Assay for detection of coronavirus neutralizing antibodies

Abstract

The present disclosure provides methods, as well as related compositions and kits, for determining the presence of coronavirus-neutralizing antibodies in a sample. The methods of the disclosure use recombinant vesicular stomatitis virus (VSV) particles, in which the VSV glycoprotein (G) is replaced with the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof. In a specific embodiment, the S glycoprotein is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the methods are used to determine the presence of SARS-CoV-2-neutralizing antibodies.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 151,623, filed February 19, 2021, U.S. Provisional Patent Application No. 63 / 078,214, filed September 14, 2020, U.S. Provisional Patent Application No. 63 / 029,267, filed May 22, 2020, U.S. Provisional Patent Application No. 63 / 020,445, filed May 5, 2020, U.S. Provisional Patent Application No. 63 / 012,066, filed April 17, 2020, and U.S. Provisional Patent Application No. 63 / 012,074, filed April 17, 2020, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 15, 2021, is titled 250298_000213_SL.txt and is 147,568 bytes in size. [Technical Field]

[0003] Described herein are methods, as well as related compositions and kits, for determining the presence of coronavirus-neutralizing antibodies in a sample. The disclosed methods use recombinant vesicular stomatitis virus (VSV) particles in which the VSV glycoprotein (G) is replaced by the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof. In a specific embodiment, the S glycoprotein is derived from Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), and the methods are used to determine the presence of SARS-CoV-2-neutralizing antibodies. [Background technology]

[0004] Three coronaviruses are known to cause severe pneumonia in humans: Severe Acute Respiratory Syndrome coronavirus (SARS-CoV or SARS-CoV-1), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-1 emerged in the People's Republic of China in 2002 and spread across five continents, infecting more than 8,000 people and killing 774. MERS-CoV emerged in the Arabian Peninsula in 2012, infecting more than 2,500 people in 27 countries and killing 858. In December 2019, a novel coronavirus emerged in Wuhan, People's Republic of China, causing an acute respiratory syndrome known as coronavirus disease 2019 (COVID-19) (Non-Patent Document 1, published online February 3, 2020, available at doi.org / 10.1038 / s41586-020-2012-7; Non-Patent Document 2). Symptoms of COVID-19 include fever, cough, shortness of breath, pneumonia, acute respiratory distress syndrome (ARDS), acute pulmonary syndrome, loss of smell, loss of taste, sore throat, runny nose, gastrointestinal symptoms (e.g., diarrhea), organ failure (e.g., renal failure and dysfunction), septic shock, and, in severe cases, death. The virus that causes COVID-19 has been identified as related to SARS-CoV-1 and has therefore been named SARS-CoV-2 (sometimes referred to as nCov-2019, Wuhan coronavirus, or SARS-nCoV19). Although some individuals are asymptomatic or experience very mild symptoms, many become severely ill and require hospitalization. SARS-CoV-2 spread rapidly around the world and was declared a global pandemic by the World Health Organization on March 11, 2020. As of May 20, 2020, the virus had infected approximately 5 million people worldwide and caused over 328,000 deaths.While most patients who develop COVID-19 experience mild upper respiratory symptoms, some (especially the elderly and those with underlying conditions such as chronic lung disease, asthma, heart disease, diabetes, or immunocompromised individuals) develop atypical pneumonia and related complications (Non-Patent Document 3, published online April 1, 2020, available at doi.org / 10.1038 / s41586-020-2196-x). SARS-CoV-2 is highly contagious and can be spread by asymptomatic carriers.

[0005] PCR assays that detect active SARS-CoV-2 infection play an important role in tracking the spread of the disease, while serological tests that detect antibodies to SARS-CoV-2 are used to detect and measure past infection, identify individuals who may be immune to SARS-CoV-2, and evaluate the effectiveness of vaccines and treatments. However, it remains unclear how accurately available serological tests reflect antibody-mediated immunity.

[0006] The generation of virus-neutralizing antibodies is important for blocking subsequent viral infection, and their presence correlates with protective immunity after vaccination (Non-Patent Document 4). However, typically, only a small subset of virus-specific antibodies are neutralizing. With regard to SARS-CoV-2, it is currently unknown how total antibody levels correlate with neutralizing antibody levels, and improved testing of SARS-CoV-2 neutralizing antibody responses is urgently needed. While early data from convalescent plasma therapy trials have shown promising results (Non-Patent Document 5), plasma donor screening is insufficient if acceptance criteria are based on total anti-SARS-CoV-2 antibody levels, rather than specifically on neutralizing antibody levels. Similarly, studies of the efficacy of novel SARS-CoV-2 vaccines must consider not only total antibody responses but also neutralizing antibody responses. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Zhou et al., Nature(https: / / doi.org / 10.1038 / s41586-020-2012-7) [Non-patent document 2] Zhu et al., New Engl J Med, 2020, 382:727-733 [Non-patent document 3] Woelfel et al., Nature(https: / / doi.org / 10.1038 / s41586-020-2196-x) [Non-patent document 4] Koff WC et al., Science. 2013;340(6136) [Non-patent document 5] Casadevall A et al., 379 J Clin Invest 2020 Mar 13 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there is a high demand in the art for robust and efficient seropositivity assays for SARS-CoV-2 and other coronaviruses, particularly assays that evaluate virus-neutralizing antibodies. [Means for solving the problem]

[0009] As noted in the "Background" section above, there is a high demand for the development of effective seropositivity assays for SARS-CoV-2 and other coronaviruses, particularly assays that evaluate virus-neutralizing antibodies. The present disclosure addresses these and other needs. The present disclosure provides assays for determining the presence of coronavirus-neutralizing antibodies in a sample, as well as related compositions and kits. The seropositivity assays of the present disclosure use recombinant vesicular stomatitis virus (VSV) particles in which the VSV glycoprotein (G) is replaced by the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof. In a specific embodiment, the S glycoprotein is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the methods are used to determine the presence of SARS-CoV-2-neutralizing antibodies.

[0010] In one aspect, a method is provided for determining the presence of coronavirus neutralizing antibodies in a sample, the method comprising: a) contacting the sample with a recombinant rhabdovirus particle, wherein the rhabdovirus glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein, or a fragment or derivative thereof, and wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a first target cell and a second target cell; b) after step (a), contacting the recombinant rhabdovirus particles with the first target cell expressing a first portion of a reporter protein and the second target cell expressing a second portion of the reporter protein to form a syncytium containing both the first and second portions of the reporter protein and producing a detectable reporter signal, wherein the first target cell and the second target cell are capable of fusing with each other upon contact with the recombinant rhabdovirus particles; c) after step (b), measuring the reporter signal in the cell; and d) comparing the reporter signal measured in step (c) with a control. Includes.

[0011] In one embodiment, both the first target cell and / or the second target cell comprise angiotensin-converting enzyme 2 (ACE2).

[0012] In one embodiment, the first target cells are Vero-DSP1 (Vero-DSP-1-Puro; CLR-73) and the second target cells are Vero-DSP2 (Vero-DSP-2-Puro; CLR-74).

[0013] In one embodiment, the first portion of the reporter protein comprises amino acids 1-229 of Renilla luciferase or a variant thereof, and the second portion of the reporter protein comprises amino acids 230-311 of Renilla luciferase or a variant thereof. In another embodiment, the first portion of the reporter protein comprises amino acids 1-155 of Renilla luciferase or a variant thereof, and the second portion of the reporter protein comprises amino acids 156-311 of Renilla luciferase or a variant thereof. In a specific embodiment, the first portion of the reporter protein comprises amino acids 1-155 of Renilla luciferase mutant RLuc8, and the second portion of the reporter protein comprises amino acids 156-311 of Renilla luciferase mutant RLuc8.

[0014] In one embodiment, the first portion of the reporter protein comprises amino acids 1-156 of green fluorescent protein (GFP) or a variant thereof, and the second portion of the reporter protein comprises amino acids 157-231 of GFP or a variant thereof. In another embodiment, the first portion of the reporter protein comprises amino acids 1-213 of superfolder GFP, and the second portion of the reporter protein comprises amino acids 214-230 of superfolder GFP. In yet another embodiment, the first portion of the reporter protein comprises amino acids 1-154 of superfolder yellow fluorescent protein (YFP), and the second portion of the reporter protein comprises amino acids 155-262 of superfolder YFP.

[0015] In another aspect, a method is provided for determining the presence of coronavirus neutralizing antibodies in a sample, the method comprising: a) contacting the sample with recombinant rhabdovirus particles, wherein (i) the rhabdovirus glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein or a fragment or derivative thereof, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell, and (ii) the rhabdovirus particles comprise a reporter protein and / or a nucleic acid molecule encoding the reporter protein; b) after step (a), contacting said recombinant rhabdovirus particles with said target cells; c) after step (b), measuring the reporter signal in the cell; and d) comparing the reporter signal measured in step (c) with a control. Includes.

[0016] In one embodiment, the recombinant rhabdovirus particle comprises a nucleic acid molecule encoding the reporter protein. In a specific embodiment, the nucleic acid sequence encoding the reporter protein is inserted between the nucleic acid sequence encoding the coronavirus S glycoprotein or a fragment or derivative thereof and the nucleic acid sequence encoding the rhabdovirus large (L) protein. In one embodiment, the target cell is a Vero cell (including a Vero-αHis cell), a Vero-Ace-2 cell, a Vero-TRMPSS2 cell, or a Vero-E6 cell. In one embodiment, the target cell comprises angiotensin-converting enzyme 2 (ACE2). In one embodiment, the genome of the recombinant rhabdovirus particle lacks a functional rhabdovirus G gene and encodes the coronavirus S glycoprotein, a fragment or derivative thereof. In one embodiment, the genome of the recombinant rhabdovirus particle lacks a rhabdovirus G gene and encodes the coronavirus S glycoprotein, a fragment or derivative thereof.

[0017] In one embodiment of any of the above-mentioned methods, the recombinant rhabdovirus particle is a recombinant vesiculovirus particle.In one embodiment of any of the above-mentioned methods, the recombinant vesiculovirus particle is a recombinant vesicular stomatitis virus (VSV) particle.

[0018] In one embodiment of any of the above-described methods, the reporter protein comprises a luciferase, non-limiting examples of luciferases that can be used include Renilla luciferase, RLuc8 mutant Renilla luciferase, (dCpG) luciferase, NanoLuc reporter, firefly luciferase, Gaussia luciferase (gLuc), MetLuc, Vibrio fischeri lumazine protein, Vibrio harveyi luminase protein, dinoflagellate luciferase, firefly luciferase YY5 mutant, firefly luciferase LGR mutant, firefly luciferase mutant E, and fragments or derivatives thereof.

[0019] In one embodiment of any of the above-described methods, the reporter protein comprises a fluorescent protein. Non-limiting examples of fluorescent proteins that can be used include green fluorescent protein (GFP), GFP-like fluorescent protein (GFP-like), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP); red fluorescent protein, superfolder GFP, superfolder YFP, orange fluorescent protein, red fluorescent protein, small super red fluorescent protein, FMN-binding fluorescent protein, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGr1, emerald fluorescent protein, and Azami. Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and fragments or derivatives thereof.

[0020] In one embodiment of any of the above-described methods, the method includes adding a reporter protein substrate to obtain the reporter signal. Non-limiting examples of reporter protein substrates that can be used for luciferase include luciferin (e.g., d-luciferin), EnduRen, and coelenterazine luciferase substrates.

[0021] In one embodiment of any of the aforementioned methods, the recombinant rhabdovirus particle is a replication-competent rhabdovirus particle.

[0022] In one embodiment of any of the above-described methods, the coronavirus S glycoprotein, fragment, or derivative thereof is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In one embodiment, the coronavirus S glycoprotein is a full-length SARS-CoV-2 S glycoprotein. In a specific embodiment, the SARS-CoV-2 S glycoprotein comprises the amino acid sequence of SEQ ID NO: 1. In a specific embodiment, the SARS-CoV-2 S glycoprotein consists of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the coronavirus S glycoprotein fragment is a SARS-CoV-2 S glycoprotein fragment lacking the 19 C-terminal amino acids. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment comprises the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 77% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to the S1 subunit of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to amino acids 14 to 684 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to the RBD domain of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to amino acids 319 to 541 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative results in recombinant rhabdovirus particles that are more fusogenic than equivalent recombinant rhabdoviruses comprising a rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome. In certain embodiments, the SARS-CoV-2An S glycoprotein derivative, or fragment thereof, may comprise or consist of an insertion, deletion, and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 residues of the SARS-CoV-2 S glycoprotein as described above. Non-limiting examples of amino acids for possible deletion include, for example, tyrosine at position 145, asparagine at position 679, serine at position 680, proline at position 681, arginine at position 682, arginine at position 683, alanine at position 684, and / or arginine at position 685 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2Non-limiting examples of amino acids for substitutions that may occur include, for example, leucine to phenylalanine at position 5, tyrosine to asparagine at position 28, threonine to isoleucine at position 29, histidine to tyrosine at position 49, leucine to phenylalanine at position 54, asparagine to lysine at position 74, glutamic acid to aspartic acid at position 96, aspartic acid to asparagine at position 111, phenylalanine to leucine at position 157, glycine to valine at position 181, serine to tryptophan at position 221, serine to arginine at position 247, alanine to threonine at position 348, arginine to isoleucine at position 408, glycine to serine at position 476, valine to alanine at position 483, and 519. histidine changed to glutamine, alanine changed to serine at position 520, aspartic acid changed to asparagine at position 614, aspartic acid changed to glycine at position 614, asparagine changed to isoleucine at position 679, serine changed to leucine at position 680, arginine changed to glycine at position 682, arginine changed to serine at position 683, arginine changed to glutamine at position 685, arginine changed to serine at position 685, phenylalanine changed to cysteine ​​at position 797, alanine changed to valine at position 930, aspartic acid changed to tyrosine at position 936, alanine changed to valine at position 1078, aspartic acid changed to histidine at position 1168, and / or aspartic acid changed to histidine at position 1259 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2 The equivalent amino acid residues in the S glycoprotein sequence are listed. Becerra-Flores and Cardozo, “SARS-CoV-2 viral spike G614 mutation exhibits highercase fatality rate,” The International Journal of Clinical Practice, published online May 6, 2020; Eaaswarkhanth et al., “Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality?” International Journal of Infectious Diseases, 96: July 2020, Pages 459-460; Tang et al., “The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain,” Preprints 2020, 2020050407 (doi: 10.20944 / preprints202005.0407.v1); Hansen et. al., “Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail” Science, published online June 15, 2020; Lokman et al., “Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach”, Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 2020 Jun;84:104389. DOI:10.1016 / j.meegid.2020.104389, each of which is incorporated herein by reference in its entirety for all intended purposes. Further non-limiting examples of amino acid residues for insertion, deletion, and / or substitution include those listed in Tables 8 and 9 (the amino acid residue positions are shown using SEQ ID NO: 1 as a reference sequence, which can be used as a reference for identifying the equivalent amino acid residue in any SARS-CoV-2 S glycoprotein sequence (as above); the references in Table 8 are incorporated herein by reference in their entirety for all intended purposes). Each of the residue modifications listed in Table 8 can be used individually, alone or in combination with others, to generate variants of recombinant vesicular stomatitis virus (VSV) particles. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and / or arginine to glutamine at position 685 (positions set forth in SEQ ID NO: 1), or by changing equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing arginine to glutamine at position 685.The S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, confers a more soluble phenotype. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 42, or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may be encoded by a codon-optimized nucleotide sequence. In various embodiments, the SARS-CoV-2The S glycoprotein fragment or derivative may be encoded by the polynucleotide sequence of SEQ ID NO:43 or a sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% polynucleotide sequence identity to SEQ ID NO:43. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 44 or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, may comprise an asparagine to tyrosine change at position 501, and / or a glutamic acid to lysine at position 484, and / or an aspartic acid to glycine change at position 614, and / or a nucleotide change at position 622. The amino acid sequence differs from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by the deletion of positions 69-70 (as set forth in SEQ ID NO: 1) or by the alteration or deletion of the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by the asparagine to tyrosine change at position 501 and the deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a aspartic acid to glycine change at position 614 and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and a deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, comprises the Q shown in SEQ ID NO:1. 677 TNSPRRARSV 687 (SEQ ID NO: 65), or the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence, to QTILRSV (SEQ ID NO: 66) or QTNSPGSASSV (SEQ ID NO: 67) results in an amino acid sequence that differs from the reference peptide or polypeptide (e.g., wild-type SARS-CoV-2 spike protein). In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, results in the phenotypic deletion of the monobasic furin cleavage site (QTILRSV (SEQ ID NO: 66)) or furin cleavage site (QTNSPGSASSV (SEQ ID NO: 67)) at the S1 / S2 boundary. In certain embodiments, modifications to the furin cleavage site can result in spike-stabilized pseudoparticles. See Hansen et al., "Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail," Science (published online June 15, 2020). The above references are incorporated by reference in their entirety for all purposes intended.

[0023] In certain embodiments of any of the above-mentioned methods, step (a) comprises contacting the sample with two or more different recombinant rhabdovirus particles (e.g., simultaneously or sequentially), wherein the two or more different recombinant rhabdovirus particles comprise different coronavirus spike (S) glycoproteins, fragments or derivatives thereof. In certain embodiments, at least one of the two or more different coronavirus spike (S) glycoproteins, fragments or derivatives thereof comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 44, or comprises one or more amino acid insertions, deletions, and / or substitutions listed in Tables 8 and 9, wherein the positions of the insertions, deletions, and / or substitutions are identified relative to SEQ ID NO: 1. In certain embodiments of the above-mentioned methods, wherein the rhabdovirus particles comprise a reporter protein and / or a nucleic acid molecule encoding the reporter protein, the two or more different recombinant rhabdovirus particles comprise different reporter proteins and / or different nucleic acid molecules encoding the different reporter proteins. In certain embodiments of the methods described above, wherein the rhabdovirus particles comprise a reporter protein and / or a nucleic acid molecule encoding the reporter protein, the two or more different recombinant rhabdovirus particles comprise the same reporter protein and / or the same nucleic acid molecule encoding the reporter protein.

[0024] In one embodiment, a method is provided for determining the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in a sample, the method comprising: a) contacting the sample with a replication-competent recombinant vesicular stomatitis virus (VSV) particle, the genome of which lacks a functional VSV glycoprotein (G) gene and encodes a full-length SARS-CoV-2 spike (S) glycoprotein or a fragment thereof lacking 19 C-terminal amino acids; b) after step (a), contacting the recombinant VSV particles with a mixture of Vero-DSP1 and Vero-DSP2 cells; c) measuring the luciferase signal and / or GFP signal of the cells after step (b); and d) comparing the signal measured in step (c) with a control. Includes.

[0025] In another embodiment, a method is provided for determining the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in a sample, the method comprising: a) contacting the sample with a replication-competent recombinant vesicular stomatitis virus (VSV) particle, wherein (i) the genome of the recombinant VSV particle lacks a functional VSV glycoprotein (G) gene and encodes a full-length SARS-CoV-2 spike (S) glycoprotein or a fragment thereof lacking 19 C-terminal amino acids, and (ii) the genome of the recombinant VSV particle further encodes a luciferase protein; b) after step (a), contacting the recombinant VSV particles with cells selected from Vero cells, Vero-Ace-2 cells, and Vero-E6 cells; c) measuring the luciferase signal of the cells after step (b); and d) comparing the signal measured in step (c) with a control. Includes.

[0026] In one embodiment of the two methods described above, the full-length SARS-CoV-2 S glycoprotein consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment of the two methods described above, the SARS-CoV-2 S glycoprotein fragment lacking the 19 C-terminal amino acids consists of the amino acid sequence of SEQ ID NO: 3.

[0027] In certain embodiments of the two methods described above, step (a) comprises contacting the sample with two or more different recombinant VSV particles (e.g., simultaneously or sequentially), wherein the two or more different recombinant VSV particles comprise different full-length SARS-CoV-2 S glycoproteins or fragments thereof lacking the 19 C-terminal amino acids. In certain embodiments, at least one of the two or more different SARS-CoV-2 S glycoproteins or fragments thereof lacking the 19 C-terminal amino acids comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:44, or comprises one or more amino acid insertions, deletions, and / or substitutions listed in Tables 8 and 9, wherein the positions of the insertions, deletions, and / or substitutions are identified relative to SEQ ID NO:1. In certain embodiments of the methods described above, wherein the VSV particles comprise a reporter protein and / or a nucleic acid molecule encoding the reporter protein, the two or more different recombinant VSV particles comprise different reporter proteins and / or different nucleic acid molecules encoding the different reporter proteins. In certain embodiments of the above method, in which the VSV particles comprise a reporter protein and / or a nucleic acid molecule encoding the reporter protein, the two or more different recombinant VSV particles comprise the same reporter protein and / or the same nucleic acid molecule encoding the reporter protein.

[0028] In one embodiment of any of the above methods, after step (b) and before step (c), the cells are exposed to trypsin.

[0029] In one embodiment of any of the above-mentioned methods, the recombinant rhabdovirus particle comprises a mutant rhabdovirus matrix (M) protein. In one embodiment, the genome of the recombinant rhabdovirus particle encodes a mutant rhabdovirus M protein. In one embodiment, the recombinant rhabdovirus particle is a recombinant VSV particle comprising a mutant VSV M protein comprising a mutation at methionine 51. In a specific embodiment, the mutation at methionine 51 is from methionine (M) to arginine (R). In a specific embodiment, the mutant VSV M protein comprises the amino acid sequence of SEQ ID NO: 7. In a specific embodiment, the mutant VSV M protein consists of the amino acid sequence of SEQ ID NO: 7.

[0030] In one embodiment of any of the above-mentioned methods, the recombinant rhabdovirus particle comprises wild-type rhabdovirus matrix (M) protein.In one embodiment, the genome of the recombinant rhabdovirus particle encodes wild-type rhabdovirus M protein.In one embodiment, the recombinant rhabdovirus particle is a recombinant VSV particle, comprising the wild-type VSV M protein comprising the amino acid sequence of SEQ ID NO:9.In a specific embodiment, the wild-type VSV M protein consists of the amino acid sequence of SEQ ID NO:9.

[0031] In one embodiment of any of the above methods, the sample is serum or plasma. In one embodiment of any of the above methods, the sample is saliva. In one embodiment of any of the above methods, the sample is a dried blood spot. In one embodiment, the method further comprises diluting the sample to about 1:10 to about 1:320. For example, the sample may be diluted to about 1:10, about 1:16, about 1:20, about 1:32, about 1:64, about 1:80, about 1:100, about 1:128, or about 1:160. In one embodiment, the method further comprises diluting the sample to about 1:100. In one embodiment, the method further comprises diluting the sample to about 1:20. In some embodiments, the method further comprises diluting the recombinant rhabdovirus particles to about 200 to 800 pfu / well. For example, the recombinant rhabdovirus particles can be diluted to about 200, about 300, about 400, about 500, about 600, about 700, about 720 or about 800 pfu / well.In one embodiment, the sample is heat-inactivated.In another embodiment, the sample is not heat-inactivated.In one embodiment, the method further comprises treating the sample with antibiotics and / or filtering the sample to prevent bacterial contamination.

[0032] In one embodiment of any of the above-described methods, the control is a reporter signal obtained using a control sample that does not contain coronavirus neutralizing antibodies, and the method includes concluding that the tested sample contains coronavirus neutralizing antibodies if the reporter signal obtained in step (c) is reduced relative to the control. In one embodiment, the method includes concluding that the tested sample contains coronavirus neutralizing antibodies if the reporter signal obtained in step (c) is reduced by more than 50% relative to the control. In one embodiment, the method further includes comparing the reporter signal obtained in step (c) to a reporter signal obtained using a control sample that contains: a coronavirus neutralizing antibody; or a molecule that blocks the interaction between a coronavirus S glycoprotein and a protein with which the coronavirus S glycoprotein interacts on the target cell; or a molecule that blocks fusion of the target cell; or any combination thereof. In one embodiment, the method includes determining the concentration of a coronavirus-neutralizing antibody in the tested sample by comparing the reporter signal to a standard curve determined from serial dilutions of a control sample comprising a coronavirus-neutralizing antibody, or a molecule that blocks the interaction between a coronavirus S glycoprotein and a protein with which the coronavirus S glycoprotein interacts on the target cell, or any combination thereof. In a specific embodiment, the control sample comprises mAb10914. In a specific embodiment, the serial dilutions of the control sample comprise from about 0.01 μg / mL to about 3 μg / mL of mAb10914. In a specific embodiment, the control sample comprises mAb10922. In a specific embodiment, the serial dilutions of the control sample comprise from about 0.01 μg / mL to about 3 μg / mL of mAb10922.

[0033] In one embodiment of any of the above methods, the reporter signal is corrected to remove a background signal measured from a control sample not contacted with the recombinant rhabdovirus particles.

[0034] In one embodiment of any of the above methods, the reporter signal is measured about 18 to 30 hours after step (b). In one embodiment of any of the above methods, the reporter signal is measured about 24 to 30 hours after step (b).

[0035] In one embodiment of any of the above-described methods, in step (a), the sample is contacted with the recombinant rhabdovirus particles at room temperature for about 30 minutes.

[0036] In one embodiment of any of the above-described methods, the method is performed in a high-throughput format. In a specific embodiment, the method is performed in a 96-well plate. In a specific embodiment, when the method is performed in a 96-well plate, the density of the first target cells and the second target cells is about 6 x 10 4 cells / well.

[0037] In another aspect, a recombinant rhabdovirus particle is provided, wherein the rhabdovirus glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein, or a fragment or derivative thereof, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell.

[0038] In one embodiment, the rhabdovirus particle further comprises a reporter protein and / or the nucleic acid molecule that encodes the reporter protein.In one embodiment, the rhabdovirus particle comprises the nucleic acid molecule that encodes the reporter protein.In a specific embodiment, the nucleic acid sequence that encodes the reporter protein is inserted between the nucleic acid sequence that encodes the coronavirus S glycoprotein or its fragment or derivative and the nucleic acid sequence that encodes the rhabdovirus large (L) protein.

[0039] In one embodiment, the recombinant rhabdovirus particle is a replication-competent rhabdovirus particle.

[0040] In one embodiment, the recombinant rhabdovirus particle is a recombinant vesiculovirus particle. In one embodiment, the recombinant vesiculovirus particle is a recombinant vesicular stomatitis virus (VSV) particle.

[0041] In one embodiment, the coronavirus S glycoprotein, fragment, or derivative thereof is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In one embodiment, the coronavirus S glycoprotein is a full-length SARS-CoV-2 S glycoprotein. In a specific embodiment, the SARS-CoV-2 S glycoprotein comprises the amino acid sequence of SEQ ID NO: 1. In a specific embodiment, the SARS-CoV-2 S glycoprotein consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment is a SARS-CoV-2 S glycoprotein fragment lacking the 19 C-terminal amino acids. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment comprises the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 77% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to the S1 subunit of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to amino acids 14-684 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to the RBD domain of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to amino acids 319-541 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the coronavirus S glycoprotein fragment or derivative results in a recombinant rhabdovirus particle that is more fusogenic than a comparable recombinant rhabdovirus comprising a rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome. In a specific embodiment, the SARS-CoV-2 S glycoprotein derivative, or a fragment thereof, is capable of binding to the SARS-CoV-2 S glycoprotein.It may comprise or consist of insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 residues of the S glycoprotein. Non-limiting examples of amino acids for possible deletion include, for example, tyrosine at position 145, asparagine at position 679, serine at position 680, proline at position 681, arginine at position 682, arginine at position 683, alanine at position 684, and / or arginine at position 685 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2Non-limiting examples of amino acids for substitutions that may occur include, for example, leucine to phenylalanine at position 5, tyrosine to asparagine at position 28, threonine to isoleucine at position 29, histidine to tyrosine at position 49, leucine to phenylalanine at position 54, asparagine to lysine at position 74, glutamic acid to aspartic acid at position 96, aspartic acid to asparagine at position 111, phenylalanine to leucine at position 157, glycine to valine at position 181, serine to tryptophan at position 221, serine to arginine at position 247, alanine to threonine at position 348, arginine to isoleucine at position 408, glycine to serine at position 476, valine to alanine at position 483, and 519. histidine changed to glutamine, alanine changed to serine at position 520, aspartic acid changed to asparagine at position 614, aspartic acid changed to glycine at position 614, asparagine changed to isoleucine at position 679, serine changed to leucine at position 680, arginine changed to glycine at position 682, arginine changed to serine at position 683, arginine changed to glutamine at position 685, arginine changed to serine at position 685, phenylalanine changed to cysteine ​​at position 797, alanine changed to valine at position 930, aspartic acid changed to tyrosine at position 936, alanine changed to valine at position 1078, aspartic acid changed to histidine at position 1168, and / or aspartic acid changed to histidine at position 1259 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2 The equivalent amino acid residues in the S glycoprotein sequence are listed. Becerra-Flores and Cardozo, “SARS-CoV-2 viral spike G614 mutation exhibits highercase fatality rate,” The International Journal of Clinical Practice, published online May 6, 2020; Eaaswarkhanth et al., “Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality?” International Journal of Infectious Diseases, 96: July 2020, Pages 459-460; Tang et al., “The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain,” Preprints 2020, 2020050407 (doi: 10.20944 / preprints202005.0407.v1); Hansen et. al., “Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail” Science, published online June 15, 2020; Lokman et al., “Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach”, Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 2020 Jun;84:104389. DOI:10.1016 / j.meegid.2020.104389, each of which is incorporated herein by reference in its entirety for all intended purposes. Further non-limiting examples of amino acid residues for insertion, deletion, and / or substitution include those listed in Tables 8 and 9 (the amino acid residue positions are shown using SEQ ID NO: 1 as a reference sequence, which can be used as a reference for identifying the equivalent amino acid residue in any SARS-CoV-2 S glycoprotein sequence (as above); the references in Table 8 are incorporated herein by reference in their entirety for all intended purposes). Each of the residue modifications listed in Table 8 can be used individually, alone or in combination with others, to generate variants of recombinant vesicular stomatitis virus (VSV) particles. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and / or arginine to glutamine at position 685 (positions set forth in SEQ ID NO: 1), or by changing equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing arginine to glutamine at position 685.The S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, confers a more soluble phenotype. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 42, or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may be encoded by a codon-optimized nucleotide sequence. In various embodiments, the SARS-CoV-2The S glycoprotein fragment or derivative may be encoded by the polynucleotide sequence of SEQ ID NO:43 or a sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% polynucleotide sequence identity to SEQ ID NO:43. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 44 or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, may comprise an asparagine to tyrosine change at position 501, and / or a glutamic acid to lysine at position 484, and / or an aspartic acid to glycine change at position 614, and / or a deletion of residues 69-70 (SEQ ID NO: 1) or by changing or deleting the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by changing an asparagine to a tyrosine at position 501. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing a glutamic acid to a lysine at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing an aspartic acid to a glycine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by the asparagine to tyrosine change at position 501 and the deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a aspartic acid to glycine change at position 614 and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and a deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, comprises the Q shown in SEQ ID NO:1. 677 TNSPRRARSV 687 (SEQ ID NO: 65), or the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence, to QTILRSV (SEQ ID NO: 66) or QTNSPGSASSV (SEQ ID NO: 67) results in an amino acid sequence that differs from the reference peptide or polypeptide (e.g., wild-type SARS-CoV-2 spike protein). In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, results in the phenotypic deletion of the monobasic furin cleavage site (QTILRSV (SEQ ID NO: 66)) or furin cleavage site (QTNSPGSASSV (SEQ ID NO: 67)) at the S1 / S2 boundary. In certain embodiments, modifications to the furin cleavage site can result in spike-stabilized pseudoparticles. See Hansen et al., "Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail," Science (published online June 15, 2020). The above references are incorporated by reference in their entirety for all purposes intended.

[0042] In one embodiment, the recombinant rhabdovirus particle comprises a mutant rhabdovirus matrix (M) protein. In one embodiment, the genome of the recombinant rhabdovirus particle encodes a mutant VSV M protein. In a specific embodiment, the recombinant rhabdovirus particle is a recombinant VSV particle comprising a mutant VSV M protein comprising a mutation at methionine 51. In a specific embodiment, the mutation at methionine 51 is from methionine (M) to arginine (R). In a specific embodiment, the mutant VSV M protein comprises the amino acid sequence of SEQ ID NO: 7. In a specific embodiment, the mutant VSV M protein consists of the amino acid sequence of SEQ ID NO: 7.

[0043] In one embodiment, the recombinant rhabdovirus particle comprises wild-type rhabdovirus matrix (M) protein.In one embodiment, the genome of the recombinant rhabdovirus particle encodes wild-type rhabdovirus M protein.In a specific embodiment, the recombinant rhabdovirus particle is a recombinant VSV particle, comprising wild-type VSV M protein comprising the amino acid sequence of SEQ ID NO:9.In a specific embodiment, the wild-type VSV M protein consists of the amino acid sequence of SEQ ID NO:9.

[0044] In another aspect, polynucleotides are provided that encode rhabdovirus nucleoprotein (N), rhabdovirus phosphoprotein (P), and rhabdovirus large protein (L), or functional fragments or derivatives thereof, for expression in the viral envelope of a recombinant rhabdovirus particle, and further encode severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, or a fragment or derivative thereof.

[0045] In one embodiment, the polynucleotide further encodes a reporter protein. In a specific embodiment, the nucleic acid sequence encoding the reporter protein is inserted between the nucleic acid sequence encoding the coronavirus S glycoprotein or a fragment or derivative thereof and the nucleic acid sequence encoding the rhabdovirus large (L) protein.

[0046] In one embodiment, the recombinant rhabdovirus particle is a replication-competent rhabdovirus particle. In one embodiment, the coronavirus S glycoprotein, fragment, or derivative thereof is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In one embodiment, the coronavirus S glycoprotein is a full-length SARS-CoV-2 S glycoprotein. In a specific embodiment, the SARS-CoV-2 S glycoprotein comprises the amino acid sequence of SEQ ID NO: 1. In a specific embodiment, the SARS-CoV-2 S glycoprotein consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the nucleotide sequence encoding the SARS-CoV-2 S glycoprotein comprises the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the coronavirus S glycoprotein fragment is a SARS-CoV-2 S glycoprotein fragment lacking the 19 C-terminal amino acids. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment comprises the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the SARS-CoV-2The nucleotide sequence encoding the S glycoprotein comprises the nucleotide sequence of SEQ ID NO:4. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 77% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to the S1 subunit of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to amino acids 14 to 684 of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to the RBD domain of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to amino acids 319 to 541 of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative results in a recombinant rhabdovirus particle that is more fusogenic than an equivalent recombinant rhabdovirus comprising a rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome. In one embodiment, the coronavirus S glycoprotein fragment or derivative results in a recombinant rhabdovirus particle that is more fusogenic than an equivalent recombinant rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome. In one embodiment, the coronavirus S glycoprotein, fragment, or derivative comprises one or more amino acid insertions, deletions, and / or substitutions listed in Tables 8 and 9, where the positions of the insertions, deletions, and / or substitutions are identified relative to SEQ ID NO: 1. In one embodiment, the SARS-CoV-2The S glycoprotein comprises the amino acid sequence of SEQ ID NO: 44. In one embodiment of any of the above-described methods, step (a) comprises contacting the sample with two or more different recombinant rhabdovirus particles, wherein the two or more different recombinant rhabdovirus particles comprise different coronavirus spike (S) glycoproteins, fragments, or derivatives thereof. At least one of the two or more different coronavirus spike (S) glycoproteins, fragments, or derivatives thereof comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 44, or comprises one or more amino acid insertions, deletions, and / or substitutions listed in Tables 8 and 9, wherein the positions of the insertions, deletions, and / or substitutions are identified relative to SEQ ID NO: 1. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, may comprise or consist of an insertion, deletion, and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 residues of the SARS-CoV-2 S glycoprotein. Non-limiting examples of amino acids for possible deletion include, for example, tyrosine at position 145, asparagine at position 679, serine at position 680, proline at position 681, arginine at position 682, arginine at position 683, alanine at position 684, and / or arginine at position 685 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2Non-limiting examples of amino acids for substitutions that may occur include, for example, leucine to phenylalanine at position 5, tyrosine to asparagine at position 28, threonine to isoleucine at position 29, histidine to tyrosine at position 49, leucine to phenylalanine at position 54, asparagine to lysine at position 74, glutamic acid to aspartic acid at position 96, aspartic acid to asparagine at position 111, phenylalanine to leucine at position 157, glycine to valine at position 181, serine to tryptophan at position 221, serine to arginine at position 247, alanine to threonine at position 348, arginine to isoleucine at position 408, glycine to serine at position 476, valine to alanine at position 483, and 519. histidine changed to glutamine, alanine changed to serine at position 520, aspartic acid changed to asparagine at position 614, aspartic acid changed to glycine at position 614, asparagine changed to isoleucine at position 679, serine changed to leucine at position 680, arginine changed to glycine at position 682, arginine changed to serine at position 683, arginine changed to glutamine at position 685, arginine changed to serine at position 685, phenylalanine changed to cysteine ​​at position 797, alanine changed to valine at position 930, aspartic acid changed to tyrosine at position 936, alanine changed to valine at position 1078, aspartic acid changed to histidine at position 1168, and / or aspartic acid changed to histidine at position 1259 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2 The equivalent amino acid residues in the S glycoprotein sequence are listed. Becerra-Flores and Cardozo, “SARS-CoV-2 viral spike G614 mutation exhibits highercase fatality rate,” The International Journal of Clinical Practice, published online May 6, 2020; Eaaswarkhanth et al., “Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality?” International Journal of Infectious Diseases, 96: July 2020, Pages 459-460; Tang et al., “The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain,” Preprints 2020, 2020050407 (doi: 10.20944 / preprints202005.0407.v1); Hansen et. al., “Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail” Science, published online June 15, 2020; Lokman et al., “Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach”, Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 2020 Jun;84:104389. DOI:10.1016 / j.meegid.2020.104389, each of which is incorporated herein by reference in its entirety for all intended purposes. Further non-limiting examples of amino acid residues for insertion, deletion, and / or substitution include those listed in Tables 8 and 9 (the amino acid residue positions are shown using SEQ ID NO: 1 as a reference sequence, which can be used as a reference for identifying the equivalent amino acid residue in any SARS-CoV-2 S glycoprotein sequence (as above); the references in Table 8 are incorporated herein by reference in their entirety for all intended purposes). Each of the residue modifications listed in Table 8 can be used individually, alone or in combination with others, to generate variants of recombinant vesicular stomatitis virus (VSV) particles. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and / or arginine to glutamine at position 685 (positions set forth in SEQ ID NO: 1), or by changing equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing arginine to glutamine at position 685.The S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, confers a more soluble phenotype. In certain embodiments, the SARS-CoV-2 The S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 42 or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may be encoded by a codon-optimized nucleotide sequence. In various embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may be encoded by the polynucleotide sequence of SEQ ID NO:43 or a sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% polynucleotide sequence identity to SEQ ID NO:43. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO:44 or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO:44. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., wild-type SARS-CoV-2 spike protein) by changing asparagine to tyrosine at position 501, and / or glutamic acid to lysine at position 484, and / or aspartic acid to glycine at position 614, and / or by deleting residues 69-70 (positions set forth in SEQ ID NO: 1), or by altering or deleting the equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and a deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing glutamic acid to lysine at position 484 and aspartic acid to glycine at position 614.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and a deletion of residues 69 to 70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an aspartic acid to glycine change at position 614 and a deletion of residues 69 to 70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine at position 484, and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from the reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and a deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, is Q as set forth in SEQ ID NO:1. 677 TNSPRRARSV 687 (SEQ ID NO: 65), or the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence, to QTILRSV (SEQ ID NO: 66) or QTNSPGSASSV (SEQ ID NO: 67) results in an amino acid sequence that differs from the reference peptide or polypeptide (e.g., wild-type SARS-CoV-2 spike protein). In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, results in the phenotypic deletion of the monobasic furin cleavage site (QTILRSV (SEQ ID NO: 66)) or furin cleavage site (QTNSPGSASSV (SEQ ID NO: 67)) at the S1 / S2 boundary. In certain embodiments, modifications to the furin cleavage site can result in spike-stabilized pseudoparticles. See Hansen et al., "Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail," Science (published online June 15, 2020). The above references are incorporated by reference in their entirety for all purposes intended.

[0047] In one embodiment, the polynucleotide further comprises a Kozak sequence 3' to the sequence encoding the SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof. In one embodiment, the Kozak sequence is a wild-type Kozak sequence. In a specific embodiment, the wild-type Kozak sequence comprises SEQ ID NO: 11 or a derivative thereof. In one embodiment, the Kozak sequence is an optimized Kozak sequence. In a specific embodiment, the optimized Kozak sequence comprises SEQ ID NO: 12 or a derivative thereof.

[0048] In one embodiment, the recombinant rhabdovirus particle is a recombinant vesiculovirus particle. In one embodiment, the recombinant vesiculovirus particle is a recombinant vesicular stomatitis virus (VSV) particle.

[0049] In a further aspect, a kit for determining the presence of coronavirus neutralizing antibodies in a sample is provided, the kit comprising: a) a recombinant rhabdovirus particle, wherein the rhabdovirus glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein or a fragment or derivative thereof, and wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a first target cell and a second target cell; b) (i) the first target cell expressing a first portion of a reporter protein, and (ii) the second cell expressing a second portion of the reporter protein, wherein the first target cell and the second target cell are capable of fusing with each other upon contact with the recombinant rhabdovirus particle, and wherein the fusion results in the production of a detectable reporter signal; c) optionally, a control sample containing no coronavirus-neutralizing antibodies; d) optionally, a control sample comprising a coronavirus neutralizing antibody; or a molecule that blocks the interaction between the coronavirus S glycoprotein and a protein with which said coronavirus S glycoprotein interacts on a target cell; or a molecule that blocks target cell fusion; or any combination thereof; e) optionally, a substrate for the reporter protein; and f) optionally, instructions for use Includes.

[0050] In yet another aspect, a kit for determining the presence of coronavirus neutralizing antibodies in a sample is provided, the kit comprising: a) a polynucleotide encoding a rhabdovirus nucleoprotein (N), a rhabdovirus phosphoprotein (P), and a rhabdovirus large protein (L), or functional fragments or derivatives thereof, for expression in the viral envelope of a recombinant rhabdovirus particle, and further encoding a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, or a fragment or derivative thereof, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a first target cell and a second target cell; b) (i) the first target cell expressing a first portion of a reporter protein, and (ii) the second cell expressing a second portion of the reporter protein, wherein the first target cell and the second target cell are capable of fusing with each other when contacted with the recombinant rhabdovirus particle, and wherein the fusion results in the production of a detectable reporter signal; c) optionally, a control sample containing no coronavirus-neutralizing antibodies; d) optionally, a control sample comprising a coronavirus neutralizing antibody; or a molecule that blocks the interaction between the coronavirus S glycoprotein and a protein with which said coronavirus S glycoprotein interacts on a target cell; or a molecule that blocks target cell fusion; or any combination thereof; e) optionally, a substrate for the reporter protein; and f) optionally, instructions for use Includes.

[0051] In one embodiment, both the first target cells and / or the second target cells contain angiotensin-converting enzyme 2 (ACE2). In one embodiment, the first target cells are Vero-DSP1 (Vero-DSP-1-Puro; CLR-73) and the second target cells are Vero-DSP2 (Vero-DSP-2-Puro; CLR-74).

[0052] In one embodiment, the first portion of the reporter protein comprises amino acids 1-229 of Renilla luciferase or a variant thereof, and the second portion of the reporter protein comprises amino acids 230-311 of Renilla luciferase or a variant thereof. In another embodiment, the first portion of the reporter protein comprises amino acids 1-155 of Renilla luciferase or a variant thereof, and the second portion of the reporter protein comprises amino acids 156-311 of Renilla luciferase or a variant thereof. In a specific embodiment, the first portion of the reporter protein comprises amino acids 1-155 of Renilla luciferase mutant RLuc8, and the second portion of the reporter protein comprises amino acids 156-311 of Renilla luciferase mutant RLuc8.

[0053] In one embodiment, the first portion of the reporter protein comprises amino acids 1-156 of green fluorescent protein (GFP) or a variant thereof, and the second portion of the reporter protein comprises amino acids 157-231 of GFP or a variant thereof. In another embodiment, the first portion of the reporter protein comprises amino acids 1-213 of superfolder GFP, and the second portion of the reporter protein comprises amino acids 214-230 of superfolder GFP. In yet another embodiment, the first portion of the reporter protein comprises amino acids 1-154 of superfolder yellow fluorescent protein (YFP), and the second portion of the reporter protein comprises amino acids 155-262 of superfolder YFP.

[0054] In another aspect, a kit for determining the presence of coronavirus neutralizing antibodies in a sample is provided, the kit comprising: a) a recombinant rhabdovirus particle, wherein (i) the rhabdovirus glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein or a fragment or derivative thereof, wherein said S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell, and (ii) said recombinant rhabdovirus particle comprises a reporter protein and / or a nucleic acid molecule encoding said reporter protein; b) the target cells; c) optionally, a control sample containing no coronavirus-neutralizing antibodies; d) optionally, a control sample comprising a coronavirus neutralizing antibody; or a molecule that blocks the interaction between the coronavirus S glycoprotein and a protein with which said coronavirus S glycoprotein interacts on the target cell; or any combination thereof; e) optionally, a substrate for the reporter protein; and f) optionally, instructions for use Includes.

[0055] In one embodiment, the recombinant rhabdovirus particle comprises a nucleic acid molecule encoding the reporter protein.

[0056] In a further aspect, a kit for determining the presence of coronavirus neutralizing antibodies in a sample is provided, the kit comprising: a) a polynucleotide encoding a rhabdovirus nucleoprotein (N), a rhabdovirus phosphoprotein (P), and a rhabdovirus large protein (L), or functional fragments or derivatives thereof, for expression in the viral envelope of a recombinant rhabdovirus particle, and further encoding a reporter protein and a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, or a fragment or derivative thereof, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell; b) the target cells; c) optionally, a control sample containing no coronavirus-neutralizing antibodies; d) optionally, a control sample comprising a coronavirus neutralizing antibody; or a molecule that blocks the interaction between the coronavirus S glycoprotein and a protein with which said coronavirus S glycoprotein interacts on the target cell; or any combination thereof; e) optionally, a substrate for the reporter protein; and f) optionally, instructions for use Includes.

[0057] In one embodiment, the nucleic acid sequence encoding the reporter protein is inserted between the nucleic acid sequence encoding the coronavirus S glycoprotein or a fragment or derivative thereof and the nucleic acid sequence encoding the rhabdovirus large (L) protein.

[0058] In one embodiment, the target cells are Vero cells (including Vero-αHis cells), Vero-Ace-2 cells, Vero-TRMPSS2 cells, or Vero-E6 cells. In one embodiment, the target cells comprise angiotensin-converting enzyme 2 (ACE2).

[0059] In one embodiment of any of the above-described kits comprising recombinant rhabdovirus particles, the recombinant rhabdovirus particles are replication-competent rhabdovirus particles.

[0060] In one embodiment of any of the above-described kits, the coronavirus S glycoprotein, fragment, or derivative thereof is derived from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In one embodiment, the coronavirus S glycoprotein is a full-length SARS-CoV-2 S glycoprotein. In a specific embodiment, the SARS-CoV-2 S glycoprotein comprises the amino acid sequence of SEQ ID NO: 1. In a specific embodiment, the SARS-CoV-2 S glycoprotein consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the nucleotide sequence encoding the SARS-CoV-2 S glycoprotein comprises the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the coronavirus S glycoprotein fragment is a SARS-CoV-2 S glycoprotein fragment lacking the 19 C-terminal amino acids. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment comprises the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the SARS-CoV-2 S glycoprotein fragment consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the SARS-CoV-2The nucleotide sequence encoding the S glycoprotein comprises the nucleotide sequence of SEQ ID NO:4. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 77% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to the S1 subunit of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 64% amino acid sequence identity to amino acids 14 to 684 of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to the RBD domain of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative has at least 74% amino acid sequence identity to amino acids 319 to 541 of the amino acid sequence of SEQ ID NO:1. In one embodiment, the coronavirus S glycoprotein fragment or derivative results in a more fusogenic recombinant rhabdovirus particle compared to a comparable recombinant rhabdovirus comprising a rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome. In one embodiment, the coronavirus S glycoprotein fragment or derivative results in a more fusogenic recombinant rhabdovirus particle compared to a comparable recombinant rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome. In a specific embodiment, the SARS-CoV-2 S glycoprotein derivative, or a fragment thereof, results in a more fusogenic recombinant rhabdovirus particle compared to a comparable recombinant rhabdovirus genome expressing a full-length wild-type SARS-CoV-2 S glycoprotein inserted at the same position in the rhabdovirus genome.It may comprise or consist of insertions, deletions, and / or substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 residues of the S glycoprotein. Non-limiting examples of amino acids for possible deletion include, for example, tyrosine at position 145, asparagine at position 679, serine at position 680, proline at position 681, arginine at position 682, arginine at position 683, alanine at position 684, and / or arginine at position 685 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2Non-limiting examples of amino acids for substitutions that may occur include, for example, leucine to phenylalanine at position 5, tyrosine to asparagine at position 28, threonine to isoleucine at position 29, histidine to tyrosine at position 49, leucine to phenylalanine at position 54, asparagine to lysine at position 74, glutamic acid to aspartic acid at position 96, aspartic acid to asparagine at position 111, phenylalanine to leucine at position 157, glycine to valine at position 181, serine to tryptophan at position 221, serine to arginine at position 247, alanine to threonine at position 348, arginine to isoleucine at position 408, glycine to serine at position 476, valine to alanine at position 483, and 519. histidine changed to glutamine, alanine changed to serine at position 520, aspartic acid changed to asparagine at position 614, aspartic acid changed to glycine at position 614, asparagine changed to isoleucine at position 679, serine changed to leucine at position 680, arginine changed to glycine at position 682, arginine changed to serine at position 683, arginine changed to glutamine at position 685, arginine changed to serine at position 685, phenylalanine changed to cysteine ​​at position 797, alanine changed to valine at position 930, aspartic acid changed to tyrosine at position 936, alanine changed to valine at position 1078, aspartic acid changed to histidine at position 1168, and / or aspartic acid changed to histidine at position 1259 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2 The equivalent amino acid residues in the S glycoprotein sequence are listed. Becerra-Flores and Cardozo, “SARS-CoV-2 viral spike G614 mutation exhibits highercase fatality rate,” The International Journal of Clinical Practice, published online May 6, 2020; Eaaswarkhanth et al., “Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality?” International Journal of Infectious Diseases, 96: July 2020, Pages 459-460; Tang et al., “The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain,” Preprints 2020, 2020050407 (doi: 10.20944 / preprints202005.0407.v1); Hansen et. al., “Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail” Science, published online June 15, 2020; Lokman et al., “Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach”, Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 2020 Jun;84:104389. DOI:10.1016 / j.meegid.2020.104389, each of which is incorporated herein by reference in its entirety for all intended purposes. Further non-limiting examples of amino acid residues for insertion, deletion, and / or substitution include those listed in Tables 8 and 9 (the amino acid residue positions are shown using SEQ ID NO: 1 as a reference sequence, which can be used as a reference for identifying the equivalent amino acid residue in any SARS-CoV-2 S glycoprotein sequence (as above); the references in Table 8 are incorporated herein by reference in their entirety for all intended purposes). Each of the residue modifications listed in Table 8 can be used individually, alone or in combination with others, to generate variants of recombinant vesicular stomatitis virus (VSV) particles. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and / or arginine to glutamine at position 685 (positions set forth in SEQ ID NO: 1), or by changing equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing arginine to glutamine at position 685.The S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, confers a more soluble phenotype. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 42, or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may be encoded by a codon-optimized nucleotide sequence. In various embodiments, the SARS-CoV-2The S glycoprotein fragment or derivative may be encoded by the polynucleotide sequence of SEQ ID NO: 43 or a sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% polynucleotide sequence identity to SEQ ID NO: 43. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative has the amino acid sequence of SEQ ID NO: 44. or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO:44. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by an asparagine to tyrosine change at position 501, and / or a glutamic acid to lysine change at position 484, and / or an aspartic acid to glycine change at position 614, and / or a deletion of residues 69-70 (positions set forth in SEQ ID NO: 1), or by alteration or deletion of equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and a glutamic acid to lysine change at position 484.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501 and a deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484 and a deletion of residues 69 to 70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an aspartic acid to glycine change at position 614 and a deletion of residues 69 to 70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine at position 484, and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and a deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, an aspartic acid to glycine change at position 614, and the deletion of residues 69 to 70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, an aspartic acid to glycine change at position 614, and the deletion of residues 69 to 70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, comprises the Q shown in SEQ ID NO:1. 677 TNSPRRARSV 687(SEQ ID NO: 65), or the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence, to QTILRSV (SEQ ID NO: 66) or QTNSPGSASSV (SEQ ID NO: 67) results in an amino acid sequence that differs from the reference peptide or polypeptide (e.g., wild-type SARS-CoV-2 spike protein). In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, results in the phenotypic deletion of the monobasic furin cleavage site (QTILRSV (SEQ ID NO: 66)) or furin cleavage site (QTNSPGSASSV (SEQ ID NO: 67)) at the S1 / S2 boundary. In certain embodiments, modifications to the furin cleavage site can result in spike-stabilized pseudoparticles. See Hansen et al., "Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail," Science (published online June 15, 2020). The above references are incorporated by reference in their entirety for all purposes intended.

[0061] In one embodiment of any of the above kits, the recombinant rhabdovirus particles are recombinant vesiculovirus particles. In one embodiment, the recombinant vesiculovirus particles are recombinant vesicular stomatitis virus (VSV) particles.

[0062] In one embodiment of any of the above-mentioned kits comprising recombinant rhabdovirus particles, the recombinant rhabdovirus particle comprises a mutant VSV matrix (M) protein.In one embodiment, the genome of the recombinant rhabdovirus particle encodes a mutant VSV M protein.In a specific embodiment, the recombinant rhabdovirus particle is a recombinant VSV particle comprising a mutant VSV M protein comprising a mutation at methionine 51.In a specific embodiment, the mutation at methionine 51 is from methionine (M) to arginine (R).In a specific embodiment, the mutant VSV M protein comprises the amino acid sequence of SEQ ID NO: 7.In a specific embodiment, the mutant VSV M protein consists of the amino acid sequence of SEQ ID NO: 7.

[0063] In one embodiment of any of the above-mentioned kits that comprise recombinant rhabdovirus particles, the recombinant rhabdovirus particle comprises wild-type rhabdovirus matrix (M) protein.In one embodiment, the genome of the recombinant rhabdovirus particle encodes wild-type rhabdovirus M protein.In a specific embodiment, the recombinant rhabdovirus particle is a recombinant VSV particle that comprises wild-type VSV M protein comprising the amino acid sequence of SEQ ID NO:9.In a specific embodiment, the wild-type VSV M protein consists of the amino acid sequence of SEQ ID NO:9.

[0064] In one embodiment of any of the above-described kits, the reporter protein comprises a luciferase, non-limiting examples of luciferases that can be used include Renilla luciferase, RLuc8 mutant Renilla luciferase, (dCpG) luciferase, NanoLuc reporter, firefly luciferase, Gaussia luciferase (gLuc), MetLuc, Vibrio fischeri lumazine protein, Vibrio harveyi luminase protein, dinoflagellate luciferase, firefly luciferase YY5 mutant, firefly luciferase LGR mutant, firefly luciferase mutant E, and derivatives thereof.

[0065] In one embodiment of any of the above-described kits, the reporter protein comprises a fluorescent protein, non-limiting examples of which include green fluorescent protein (GFP), GFP-like fluorescent protein (GFP-like), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP); red fluorescent protein, superfolder GFP, superfolder YFP, orange fluorescent protein, red fluorescent protein, small super red fluorescent protein, FMN-binding fluorescent protein, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGr1, bright green fluorescent protein, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and derivatives thereof.

[0066] In certain specific embodiments of any of the above-described kits, the reporter protein comprises luciferase, and the substrate for the reporter protein comprises luciferin (e.g., d-luciferin), coelenterazine, or EnduRen luciferase substrate.

[0067] In certain specific embodiments of any of the above-described kits, the control sample comprising a coronavirus-neutralizing antibody is a control sample comprising mAb 10914. In certain specific embodiments of any of the above-described kits, the control sample comprising a coronavirus-neutralizing antibody is a control sample comprising mAb 10922.

[0068] These and other aspects described herein will become apparent to those skilled in the art in light of the following description, claims, and drawings. [Brief explanation of the drawings]

[0069] [Figure 1] FIG. 1 shows the VSV SARS-CoV-2 constructs (variants 1-4) tested in the examples. In these constructs, the VSV G glycoprotein was modified to: (1) the full-length SARS-CoV-2 spike (S) glycoprotein sequence (variant 1; VSV SARS-CoV-2 dG = VSV-SARS-CoV-2-S; amino acid sequence of SEQ ID NO: 1; codon-optimized nucleotide sequence of SEQ ID NO: 2), (2) a SARS-CoV-2 S glycoprotein sequence with the 19 amino acids KFDEDDSEPVLKGVKLHYT (SEQ ID NO: 20) deleted from the cytoplasmic tail (variant 2; VSV SARS-CoV-2 Δ19CT dG = VSV-SARS-CoV-2-S-Δ19CT; amino acid sequence of SEQ ID NO: 3; codon-optimized nucleotide sequence of SEQ ID NO: 4), or (3) a SARS-CoV-2 S glycoprotein sequence with the S cytoplasmic tail replaced with the VSV G glycoprotein cytoplasmic tail sequence KLKHTKKRQIYTDIEMNRLGK (SEQ ID NO: 21) (variant 3; VSV (4) the full-length SARS-CoV-2 S glycoprotein sequence (variant 4; VSV-SARS-CoV-2-S; amino acid sequence of SEQ ID NO: 1; codon-optimized nucleotide sequence of SEQ ID NO: 2) in which the wild-type M protein (amino acid sequence of SEQ ID NO: 9; nucleotide sequence of SEQ ID NO: 10) was substituted with the SARS-CoV-2 VSV-G CT dG; amino acid sequence of SEQ ID NO: 5; codon-optimized nucleotide sequence of SEQ ID NO: 6), or (5) the full-length SARS-CoV-2 S glycoprotein sequence, but in which the wild-type VSV Kozak sequence (cActATG; SEQ ID NO: 11) was used instead of the optimized Kozak sequence (caccATG; SEQ ID NO: 12) used in the other three constructs. A first set of constructs for variants 1-4 was prepared, encoding a wild-type M protein (amino acid sequence of SEQ ID NO: 9; nucleotide sequence of SEQ ID NO: 10). A second set of constructs for variants 1-4 was prepared, encoding an M protein (amino acid sequence of SEQ ID NO: 7; nucleotide sequence of SEQ ID NO: 8) with the substitution M51R, which confers viral attenuation. [Figure 2]Figure 2 shows an overview of the DSP Vero luciferase assay of the present invention. VSV expressing the SARS-CoV-2 spike (e.g., VSV-SARS-CoV-2-S-Δ19CT or VSV-SARS-CoV-2-S) is incubated with a test serum sample. In the absence of SARS-CoV-2 neutralizing antibodies (top panel), the virus remains infectious and infects Vero-DSP1 / Vero-DSP2 monolayers. When the test sample contains SARS-CoV-2 neutralizing antibodies (bottom panel), the antibodies bind to the spike protein, blocking cell entry and neutralizing the virus. VSV-SARS-CoV-2-S-Δ19CT induces syncytium formation in Vero-DSP1 / Vero-DSP2 monolayers, thereby reconstituting a fully functional luciferase reporter, which is used to quantify virus-induced syncytium formation. A high luciferase signal indicates that the test sample did not neutralize the virus, and a decrease in luciferase indicates the presence of SARS-CoV-2-neutralizing antibodies in the test sample. [Figure 3](A) Schematic diagram of the VSV-SARS-CoV-2-S-Δ19CT genome used in this assay. The locations of the VSV N, P, M, and L genes are indicated. VSV-G has been replaced with a codon-optimized SARS-CoV-2 spike gene with a 19-amino acid C-terminal (CT) deletion (Δ19CT). TM is the transmembrane domain, and CT is the C-terminal domain. Drawings are not to scale. (B) VSV-SARS-CoV-2-S-Δ19CT induces syncytia formation in Vero cell monolayers. Vero monolayers were infected with VSV-SARS-CoV-2-S-Δ19CT or mock-infected. After 4 hours, the inoculum was removed and replaced with fresh medium ± 0.4 μg / mL trypsin. Images were taken at 100x magnification 20 hours post-infection. (C) Immunoblot analysis. Viral supernatants prepared from Vero cells infected with VSV-GFP (encoding VSV-G) or VSV-SARS-CoV-2-S-Δ19CT, or mock-infected, were subjected to immunoblot analysis using anti-SARS-CoV-2-spike antibody (left) and rabbit anti-VSV antiserum (right). Arrows indicate the full-length S1 / S2 variant and proteolytically cleaved S2 variant of the SARS-CoV-2 spike protein, as well as VSV-G, VSV-N, and VSV-M proteins. (D and E) Flow cytometry. Vero-DSP1 cell monolayers were stripped with Versene, permeabilized (for TMPRSS2 staining only), stained with isotype control (gray), anti-ACE2 (D, black), or anti-TMPRSS2 (E, black) antibodies, fixed, and subjected to flow cytometry analysis. (F) VSV-SARS-CoV-2-S-Δ19CT induced luciferase activity. Monolayers of Vero-DSP1, Vero-DSP2, or mixed Vero-DSP1 / Vero-DSP2 cells were infected with VSV-SARS-CoV-2-S-Δ19CT (10,000 TCID50 units / well) or mock-infected. Luciferase activity was measured as a marker of cell fusion 24 hours postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. [Figure 4]Figures 4A-B show optimization of the luciferase signal of the assay. (A) Kinetics of VSV-SARS-CoV-2-S-Δ19CT-induced luciferase activity. VSV-SARS-CoV-2-S-Δ19CT (2360 TCID units / well) or OptiMEM alone (mock) was layered onto a monolayer of Vero-DSP1 / Vero-DSP2 cells. The luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured at the indicated time points (in hours). Values ​​represent the mean RLU ± standard deviation from duplicate wells. (B) Optimization of Vero-DSP1 / Vero-DSP2 cell density. The indicated number of Vero-DSP1 / Vero-DSP2 cells was seeded into a 96-well plate. The next day, VSV-SARS-CoV-2-S-Δ19CT was diluted in OptiMEM to the indicated dilution and layered onto the cell monolayer. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 28 hours post-infection. Values ​​represent the mean RLU ± standard deviation from two separate experiments performed in duplicate. [Figure 5]Figures 5A-B show neutralization of VSV-SARS-CoV-2-S-Δ19CT-induced luciferase activity. (A) Inhibition by purified molecules. VSV-SARS-CoV-2-S-Δ19CT was incubated with medium alone or the indicated concentrations of polyclonal anti-SARS-CoV-2-spike antibody, monoclonal anti-ACE2 antibody, or recombinant ACE2. After 1 h at 37°C, the virus mixture was layered onto Vero-DSP1 / Vero-DSP2 monolayers. Control wells received medium only (no virus). Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 24 h postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. (B) Inhibition by COVID-19 convalescent plasma. VSV-SARS-CoV-2-S-Δ19CT was incubated with the indicated dilutions of plasma from a COVID-19 convalescent individual (NL1) or pooled plasma from three presumed SARS-CoV-2 seronegative individuals. Plasma dilutions represent the final dilution after virus addition. After 1 hour at 37°C, the virus mixture was layered onto Vero-DSP1 / Vero-DSP2 monolayers. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 28 hours postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. [Figure 6]Figures 6A-E show optimization of neutralization conditions for the assay. (A) Virus incubation reduces syncytia formation. VSV-SARS-CoV-2-S-Δ19CT was thawed and diluted in OptiMEM to the indicated TCID50 units / 100 μL. Samples were prepared in duplicate; one set was immediately layered onto Vero-DSP1 / Vero-DSP2 monolayers, and the other was incubated at 37°C for 1 hour before layering onto Vero-DSP1 / Vero-DSP2 monolayers. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 27 hours postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. (B) Inhibition of NL1 under different conditions. Dilutions of NL1 convalescent plasma were prepared and incubated with VSV-SARS-CoV-2-S-Δ19CT for the indicated times at the indicated temperatures before layering onto Vero-DSP1 / Vero-DSP2 monolayers. Dilutions represent the NL1 dilution in the final virus / plasma mixture. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 25 hours postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. (C and D) Effect of heat inactivation on negative sera. Presumably negative serum samples A, B, C, D, E, F, and G, and a commercially available SARS-CoV-2 seronegative pooled serum (pool), were prepared in duplicate aliquots. One aliquot was kept on ice, and the other was incubated at 56°C for 30 minutes. Each aliquot was then diluted in OptiMEM and mixed with VSV-SARS-CoV-2-S-Δ19CT to a final dilution of 1:100 (C) or the indicated dilution (D). After a 30-minute incubation at room temperature, the virus / serum mixture was layered onto the Vero-DSP1 / Vero-DSP2 monolayer. The luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 23 hours postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. (E) Effect of heat inactivation on COVID-19 convalescent serum. Presumed SARS-CoV-2 seropositive samples were thawed and assayed as described in panel D. [Figure 7]Figures 7A-D show the development of a contrived positive control. (A) Recommended minimum dilution. Thirty-nine sera from presumed SARS-CoV-2 seronegative individuals were serially diluted and mixed one-by-one with VSV-SARS-CoV-2-S-Δ19CT. Dilutions represent the serum dilution in the virus / serum mixture. After 30 minutes at room temperature, the virus / serum mixture was layered onto the Vero-DSP1 / Vero-DSP2 monolayer. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 22 hours postinfection. Values ​​represent the mean RLU ± standard deviation fitted to a nonlinear regression curve. (B and C) Neutralization of VSV-SARS-CoV-2-S-Δ19CT by mAb10914. mAb10914 or an isotype control antibody was diluted in OptiMEM (B) or pooled SARS-CoV-2 seronegative serum (C) and incubated with VSV-SARS-CoV-2-S-Δ19CT. Concentrations represent the concentration in the virus / antibody mixture; serum concentrations were 1:100. After 30 minutes at room temperature, virus was layered onto Vero-DSP1 / Vero-DSP2 monolayers. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured 24 hours postinfection. Values ​​represent the mean RLU ± standard deviation from duplicate wells. (D) Establishment of an artificial positive control value. mAb10914 was diluted in pooled SARS-CoV-2 seronegative serum as described in panel C and subjected to the same assay as panel C, performed in triplicate by three different operators. A dilution of pooled negative serum was used as a control (0 μg / mL mAb10914). The % signal relative to the control was determined for each sample. Values ​​represent the mean % signal ± standard deviation from three independent experiments performed in duplicate, fitted to a nonlinear regression curve. % luciferase signal: luciferase signal from test samples relative to that of the negative control (pooled negative serum): >50% = negative sample; <50% = positive or borderline. Virus neutralizing unit (VNU): an arbitrary unit equal to 100 times the concentration of mAb10914 that results in an equivalent % luciferase signal.To be positive, a sample must have a VNU of 30. [Figure 8-1] Figures 8A-D show the correlation of virus neutralization units to PRNTEC50 and VNTEC50. SARS-CoV-2 seropositive serum samples were serially diluted in OptiMEM and mixed with VSV-SARS-CoV-2-S-Δ19CT. After 30 minutes at room temperature, the virus / serum mixture was layered onto the Vero-DSP1 / Vero-DSP2 monolayer. Luciferase substrate EnduRen™ was added to the wells, and luciferase activity was measured at 23 and 27 hours postinfection. Each assay plate also contained a standard curve in which mAb10914 was spiked into pooled SARS-CoV-2 seronegative serum and mixed with the virus. The concentrations of mAb10914 in the virus mixture for the standard curve were 3, 1, 0.33, 0.11, and 0.037 μg / mL. The % signal for each test sample and standard curve point was determined. Subsequently, virus neutralization units (VNU) were determined for each sample based on its % signal relative to the standard curve. Here, VNU is equal to 100 times the concentration of mAb10914 for a given % signal. To be positive, a sample must have a VNU of 30. (A) Comparison with PRNTEC50. Fifteen serum samples from the tested positive samples were subjected to a plaque reduction neutralization test (PRNT) using a clinical isolate of SARS-CoV-2. Two-fold serial dilutions of the samples were tested in the PRNT50 assay, from 1:20 to 1:40960. For each sample, the number of plaques at each dilution was plotted and used to determine the PRNTEC50 value for each sample. Statistical comparison of VNU to PRNTEC50 was performed. Data were transformed due to non-normal distribution (p<0.0001). (B) Comparison with VNTEC50. The % signal for each sample dilution was plotted and used to determine VNTEC50. Statistical comparisons of VNU to VNTEC50 were performed. Data were transformed due to non-normal distribution (p<0.0001). [Figure 8-2]Continued from Figure 8-1. (C) Representative distribution of VNU in the PCR-positive cohort. (D) Correlation between VNU values ​​and symptom severity in 31 positive samples with self-reported clinical symptoms. [Figure 9] Figure 9 shows an example of an assay plate layout for the DSP Vero luciferase assay. Exemplary assay plate layout. All controls and samples were assayed in duplicate. A total of 41 samples (S1–S41) can be analyzed on a single plate using a single dilution for each sample. The background control (BC) consists of 1:100 pooled SARS-CoV-2 seronegative sera containing no virus. The negative control (NC) consists of 1:100 pooled SARS-CoV-2 seronegative sera containing VSV-SARS-CoV-2-S-Δ19CT. The NC is used to determine 100% luciferase signal in the assay. St1–St5 are standards for the calibration curve. All standards were 1:100 pooled SARS-CoV-2 seronegative sera containing VSV-SARS-CoV-2-S-Δ19CT and mAb10914, spiked into each standard at different concentrations: Std1 at 3 μg / mL, Std2 at 1 μg / mL, Std3 at 0.33 μg / mL, Std4 at 0.11 μg / mL, and Std5 at 0.037 μg / mL. [Figure 10] Figure 10 is a schematic diagram of the DSP Vero luciferase assay workflow. [Figure 11-1] Figures 11A-C show luciferase activity in plasma and serum from three subjects at different dilutions. Plasma and serum from three COVID-19 convalescent individuals were heat-inactivated at 56°C for 30 minutes. Serial dilutions were then prepared and incubated with VSV-SARS-CoV-2-S-Δ19CT for 30 minutes at room temperature before being layered onto Vero-DSP1 / DPS2 monolayers. Luciferase activity was measured approximately 24 hours later. [Figure 11-2]Continuation of Figure 11-1. Figure 11D shows luciferase activity in pooled SARS-CoV-2 seronegative plasma containing graded concentrations of the anti-SARS-CoV-2 spike antibody mAb 10914 mixed with VSV-SARS-CoV2 v1.0 (VSV-SARS-CoV-2-S-Δ19CT) virus. After a 30-minute incubation at room temperature, the plasma / virus mixture was layered onto Vero-DSP1 / DSP2 monolayers. Luciferase activity was measured approximately 24 hours later. Figure 11E shows luciferase activity in pooled SARS-CoV-2 seronegative plasma or dilutions of plasma from a COVID-19 convalescent individual (NL1) mixed with VSV-SARS-CoV2 v.1.0 (VSV-SARS-CoV-2-S-Δ19CT) virus. After 1 hour of incubation at 37°C, the plasma / virus mixture was layered onto the Vero-DSP1 / DSP2 cell monolayer. Luciferase activity was measured approximately 24 hours later. Figure 11F shows the effect of heat inactivation on plasma samples. One aliquot was kept on ice, while the other was incubated at 56°C for 30 minutes. Each aliquot was then diluted in OptiMEM and mixed with VSV-SARS-CoV-2-S-Δ19CT to the final dilution indicated in the graph. After 30 minutes of incubation at room temperature, the virus / plasma mixture was layered onto the Vero-DSP1 / Vero-DSP2 monolayer. Luciferase activity was measured approximately 24 hours later. [Figure 12]Figure 12 shows luciferase activity in pooled saliva prepared from pre-COVID pandemic individuals spiked with graded concentrations of mAb10914 (2 μg / mL (QC-High), 0.6 μg / mL (QC-Mid), and 0.4 μg / mL (QC-Low)) mixed with a 1:100 dilution of VSV-SARS-CoV2 v.1.0 (VSV-SARS-CoV-2-S-Δ19CT) virus. After a 30-minute incubation at room temperature, the saliva / virus mixture was layered onto a Vero-DSP1 / Vero-DSP2 monolayer. Luciferase activity was measured approximately 24 hours later. Bars corresponding to data generated using no spike, QC-Low, QC-Mid, and QC-High are shown in the order listed above (left to right) for each dilution tested. [Figure 13] Figure 13A is a schematic diagram of VSV-SARS-CoV-2-Fluc and VSV-SARS-CoV-2-S-Δ19CT-gLuc. VSV-SARS-CoV-2-Fluc encodes the mutant Luc2 variant of firefly luciferase (GenBank accession number AY738222). VSV-SARS-CoV-2-S-Δ19CT-gLuc encodes secreted Gaussia luciferase (gLuc). Figure 13B shows the workflow for version 2 of the IMMUNO-COV assay. [Figure 14]Figure 14A shows luciferase activity when VSV-SARS-CoV-2-S-Δ19CT-Fluc, encoding the mutant Luc2 variant of firefly luciferase, was incubated with pooled negative sera (diluted 1:80), COVID-19 convalescent sera (diluted 1:80), or pooled negative sera (diluted 1:80) mixed with mAb10914 at 10, 2, or 0.2 μg / mL. After 45 minutes of incubation at room temperature, the virus / serum mixture was layered onto a Vero cell monolayer. After an additional 18 hours, d-luciferin substrate was added to the wells, and luciferase activity (RLU) was measured using a luminometer. Figure 14B shows luciferase activity when VSV-SARS-CoV-2-S-Δ19CT-gLuc (1:48 dilution of virus stock) encoding secreted Gaussia luciferase was incubated alone or with pooled SARS-CoV-2 seronegative sera (1:80 dilution) spiked with 0.2 or 2 μg / mL of mAb10914. After 30 minutes of incubation at room temperature, the virus / serum mixture was layered onto Vero-Ace-2 monolayers. After an additional 24 hours, coelenterazine (5 μM stock, 20 μL) was added, and luciferase activity was measured. [Figure 15]Figures 15A-B demonstrate that Vero-Ace-2 cells are particularly effective in the IMMUNO-COV Assay v.2. Figure 15A shows luciferase activity when VSV-SARS-CoV-2-S-Δ19CT-Fluc or medium alone (no virus control) was incubated with pooled SARS-CoV-2 seronegative sera (1:80 dilution) mixed with 2 or 0.2 μg / mL of mAb 10914. After a 30-minute incubation at room temperature, the virus / serum mixture was layered onto a monolayer of cells (Vero-αHis, Vero-E6, or Vero-Ace-2). After an additional 26 hours, d-luciferin substrate was added to the wells, and luciferase activity (RLU) was measured using a luminometer. Figure 15B shows luciferase activity when pooled SARS-CoV-2 seronegative sera (diluted 1:80) were incubated with VSV-SARS-CoV-2-S-Δ19CT-gLuc (4800 TCID / well) alone or spiked with 0.2 μg / mL mAb 10914. After 30 minutes of incubation at room temperature, the virus / serum mixture was layered onto Vero-αHis, Vero-Ace-2, or Vero-Ace-2 / TMPRSS2 cell monolayers seeded the previous day at 1e4 cells / well in black, clear-bottom 96-well plates. After an additional 24 hours, d-luciferin was added and luciferase activity in the wells was measured. [Figure 16]Figures 16A-B show that when cells are plated simultaneously with virus, the luciferase signal is significantly lower (compared to pre-plated cells), but recovers significantly after 4 hours of recovery. (A) Virus was incubated with pooled SARS-CoV-2 seronegative sera (1:80) at room temperature for 30 minutes before being overlaid onto Vero cell monolayers or mixed with Vero cells at an equivalent density. Luciferase activity was measured at the indicated times after overlaying onto the cells. (B) Virus was incubated with pooled SARS-CoV-2 seronegative sera (1:80) at room temperature for 30 minutes before being overlaid onto Vero cell monolayers plated 4 hours or 24 hours earlier. Luciferase activity was measured after an additional 24 hours. [Figure 17] Figures 17A-B show that luciferase activity increases with increasing cell density (A) and virus dose / well (B). Virus was diluted in OptiMEM to the indicated TCID50 per well and layered onto Vero cell monolayers plated the previous day at the indicated cells / well. Luciferase activity was measured after an additional 16, 20, and 24 hours. Data in panel A are from 24 hours. In panel A, bars corresponding to data generated using 7,000, 10,000, 15,000, and 20,000 cells / well are shown in the order listed (left to right) for each virus TCID50 condition tested. In panel B, bars corresponding to data generated using 2,400, 1,600, 800, and 400 PFU / well are shown in the order listed (left to right) for each time point tested. [Figure 18]Figure 18 shows that the IMMUNO-COV Assay v.2 is highly sensitive and exhibits dose-dependent inhibition of VSV-SARS-CoV-2-S-Δ19CT-Fluc. Virus was incubated with pooled negative sera containing graded concentrations of mAb 10914 at room temperature for 30 minutes. The virus / serum mixture was then layered onto Vero cell monolayers. After an additional 24 hours, luciferase activity was measured. [Figure 19] Figure 19 shows the kinetic curve of Fluc activity after the addition of d-luciferin in Vero-Ace-2 cells infected with VSV-SARS-CoV-2-S-Δ19CT-Fluc. Vero-Ace-2 cell monolayers (seeded at 1e4 cells / well the day before infection) were infected with 4800 TCID50 units of VSV-SARS-CoV-2-S-Δ19CT-Fluc. After 24 hours, the plate was loaded into a TECAN instrument (luminescence plate reader) equipped with an injector. The injector was programmed to inject the desired amount of substrate (20 μL of a 3.75 mg / mL solution of d-luciferin) into each well, and luminescence was read after 0.5 seconds (1000 ms integration time). Further luciferase readings were taken every 5 seconds for 3 minutes to generate a kinetic curve of luciferase activity (two readings were taken, d-luciferin was added at 9 seconds (arrow) (final concentration in the well: 0.44 mg / mL), and further readings were taken for 3 minutes). [Figure 20] Figure 20 shows a description of an exemplary dried blood spot (DBS) collection card. A drop of blood is placed on a filter paper with five circles printed on it. [Figure 21] 21 shows a graph summarizing % relative luciferase response data generated by extracting blood spots with OptiMEM across a range of sample dilutions. OptiMEM A and B are independent samples of OptiMEM. [Figure 22]Figures 22A-B show exemplary micrographs of blood spot matrices: (A) B7 Opti-20x Positive Matrix A, and (B) A7 Opti-20x Negative Matrix A. The blood spot matrices were compatible at a 1:20 dilution of the matrix. No issues with cell health or viral infectivity were observed in the blood spot samples tested. [Figure 23] FIG. 23 shows an exemplary layout map for a 96-well U-well (or U-bottom) plate. [Figure 24] FIG. 24 shows an exemplary layout map for a 96-well assay plate. DETAILED DESCRIPTION OF THE INVENTION

[0070] definition Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0071] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0072] The terms "about" or "approximately" include within a statistically significant range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the terms "about" or "approximately" depends on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0073] The terms "comprise(s)," "include(s)," "having (has)," and "contain(s)" are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures.

[0074] As used herein, the terms "assay" or "test" may be used interchangeably with the term "method."

[0075] As used herein, "antibody" encompasses polyclonal and monoclonal antibodies and refers to immunoglobulin molecules of the classes IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and IgG4), or IgM, or fragments or derivatives thereof, including, but not limited to, Fab, F(ab'), Fd, single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, humanized antibodies, and various derivatives thereof.

[0076] In the context of the present disclosure, the term "neutralizing antibody" refers to an antibody that binds to a pathogen (e.g., a virus) and interferes with the pathogen's ability to infect cells. Non-limiting examples of neutralizing antibodies include antibodies that bind to viral particles and inhibit successful transduction, such as one or more steps selected from binding, entry, transport to the nucleus, and transcription of the viral genome. Some neutralizing antibodies can block viruses at steps after entry.

[0077] The term "immune response" refers to the response of cells of the immune system (e.g., B cells, T cells, macrophages, or polymorphonuclear cells) to a stimulus such as an antigen (e.g., a viral antigen). An active immune response can involve the differentiation and proliferation of immunocompetent cells, which leads to the synthesis of antibodies or the development of cell-mediated reactivity, or both. An active immune response can be initiated by a host after exposure to an antigen (e.g., by infection or vaccination). An active immune response can be contrasted with passive immunity, which can be achieved by the transfer of substances such as antibodies, transfer factors, thymic grafts, and / or cytokines from an actively immunized host to a non-immunized host.

[0078] As used herein in relation to viral infection and vaccination, the term "protective immune response" or "protective immunity" refers to an immune response that provides some benefit to a subject in that it prevents or reduces infection or prevents or reduces the development of disease associated with the infection. Without wishing to be bound by theory, the presence of SARS-CoV-2 neutralizing antibodies in a subject may indicate the presence of a protective immune response in the subject.

[0079] The terms "immunogenic composition," "vaccine composition," or "vaccine" are used interchangeably and refer to a composition comprising at least one immunogenic and / or antigenic component that induces an immune response (e.g., a humoral and / or cellular response) in a subject. In certain embodiments, the immune response is a protective immune response. Vaccines can be administered for the prevention or treatment of diseases, such as infectious diseases. Vaccine compositions can include, for example, live or killed infectious pathogens, recombinant infectious pathogens (e.g., recombinant virus particles, virus-like particles, nanoparticles, liposomes, or cells expressing immunogenic and / or antigenic components), antigenic proteins or peptides, nucleic acids, etc. Vaccines can also be administered with adjuvants to enhance the immune response.

[0080] The term "operably linked" includes the linkage of two or more nucleic acid elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer, or a 5' regulatory region containing a promoter or enhancer, is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0081] As used herein, the term "promoter" refers to a genetic sequence that is generally located in cis and upstream of a gene and promotes transcription of that gene. Promoters can be regulatable (developmentally, tissue-specific, or (chemically, temperature) inducible) or constitutively active. In certain embodiments, the promoter is the ubiquitin C promoter (see Schorpp et al., Nucl. Acids Res. 24(9): 1787-1788, 1996; Byun et al., Biochem. Biophys. Res. Comm 332(2): 518-523, 2005), or the CMV-IE promoter (see Addison et al., J. Gen. Virol. 78(7): 1653-1661, 1997; Hunninghake et al., J. Virol. 63(7): 3026-3033, 1989), or the hCMV-IE promoter (human cytomegalovirus immediate early gene promoter) (Stinski & Roehr, J. Virol. 55(2): 431-441, 1985; Hunninghake et al., J. Virol. 63(7): 3026-3033, 1989). 3026-3033, 1989). Promoters can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. Promoters can also contain distal enhancer or repressor elements, which can be located as many as thousands of base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate expression of genetic components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers.Non-limiting representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

[0082] The terms "signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can direct the localization of a polypeptide. A signal peptide can direct a polypeptide into the secretory pathway of a cell. A signal peptide is often located at the N-terminus of a polypeptide and is often cleaved from the remainder of the polypeptide (often referred to as the "mature protein") upon secretion from the cell.

[0083] The terms "derivative" and "variant" are used interchangeably herein to refer to an entity that has substantial structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties relative to the reference entity. In many embodiments, a derivative also differs functionally from the reference entity. Generally, whether a particular entity is properly considered a "derivative" of a reference entity is based on the degree of structural identity of the particular entity with the reference entity. As will be understood by those of skill in the art, any biological or chemical reference entity will have certain characteristic structural elements. A derivative is, by definition, a distinct entity that shares one or more such characteristic structural elements. To give just a few examples, a small molecule can have a characteristic core structural element (e.g., a macrocycle core) and / or one or more characteristic pendant moieties; thus, derivatives of this small molecule will share the core structural element and the characteristic pendant moieties but differ in other pendant moieties and / or the type of bond (single or double bond, E or Z, etc.) present within the core. Nucleic acid and polypeptide / protein derivatives encompass variants. Derivative nucleic acids can have characteristic sequence elements made up of multiple nucleotide residues with designated positions relative to each other in linear or three-dimensional space. In some embodiments, the nucleic acid sequence of a derivative can be 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage identical over the entire length of a reference sequence or a fragment thereof. Derivative peptides or polypeptides can have characteristic sequence elements made up of multiple amino acids with designated positions relative to each other in linear or three-dimensional space and / or that contribute to a specific biological function.Derivative peptides and polypeptides include peptides and polypeptides that differ in amino acid sequence from a reference peptide or polypeptide by the insertion, deletion, and / or substitution of one or more amino acids, but retain at least one biological activity of the reference peptide or polypeptide (e.g., the ability to mediate cell infection by a virus, the ability to mediate membrane fusion, the ability to be bound by a specific antibody or to stimulate an immune response, etc.). In some non-limiting embodiments, a derivative peptide or polypeptide exhibits at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage identity over its entire length with the reference peptide or polypeptide (or a fragment thereof). Alternatively, or in addition, a derivative peptide or polypeptide may differ from a reference peptide or polypeptide as a result of one or more chemical moieties attached to the polypeptide backbone (e.g., in glycosylation, phosphorylation, acetylation, myristoylation, palmitoylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.) and / or one or more differences in one or more chemical moieties. In some embodiments, a derivative peptide or polypeptide lacks one or more of the biological activities of the reference polypeptide, or has a reduced or increased level of one or more biological activities compared to the reference polypeptide. Multiple derivatives of a particular peptide or polypeptide may be found in nature or may be produced synthetically or recombinantly. As used herein, the term "derivative" or "variant" also encompasses various chimeras, fusion proteins, and conjugates, including fusions or conjugates with detection tags (e.g., HA tags, histidine tags, biotin, fusions with fluorescent or luminescent domains, etc.), dimerization / multimerization sequences, Fc, signaling sequences, etc.

[0084] As used herein, the term "coronavirus" refers to the subfamily Coronavirinae within the family Coronaviridae within the order Nidovirales. Based on phylogenetic relationships and genome structure, this subfamily consists of four genera: αcoronavirus, βcoronavirus, γcoronavirus, and δcoronavirus. αcoronavirus and βcoronavirus only infect mammals. γcoronavirus and δcoronavirus infect birds, although some also infect mammals. αcoronavirus and βcoronavirus typically cause respiratory disease in humans and gastroenteritis in animals. Three highly pathogenic viruses, namely SARS-CoV-2, SARS-CoV-1, and MERS-CoV, cause severe respiratory syndrome in humans. Four other human coronaviruses, namely HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1, cause only mild upper respiratory tract disease in immunocompetent hosts, although some can cause severe infections in infants, young children, and elderly individuals. Further non-limiting examples of commercially important coronaviruses include transmissible gastroenteritis coronavirus (TGEV), porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis coronavirus, rabbit coronavirus, murine hepatitis virus, sialadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus (Cui et al., Nature Reviews Microbiology, 2019, 17:181-192; Fung et al., Annu. Rev. Microbiol., 2019, 73:529-557).

[0085] Coronaviruses form enveloped, spherical particles with diameters of 80 to 160 nm. These particles contain a positive-sense, nonsegmented, single-stranded RNA (ssRNA) genome ranging in size from 27 to 32 kb. The 5'-terminal two-thirds of this genome encodes the polyproteins pp1a and pp1ab. The 3'-terminal end encodes structural proteins, including the envelope glycoprotein spike (S), envelope (E), membrane (M), and nucleocapsid (N). The genomic RNA is 5'-capped and 3'-polyadenylated and contains multiple open reading frames (ORFs). The invariant gene order is 5'-replicase-SEMN-3', with numerous small ORFs (encoding accessory proteins) scattered among the structural genes. Coronavirus replicases are encoded by two large, overlapping open reading frames (ORF1a and ORF1b), which occupy approximately two-thirds of the genome, and are translated directly from the genomic RNA (gRNA). However, structural and accessory genes are translated from subgenomic RNA (sgRNA), which is generated during genome transcription / replication. Infection is initiated by coronavirus attachment to a cognate cellular receptor, which induces endocytosis. Membrane fusion typically occurs in endosomes, releasing the viral nucleocapsid into the cytoplasm. The genomic RNA (gRNA) serves as a translation template for the polyproteins pp1a and pp1ab, which are cleaved to form nonstructural proteins (nsp). The nsp induces membrane rearrangements to form double-membrane vesicles (DMVs), where the viral replication transcription complex (RTC) is anchored. The full-length gRNA is replicated via a negative-sense intermediate, and a set of nested subgenomic RNA (sgRNA) species are synthesized by discontinuous transcription. These sgRNAs encode viral structural and accessory proteins.Particle assembly occurs in the ER-Golgi intermediate complex (ERGIC), and mature virions are released into smooth-walled vesicles via the secretory pathway.

[0086] Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein (interchangeably referred to as "spike glycoprotein," "S glycoprotein," "S protein," or "spike protein"), which is the primary target of antiviral neutralizing antibodies and the focus of therapeutic and vaccine design. The S glycoprotein forms a homotrimer that protrudes from the surface of the virus. The S glycoprotein contains two functional subunits, which are involved in binding to host cell receptors (the S1 subunit) and in fusing the viral membrane with the cellular membrane (the S2 subunit). For many coronaviruses, including SARS-CoV-1 and SARS-CoV-2, the S glycoprotein is cleaved at the interface between the S1 and S2 subunits, which remain noncovalently bound in a prefusion conformation. The distal S1 subunit contains one or more receptor-binding domains (RBDs) and contributes to stabilizing the prefusion state of the membrane-anchored S2 subunit, which contains the fusion machinery. S is further cleaved by host proteases at the S2' site, located immediately upstream of the fusion peptide. This cleavage is proposed to activate the protein for membrane fusion by changing its conformation. Walls et al., Cell (published online March 9, 2020, available at doi.org / 10.1016 / j.cell.2020.02.058).

[0087] SARS-CoV-1 and SARS-CoV-2 interact directly with angiotensin-converting enzyme 2 (ACE2) to enter target cells and can use the cellular serine protease, type II transmembrane protease (TMPRSS2), to prime the S protein (Hoffmann et al., Cell, 2020, 181:1-10; available at doi.org / 10.1016 / j.cell.2020.02.052). SARS-CoV-S and SARS-CoV-2-S share 76% amino acid identity. The receptor-binding domain (RBD) of the S glycoprotein is involved in binding to the ACE2 receptor. Six RBD amino acids have been shown to be important for binding to the ACE2 receptor and for determining the host range of SARS-CoV-like viruses. These are Y442, L472, N479, D480, T487, and Y4911 in SARS-CoV, which correspond to L455, F486, Q493, S494, N501, and Y505 in SARS-CoV-2 (Andersen et al., Nature Medicine, 2020; available at doi.org / 10.1038 / s41591-020-0820-9).

[0088] As used herein, the term "rhabdovirus" refers to viruses of the family Rhabdoviridae in the order Mononegavirales, which encompasses over 150 viruses of vertebrates, invertebrates, and plants. Examples of rhabdoviruses include rabies virus (RABV) of the genus Lyssavirus, vesiculovirus of the genus Vesiculovirus, and viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus of the genus Novirhabdovirus. Rhabdoviruses are bullet-shaped, enveloped viruses with a negative-sense, single-stranded RNA genome measuring 11 to 15 kb in length. The genome of rhabdoviruses contains up to 10 genes, only five of which are common to all members of the family. These genes encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L). The genome associates with N, L, and P to form a nucleocapsid, which is condensed into a tightly coiled helix by the M protein. This condensed nucleocapsid is surrounded by a lipid bilayer containing the viral glycoprotein G, which constitutes the spikes that protrude from the viral surface. After rhabdoviruses enter cells via the endocytic pathway, they fuse with the cell membrane in the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion between the viral envelope and the endosomal membrane is triggered by structural rearrangements of G induced by low pH (in the endosome), resulting in the release of the viral genome and associated proteins into the cytoplasm of the target cell.

[0089] As used herein, the term "vesiculovirus" refers to any virus in the genus Vesiculovirus. Non-limiting examples of vesiculoviruses include vesicular stomatitis virus (VSV) (e.g., VSV-New Jersey, VSV-Indiana), Alagoas vesiculovirus, Cocal vesiculovirus, Jurona vesiculovirus, Carajas vesiculovirus, Maraba vesiculovirus, Piry vesiculovirus, Calchaqui vesiculovirus, Yug Bogdanovac vesiculovirus, Isfahan vesiculovirus, Chandipura vesiculovirus, Perinct vesiculovirus, and Porton-S vesiculovirus. Vesiculovirus (VSV) of the genus Vesiculovirus is the prototypic rhabdovirus. While VSV is used as an example in this disclosure, the disclosure can also be used with other vesiculoviruses and other rhabdoviruses. VSV has two major serotypes, New Jersey and Indiana, both of which infect insects and mammals and cause economically important diseases in cattle, horses, and swine. The VSV genome consists of a single-stranded, negative-sense RNA of 11–12 kB, which encodes five viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L). Multiple G monomers associate to form trimeric spikes that anchor the viral membrane. For example, see Sun et al., Future Virol., 2010, 5(1):85–96 and Aurelie et al., Viruses 2012, 4:117–139.

[0090] As used herein, the phrase "non-essential portion(s) of the recombinant VSV genome" refers to regions of the VSV genome that can be modified without affecting the development and / or growth of the virus in vitro and / or in vivo and without affecting the functions of the virus necessary to function as an immunogenic composition or vaccine.

[0091] As used herein in reference to various recombinant enveloped viral particles, the term "pseudotyped" refers to a viral particle that includes, e.g., proteins, glycoproteins, etc., in its lipid envelope molecules that are mutated and / or heterologous relative to molecules typically found on the surface of the virus from which the particle is derived (i.e., the "reference virus") and that affect, contribute to, determine, redirect, and / or completely change the tropism of the viral particle relative to the reference virus. In some embodiments, the viral particle is pseudotyped to recognize, bind to, and / or infect a different target (ligand or cell) than the reference virus. In some embodiments, the viral particle is pseudotyped to not recognize, bind to, and / or infect a different target (ligand or cell) than the reference virus.

[0092] The term "fusogen" or "fusogenic molecule," as used herein, refers to any molecule that can induce membrane fusion when present on the surface of a viral particle. A fusogen can be, for example, a protein (e.g., a viral glycoprotein), or a fragment or derivative thereof.

[0093] The term "replication-competent" refers to viruses (including wild-type and recombinant viral particles) that are capable of infecting and multiplying in susceptible cells.

[0094] The term "encoding" can refer to encoding from either the (+) or (-) sense strand of a polynucleotide for expression in a viral particle.

[0095] The terms "specifically binds," "specifically adheres," "specifically targets," "selectively binds," or "binding specificity" refer to the ability of a spike (S) glycoprotein, recombinant viral particle, vaccine, neutralizing antibody, or other molecule described herein to bind to a target. In certain embodiments, specific binding refers to binding to a target with an affinity that is at least 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold greater than the affinity for a non-target. In certain embodiments, the affinity is determined by an affinity ELISA assay. In certain embodiments, the affinity is determined by a BIAcore assay. In certain embodiments, the affinity is determined by a kinetic method. In certain embodiments, the affinity is determined by an equilibrium / solution method.

[0096] As used herein, the term "reporter protein" refers to any protein that produces a detectable, quantifiable signal when present in a cell. Non-limiting examples of reporter proteins that can be used in the assays of the present disclosure include luciferases (including, but not limited to, Renilla luciferase, mutant Renilla luciferase RLuc8, (dCpG) luciferase, NanoLuc reporter, firefly luciferase, Gaussia luciferase (gLuc), MetLuc, Vibrio fischeri lumazine protein, Vibrio harveyi luminase protein, dinoflagellate luciferase, firefly luciferase YY5 mutant, firefly luciferase LGR mutant, firefly luciferase mutant E, and derivatives thereof), and fluorescent proteins (including, but not limited to, green fluorescent protein (GFP) [e.g., Aequoria victoria GFP, Renilla muelleri GFP, Renilla reniformis GFP, Renilla ptilosarcus GFP, GFP], GFP-like fluorescent protein, (GFP-like), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP) [e.g., Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana], enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP) [e.g., EBFP2, Azurite, GFP2, GFP10, and mTagBFP], enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP) [e.g., mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mCFPmm, mTFP1(Teal)], enhanced cyan fluorescent protein (ECFP), superfolder GFP, superfolder YFP, orange fluorescent protein [e.g., Kusabira Orange, KusabiraOrange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine], red fluorescent proteins [e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, tdTomato, AQ143], small ultra-red fluorescent proteins, FMN-binding fluorescent proteins, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGr1, bright green fluorescent protein, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and fragments or derivatives thereof), β-galactosidase, β-glucuronidase, β-geo, and the like.

[0097] The term "effective" as applied to dose or amount refers to an amount of a compound (e.g., a recombinant virus) or composition (e.g., a pharmaceutical, vaccine, or immunogenic composition) sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that was effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug(s) employed, the mode of administration, etc.

[0098] In the context of the present disclosure, the terms "treat," "treatment," and the like, as they relate to any of the conditions listed herein, mean alleviating or alleviating at least one symptom associated with said condition, or slowing or reversing the progression of said condition. Within the meaning of the present disclosure, the term "treat" also refers to preventing disease, delaying the onset of disease (i.e., the period before clinical manifestation of disease), and / or reducing the risk of disease progression or worsening. The terms "treat," "treatment," and the like, with respect to a condition, disorder, or medical condition, can also include (1) preventing or delaying the onset of at least one clinical or subclinical symptom of the condition, disorder, or medical condition occurring in a subject who may be suffering from or predisposed to the condition, disorder, or medical condition, but who has not yet experienced or exhibited a clinical or subclinical symptom of the condition, disorder, or medical condition; or (2) inhibiting the condition, disorder, or medical condition, i.e., preventing, reducing, or delaying the onset of the disease, or its recurrence (in the case of maintenance therapy), or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of the condition, disorder, or medical condition, or at least one of its clinical or subclinical symptoms. Non-limiting examples of symptoms of COVID-19 disease include, but are not limited to, fever, cough, shortness of breath, pneumonia, acute respiratory distress syndrome (ARDS), acute pulmonary syndrome, loss of smell, loss of taste, sore throat, runny nose, gastrointestinal symptoms (e.g., diarrhea), organ failure (e.g., renal failure and dysfunction), septic shock, and death. When used in reference to a disease caused by a viral infection (e.g., SARS-CoV-2 infection), the terms "prevent," "preventing," or "prevention" refer to preventing the spread of infection in a subject exposed to the virus, e.g., preventing the virus from entering the subject's cells.

[0099] The terms "individual," "subject," or "patient" or "animal" refer to humans, livestock (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), poultry species, and experimental animal models of disease (e.g., mice, rats, ferrets, monkeys, etc.). In a preferred embodiment, the subject is a human.

[0100] The terms "nucleic acid," "polynucleotide," and "nucleotide" are used interchangeably and include both DNA and RNA, which, unless otherwise specified, includes positive and negative strands, single and double strands.

[0101] The phrase "pharmaceutically acceptable," when used in connection with the compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a subject (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias, for use in mammals, more particularly humans.

[0102] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), virology, microbiology, cell biology, chemistry, and biochemistry, which are within the skill of the art. Such tools and techniques are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques are described in detail in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985); U.S. Patent No. 5,071,743; Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech.28: 196-198 (2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14. 3: 74-75 (2001), U.S. Patent Nos. 5,702,931, 5,780,270 and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nucl. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrentino et al., Nuc. Acids. Res. 22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec. Biol. 67: 209-218.

[0103] virus particles One aspect of the present disclosure provides recombinant vesicular stomatitis virus (VSV) particles. VSV is an attractive virus for producing recombinant viral particles because it can be produced at high titers and does not cause serious pathology in humans. In certain embodiments, in the recombinant VSV particles of the present disclosure, the VSV glycoprotein (G) is replaced with the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof.

[0104] Recombinant VSV particles of the present disclosure can be produced using methods known in the art, for example, by providing a suitable host cell with: (a) DNA that can be transcribed to encode a VSV antigenome (+) RNA (complementary to the VSV genome), (b) a recombinant source of VSV nucleoprotein (N), (c) a recombinant source of VSV phosphoprotein (P), (d) a recombinant source of VSV large protein (L), and (e) foreign DNA under conditions such that the DNA is transcribed to produce antigenome RNA, thereby producing VSV containing genomic RNA complementary to the generated antigenome RNA and foreign RNA derived from the DNA that is not naturally part of the VSV genome. Methods and compositions that can be used to generate recombinant VSV particles can be found, for example, in U.S. Patent Nos. 7,153,510, 9,861,668, 8,012,489, 9,630,996, 8,287,878, and 9,248,178; U.S. Patent Application Publication Nos. 2014 / 0271564 and 2012 / 0121650; and Fukishi et al., J. Gen. Virol.l 2005, 86:2269-2274, all of which are incorporated herein by reference in their entireties.

[0105] In certain embodiments, the foreign RNA contained in the genome of the recombinant VSV particle, upon expression in a suitable host cell, produces one or more foreign proteins, hi certain embodiments, a foreign protein is a coronavirus spike (S) glycoprotein (e.g., the S glycoprotein from SARS-CoV-2), or a fragment or derivative thereof, as described in further detail below.

[0106] In certain embodiments, in addition to encoding a coronavirus spike (S) glycoprotein (e.g., the S glycoprotein from SARS-CoV-2) fragment or derivative thereof, the genome of the recombinant VSV encodes a reporter protein. Non-limiting examples of reporter proteins include luciferases (including, but not limited to, Renilla luciferase, RLuc8 mutant Renilla luciferase, (dCpG) luciferase, NanoLuc reporter, firefly luciferase, Gaussia luciferase (gLuc), MetLuc, Vibrio fischeri lumazine protein, Vibrio harveyi luminase protein, dinoflagellate luciferase, firefly luciferase YY5 mutant, firefly luciferase LGR mutant, firefly luciferase mutant E, and derivatives thereof), and fluorescent proteins (including, but not limited to, green fluorescent protein (GFP) [e.g., Aequoria victoria GFP, Renilla muelleri GFP, Renilla reniformis GFP, Renilla ptilosarcus GFP, GFP], GFP-like fluorescent protein, (GFP-like), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP) [e.g., Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana], enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP) [e.g., EBFP2, Azurite, GFP2, GFP10, and mTagBFP], enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP) [e.g., mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mCFPmm, mTFP1(Teal)], enhanced cyan fluorescent protein (ECFP), superfolder GFP, superfolder YFP, orange fluorescent protein [e.g., Kusabira Orange, KusabiraOrange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine], red fluorescent proteins [e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, tdTomato, AQ143], small ultra-red fluorescent proteins, FMN-binding fluorescent proteins, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGr1, bright green fluorescent protein, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and fragments or derivatives thereof), β-galactosidase, β-glucuronidase, β-geo, and the like.

[0107] In certain alternative embodiments, the one or more foreign proteins (e.g., coronavirus S glycoprotein and / or reporter protein) are not encoded by the genome of the recombinant VSV particle, but are incorporated into the VSV particle as proteins during production of the recombinant viral particle.

[0108] In certain embodiments, the recombinant VSV particles are replication-competent viral particles. In certain embodiments, the recombinant VSV particles are replication-defective viral particles.

[0109] In certain embodiments, the recombinant VSV particles of the present disclosure are used in seropositivity assays to determine the presence of coronavirus-neutralizing antibodies (e.g., SARS-CoV-2 neutralizing antibodies) in a sample from a subject. Such seropositivity assays can be used, for example, to determine whether a subject has previously been infected with the coronavirus and whether the infection has produced neutralizing antibodies that protect the subject (and therefore whether the subject can recover). Seropositivity assays of the present disclosure can also be used, for example, to determine vaccine efficacy, for therapeutic antibody development, for selecting the best donors for convalescent plasma therapy, for patient contact tracing, for identifying hosts that carry the virus, for determining disease burden, for determining asymptomatic infection rates, for identifying the extent of viral spread in households, communities, and specific environments, and the like.

[0110] In certain embodiments, the seropositivity assays of the present disclosure may use two or more types of recombinant VSV particles, hi certain embodiments, the assays use a mixture of two or more recombinant VSV particles encoding different coronavirus S glycoproteins (e.g., SARS-CoV-2 S glycoproteins from different strains, variants, or mutants).

[0111] In certain aspects, the present disclosure provides cells for producing the recombinant VSV particles of the present disclosure. Exemplary cells include, but are not limited to, any cell in which VSV grows, such as mammalian cells and some insect (e.g., Drosophila) cells. Many primary cells and cell lines commonly known in the art can be used as host cells. For example, cell lines that can be used include, but are not limited to, BHK (baby hamster kidney) cells, CHO (Chinese hamster ovary) cells, HeLA (human) cells, mouse L cells, Vero (monkey) cells (including Vero-αHis cells), Vero-Ace-2 cells, Vero-TRMPSS2 cells, Vero-E6 cells, ESK-4, PK-15, EMSK cells, MDCK (Madin-Darby canine kidney) cells, MDBK (Madin-Darby bovine kidney) cells, 293 (human) cells, Hep-2 cells, primary chicken embryo fibroblasts, primary chicken embryo fibroblasts, and semi-primary continuous cell lines (e.g., AGMK: African green monkey kidney cells). cell), human diploid primary cell lines (e.g., WI-38 and MRC5 cells), and monkey diploid cell lines (e.g., FRhL: Fetal Rhesus Lung cells).

[0112] VSV DNA for transcription to generate VSV antigenomic (+)RNA Vesicular stomatitis virus (VSV) is a rhabdovirus containing a single (non-segmented) negative-strand genomic RNA that is transcribed by the virion polymerase into five mRNAs encoding five structural proteins. These five structural proteins include glycoprotein (G), large protein (L), phosphoprotein (P), matrix protein (M), and nucleoprotein (N). The nucleocapsid protein encapsidates the RNA genome. Two proteins that form the polymerase complex bind to the nucleocapsid. The matrix (M) protein associates with the nucleocapsid and membrane. A single (transmembrane) envelope spike glycoprotein (G) extends from the viral envelope. The G protein functions to bind the virus to cellular receptors and catalyze the fusion of the viral membrane with the cellular membrane, initiating the infectious cycle. The VSV genome is approximately 11 kilobases in size.

[0113] VSV can be transmitted to a variety of mammalian hosts, commonly cattle, horses, pigs, and rodents. Human VSV infections are rare and generally asymptomatic or characterized by mild, flu-like symptoms that resolve within 3–8 days without complications. VSV is not considered a human pathogen, and pre-existing immunity to VSV is rare in the human population, making it an attractive viral vector for vaccine and therapeutic applications. Other beneficial features of VSV include, but are not limited to, (i) its ability to replicate reliably in cell culture, (ii) its inability to integrate or recombine with host cell DNA, (iii) its multiple serotypes, enabling prime-boost immunization strategies, and (iv) its ability to insert foreign genes of interest into the VSV genome and express them abundantly via viral transcriptase.

[0114] Fusion and subsequent uptake of rhabdoviruses (e.g., VSV) into cells is described in Belot, L. et al., "Structural and cellular biology of rhabdovirus entry," Adv. Virus Res., 2019, 104:147-183 (incorporated herein by reference in its entirety), and Albertini, AAV et al., "Molecular and Cellular Aspects of Rhabdovirus Entry," Viruses, 2012, 4:117-139 (incorporated herein by reference in its entirety). Further description of VSV endocytosis can be found in Sun, X. et al., "Internalization and fusion mechanism of vesicular stomatitis virus and related rhabdoviruses," Future Virol., 2010, 5(1):85-96 (incorporated herein by reference in its entirety). For general information regarding virus-cell fusion, see Igonet, S. et al., "SnapShot: Viral and Eukaryotic Protein Fusogens" Cell, 2012, 151:1634e1, which is incorporated herein by reference in its entirety.

[0115] Cell-cell fusion mediated by other viruses, such as the HIV virus, is described in Kondo, N. et al., "Conformational changes of the HIV-1 envelope protein during membrane fusion are inhibited by the replacement of its membrane-spanning domain," Journ. Biol. Chem., 2010, 285(19):14681-88, which is incorporated herein by reference in its entirety.

[0116] Numerous vesiculoviruses are known in the art and can be recombined according to the methods disclosed herein. Non-limiting examples of such vesiculoviruses are listed below:

[0117] TIFF0007617140000001.tif109123

[0118] In certain embodiments, VSV is the vesiculovirus used to generate the recombinant viruses of the present disclosure. In certain embodiments, the recombinant VSV is recombinant VSV-New Jersey or VSV-Indiana. In certain embodiments, the recombinant VSV is recombinant VSV-New Jersey. While the following disclosure uses VSV as an example, the disclosure can also be used with other vesiculoviruses.

[0119] Any DNA that can be transcribed to produce a VSV antigenomic (+)RNA (complementary to the VSV genome) can be used to construct recombinant DNA containing foreign DNA encoding a heterologous (foreign) protein or peptide for use in generating the recombinant VSV particles of the present disclosure. In certain embodiments, the DNA that can be transcribed to encode a VSV antigenomic (+)RNA includes at least the genes for the VSV nucleoprotein (N), VSV phosphoprotein (P), and VSV large protein (L).

[0120] VSV vectors can be genetically modified to contain one or more mutations or "mutation classes" in their genome. "Mutation classes" or "classes of mutations" are used interchangeably and refer to mutations known in the art to attenuate VSV when used alone. Exemplary mutation classes include, but are not limited to, VSV temperature-sensitive N gene mutations (hereinafter "N(ts)"), temperature-sensitive L gene mutations (hereinafter "L(ts):"), point mutations, G stem mutations (hereinafter "G(stem)"), non-cytopathic M gene mutations (hereinafter "M(ncp)"), gene shuffling or rearrangement mutations, truncated G gene mutations (hereinafter "G(ct)"), ambisense RNA mutations, G gene insertion mutations, gene deletion mutations, and the like. Mutations can be insertions, deletions, substitutions, gene rearrangements, or shuffling modifications.

[0121] In certain embodiments of the methods disclosed herein, the recombinant VSV particles are replication competent. In other specific embodiments, the recombinant VSV particles are replication deficient.

[0122] In certain embodiments of the methods and compositions disclosed herein, the VSV G glycoprotein is replaced with the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof, in a recombinant VSV particle, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell. In certain embodiments, the genome of the recombinant VSV particle lacks a functional VSV G gene and encodes the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof. Also provided are VSV particles, wherein (i) the VSV glycoprotein (G) is replaced with the coronavirus spike (S) glycoprotein, or a fragment or derivative thereof, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell, and (ii) the VSV particle comprises a reporter protein and / or a nucleic acid molecule encoding the reporter protein. The nucleic acid sequence encoding the reporter protein may be inserted between the nucleic acid sequence encoding the S glycoprotein and the nucleic acid sequence encoding the VSV large (L) protein.

[0123] The mutations can reduce the infectivity, virulence, or pathogenicity of VSV. This attenuation can be additive or synergistic. In synergistic attenuation, the level of VSV attenuation is greater than additive. Synergistic attenuation of VSV can be achieved by combining at least two classes of mutations within the same VSV genome, resulting in a reduction in VSV pathogenicity that far exceeds the additive level of attenuation observed with each VSV mutation class alone. Synergistic attenuation of VSV can provide an LD50 that is at least greater than the additive level of attenuation observed with each VSV mutation class alone (i.e., the sum of the two mutation classes), where the level of attenuation (i.e., LD50) is determined in a small animal neurovirulence model.

[0124] The VSV M gene encodes the viral matrix (M) protein and two smaller in-frame polypeptides (M2 and M3). The M2 and M3 polypeptides can be translated from the same open reading frame (ORF) as the M protein and lack the first 33 and 51 amino acids, respectively. Recombinant VSV vectors containing non-cytopathic M gene mutations (i.e., VSV vectors that do not express the M2 and M3 proteins) can be generated, which can also contain one or more additional mutations, resulting in VSV vectors that are significantly attenuated in cell culture and in animals.

[0125] In certain embodiments, recombinant VSV particles of the present disclosure comprise non-cytopathic mutations in the M gene. The VSV (Indiana serotype) M gene encodes a 229-amino acid M (matrix) protein, of which the first 30 amino acids at the NH2-terminus contain a proline-rich PPPY (PY) motif (SEQ ID NO: 68). The PY motif of the VSV M protein is located at amino acid positions 24-27 in both the VSV Indiana (Genbank Accession No. X04452) and New Jersey (Genbank Accession No. M14553) serotypes. VSV may comprise mutations in the PY motif (e.g., APPY (SEQ ID NO: 69), AAPY (SEQ ID NO: 70), PPAY (SEQ ID NO: 71), APPA (SEQ ID NO: 72), AAPA (SEQ ID NO: 73), and PPPA (SEQ ID NO: 74)). VSV may comprise any of a variety of amino acid mutations (e.g., deletions, substitutions, insertions, etc.) in the M protein PSAP (PS) motif (SEQ ID NO: 75). These and other mutations in the PY motif may be effective to reduce virus yield by blocking the late steps of virus budding.

[0126] The recombinant VSV particles of the present disclosure may contain one or more mutations in the M gene. Non-limiting examples of M protein mutations include, for example, a glycine changed to glutamic acid at position 21, a leucine changed to phenylalanine at position 111, a methionine changed to arginine at position 51, a glycine changed to glutamic acid at position 22, a methionine changed to arginine at position 48, a leucine changed to phenylalanine at position 110, a methionine changed to alanine at position 51, and a methionine changed to alanine at position 33. See, e.g., U.S. Patent No. 9,630,996. In various embodiments of the methods described herein, the genome of the recombinant VSV encodes a mutant VSV matrix (M) protein containing the M51R mutation. The M51R mutation eliminates the ability of the M protein to block cellular nucleocytoplasmic transport, thereby significantly attenuating VSV infectivity.

[0127] In certain embodiments, the DNA capable of being transcribed to encode a VSV antigenomic (+)RNA comprises a gene encoding a VSV M protein. In certain embodiments, the VSV M protein used in the methods and compositions described herein may comprise the amino acid sequence of SEQ ID NO:9 or a sequence that is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher, identical to the amino acid sequence of SEQ ID NO:9. In certain embodiments, the VSV M protein used in the vaccines or methods described herein may consist of the amino acid sequence of SEQ ID NO:9 or a sequence that is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher, identical to the amino acid sequence of SEQ ID NO:9. In certain embodiments, the mutated VSV M protein used in the vaccines or methods described herein may comprise the amino acid sequence of SEQ ID NO:7 or a sequence that is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher, identical to the amino acid sequence of SEQ ID NO:7. In certain embodiments, the mutant VSV M protein used in the vaccines or methods described herein may consist of the amino acid sequence of SEQ ID NO:7 or a sequence that is at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher, identical to the amino acid sequence of SEQ ID NO:7.

[0128] DNA capable of being transcribed to produce (e.g.,) antigenomic (+) RNA of VSV (such DNA is referred to herein as "VSV(-) DNA") is available in the art and / or can be obtained by standard methods. VSV(-) DNA for any serotype or strain known in the art, e.g., the New Jersey or Indiana serotype of VSV, can be used. The complete nucleotide and deduced protein sequence of the VSV genome is known and available as Genbank VSVCG, accession number J02428; NCBI Seq ID 335873, and is published in Rose and Schubert, 1987, in *The Viruses: The Rhabdoviruses*, Plenum Press, NY, pp. 129-166. An example of the complete sequence of VSV(-) DNA contained in plasmid pVSVFL(+) is shown, for example, in U.S. Pat. No. 7,153,510, which is incorporated herein by reference in its entirety for all purposes. Sequences of other vesiculovirus genomes have also been published and are available in the art.

[0129] If VSV(-) DNA is not already available, it can be prepared by standard methods: VSV genomic RNA can be purified from a virus preparation and reverse transcribed using the long-range polymerase chain reaction used to generate v(-) DNA. Alternatively, after purification of the genomic RNA, VSV mRNA can be synthesized in vitro, and cDNA can be prepared by standard methods and then inserted into a cloning vector (see, e.g., Rose and Gallione, 1981, J. Virol. 39(2):519-528). Individual cDNA clones of VSV RNA can be joined using small DNA fragments covering the gene junctions generated from VSV genomic RNA by reverse transcription and polymerase chain reaction (RT-PCR) (Mullis and Faloona, 1987, Meth. Enzymol. 155:335-350) (see Section 6, infra). VSV and other vesiculoviruses are available in the art.

[0130] In certain embodiments, insertion of foreign DNA is facilitated by introducing one or more, usually unique, restriction sites (e.g., in a polylinker) into VSV(-) DNA, an intergenic region, or 5' of a sequence complementary to the 3' end of the VSV genome, or 3' of a sequence complementary to the 5' end of the VSV genome.

[0131] In certain embodiments, the VSV (-) DNA is constructed to have a promoter operably linked to it, which promoter must be capable of initiating transcription of the (-) DNA in the cells of the animal or insect in which production of recombinant VSV is desired. Promoters that can be used include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequence of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); heat shock promoters (e.g., hsp70 for use in Drosophila S2 cells); the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol Examples of animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals include the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, which is active in pancreatic β cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region active in testis, breast, lymphocytes, and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); alpha-fetoprotein gene control region active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1 antitrypsin gene control region active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta globin gene control region active in bone marrow cells (Mogram Examples of suitable promoters include the myelin basic protein gene control region, which is active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48:703-712); and the myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286). Preferably, the promoter is an RNA polymerase promoter, preferably a bacteriophage, viral, or insect RNA polymerase promoter, including, but not limited to, promoters for T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase. When using an RNA polymerase promoter in which RNA polymerase is not endogenously produced by the host cell in which recombinant VSV production is desired, a recombinant source of RNA must also be provided to the host cell.

[0132] The VSV(-) DNA can be operably linked to a promoter before or after insertion of the foreign DNA. In certain embodiments, a transcription terminator is located downstream of the VSV(-) DNA.

[0133] In another embodiment, a DNA sequence that can be transcribed to generate a ribozyme sequence is located immediately 3' to the VSV(-) DNA before the transcription termination signal, such that upon transcription, a self-cleaving ribozyme sequence is generated at the 3' end of the antigenome RNA, which autolytically cleaves the fusion transcript (after the U) to release the precise 3' end of the VSV antigenome (+) RNA. Any ribozyme sequence known in the art may be used, as long as the correct sequence is recognized and cleaved. In a preferred embodiment, a hepatitis delta virus (HDV) ribozyme is used (Perrotta and Been, 1991, Nature 350:434-436; Pattnaik et al., 1992, Cell 69:1011-1020).

[0134] Thus, an example of a VSV(-) DNA for use in inserting foreign DNA includes the following operably linked components: a T7 RNA polymerase promoter, VSV(-) DNA, a DNA sequence to be transcribed to produce an HDV ribozyme sequence (immediately downstream of the VSV(-) DNA), and a T7 RNA polymerase transcription termination site.

[0135] Examples of plasmids that can be used are pVSVFL(+) or pVSVSS1.

[0136] In certain embodiments, the coronavirus spike (S) protein can replace the endogenous VSV G protein in recombinant VSV particles, or can be expressed as a fusion with the endogenous VSV G protein, or can be expressed in addition to the endogenous VSV G protein, either as a fusion protein or a non-fusion protein. For example, the VSV G gene in the VSV(-) DNA of the plasmid pVSVFL(+) can be excised and replaced by cleaving the NheI and MluI sites adjacent to the G gene and inserting the desired sequence. In other embodiments, the coronavirus spike (S) protein is expressed as a fusion protein containing the cytoplasmic domain (and optionally the transmembrane region) of the VSV G protein. In certain embodiments, the coronavirus spike (S) protein forms part of the VSV envelope and is therefore surface-displayed on VSV particles.

[0137] In certain embodiments, the foreign DNA is inserted into an intergenic region or into a portion of VSV(-) DNA that is transcribed to form the non-coding region of viral mRNA. In certain embodiments, the foreign DNA is inserted into a coding region of the VSV genome that is not essential for viral development, growth, and / or functions necessary to act as a vaccine. In certain embodiments, the VSV G gene is disrupted. In certain embodiments, the foreign DNA insertion does not disrupt the function of the G gene or G protein.

[0138] Coronavirus spike (S) glycoprotein Coronavirus entry into host cells is mediated by the transmembrane spike (S) glycoprotein (interchangeably referred to as "spike glycoprotein," "S glycoprotein," "S protein," or "spike protein"), which is the primary target of antiviral neutralizing antibodies and the focus of therapeutic and vaccine design. The S glycoprotein forms a homotrimer that protrudes from the surface of the virus. The S glycoprotein contains two functional subunits, which are involved in binding to host cell receptors (the S1 subunit) and in fusing the viral membrane with the cellular membrane (the S2 subunit). For many coronaviruses, including SARS-CoV-1 and SARS-CoV-2, the S glycoprotein is cleaved at the interface between the S1 and S2 subunits, which remain noncovalently bound in a prefusion conformation. The distal S1 subunit contains one or more receptor-binding domains (RBDs) and contributes to stabilizing the prefusion state of the membrane-anchored S2 subunit, which contains the fusion machinery. S is further cleaved by host proteases at the S2' site, located immediately upstream of the fusion peptide. This cleavage is proposed to activate the protein for membrane fusion by changing its conformation. Walls et al., Cell (published online March 9, 2020, available at doi.org / 10.1016 / j.cell.2020.02.058).

[0139] SARS-CoV-1 and SARS-CoV-2 can enter target cells by directly interacting with angiotensin-converting enzyme 2 (ACE2) and can use the cellular serine protease, transmembrane protease, serine 2 (TMPRSS2) to prime the S protein (Hoffmann et al., Cell, 2020, 181:1-10; available at doi.org / 10.1016 / j.cell.2020.02.052). SARS-CoV-S and SARS-CoV-2-S share approximately 76% amino acid identity. The receptor-binding domain (RBD) of the S glycoprotein is the most variable part of the coronavirus genome. Six RBD amino acids have been shown to be important for binding to the ACE2 receptor and for determining the host range of SARS-CoV-like viruses. These are Y442, L472, N479, D480, T487, and Y4911 in SARS-CoV, which correspond to L455, F486, Q493, S494, N501, and Y505 in SARS-CoV-2 (Andersen et al., Nature Medicine, 2020; available at doi.org / 10.1038 / s41591-020-0820-9).

[0140] In certain embodiments of the present disclosure, a VSV particle comprises the SARS-CoV-2 S glycoprotein or a fragment or derivative thereof, wherein the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell. In various embodiments, the S glycoprotein can be the full-length SARS-CoV-2 S glycoprotein (including or consisting of SEQ ID NO:1) or a fragment or derivative thereof having at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage amino acid sequence identity to SEQ ID NO:1.

[0141] Wild-type coronavirus S glycoproteins contain an S1 subunit that promotes binding of the coronavirus to cell surface proteins. Without wishing to be bound by theory, the S1 subunit of wild-type S glycoproteins controls which cells become infected by the coronavirus. Wild-type S glycoproteins also contain an S2 subunit, which is a transmembrane subunit that promotes fusion of the viral membrane with the cellular membrane. In various aspects and embodiments described herein, a fragment or derivative of a SARS-CoV-2 S glycoprotein can include the S1 subunit of said SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof having at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage amino acid sequence identity to said S1 subunit of said SARS-CoV-2 S glycoprotein. In some embodiments described herein, a fragment or derivative of the SARS-CoV-2 S glycoprotein can include a sequence having at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage amino acid sequence identity to amino acids 14-168 of SEQ ID NO:1. In various aspects and embodiments described herein, a fragment or derivative of the SARS-CoV-2 S glycoprotein can include the S2 subunit of said SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage amino acid sequence identity to said S2 subunit of said SARS-CoV-2 S glycoprotein.

[0142] The wild-type coronavirus S glycoprotein contains a receptor-binding domain (RBD) that facilitates binding of the coronavirus to a receptor on the host cell. The RBD of the SARS-CoV-2 spike (S) glycoprotein is described, for example, in Anderson et al., Nature Medicine, 2020 (available at doi.org / 10.1038 / s41591-020-0820-9). In various aspects and embodiments described herein, a fragment or derivative of a SARS-CoV-2 S glycoprotein can comprise the RBD of said SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof having at least 74%, 75%, 76%, 77%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage amino acid sequence identity to said RBD of said SARS-CoV-2 S glycoprotein. In some embodiments described herein, a fragment or derivative of the SARS-CoV-2 S glycoprotein can include a sequence having at least 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even higher percentage amino acid sequence identity to amino acids 319-541 of SEQ ID NO:1.

[0143] In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, may comprise or consist of an insertion, deletion, and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 residues of the SARS-CoV-2 S glycoprotein. Non-limiting examples of amino acids for possible deletion include, for example, tyrosine at position 145, asparagine at position 679, serine at position 680, proline at position 681, arginine at position 682, arginine at position 683, alanine at position 684, and / or arginine at position 685 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2Non-limiting examples of amino acids for substitutions that may occur include, for example, leucine to phenylalanine at position 5, tyrosine to asparagine at position 28, threonine to isoleucine at position 29, histidine to tyrosine at position 49, leucine to phenylalanine at position 54, asparagine to lysine at position 74, glutamic acid to aspartic acid at position 96, aspartic acid to asparagine at position 111, phenylalanine to leucine at position 157, glycine to valine at position 181, serine to tryptophan at position 221, serine to arginine at position 247, alanine to threonine at position 348, arginine to isoleucine at position 408, glycine to serine at position 476, valine to alanine at position 483, and 519. histidine changed to glutamine, alanine changed to serine at position 520, aspartic acid changed to asparagine at position 614, aspartic acid changed to glycine at position 614, asparagine changed to isoleucine at position 679, serine changed to leucine at position 680, arginine changed to glycine at position 682, arginine changed to serine at position 683, arginine changed to glutamine at position 685, arginine changed to serine at position 685, phenylalanine changed to cysteine ​​at position 797, alanine changed to valine at position 930, aspartic acid changed to tyrosine at position 936, alanine changed to valine at position 1078, aspartic acid changed to histidine at position 1168, and / or aspartic acid changed to histidine at position 1259 (positions shown in SEQ ID NO: 1), or a mutant SARS-CoV-2 The equivalent amino acid residues in the S glycoprotein sequence are listed. Becerra-Flores and Cardozo, “SARS-CoV-2 viral spike G614 mutation exhibits highercase fatality rate,” The International Journal of Clinical Practice, published online May 6, 2020; Eaaswarkhanth et al., “Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality?” International Journal of Infectious Diseases, 96: July 2020, Pages 459-460; Tang et al., “The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain,” Preprints 2020, 2020050407 (doi: 10.20944 / preprints202005.0407.v1); Hansen et. al., “Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail” Science, published online June 15, 2020; Lokman et al., “Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: A computational biology approach”, Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 2020 Jun;84:104389. DOI:10.1016 / j.meegid.2020.104389, each of which is incorporated herein by reference in its entirety for all intended purposes. Further non-limiting examples of amino acid residues for insertion, deletion, and / or substitution include those listed in Tables 8 and 9 (the amino acid residue positions are shown using SEQ ID NO: 1 as a reference sequence, which can be used as a reference for identifying the equivalent amino acid residue in any SARS-CoV-2 S glycoprotein sequence (as above); the references in Table 8 are incorporated herein by reference in their entirety for all intended purposes). Each of the residue modifications listed in Table 8 can be used individually, alone or in combination with others, to generate variants of recombinant vesicular stomatitis virus (VSV) particles. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and / or arginine to glutamine at position 685 (positions set forth in SEQ ID NO: 1), or by changing equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing arginine to glutamine at position 685.The S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and aspartic acid to asparagine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing aspartic acid to asparagine at position 614 and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing serine to arginine at position 247, aspartic acid to asparagine at position 614, and arginine to glutamine at position 685. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, confers a more soluble phenotype. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO: 42, or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may be encoded by a codon-optimized nucleotide sequence. In various embodiments, the SARS-CoV-2The S glycoprotein fragment or derivative may be encoded by the polynucleotide sequence of SEQ ID NO:43 or a sequence having at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% polynucleotide sequence identity to SEQ ID NO:43. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative may comprise the amino acid sequence of SEQ ID NO:44 or a sequence at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO:44. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by an asparagine to tyrosine change at position 501, and / or a glutamic acid to lysine change at position 484, and / or an aspartic acid to glycine change at position 614, and / or a deletion of residues 69-70 (positions set forth in SEQ ID NO: 1), or by alteration or deletion of equivalent amino acid residues in the mutant SARS-CoV-2 S glycoprotein sequence. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing the aspartic acid at position 614 to a glycine.The S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing asparagine to tyrosine at position 501 and glutamic acid to lysine at position 484. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing asparagine to tyrosine at position 501 and aspartic acid to glycine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing asparagine to tyrosine at position 501 and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by changing asparagine to tyrosine at position 501 and the deletion of residues 69-70. The SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from the reference peptide or polypeptide by changing glutamic acid to lysine at position 484 and aspartic acid to glycine at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from the reference peptide or polypeptide by changing glutamic acid to lysine at position 484 and by deleting residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from the reference peptide or polypeptide by changing aspartic acid to glycine at position 614 and by deleting residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and an aspartic acid to glycine change at position 614. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and a deletion of residues 69-70.In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide by an asparagine to tyrosine change at position 501, a glutamic acid to lysine change at position 484, an aspartic acid to glycine change at position 614, and the deletion of residues 69-70. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, differs in amino acid sequence from a reference peptide or polypeptide (e.g., a wild-type SARS-CoV-2 spike protein) by inactivating a furin cleavage site within the spike protein. In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, comprises the Q shown in SEQ ID NO:1. 677 TNSPRRARSV 687 (SEQ ID NO: 65), or the equivalent amino acid residue in the mutant SARS-CoV-2 S glycoprotein sequence, to QTILRSV (SEQ ID NO: 66) or QTNSPGSASSV (SEQ ID NO: 67) results in an amino acid sequence that differs from the reference peptide or polypeptide (e.g., wild-type SARS-CoV-2 spike protein). In certain embodiments, the SARS-CoV-2 S glycoprotein derivative, or fragment thereof, results in the phenotypic deletion of the monobasic furin cleavage site (QTILRSV (SEQ ID NO: 66)) or furin cleavage site (QTNSPGSASSV (SEQ ID NO: 67)) at the S1 / S2 boundary. In certain embodiments, modifications to the furin cleavage site can result in spike-stabilized pseudoparticles. See Hansen et al., "Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail," Science (published online June 15, 2020). The above references are incorporated by reference in their entirety for all purposes intended.

[0144] In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative lacks one or more C-terminal residues of the full-length SARS-CoV-2 S glycoprotein. For example, the SARS-CoV-2 S glycoprotein fragment may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 of the C-terminal residues of the SARS-CoV-2 S glycoprotein. In certain embodiments, the SARS-CoV-2 S glycoprotein fragment or derivative lacks the 19 C-terminal residues of the SARS-CoV-2 S glycoprotein. The SARS-CoV-2 S glycoprotein fragment or derivative may consist of the amino acid sequence of SEQ ID NO: 3 or a sequence that is at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher identical to the amino acid sequence of SEQ ID NO: 3.

[0145] In certain embodiments, the SARS-CoV-2 S glycoprotein derivative is a chimeric or fusion molecule comprising a fusogenic sequence from a virus other than SARS-CoV-2. In certain embodiments, such a chimera comprises the S1 or RBD sequence of the SARS-CoV-2 S glycoprotein. In certain embodiments, the fusion protein is a fusion of the extracellular transmembrane sequence of the SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof, with the cytoplasmic domain of a non-SARS-CoV-2 fusogen, or a fragment or derivative thereof. In certain embodiments, the fusion protein is a fusion of the extracellular sequence of the SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof, with the transmembrane and cytoplasmic domains of a non-SARS-CoV-2 fusogen, or a fragment or derivative thereof. Non-limiting examples of non-SARS-CoV-2 fusogens that can be used in the above-described chimeric and fusion molecules include, for example, coronavirus fusogens (e.g., from SARS-CoV-1 or MERS-CoV); VSV or other vesiculoviruses, or other viruses from the Rhabdoviridae family, viruses from the Retroviridae family (e.g., human immunodeficiency virus (HIV), murine leukemia virus (MLV), avian sarcoma leukosis virus (ASLV), Jaagsiekte sheep retrovirus (JSRV)), viruses from the Paramyxoviridae family (e.g., parainfluenza virus 5 (PIV5)), viruses from the Herpesviridae family (e.g., herpes simplex virus (HSV)), viruses from the Togaviridae family (e.g., Semliki Forest virus (SMV)), viruses from the Hepatitis B virus (HBV ... viruses of the Flaviviridae family (e.g., tick-borne encephalitis virus (TBE) and dengue virus), viruses of the Orthomyxoviridae family (e.g., influenza virus), viruses of the Arenaviridae family (e.g., lymphocytic choriomeningitis virus (LCMV) and rubella virus), ...Examples of fusogens include those derived from viruses of the Bunyaviridae family (e.g., Uukuniemi virus (UUKV)), Filoviridae family (e.g., Ebola virus (EBOV)), Poxviridae family (e.g., Vaccinia virus (VV)), Asfarviridae family (e.g., African swine fever virus (ASFV)), Arteriviridae family (e.g., porcine reproductive and respiratory syndrome virus (PRRSV)), Bornaviridae family (e.g., Borna disease virus (BDV)), Hepadnaviridae family (e.g., Hepatitis B virus (HBV)), and Hantaviridae family (e.g., Andes virus).

[0146] In certain embodiments, the fusion protein is a fusion of the SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof, with the VSV glycoprotein G protein, or a fragment or derivative thereof. In certain embodiments, the fusion protein is a fusion of the SARS-CoV-2 S glycoprotein, or a fragment or derivative thereof, with the cytoplasmic portion of the VSV G glycoprotein, or a fragment or derivative thereof. In a specific embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:5.

[0147] In certain embodiments of the assays of the present disclosure, the SARS-CoV-2 S glycoprotein or fragment or derivative thereof can comprise a consensus sequence derived from two or more different strains, mutants, or variants of SARS-CoV-2 to ensure detection of a broader range of SARS-CoV-2 neutralizing antibodies. In other embodiments, to ensure detection of a broader range of SARS-CoV-2 neutralizing antibodies, the methods of the present disclosure use a mixture of SARS-CoV-2 S glycoproteins (or fragments or derivatives thereof) derived from two or more different strains, mutants, or variants of SARS-CoV-2.

[0148] A polynucleotide molecule encoding the SARS-CoV-2 S glycoprotein or a fragment or derivative thereof can include a consensus sequence and / or one or more modifications to improve expression of the SARS-CoV-2 S glycoprotein or a fragment or derivative thereof. Modifications can include codon optimization, the addition of a Kozak sequence or a modified (e.g., optimized) Kozak sequence to increase translation initiation, and / or the addition of a signal peptide / leader sequence (e.g., an immunoglobulin signal peptide, such as an IgE or IgG signal peptide). In certain embodiments, the Kozak sequence or modified (e.g., optimized) Kozak sequence is 3' to the foreign gene. In certain embodiments, the Kozak sequence or modified (e.g., optimized) Kozak sequence is immediately 3' to the foreign gene. In certain embodiments, the Kozak sequence or modified (e.g., optimized) Kozak sequence is immediately 5' to the foreign gene. In certain embodiments, the Kozak sequence or modified (e.g., optimized) Kozak sequence is immediately 5' to the foreign gene.

[0149] In some embodiments, the SARS-CoV-2 S glycoprotein or a fragment or derivative thereof comprises a fusion or conjugate with a detection tag (e.g., an HA tag, a histidine tag, biotin), a reporter protein or a fragment thereof, a dimerization / multimerization sequence, Fc, a signaling sequence, etc. In some embodiments, the recombinant VSV particles of the present disclosure comprise, in addition to the SARS-CoV-2 S glycoprotein or a fragment or derivative thereof, a reporter protein or a fragment thereof, wherein the reporter protein or a fragment thereof is encoded by or included as a protein in the VSV particle genome. Non-limiting examples of reporter proteins include luciferases (including, but not limited to, Renilla luciferase, RLuc8 mutant Renilla luciferase, (dCpG) luciferase, NanoLuc reporter, firefly luciferase, Gaussia luciferase (gLuc), MetLuc, Vibrio fischeri lumazine protein, Vibrio harveyi luminase protein, dinoflagellate luciferase, firefly luciferase YY5 mutant, firefly luciferase LGR mutant, firefly luciferase mutant E, and derivatives thereof), and fluorescent proteins (including, but not limited to, green fluorescent protein (GFP) [e.g., Aequoria victoria GFP, Renilla muelleri GFP, Renilla reniformis GFP, Renilla ptilosarcus GFP, GFP], GFP-like fluorescent protein, (GFP-like), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP) [e.g., Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana], enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP) [e.g., EBFP2, Azurite, GFP2, GFP10, and mTagBFP], enhanced blue fluorescent protein (EBFP), cyan-green fluorescent protein (CFP) [e.g., mECFP, Cerulean, CyPet, AmCyan1, Midori-IshiCyan, TagCFP, mCFPmm, mTFP1(Teal)], enhanced cyan-green fluorescent protein (ECFP), superfolder GFP, superfolder YFP, orange fluorescent protein [e.g., Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine], red fluorescent proteins [e.g., mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, tdTomato, AQ143], small ultra-red fluorescent proteins, FMN-binding fluorescent proteins, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGr1, bright green fluorescent protein, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, and fragments or derivatives thereof), β-galactosidase, β-glucuronidase, β-geo, and fragments thereof.

[0150] Generation of recombinant VSV particles The following disclosure uses recombinant VSV particles as an example, but the disclosure can also be used with other vesiculoviruses.

[0151] The recombinant VSV particles of the present disclosure are produced by providing a suitable host cell: a recombinant source of VSV(-) DNA, in which regions nonessential for replication have been inserted into or replaced by foreign DNA comprising a sequence encoding the coronavirus S glycoprotein or a fragment or derivative thereof, and optionally other sequences as described above, and the VSV N protein, P protein, L protein, and any additional desired VSV proteins (e.g., M protein and / or G glycoprotein). In certain embodiments, such production is preferably in vitro (e.g., in cell culture).

[0152] Host cells used to generate recombinant VSV can be any cell in which VSV grows. Non-limiting sources of host cells include prokaryotic or eukaryotic cells, vertebrate cells, mammalian cells, and some insect (e.g., Drosophila) cells, primary cells (e.g., primary chicken embryo fibroblasts), or cell lines (e.g., BHK (baby hamster kidney) cells, CHO (Chinese hamster ovary) cells, HeLA (human) cells, mouse L cells, Vero (monkey) cells (including Vero-αHis cells), Vero-Ace-2 cells, Vero-TRMPSS2 cells, Vero-E6 cells, ESK-4, PK-15, EMSK cells, MDCK (Madin-Darby) cells, and the like). canine kidney) cells, MDBK (Madin-Darby bovine kidney) cells, 293 (human) cells, Hep-2 cells, human diploid primary cell lines (e.g., WI-38 and MRC5 cells), monkey diploid cell lines (e.g., FRhL: fetal rhesus lung cells), and semi-primary continuous cell lines (e.g., AGMK: African green monkey kidney cells).

[0153] The source of the N, P, and L proteins, and any additional desired VSV proteins (e.g., M protein and / or G glycoprotein), can be the same or different recombinant nucleic acids that encode and are capable of expressing these proteins in a host cell in which recombinant VSV production is desired. Nucleic acids encoding the N, P, and L proteins, and any additional desired VSV proteins (e.g., M protein and / or G glycoprotein), can be obtained by any means available in the art. VSV N, P, L, M, and G-encoding nucleic acid sequences have been previously disclosed and are available for use. See, e.g., Genbank Accession No. J02428; Rose and Schubert, 1987, in *The Viruses: The Rhabdoviruses*, *Plenum Press, NY*, pp. 129-166. Sequences encoding the N, P, and L genes can also be obtained, for example, by PCR amplification of the desired genes from the plasmid pVSVFL(+), which has been deposited with the ATCC and assigned accession number 97134 (see also U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. USA 85:7652-7656; Ochman et al., 1988, Genetics 120:621-623; Loh et al., 1989, Science 243:217-220). If a nucleic acid clone of any of the N, P, L, M, or G genes is not already available, the clone can be obtained using standard recombinant DNA techniques. For example, the DNA can be obtained by standard procedures known in the art, such as by purification of RNA from VSV virions, followed by reverse transcription and PCR (Mullis and Faloona, 1987, Meth. Enzymol. 155:335-350). Alternatives include, but are not limited to, chemical synthesis of the gene sequence itself. Other methods are possible and within the scope of this disclosure.

[0154] Nucleic acids encoding fragments and derivatives of the VSV N, P, L, M, and / or G genes, as well as fragments and derivatives of VSV(-) DNA, can also be used in the present disclosure, so long as such fragments and derivatives retain the required functionality (e.g., the ability to generate replicating or non-replicating VSV particles that can be used in one or more of the methods of the present disclosure). In particular, derivatives can be created by altering the sequence through substitution, addition, or deletion. Furthermore, due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences may be used in practicing the methods of the present disclosure. Amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved.

[0155] A vector capable of directing the synthesis of VSV proteins can be generated by inserting the desired N / P / L / M / G-encoding nucleic acid into an appropriate expression vector, i.e., a vector containing the elements necessary for the transcription and translation of the inserted protein-encoding sequence in a host cell in which production of recombinant VSV particles is desired, which vector is subsequently assembled with VSV genomic RNA (e.g., produced in the host cell from antigenomic VSV(+)RNA generated, for example, by transcription of VSV(-)DNA).

[0156] A variety of vector systems can be utilized to express the N, P, and LVSV proteins, as well as any additional desired VSV proteins (e.g., M and / or G), and to transcribe VSV(-) DNA (e.g., containing foreign DNA), so long as the vector functions in the host cell and is compatible with any other vectors present. Vector expression elements vary in their strengths and specificities. Any of a number of suitable transcription and translation elements may be used, so long as they function in the host cell.

[0157] Standard recombinant DNA techniques can be used to construct expression vectors containing DNA encoding VSV proteins and VSV(-) DNA containing foreign DNA with appropriate transcriptional / translational control signals (see, e.g., Sambrook et al., 1989, supra, and the methods described above). Expression can be controlled by any promoter / enhancer known in the art. Promoters that can be used to control expression can be constitutive or inducible. In a specific embodiment, the promoter is an RNA polymerase promoter.

[0158] Transcription termination signals (downstream of the gene) and selectable markers are also preferably included in the expression vector. In addition to promoter sequences, expression vectors for N, P, L, and any additional desired VSV proteins, as well as any coronavirus protein, may contain specific initiation signals for efficient translation of inserted sequences, such as ribosome binding sites.

[0159] Specific initiation signals may be required for efficient translation of protein-coding sequences. These signals include the ATG initiation codon and adjacent sequences. If the entire N, P, L, or other (e.g., M and / or G) VSV gene, including its own initiation codon and adjacent sequences, is inserted into an appropriate vector, additional translational control signals may not be necessary. However, if only a portion of the gene sequence is inserted, exogenous control signals, including the ATG initiation codon, must be provided. The initiation codon must also be in phase with the reading frame of the protein-coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.

[0160] In a specific embodiment, the recombinant expression vectors provided herein encoding the N, P, L, and / or other (e.g., M and / or G) proteins, or functional derivatives thereof, comprise the following operably linked components: a promoter controlling expression of a protein (e.g., N, P, L, and / or other VSV proteins (e.g., M and / or G), a coronavirus protein (e.g., a spike glycoprotein such as the SARS-CoV-2 spike glycoprotein), or a fragment or derivative thereof; a translation initiation signal; a DNA sequence encoding the VSV protein or a functional fragment or derivative thereof; and a transcription termination signal. In embodiments, the above components are located 5' to 3' apart as listed above. In certain embodiments, the genes encoding the M protein, G protein, and / or coronavirus S glycoprotein, or fragments or derivatives thereof, are interspersed among the N, P, and / or L proteins. In certain embodiments, the genes for the M protein, G protein, and / or coronavirus S glycoprotein, or fragments or derivatives thereof, are present between the gene for the P protein and the gene for the L protein (see Figure 1). In certain embodiments, the N, P, and L proteins, or functional fragments or derivatives thereof, are not present in the 5' to 3' order listed above. In certain embodiments, the order is altered (e.g., to attenuate the recombinant VSV).

[0161] In certain embodiments, genes encoding N, P, L, and other (e.g., M and / or G) VSV proteins are inserted downstream of the T7 RNA polymerase promoter from phage T7 gene 10, located at position -3 with A. A T7 RNA polymerase terminator and replicon can also be included in the expression vector. T7 RNA polymerase can be provided to transcribe the VSV protein sequence. T7 RNA polymerase can be produced from a chromosomally integrated sequence or an episomal vector. In certain embodiments, T7 RNA polymerase can be provided by intracellular expression from a recombinant vaccinia virus vector encoding T7 RNA polymerase. In certain embodiments, the N, P, L, and / or other (e.g., M and / or G) VSV proteins are each encoded by a DNA sequence operably linked to a promoter within an expression plasmid, which contains the necessary regulatory signals for transcription and translation of the encoded protein. Such expression plasmids preferably contain a promoter, coding sequence, and transcription termination / polyadenylation signal, and optionally a selectable marker (e.g., β-galactosidase).

[0162] In certain embodiments, the N, P, L, and / or other (e.g., M and / or G) proteins can be encoded by the same plasmid, different plasmids, or a combination thereof. In other embodiments, one or more of the N, P, L, and / or other (e.g., M and / or G) VSV proteins can be expressed intrachromosomally.

[0163] Cloned sequences containing VSV(-) DNA containing foreign DNA, as well as cloned sequences containing sequences encoding VSV and foreign proteins, can be introduced into desired host cells by any method known in the art, such as transfection, electroporation, infection (if the sequences are contained in a viral vector), microinjection, etc. In certain embodiments, DNA uptake by cells is enhanced by the addition of transfection-enhancing reagents. Many of these reagents are known in the art (e.g., non-limiting examples are calcium phosphate; LipofectACE (Life Technologies, Gaithersburg, MD); and Effectene (Qiagen, Valencia, CA)).

[0164] In certain embodiments, DNA comprising VSV(-) DNA containing foreign DNA encoding coronavirus S glycoprotein or a fragment or derivative thereof operably linked to an RNA polymerase promoter (e.g., a bacteriophage RNA polymerase promoter); DNA encoding N operably linked to the same RNA polymerase promoter; DNA encoding P operably linked to the same polymerase promoter; and DNA encoding L operably linked to the same polymerase promoter are all introduced (e.g., by transfection) into the same host cell, in which RNA polymerase is provided in the cytoplasm. In certain embodiments, RNA polymerase is provided in the cytoplasm by expression from a recombinant viral vector, most preferably a vaccinia virus vector, introduced (e.g., by infection) into the same host cell, which replicates in the cytoplasm and expresses RNA polymerase. Providing RNA polymerase in the cytoplasm can be used to: enable cytoplasmic transcription and processing of VSV(-) DNA, including foreign DNA, and of N, P, L, and other (e.g., M and / or G) VSV proteins; bypass the splicing machinery in the cell nucleus; thereby maximizing proper processing and production of N, P, L, and other (e.g., M and / or G) VSV proteins; and result in assembly of recombinant VSV. Vaccinia virus vectors also provide enzymes for mRNA processing (capping and polyadenylation) in the cytoplasm to promote proper translation. In a most preferred embodiment, a T7 RNA polymerase promoter is employed, and a cytoplasmic source of T7 RNA polymerase is provided by also introducing a recombinant vaccinia virus vector encoding T7 RNA polymerase into the host cell. Such vaccinia virus vectors can be obtained by known methods. In certain embodiments, a recombinant vaccinia virus vector such as vTF7-3 (Fuerst et al., 1986, Proc. Natl. Acad. Sci. USA 83:8122-8126) can be used.

[0165] In other embodiments, an RNA polymerase (e.g., T7 RNA polymerase) can be provided by the use of a host cell that expresses T7 RNA polymerase, optionally constitutively, from a sequence integrated into the chromosome (e.g., originally inserted into the chromosome by homologous recombination) or that expresses T7 RNA polymerase episomally from a plasmid.

[0166] In other embodiments, VSV(-) DNA encoding the coronavirus S glycoprotein or a fragment or derivative thereof operably linked to a promoter can be transfected into host cells that stably recombinantly express the N, P, L, and any other (e.g., M and / or G) VSV proteins from chromosomally integrated sequences.

[0167] The cells are cultured and recombinant VSV particles can be harvested, for example, using standard methods. For example, but not limited to, after approximately 24 hours, the cells and medium can be harvested, frozen, and thawed. The lysate can be clarified to obtain a virus preparation. Alternatively, the cells and medium can be harvested and the cells and debris can be simply removed by low-speed elution.

[0168] Verification can be performed that the appropriate foreign sequences are present in the genome of the recombinant VSV and direct the production of one or more desired proteins in infected cells. Standard procedures known in the art can be used for this purpose. For example, but not limited to, genomic RNA can be obtained from VSV by SDS-phenol extraction from virus preparations, which can be subjected to reverse transcription (and / or PCR), followed by subsequent procedures such as sequencing, Southern hybridization using probes specific for the foreign DNA, or restriction enzyme mapping. The virus can be used to infect host cells, after which expression of the desired protein can be assayed by standard immunoassay techniques (e.g., Western blotting) using antibodies against the protein or by assays based on the functional activity of the protein. Other techniques are known in the art and can be used.

[0169] Large-scale growth and purification of recombinant viruses The following disclosure uses VSV as an example, but the disclosure can be used for other vesiculoviruses.

[0170] A non-limiting example of large-scale production of recombinant VSV virus after plaque purification is shown below. 5 pfu), and used to recover the virus. 7 By infecting cells (e.g., BHK cells), typically 10 9 ~10 10 10 ml with a titer of pfu / ml, totaling approximately 10 11 pfu is obtained. Then (for example, at a multiplicity of infection of 0.1) about 10 12 Infection of the cells can be carried out and the cells grown in suspension culture, large dishes, or roller bottles by standard methods known in the art.

[0171] Virus for vaccine preparation can then be recovered from the cell supernatant, which has been clarified to remove cellular debris. If necessary, one method that can be used to isolate and concentrate the virus is by concentrating the supernatant through a tangential flow membrane. The volume of the product can be further reduced by pelleting through a glycerol cushion and concentrating on a sucrose step gradient. Another concentration method is affinity column purification (Daniel et al., 1988, Int. J. Cancer 41:601-608). However, other methods for purification can also be used (see, e.g., Arthur et al., 1986, J. Cell. Biochem. Suppl. 10A:226), and any possible modifications of the above procedures will be readily apparent to those skilled in the art. To maintain the integrity of the virus particles, purification should be as gentle as possible.

[0172] Assay In one aspect, the present disclosure provides a method for determining the presence of coronavirus-neutralizing antibodies in a sample. In one embodiment, the sample is contacted with or incubated with recombinant vesicular stomatitis virus (VSV) particles, in which the VSV glycoprotein (G) is replaced by the coronavirus spike (S) glycoprotein or a fragment or derivative thereof, and the S glycoprotein, fragment, or derivative is capable of mediating infection of susceptible target cells. Simultaneously or subsequently, the recombinant VSV particles are contacted with a first target cell expressing a first portion of a reporter protein and a second target cell expressing a second portion of the reporter protein to form a syncytium, which contains both the first and second portions of the reporter protein and produces a detectable reporter signal. The first and second target cells must be capable of fusing with each other upon contact with the recombinant VSV particles. The reporter signal is measured in the syncytium and compared to a control.

[0173] In a specific embodiment, the first cell is Vero-DSP1 (Vero-DSP-1-Puro; CLR-73) and the second cell is Vero-DSP2 (Vero-DSP-2-Puro; CLR-74). Vero-DSP1 and Vero-DSP2 are generated by lentiviral transduction of Vero cells. Vero-DSP1 cells express the Rluc8 DSP 1-7 luciferase-GFP fusion protein (SEQ ID NO: 14), which contains amino acids 1-155 of a fragment of mutant Renilla luciferase RLuc8 and amino acids 1-156 of a fragment of modified GFP. Vero-DSP2 cells express the Rluc8 DSP 8-11 luciferase-GFP fusion protein (SEQ ID NO: 16), which contains amino acids 156-311 of a fragment of mutant Renilla luciferase RLuc8 and amino acids 157-231 of a fragment of modified GFP. The RLuc8 mutant Renilla luciferase contains the mutations A55T, C124A, S130A, K136R, A143M, M185V, M253L, and S287L (see SEQ ID NO: 19). The sequence of the modified GFP is provided in SEQ ID NO: 18.

[0174] The first portion of the reporter protein used in any of the aforementioned methods of the present disclosure may comprise amino acids 1-229 of Renilla luciferase or a variant thereof, and the second portion of the reporter protein may comprise amino acids 230-311 of Renilla luciferase or a variant thereof. Alternatively, the first portion of the reporter protein may comprise amino acids 1-155 of Renilla luciferase or a variant thereof, and the second portion of the reporter protein may comprise amino acids 156-311 of Renilla luciferase or a variant thereof. Alternatively, or in addition, the first portion of the reporter protein may comprise amino acids 1-156 of green fluorescent protein (GFP) or a variant thereof, and the second portion of the reporter protein may comprise amino acids 157-231 of GFP or a variant thereof. Alternatively, the first portion of the reporter protein may comprise amino acids 1-213 of superfolder GFP and the second portion of the reporter protein may comprise amino acids 214-230 of superfolder GFP. Alternatively, the first portion of the reporter protein may comprise amino acids 1-154 of superfolder yellow fluorescent protein (YFP) and the second portion of the reporter protein may comprise amino acids 155-262 of superfolder YFP.

[0175] In another aspect, the present disclosure provides a method for determining the presence of coronavirus-neutralizing antibodies in a sample, comprising contacting or incubating the sample with recombinant vesicular stomatitis virus (VSV) particles, in which the VSV glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein or a fragment or derivative thereof, and the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell, and the VSV particles comprise a reporter protein or a nucleic acid molecule encoding the reporter protein. The recombinant VSV particles are then contacted with the target cell. A reporter signal is measured and compared with a control. In certain embodiments, the reporter protein is encoded by the genome of the recombinant VSV particle. In certain embodiments, the reporter protein is incorporated into the recombinant VSV particle without being encoded by the genome of the virus particle. The nucleic acid sequence encoding the reporter protein may be inserted between the nucleic acid sequence encoding the S glycoprotein and the nucleic acid sequence encoding the VSV L protein. The target cells may be Vero cells (including Vero-αHis cells), Vero-Ace-2 cells, Vero-TRMPSS2 cells, Vero-E6 cells, or any other cells that contain angiotensin-converting enzyme 2 (ACE2).

[0176] In the above-described methods of the present disclosure, the recombinant VSV particles can encode the coronavirus S glycoprotein in the VSV viral genome, or the VSV particles can be pseudotyped (e.g., by using a separate plasmid in the packaging cell) without the coronavirus S glycoprotein being encoded within the genome.

[0177] The sample used in the above-described methods of the present disclosure can be, for example, serum or plasma (e.g., heat-inactivated serum or plasma).

[0178] The first step of contacting the sample with the recombinant VSV particles can be carried out for 1 hour at about 37° C. The second step of contacting the recombinant VSV particles with the target cells can be carried out for 1 to 12, 1 to 3, 2 to 4, 3 to 5, 4 to 6, 5 to 7, 6 to 8, 7 to 9, or 8 to 10 hours at about 37° C.

[0179] In various embodiments of the above-described methods of the present disclosure, the method includes adding a reporter protein substrate to obtain the reporter signal. The reporter protein may be luciferase, and the reporter protein substrate may be luciferin (e.g., d-luciferin), coelenterazine, or an EnduRen luciferase substrate.

[0180] In some embodiments, the coronavirus S protein, fragment, or derivative thereof is derived from SARS-CoV-2. In specific embodiments, the coronavirus S protein is a full-length SARS-CoV-2 S protein (e.g., a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 1). In specific embodiments, the coronavirus S protein is a SARS-CoV-2 S protein lacking the 19 C-terminal amino acids (e.g., a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3). In specific embodiments, the coronavirus S protein, fragment, or derivative has at least 77% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In specific embodiments, the coronavirus S protein, fragment, or derivative has at least 64% amino acid sequence identity to the S1 subunit of the amino acid sequence of SEQ ID NO: 1. In specific embodiments, the coronavirus S protein, fragment, or derivative has at least 64% amino acid sequence identity to amino acids 14 to 684 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the coronavirus S protein, fragment, or derivative has at least 74% amino acid sequence identity to the RBD domain of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the coronavirus S protein, fragment, or derivative has at least 74% amino acid sequence identity to amino acids 319-541 of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the recombinant VSV particle comprises a mutant VSV matrix (M) protein. In certain embodiments, the genome of the recombinant VSV encodes a mutant VSV matrix (M) protein. In certain embodiments, the mutant matrix (M) protein comprises a mutation at methionine (M) 51 (e.g., a change from methionine (M) to arginine (R)). In certain embodiments, the mutant VSV matrix (M) protein comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0181] In certain embodiments of the above-described methods of the present disclosure, the control is a reporter signal obtained using a control sample that does not contain coronavirus-neutralizing antibodies, and the method includes concluding that the tested sample contains coronavirus-neutralizing antibodies if the reporter signal obtained with the sample is reduced relative to the control. In certain embodiments, the control sample contains VSV particles that do not contain the coronavirus S glycoprotein or VSV particles that contain a mutant S glycoprotein that does not mediate infection of target cells. In certain embodiments of the above-described methods of the present disclosure, the method further includes comparing the reporter signal obtained with the sample to a reporter signal obtained with a control sample that contains coronavirus-neutralizing antibodies or a molecule that blocks the interaction between the coronavirus S glycoprotein and a protein with which the coronavirus S glycoprotein interacts on the target cell.

[0182] In certain embodiments of the above-described methods of the present disclosure, the methods are performed in a high-throughput format.

[0183] In a specific embodiment, a method is provided for determining the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in a sample, the method comprising: a) contacting the sample with recombinant vesicular stomatitis virus (VSV) particles, in which the VSV glycoprotein (G) has been replaced by the full-length SARS-CoV-2 spike (S) glycoprotein lacking the 19 C-terminal amino acids or a fragment thereof; b) after step (a), contacting the recombinant VSV particles with a mixture of Vero-DSP1 (Vero-DSP-1-Puro; CLR-73) cells and Vero-DSP2 (Vero-DSP-2-Puro; CLR-74) cells; c) measuring the luciferase signal and / or GFP signal of the cells after step (b); and d) comparing the signal measured in step (c) with a control. Includes.

[0184] In another specific embodiment, a method is provided for determining the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies in a sample, the method comprising: a) contacting the sample with recombinant vesicular stomatitis virus (VSV) particles, wherein (i) the VSV glycoprotein (G) gene has been replaced by a full-length SARS-CoV-2 spike (S) glycoprotein lacking 19 C-terminal amino acids or a fragment thereof, and (ii) the VSV particles further comprise a luciferase protein and / or a nucleic acid molecule encoding the luciferase protein; b) after step (a), contacting the recombinant VSV particles with cells selected from Vero cells, Vero-Ace-2 cells, and Vero-E6 cells; c) measuring the luciferase signal of the cells after step (b); and d) comparing the signal measured in step (c) with a control. Includes.

[0185] Cells used in any of the methods disclosed herein may comprise angiotensin-converting enzyme 2 (ACE2). ACE2 is a functional receptor for coronaviruses, particularly SARS-CoV-1 and SARS-CoV-2. Cells used in any of the methods disclosed herein may further comprise a type II transmembrane serine protease (TMPRSS2). Non-limiting examples of susceptible target cells that can be used in the methods of the present disclosure include, for example, Vero green monkey kidney epithelial cells (including Vero-αHis cells), Vero-Ace-2 cells, Vero-TRMPSS2 cells, Vero-E6 cells, avian erythrocytes, and Madin-Darby canine kidney epithelial cells.

[0186] In some embodiments, the addition of trypsin to the virus-cell mixture can be used to enhance virus-mediated cell fusion. Trypsin may be added before measuring the reporter signal in the cells. Trypsin may be added after incubating the virus-cell mixture, for example, 2-6 hours after the start of incubation, 3-5 hours after the start of incubation, about 4 hours after the start of incubation, or 4 hours after the start of incubation. Trypsin does not need to be removed from the virus-cell mixture after addition. As shown in the data below, the addition of trypsin can promote cell fusion. The concentration of trypsin used can be 0.1-4.0 μL per mL of medium (e.g., OptiMEM), or 0.1-0.6 μL per mL of medium, 0.2-0.7 μL per mL of medium, 0.3-1.2 μL per mL of medium, 0.5-2.0 μL per mL of medium, 1.0-2.5 μL per mL of medium, 1.5-3.0 μL per mL of medium, or 2.0-4.0 μL per mL of medium. Adding trypsin to the medium during the fusion assay can increase the sensitivity of the assay. Additional trypsin allows cell fusion to occur more quickly. Additional trypsin allows for more rapid assay readout (e.g., luciferase activity) than with less or no trypsin.

[0187] In some embodiments, trypsin is not used in the assay. If trypsin is omitted, additional washing steps may not be necessary, even if the serum / virus or plasma / virus mixture is removed and replaced with fresh serum medium or medium suitable for plasma.

[0188] Reporter proteins A variety of split reporter proteins can be used in any of the aspects and embodiments described herein, for example, an enzyme that catalyzes the conversion of a substrate into a detectable product. Several such systems for reassembly of split polypeptides include, but are not limited to, β-galactosidase (Rossi et al., PNAS, 1997, 94:8405-8410), dihydrofolate reductase (DHFR) (Pelletier et al., PNAS, 1998, 95:12141-12146), TEM-1 β-lactamase (LAC) (Galarneau at al., Nat. Biotech. 2002; 20: 619-622), and firefly luciferase (Ray et al., PNAS, 2002, 99: 3105-3110 and Paulmurugan et al., 2002; PNAS, 99: 15608-15613). For example, split β-lactamase has been used to detect double-stranded DNA (see Ooi et al., Biochemistry, 2006, 45:3620-3525). Split polypeptide fragments can be used for real-time signal detection, where the fragments are fully folded into their mature conformation, allowing for rapid signal detection upon completion.

[0189] The split polypeptide fragments can be any polypeptides that, when brought into close proximity with one another, associate to produce a protein that can be detected by any means that recognizes the assembled polypeptide fragments but not the individual polypeptide fragments. In one embodiment of the present disclosure, the method involves designing the split polypeptide fragments to be active upon reconstitution.

[0190] The split polypeptide fragments can be any polypeptides that, when brought into close proximity, associate to produce an active protein that can be detected by any means that recognizes the assembled active protein but not the individual fragments. For example, two polypeptides can reassociate to produce a protein with enzymatic activity, to produce a protein with luciferase activity, chromogenic activity, or fluorogenic activity, or to create a protein that is recognized by an antibody. Furthermore, they are designed to be active and primed (i.e., ready) for reconstitution of an active protein, thereby minimizing the lag time previously observed with protein complementation in vitro and in vivo.

[0191] The split polypeptide fragments can be fluorescent proteins or polypeptides. In such embodiments, one of the activated split fluorescent protein fragments contains a preformed mature chromophore that is primed and ready to fluoresce immediately upon complementation with one or more cognate activated split fluorescent fragments. For example, using inclusion bodies containing such split fluorescent fragments involves having approximately half of the fully folded fluorescent protein accompanied by a correctly folded mature chromophore that is not fluorescent by itself but is primed to fluoresce upon association with its cognate pair.

[0192] In the methods described herein, the reporter protein may be luciferase. Various luciferase enzymes known to those skilled in the art may be used, and exemplary luciferases include, but are not limited to, Renilla luciferase, RLuc8 mutant Renilla luciferase, (dCpG) luciferase, NanoLuc reporter, firefly luciferase, Gaussia luciferase (gLuc), MetLuc, Vibrio fischeri lumazine protein, Vibrio harveyi luminase protein, dinoflagellate luciferase, firefly luciferase YY5 mutant, firefly luciferase LGR mutant, firefly luciferase mutant E, and derivatives thereof.

[0193] In the methods described herein, in which recombinant VSV particles contact a first cell expressing a first portion of a reporter protein and a second cell expressing a second portion of the reporter protein to form a syncytium, the reporter protein can be luciferase. The first and second portions of the luciferase can be split polypeptide luciferase fragments. When both split polypeptide luciferase fragments are introduced into an assay system, the first and second proteins bind to each other, resulting in reconstitution of the luciferase and restoration of luciferase activity, allowing the luciferase to emit light under appropriate light-emitting conditions. When the assay system is a cell, the luciferase activity can be measured by adding luciferin to the cell culture, preparing a cell extract, and measuring the luciferase activity. In this case, the activity can be easily measured using a commercially available Emerald Luc luciferase assay reagent / cell lysate (Toyobo Co., Ltd.), or the like.

[0194] Fusion proteins containing luciferase fragments can be detected. For example, if a first fusion protein, created by fusing a target protein to be detected with a luciferase fragment, is present in an assay system, a second fusion protein, created by fusing a binding protein that binds to the target protein with the luciferase fragment, is prepared as a probe and introduced into the assay system. The binding protein in the second fusion protein then binds to the target protein in the first fusion protein, thereby interacting with the luciferase fragment and acquiring luciferase activity. By detecting this luciferase activity, the target fusion protein containing the luciferase fragment can be detected. Specifically, an expression vector expressing the first fusion protein can be prepared and introduced into cells. Next, an expression vector expressing the second fusion protein can be prepared and introduced into the cells expressing the first fusion protein. Then, by measuring luciferase activity as described above, the fusion protein containing the luciferase fragment can be detected.

[0195] Split luciferase assays are further described, for example, in Saw, WT et al., "Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins," Methods, 2015, 90:68-75, which is incorporated herein by reference in its entirety. The methods described herein can be performed when both luciferase and a fluorescent protein are fused together as reporter proteins.

[0196] The split polypeptide fragments can comprise fragments of an active enzyme that can be detected using an enzyme activity assay. In such embodiments, the enzyme activity is detected by a colorimetric or fluorogenic reaction. In one embodiment, the enzyme is dihydrofolate reductase or β-lactamase.

[0197] The enzyme can be dihydrofolate reductase (DHFR). For example, Michnick et al. have developed a "protein complementation assay" consisting of N- and C-terminal fragments of DHFR, which lack either enzymatic activity alone but form a functional enzyme when brought into close proximity. See, e.g., U.S. Patent Nos. 6,428,951, 6,294,330, and 6,270,964, which are incorporated herein by reference in their entireties. Methods for detecting DHFR activity, including chromogenic and fluorogenic methods, are known in the art.

[0198] A variety of fluorescent proteins may be used in any of the methods and compositions described herein. In the methods disclosed herein, the reporter protein may comprise a fluorescent protein.

[0199] The fluorescent protein may be green fluorescent protein (GFP), GFP-like fluorescent protein (GFP-like), or enhanced green fluorescent protein (EGFP). In alternative embodiments, the fluorescent protein is yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), red fluorescent protein (dsRED), superfolder GFP, superfolder YFP, orange fluorescent protein, red fluorescent protein, small very red fluorescent protein, FMN-binding fluorescent protein, dsRed, qFP611, Dronpa, TagRFP, KFP, EosFP, IrisFP, Dendra, Kaede, KikGr1, and derivatives thereof, or any other naturally occurring or genetically engineered version of the above fluorescent proteins. In yet further embodiments, the reconstituted fluorescent protein may be composed of a mixture of fragments from any of the above fluorescent proteins or combinations thereof.

[0200] Green fluorescent protein (GFP) is a 238 amino acid protein derived from the jellyfish Aequorea Victoria. The modified GFP used in the split constructs of the present disclosure is 231 amino acids in length and consists of SEQ ID NO: 18. Fluorescent proteins have also been isolated from other members of the coelenterate family, such as the red fluorescent protein from Discosoma species (Matz, MV et al. 1999, Nature Biotechnology 17: 969-973), the GFP from Renilla reniformis, the GFP from Renilla Muelleri, or fluorescent proteins from other animals, fungi, or plants. Various modified forms of GFP exist. A blue fluorescent variant of GFP (BFP) was described by Heim et al., Proc. Natl. Acad. Sci. 91:26, 1994, pp 12501-12504, and is a Y66H variant of wild-type GFP. Yellow fluorescent variants of GFP (YFP) contain S65G, S72A, and T203Y mutations, as disclosed in International Patent Publication No. WO 98 / 06737. Blue-green fluorescent variants of GFP (CFP) contain the Y66W color mutation and, optionally, the F64L, S65T, N1461, M153T, and V163A folding / solubility mutations. CFP is described in Heim, R., Tsien, R.Y., Curr. Biol. 6, 1996, 178-182. EGFP variants contain the F64L and S65T mutations of GFP and the insertion of one valine residue after the first methionine. EGFP is described in International Patent Publication Nos. WO 97 / 11094 and WO 96 / 23810. The F64L mutation is the amino acid immediately upstream of the chromophore. GFP or GFP variants containing this folding mutation provide increased fluorescence intensity when the GFP or GFP variants are expressed in cells at temperatures above about 30°C (WO 97 / 11094).

[0201] Non-limiting examples of GFPs include: Aequoria victoria GFP, Renilla muelleri GFP, Renilla reniformis GFP, Renilla ptilosarcus GFP, Emerald, superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire.

[0202] Non-limiting examples of BFPs include: EBFP2, Azurite, GFP2, GFP10, and mTagBFP.

[0203] Non-limiting examples of CFPs include: mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mCFPmm, and mTFP1 (Teal).

[0204] Non-limiting examples of YFPs include: Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, and mBanana.

[0205] Non-limiting examples of orange fluorescent proteins include: Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, and mTangerine.

[0206] Non-limiting examples of red fluorescent proteins include: mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, tdTomato, and AQ143.

[0207] Also included are fragments and derivatives of the above-mentioned reporter proteins.

[0208] The fluorescent protein can be split into two or more polypeptide or protein fragments that can be activated. One or more of these activated split fluorescent protein fragments can contain a preformed mature chromophore that is primed and ready to fluoresce immediately upon complementation with one or more cognate activated split fluorescent fragments. For example, using inclusion bodies containing such split fluorescent fragments involves approximately half of the fully folded fluorescent protein containing a correctly folded mature chromophore that is not fluorescent by itself but is primed to fluoresce upon association with its cognate pair.

[0209] One or more of the activated split-fluorescent protein fragments may contain a preformed mature chromophore in an active but non-fluorescent state, where isolation of the chromophore in its mature but still inactive state allows for immediate fluorescence detection upon complementation with the corresponding fragment.

[0210] The first portion of the reporter protein may comprise amino acids 1-157 of green fluorescent protein (GFP), and the second portion of the reporter protein may comprise amino acids 158-231 of GFP. If GFP is incomplete, the amino acid numbering must be adjusted accordingly. Adding a C-terminal cysteine ​​to the first portion can facilitate conjugation of various nucleic acid binding motifs prior to expression. Adding an N-terminal cysteine ​​to the second portion can facilitate conjugation of various nucleic acid binding motifs prior to expression. The first portion includes a mature chromophore-containing α-fragment, which does not fluoresce alone but is primed to fluoresce when paired with the second portion, which includes a β-fragment. Without wishing to be bound by theory, the preformed chromophore allows it to fluoresce immediately. The α-fragment and the β-fragment cannot reassociate or fluoresce unless association is promoted.

[0211] The first portion of the reporter protein may comprise amino acids 1-213 of superfolder GFP, and the second portion of the reporter protein may comprise amino acids 214-230 of superfolder GFP. If the superfolder GFP is incomplete, the amino acid numbering must be adjusted accordingly. Adding a C-terminal cysteine ​​to the first portion can facilitate conjugation of various nucleic acid binding motifs prior to expression. Adding an N-terminal cysteine ​​to the second portion can facilitate conjugation of various nucleic acid binding motifs prior to expression. The first portion contains a mature chromophore-containing α-fragment, which is not fluorescent by itself but is primed to fluoresce when paired with the second portion containing the β-fragment.

[0212] The first portion of the reporter protein may comprise amino acids 1-154 of superfolder yellow fluorescent protein (YFP), and the second portion of the reporter protein may comprise amino acids 155-262 of superfolder YFP. If the superfolder YFP is incomplete, the amino acid numbering must be adjusted accordingly. Adding a C-terminal cysteine ​​to the first portion can support conjugation of various nucleic acid binding motifs prior to expression. Adding an N-terminal cysteine ​​to the second portion can support conjugation of various nucleic acid binding motifs prior to expression. The first portion contains a mature chromophore-containing α-fragment, which is not fluorescent by itself but is primed to fluoresce when paired with the second portion containing the β-fragment.

[0213] Fluorescent proteins can be detected by one or more of flow cytometry, a fluorescence plate reader, a fluorometer, microscopy, fluorescence resonance energy transfer (FRET), the naked eye, or other methods known to those skilled in the art. When using flow cytometry, fluorescence can be detected using a fluorescence activated cell sorter (FACS) or time-lapse microscopy.

[0214] In another embodiment of the present disclosure, association of activated split polypeptide fragments can form an assembled protein containing discontinuous epitopes that can be detected by antibodies that specifically recognize the discontinuous epitopes on the assembled protein but do not recognize the partial epitopes that were present on the individual polypeptides. These and other such derivatives can be readily made by those skilled in the art using known methods and can be screened for antibodies that do not recognize the assembled protein through any of the protein fragments themselves.

[0215] The split polypeptide may be a split fluorescent molecule. The molecule may comprise at least two activated split fluorescent fragments selected from the group consisting of GFP, GFP-like fluorescent proteins, fluorescent proteins, and variants thereof. One of the split fluorescent fragments may comprise a preformed mature chromophore, which is active in a non-fluorescent state in the dissociated fragment. The activated fluorescent fragments may contain the full complement of beta strands necessary for fluorescence when associated with each other, but are not fluorescent by themselves. Each activated split fluorescent fragment of the molecule further comprises a nucleic acid-binding motif. Binding of the nucleic acid-binding motif to a target nucleic acid can promote association of the at least two active split fluorescent fragments and real-time reconstitution of the active fluorescent protein and fluorescent phenotype.

[0216] Split fluorescent protein assays are also described in International Patent Publication No. WO 2010 / 066113, published June 17, 2010, which is incorporated herein by reference in its entirety, and U.S. Patent Application Publication No. 2009 / 0220942, which is incorporated herein by reference in its entirety.

[0217] The combined split luciferase-split fluorescent protein assay is further described in Nakane, S. et al., "Dual Split Protein (DSP) Assay to Monitor Cell-Cell Membrane Fusion", Methods Mol. Biol., 2015, 1313:229-36, which is incorporated herein by reference in its entirety.

[0218] In various embodiments, fluorescence can be measured using a flow cytometer or a bead array reader, for example, a BioPlex-100, BioPlex-200, Luminex-100, or Luminex-200 bead array reader may be used.

[0219] In some embodiments, the plating medium is removed and replaced with serum medium (e.g., 50 μL / well of OptiMEM) before adding the VSV particles to the cells. In other embodiments, the plating medium is not changed before adding the virus / serum mixture to the cells.

[0220] The VSV particles may be present in serum as a virus / serum mixture. In other embodiments, the plating medium is not changed before adding the virus / serum mixture to the cells. The VSV particles may be present in plasma as a virus / plasma mixture. In other embodiments, the plating medium is not changed before adding the virus / plasma mixture to the cells.

[0221] The reporter signal in the cells is measured and compared to a control.

[0222] The period of time for which the recombinant VSV particles are incubated with the virus / serum or virus / plasma mixture may be set as a suitable period for such incubation, such as, for example, about 30 minutes to 3 days, or about 30 minutes to 75 minutes, 45 minutes to 90 minutes, 60 minutes to 2 hours, or 90 minutes to 3 hours, etc. An exemplary period may be about 1 hour.

[0223] In some embodiments, after adding recombinant VSV particles (e.g., in a mixture with serum or plasma) to target cells, the cells with virus are incubated for an additional 2-6 hours, about 4 hours, or 4 hours. If serum or plasma is present after incubation, it can be removed to enhance cell fusion or luciferase activity.

[0224] The sample (e.g., serum or plasma) can be obtained from a control, e.g., a human who has been exposed to SARS-CoV-2 infection or vaccinated with a SARS-CoV-2 vaccine or another immunogenic composition. The sample may or may not contain some levels of SARS-CoV-2 neutralizing antibodies. Antibody responses in COVID-19 patients are described, for example, in Okba, NMA et al., "SARS-CoV-2 specific antibody responses in COVID-19 patients," Emerg. Infect. Dis., 2020, 26(7) (available at doi.org / 10.3201 / eid2607.200841). There is a need to rapidly determine the level of neutralizing antibodies in patient samples, and the methods described herein allow for the rapid assessment of the presence and / or level of SARS-CoV-2 neutralizing antibodies.

[0225] The method described in Montefiori, DC, "Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays." Curr. Protoc. Immunol., 2005, Chapter 12, Unit 12.11, which is incorporated herein by reference in its entirety, may also be used as part of the methods described herein.

[0226] kit The present disclosure also provides kits related to the various methods disclosed herein. In certain embodiments, the kits include, in one or more containers (e.g., separate containers): (a) a first recombinant DNA capable of being transcribed in a suitable host cell to produce a VSV antigenomic (+)RNA having a foreign RNA sequence inserted therein; (b) a second recombinant DNA including a sequence encoding a VSV N protein or a functional fragment or derivative thereof; (c) a third recombinant DNA including a sequence encoding a VSV L protein or a functional fragment or derivative thereof; and (d) a fourth recombinant DNA including a sequence encoding a VSV P protein or a functional fragment or derivative thereof; and optionally (e) a fifth recombinant DNA including a sequence encoding a VSV M protein or a functional fragment or derivative thereof; and optionally (f) a sixth recombinant DNA including a sequence encoding a VSV G protein or a functional fragment or derivative thereof. The second, third, fourth, and optionally the fifth and / or sixth recombinant DNAs may be part of the same DNA molecule or different DNA molecules. In a preferred embodiment, the sequences encoding the N, L, P, and other proteins are each operably linked to a promoter that controls expression of the N, L, P, and other proteins in a suitable host cell. In various embodiments, the kits can contain various nucleic acids, e.g., plasmid expression vectors, as described above for use in generating recombinant VSV particles.

[0227] In another embodiment, the kit of the present disclosure includes (a) a first recombinant DNA capable of being transcribed in a suitable host cell to produce a VSV antigenome DNA in which a portion of the RNA has been inserted into or replaced by a foreign RNA sequence; and (b) a host cell recombinantly expressing the VSV N, P, and L proteins, and optionally the M and / or G protein. In certain embodiments, the foreign RNA sequence is inserted into a non-essential portion of the recombinant VSV genome. In certain embodiments, the foreign RNA sequence replaces the VSV G protein.

[0228] In certain embodiments, the kit of the present disclosure comprises, in separate containers: (a) a first plasmid comprising the following operably linked components: (i) a bacteriophage RNA polymerase promoter, (ii) DNA comprising a sequence capable of being transcribed in a suitable host cell to produce an RNA molecule comprising a VSV antigenome RNA, wherein within the VSV antigenome RNA, a portion of VSV RNA has been inserted into or replaced by a foreign RNA sequence, and the 3' end of the antigenome RNA is immediately adjacent to a ribozyme sequence that cleaves the 3' end of the antigenome RNA, and (iii) a transcription termination signal for a bacteriophage RNA polymerase; (b) a second plasmid comprising the following operably linked components: (i) the bacteriophage RNA polymerase promoter described above, (ii) DNA comprising a sequence encoding a VSV N protein, or a functional fragment or derivative thereof, and (iii) a transcription termination signal for a bacteriophage RNA polymerase; (c) a third plasmid comprising the following operably linked components: (i) the bacteriophage RNA polymerase promoter described above, (ii) DNA comprising a sequence encoding a VSV P protein, or a functional fragment or derivative thereof, and (iii) a transcription termination signal for a bacteriophage RNA polymerase; (d) a fourth plasmid comprising the following operably linked components: (i) the bacteriophage RNA polymerase promoter described above, (ii) DNA comprising a sequence encoding a VSV L protein, or a functional fragment or derivative thereof, and (iii) a transcription termination signal for a bacteriophage RNA polymerase; optionally, (e) a fifth plasmid comprising the following operably linked components: (i) the bacteriophage RNA polymerase promoter described above, (ii) DNA comprising a sequence encoding a VSV M protein, or a functional fragment or derivative thereof, and (iii) a transcription termination signal for the bacteriophage RNA polymerase; and and optionally (f) a sixth plasmid comprising the following operably linked components: (i) the bacteriophage RNA polymerase promoter described above, (ii) DNA comprising a sequence encoding a VSV G protein, or a functional fragment or derivative thereof, and (iii) a transcription termination signal for the bacteriophage RNA polymerase. In certain embodiments, the foreign RNA sequence is inserted into a non-essential portion of the recombinant VSV genome. In certain embodiments, the foreign RNA sequence replaces the G protein of VSV.

[0229] In another embodiment, the kit of the present disclosure further comprises, in a separate container, a recombinant vaccinia virus encoding and capable of expressing a bacteriophage RNA polymerase.

[0230] In certain embodiments, the components in the container are in purified form.

[0231] The present disclosure further provides a kit for detecting coronavirus-neutralizing antibodies (e.g., neutralizing antibodies targeting the SARS-CoV-2 S glycoprotein) in a sample, the kit comprising: a) recombinant vesicular stomatitis virus (VSV) particles, in which the VSV glycoprotein (G) is replaced by the coronavirus spike (S) glycoprotein or a fragment or derivative thereof, and the S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell; b) a first cell expressing a first portion of a reporter protein and a second cell expressing a second portion of the reporter protein, wherein the first cell and the second cell are capable of fusing with each other when contacted with the recombinant VSV particle, and wherein the fusion results in the production of a detectable reporter signal; c) optionally, a positive control and / or a negative control; d) optionally a substrate for the reporter protein; and e) Optionally, instructions for use may include:

[0232] The reporter protein can be any of the enzymes (e.g., luciferase) or fluorescent proteins (e.g., GFP) described herein. In various embodiments, the reporter protein can be incorporated into the VSV particle without being encoded by the genome in the VSV particle. In various embodiments, the reporter protein is encoded by the genome of the VSV particle.

[0233] In some embodiments, the positive control may include a neutralizing antibody (e.g., an antibody against the SARS-CoV-2 spike (S) glycoprotein). Non-limiting examples of neutralizing antibodies against the SARS-CoV-2 S glycoprotein are mAb10914 or mAb10922 (the amino acid and nucleotide sequences of mAb10914 and mAb10922 are provided in the Sequences section below). A positive control can be generated by adding a known amount of a neutralizing antibody to serum or plasma that does not contain the neutralizing antibody. In some embodiments, the positive control may include a molecule that blocks the interaction between the SARS-CoV-2 spike (S) glycoprotein and a protein with which it interacts on the cell surface, such as ACE2 or TMPRSS2. Non-limiting examples of such molecules include, for example, anti-ACE2 antibodies (e.g., CL4013, AC18F, 881CT16.4.4, MA5-31395, and OTI1D2 antibodies), soluble ACE2 proteins (e.g., as disclosed in Fukuski et al., Journal of General Virology, 2005, 86:2269-74, incorporated herein by reference in its entirety), and anti-TMPRSS2 antibodies (e.g., the H1H7017N antibody described in International Patent Application Publication No. 2019 / 147831, incorporated herein by reference in its entirety). In some embodiments, the positive control may include a molecule (e.g., an antibody) that blocks cell-cell fusion. Non-limiting examples of such molecules include, for example, the MC5 and DL11 antibodies described in Saw, WT et al., Methods, 2015, 90:68-75, incorporated herein by reference in its entirety.

[0234] The negative control can include serum or plasma that does not contain the neutralizing antibody, or serum or plasma that does not contain the neutralizing antibody but contains other antibodies, such as an antibody that specifically targets the VSV glycoprotein (G), an antibody that specifically targets the VSV matrix protein (M), an antibody that specifically targets another coronavirus spike (S) glycoprotein different from the one being tested in the method (e.g., an antibody that specifically targets the SARS-CoV-1 spike (S) glycoprotein but does not bind to the SARS-CoV-2 spike (S) glycoprotein), or a monoclonal antibody specific for an HIV antigen (e.g., IgG1b12, 2G12, X5, 2F5, 4E10, as described in Montefiori, DC, Current Protocols in Immunology, 2004, Chapter 12: Unit 12.11.1, which is incorporated herein by reference in its entirety), or any isotype control antibody.

[0235] In yet another embodiment, the kit comprises: a) a recombinant vesicular stomatitis virus (VSV) particle, wherein (i) the VSV glycoprotein (G) is replaced by a coronavirus spike (S) glycoprotein or a fragment or derivative thereof, wherein said S glycoprotein, fragment, or derivative is capable of mediating infection of a target cell, and (ii) the VSV particle comprises a reporter protein and / or a nucleic acid molecule encoding said reporter protein; b) cells susceptible to infection mediated by coronavirus S glycoprotein; c) optionally, a positive control and / or a negative control; d) optionally a substrate for the reporter protein; and e) Optionally, instructions for use may include:

[0236] Any of the above kits may optionally include cell growth media and / or fixatives. [Example]

[0237] The present disclosure is also described and demonstrated by the following example. However, the use of this example, and other examples elsewhere in the specification, is merely illustrative and in no way limits the scope and meaning of the present disclosure or of any exemplified term. Likewise, the present disclosure is not limited to any particular preferred embodiment described herein. Indeed, numerous modifications and variations of the present disclosure may become apparent to those skilled in the art upon reading this specification, and these variations can be made without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to be limited only by the terms of the appended claims, along with the full range of equivalents to which such claims are entitled.

[0238] Example 1: Preparation of VSV expressing spike (S) glycoprotein constructs An infectious clone of the Indiana strain of VSV was used to generate four recombinant VSV constructs in which the VSV (G) glycoprotein was deleted and replaced with a codon-optimized sequence suitable for expression in human cells and containing: (1) the full-length SARS-CoV-2 spike (S) glycoprotein sequence (NCBI Reference Sequence: NC_045512.2; Protein ID: YP_009724390.1) (variant 1; VSV SARS-CoV-2 dG = VSV-SARS-CoV-2-S; amino acid sequence of SEQ ID NO: 1; codon-optimized coding nucleotide sequence of SEQ ID NO: 2), (2) the SARS-CoV-2 S glycoprotein sequence with the C-terminal 19 amino acids KFDEDDSEPVLKGVKLHYT (SEQ ID NO: 20) deleted (variant 2; VSV SARS-CoV-2 Δ19CT (3) a SARS-CoV-2 S glycoprotein sequence in which the S glycoprotein cytoplasmic tail is replaced with the VSV G cytoplasmic tail KLKHTKKRQIYTDIEMNRLGK (SEQ ID NO:21) (variant 3; VSV SARS-CoV-2 VSV-G CT dG; amino acid sequence of SEQ ID NO:5; codon-optimized coding nucleotide sequence of SEQ ID NO:6), or (4) variant 4—full-length SARS-CoV-2 S glycoprotein sequence (VSV-SARS-CoV-2-S; amino acid sequence of SEQ ID NO:1; codon-optimized coding nucleotide sequence of SEQ ID NO:2) with the wild-type VSV Kozak sequence (cActATG; SEQ ID NO:11) instead of the optimized Kozak sequence (caccATG; SEQ ID NO:12) used in the other three constructs. A first set of constructs (constructs 1-4) of variants 1-4 were prepared, encoding a wild-type VSV M protein (amino acid sequence of SEQ ID NO:9; nucleotide sequence of SEQ ID NO:10). A second set of constructs (constructs 5-8) of variants 1-4 were prepared, encoding an M protein with the substitution M51R (amino acid sequence of SEQ ID NO:7; nucleotide sequence of SEQ ID NO:8), which confers viral attenuation. See Figure 1.

[0239] Variant 1-4 recombinant viral particles were generated using standard published protocols, involving transfection with vaccinia-T7 virus (expressing T7 polymerase) and cotransfection with N, P, and L expression plasmids (each carrying a gene under the control of a T7 promoter) and the viral genome plasmid. To facilitate rescue, cells were also transfected with a plasmid expressing VSV G. Viruses were amplified and propagated in Vero cells. The amplified recombinant viruses lack the VSV (G) glycoprotein and rely on the SARS-CoV-2 spike (S) glycoprotein for entry and infection.

[0240] The correct incorporation of VSV G, N, and M proteins and SARS-CoV-2 S glycoprotein into recombinant variant 2 VSV-SARS-CoV-2-S-Δ19CT construct 6 (VSV-M51R-nCoV19-S Δ19CT) virions was analyzed by Western blotting. The results are shown in Figure 3C. Viral supernatants prepared from Vero cells infected with VSV-GFP (encoding VSV-G) or VSV-SARS-CoV-2-S-Δ19CT, or mock-infected, were subjected to immunoblot analysis using anti-SARS-CoV-2-spike antibody (left) and rabbit anti-VSV antiserum (right). The SARS-CoV-2-S-Δ19CT S glycoprotein generated two bands, corresponding to the full-length S1 / S2 variant (180 kDa) and the proteolytically cleaved S2 variant (75 kDa) glycoproteins. Western blots show the presence of VSV N, M, and G proteins in the VSV-GFP virus and the presence of VSV N and M proteins (but not VSV G glycoprotein) in the variant 2 VSV-SARS-CoV-2-S-Δ19CT virus. Western blots of the variant 2 VSV-SARS-CoV-2-S-Δ19CT virus also show efficient incorporation of the VSV-SARS-CoV-2-S-Δ19CT S glycoprotein in place of the VSV G glycoprotein.

[0241] Example 2: Development of a high-throughput IMMUNO-COV v.1 assay to detect antibodies that neutralize SARS-CoV-2 introduction A laboratory assay for detecting SARS-CoV-2 neutralizing antibodies was developed using a clinical isolate of SARS-CoV-2, a pseudotyped lentiviral vector, and pseudotyped vesicular stomatitis virus (VSV). Described here is a high-throughput, scalable clinical assay for detecting and quantifying SARS-CoV-2-neutralizing antibodies using a recombinant VSV engineered to express the SARS-CoV-2 spike (S) glycoprotein in place of the VSV-G glycoprotein. The SARS-CoV-2 spike (S) glycoprotein binds to angiotensin-converting enzyme-2 (ACE2) on the cell surface, initiating viral entry into cells (Hoffman 2020, Walls 2020, Wan 2020). As the primary surface-exposed viral protein, the coronavirus spike is a primary target of the host immune system (Corman 2016, Liu 2006, Nie 2004, Zhao 2017). Blocking the spike's interaction with ACE2 prevents viral entry, making the spike a primary target for neutralizing antibodies (Walls 2020, Wu 2020). Therefore, we hypothesized that detecting antibodies capable of neutralizing VSV expressing the spike (S) glycoprotein should directly reflect the level of SARS-CoV-2 neutralizing antibodies present in a sample. Importantly, using spike-expressing VSV instead of SARS-CoV-2 significantly improves the safety and scalability of the assay.

[0242] The assay of the present invention (IMMUNO-COV v.1) utilizes a dual split protein (DSP) luciferase reporter system to exploit the fusion phenotype of SARS-CoV-2 spike glycoprotein-expressing VSV to quantify virus-induced cell fusion and, consequently, the level of virus neutralization (Figure 2). The DSP system of this embodiment utilizes a chimeric split-modified green fluorescent protein (GFP) and a split Renilla luciferase mutant, Rluc8. Fusion between two cell lines expressing complementary split reporters promotes the reassociation of fully functional GFP and Rluc8 luciferase. Thus, luciferase activity can be used to measure virus-induced cell fusion in a high-throughput 96-well plate format. Described here is the development of the assay, including optimization of assay conditions and eventual clinical assay validation. Excellent correlation was observed between the results of this assay, clinical symptoms, and results from other serological tests. Notably, quantification of SARS-CoV-2 neutralizing antibody levels closely correlated with plaque reduction neutralization IC50 values ​​from assays using clinical isolates of SARS-CoV-2. Therefore, the assay of the present invention accurately quantifies SARS-CoV-2 neutralizing antibodies, making it a valuable tool for evaluating immune responses to plasma therapy and SARS-CoV-2 vaccines.

[0243] The IMMUNO-COV v.1 assay demonstrated 100% specificity in validation studies, with no false positives detected across all tests. In blinded analyses, assay results showed near-perfect correlation (196 / 197) with available clinical data and with results from qRT-PCR or other serological tests (commercially available ELISAs). To quantify the strength of the antiviral response, a standard curve was constructed consisting of different concentrations of anti-SARS-CoV-2 spiked monoclonal antibody spiked into a SARS-CoV-2 seronegative serum or plasma matrix. This standard curve was used to quantify neutralizing antibody levels in the assay from a single 1:100 serum test dilution, determined to be the minimum recommended dilution. For samples containing high levels of SARS-CoV-2 neutralizing antibodies, a second dilution facilitated more accurate quantification. The virus neutralization unit (VNU) calculated using this calibrator method is the plaque reduction neutralization titer (EC50) determined by plaque reduction neutralization testing against clinical isolates of SARS-CoV-2. EC50 ) values ​​correlated closely with those of the IMMUNO-COV v.1 assay (p<0.0001). Together, these results demonstrate that the IMMUNO-COV v.1 assay can accurately quantify SARS-CoV-2 neutralizing antibodies in samples (e.g., serum or plasma) in a biosafety level 2 (BSL2) high-throughput format, providing important information for, for example, assessing donor eligibility for convalescent plasma programs and participant eligibility for various clinical trials, as well as evaluating immune responses to candidate SARS-CoV-2 vaccines.

[0244] result Measurement of VSV-SARS-CoV-2-S-Δ19CT fusion by luciferase assay Safe implementation of SARS-CoV-2 neutralization assays requires biosafety level 3 (BSL3) contaminants and practices, making widespread testing of patient samples with these assays impractical. Therefore, we sought to develop a safer, scalable assay that could be used to detect SARS-CoV-2 neutralizing antibodies in blood samples. We designed an assay to detect syncytia formation in Vero cells induced by infection with SARS-CoV-2 spike (S) glycoprotein-expressing VSV using a dual split protein (DSP) luciferase reporter (Figure 2). Using reverse genetics, we generated a recombinant vesicular stomatitis virus (VSV) in which the VSV glycoprotein (G) was replaced with the SARS-CoV-2 spike (S) glycoprotein (Figures 1 and 3A). Deletion of the last 19 amino acids of the C-terminal (cytoplasmic) domain of the SARS-CoV-2 spike (S) glycoprotein improved virus rescue and amplification. The resulting virus (variant 2 VSV-SARS-CoV-2-S-Δ19CT) induced syncytia formation in Vero cell monolayers, which was enhanced in the presence of 0.4 μg / mL trypsin (Figure 3B). Incorporation of the SARS-CoV-2-Δ19CT spike glycoprotein into virions was confirmed by immunoblot analysis in viral supernatants (Figure 3C). The SARS-CoV-2-Δ19CT spike glycoprotein, but not VSV-G, was detected, confirming that VSV-G was efficiently replaced by SARS-CoV-2-S-Δ19CT in the recombinant virus.

[0245] Vero cells were chosen as the reporter cell line for this assay because they are infected with VSV-SARS-CoV-2-S-Δ19CT and express both ACE2 and TMPRSS2 (Figure 3D and E). Parental Vero cells were transfected with the self-inactivating lentiviral vectors SFFV-DSP1-7 and SFFV-DSP8-11, respectively, under the control of the spleen focus-forming virus (SFFV) promoter. 1-7 -P2A-Puro or SFFV-DSP 8-11 Vero-DSP1 cells were transduced with Rluc8 DSP 1-7 luciferase-GFP fusion protein (SEQ ID NO: 14), which contains amino acids 1-155 of the Rluc8 mutant Renilla luciferase fragment and amino acids 1-156 of the modified GFP fragment. Vero-DSP2 cells expressed Rluc8 DSP 8-11 luciferase-GFP fusion protein (SEQ ID NO: 16), which contains amino acids 157-311 of the Rluc8 mutant Renilla luciferase fragment and amino acids 157-231 of the modified GFP fragment. The RLuc8 mutant Renilla luciferase contains the mutations A55T, C124A, S130A, K136R, A143M, M185V, M253L, and S287L (see SEQ ID NO: 19). The sequence of the modified GFP is provided in SEQ ID NO: 18.

[0246] When mixed monolayers of Vero-DSP1 and Vero-DSP2 cells seeded at a 1:1 ratio were infected with VSV-SARS-CoV-2-S-Δ19CT, syncytia formation was observed, and luciferase activity was readily detected 24 h postinfection (Figure 3F). In contrast, no luciferase activity was detected in monolayers infected with Vero-DSP1 alone or Vero-DSP2 alone, or in mock-infected Vero-DSP1 / Vero-DSP2 monolayers. Thus, luciferase activity was specific to virus-induced cell fusion in mixed Vero-DSP1 / Vero-DSP2 monolayers.

[0247] Optimization of VSV-SARS-CoV-2-S-Δ19CT fusion Syncytium formation is necessary to generate functional luciferase in our assay. However, complete disruption of the cell monolayer following syncytium formation reduces luciferase activity due to loss of cell viability. Therefore, we determined the optimal time point after VSV-SARS-CoV-2-S-Δ19CT infection for measuring luciferase activity. EnduRen™ Live Cell Substrate (EnduRen™) is a modified "pro-substrate" that must be cleaved by cellular esterases to generate the luciferase substrate, coelenterazine. Because this pro-substrate is highly stable, luciferase activity can be repeatedly measured anywhere from 2 to 24 hours after adding EnduRen™ to cells. By 15 hours post-infection, luciferase activity in infected Vero-DSP1 / Vero-DSP2 monolayers was readily distinguishable from the background signal of mock-infected cells (Figure 4A). Luciferase activity continued to increase, reaching peak levels approximately 26 hours post-infection, so the optimal luciferase readout in this assay was 24-30 hours post-infection.

[0248] The optimal cell density and virus concentration were also determined to enhance cell fusion and luciferase activity, thereby improving assay sensitivity. Vero-DSP1 / Vero-DSP2 cells were seeded at increasing cell densities, and 24 hours later, the monolayers were infected with increasing concentrations of VSV-SARS-CoV-2-S-Δ19CT. Increasing cell density improved the luciferase signal at all virus dilutions (Figure 4B). As expected, syncytia formation and luciferase activity also improved when more virus was used to inoculate the monolayer. For this assay, 6 × 10 4 It was concluded that 100 cells / well should be used. It was also noted that for best results, cells should be seeded from an "under-confluent" flask, and that cells can be seeded directly from frozen stocks if two days are allowed for recovery.

[0249] Not surprisingly, further studies across multiple experiments showed that different lots of VSV-SARS-CoV-2-S-Δ19CT exhibited different efficacies for inducing fusion. Therefore, the appropriate concentration of virus for the assay was determined for each lot of virus based on its performance against a series of artificial controls (see below).

[0250] Detection of neutralization of VSV-SARS-CoV-2-S-Δ19CT using the Vero-DSP reporter assay To demonstrate that the assay of the present invention can detect virus neutralization, we first tested the ability of several purified proteins to inhibit virus-induced fusion. Affinity-purified polyclonal anti-SARS-CoV-spike antibodies and soluble recombinant Ace2 protein bind to spikes on the viral surface and block cell entry. Similarly, monoclonal anti-Ace2 antibodies block cellular receptors, limiting virus entry. All three inhibitors successfully blocked VSV-SARS-CoV-2-S-Δ19CT-induced fusion in a dose-dependent manner (Figure 5A). This demonstrates that the assay of the present invention can detect inhibition of VSV-SARS-CoV-2-S-Δ19CT infection. Importantly, neutralization of VSV-SARS-CoV-2-S-Δ19CT by plasma obtained from a SARS-CoV-2 convalescent individual (NL1) was also detected. Compared to pooled plasma from three presumed SARS-CoV-2 seronegative individuals, heat-inactivated NL1 plasma neutralized VSV-SARS-CoV-2-S-Δ19CT in a dose-dependent manner (Figure 5B). Thus, our assay was able to detect VSV-SARS-CoV-2-S-Δ19CT-neutralizing antibodies in convalescent plasma.

[0251] Optimization of VSV-SARS-CoV-2-S-Δ19CT neutralization time In initial experiments, we observed that incubating VSV-SARS-CoV-2-S-Δ19CT at 37°C for 1 hour resulted in a loss of luciferase activity, even in the absence of any neutralizing serum or inhibitors (Figure 6A). This loss of activity was partially abolished when the virus concentration was relatively high. Nevertheless, we investigated whether shorter incubation times at room temperature could promote efficient virus neutralization. Two-fold serial dilutions of neutralized NL1 plasma were prepared and incubated with VSV-SARS-CoV-2-S-Δ19CT for 60 minutes at 37°C or for 15, 30, or 60 minutes at room temperature. Compared to the other conditions, luciferase activity was slightly higher in the 1:160 dilution of the sample incubated for 15 minutes at room temperature (Figure 6B), suggesting that this condition may not be sufficient for complete neutralization. However, NL1 neutralized VSV-SARS-CoV-2-S-Δ19CT when incubated at room temperature for 30 or 60 min, although less efficiently than when incubated at 37°C for 1 h. It was concluded that a 30-min incubation at room temperature was sufficient for virus neutralization.

[0252] Heat inactivation is not required for assay suitability To further streamline the assay workflow, we determined whether heat inactivation of samples was necessary to achieve reliable results. Serum samples were used for this study because they are more readily available than plasma samples. We first tested three presumptive SARS-CoV-2 seronegative samples and a commercially available serum pool (produced up until fall 2019) at a dilution of 1:100. Heat inactivation slightly reduced luciferase activity in the serum pool and two of the three negative sera (Figure 6C), likely due to low levels of complement-mediated viral inactivation. Overall, however, heat inactivation had little effect on VSV-SARS-CoV-2-S-Δ19CT-induced cell fusion and luciferase activity. Even when three additional presumptive negative sera were tested at multiple dilutions, there was little difference in luciferase activity between heat-inactivated and non-heat-inactivated samples (Figure 6D). In contrast, heat inactivation reduced the ability of SARS-CoV-2 convalescent sera to neutralize VSV-SARS-CoV-2-S-Δ19CT (Figure 6E). Therefore, we conclude that the use of non-heat-inactivated samples is preferable because it streamlines the assay workflow and improves assay sensitivity.

[0253] Determining the minimum recommended dilution Sample interference is common in serological assays at high sample concentrations. Therefore, we defined a minimum recommended dilution of the assay sample that nearly eliminates false positives due to sample interference. To this end, 39 presumed SARS-CoV-2 seronegative samples were subjected to the assay at two-fold serial dilutions from 1:10 to 1:320. As expected, viral fusion and interference with luciferase activity were observed at relatively high serum concentrations (Figure 7A), but disappeared upon further dilution of the samples. Statistical analysis indicated that the minimum recommended dilution was approximately 1:100. At this dilution, the luciferase signal from negative serum samples was greater than 50% with a confidence level of >95%. Therefore, in all subsequent studies, we used a dilution of 1:100 for both the test serum samples and the pooled SARS-CoV-2 seronegative serum control.

[0254] Development of an artificial positive control Due to the limited availability of large quantities of qualified SARS-CoV-2 seropositive sera, we attempted to develop an artificial positive control for the assay. To this end, various concentrations of mAb10914 (a monoclonal antibody that binds to the SARS-CoV-2 spike (S) glycoprotein) were spiked into pooled SARS-CoV-2 seronegative sera or into culture medium alone. An isotype antibody was also used as a control. As expected, mAb10914 caused dose-dependent neutralization of VSV-SARS-CoV-2-S-Δ19CT in both culture medium and pooled negative sera (Figures 7B and C). In contrast, the isotype control antibody did not significantly alter virus-induced luciferase activity, indicating that neutralization by mAb10914 was specific to spike binding. To establish the appropriate concentration to use for the artificial control in validation studies, a total of nine 3-fold serial dilutions of mAb10914 (10 μg / mL to 0.0015 μg / mL) were tested in duplicate in three separate experiments performed by three independent analysts. Due to plate-to-plate variability in raw RLU (relative light units), results were normalized as the % luciferase signal relative to the luciferase signal in control wells containing pooled SARS-CoV-2 seronegative sera. The mean % luciferase signal at each concentration was plotted, and a nonlinear regression curve was generated (Figure 7D). The linear range of the assay was approximately 10% to 80% of the signal, or from 0.01 μg / mL to 0.6 μg / mL of mAb10914. 2 μg / mL of mAb10914 was selected as the artificial positive control: high (CPC High), which consistently reduced the signal by less than 20%. Based on a 95% confidence level of the response curve, the concentration of mAb10914 that should induce a signal of less than 50% was calculated to be 0.18 μg / mL, thus representing the approximate limit of detection of the assay.

[0255] Testing assay specificity and sensitivity The CPC High value was used to define the specificity and sensitivity of the assay. To measure specificity, 30 presumed SARS-CoV-2 seronegative samples were tested alone or spiked with mAb 10914 at 2 μg / mL (CPC High). This evaluation verified that no false positives were observed at the optimized serum dilution of 1:100 and that all artificial positive controls showed virus neutralization. No false positives or false negatives were observed in two separate analyses performed by different analysts (Table 1). One negative sample was qualified as indeterminate in the first assay because one replicate had a signal less than 50%. However, in the second assay, this sample was correctly identified as negative. Therefore, in this validation, the sensitivity and specificity of the IMMUNO-COV v.1 assay were 100%. To measure assay specificity near the limit of detection, 30 presumed SARS-CoV-2 seronegative samples (1:100 serum dilution) were tested alone, and 19 of these samples were also measured after spiking with 0.35 μg / mL of mAb10914. Eleven of these tests were spiked with 2 μg / mL of mAb10914, which is not near the limit of detection. This experiment demonstrated a sensitivity of 68% near the limit of detection (Table 2).

[0256] To assess the sensitivity of the assay, a similar single-blind analysis was performed using the same 30 presumed SARS-CoV-2 seronegative samples described above. Duplicate samples were run at a 1:100 ratio, with one replicate spiked with mAb 10914 at 2 μg / mL (CPC High) or 0.35 μg / mL (borderline inhibition) and subsequently assigned a blinded ID. Of the 30 negative samples, 26 were identified as negative and 4 were identified as indeterminate (Table 2). Of the 11 CPC High samples, 10 were detected, and the one unidentified positive was suspected to be a sample preparation error. Of the 19 borderline samples, 13 were identified as positive, 4 as negative, and 2 as indeterminate, consistent with samples containing only low levels of neutralizing antibodies, with 0.35 μg / mL being close to the previously estimated detection limit of the assay.

[0257] To further evaluate the specificity of the assay, samples from patients who had documented and recovered from infection with four common coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1) before December 2019 were tested. No cross-reactivity with these common coronaviruses was observed, demonstrating the strong specificity of the IMMUNO-COV v.1 assay for SARS-CoV-2.

[0258] Demonstration of clinical concordance Given that our assay performed well in artificial controls, we proceeded with a blinded study to determine whether results correlated well with clinical symptoms. Serum samples were obtained from negative donors (asymptomatic, negative for SARS-CoV-2 by PCR, or SARS-CoV-2 seronegative by ELISA), positive donors (positive for SARS-CoV-2 by PCR, or SARS-CoV-2 seropositive by ELISA), and contact-positive donors (untested but in direct contact with someone who tested positive for SARS-CoV-2 by PCR). A total of 230 samples were obtained, which were blinded to the assay analysts until study completion. Of the 125 negative samples obtained, all 125 analyzed as negative in the assay (Table 3), providing further evidence of the assay's excellent specificity. Again, all 38 of the known SARS-CoV-2 seropositive samples analyzed as positive in the assay. Of 34 samples obtained from donors previously tested positive for SARS-CoV-2 by PCR, neutralizing antibodies were identified in 31 samples. On closer examination, one sample was predicted negative because it was collected only 8 days after a positive PCR test (not enough time for a strong antibody response to develop). From donors who had contact with SARS-CoV-2-positive individuals, 13 of 33 samples (39.4%) were identified as positive for neutralizing antibodies. Together, these data demonstrate that the assay has excellent clinical concordance.

[0259] Correlation between neutralization of VSV-SARS-CoV-2-S-Δ19CT and neutralization of SARS-CoV-2 The excellent clinical concordance observed with this assay supported our premise that neutralization of VSV-SARS-CoV-2-S-Δ19CT accurately reflects neutralization of SARS-CoV-2. To directly demonstrate this correlation, 20 of the serum samples described above were also tested against clinical isolates of SARS-CoV-2 in a BSL-3 plaque reduction neutralization test (PRNT) (PRNT is described, e.g., in Wang et al., Nat Commun 11, 2251 (2020) doi.org / 10.1038 / s41467-020-16256-y; Okba et al., Emerg Infect Dis. 2020 Jul. doi.org / 10.3201 / eid2607.200841; Ranawaka et al., Euro Surveill. 2020;25(16):pii=2000421. doi.org / 10.2807 / 1560-7917.ES.2020.25.16.2000421). Each of the five samples that tested negative in the assay of the present invention was also below the limit of detection of the PRNT assay (Table 4). Similarly, the 15 samples that tested positive in the assay of the present invention were also positive in the PRNT assay. Furthermore, the quantitative virus neutralization units (VNU) determined in the assay of the present invention were below the limits of PRNT50% and PRNT EC50 The values ​​correlated closely with those of the mAb10914 (Tables 4-5 and Figure 8A). To calculate VNU, a standard curve consisting of a 1:100 five-point dilution series of mAb10914 in pooled SARS-CoV-2 seronegative sera was included on each assay plate. Based on the % luciferase signal, the equivalent mAb10914 concentration was determined for each sample and multiplied by 100 to obtain the VNU for that sample. VNU and PRNT EC50A strong correlation (Pearson ρ = 0.8902, p < 0.0001) was observed between VNU and virus neutralization titer (VNT) EC50 values. The relationship between VNU and virus neutralization titer (VNT) EC50 values ​​was also examined. Five two-fold serial dilutions of 44 SARS-CoV-2 positive samples were prepared and tested in the assay. The % luciferase signal for each dilution was plotted, and the resulting curve was used to calculate VNT EC50 The values ​​of VNU and VNT were determined. EC50 Statistical comparison with the VNU value showed an excellent correlation between these two values ​​(Spearman ρ = 0.8327, p < 0.0001) (Figure 8B), providing further validation of the accuracy of the VNU value. It was concluded that neutralization of VSV-SARS-CoV-2-S-Δ19CT closely replicates neutralization of SARS-CoV-2, and that VNU provides an accurate method for quantifying the strength of the anti-SARS-CoV-2 antibody response. Figure 8C shows representative VNU values ​​from PCR-positive samples, demonstrating that convalescent samples exhibit a range of VNU values.

[0260] The correlation between VNU values ​​and disease severity was examined for 31 positive samples in which clinical symptoms were self-reported (Figure 8D). Three patients reported being asymptomatic: two in the lowest VNU quadrant and one in the second-lowest VNU quadrant. One patient who experienced only chills generated neutralizing antibodies exceeding 1000 VNU. Thus, although individuals with severe disease are more likely to mount a strong neutralizing antibody response, severe disease is not required for a strong neutralizing antibody response.

[0261] Example of a 96-well plate layout for the assay A non-limiting example of a 96-well plate layout for the assay is shown in Figure 9. Each sample and control is prepared individually in a U-well plate or tube and transferred to duplicate wells of Vero-DSP1 / DSP2 mixed cells in a black, clear-bottom 96-well plate. Samples S1-S41 are shown. Each sample is assayed at 1:100 in OptiMEM. The plate position of samples and controls can be varied. It is important to ensure that all controls are included on each plate. Controls include BC, NC, and Standards (St) 1-5. BC is pooled negative (matrix) sera at a dilution of 1:100 and contains no virus. NC is pooled negative (matrix) sera at a dilution of 1:100 + VSV-SARS-CoV-2-S-Δ19CT Each standard contains the following three components: Pooled negative (matrix) sera at a dilution of 1:100 VSV-SARS-CoV-2-S-Δ19CT Positive control (e.g., mAb10914) antibody at the specified concentration The final concentrations of mAb10914 in each standard are currently: St1 = 3 μg / mL, St2 = 1 μg / mL, St3 = 0.3333 μg / mL, St4 = 0.1111 μg / mL, St5 = 0.0370 μg / mL (an alternative standard curve could include 6 standards, with the ranges: St1 = 2 μg / mL, St2 = 1 μg / mL, St3 = 0.5 μg / mL, St4 = 0.25 μg / mL, St5 = 0.125 μg / mL, St6 = 0.0625 μg / mL).

[0262] To determine the results, the following steps are performed: 1. Luciferase activity is measured (integration time per well 2000 ms, settling time between wells 200 ms), and the output is in relative light units (RLU). 2. Determine the average ...

Claims

1. 1. A replication-competent recombinant rhabdovirus particle, wherein (i) the rhabdovirus glycoprotein (G) is replaced by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein or a fragment of SARS-CoV-2 S glycoprotein comprising a receptor binding domain thereof, said SARS-CoV-2 S glycoprotein or fragment thereof being capable of mediating infection of a target cell, and (ii) said recombinant rhabdovirus particle comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a reporter protein, said nucleic acid sequence encoding the reporter protein being inserted between the nucleic acid sequence encoding the SARS-CoV-2 S glycoprotein or fragment thereof and the nucleic acid sequence encoding the rhabdovirus large (L) protein.

2. The replication-competent recombinant rhabdovirus particle of claim 1, wherein the reporter protein is luciferase or a fluorescent protein.

3. The replication-competent recombinant rhabdovirus particle of claim 1 or 2, wherein the recombinant rhabdovirus particle is a recombinant vesiculovirus particle.

4. The replication-competent recombinant rhabdovirus particle of claim 3, wherein the recombinant vesicular stomatitis virus particle is a recombinant vesicular stomatitis virus (VSV) particle.

5. (i) the SARS-CoV-2 S glycoprotein is a full-length SARS-CoV-2 S glycoprotein; or (ii) the fragment is a SARS-CoV-2 S glycoprotein lacking 19 C-terminal amino acids relative to the amino acid sequence of the full-length SARS-CoV-2 S glycoprotein of SEQ ID NO:1; or (iii) The replication-competent recombinant rhabdovirus particle according to any one of claims 1 to 4, wherein the SARS-CoV-2 S glycoprotein comprises one or more amino acid insertions, deletions, and / or substitutions as listed in the table below, and the positions of the insertions, deletions, and / or substitutions are specified relative to the amino acid sequence of the full-length SARS-CoV-2 S glycoprotein of SEQ ID NO:

1. 【Table 1-1】 【Table 1-2】

6. A replication-competent recombinant rhabdovirus particle according to any one of claims 1 to 5, comprising a mutant rhabdovirus matrix (M) protein.

7. The replication-competent recombinant rhabdovirus particle of claim 6, which is a recombinant VSV particle comprising a mutant VSV M protein comprising a mutation at methionine 51.

8. The replication-competent recombinant rhabdovirus particle of claim 7, wherein the mutation at methionine 51 is a mutation from methionine (M) to arginine (R).

9. 1. A method for determining the presence of SARS-CoV-2 neutralizing antibodies in a sample, the method comprising: (a) contacting a sample with a replication-competent recombinant rhabdovirus particle according to any one of claims 1 to 8; (b) after step (a), contacting the replication-competent recombinant rhabdovirus particle with a target cell; (c) after step (b), measuring a reporter signal in the target cells; and (d) comparing the reporter signal measured in step (c) with a control. A method comprising:

10. The method of claim 9, wherein the target cells are Vero cells or Vero-E6 cells.

11. The method of claim 9 or 10, further comprising the step of adding a substrate for the reporter protein to obtain the reporter signal.

12. 12. The method of claim 11, wherein the reporter protein comprises luciferase and the substrate of the reporter protein comprises luciferin or an EnduRen luciferase substrate.

13. The sample is (i) serum or plasma; and / or (ii) heat-inactivated The method according to any one of claims 9 to 12.

14. 14. The method of any one of claims 9 to 13, further comprising diluting the sample from 1:10 to 1:

320.

15. 15. The method of any one of claims 9 to 14, wherein the control is a reporter signal obtained with a control sample that does not contain coronavirus neutralizing antibodies, the method comprising concluding that the tested sample contains SARS-CoV-2 neutralizing antibodies if the reporter signal obtained in step (c) is reduced compared to the control.

16. 16. The method of any one of claims 9 to 15, comprising determining the concentration of SARS-CoV-2 neutralizing antibodies in the tested sample by comparing the reporter signal to a standard curve determined from serial dilutions of a control sample comprising a SARS-CoV-2 neutralizing antibody, or a molecule that blocks the interaction between the SARS-CoV-2 S glycoprotein and a protein with which the coronavirus S glycoprotein interacts on the target cell, or any combination thereof.

17. (i) the reporter signal is measured 24 to 30 hours after step (b); and / or (ii) In step (a), the sample is contacted with the recombinant rhabdovirus particles at room temperature for 30 minutes.

18. The method of any one of claims 9 to 17, carried out in a high throughput format.

19. A polynucleotide encoding a replicable recombinant rhabdovirus particle according to any one of claims 1 to 8, comprising a nucleic acid sequence encoding a rhabdovirus nucleoprotein (N), a nucleic acid sequence encoding a rhabdovirus phosphoprotein (P), a nucleic acid sequence encoding a rhabdovirus L protein, a nucleic acid sequence encoding a SARS-CoV-2 S glycoprotein or a fragment thereof, and a nucleic acid sequence encoding a reporter protein inserted as specified in claim 1.

20. A kit for determining the presence of SARS-CoV-2 neutralizing antibodies in a sample, comprising: a) a replication-competent recombinant rhabdovirus particle according to any one of claims 1 to 8, or a polynucleotide according to claim 19; and b) target cell Including the kit.

21. c) a control sample that does not contain coronavirus neutralizing antibodies; and / or d) a control sample comprising a SARS-CoV-2 neutralizing antibody; or a molecule that blocks the interaction between the SARS-CoV-2 S glycoprotein and a protein with which the SARS-CoV-2 S glycoprotein interacts on a target cell; or any combination thereof; and / or e) a substrate for the reporter protein; and / or d) Instructions for use 21. The kit of claim 20, further comprising:

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