Pseudovirus-based neutralization assays for evaluating vaccine immunogenicity

Abstract

Provided herein is a pseudovirus that expresses an SARS-CoV-2S glycoprotein. Also provided herein are assays that employ the pseudoviruses to evaluate the immunogenicity of a biological sample against the SARS-CoV-2 virus or a variant thereof. Also provided herein are methods of evaluating the immunogenicity of a COVID-19 vaccine using the assay.

Description

[0001] Citation of Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 392,397, filed on July 26, 2022, and U.S. Provisional Application No. 63 / 407,371, filed on September 16, 2022. These applications are hereby incorporated by reference in their entirety for all purposes.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (NOVV-101_02WO_SeqList_ST26.xml; size: 203,198 bytes; and creation date: July 26, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure generally relates to pseudoviruses expressing the SARS-CoV-2 S glycoprotein. The present disclosure also relates to assays that use pseudoviruses to evaluate the immunogenicity of biological samples against the SARS-CoV-2 virus or variants thereof (e.g., whether the biological sample contains neutralizing antibodies). The present disclosure also relates to methods for evaluating the ability of COVID-19 vaccines to stimulate an immune response against the SARS-CoV-2 virus or variants thereof using the assays. Background Art

[0006] Neutralizing antibody responses induced by COVID-19 vaccines play a key role in conferring protection against the 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 measures include restricted / controlled laboratory access, medical monitoring and / or immunization of personnel, appropriate laboratory design (including automatically closing and locking doors, continuous directional airflow, and sink / eyewash stations), use of appropriate biosafety cabinets, and appropriate personal protective equipment (which may include respirators).

[0007] Due to these safety precautions, BSL-3 laboratories are expensive to operate and difficult to access. There is a need in the art for new assays that enable the measurement of SARS-CoV-2 neutralizing antibodies outside of BSL-3 laboratories. Summary of the invention

[0008] A pseudovirus-based neutralization assay for measuring neutralizing antibodies against SARS-CoV-2 virus is provided herein. The assay described herein can be performed in a biosafety level 2 (BSL-2) laboratory. Therefore, the assay is more cost-effective than conventional assays that require the use of SARS-CoV-2 virus.

[0009] Provided herein is a pseudovirus encoding: (i) SARS-CoV-2 spike (S) glycoprotein; (ii) one or more of Rev, Tat, Gag, and Pol proteins; and (iii) a reporter protein.

[0010] Provided herein is a method for producing a pseudovirus, comprising transfecting cells 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 a SARS-CoV-2 spike (S) glycoprotein.

[0011] Provided herein is a method for measuring the ability of a biological sample to stimulate an immune response against the SARS-CoV-2 virus or a variant thereof, comprising:

[0012] (a) contacting the biological sample with a pseudovirus expressing the SARS-CoV-2 S glycoprotein and a reporter protein;

[0013] (b) contacting cells expressing angiotensin converting enzyme 2 with pseudovirus; and

[0014] (c) Quantification of reporter protein expression.

[0015] Provided herein is a pseudovirus encoding: (i) SARS-CoV-2 spike (S) glycoprotein; (ii) one or more of Rev, Tat, Gag, and Pol proteins; and (iii) a reporter protein. In embodiments, the pseudovirus encodes 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 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-2S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In embodiments, the SARS-CoV-2S glycoprotein has an inactive furin cleavage site. In embodiments, the SARS-CoV-2S glycoprotein has an inactive furin cleavage site having an amino acid sequence of QQAQ (SEQ ID NO: 68). In an embodiment, amino acids 973 and 974 of the SARS-CoV-2 S glycoprotein are proline compared to a 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 a polypeptide of any one of SEQ ID NOs: 3, 5-13, 15, 17-19, 21, and 23-66.In embodiments, the SARS-CoV-2 S glycoprotein 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 the polypeptide of any one of SEQ ID NOs: 3-13, 20, and 22-66. In embodiments, the SARS-CoV-2 S glycoprotein 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 the polypeptide of any one of SEQ ID NOs: 3, 5-13, and 23-66. In embodiments, the SARS-CoV-2 S glycoprotein is from a SARS-CoV-2 virus or a variant of SARS-CoV-2. In the implementation scheme, the variant of SARS-CoV-2 is B.1.1.7SARS-CoV-2 strain; B.1.351SARS-CoV-2 strain; P.1SARS-CoV-2 strain; Cl.20C SARS-CoV-2 strain; B.1.617.2SARS-CoV-2 strain; B.1.525SARS-CoV-2 strain; B.1.526SARS-CoV-2 strain; B.1.617.1SARS-CoV-2 strain; C.37SARS-CoV-2 strain; B.1.621SARS-CoV-2 strain; or B.1.1.529SARS-CoV-2 strain. In embodiments, the SARS-CoV-2 S glycoprotein 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 a SARS-CoV-2 S glycoprotein from a SARS-CoV-2 S 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 embodiments, the pseudovirus comprises a Gag protein and a Pol protein. In embodiments, the fusion protein encodes a Gag protein and a Pol protein.In an embodiment, the Gag protein and the Pol protein are from Moloney murine leukemia virus (MLV).In an embodiment, the Rev, Tat, Gag and Pol proteins are from a lentivirus.

[0016] In embodiments, provided herein is a method for producing a pseudovirus as described herein, comprising 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, Pol protein; or a combination thereof; and (iii) a plasmid encoding the SARS-CoV-2 spike (S) glycoprotein. In embodiments, the host cell is a human cell. In embodiments, the host cell is a HEK293T (also referred to as a "293T" cell).

[0017] In embodiments, provided herein is a method for determining whether a biological sample contains neutralizing antibodies against a SARS-CoV-2 virus or a variant thereof, comprising: (a) contacting a biological sample with 1 to about 20 pseudoviruses as described herein; (b) contacting a cell expressing angiotensin-converting enzyme 2 (ACE2) with 1 to about 20 pseudoviruses; (c) quantifying the expression of a reporter protein in a cell expressing ACE2; (d) contacting a control cell expressing ACE2 with 1 to about 20 pseudoviruses, wherein 1 to about 20 pseudoviruses have not been contacted with the biological sample; and (e) quantifying the expression of a reporter protein in a control cell expressing ACE2; wherein if the expression of the reporter protein in the cell of (c) is lower than the expression of the reporter protein in the control cell of (e), the biological sample contains neutralizing antibodies against a SARS-CoV-2 virus or a variant thereof. In embodiments, the method comprises contacting the biological sample and the pseudovirus for 1 hour to about 7 hours. In embodiments, the method comprises contacting the biological sample and the pseudovirus for about two hours. In embodiments, the method comprises contacting the biological sample and the pseudovirus at about 37°C. In embodiments, the method includes heat-inactivating biological samples. In embodiments, the method includes contacting cells with pseudoviruses for 1 day to about 7 days. In embodiments, the method includes contacting cells with pseudoviruses for about 3 days. In embodiments, cells are selected from the group consisting of: Vero E6, A549 and HEK293T cells. In embodiments, cells are HEK293T cells. In embodiments, implementation of the method does not require a biosafety level 3 (BSL-3) laboratory. In embodiments, biological samples are saliva, nasopharyngeal swabs, sputum, saliva, urine, fecal samples, cerebrospinal fluid, synovial fluid, serum, blood, mucosal samples or plasma. In embodiments, biological samples are from patients who have been administered an immunogenic composition for SARS-CoV-2 virus or its variants. In embodiments, biological samples are from patients who have previously suffered from COVID-19. In embodiments, the method comprises 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 pseudoviruses. In embodiments, each pseudovirus expresses a different reporter gene and a different SARS-CoV-2 S glycoprotein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the pseudovirus neutralization assay of Example 1 is provided.

[0019] Figure 2A-2B The dependence of the luminescence readout in the pseudovirus neutralization assay of Example 1 on the amount of pseudovirus used for infection is shown. Figure 2AShown is the dependence of the luminescence readout on the amount of pseudovirus used for infection when the pseudovirus expresses the SARS-CoV-2 S protein from the prototype strain. Figure 2B The dependence of the luminescence reading on the amount of pseudovirus used for infection is shown when the pseudovirus expresses the SARS-CoV-2S protein from Omicron BA.1, BA.2 or BA.5 variants. HEK293T cells were infected with serial dilutions of pseudovirus expressing the SARS-CoV-2S protein of the prototype SARS-CoV-2 strain or the SARS-CoV-2S protein of SARS-CoV-2 variants (Omicron BA.1, BA.2, BA.5), and then the luciferase level was measured 2 days after infection. The dose-dependent luciferase level as the amount of pseudovirus used for infection decreased is shown in the figure.

[0020] Figure 3A-3C The pseudovirus of Example 1 was shown to infect A549 cells ( Figure 3A )、293T cells( Figure 3B ) and VeroE6 cells ( Figure 3C ) ability.

[0021] Figure 4A-4B The ID50 of pseudovirus infection of 293T cells and A549 / ACE2-TMPRESS cells is shown. Figure 4A The ID50 of pseudovirus infection in is for the pseudovirus expressing the Omicron BA.1S protein. Figure 4B The ID50 of pseudovirus infection in is for the pseudovirus expressing the Omicron BA.5S protein.

[0022] Figure 5A-Figure 5B It shows that 48 hours after the HEK293T cells expressing ACE2 in the experiment described in Example 1 were infected with pseudovirus ( Figure 5A ) and 72 hours ( Figure 5B )'s luminous output.

[0023] Figure 6A-6C The inter-assay and intra-assay precision of the pseudovirus neutralization assay of Example 1 is shown. Fig. 6A Showing the accuracy of pseudoviruses encoding SARS-CoV-2 S protein from the prototype strain. Figure 6B The accuracy of the pseudovirus encoding the SARS-CoV-2 S protein from the Omicron BA.1 strain is shown. Figure 6C The accuracy of the pseudovirus encoding the SARS-CoV-2 S protein from the Omicron BA.5 strain is shown.

[0024] Figure 7A-7B The pseudovirus-based neutralization assay of Example 1 is shown for two different operators (Operator No. Fig. 7A ) and operator 2 ( Figure 7B ))'s linearity.

[0025] Figure 8 The results of the pseudovirus neutralization assay of Example 1 showed significant correlation with the live virus MN assay for the prototype strain (Pearson r = 0.9304, R 2 =0.8657, P<0.0001). Test serum samples (n=13) from clinical studies were evaluated against the prototype strain in PNT as described above, followed by regression analysis using a validated live virus microneutralization assay. Analysis was performed using GraphPad Prism software (9.3.1). Dashed lines show 95% confidence intervals (CI). Pearson correlation coefficients (Pearson r) and two-tailed P values ​​are shown.

[0026] Figure 9A-9B The ability of serum samples to resist pseudovirus infection is shown to be related to the amount of anti-SARS-CoV-2 S protein immunoglobulin ( Fig.9A ) and hACE2 binding inhibition ( Fig. 9B ) related.

[0027] Figure 10A-10C Shown are samples from patients 1 and 2 ( Fig. 10A ), patients 3 and 4 ( Fig. 10B ) and patients 6 and 7 ( Fig. 10C )'s neutralizing titer (ID50). Fig. 10D Patient 5 was shown to have received the primary NVX-CoV2373 vaccine series ( Fig. 10D ) and after administration of the NVX-CoV2373 booster ( Fig. 10D Neutralization titer 14 days after (“3x” in the figure).

[0028] Figures 11A-11D Shown is the fluorescence (mean + / - standard deviation from duplicate wells) of ACE2-expressing 293T cells infected with a pseudovirus expressing the SARS-CoV-2 S glycoprotein and red fluorescent protein from the Omicron BA.1 strain ("BA.1RFP"), a pseudovirus expressing the SARS-CoV-2 S glycoprotein and green fluorescent protein from the Omicron BA.5 strain ("BA.5GFP"), or both pseudoviruses ("double adj"). Fig.11A Red fluorescent protein (RFP) fluorescence of the cells as determined by a fluorescence plate reader (CELIGO) is shown. Fig. 11B Green fluorescent protein (GFP) fluorescence of the cells as determined by a fluorescence plate reader (CELIGO) is shown. Fig. 11C Displayed as measured by a fluorescent plate reader ( iD3) determines the red fluorescent protein (RFP) fluorescence of the cells. Fig.11D Displayed as measured by a fluorescent plate reader ( iD3) determined the green fluorescent protein (GFP) fluorescence of the cells.

[0029] Figure 12A-12B A linear regression plot of pseudoneutralization titers from two samples is shown, where the pseudovirus expresses a SARS-CoV-2 S glycoprotein with a D614G mutation compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO:2.

[0030] Figure 13A-13B Linear regression plot showing pseudoneutralization titers from two samples where the pseudovirus expressed the SARS-CoV-2 S glycoprotein from the SARS-CoV-2 Omicron BA.5 strain.

[0031] Figure 14A-14B Linear regression plot showing pseudoneutralization titers from two samples where the pseudovirus expressed the SARS-CoV-2 S glycoprotein from the SARS-CoV-2 Omicron XBB.1.5 strain. DETAILED DESCRIPTION

[0032] definition

[0033] 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" may refer to one protein or a mixture of such proteins, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0034] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response induced to the immunogen. Modification of the immune response may include enhancing or amplifying the specificity of either or both of the antibody and cellular immune responses.

[0035] As used herein, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 10% of the value. For example, "about 100" encompasses 90 and 110.

[0036] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances, such as proteins (including glycoproteins) and peptides, that are capable of inducing an immune response.

[0037] As used herein, an "immunogenic composition" is a composition comprising an antigen, wherein administration of the composition to a subject results in the subject generating a humoral and / or cellular immune response to the antigen.

[0038] As used herein, the term "vaccine" refers to an immunogenic composition for inducing an immune response against a pathogen, such as an immunogen derived from the pathogen, which provides protective immunity (e.g., immunity that protects a subject from infection by the pathogen and / or reduces the severity of a disease or condition caused by infection with the pathogen). A protective immune response may include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen administered to a subject to produce protective immunity.

[0039] As used herein, the term "modification" when it refers to a CoV S polypeptide refers to a mutation, deletion or addition of an amino acid of the CoV S polypeptide. The position of the modification within the CoV S polypeptide can be determined based on the alignment of the polypeptide sequence with 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 sequence).

[0040]

[0041]

[0042]

[0043] The term "variant of SARS-CoV-2", which is used interchangeably herein with "heterologous SARS-CoV-2 strain", refers to a SARS-CoV-2 virus comprising the following SARS-CoV-2 S glycoprotein (also referred to as "CoV S polypeptide"), which is different from a CoV having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Compared to the S polypeptide, the SARS-CoV-2 S glycoprotein has at least about 2 modifications, at least about 3 modifications, at least about 4 modifications, at least about 5 modifications, at least about 6 modifications, at least about 7 modifications, at least about 8 modifications, at least about 9 modifications, at least about 10 modifications, at least about 11 modifications, at least about 12 modifications, at least about 13 modifications, at least about 14 modifications, at least about 15 modifications, at least about 16 modifications, at least about 17 modifications, at least about 18 modifications, at least about 19 modifications, at least about 20 modifications, at least about 21 modifications, at least about 22 modifications, at least about 23 modifications, at least about 24 modifications, at least about 25 modifications, at least about 26 modifications, at least about 27 modifications, at least about 28 modifications, at least about 29 modifications, at least about 30 modifications, at least about 31 modifications, at least about 32 modifications, at least about 33 modifications, at least about 34 modifications, at least about 35 modifications, at least about 36 modifications, at least about 37 modifications, at least about 38 modifications, at least about 39 modifications, at least about 40 modifications, at least about 41 modifications, at least about 42 modifications, at least about 43 modifications, at least about 44 modifications about 27 modifications, at least about 28 modifications, at least about 29 modifications, at least about 30 modifications, at least about 31 modifications, at least about 32 modifications, at least about 33 modifications, at least about 34 modifications, 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.

[0044] In an embodiment, a 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 a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 70% to about 99.9% identity to a CoV S polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 70% to about 99.5% identity to a CoV S polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 90% to about 99.9% identity to a CoV S polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 90% to about 99.8% identity to a CoV S polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 95% to about 99.9% identity to a CoV S polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 95% to about 99.8% identity to a CoV S polypeptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.In an embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having about 95% to about 99% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0045] In embodiments, the heterologous SARS-CoV-2 strain has a World Health Organization Label of alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omega. In embodiments, the 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 the Pango lineage nomenclature and is incorporated herein by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0046] In embodiments, the heterologous SARS-CoV-2 strain has a World Health Organization signature of Omicron. In embodiments, the heterologous SARS-CoV-2 strain having a World Health Organization signature of Omicron has at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO:2. In embodiments, the heterologous SARS-CoV-2 strain having a World Health Organization signature of Omicron has 35 to 55, 35 to 65, 35 to 75, 35 to 85, 35 to 95, or 35 to 105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO:2. In an embodiment, the modification is 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, G32 6H, R333T, L355I, S358F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K , K431T, V432P, G433S, L439R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F4 77S, 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.

[0047] In an embodiment, the variant CoV S polypeptide comprises a combination of modifications selected from the group consisting of:

[0048] (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;

[0049] (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;

[0050] (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, a deletion of amino acid 144, a deletion of amino acid 145, a deletion of amino acid 198, and an insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215;

[0051] (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;

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (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;

[0056] (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;

[0057] (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, a deletion of amino acid 11, a deletion of amino acid 12, and a deletion of amino acid 13;

[0058] (xi)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333 T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447 K, 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;

[0059] (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, a deletion of amino acid 11, a deletion of amino acid 12, a deletion of amino acid 13, a deletion of amino acid 56, and a deletion of amino acid 57;

[0060] (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;

[0061] (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;

[0062] (xv)T6I, A14S, G129D, H133Q, Q170E, V200E, G326H, R333T, L355I, S358F, S360P, S 362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E47 1A, 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;

[0063] (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;

[0064] (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, a deletion of amino acid 11, a deletion of amino acid 12, a deletion of amino acid 13, a deletion of amino acid 56, and a deletion of amino acid 57; and

[0065] (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;

[0066] (xix) a deletion of amino acid 56, a deletion of amino acid 57, and a deletion of amino acid 131, N488Y, A557D, D601G, P668H or P668R, T703I, S969A, and D1105H;

[0067] (xx) D67A, K404N, E471K, N488Y, D601G and A688V;

[0068] (xxi) D67A, D202G, L229H, K404N, E471K, N488Y, D601G and A688V;

[0069] (xxii) D67A, D202G, deletion of 1, 2 or 3 amino acids from amino acids 228-230, K404N, E471K, N488Y, D601G and A688V;

[0070] (xxiii) D67A, L229H, R233I, N488Y, K404N, E471K, D601G and A688V;

[0071] (xxiv) L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F;

[0072] (xxv) W139C and L439;

[0073] (xxvi) deletion of amino acid 144, deletion of amino acid 145, T6R, E143G, L439R, T465K, D601G, P668R, and D937N;

[0074] (xxvii) deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, L439R, T465K, D601G, P668R, and D937N;

[0075] (xxviii) deletion of amino acid 144, deletion of amino acid 145, T6R, T82I, G129D, Y132H, E143G, A209V, K404N, L439R, T465K, D601G, P668R, and D937N;

[0076] (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;

[0077] (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;

[0078] (xxxi) deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, K404N, L439R, T465K, E471Q, D601G, P668R, and D937N;

[0079] (xxxii) Q39R, A54V, E471K; D601G, Q664H, F875L and deletion of 1, 2, 3 or 4 of amino acids 56, 57, 131, 132;

[0080] (xxxiii) T82I, D240G, E471K, D601G and A688V;

[0081] (xxxiv) L439R, E471Q, D601G, P668R and Q1058H;

[0082] (xxxv) G62V, T63I, R233N, L439Q, F477S, D601G, T846N and deletion of 1, 2, 3, 4, 5 or 6 of amino acids 234-240;

[0083] (xxxvi) T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H, and D937N; and

[0084] (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;

[0085] The amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the sequence of SEQ ID NO: 2.

[0086] As used herein, the term "NVX-CoV2373" refers to a vector comprising the BV2373 spike glycoprotein (SEQ ID NO: 3) and fraction A and fraction C iscom matrix (e.g., MATRIX-M TM ) vaccine composition.

[0087] The term "pseudovirus" refers to a recombinant virus particle encoding one or more proteins from a first virus and an envelope protein from a second virus, wherein the first virus and the second virus are different. 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 a SARS-CoV-2S virus or a variant thereof. The following references describe pseudoviruses and are incorporated by reference in their entirety: Li et al. Rev Med Virol. 2018 Jan; 28(1): e1963.

[0088] 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.

[0089] Assays for evaluating the immunogenicity of vaccine compositions against SARS-CoV-2

[0090] The present disclosure provides pseudoviruses and methods for evaluating the immunogenicity of immunogenic compositions and vaccine compositions against SARS-CoV-2 or its variants using pseudoviruses. Pseudoviruses are also used to determine whether a biological sample contains antibodies that neutralize SARS-CoV-2 or its variants.

[0091] Pseudovirus

[0092] In embodiments, provided herein are pseudoviruses encoding: (i) SARS-CoV-2 spike (S) glycoprotein; and (ii) one or more of Rev, Tat, Gag, and Pol proteins. In embodiments, the pseudovirus further encodes a reporter protein.

[0093] In embodiments, the pseudovirus encodes 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 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 SARS-CoV-2 S glycoproteins.

[0094] Suitable SARS-CoV-2 S glycoproteins include SARS-CoV-2 S glycoproteins associated with any of the following Protein DataBank (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, 6XS7 YBB, 6YLA, 6YM0, 6YOR, 6YZ5, 6YZ7, 6Z2M, 6Z43, 6Z97, 6ZB4, 6ZB5, 6ZBP, 6ZCZ, 6ZDG, 6ZDH, 6ZER, 6ZFO, 6ZGE, 6ZGG, 6ZGH, 6ZGI, 6ZH9, 6ZHD, 6ZLR, 6 ZOW, 6ZOX, 6ZOY, 6ZOZ, 6ZP0, 6ZP1, 6ZP2, 6ZP5, 6ZP7, 6ZWV, 6ZXN, 7A25, 7 A29, 7A4N, 7A5R, 7A5S, 7A91, 7A92, 7A93, 7A94, 7A95, 7A96, 7A97, 7A98, 7 AD1, 7AKD, 7B0B, 7B14, 7B17, 7B18, 7B27, 7B3O, 7B62, 7BEH, 7BEI, 7BEJ, 7BEK, 7BEL, 7BEM, 7BEN, 7BEO, 7BEP, 7BH9, 7BNM, 7BNN, 7BNO, 7BNV, 7BWJ, 7BYR, 7BZ5, 7C01, 7C2L, 7C53, 7C8D, 7C8J, 7C8V, 7C8W, 7CAB, 7CAC, 7CAH, 7CAI, 7CAK, 7CAN, 7CDI, 7CDJ, 7CH4, 7CH5, 7CHB, 7CHC, 7CHE, 7CHF, 7CHH, 7CHO, 7CHP, 7CHS, 7CJF, 7CM4, 7CN9, 7COT, 7CT5, 7CWL, 7CWM, 7CWN, 7CWO, 7CWS, 7CWT, 7CWU, 7CYH, 7CYP, 7CYV, 7CZP, 7CZQ, 7CZR, 7CZS, 7CZT, 7CZU, 7CZV, 7CZW, 7CZX, 7CZY, 7CZZ, 7D00, 7D03, 7D0B, 7D0C, 7D0D, 7D2Z, 7D30, 7D4G, 7D6I, 7DCC, 7DCX, 7DD2, 7DD8, 7DDD, 7DDN, 7DEO, 7DET, 7DEU, 7DF3,<h2 style=";text-align:left;direction:ltr">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, 8E8M, 7E9N, 7E9O, 7E9P, 7E9Q, 7E9T, 7EAM, 7EAN, 7EAZ, 7EB0, 7EB3, 7EB4, 7EB5, 7EDF, 7EDG, 7EDH, 7EDI, 7EDJ, 7EFP, 7EFR, 7EH5, 7EJ4, 7EJ5, 7EJL, 7EJY, 7EJZ, 7EK0, 7EK6, 7EKC, 7EKE, 7EKF, 7EKG, 7EKH, 7ENF, 7ENG, 7EPX, 7EY0, 7EY4, 7EY5, 7EYA, 7EZV, 7F0X, 7F12, 7F15, 7F3Q, 7F46, 7F5H, 7F5R, 7F62, 7F63, 7F6Y, 7F6Z, 7F7E, 7F7H, 7FAE, 7FAF, 7FAT, 7FAU, 7FB0, 7FB1, 7FB3, 7FB4, 7FBJ, 7FBK, 7FC5, 7FCD, 7FCE, 7FCP, 7FCQ, 7FDG, 7FDH, 7FDI, 7FDK, 7FEM, 7FET 7FG2, 7FG3, 7FG7, 7FH0, 7FJC, 7FJN, 7FJO, 7FJS, 7JJC, 7JJI, 7JJJ, 7JMO, 7JMP, 7JMW, 7JV2, 7JV4, 7JV6, 7JVA, 7JVB, 7JVC, 7JW0, 7JWB, 7JWY, 7JX3, 7JZL 7JZM, 7JZN, 7JZU, 7K43, 7K45, 7K4N, 7K8M, 7K8S, 7K8T, 7K8U, 7K8V, 7K8W, 7K8X, 7K8Y, 7K8Z, 7K90, 7K9H, 7K9I, 7K9J, 7K9K, 7K9Z, 7KDG, 7KDH, 7KDI, 7KDJ, 7KDK, 7KDL, 7KE4, 7KE6, 7KE7, 7KE8, 7KE9, 7KEA, 7KEB, 7KEC, 7KFV, 7KFW, 7KFX, 7KFY, 7KGJ, 7KGK, 7KJ2, 7KJ3, 7KJ4, 7KJ5, 7KKK, 7KKL, 7KL9, 7KLG, 7KLH<h2 style=";text-align:left;direction:ltr">7KLW, 7KM5, 7KMB, 7KMG, 7KMH, 7KMI, 7KMK, 7KML, 7KMS, 7KMZ, 7KN3, 7KN4, 7KN5, 7KN6, 7KN7, 7KNB, 7KNE, 7KNH, 7KNI, 7KQE, 7KRQ, 7KRR, 7KRS, 7KS9, 7KSG, 7KXJ, 7KXK, 7KZB, 7L02, 7L06, 7L09, 7L0N, 7L2C, 7L2D, 7L2E, 7L2F, 7L3N, 7L4Z, 7L56, 7L57, 7L58, 7L5B, 7L7D, 7L7E, 7L7F, 7L7K, 7LAA, 7LAB, 7LC8, 7LCN 7LD1, 7LDJ, 7LJR, 7LM8, 7LO4, 7LOP, 7LQ7, 7LQV, 7LQW, 7LRS, 7LRT, 7LS9, 7LSS, 7LWI, 7LWJ, 7LWK, 7LWL, 7LWM, 7LWN, 7LWO, 7LWP, 7LWQ, 7LWS, 7LWT, 7LWU 7LWV, 7LWW, 7LX5, 7LXW, 7LXX, 7LXY, 7LXZ, 7LY0, 7LY2, 7LY3, 7LYK, 7LYL, 7LYM, 7LYN, 7LYO, 7LYP, 7LYQ, 7M0J, 7M3I, 7M42, 7M53, 7M6D, 7M6E, 7M6F, 7M6G 7M6H, 7M6I, 7M71, 7M7B, 7M7W, 7M8J, 7M8K, 7M8S, 7M8T, 7M8U, 7MDW, 7ME7, 7MEJ, 7MF1, 7MFU, 7MJG, 7MJH, 7MJI, 7MJJ, 7MJK, 7MJL, 7MJM, 7MJN, 7MKL, 7MKM 7MLZ, 7MM0, 7MMO, 7MSQ, 7MTC, 7MTD, 7MTE, 7MW2, 7MW3, 7MW4, 7MW5, 7MW6, 7MY2, 7MY3, 7MY8, 7MZF, 7MZG, 7MZH, 7MZI, 7MZJ, 7MZK, 7MZL, 7MZM, 7MZN, 7N0G 7N0H, 7N1A, 7N1B, 7N1E, 7N1F, 7N1Q, 7N1T, 7N1U, 7N1V, 7N1W, 7N1X, 7N1Y, 7N3I, 7N4I, 7N4J, 7N4L, 7N4M, 7N5H, 7N62, 7N64, 7N6D, 7N6E, 7N8H, 7N8I, 7N9A 7N9B, 7N9C, 7N9E, 7N9T, 7NAB, 7ND3, 7ND4, 7ND5, 7ND6, 7ND7, 7ND8, 7ND9, 7NDA, 7NDB, 7NDC, 7NDD, 7NEG, 7NEH, 7NKT, 7NLL, 7NP1, 7NS6, 7NT9, 7NTA, 7NTC<h2 style=";text-align:left;direction:ltr">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、7S O9、7SOA、7SOB、7SOC、7SOD、7SOE、7SOF、7SPO、7SPP、7SWN、7SWO、7SWP、7SWW、 7SWX, 7SXR, 7SXS, 7SXT, 7SXU, 7SXV, 7SXW, 7SXX, 7SXY, 7SXZ, 7SY0, 7SY1, 7SY2, 7SY3, 7SY4, 7SY5, 7SY6, 7SY7, 7SY8, 7T01, 7T3M, 7T67, 7T72, 7T7B, 7T9J<h2 style=";text-align:left;direction:ltr">7T9K, 7T9L, 7TAS, 7TAT, 7TB4, 7TB8, 7TBF, 7TCA, 7TCC, 7TCQ, 7TEI, 7TEW, 7TEX, 7TEY, 7TEZ, 7TF0, 7TF1, 7TF2, ​​7TF3, 7TF4, 7TF5, 7TF8, 7TGE, 7TGW, 7TGX 7TGY、7THE、7THK、7THT、7TIK、7TL1、7TL9、7TLA、7TLB、7TLC、7TLD、7TLT、7T LY、7TM0、7TN0、7TNW、7TO4、7TOU、7TOV、7TOW、7TOX、7TOY、7TOZ、7TP0、7TP1、 7TP2、7TP3、7TP4、7TP7、7TP8、7TP9、7TPA、7TPC、7TPE、7TPF、7TPH、7TPK、7T PL、7TPR、7TYZ、7TZ0、7U09、7U0A、7U0D、7U0E、7U0N、7U0P、7U0Q、7U0X、7U1R、 7U2D, 7U2E, 7U8E, 7U9O, 7U9P, 7UAP, 7UAQ, 7UAR, 7UB0, 7UB5, 7UB6, 7UFK, 7UFL, 7UHC, 7UL0, 7UM2, 7UPL, 7UR1, 7URQ, 7URS, 7UZ4, 7UZ5, 7UZ6, 7UZ7, 7UZ8, 7UZ9, 7UZA, 7UZB, 7UZC, 7UZD, 7V20, 7V22, 7V23, 7V24, 7V26, 7V27, 7V2A, 7V76, 7V77, 7V78, 7V79, 7V7A, 7V7D, 7V7E, 7V7F, 7V7G, 7V7H, 7V7I, 7V7J, 7V7N, 7V7O, 7V7P, 7V7Q, 7V7R, 7V7S, 7V7T, 7V7U, 7V7V, 7V7Z, 7V80, 7V81, 7V82, 7V83, 7V84, 7V85, 7V86, 7V87, 7V88, 7V89, 7V8A, 7V8B, 7V8C, 7VHH, 7VHJ, 7VHK, 7VHL, 7VHM, 7VHN, 7VMU, 7VNB, 7VNC, 7VND, 7VNE, 7VOA, 7VQ0, 7VRV, 7VRW, 7VX1, 7VX4, 7VX5, 7VX9, 7VXA, 7VXB, 7VXC, 7VXD, 7VXE, 7VXF, 7VXI, 7VXK, 7VXM, 7VYR, 7VZT, 7W1S, 7W6U, 7W8S, 7W92, 7W94, 7W99, 7W9B, 7W9C, 7W9E, 7W9F, 7WA1, 7WB5, 7WBL, 7WBP, 7WBQ, 7WBZ, 7WCD, 7WCH, 7WCK, 7WCP, 7WCR, 7WCU, 7WCZ<h2 style=";text-align:left;direction:ltr">7WD0, 7WD1, 7WD2, 7WD7, 7WD8, 7WD9, 7WDF, 7WE7, 7WE8, 7WE9, 7WEA, 7WEB, 7WEC, 7WED, 7WEE, 7WEF, 7WEV, 7WG6, 7WG7, 7WG8, 7WG9, 7WGB, 7WGC, 7WGV, 7WGX 7WGY, 7WGZ, 7WH8, 7WHB, 7WHD, 7WHH, 7WHI, 7WHJ, 7WHK, 7WHZ, 7WJY, 7WJZ, 7WK0, 7WK2, 7WK3, 7WK4, 7WK5, 7WK6, 7WK8, 7WK9, 7WKA, 7WLC, 7WM0, 7WN2, 7WNB 7WNM, 7WO4, 7WO5, 7WO7, 7WOA, 7WOB, 7WOC, 7WOG, 7WON, 7WOP, 7WOQ, 7WOR, 7WOS, 7WOU, 7WOV, 7WOW, 7WP0, 7WP1, 7WP2, 7WP5, 7WP6, 7WP8, 7WP9, 7WPA, 7WPB, 7WPC, 7WPD, 7WPE, 7WPF, 7WPH, 7WQV, 7WR8, 7WRH, 7WRI, 7WRJ, 7WRV, 7WS0, 7WS1, 7WS2, 7WS3, 7WS4, 7WS5, 7WS6, 7WS7, 7WS8, 7WS9, 7WSA, 7WSE, 7WSH, 7WSK 7WT7, 7WT8, 7WT9, 7WTF, 7WTG, 7WTH, 7WTI, 7WTJ, 7WTK, 7WUE, 7WUH, 7WVL, 7WVP, 7WVQ, 7WWI, 7WWJ, 7WWK, 7WWL, 7WWM, 7WXZ, 7WZ1, 7WZ2, 7X1M, 7X25, 7X2H 7X2K, 7X2L, 7X2M, 7X63, 7X66, 7X6A, 7X7D, 7X7E, 7X7N, 7X7T, 7X7U, 7X8W, 7X8Y, 7X8Z, 7X90, 7X91, 7X92, 7X93, 7X94, 7X95, 7X96, 7X9E, 7XA7, 7XAZ, 7XB0, 7XB1, 7XBY, 7XCH, 7XCI, 7XCK, 7XCO, 7XCP, 7XCZ, 7XD2, 7XDA, 7XDB, 7XDK, 7XDL, 7XEG, 7XEI, 7XH8, 7XIC, 7XID, 7XIK, 7XIL, 7XIW, 7XIX, 7XIY, 7XIZ, 7XJ6, 7XJ8, 7XJ9, 7XMX, 7XMZ, 7XNQ, 7XNR, 7XNS, 7XO4, 7XO5, 7XO6, 7XO7, 7XO8, 7XO9, 7XOA, 7XOB, 7XOC, 7XOD, 7XRP, 7XS8, 7XSA, 7XSB, 7XSC, 7XST, 7XTZ, 7XU0,<h2 style=";text-align:left;direction:ltr">7XU1, 7XU2, 7XU3, 7XU4, 7XU5, 7XU6, 7XWA, 7XXL, 7Y0C, 7Y0V, 7Y1Y, 7Y1Z, 7Y42, 7Y6D, 7Y6K, 7Y6L, 7Y6N, 7Y75, 7Y76, 7Y7J, 7Y7K, 7Y8J, 7Y9N, 7Y9S, 7Y9Z, 7Y75 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, 7YV 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、8C WI、8CWK、8CWU、8CWV、8CXN、8CXQ、8CY6、8CY7、8CY9、8CYA、8CYB、8CYC、8CYD、 8CYJ、8CZI、8D0Z、8D36、8D47、8D48、8D55、8D56、8D5A、8D6Z、8D8Q、8D8R、8D AD、8DAO、8DCC、8DCE、8DF5、8DGU、8DI5、8DLI、8DLJ、8DLK、8DLL、8DLM、8DLN、 8DLO、8DLP、8DLQ、8DLR、8DLS、8DLT、8DLU、8DLV、8DLW、8DLX、8DLY、8DLZ、8D M0、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, 8 EPN, 8EPP, 8EPQ, 8ERQ, 8ERR, 8F0G, 8F0H, 8FA1, 8FA2, 8FEZ, 8FU7, 8FU8, 8FU9, 8GB0, 8GB5, 8GB6, 8GB7, 8GB8, 8GJM, 8GJN, 8GOM, 8GON, 8GOU, 8GPY, 8G 8HN6, 8HN7, 8I5H, 8I5I, 8IDN, 8IF2, 8IOS, 8IOT, 8IOU, 8IOV, 8ITU, 8J1Q, 8J26, 8SMT. Each of these PDB IDs is incorporated herein by reference in its entirety. The amino acid sequence of the SARS-CoV-2 S glycoprotein associated with each entry can be accessed by downloading the FASTA file associated with the PDB ID at www.rcsb.org. In an embodiment, the pseudovirus expresses a nucleic acid encoding the SARS-CoV-2 S glycoprotein of any of the above PDB IDs.

[0095] 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 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), which is located at positions 669-672 of the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In an embodiment, the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site. In an embodiment, the inactive furin cleavage site has an amino acid sequence of any one of SEQ ID NO: 68-97. In an embodiment, the amino acid sequence of the inactive furin cleavage site is GG. In an embodiment, non-limiting examples of SARS-CoV-2 S glycoproteins having an inactive furin cleavage site of GG include glycoproteins of SEQ ID NO: 26-28 and 30.

[0096] In embodiments, the amino acid sequence of the inactive furin cleavage site is QQQAQ (SEQ ID NO: 68). In embodiments, non-limiting examples of SARS-CoV-2S glycoproteins having an inactive furin cleavage site of QQQAQ (SEQ ID NO: 68) include glycoproteins of SEQ ID NO: 3, 5-13, 15, 17-19, 21, 23-25, 29, and 31-66.

[0097] In an embodiment, one or more of the amino acids that make up the native furin cleavage site are mutated to any native amino acid. In an embodiment, one or more of the amino acids that make up the native furin cleavage site are deleted.

[0098] In embodiments, one or more of the amino acids constituting the natural furin cleavage site are mutated to glutamine. In embodiments, 1, 2, 3 or 4 amino acids may be mutated to glutamine. In embodiments, one of the arginines constituting the natural furin cleavage site is mutated to glutamine. In embodiments, two of the arginines constituting the natural furin cleavage site are mutated to glutamine. In embodiments, three of the arginines constituting the natural furin cleavage site are mutated to glutamine.

[0099] In embodiments, one or more of the amino acids constituting the native furin cleavage site are mutated to alanine. In embodiments, 1, 2, 3 or 4 amino acids may be mutated to alanine. In embodiments, one of the arginines constituting the native furin cleavage site is mutated to alanine. In embodiments, two of the arginines constituting the native furin cleavage site are mutated to alanine. In embodiments, three of the arginines constituting the native furin cleavage site are mutated to alanine.

[0100] In embodiments, one or more mutations in the amino acids constituting the native furin cleavage site are glycine. In embodiments, 1, 2, 3 or 4 amino acids may mutate to glycine. In embodiments, one mutation in the arginine of the native furin cleavage site is glycine. In embodiments, two mutations in the arginine constituting the native furin cleavage site are glycine. In embodiments, three mutations in the arginine constituting the native furin cleavage site are glycine.

[0101] In embodiments, one or more of the amino acids constituting the natural furin cleavage site are mutated to asparagine. For example, 1, 2, 3 or 4 amino acids may be mutated to asparagine. In embodiments, one of the arginines constituting the natural furin cleavage site is mutated to asparagine. In embodiments, two of the arginines constituting the natural furin cleavage site are mutated to asparagine. In embodiments, three of the arginines constituting the natural furin cleavage site are mutated to asparagine.

[0102] In an embodiment, the active furin cleavage site (SEQ ID NO: 67) of the SARS-CoV-2 S glycoprotein described herein is replaced with an inactivated furin cleavage site of the table below.

[0103] Inactivated furin cleavage site

[0104]

[0105]

[0106] In embodiments, 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 embodiments, Lys-973 is mutated to any native amino acid. In embodiments, Lys-973 is mutated to proline. In embodiments, Lys-973 is mutated to glycine.

[0107] In embodiments, 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 embodiments, Val-974 is mutated to any native amino acid compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In embodiments, Val-974 is mutated to proline. In embodiments, Val-974 is mutated to glycine compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2.

[0108] In an embodiment, the SARS-CoV-2 S glycoprotein contains mutations at Lys-973 and Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In an embodiment, Lys-973 and Val-974 are mutated to any natural amino acid compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. In an embodiment, Lys-973 and Val-974 are mutated to proline compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 2. Non-limiting examples of SARS-CoV-2 S glycoproteins in which amino acids Lys-973 and Val-974 are mutated to proline include glycoproteins of SEQ ID NOs: 3-13, 20, and 22-60.

[0109] In embodiments, the SARS-CoV-2 S glycoprotein 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 the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0110] In embodiments, the SARS-CoV-2 S glycoprotein 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 the polypeptide of any one of SEQ ID NOs: 3-66.

[0111] In an embodiment, the pseudovirus encodes Gag protein and Pol protein. In an embodiment, the pseudovirus encodes a fusion protein, the fusion protein encoding Gag protein and Pol protein. In an embodiment, the pseudovirus encodes Rev protein and Tat protein. In an embodiment, Gag protein and Pol protein are from murine leukemia virus (MLV). In an embodiment, one or more of Rev, Tat, Gag and Pol proteins are from a lentivirus.

[0112] In embodiments, the pseudovirus encodes one or more reporter proteins. In embodiments, the pseudovirus encodes 1 to 10 reporter proteins. In embodiments, the one or more reporter proteins are fluorescent proteins. In embodiments, the fluorescent proteins are green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, enhanced green fluorescent protein and ZsGreen. In embodiments, one or more reporter proteins are labeled with fluorescent dyes. In embodiments, the one or more reporter proteins are luciferase.

[0113] Methods for generating pseudoviruses

[0114] In embodiments, a method for producing a pseudovirus as described herein is provided herein, 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 embodiments, the cell is a HEK293T (also referred to as "293T") cell. The SARS-CoV-2S glycoprotein can be any SARS-CoV-2S glycoprotein described herein. In embodiments, the SARS-CoV-2S glycoprotein contains mutations from amino acids 973 and 974 to proline, wherein the SARS-CoV-2S glycoprotein is numbered according to the SARS-CoV-2S glycoprotein of SEQ ID NO:2. In embodiments, the SARS-CoV-2S glycoprotein contains an inactive furin cleavage site. Many examples of inactive furin cleavage sites are described herein.

[0115] In embodiments, pseudoviruses are produced in human host cells. In embodiments, pseudoviruses are produced in HEK293T cells (also referred to as "293T" cells).

[0116] In an embodiment, the plasmid encoding the Rev protein 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 the plasmid of SEQ ID NO:100. In an embodiment, the plasmid encoding the fusion protein of Gag protein and 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 embodiments, the plasmid encoding the Tat protein 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 the plasmid of SEQ ID NO:98.

[0117] Methods for identifying neutralizing antibodies against SARS-CoV-2 or its variants

[0118] In an embodiment, provided herein is a method for determining whether a biological sample contains neutralizing antibodies against 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 cells expressing ACE2; (d) contacting control cells expressing ACE2 with 1 to about 20 pseudoviruses, wherein 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 SARS-CoV-2 virus or a variant thereof.

[0119] In embodiments, the method comprises contacting a 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 embodiments, each pseudovirus expresses a different SARS-CoV-2 S glycoprotein. In embodiments, each pseudovirus expresses a different reporter protein.

[0120] In an embodiment, cells expressing ACE2 are produced by transfecting cells with a plasmid 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 the plasmid of SEQ ID NO: 101. In an embodiment, ACE2 has an amino acid 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 the polypeptide of SEQ ID NO: 102 or 103.

[0121] In embodiments, the biological sample and 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 embodiments, the biological sample and 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 embodiments, the biological sample and pseudovirus are incubated for 2 or 3 hours. In embodiments, the biological sample and pseudovirus are incubated for 1 day to about 7 days. In embodiments, the biological sample and pseudovirus are incubated for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In embodiments, the biological sample and pseudovirus are incubated for three days.

[0122] In embodiments, cells are contacted with pseudoviruses for 1 hour to about 24 hours. In embodiments, cells are contacted with pseudoviruses 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 embodiments, cells are contacted with pseudoviruses for 1 day to about 7 days. In embodiments, cells are contacted with pseudoviruses for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In embodiments, cells are contacted with pseudoviruses for three days.

[0123] In embodiments, cells expressing ACE2 are human cells. In embodiments, cells expressing ACE2 are 293T cells. In embodiments, cells are A549 cells or Vero E6 cells. In embodiments, the methods described herein do not require a biosafety level 3 (BSL-3) laboratory.

[0124] In embodiments, the cell is selected from the group consisting of: Vero E6, A549 and HEK293T cells. In embodiments, the biological sample is serum. In embodiments, the serum is human serum. In embodiments, the serum is from an individual infected with SARS-CoV-2 or a variant thereof. In embodiments, the serum is from an individual who has received an immunogenic composition or vaccine for SARS-CoV-2 or a variant thereof. In embodiments, serum, plasma or blood is heat inactivated prior to use in the methods described herein. In embodiments, heat inactivation includes incubating serum, plasma or blood at a temperature of about 37°C to about 90°C. In embodiments, heat inactivation includes incubating serum, plasma or blood at a temperature of about 56°C. In embodiments, heat inactivation includes incubating serum, plasma or blood at a temperature of about 37°C to about 90°C for about 15 minutes to about 2 hours. In embodiments, 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 embodiments, 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 embodiments, serum, plasma or blood is incubated at about 37° C. to about 90° C. for about 30 minutes.

[0125] In embodiments, the immunogenic compositions and vaccine compositions contain non-naturally occurring coronavirus (CoV) spike (S) polypeptides or nanoparticles containing CoV S polypeptides.

[0126] In embodiments, 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.

[0127] In embodiments, 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.

[0128] In embodiments, 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.

[0129] The sequence of SEQ ID NO: 3 is in the table below.

[0130] SEQ ID NO:3

[0131]

[0132] In an embodiment, the immunogenic composition or vaccine composition comprises an adjuvant. Exemplary adjuvants are described throughout this disclosure.

[0133] Aluminum-based adjuvants

[0134] In embodiments, the adjuvant may be an aluminum adjuvant (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the aluminum adjuvant. For example, the nanoparticles may be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to the aluminum adjuvant. Typically, in the composition, the nanoparticles are 92% to 97% bound to the aluminum adjuvant. The amount of aluminum adjuvant present per dose is typically in the range of about 400 μg to about 1250 μg. For example, the aluminum adjuvant may be present in an amount of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg per dose. Typically, for a dose of 120 μg of protein nanoparticles, the aluminum adjuvant is present at about 400 μg.

[0135] Saponin adjuvant

[0136] Adjuvants containing saponins can also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja tree. Typically, saponins are prepared using a multi-step purification process to produce a variety of fractions. As used herein, the term "saponin fraction from Quillaja tree" is generally used to describe a semi-purified or defined saponin fraction of the Quillaja tree or its substantially pure fraction.

[0137] Saponin fraction

[0138] Several methods for producing saponin fractions are suitable. 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, i.e., a crude aqueous Quillaja extract, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. The lipophilic fraction is then separated by semi-preparative HPLC, eluting with a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "fraction A" or "QH-A" is or corresponds to a fraction eluted with about 39% acetonitrile. The fraction referred to herein as "fraction B" or "QH-B" is or corresponds to a fraction eluted with about 47% acetonitrile. The fraction referred to herein as "fraction C" or "QH-C" is or corresponds to a fraction eluted with about 49% acetonitrile. Additional information about fraction purification is found in U.S. Patent No. 5,057,540. When prepared as described herein, fractions A, B and C of Quillaja each represent a group or family of chemically closely related molecules with definable properties. The chromatographic conditions under which the fractions were obtained allow for a high degree of batch-to-batch reproducibility with respect to elution profiles and biological activity.

[0139] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja Spikoside (lsconova AB, Ultunaallén 2B, 756 51 Uppsala, Sweden). The fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21 and QA-22, especially QA-7, QA-17, QA-18 and QA-21 of EP 0 3632 279 B2 can be used. They are obtained as described in Example 1 on pages 6 and 8 and 9 of EP 03632 279 B2.

[0140] The saponin fractions described herein and used to form adjuvants are generally substantially pure fractions; that is, the fractions are substantially free of contamination from other materials. In particular aspects, substantially pure saponin fractions may 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.

[0141] ISCOM Structure

[0142] The saponin fraction can be administered in the form of caged particles known as ISCOM (immunostimulatory COMplex). ISCOM can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546 B1. In a particular embodiment, transport and / or passenger antigens can be used as described in EP9600647-3 (PCT / SE97 / 00289).

[0143] Matrix adjuvant

[0144] In an embodiment, 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 a particular aspect, the ISCOM matrix complex also contains a phospholipid. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant active) substances, not necessarily glycosides, and may be produced as described in EP0436620B1, which is incorporated herein by reference in its entirety.

[0145] In other aspects, 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 that is associated by detergent 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.

[0146] According to one embodiment, the saponin fraction incorporated into the ISCOM matrix complex or ISCOM complex, or at least one additional adjuvant also incorporated into or mixed with the ISCOM or ISCOM matrix complex, is selected from Fraction A, Fraction B or Fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any purified subfraction, e.g., QA 1-21.

[0147] In certain aspects, 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 particle may contain any weight % of fraction A and any weight % of another saponin fraction, such as a crude saponin fraction or fraction C, respectively. Thus, in certain aspects, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1 wt% to 99.9 wt%, 5 wt% to 95 wt%, 10 wt% to 90 wt%, 15 wt% to 85 wt%, 20 wt% to 80 wt%, 25 wt% to 75 wt%, 30 wt% to 70 wt%, 35 wt% to 65 wt%, 40 wt% to 60 wt%, 45 wt% to 55 wt%, 40 wt% to 60 wt%, or 50 wt% of one saponin fraction (e.g., Fraction A) and the remainder to 100 wt% of another saponin (e.g., any crude fraction or any other fraction, e.g., Fraction C), in each case. The weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in U.S. Published Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0148] In certain embodiments, the ISCOM matrix or ISCOM complex comprises 5-99 wt% of one fraction (eg, Fraction A) and the remainder to 100 wt% of another fraction (eg, crude saponin fraction or Fraction C). The weight is calculated as the total weight of the saponin fractions.

[0149] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40 to 99 wt% of one fraction (eg, Fraction A) and 1 to 60 wt% of another fraction (eg, crude saponin fraction or Fraction C). The weights are calculated as the total weight of the saponin fractions.

[0150] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction (e.g., Fraction A) and 30% to 5% by weight of another fraction (e.g., crude saponin fraction or fraction C). The weight is calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction from Quillaja saponaria is selected from any one of QA 1-21.

[0151] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles can each be formed using only one saponin fraction. The compositions disclosed herein can contain a plurality of particles, wherein each particle contains only one saponin fraction. That is, certain compositions can contain one or more different types of ISCOM-matrix complex particles and / or one or more different types of ISCOM complex particles, wherein each individual particle contains a saponin fraction from Quillaja saponaria, wherein the saponin fraction in one complex is different from the saponin fraction in the other complex particles.

[0152] In certain aspects, one type of saponin fraction or crude saponin fraction can be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction can be incorporated into another ISCOM matrix complex or particle. The composition or vaccine can comprise at least two types of complexes or particles, each type having one type of saponin incorporated into physically distinct particles.

[0153] In the composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used, wherein one Quillaja saponin fraction and another Quillaja saponin fraction are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0154] ISCOM matrix or ISCOM complex particles each having a saponin fraction can be present in the composition in any combination of wt%. In particular aspects, the composition may contain 0.1 wt% to 99.9 wt%, 5 wt% to 95 wt%, 10 wt% to 90 wt%, 15 wt% to 85 wt%, 20 wt% to 80 wt%, 25 wt% to 75 wt%, 30 wt% to 70 wt%, 35 wt% to 65 wt%, 40 wt% to 60 wt%, 45 wt% to 55 wt%, 40 wt% to 60 wt% or 50 wt% of an ISCOM matrix or complex containing a first saponin fraction, the remainder being composed of an ISCOM matrix or complex containing a different saponin fraction. In various aspects, the remainder is one or more ISCOM matrices or complexes, wherein each matrix or complex particle contains only one saponin fraction. In other aspects, the ISCOM matrix or complex particle may contain more than one saponin fraction.

[0155] In certain compositions, the only saponin fraction in the first ISCOM matrix or ISCOM complex particle is Fraction A, and the only saponin fraction in the second ISCOM matrix or ISCOM complex particle is Fraction C.

[0156] In embodiments, Quillaja Fraction A comprises 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 of the sum of the weights of Quillaja Fraction A and Quillaja Fraction C in the adjuvant, respectively, and Quillaja Fraction C comprises the remainder.

[0157] A preferred composition comprises a first ISCOM matrix comprising fraction A and a second ISCOM matrix comprising fraction C, wherein the fraction A ISCOM matrix constitutes about 70% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 30% by weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix constitutes about 85% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 15% by weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix constitutes about 92% by weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 8% by weight of the total saponin adjuvant. Thus, in certain compositions, the fraction A ISCOM matrix is ​​present in a range of about 70% to about 85% of the total weight of the saponin adjuvant in the composition, and the fraction C ISCOM matrix is ​​present in a range of about 15% to about 30% of the total weight of the saponin adjuvant in the composition. In certain compositions, the fraction A ISCOM matrix is ​​present in the range of about 70% to about 92% of the total weight of the saponin adjuvant in the composition, and the fraction C ISCOM matrix is ​​present in the range of about 8% to about 30% of the total weight of the saponin adjuvant in the composition. In embodiments, the fraction A ISCOM matrix comprises 50-96% by weight of the sum of the weight of the fraction A ISCOM matrix and the fraction C ISCOM matrix in the adjuvant, respectively, and the fraction C ISCOM matrix comprises the remainder of the sum of the weight of the fraction A ISCOM matrix and the fraction C ISCOM matrix in the adjuvant. TM In a particularly preferred composition, the Fraction A ISCOM matrix is ​​present at about 85% by weight of the total saponin adjuvant in the composition and the Fraction C ISCOM matrix is ​​present at about 15% by weight of the total saponin adjuvant in the composition. TM May be referred to interchangeably as Matrix-M1.

[0158] Exemplary QS-7 and QS-21 fractions, their production, and their uses 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.

[0159] In embodiments, other adjuvants may be used in addition or alternatively. It is contemplated that any adjuvant described in Vogel et al. "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)" (incorporated herein by reference in its entirety for all purposes) is included within the scope of the present disclosure. Other adjuvants include complete Freund's adjuvant (a non-specific stimulator 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 (which contains three components extracted from bacteria), MPL, trehalose dimycolate (TDM), and in 2% squalene / Cell wall skeleton (CWS) in polysorbate 80 emulsion. In embodiments, the adjuvant may be a paucilamellar lipid vesicle; e.g. They are paucilamellar, non-phospholipid vesicles ranging from about 100 nm to about 500 nm. They contain Alcohol ethoxylate 72, cholesterol, oleic acid and squalene. It has been shown to be an effective adjuvant (see US Pat. Nos. 5,629,021, 6,387,373 and 4,911,928).

[0160] Example

[0161] Example 1: Pseudovirus-based neutralization assay for SARS-CoV-2 variants: A rapid, cost-effective, high-throughput BSL2-based assay that can be used for vaccine immunogenicity evaluation

[0162] Introduction: We have discovered a novel pseudovirus-based neutralization assay for evaluating the immunogenicity of vaccines against SARS-CoV-2 variants. The assay is a BSL-2-based assay that is cost-effective, results in a rapid turnaround, and enables the evaluation of the immunogenicity (e.g., neutralizing antibody production) of SARS-CoV-2 vaccines against emerging SARS-CoV-2 variants. The assay is an alternative to live virus neutralization assays and does not require the use of a BSL-3 laboratory.

[0163] Methods: We developed a BSL-2-based pseudovirus-based neutralization assay for SARS-CoV-2 prototypes and variants (Omicron BA.1 and BA.2). The assay is suitable for measuring neutralization capacity in clinical samples (sera from human subjects infected with SARS-CoV-2 or immunized with COVID-19 vaccines). Notably, the assay can be performed in BSL-2 laboratories rather than BSL-3 laboratories. The 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 a SARS-CoV-2 S protein having an amino acid sequence of SEQ ID NO:2. The assay can also be used to evaluate the ability of SARS-CoV-2 vaccines to stimulate an immune response against the prototype SARS-CoV-2 virus (including Omicron BA.1, BA.2, and BA.5 variants). The assay can also be used to evaluate the ability of human sera to neutralize SARS-CoV-2 and SARS-CoV-2 variants.

[0164] SARS-CoV-2 pseudoviruses expressing the SARS-CoV-2 spike protein were generated by co-transfecting cells with a plasmid encoding a lentiviral backbone expressing a luciferase reporter, a plasmid encoding a non-surface protein for lentiviral production, and a plasmid expressing the SARS-CoV-2 spike protein (prototype strain or Omicron strain) under the control of a mammalian promoter.

[0165] A schematic diagram of the pseudovirus neutralization assay used in this example is shown in Figure 1 .

[0166] SARS-CoV-2 pseudoviruses were generated by transfecting HEK293T cells with the following plasmids: (i) a plasmid encoding a lentiviral backbone expressing 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. The plasmids expressed the gene of interest under the control of a cytomegalovirus (CMV) promoter. Pseudoviruses were harvested from cell culture supernatants.

[0167] The pseudoviruses were then incubated with serum from patients who had recovered from SARS-CoV-2 infection or from patients immunized with the SARS-CoV-2 vaccine NVX-CoV2373. As a control, the pseudoviruses were incubated with cell culture medium without serum (virus-only "VC" control).

[0168] Cells expressing ACE2 receptors were infected with pseudoviruses and incubated at 37°C for 48 to 72 hours. As a negative control, cells were infected with cell culture medium in the absence of pseudoviruses. The luminescence of the cells (from luciferase) was measured. The reduction of the luciferase signal indicated neutralization of the infection.

[0169] The dose dependence of luminescence on pseudovirus levels was determined by infecting cells expressing the ACE2 receptor with different amounts of pseudovirus and measuring luciferase luminescence (RLU) 2 days after infection.

[0170] The best cell line for pseudovirus-based neutralization assays 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 pseudoviruses, and the 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 pseudovirus neutralization assays was further confirmed by infecting 293T / ACE2 and A549 / ACE2-TMPRESS cells with or without serum, and then measuring luminescence. The control group was cells infected but not treated with serum (virus control) and cells without pseudovirus (cell control). The inhibition / neutralization percentage (%) of serum relative to the virus control for pseudovirus infection was determined.

[0171] Pseudovirus-based neutralization titers using different amounts of pseudovirus were evaluated to optimize the assay procedure.

[0172] The kinetics of the pseudovirus neutralization assay endpoint were evaluated by measuring luminescence at 48 and 72 hours and comparing the signal to background (S / B) ratios.

[0173] Assay quality - precision: Quality control (QC) samples were tested by 2 operators (analysts) on two different days, and replicates of each sample were tested in the same assay. For prototype strains, high, medium and low quality QC samples were used. For variants, 2 or 3 QC samples were used.

[0174] Assay quality - linearity: One sample was serially diluted (6 times, 4-fold dilution series) and evaluated in duplicate by 2 different operators.

[0175] Correlation analysis with live virus based microneutralization (MN) assay: Samples were tested in a validated live virus MN assay (360biolabs) (n=13) and the data were then compared to that of the pseudovirus neutralization assay using linear regression analysis.

[0176] Correlation with anti-S IgG antibodies 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), after which the data were compared to the pseudovirus neutralization assay results using linear regression analysis.

[0177] Clinical utility: To evaluate the clinical utility of the assay, clinical serum samples from participants administered the NVX-CoV2373 vaccine were tested against the prototype strain and variants (Omicron BA.1, BA.2, BA.5) using a pseudovirus neutralization assay. Neutralizing antibody responses against the prototype strain and its variants were profiled in clinical serum samples. To confirm the effect of vaccine boosters on neutralizing antibody responses (using PNT), sera from patients before and after they were administered a booster dose of NVX-CoV2373 were evaluated in a pseudovirus neutralization assay.

[0178] Results: A pseudovirus-based neutralization assay was developed in a 96-well plate format and tested in HEK293 / ACE2 cells at doses (TCID 50 , pseudovirus amount / well) and kinetic (assay duration) experiments were optimized. Assay positive and negative controls exhibited a wide dynamic range. Neutralization data from the pseudovirus assay showed robust correlation with validated anti-r-spike IgG levels and ACE2 inhibition titers (prototype). Currently, a pseudovirus assay is being developed for Omicron BA.5, and sera from patients who received the NVX-CoV2373 vaccine are being evaluated in the assay.

[0179] The luminescent signal from the pseudovirus neutralization assay showed a dose-dependency on the amount of pseudovirus used for infection ( Figure 2A , Figure 2B ). The luciferase endpoint showed robust dynamic range and user-friendliness (ease of use and throughput).

[0180] Results - Effect of ACE2-expressing cell lines on pseudovirus infection: To evaluate the suitability 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 different amounts of pseudovirus expressing SARS-CoV-2 S protein from the prototype strain (also referred to as "prototype") or S protein from SARS-CoV-2 variants (Omicron BA.1, BA.2, BA.5). Luciferase levels in infected cells after two days are shown on the Y axis. The TCID50 / mL of pseudoviruses calculated based on luciferase levels are shown in the table below.

[0181]

[0182] Cell lines expressing ACE2 (e.g., Vero-E6, A549-ACE2-TMPRESS, and 293T-ACE2 cell lines) have different susceptibilities to pseudovirus infection ( Figure 3A-3C Regardless of the identity of the expressed SARS-CoV-2 S protein, Vero-E6 cells were less amenable to pseudovirus infection than the other cell lines evaluated ( Figure 3C ). Pseudoviruses expressing the SARS-CoV-2 S protein of the prototype strain had similar abilities to infect 293T-ACE2 cells and A549-ACE2-TMPRESS cells ( Figure 3A , Figure 3B ). However, when the pseudovirus expressed the SARS-CoV-2 S protein from the SARS-CoV-2 Omicron strain, the pseudovirus was more infectious in 293T / ACE2 cells.

[0183] In order to compare the suitability of 293T / ACE2 and A549 / ACE2 cell lines for pseudovirus neutralization assay (PNT), cells were infected with Omicron BA.1 and BA.5 pseudoviruses in the presence / absence of increasing serum (No. 2127) dilutions, and luciferase measurements were performed as described in the method. Cells infected with pseudoviruses (BA.1 and BA.5) in the absence of test serum (No. 2127) (virus control) and cells without pseudoviruses (cell control) were used as controls. The inhibition percentage (%) compared to the virus control is shown on the Y axis. Both cell lines show similar (overlapping) luciferase inhibition curves (ID50). Figure 4A-4B The ID50 of pseudovirus infection of 293T cells and A549 / ACE2-TMPRESS cells is shown. The table below shows the pseudovirus equivalent neutralization titer (ID50) of different serum samples.

[0184] These data show that both cell lines are suitable for pseudovirus neutralization assays.

[0185]

[0186] In order to evaluate the effect of dose and time on PNT, four (4) test serum samples were evaluated in PNT, and the amount of pseudovirus (prototype strain) was gradually increased: 50, 125 and 250TCID50 / well. The ID50 values ​​of serum are plotted on the Y axis. It was observed that the neutralization capacity measured by ID50 level decreased with the increase in the amount of pseudovirus used for infection. The following table shows the effect of pseudovirus dose (ranging from 50 to 3.13uL / well) and kinetics (48 hours (48h) relative to 72 hours (72h) determination endpoint) on signal / background (S / B) levels in 293T / ACE2 cells.

[0187]

[0188] Since the 48 h time point was found to provide a better S / B ratio for all pseudovirus loads and strains, it was chosen for subsequent experiments. Figure 5A-Figure 5B The cells were infected with pseudovirus 48 hours later ( Figure 5A ) and 72 hours ( Figure 5B ) luminescence output. Three of the four serum samples showed a dose-dependent ( Figure 5A-Figure 5B ).

[0189] To evaluate the precision of the assay, quality control serum samples were evaluated in PNT by two different operators on two different days. Replicate samples were also tested in the same assay to evaluate intra-assay precision. Neutralization titers (ID50) were plotted on the Y-axis (GMT, 95% CI). Figure 6A-6C Inter-assay and intra-assay precision of the pseudovirus neutralization assay are shown. Fig. 6A Showing the accuracy of pseudoviruses encoding SARS-CoV-2 S protein from the prototype strain. Figure 6B The accuracy of the pseudovirus encoding the SARS-CoV-2 S protein from the Omicron BA.1 strain is shown. Figure 6C The accuracy of the pseudovirus encoding the SARS-CoV-2 S protein from the Omicron BA.5 strain is shown.

[0190] To evaluate the linearity of PNT, the test serum (serum No. 2127) was serially diluted (6 times, 4-fold dilution series) and then evaluated in duplicate at each dilution in PNT (prototype strain) by two different operators (1 and 2) as described in the methods. The neutralization titers (ID50) of the different dilutions were plotted on the Y axis. Figure 7A-7B Display different operators ( Fig. 7A -Operator No. 1, Figure 7B - Operator 2) Dose proportional neutralization with serum dilutions. Assay linearity was confirmed for the prototype strain-based pseudovirus (R2 = 0.9978 and 0.9764 for both operators).

[0191] Figure 8 Results of the pseudovirus neutralization assays were shown to be significantly correlated with the live virus MN assay for the prototype strain (Pearson r = 0.9304, R2 = 0.8657, P < 0.0001). Test serum samples (n = 13) from clinical studies were evaluated for the prototype strain in the PNT as described above, followed by regression analysis using a validated live virus microneutralization assay. GraphPad Prism software (9.3.1) was used for analysis. The dotted lines show the 95% confidence interval (CI). Pearson correlation coefficients (Pearson r) and two-tailed P values ​​are shown in the figure.

[0192] Figure 9A-9B The ability of serum samples to resist pseudovirus infection was shown to be related to the amount of anti-SARS-CoV-2 S protein immunoglobulin (Pearson r = 0.7133, R 2 =0.5088, P = 0.0028)( Fig.9A ) and hACE2 binding inhibition titer (Pearson r = 0.8949, R 2 =0.8009, P=0.0027)( Fig. 9B ) correlation. GraphPad Prism software (9.3.1) was used for analysis. The dotted line shows the 95% confidence interval (CI). The Pearson correlation coefficient (Pearson r) and two-tailed P value are shown in the figure.

[0193] Figure 10A-10C Shown are samples from patients 1 and 2 ( Fig. 10A ), patients 3 and 4 ( Fig. 10B ) and patients 6 and 7 ( Fig. 10C )'s neutralizing titer (ID50). Fig. 10D Patient 5 was shown to have received the primary NVX-CoV2373 vaccine series ( Fig. 10D ) and after administration of the NVX-CoV2373 booster ( Fig. 10D Neutralization titers 14 days after immunization ("3x" in the figure). The primary NVX-CoV2373 vaccine series was administered on days 0 and 21. Sera were collected 28 days after the booster dose, which is shown on the X-axis as 3x [primary series (days 0 and 21) + booster (day 189)].

[0194] Abbreviations: GMT, geometric mean titer; hACE2, human angiotensin-converting enzyme 2; HQC, high quality control; IgG, immunoglobulin G; LQC, low quality control; MN, microneutralization; MQC, medium quality control; N / A, not applicable; NC, negative control; PNT, pseudovirus neutralization assay; RLU, relative luminescence 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.

[0195] 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 rapid turnaround and will further enable the discovery and development of effective vaccines against emerging COVID-19 variants.

[0196] Example 2: Multiplex detection of neutralizing antibody responses against different SARS-CoV-2 strains in the same assay plate using multiple pseudovirus assays

[0197] Introduction: As SARS-CoV-2 continues to evolve around the world, different variants are observed to be circulating. Due to the limited amount of serum collected from human subjects in vaccine clinical trials, convalescent subjects, or subjects infected with SARS-CoV-2, it is difficult to measure neutralizing antibody responses against multiple variants simultaneously. Separate measurement of neutralizing antibodies against each variant increases cost, analytical turnaround time, and complexity of the sample analysis and interpretation process to be clinically meaningful.

[0198] Methods: To address this issue, a multiplex pseudovirus neutralization assay was developed using pseudoviruses expressing spike proteins from different SARS-CoV-2 strains (e.g., prototype, Omicron BA.1, and BA5 strains) and different reporter proteins (RFP and GFP), followed by optimization of the pseudovirus neutralization assay and evaluation of serum samples positive for neutralizing antibodies against SARS-CoV-2.

[0199] Results: In the first set of experiments, pseudoviruses expressing the Omicron BA.1 spike protein and RFP reporter protein (BA.1-RFP) and the Omicron BA.5 spike protein and GFP reporter protein (BA.5-GFP) were prepared and then infected with HEK293T / ACE2 cells in 96-well plates (1.25×10 4Cells / well) were infected individually or in combination at serial dilutions (ranging from 2-fold to 54-fold dilutions using a 3-fold dilution series). Cells were incubated at 37°C and 5% CO2 for 72 hours, after which the fluorescence of GFP and RFP was measured using two different plate readers (Celigo and ID3 plate reader). Our observations showed that reporter protein (RFP and GFP) fluorescence could be observed in the same wells of a 96-well plate with little or no interference ( Figures 11A-11D ).

[0200] To further test the feasibility of multiplex methods 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-2S glycoproteins from different strains and different reporters (BA.1RFP and prototype-GFP), either alone or in combination. The serum-pseudovirus mixture was used to infect 293T / ACE2 cells in 96-well plates in duplicate, after which the fluorescence of RFP and GFP was measured after 72 hours. The table below shows the neutralization percentages of Omicron BA.1RFP and BA.5GFP observed in a single pseudovirus neutralization test (PNT) and both of them together.

[0201]

[0202] The data from this experiment demonstrates the feasibility of measuring neutralizing activity present in human serum samples in multiplex pseudovirus assays in the same wells. Some advantages of this multiplex assay are: 1. Low volume test serum sample requirements. 2. Reduced assay costs (reagent and labor costs) 3. Fast turnaround time 4. Improved neutralization assay measurement efficiency.

[0203] Example 3: Validation of pseudovirus-based neutralization assay

[0204] Objective: To validate a pseudovirus-based neutralization assay to confirm its suitability for the clinical samples tested.

[0205] Methods: Sera from patients immunized against SARS-CoV-2 were heat inactivated at 56°C for approximately 30 minutes. As a negative control, patient sera collected before the SARS-CoV-2 outbreak were used. Positive controls were human sera with high, medium, and low pseudovirus neutralization (PNT) titers. Each heat-inactivated serum sample was diluted 1:10 in infection medium before use in the assay. Pseudoviruses were added to the samples and incubated to allow virus-specific neutralizing antibodies to neutralize the virus. The pseudovirus was a replication-defective Moloney murine leukemia virus (MLV). The pseudovirus expressed one of the following SARS-CoV-2 glycoproteins: (i) 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 "prototype D614 pseudovirus"); (ii) 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 "prototype D614 pseudovirus"); Compared with the glycoprotein of NO:2, amino acid 54 is V, amino acid 56 is missing, amino acid 57 is missing, amino acid 82 is I, amino acid 129 is D, amino acid 130 is missing, amino acid 131 is missing, amino acid 132 is missing, amino acid 198 is missing, 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, and 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, and amino acid 492 is Y (referred to as the “BA.5 pseudovirus”); and (iii) SARS-CoV-2 S glycoprotein of the Omicron XBB.1.5 subtype (referred to as the “XBB.1.5 pseudovirus”).

[0206] HEK293T cells expressing ACE2 (HEK293T / ACE2 cells) were added to a mixture of pseudovirus and sample and incubated for three days to infect HEK293T / ACE2 cells with unneutralized viruses. On the fourth day, cells were lysed and the luminescence (relative light units) of the cells was measured to determine the level of infection that occurs when antibodies are present in serum. The parameters of assay accuracy, specificity, LLOQ and ULOQ were evaluated.

[0207] Precision: Precision is the closeness (dispersion) of a series of measurements obtained from multiple tests of the same sample. Total or overall precision consists of intra-assay and inter-assay precision. Intra-assay (within-run) precision is the closeness of multiple determinations of a single sample made within a single assay run under the same operating conditions over a short time interval. Inter-assay (between-run) precision is the closeness of replicate measurements within a laboratory taking into account all relevant sources of variation that affect the results (e.g., runs, analysts, equipment, and reagents).

[0208] A set of 40 serum samples ranging from negative, low to high PNT titers were used for each pseudovirus to determine the intra- and inter-assay precision of the PNT assay. Assay precision data were generated by 2 analysts on 3 different days in 6 runs, with each sample tested twice in each run. A total of 24 results from 6 runs were generated and used for precision evaluation. In 9 samples, fewer than 24 (16 to 23) values ​​were available for precision evaluation because titers could not be generated or sample amounts were insufficient for testing in all runs.

[0209] Intra-assay and inter-assay precision were estimated by calculating the geometric coefficient of variation percentage (GCV %) using a variance component analysis model with sample as a fixed effect, and analyst and day as random effects.

[0210] Intra-assay and inter-assay GCV% were calculated based on the natural log-transformed values ​​of PNT titers:

[0211]

[0212] The total GCV% of intermediate accuracy is calculated as follows:

[0213]

[0214] Intra-assay, inter-assay and overall assay precision, ie the overall assay variance of all 40 tested samples, were evaluated at the individual sample level and also at the strain level.

[0215] Specificity / Selectivity: Specificity is the ability of an analytical method to measure and distinguish an analyte in the presence of components that might be expected to be present. Selectivity is the extent to which the method can determine specific compounds in the matrix being analyzed without interference from matrix components. The selectivity of the method is confirmed by analyzing negative control samples of appropriate biological matrices (e.g., serum) from multiple sources. Negative control human serum samples from before the SARS-CoV-2 pandemic era included sera from influenza vaccine studies and RSV vaccine studies that showed very strong specific immune responses to influenza virus and RSV F protein, as well as normal human serum samples collected before the SARS-CoV-2 pandemic were tested.

[0216] Linearity: The linearity of an analytical method measures its ability to obtain test results that are proportional to the concentration (amount) of the analyte in the sample (over a given range). The linearity of the PNT assay in the prototype virus was evaluated by testing two SARS-CoV-2 PNT-positive samples undiluted and diluted in negative serum. The number of dilutions and the dilution factor depended on the titer of the undiluted serum (i.e., 1:2, 1:8, more or less dilutions were performed where appropriate and feasible). Linearity samples were tested twice in duplicate in the same run by 2 different analysts over 3 days for a total of 6 runs. For each dilution, the sample was diluted independently in negative serum and then heat inactivated.

[0217] A simple linear regression was performed on the data set. The independent variable was the log10 expected PNT titer, and the dependent variable was the log10 observed PNT titer. The slope and coefficient of determination (R 2 ) and 95% confidence intervals (CI).

[0218] The expected PNT titer at each dilution was calculated by dividing the overall GMT from all runs of undiluted samples by the dilution factor for each dilution of each sample. The observed PNT titer at each dilution was the overall GMT at each dilution of each sample in all runs.

[0219] The accuracy of an analytical procedure is defined as the closeness of agreement between the measured value and a value accepted as the agreed true value or recognized reference value. Accuracy in linearity studies is evaluated by comparing the expected and observed values ​​and calculating the relative percent deviation. Relative percent deviation for each dilution:

[0220]

[0221] A relative deviation percentage of 150% or -60% corresponds to a titer 2.5-fold higher or 2.5-fold lower than the expected titer, respectively.

[0222] PNT assay lower and upper limits of quantitation: The lower limit of quantitation (LLOQ) and upper limit of quantitation (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 of these samples during each run, and accuracy was estimated by calculating the relative deviation percentage of these samples during each run. The precision and accuracy of the LLOQ determination were evaluated at dilutions with expected PNT titers of 20-100.

[0223] Results: Accuracy: The table below contains the accuracy of the pseudovirus-based neutralization assay for pseudoviruses expressing each SARS-CoV-2 S glycoprotein.

[0224]

[0225]

[0226] Overall precision was calculated by analyzing the assay variance from all 40 samples using the prototype D614 prototype virus. The overall inter-assay, intra-assay, and total assay GCV% for pseudovirus neutralization assays performed using pseudoviruses expressing the prototype D614 SARS-CoV-2 S glycoprotein were 6.6%, 42.8%, and 43.4%, respectively. At the individual sample level, the lowest GCV% for inter-assay, intra-assay, and total assay precision was 0.0% across all 40 samples. The highest GCV% for inter-assay, intra-assay, and total assay precision were 60.5%, 59.8%, and 76.1%, respectively. Ninety-five percent (95%, 38 of 40), 90% (36 of 40), and 65% (26 of 40) of the samples showed an inter-assay, intra-assay, and overall GCV% of less than 50, respectively; and 97.5% (39 of 40), 100% (40 of 40), and 85% (34 of 40) of the samples showed an inter-assay, intra-assay, and overall GCV% of less than or equal to 60, respectively. A GCV% of less than 60 for the prototype D614 virus of at least 80% of the samples is acceptable.

[0227]

[0228]

[0229] Overall accuracy was calculated by using BA.5 subvariant pseudovirus analysis of the assay variance from all 40 samples. Overall assay inter-, intra- and total assay GCV% were 15.1%, 26.4% and 30.7% respectively. Under the single sample level, in all 40 samples, the lowest GCV% between assays, within assays and total assay accuracy was 0.0%. The highest GCV% between assays, within assays and total assay accuracy was 63.1%, 48.3% and 71.5% respectively. Thirty-nine (98%) of the forty samples, all forty samples (100%) and thirty-eight (95%) of the forty samples showed less than 50 between assays, within assays and overall GCV% respectively. Assay accuracy meets the target acceptance criteria.

[0230]

[0231]

[0232] The overall precision was calculated by analyzing the variance of the assays from all 40 samples using the XBB.1.5 subvariant pseudovirus. The between-assay, within-assay, and total-assay GCV% were 20.0%, 29.5%, and 36.1%, respectively. At the individual sample level, the lowest GCV% for between-assay, within-assay, and total-assay precision was 0.0% in all 40 samples. The highest GCV% for between-assay, within-assay, and total-assay precision was 65.7%, 55.9%, and 72.1%, respectively. Thirty-eight (95%), thirty-nine (97.5%), and thirty-two (80%) of the forty samples showed between-assay, within-assay, and overall GCV% less than 50, respectively. The assay precision met the target acceptance criteria.

[0233] Results - Specificity: The assay confirmed the specificity of detecting neutralizing antibodies against SARS-CoV-2 antigens. No detectable neutralizing antibodies (<LLOQ) against the prototype D614 pseudovirus, XBB1.5 pseudovirus, or BA.5 pseudovirus were found in human sera collected before the SARS-CoV-2 pandemic era. Additionally, 5 pairs of pre- and post-vaccination sera from the phase 3 clinical trial RSV-M-301 of the RSV F vaccine and 5 pairs of pre- and post-vaccination sera from the phase 3 clinical trial qNIV-E-301 of the nanoparticle influenza vaccine, which were collected before the SARS-CoV-2 pandemic, were tested in the PNT assay. All samples tested negative (<LLOQ) in the prototype D614, XBB1.5, and BA.5 PNT assays, while having a very strong specific immune response to influenza or RSV post-vaccination, confirming the specificity of the SARS-CoV-2 PNT assay.

[0234] Results - Linearity: The relative deviation percentage, LLOQ, and ULOQ of the pseudo-neutralizing antibody titers for each pseudovirus were determined. The accuracy of the PNT assay was evaluated by the relative deviation percentage (RB%). The precision was determined by the GCV% value of all runs from the dilution points.

[0235]

[0236]

[0237]

[0238] The above table shows the linearity test data of the prototype D614 prototype pseudovirus assay. The linearity test data of the prototype D614 prototype pseudovirus assay showed an acceptable linear response. The RB% of all 3 HMN865539 dilution points with an expected GMT higher than 20 were within the target range (-60% to 150%); the RB% of all 6 HMN865536 dilution points with an expected GMT higher than 20 were within the target range. The GCV% of 6 (75%) of the 8 dilution points of sample HMN865539 was <60, and the GCV% of 7 (87.5%) of the 8 dilution points of sample HMN865536 was <60. The point estimates of the slopes of the linear regression plots of 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 of the slope for sample HMN865539 were 0.521–1.080, which was outside the target range of 0.7–1.43. This may be attributed to the lower titer of the sample and the fewer dilution points used for the regression plot (only 4 dilution points). Sample HMN865536, which had 7 dilution points for the regression plot, had a 95% LCL and UCL of 0.847–1.153, which was within the acceptable range of 0.7–1.43. The R values ​​of the regression plots for samples HMN865539 and HMN865536 were 2 The results of the present invention show that the present invention can measure the neutralizing antibody titer of the prototype pseudovirus at the expected PNT titer of 20 and above 20. The RB% of the two samples is within -42.6% to 75.8%, which is within 2.5 times of the expected titer (-60% to 150%). The GCV% of sample HMN865539 under the expected GMT of 41.6 is 60.5 and 39.6 respectively for the inter-assay and intra-assay precision, although the total GCV% is higher (76.1). For sample HMN865536, under the expected GMT of 29.0 and the observed GMT of 41.6, the GCV% between the assay and the intra-assay is 0 and 46.1 respectively, and the total GCV% is 46.1. Therefore, a GMT of 42 had acceptable precision (GCV% ≤ 60) and relative accuracy (RB% of -60% to 150%) and was set as the assay LLOQ. The highest PNT GMT titer was 14863.2, with inter-assay, intra-assay, and overall GCVs less than 60. Therefore, the assay provisional ULOQ for the PNT assay using the prototype virus was 14863. The assay ULOQ may be further evaluated during clinical testing when higher titer serum samples become available.

[0239]

[0240]

[0241] The table above shows the linearity test data for the BA.5 pseudovirus assay. Linear analysis of the BA.5PNT assay was performed using 2 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 for the slope was 0.872. The highest upper confidence limit (UCL) of the 95% confidence interval for the slope was 1.256. The slopes of both samples passed the acceptance criteria of 95% CI 0.7-1.43. The R of the regression plots 2 The relative deviation percentage (RB%) of HMN934977 at 6 dilutions was -60% to 150%, which passed the acceptance criteria. Six (6) of the seven (7) dilutions of ZA018-0389 (85.7% of the samples) had a RB of -60% to 150%. It met the acceptance criteria. Since 12 of the 13 samples (dilutions) tested showed a relative deviation percentage of -60% to 150%, and the slope and R from the 2 linear samples were 2 The acceptance criteria were passed, so the method was considered to be able to measure neutralization of the Omicron BA.5 pseudovirus 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 linear evaluation. Precision was estimated by calculating the GCV% of the PNT titer of these samples during each run, and accuracy was estimated by calculating the relative deviation percentage of these samples during each run. The precision and accuracy of the LLOQ determination were evaluated at dilutions with expected PNT titers of 20-100.

[0242] The accuracy and precision of 2 linear samples for BA.5 virus PNT determination were evaluated. When the expected PNT titer was 20 and above 20, except for one sample (12 of 13) with a relative deviation of -62.7%, the relative deviation percentages of both samples were within -55% to -8.2%, which was within 2.5 times the difference (-60% to 150%) of the expected titer. At 14 of the 15 dilutions (from two samples), GCV% <50.0%. The lowest expected PNT GMT titers above 20 were 27.2 (from HMN934977) and 21.9 (from ZA018-0389), respectively. They are related to the HMN934977 1:192, ZA018-0389 1:1280 dilutions. At these 2 dilutions, 91.3% (21 out of 23) of the GMT titers (Appendix 3) were less than 20. They are defined as 20 when calculating GCV% and RB%, so they have the lowest variability of all. They 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% less than 50 and RB% between -60 and 150 was 15856.3. For this type of semi-quantitative assay, a GCV% ≤ 50% is considered acceptable. Therefore, an LLOQ of 36 and a ULOQ of 15856 are acceptable for the PNT assay using BA.5 virus. The assay ULOQ may be further evaluated during clinical testing when higher titer serum samples become available.

[0243]

[0244]

[0245] The above table shows the linearity test data for the XBB.1.5 pseudovirus assay. Linearity analysis of the BA.5PNT assay was performed using 2 serum samples. Linearity analysis of the XBB.1.5PNT assay was performed using 2 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 95% confidence interval for the slope of HMN934977 were 1.011 and 1.361, respectively. They passed the target acceptance criteria (0.70-1.43). The LCL and UCL of the 95% confidence interval for the slope of HMN865550 were 0.879 and 1.479, respectively. The UCL was slightly higher than the target acceptance criteria (1.43), but was considered acceptable (approximately 32% higher than the point estimate of the slope, rather than 30% higher). The R of the regression plot 2The relative deviation percentage (RB%) of HMN865550 was -60% to 150%, which passed the acceptance criteria. Five (5) of the six (6) dilutions of HMN934977 (83.3% of the samples) had a RB% of -60% to 150%. It met the acceptance criteria. Since 11 of the 12 samples tested showed a relative deviation percentage of -60% to 150%, and the slope and R from the two linear samples were 2 The acceptance criteria were passed, so the method was considered capable of measuring Omicron XBB.1.5 pseudovirus neutralization within 2.5 times the expected PNT titer. The lower limit of quantitation (LLOQ) and upper limit of quantitation (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 of these samples during each run, and accuracy was estimated by calculating the relative deviation percentage of these samples during each run. The precision and accuracy of the LLOQ determination were evaluated at dilutions with expected PNT titers of 20-100.

[0246] Describe the summary of the accuracy and precision of two linear samples for XBB.1.5 virus PNT determination. At the dilution 216 of sample HMN934977, 10 of 12 GMT titers (83.3%) <20 and are defined as 20 for calculation. Therefore, GCV% is low. The PNT GMT titer (20.6) at this dilution is not suitable for definition as LLOQ. The observed second lowest GMT titer is 35.5. The inter-assay and total GCV% are 53.4% ​​and 60.8%, respectively, which do not pass the target acceptance standard (GCV% <50). At the dilution 144 of HMN865550, all 12 GMT titers are <20, and at the dilution 72 of HMN865550, 11 GMT titers <20 and are defined as 20 for calculation. Therefore, GCV% is low. The PNT GMT titer (20.0) at these two dilutions is not suitable for definition as LLOQ. The next lowest GMT titer was 37.4. Both inter-assay and intra-assay precision passed the acceptance criteria (GCV%<50), but the overall GCV% failed at 50.6%. Since the GMT is below 100, it is close to the LLOQ. 50.6% GCV is acceptable. Therefore, a GMT titer of 37 is defined as the assay LLOQ. The highest PNT GMT titer was 7560.9, with a GCV% (inter-assay, intra-assay, and overall) less than 50%. For this type of semi-quantitative assay, a GCV%≤50% is considered acceptable. For the PNT assay performed using the XBB.1.5 virus, a ULOQ of 7561 is acceptable. The assay ULOQ can be further evaluated during clinical testing when higher titer serum samples become available.

[0247] Conclusion: The results show that the pseudoneutralization assay described herein is suitable for measuring neutralizing antibodies induced by SARS-CoV-2 and its variants.

[0248] The results obtained from the assay validation experiments confirmed that the pseudovirus neutralization assay in the prototype D614 prototype virus was precise and accurate in measuring PNT titers in human serum, with an overall assay GCV% ≤ 60% and a relative deviation percentage within -60% and 150%. The PNT assay performed a semi-quantitative assessment of PNT antibody titers in a reliable and reproducible manner. The method showed specificity for the SARS-CoV-2 virus used for validation. The method was linear with respect to antiserum dilution. LLOQ and ULOQ were established, with sample GCV% below 60 and sample fit within the linear range. The method is therefore suitable for the analysis of SARS-CoV-2 vaccine clinical trial samples for the prototype D614 prototype virus to assess immunogenicity.

[0249] The results obtained from the assay validation experiments confirmed that the pseudovirus neutralization assay in Omicron BA.5 was precise and accurate in measuring PNT titers in human sera, with an overall assay GCV% ≤ 50% and relative deviation percentages within -60% and 150%. The PNT assay performed a semi-quantitative assessment of PNT antibody titers in a reliable and reproducible manner. The method showed specificity for the SARS-CoV-2 virus used for validation. The method was linear with respect to antiserum dilution, and LLOQ and ULOQ were established, with sample GCV% below 50 and sample fit within the linear range. Therefore, this method is suitable for analyzing samples from patients who have been administered a SARS-CoV-2 vaccine against Omicron BA.5 to assess immunogenicity.

[0250] Results obtained from assay validation experiments confirmed that the pseudovirus neutralization assay in Omicron XBB.1.5 was precise and accurate in measuring PNT titers in human serum, with overall assay GCV% ≤ 50% and relative percent deviations within -60% and 150%. The PNT assay performed semi-quantitative assessment of PNT antibody titers in a reliable and reproducible manner. The method showed specificity for the SARS-CoV-2 virus used for validation. The method was linear with respect to antiserum dilution, and LLOQ and ULOQ were established, with sample GCV% below 50 and sample fit within the linear range.

[0251] Incorporated by Reference

[0252] All references, articles, publications, patents, patent disclosures and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent disclosure and patent application cited herein is not and should not be regarded as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. The following patent documents are incorporated by reference in their entirety: International Publication No. 2021 / 154812; and International Publication No. 2022 / 203963; International Publication No. 2023 / 102448.

Claims

1. A pseudovirus encoding: ( i) SARS-CoV-2 spike (S) glycoprotein; (ii) one or more of Rev, Tat, Gag and Pol proteins; and (iii) Reporter protein.

2. The pseudovirus according to claim 1, encoding 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 SARS-CoV-2 S glycoproteins.

3. The pseudovirus according to claim 1, wherein the reporter protein is luciferase.

4. The pseudovirus according to claim 1, wherein the reporter protein is a fluorescent protein.

5. The pseudovirus according to claim 4, wherein the fluorescent protein is ZsGreen.

6. The pseudovirus according to any one of claims 1 to 5, wherein the SARS-CoV-2 S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

7. The pseudovirus according to any one of claims 1 to 6, wherein the SARS-CoV-2 S glycoprotein has an inactive furin cleavage site.

8. The pseudovirus according to claim 7, wherein the SARS-CoV-2S glycoprotein has an inactive furin cleavage site, and the inactive furin cleavage site has an amino acid sequence of QQAQ (SEQ ID NO: 68).

9. The pseudovirus according to any one of claims 1 to 8, 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.

10. The pseudovirus of any one of claims 1-8, wherein the SARS-CoV-2 S glycoprotein 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 the polypeptide of any one of SEQ ID NOs: 3, 5-13, 15, 17-19, 21 and 23-66.

11. The pseudovirus according to any one of claims 1-6 and 10, wherein the SARS-CoV-2 S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polypeptide of any one of SEQ ID NOs: 3-13, 20 and 22-66.

12. The pseudovirus according to any one of claims 1-11, wherein the SARS-CoV-2 S glycoprotein has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polypeptide of any one of SEQ ID NOs: 3, 5-13 and 23-66.

13. The pseudovirus according to any one of claims 1 to 5, wherein the SARS-CoV-2 S glycoprotein is from the SARS-CoV-2 virus or a variant of SARS-CoV-2.

14. A pseudovirus according to claim 13, wherein the variant of SARS-CoV-2 is B.1.1.7SARS-CoV-2 strain; B.1.351SARS-CoV-2 strain; P.1SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2SARS-CoV-2 strain; B.1.525SARS-CoV-2 strain; B.1.526SARS-CoV-2 strain; B.1.617.1SARS-CoV-2 strain; C.37SARS-CoV-2 strain; B.1.621SARS-CoV-2 strain; or B.1.1.529SARS-CoV-2 strain.

15. The pseudovirus of any one of claims 1-14, wherein the SARS-CoV-2 S glycoprotein 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 a SARS-CoV-2 S glycoprotein from a SARS-CoV-2 S 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.

16. The pseudovirus according to any one of claims 1 to 15, comprising Gag protein and Pol protein.

17. The pseudovirus according to claim 16, wherein the fusion protein encodes the Gag protein and the Pol protein.

18. The pseudovirus according to any one of claims 1 to 17, wherein the Gag protein and the Pol protein are from murine leukemia virus (MLV).

19. The pseudovirus according to any one of claims 1 to 17, wherein the Rev, Tat, Gag and Pol proteins are from a lentivirus.

20. A method for producing a pseudovirus according to any one of claims 1 to 19, comprising 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, Pol protein, or a combination thereof; and (iii) A plasmid encoding the SARS-CoV-2 spike (S) glycoprotein.

21. The method of claim 20, wherein the host cell is human.

22. The method of claim 9, wherein the host cell is a HEK293T (also referred to as "293T") cell.

23. A method for determining whether a biological sample contains neutralizing antibodies against the SARS-CoV-2 virus or a variant thereof, comprising: (a) contacting a biological sample with 1 to about 20 pseudoviruses according to any one of claims 1 to 19; (b) contacting cells expressing angiotensin-converting enzyme 2 (ACE2) with the 1 to about 20 pseudoviruses; (c) quantifying the expression of the reporter protein in the ACE2-expressing cells; (d) contacting a control cell expressing ACE2 with the 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 ACE2-expressing control cells; 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 its variant.

24. The method of claim 23, comprising contacting the biological sample and pseudovirus for a period of 1 hour to about 7 hours.

25. The method of claim 24, comprising contacting the biological sample and pseudovirus for about two hours.

26. The method of any one of claims 23-25, comprising contacting the biological sample and the pseudovirus at about 37°C.

27. The method of any one of claims 23-26, comprising heat inactivating the biological sample.

28. The method of any one of claims 23-27, comprising contacting the cells with the pseudovirus for 1 day to about 7 days.

29. The method of any one of claims 23-28, comprising contacting the cells with the pseudovirus for about 3 days.

30. The method of any one of claims 23-29, wherein the cells are selected from the group consisting of Vero E6, A549 and HEK293T cells.

31. The method of any one of claims 23-30, wherein the cell is a HEK293T cell.

32. The method of any one of claims 23-30, wherein a Biosafety Level 3 (BSL-3) laboratory is not required to perform the method.

33. The method of any one of claims 23-32, wherein the biological sample is serum, plasma or blood from a patient who has been administered an immunogenic composition against the SARS-CoV-2 virus or a variant thereof.

34. The method of any one of claims 23-32, wherein the biological sample is serum, plasma, or blood from a patient who previously had COVID-19.

35. The method of any one of claims 23-34, comprising 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 pseudoviruses.

36. The method of claim 35, wherein each pseudovirus expresses a different reporter gene and a different SARS-CoV-2 S glycoprotein.

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