BA.1 and BA.5 pseudoviruses in the presence / absence of serially diluted serum (Lot number 2127), and then luciferase was measured as described in the method. Cells infected with pseudoviruses (BA.1 and BA.5) in the absence of the test serum (Lot number 2127) (virus control) and cells not infected with the pseudovirus (cell control) were used as controls. The percent (%) inhibition compared to the virus control is shown on the Y-axis. Both cell lines showed similar (overlapping) luciferase inhibition curves (ID50). Figures 4A - 4B show the ID50 of pseudovirus infection for 293T cells and A549 / ACE2-TMPRESS cells. The following table shows the comparable neutralization titers (ID50) of pseudoviruses for different serum samples. This data indicates that both of these cell lines are suitable for use in the pseudovirus neutralization assay.

Table 5

[0117] To evaluate the effects of dose and time in PNT, four test serum samples were evaluated in PNT using incremental amounts of pseudovirus (Prototype / Wuhan strain): 50, 125, and 250 TCID50 / well. The ID50 values of the sera were plotted on the Y-axis. A decreasing neutralizing ability was observed when measured by ID50 levels using the incremental amounts of pseudovirus used for infection. The following table shows the effects of pseudovirus dose (in the range of 50 - 3.13 μL / well) and kinetics (48-hour (48h) vs 72-hour (72h) assay endpoints) on the signal / background (S / B) levels in 293T / ACE2 cells.

Table 6

[0118] The 48-hour time point was selected for subsequent experiments as it was found to provide a better S / B ratio for all pseudovirus amounts and strains. Figures 5A - 5B show the luminescence output 48 hours (Figure 5A) and 72 hours (Figure 5B) after infecting the cells with pseudovirus. Three of the four serum samples showed a dose-dependence of the pseudovirus-based neutralizing titer (PNT ID50) (Figures 5A - 5B).

[0119] To evaluate the assay precision, quality control serum samples were evaluated in the PNT by two different operators on two different days. Duplicate samples were also tested in the same assay to evaluate the within-assay precision. The neutralization titers (ID50) were plotted on the Y-axis (GMT, 95% CI). Figures 6A - 6C show the between-assay and within-assay precision of the pseudovirus neutralization assay. Figure 6A shows the precision when the pseudovirus encodes the SARS-CoV-2 S protein from the prototype strain. Figure 6B shows the precision when the pseudovirus encodes the SARS-CoV-2 S protein from the Omicron BA.1 strain. Figure 6C shows the precision when the pseudovirus encodes the SARS-CoV-2 S protein from the Omicron BA.5 strain.

[0120] To evaluate the linearity of the PNT, test serum (serum number 2127) was serially diluted (6 times, 4-fold dilution series), and then two different operators (1 and 2) evaluated it in duplicate in the PNT (prototype / Wuhan strain) at each dilution as described in the method. The neutralization titers (ID50) of different dilutions were plotted on the Y-axis. Figures 7A - 7B show the dose-proportional neutralization by serum dilution by different operators (Figure 7A - operator number 1, Figure 7B - operator number 2). Assay linearity was demonstrated for the prototype / Wuhan strain-based pseudovirus (for two operators, R 2 = 0.9978 and 0.9764).

[0121] Figure 8 shows that the results from the pseudovirus neutralization assay are significantly correlated with the live virus MN assay for the prototype / Wuhan strain (Pearson's r = 0.9304, R 2= 0.8657, P < 0.0001). Clinical study-derived test serum samples (n = 13) were evaluated for PNT against the prototype / Wuhan strain according to the method mentioned above, and then regression analysis was performed by a validated live virus microneutralization assay. Analyses were performed using GraphPad Prism software (9.3.1). The dotted line indicates the 95% confidence interval (CI). The Pearson correlation coefficient (Pearson's r) and two-sided P value are shown in the figure.

[0122] Figures 9A - 9B show that the ability of serum samples to resist pseudovirus infection correlates with the amount of anti-SARS-CoV-2 S protein immunoglobulin (Pearson's r = 0.7133, R 2 = 0.5088, P = 0.0028) (Figure 9A) and the hACE2 binding inhibitory titer (Pearson's r = 0.8949, R 2 = 0.8009, P = 0.0027) (Figure 9B). Analyses were performed using GraphPad Prism software (9.3.1). The dotted line indicates the 95% confidence interval (CI). The Pearson correlation coefficient (Pearson's r) and two-sided P value are shown in the figure.

[0123] Figures 10A - C show the neutralization titers (ID50) for sera from patients 1 and 2 (Figure 10A), patients 3 and 4 (Figure 10B), and patients 6 and 7 (Figure 10C). Figure 10D shows the neutralization titers for patient number 5 14 days after administration of the primary NVX-CoV2373 vaccine series (''2×'' in Figure 10D) and after administration of the NVX-CoV2373 booster (''3×'' in Figure 10D). The primary NVX-CoV2373 vaccine series was administered on days 0 and 21. Sera were collected 28 days after the booster dose, shown as 3× on the X-axis [prime series (days 0 and 21) + booster (day 189)].

[0124] Abbreviations: GMT, geometric mean titer; hACE2, human angiotensin-converting enzyme 2; HQC, high-quality control sample; IgG, immunoglobulin G; LQC, low-quality control sample; MN, microneutralization; MQC, medium-quality control sample; N / A, not applicable; NC, negative control; PNT, pseudovirus neutralization assay; RLU, relative light unit; rS, recombinant spike protein; R2, coefficient of determination; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SD, standard deviation; S-protein, spike protein.

[0125] Conclusion: The pseudovirus-based neutralization assay described herein for determining the immunogenicity of COVID-19 vaccines provides a cost-effective high-throughput alternative to BSL3-based microneutralization assays with a rapid turnaround, enabling the discovery and development of effective vaccines against emerging COVID-19 variants. (Example 2) Multiplexed detection of neutralizing antibody responses against different SARS-CoV-2 strains on the same assay plate using a multiplexed pseudovirus assay

[0126] Introduction: Due to the continuous evolution of SARS-CoV-2 worldwide, different variants have been observed to spread. Given the limited amount of serum collected from human subjects in either vaccine clinical trials or from convalescent or SARS-CoV-2-infected subjects, it is difficult to simultaneously measure neutralizing antibody responses against multiple variants. Separate measurements of neutralizing antibodies against each variant increase costs, analytical turnaround time, and the complexity of the logistics to make sample analysis and interpretation clinically meaningful.

[0127] Method: To address this issue, a multiplex pseudovirus neutralization assay was developed using pseudoviruses expressing spike proteins and different reporter proteins (RFP and GFP) for different strains of SARS-CoV-2 (e.g., for the prototype Wuhan, Omicron BA.1, and BA5 strains). Subsequently, the pseudovirus neutralization assay was optimized, and serum samples were evaluated as positive for neutralizing antibodies against SARS-CoV-2.

[0128] Results: In the first set of experiments, pseudoviruses expressing the Omicron BA.1 spike protein with the RFP reporter protein (BA.1-RFP) and the Omicron BA.5 spike protein with the GFP reporter protein (BA.5-GFP) were prepared. Subsequently, serial dilutions (using a 3-fold dilution series, ranging from 2-fold to 54-fold dilution) were used to infect HEK293T / ACE2 cells (1.25×10 4 cells / well) in a 96-well plate, either alone or in combination. The cells were incubated at 37 °C and 5% CO2 for 72 hours, and then fluorescence for GFP and RFP was measured using two different plate readers (Celigo and ID3 plate readers). Our observations suggested that fluorescence of both reporter proteins (RFP and GFP) could be observed in the same wells of a 96-well plate without / with little interference (Figures 11A - 11D).

[0129] To further test the feasibility of a multiplexed approach for measuring neutralizing antibody responses in serum samples, convalescent serum samples from commercial sources were heat inactivated (56 °C for 30 minutes) and incubated at 37 °C for 2 hours with pseudoviruses expressing SARS-CoV-2 S proteins from different strains and different reporters (BA.1 RFP and Wuhan-GFP), either alone or in combination. Serum-pseudovirus mixtures were used to infect 293T / ACE2 cells in duplicate in 96-well plates, and then, after 72 hours, fluorescence for RFP and GFP was measured. The following table shows the percent neutralization observed in individual pseudovirus neutralization tests (PNTs) for either Omicron BA.1 RFP and BA.5 GFP and both of them together.

Table 7

[0130] Data from this experiment demonstrated the feasibility of measuring neutralizing activity present in human serum samples in a multiplexed pseudovirus assay in the same well. Some of the advantages of this multiplex assay are: 1. Low volume of test serum sample requirement. 2. Reduced assay cost (reagents and labor). 3. Rapid turnaround time. 4. Improved efficiency of neutralization assay measurement. (Example 3) Verification of Pseudovirus-Based Neutralization Assay

[0131] Objective: To verify a pseudovirus-based neutralization assay and demonstrate its appropriateness for the clinical samples tested.

[0132] Method: Serum from patients immunized against SARS-CoV-2 was heat-inactivated at 56 °C for approximately 30 minutes. As a negative control, patient serum collected prior to the outbreak of the SARS-CoV-2 outbreak was utilized. Positive controls were human sera with high, intermediate, and low pseudovirus neutralization (PNT) titers. Each heat-inactivated serum sample was diluted 1:10 in infection medium prior to use in the assay. Pseudovirus was added to the samples and incubated to allow virus-specific neutralizing antibodies to neutralize the virus. The pseudovirus was replication-defective Moloney murine leukemia virus (MLV).The pseudovirus expressed one of the following SARS-CoV-2 S glycoproteins: (i) the SARS-CoV-2 S glycoprotein in which amino acid 601 is glycine compared to the glycoprotein of SEQ ID NO: 2 (referred to as the "Wuhan D614 pseudovirus"); (ii) compared to the glycoprotein of SEQ ID NO: 2, amino acid 54 is V, amino acid 56 is deleted, amino acid 57 is deleted, amino acid 82 is I, amino acid 129 is D, amino acid 130 is deleted, amino acid 131 is deleted, amino acid 132 is deleted, amino acid 198 is deleted, amino acid 199 is I, the tripeptide EPE is inserted after amino acid 201, amino acid 534 is K, amino acid 601 is G, amino acid 642 is Y, amino acid 666 is K, amino acid 668 is H, amino acid 751 is K, amino acid 783 is Y, amino acid 843 is K, amino acid 941 is H, amino acid 956 is K, amino acid 968 is F, amino acid 326 is D, amino acid 358 is L, amino acid 360 is P, amino acid 362 is F, amino acid 404 is N, amino acid 427 is K, amino acid 433 is S, amino acid 464 is N, amino acid 465 is K, amino acid 471 is A, amino acid 480 is R, amino acid 485 is R, amino acid 488 is Y, amino acid 492 is Y, the SARS-CoV-2 S glycoprotein (referred to as the "BA.5 pseudovirus"); and (iii) the SARS-CoV-2 S glycoprotein of the Omicron XBB.1.5 subtype (referred to as the "XBB.1.5 pseudovirus").

[0133] HEK293T cells expressing ACE2 (HEK293T / ACE2 cells) were added to a mixture of the pseudovirus and the sample and incubated for 3 days to infect the HEK293T / ACE2 cells with the non-neutralizing virus. On the 4th day, the cells were lysed and the luminescence (relative light units) of the cells was measured to determine the level of infection that occurred in the presence of the antibody in the serum. The parameters of assay accuracy, specificity, LLOQ, and ULOQ were evaluated.

[0134] Precision: Precision is the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple tests on the same sample. Total precision or overall precision is composed of within-assay precision components and between-assay precision components. Within-assay (intra-run) precision is the closeness of multiple determinations of a single sample within one assay run under the same operating conditions over a short time interval. Between-assay (inter-run) precision is the closeness of repeated measurements within the laboratory considering all relevant sources of variation that affect the results (e.g., runs, analysts, equipment, and reagents).

[0135] The within-assay and between-assay precision of the PNT assay was determined for each pseudovirus using a panel of 40 serum samples ranging from negative, low PNT titers to high PNT titers. Assay precision data were generated by two analysts in six runs on three different days, and each sample was tested twice in each run. A total of 24 results were generated from the six runs and used for precision evaluation. For nine samples, the values available for precision evaluation were less than 24 (16 - 23) because the titers could not be generated or the sample volume was insufficient to test in all runs.

[0136] The within-assay and between-assay precision were estimated by calculating the percent geometric coefficient of variation (%GCV) using a variance components analysis model with the sample as a fixed effect and the analyst and day as random effects.

[0137] The within-assay and between-assay %GCV were calculated based on the natural logarithm-transformed values of the PNT titers:

Equation

Equation

[0138] Within-assay, between-assay, and total assay precision were evaluated at the individual sample level and also at the stock level, which is the overall assay variance of all 40 samples tested.

[0139] Specificity / Selectivity: Specificity is the ability of an analytical method to measure and identify an analyte in the presence of components that may be expected to be present. Selectivity is the degree to which the method can determine a specific compound in the analyzed matrix without interference from matrix components. The selectivity of the method is demonstrated by analyzing negative control samples of appropriate biological matrices (e.g., serum) from multiple sources. Negative control human serum samples from the period before the SARS-CoV-2 pandemic included sera from influenza vaccine studies and RSV vaccine studies that showed very strong specific immune responses against influenza virus and RSV F protein, as well as normal human serum samples collected in the pre-SARS-CoV-2 pandemic era, and they were tested.

[0140] Linearity: The linearity of an analytical method measures its ability to obtain test results that are directly proportional to the concentration (amount) of the analyte in the sample (within a given range). The linearity of the PNT assay in the Wuhan prototype virus was evaluated by testing two SARS-CoV-2 PNT positive samples diluted in undiluted and negative sera. The number of dilutions and dilution factors were dependent on the titer of the undiluted serum (i.e., 1:2, 1:8, and more or fewer dilutions were performed if appropriate and feasible). The linearity samples were tested in duplicate by two different analysts over a total of six runs over 3 days, with two tests in the same run. The samples were diluted independently for each dilution in negative serum and then heat-inactivated.

[0141] Simple linear regression was performed on the dataset. The independent variable was the log10 predicted PNT titer, and the dependent variable was the log10 observed PNT titer. The point estimate of the slope and the 95% confidence interval (CI) as well as the coefficient of determination (R 2 ) of the regression line were evaluated.

[0142] The predicted PNT titers at each dilution were calculated by dividing the overall GMT from all runs of the undiluted sample by the dilution factor for each dilution of each sample. The observed PNT titers at each dilution were the overall GMTs at each dilution for each sample across all runs.

[0143] The accuracy of the analytical procedure is defined as the closeness of agreement between the detected value and the value accepted as either the conventional true value or an accepted reference value. Accuracy was evaluated in the linearity study by comparing the predicted and observed values and calculating the percent relative bias. Percent relative bias at each dilution:

[0144]

Number

[0145] A percent relative bias of 150% or -60% corresponds to being 2.5 times higher or 2.5 times lower, respectively, than the predicted titer.

[0146] Lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the PNT assay: The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the PNT assay are the lowest and highest PNT titers, respectively, that can be quantitatively determined with acceptable precision and accuracy. The LLOQ and ULOQ were determined using samples for linearity evaluation. Precision was estimated by calculating the %GCV of the PNT titers across runs for these samples, and accuracy was estimated by calculating the percent relative bias across runs for these samples. The precision and accuracy for LLOQ determination were evaluated at dilutions where the predicted PNT titers were between 20 and 100.

[0147] Results: Precision: The following table contains the precision of the pseudovirus-based neutralization assay for pseudoviruses expressing each SARS-CoV-2 S protein.

Table 8-1

Table 8-2

[0148] The overall precision was calculated by analyzing the assay variance from all 40 samples using the Wuhan D614 prototype virus. The between-assay, within-assay, and total assay %GCV were 6.6%, 42.8%, and 43.4%, respectively, for the pseudovirus neutralization assay utilizing pseudovirus expressing the Wuhan D614 SARS-CoV-2 S protein. At the individual sample level, among all 40 samples, the lowest %GCV for between-assay, within-assay, and total assay precision (inter-, intra-, and total assay precision) was 0.0%. The highest %GCV for between-assay, within-assay, and total assay precision were 60.5%, 59.8%, and 76.1%, respectively. 95% (38 out of 40), 90% (36 out of 40), and 65% (26 out of 40) of the samples showed between-assay, within-assay, and total assay %GCV (inter-, intra-, and total %GCV) less than 50, respectively; 97.5% (39 out of 40), 100% (40 out of 40), and 85% (34 out of 40) of the samples showed between-assay, within-assay, and overall assay %GCV (inter-, intra-, and overall %GCV) less than or equal to 60, respectively. It is acceptable that at least 80% of the samples have a %GCV less than 60 for the Wuhan D614 virus.

Table 9-1

Table 9-2

[0149] Overall precision was calculated by analyzing assay variance from all 40 samples using the BA.5 subvariant pseudovirus. The overall between-assay, within-assay, and total assay %GCV were 15.1%, 26.4%, and 30.7%, respectively. At the individual sample level, among all 40 samples, the lowest %GCV for between-assay, within-assay, and total assay precision was 0.0%. The highest %GCV for between-assay, within-assay, and total assay precision were 63.1%, 48.3%, and 71.5%, respectively. Among 39 out of 40 (98%), all 40 (100%) and 38 out of 40 (95%) samples showed between-assay, within-assay, and overall assay %GCV less than 50, respectively. The assay precision met the target acceptance criteria.

Table 10-1

Table 10-2

[0150] Overall precision was calculated by analyzing assay variance from all 40 samples using the XBB.1.5 subvariant pseudovirus. The overall between-assay, within-assay, and total assay %GCV were 20.0%, 29.5%, and 36.1%, respectively. At the individual sample level, among all 40 samples, the lowest %GCV for between-assay, within-assay, and total assay precision was 0.0%. The highest %GCV for between-assay, within-assay, and total assay precision were 65.7%, 55.9%, and 72.1%, respectively. Among 38 out of 40 (95%), 39 out of 40 (97.5%) and 32 out of 40 (80%) samples showed between-assay, within-assay, and overall assay %GCV less than 50, respectively. The assay precision met the target acceptance criteria.

[0151] Results - Specificity: This assay demonstrated specificity for detecting neutralizing antibodies against SARS-CoV-2 antigens. Human sera collected in the pre-SARS-CoV-2 pandemic era had no detectable neutralizing antibodies (<LLOQ) against the Wuhan D614 pseudovirus, the XBB1.5 pseudovirus, or the BA.5 pseudovirus. Furthermore, 5 pairs of pre- and post-vaccination sera from the RSV F vaccine Phase 3 clinical trial RSV-M-301, and 5 pairs of pre- and post-vaccination sera from the nanoparticle influenza vaccine Phase 3 clinical trial qNIV-E-301 collected prior to the SARS-CoV-2 pandemic were tested in the PNT assay. All samples were tested negative (<LLOQ) in the Wuhan D614, XBB1.5, and BA.5 PNT assays, but had a very strong specific immune response against influenza or RSV after vaccination, demonstrating the specificity of the SARS-CoV-2 PNT assay.

[0152] Results - Linearity: The percent relative bias, LLOQ, and ULOQ of the false neutralizing antibody titers were determined for each pseudovirus. The accuracy of the PNT assay was evaluated by the percent relative bias (%RB). The precision was determined by the %GCV of the values from all runs for the dilution points.

Table 11-1

Table 11-2

Table 12

[0153] The above table shows the linearity test data for the Wuhan D614 prototype pseudovirus assay. The linearity test data for the Wuhan D614 prototype pseudovirus assay showed an acceptable linear response. The %RB for all three HMN865539 dilution points with predicted GMTs above 20 was within the target range (-60% to 150%); the %RB for all six HMN865536 dilution points with predicted GMTs above 20 was within the target range. The %GCV for 6 out of 8 (75%) dilution points for sample HMN865539 was <60, and the %GCV for 7 out of 8 (87.5%) dilution points for sample HMN865536 was <60. The point estimates of the slopes of the linear regression plots for samples HMN865539 and HMN865536 in the linearity test were 0.801 and 1.000, respectively. The lower confidence limit (LCL) and upper confidence limit (UCL) of the 95% confidence interval for the slope for sample HMN865539 were 0.521 to 1.080, outside the target range of 0.7 to 1.43. This can be attributed to the lower titer and fewer dilution points (only four dilution points) of the samples used in the regression plot. For sample HMN865536, which had seven dilution points for the regression plot, the 95% LCL and UCL were 0.847 to 1.153, within the acceptable range of 0.7 to 1.43. The R of the regression plot 2For samples HMN865539 and HMN865536, they were 0.987 and 0.983 respectively. The linearity test data demonstrated that an acceptable assay linear response, and that this method could measure the Wuhan prototype pseudovirus neutralizing antibody titer with acceptable precision and relative accuracy. At 20 and predicted PNT titers above it, %RB was within a difference of 2.5 times the predicted titer (-60% to 150%), within -42.6% and 75.8% for both samples. The %GCV at the predicted GMT of 41.6 for sample HMN865539 was 60.5 and 39.6 for between-assay and within-assay precision respectively, but the total %GCV was higher (76.1). For sample HMN865536, at the predicted GMT of 29.0 and the observed GMT of 41.6, the between-assay and within-assay %GCV were 0 and 46.1 respectively, and the total %GCV was 46.1. Therefore, a GMT of 42 has acceptable precision (%GCV ≤ 60) and relative accuracy (%RB between -60% and 150%) and is set as the assay LLOQ. The highest PNT GMT titer is 14863.2, and the between-assay, within-assay and total assay GCV are less than 60. Therefore, the provisional ULOQ of the assay is 14863 for the PNT assay using the Wuhan prototype virus. The assay ULOQ can be further evaluated during clinical tests when higher-titer serum samples become available.

Table 13

Table 14

[0154] The above table shows the linearity test data for the BA.5 pseudovirus assay. The linearity analysis of the BA.5 PNT assay was performed using two serum samples. The point estimates of the slopes of all linear regression plots in the linearity test were 1.063 and 1.107, respectively. The lowest lower confidence limit (LCL) of the 95% confidence interval of the slope was 0.872. The highest upper confidence limit (UCL) of the 95% confidence interval of the slope was 1.256. The slopes for both samples passed the acceptance criteria of 95% CI 0.7 to 1.43. The R 2 values were 0.984 and 0.987, respectively. The percentage relative bias (%RB) at six dilutions for HMN934977 was between -60% and 150%, passing the acceptance criteria. Six out of seven dilutions (85.7% of the samples) of ZA018 - 0389 had %RB between -60% and 150%. This met the acceptance criteria. Twelve out of the 13 samples (dilutions) tested showed percent relative bias between -60% and 150%, and the slopes and R 2 values from the two linearity samples passed the acceptance criteria. Therefore, this method is considered capable of measuring Omicron BA.5 pseudovirus neutralization within 2.5 times the expected PNT titer. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the PNT assay are the lowest and highest PNT titers that can be quantitatively determined with acceptable precision and accuracy, respectively. The LLOQ and ULOQ were determined using samples for linearity evaluation. Precision was estimated by calculating the %GCV of the PNT titer over runs for these samples, and accuracy was estimated by calculating the percent relative bias over runs for these samples. The precision and accuracy for LLOQ determination were evaluated at dilutions where the expected PNT titer was between 20 and 100.

[0155] The accuracy and precision of two linearity samples for the BA.5 virus PNT assay were evaluated. At predicted PNT titers of 20 and above, the percent relative bias was within 2.5-fold differences (-60% to 150%) of the predicted titer, and within -55% and -8.2% for both samples, except for one sample that had a relative bias of -62.7% (12 out of 13). For 14 out of 15 dilutions (from both samples), the %GCV was <50.0%. The lowest predicted PNT GMT titers above 20 were 27.2 (from HMN934977) and 21.9 (from ZA018-0389), respectively. These were related to HMN934977 1:192 and ZA018-0389 1:1280 dilutions. At these two dilution levels, 91.3% (21 out of 23) of the GMT titers (Appendix 3) were below 20. When calculating %GCV and %RB, these were defined as 20 and thus had the lowest variability among all. These should not be defined as the assay LLOQ. The lowest observed GMT titer with reliable %GCV and %RB was 35.5. The highest PNT GMT titer with %GCV <50% and %RB between -60 and 150 was 15856.3. %GCV ≤ 50% is considered acceptable for this type of semi-quantitative assay. Thus, for the PNT assay using the BA.5 virus, 36 LLOQs and 15856 ULOQs are acceptable. The assay ULOQ can be further evaluated during clinical testing if higher titer serum samples become available.

Table 15-1

Table 15-2

Table 16

[0156] The above table shows the linearity test data for the XBB.1.5 pseudovirus assay. The linearity analysis of the BA.5 PNT assay was performed using two serum samples. The linearity analysis of the XBB.1.5 PNT assay was performed using two serum samples. The point estimates of the slopes of all linear regression plots in the linearity test were 1.179 and 1.186, respectively. The lower confidence limit (LCL) and upper confidence limit (UCL) of the slope for HMN934977 were 1.011 and 1.361, respectively. These met the target acceptance criteria (0.70 - 1.43). The LCL and UCL of the slope for HMN865550 were 0.879 and 1.479, respectively. The UCL was slightly higher than the target acceptance criteria (1.43) but was considered acceptable (about 32% higher instead of 30% higher than the point estimate of the slope). The R 2 values of the regression plots were 0.981 and 0.989, respectively. The percent relative bias (%RB) for HMN865550 was between -60% and 150%, meeting the acceptance criteria. Five out of six dilutions of HMN934977 (83.3% of the samples) had a %RB between -60% and 150%. This met the acceptance criteria. Eleven out of the twelve samples tested showed a percent relative bias between -60% and 150%, with the slopes and R 2Since it passed the acceptance criteria, this method is considered capable of measuring Omicron XBB.1.5 pseudovirus neutralization within 2.5-fold of the expected PNT titer. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the PNT assay are the lowest and highest PNT titers, respectively, that can be quantitatively determined with acceptable precision and accuracy. The LLOQ and ULOQ were determined using samples for linearity evaluation. Precision was estimated by calculating the %GCV of the PNT titer over runs for these samples, and accuracy was estimated by calculating the percent relative bias over runs for these samples. The precision and accuracy for LLOQ determination were evaluated at dilutions where the expected PNT titer was between 20 and 100.

[0157] A summary of the accuracy and precision of two linearity samples for the XBB.1.5 virus PNT assay is described. At a dilution of 216 for sample HMN934977, the GMT titers of 10 out of 12 (83.3%) were <20 and were defined as 20 for calculation. Thus, the %GCV was low. The PNT GMT titer (20.6) at this dilution was not appropriate to define as the LLOQ. The next lowest observed GMT titer was 35.5. The inter-assay and total %GCV were 53.4 and 60.8%, respectively, and these did not meet the target acceptance criteria (%GCV < 50). At a dilution of 144 for HMN865550, all 12 GMT titers were <20, and at a dilution of 72 for HMN865550, 11 GMT titers were <20 and were defined as 20 for calculation. Thus, the %GCV was low. The PNT GMT titers (20.0) at these two dilutions were not appropriate to define as the LLOQ. The next lowest GMT titer was 37.4. The inter-assay and intra-assay precision met the acceptance criteria (%GCV < 50), but the total %GCV did not meet the criteria at 50.6%. Since the GMT was below 100, it was close to the LLOQ. A GCV of 50.6% is acceptable. Thus, a GMT titer of 37 was defined as the assay LLOQ. The highest PNT GMT titer was 7560.9, and the %GCV (inter-, intra-, and total assay) was less than 50%. A %GCV ≤ 50% is considered acceptable for this type of semi-quantitative assay. A ULOQ of 7561 is acceptable for the PNT assay using the XBB.1.5 virus. The assay ULOQ can be further evaluated during clinical testing if higher titer serum samples become available.

[0158] Conclusion: The results described indicate that the pseudo-neutralization assay described herein is appropriate for measuring neutralizing antibodies induced by SARS-CoV-2 and its variants.

[0159] The results obtained from the assay validation experiments demonstrated that the pseudovirus neutralization assay in the Wuhan D614 prototype virus was highly accurate and precise in measuring the PNT titers in human sera with overall assay %GCV ≤ 60% and percent relative bias within -60% and 150%. The PNT assay functions in a reliable and reproducible manner for the semi-quantitative assessment of PNT antibody titers. This method demonstrated specificity for the SARS-CoV-2 virus used in the validation. This method is linear with respect to antiserum dilution. LLOQ and ULOQ were established when the sample %GCV was below 60 and the sample fit was within the linear range. Therefore, this method is appropriate for the analysis of samples in clinical trials of SARS-CoV-2 vaccines against the Wuhan D614 prototype virus for the evaluation of immunogenicity.

[0160] The results obtained from the assay validation experiments demonstrated that the pseudovirus neutralization assay in Omicron BA.5 was highly accurate and precise in measuring the PNT titers in human sera with overall assay %GCV ≤ 50% and percent relative bias within -60% and 150%. The PNT assay functions in a reliable and reproducible manner for the semi-quantitative assessment of PNT antibody titers. This method demonstrated specificity for the SARS-CoV-2 virus used in the validation. This method is linear with respect to antiserum dilution and LLOQ, and ULOQ was established when the sample %GCV was below 50 and the sample fit was within the linear range. Therefore, this method is appropriate for the analysis of samples from patients administered with SARS-CoV-2 vaccines against Omicron BA.5 for the evaluation of immunogenicity.

[0161] The results obtained from the assay validation experiments demonstrated that the pseudovirus neutralization assay in Omicron XBB.1.5 was highly accurate and precise in measuring the PNT titer in human sera with an overall assay %GCV ≤ 50% and percent relative bias within -60% and 150%. The PNT assay functions in a reliable and reproducible manner for the semi-quantitative assessment of PNT antibody titers. This method demonstrated specificity for the SARS-CoV-2 virus used in the validation. This method was linear with respect to antiserum dilution and LLOQ, and established the ULOQ when the sample %GCV was below 50 and the sample fit was within the linear range.

[0162] 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 a reference, paper, publication, patent, patent publication, or 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 form 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

1. (i) SARS-CoV-2 spike (S) glycoprotein; (ii) The target gene that codes for the reporter protein and This includes replication-deficient mouse leukemia virus (MLV) pseudovirus.

2. The pseudovirus according to 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 with any one polypeptide of SEQ ID NOs: 1-13, 15, and 17-66.

3. The pseudovirus according to claim 1, wherein the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site.

4. The pseudovirus according to claim 1, wherein 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.

5. The SARS-CoV-2 S glycoprotein is derived from the SARS-CoV-2 virus or a variant of SARS-CoV-2, and 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. The pseudovirus according to claim 1, wherein the strain is 1.621 SARS-CoV-2; or B. 1.1.529 SARS-CoV-2.

6. The pseudovirus according to 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 with respect to the SARS-CoV-2 S glycoprotein derived from a SARS-CoV-2 S omicron variant selected from the group consisting of BA. 1, BA. 2.12.1, BA. 2, BA. 3, BA. 4, BA. S, XBB. 1.5, XBB. 2.3, and XBB. 1.

16.

7. A method for producing a pseudovirus according to any one of claims 1 to 6, wherein a host cell contains (i) an import plasmid encoding a reporter protein; (ii) One or more plasmids encoding Rev protein, Tat protein, Gag protein, Pol protein, or a combination thereof; and (iii) A method comprising the step of transfecting a plasmid encoding a SARS-CoV-2 spike (S) glycoprotein.

8. The method according to claim 7, wherein the host cell is human, or the host cell is HEK293T (also known as "293T") cell.

9. A method for determining whether a biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or its variants, (a) The step of contacting a biological sample with one to about 20 pseudoviruses as described in any one of claims 1 to 6; (b) A step of bringing cells expressing angiotensin-converting enzyme 2 (ACE2) into contact with the 1 to about 20 types of pseudoviruses; (c) A step of quantifying the expression of the reporter protein in the cells expressing ACE2; (d) A step of contacting control cells expressing ACE2 with the 1 to about 20 pseudoviruses, wherein the 1 to about 20 pseudoviruses have not been in contact with the biological sample; and (e) A step of quantifying the expression of the reporter protein in the control cells expressing ACE2. Includes, A method wherein, if the expression of the reporter protein in the cells of (c) is lower than the expression of the reporter protein in the control cells of (e), the biological sample contains a neutralizing antibody against the SARS-CoV-2 virus or a variant thereof.

10. The method according to claim 9, wherein the cells are selected from the group consisting of Vero E6, A549, and HEK293T cells.

11. A method for determining whether a biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or its variants, (a) A biological sample containing thermally inactivated plasma, A step of forming a sample-pseudovirus mixture by contacting a replication-deficient Moloney mouse leukemia virus (MLV) pseudovirus, wherein the MLV pseudovirus comprises a SARS-CoV-2 S glycoprotein and a target gene (GOI) encoding a reporter protein; (b) Adding cells expressing angiotensin-converting enzyme 2 (ACE2) to the sample-pseudovirus mixture, wherein the cells are A549 or HEK293T cells; (c) Incubating the sample-pseudovirus mixture with the cells expressing ACE2 at 37°C, and quantifying the expression of the reporter protein in the ACE2-expressing cells for 48 to 72 hours of incubation; (d) A step of forming a virus-only control by contacting control A549 or HEK293T cells expressing ACE2 with the MLV pseudovirus, wherein the MLV pseudovirus has not been in contact with the biological sample; and (e) The steps of incubating the virus-only control together with the control cells at 37°C, and quantifying the expression of the reporter protein in the control cells expressing ACE2 for 48 to 72 hours of incubation. Includes, A method wherein, if the expression of the reporter protein in the cells of (c) is lower than the expression of the reporter protein in the control cells of (e), the biological sample contains a neutralizing antibody against the SARS-CoV-2 virus or a variant thereof.

12. A method for simultaneously determining whether a biological sample contains neutralizing antibodies against two or more species of SARS-CoV-2 virus or its variants, (a) A step of contacting a biological sample containing heat-inactivated plasma with a first replication-deficient MLV pseudovirus and a second replication-deficient MLV pseudovirus to form a sample-pseudovirus mixture, wherein the first MLV pseudovirus comprises a SARS-CoV-2 S glycoprotein derived from a first SARS-CoV-2 virus or a variant thereof and a GOI encoding a first reporter protein, and the second MLV pseudovirus comprises a SARS-CoV-2 S glycoprotein derived from a second SARS-CoV-2 virus or a variant thereof and a GOI encoding a second reporter protein; (b) A step of adding cells expressing ACE2 to the sample-pseudovirus mixture, wherein the cells are A549 or HEK293T cells; (c) Incubating the sample-pseudovirus mixture with the cells expressing ACE2 at 37°C, and quantifying the expression of the first reporter protein and the second reporter protein in the cells expressing ACE2 for 48 to 72 hours of incubation; (d) A step of forming a virus-only control by contacting control A549 or HEK293T cells expressing ACE2 with the first MLV pseudovirus and the second MLV pseudovirus, wherein the first MLV pseudovirus and the second MLV pseudovirus are not in contact with the biological sample; and (e) Incubating the virus-only control together with the control cells at 37°C, and quantifying the expression of the first reporter protein and the second reporter protein in the control cells expressing ACE2 for 48 to 72 hours of incubation. Includes, If the expression of the first reporter protein in the cells of (c) is lower than the expression of the first reporter protein in the control cells of (e), then the biological sample contains a neutralizing antibody against the first SARS-CoV-2 virus or its variant. A method wherein, if the expression of the second reporter protein in the cells of (c) is lower than the expression of the second reporter protein in the control cells of (e), the biological sample contains a neutralizing antibody against the second SARS-CoV-2 virus or a variant thereof.

13. The method according to claim 11 or 12, wherein the first SARS-CoV-2 virus or its variant is an omicron variant selected from the group consisting of BA. 1, BA. 2.12.1, BA. 2, BA. 3, BA. 4, BA. S, XBB. 1.5, XBB. 2.3, and XBB. 1.

16.

14. The method according to claim 12, wherein the second SARS-CoV-2 virus or its variant is an omicron variant selected from the group consisting of BA. 1, BA. 2.12.1, BA. 2, BA. 3, BA. 4, BA. S, XBB. 1.5, XBB. 2.3, and XBB. 1.

16.

15. The method according to claim 11 or 12, wherein the SARS-CoV-2 S glycoprotein has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

16. The method according to claim 11 or 12, wherein amino acids 973 and 974 of the SARS-CoV-2 S glycoprotein are proline, compared to wild-type SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 2.

Images