Recombinant Newcastle Disease Virus And Immunogenic Compositions For Prevention Of COVID-19

Abstract

Described herein is a recombinant Newcastle disease virus ("NDV") comprising a packaging genome wherein the packaging genome comprises a transgene comprising a nucleotide sequence encoding a protein comprising a SARS-CoV-2 spike protein or a portion thereof. Also described herein are recombinant NDVs comprising a packaging genome wherein the packaging genome comprises a transgene encoding a chimeric F protein wherein the chimeric F protein comprises an SARS-CoV-2 spike protein extracellular domain and an NDV F protein transmembrane domain and a cytoplasmic domain. In addition, immunogenic compositions comprising the recombinant NDV are described herein. The recombinant NDV and immunogenic compositions can be used to immunize against SARS-CoV-2 as well as to prevent COVID-19.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 318,640, filed March 10, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Statement on Federally Funded Research

[0004] This invention was developed with government support under approval number HHSN272201400008C issued by the National Institutes of Health in the United States. The government owns certain rights to this invention.

[0005] sequence list

[0006] This application includes a computer-readable sequence list, which has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence list XML file submitted with this application is named "06923-384-228_SEQ_LISTING.xml", was created on March 9, 2023, and has a size of 173,741 bytes.

[0007] introduction

[0008] In one aspect, this document describes a recombinant Newcastle disease virus (“NDV”) comprising a packaging genome, wherein the packaging genome contains a transgene encoding a protein comprising the spike protein of severe acute respiratory syndrome coronavirus 2 (“SARS-CoV-2”) or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein). In one specific embodiment, this document describes a recombinant NDV comprising a packaging genome comprising a transgene encoding a codon-optimized nucleic acid sequence comprising the SARS-CoV-2 spike protein or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein). In one specific embodiment, this document describes a recombinant NDV comprising a packaging genome comprising a transgene encoding a chimeric F protein, wherein the chimeric F protein comprises the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein. In one specific embodiment, this document describes a recombinant NDV comprising a packaging genome containing a transgene encoding a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane and cytoplasmic domains of the NDV F protein. This document also describes compositions comprising such a recombinant NDV and the use of such a recombinant NDV and compositions in inducing an immune response against the SARS-CoV-2 spike protein, and in detecting the presence of antibodies binding to the SARS-CoV-2 spike protein in an immunoassay. Furthermore, this document provides bivalent and multivalent immunogenic compositions comprising recombinant NDV, and the use of such immunogenic compositions for immune resistance to SARS-CoV-2 and prevention of COVID-19. Background Technology

[0009] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen of the current coronavirus disease 2019 (COVID-19). Since the start of the pandemic, the emergence of new variants of concern (VOCs) has threatened the protective effect conferred by vaccination using the original strain (1). In December 2020, the Alpha variant (B.1.1.7) and the Beta variant (B.1.351) were declared VOCs and spread worldwide, followed by the Gamma strain (P.1) in January 2021. In humans, the Beta and Gamma variants showed significant resistance to neutralizing antibodies against the original strain (1,2). In May 2021, a pandemic in India gave rise to a new VOC: the Delta variant (B.1.617.2). This new VOC contained cryptic mutations in its spike protein that differed from other variants, which significantly reduced its sensitivity to neutralizing antibodies and increased its transmissibility, rapidly replacing previous variants globally (1,3). In November 2021, a new VOC called Omicron emerged in South Africa. Since then, Omicron has replaced the Delta variant globally (4). Compared to previous VOCs, Omicron exhibits the highest number of mutations in the spike protein and has shown the highest decrease-neutralizing activity (5,6). Currently, the Omicron sublineage BA.2, also known as "stealthy" Omicron, appears to show more immune evasion and transmissibility (7–9).

[0010] Despite the unprecedentedly rapid development of COVID-19 vaccines, only 63.1% of the global population has been fully vaccinated (6). Therefore, low- and middle-income countries still need COVID-19 vaccines that can be produced locally, where vaccination rates are among the lowest globally (6). Summary of the Invention

[0011] In one aspect, this document describes nucleotide sequences comprising the spike protein of severe acute respiratory syndrome coronavirus 2 (“SARS-CoV-2”) or portions thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein) or derivatives thereof. In one embodiment, this document provides nucleotide sequences encoding the extracellular domain of SARS-CoV-2 or derivatives thereof (e.g., derivatives of the extracellular domain of the SARS-CoV-2 spike protein as described herein, such as in Sections 5.1.2 or 6). In some embodiments, this document provides nucleotide sequences encoding derivatives of the extracellular domain of SARS-CoV-2. In some embodiments, the derivatives of the extracellular domain of SARS-CoV-2 comprise the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, this document provides a nucleotide sequence encoding a protein comprising the SARS-CoV-2 spike protein extracellular domain or a derivative thereof. In some embodiments, this document provides a nucleotide sequence encoding a protein comprising a derivative of the SARS-CoV-2 spike protein extracellular domain. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In one specific embodiment, this document provides a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises a SARS-CoV-2 spike protein extracellular domain or a derivative thereof, and a transmembrane domain and a cytoplasmic domain of the NDV F protein. In some embodiments, this document provides a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises a derivative of the SARS-CoV-2 spike protein extracellular domain (e.g., a derivative of the SARS-CoV-2 spike protein extracellular domain as described herein, such as in Section 5.1.2 or 6) and a transmembrane domain and a cytoplasmic domain of the NDV F protein. In some implementations, the derivative of the SARS-CoV-2 extracellular domain comprises the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29 or 30.In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the transmembrane and cytoplasmic domains of the NDV F protein belong to the LaSota or Hitchner strains. In some embodiments, the transmembrane and cytoplasmic domains of the NDV F protein comprise SEQ ID NO: 42. In some embodiments, the derivatives of the SARS-CoV-2 spike protein extracellular domain and the transmembrane and cytoplasmic domains of the NDV F protein are linked via a linker (e.g., a linker described herein, such as SEQ ID NO: 7) or directly linked. In some embodiments, the chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 11, 18, 22, 28, 39, 40, or 41. In some embodiments, the chimeric F protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 11, 18, 22, 28, 39, 40, or 41. In some embodiments, the nucleotide sequence is an RNA sequence. In some embodiments, the nucleotide sequence is a DNA sequence (e.g., cDNA). In some embodiments, the nucleotide sequence is present in a vector (e.g., a plasmid or virus). In some embodiments, immunogenic compositions comprising such nucleotide sequences are provided herein. In some embodiments, the immunogenic composition comprises two or more of the nucleotide sequences described herein (e.g., the immunogenic composition is divalent or polyvalent).

[0012] On the other hand, this document describes proteins comprising the spike protein of severe acute respiratory syndrome coronavirus 2 (“SARS-CoV-2”) or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein) or derivatives thereof. In one embodiment, this document provides proteins comprising the extracellular domain of SARS-CoV-2 or derivatives thereof (e.g., derivatives of the extracellular domain of the SARS-CoV-2 spike protein as described herein, such as in Sections 5.1.2 or 6). In some embodiments, this document provides proteins comprising derivatives of the SARS-CoV-2 extracellular domain. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In one specific embodiment, this document provides a chimeric F protein comprising the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof, and the transmembrane domain and cytoplasmic domain of the NDV F protein. In some embodiments, this document provides a chimeric F protein comprising a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., a derivative of the extracellular domain of the SARS-CoV-2 spike protein as described herein, such as in Section 5.1.2 or 6) and the transmembrane and cytoplasmic domains of the NDV F protein. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the transmembrane and cytoplasmic domains of the NDV F protein belong to the LaSota or Hitchner strains. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein comprise SEQ ID NO:42. In some embodiments, derivatives of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein are linked via a linker (e.g., a linker described herein, such as SEQ ID NO:7) or directly linked. In some embodiments, the chimeric F protein comprises the amino acid sequence of SEQ ID NO:6, 11, 18, 22, 28, 39, 40, or 41. In some embodiments, the chimeric F protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:6, 11, 18, 22, 28, 39, 40, or 41.In some embodiments, this document provides immunogenic compositions comprising such a protein or a chimeric F protein. In some embodiments, the immunogenic composition comprises two or more of the proteins or chimeric F proteins described herein (e.g., the immunogenic composition is divalent or polyvalent).

[0013] In another embodiment, this document describes a recombinant Newcastle disease virus (“NDV”) comprising a packaging genome, wherein the packaging genome comprises a transgene encoding a spike protein of severe acute respiratory syndrome coronavirus 2 (“SARS-CoV-2”) or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein) or a derivative thereof. In one specific embodiment, this document describes a recombinant NDV comprising a packaging genome, wherein the packaging genome comprises a transgene that comprises a codon-optimized nucleic acid sequence encoding a spike protein of SARS-CoV-2 or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein) or a derivative thereof. In one specific embodiment, this document describes a recombinant NDV comprising a packaging genome, wherein the packaging genome comprises a transgene that comprises a nucleic acid sequence encoding a protein containing the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof. In another specific embodiment, this document describes a recombinant NDV comprising a packaging genome, wherein the packaging genome comprises a transgene that comprises a nucleic acid sequence encoding a protein containing a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In one specific embodiment, this document describes a recombinant NDV comprising a packaging genome, wherein the packaging genome contains a transgene encoding a chimeric F protein, wherein the chimeric F protein comprises a SARS-CoV-2 spike protein extracellular domain or a derivative thereof and an NDV F protein transmembrane domain and a cytoplasmic domain. In some embodiments, the SARS-CoV-2 extracellular domain derivative comprises the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof is encoded by a codon-optimized nucleic acid sequence. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein belong to the LaSota or Hitchner strains. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein comprise SEQ ID NO:42.In some implementations, derivatives of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane and cytoplasmic domains of the NDV F protein are linked via adapters (e.g., adapters described herein, such as SEQ ID NO:7) or directly.

[0014] In another embodiment, this document provides a recombinant Newcastle disease virus (“NDV”) comprising the spike protein of severe acute respiratory syndrome coronavirus 2 (“SARS-CoV-2”) or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein) or a derivative thereof. In one embodiment, this document provides a recombinant NDV comprising a protein containing the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof. In another embodiment, this document provides a recombinant NDV comprising a protein containing a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In yet another embodiment, this document provides a recombinant NDV comprising a chimeric F protein, wherein the chimeric F protein comprises the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof, as well as the transmembrane domain and cytoplasmic domain of the NDV F protein. In one embodiment, this document provides a recombinant NDV comprising a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 spike protein, as well as the transmembrane domain and cytoplasmic domain of the NDV F protein. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises the amino acid sequence of SEQ ID NO:12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof is encoded by a codon-optimized nucleic acid sequence. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein belong to the LaSota or Hitchner strains. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein comprise SEQ ID NO:42. In some implementations, derivatives of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane and cytoplasmic domains of the NDV F protein are linked via adapters (e.g., adapters described herein, such as SEQ ID NO:7) or directly.

[0015] On the other hand, this document provides immunogenic compositions comprising the recombinant NDV described herein. The immunogenic composition may be monovalent, bivalent, or polyvalent. In some embodiments, the immunogenic composition is monovalent. In some embodiments, the immunogenic composition is bivalent. In some embodiments, the immunogenic composition is trivalent. In some embodiments, the immunogenic composition is tetravalent.

[0016] In some embodiments, this document provides an immunogenic composition comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, and wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, and wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; wherein the first derivative and the second derivative are different from each other. In a specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lacks a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with other amino acid residues). In a specific embodiment, lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, amino acid residues 682 to 685 (RRAR) corresponding to amino acid residues in GenBank accession number MN908947.3 are substituted with a single alanine. In some implementations, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) contains the following amino acid substitutions at amino acid residues corresponding to the amino acid residues in GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P.

[0017] In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12 or 16. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29, or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29, or 30. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:23 or 24, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:23 or 24. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13, 17, 29, or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13, 17, 29, or 30. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0018] In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, and wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, and wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; and (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, and wherein the third chimeric F protein comprises a third derivative of the extracellular domain of the SARS-CoV-2 spike protein, and an NDV The F protein comprises a transmembrane domain and a cytoplasmic domain; wherein the first, second, and third derivatives are distinct from each other. In a specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lacks a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with other amino acid residues). In a specific embodiment, the lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, amino acid residues 682 to 685 (RRAR) corresponding to amino acid residues MN908947.3 are substituted with a single alanine. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) contains the following amino acid substitutions at amino acid residues corresponding to the amino acid residues in GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 or 16, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12 or 16.In some embodiments, the second derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 13 or 17. In some embodiments, the third derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 29 or 30. In some embodiments, the first derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 23 or 24. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 or 16, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12 or 16. In some embodiments, the third derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30.In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17. In some embodiments, the immunogenic composition further comprises a fourth recombinant NDV, wherein the fourth recombinant NDV comprises a fourth transgene, the fourth transgene comprising a nucleotide sequence encoding a fourth chimeric F protein, and wherein the fourth chimeric F protein comprises a fourth derivative of the extracellular domain of the SARS-CoV-2 spike protein, and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the fourth derivative is distinct from the first, second, and third derivatives. In some embodiments, the fourth recombinant NDV comprises a fourth transgene, the fourth transgene comprising a nucleotide sequence encoding a fourth chimeric F protein, and wherein the fourth derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:23 or 24, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:23 or 24. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0019] In some embodiments, this document provides an immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; wherein the first derivative and the second derivative are distinct from each other. In a specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lacks a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with other amino acid residues). In specific embodiments, the lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 are replaced by a single alanine. In some embodiments, derivatives of the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) contain the following amino acid substitutions at the amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12 or 16. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29, or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29, or 30.In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:23 or 24, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:23 or 24. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13, 17, 29, or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13, 17, 29, or 30. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17.

[0020] In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises a third derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; wherein the first derivative, the second derivative, and the third derivative are different from each other. In a specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lacks a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with other amino acid residues). In a specific embodiment, lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, amino acid residues 682 to 685 (RRAR) corresponding to amino acid residues in GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) contains the following amino acid substitutions at amino acid residues corresponding to the amino acid residues in GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 or 16, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12 or 16. In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17.In some embodiments, the third derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30. In some embodiments, the first derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO:23 or 24, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:23 or 24. In some embodiments, the second derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17. In some embodiments, a second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 or 16, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12 or 16. In some embodiments, a third derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30. In some embodiments, a first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:29 or 30, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:29 or 30. In some embodiments, the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17.In some embodiments, the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:13 or 17, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13 or 17. In some embodiments, the immunogenic composition further comprises a fourth recombinant NDV comprising a fourth chimeric F protein, wherein the fourth chimeric F protein comprises a fourth derivative of the extracellular domain of the SARS-CoV-2 spike protein, and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the fourth derivative is distinct from the first, second, and third derivatives. In some embodiments, the fourth recombinant NDV comprises a fourth chimeric F protein, and wherein the fourth derivative of the SARS-CoV-2 spike protein extracellular domain comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 23 or 24. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0021] In the specific implementation scheme, the derivative of the extracellular domain of the SARS-CoV-2 spike protein contains an amino acid substitution of proline at positions 817, 892, 899, 942, 986, and 987 corresponding to the W strain spike protein (see GenBank accession number MN908947.3), and contains an RRAR substitution of alanine at positions 682 to 685 corresponding to the W strain spike protein (see GenBank accession number MN908947.3).

[0022] In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share less than 85% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share less than 90% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share less than 95% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share less than 98% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share less than 99% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share 75% to 90% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share 85% to 90% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share 90% to 95% identity with each other. In some embodiments, the first derivative and the second derivative of the SARS-CoV-2 spike protein extracellular domain share 95% to 99% identity with each other.

[0023] In some embodiments, the first derivative, the second derivative, and the third derivative of the SARS-CoV-2 spike protein extracellular domain share less than 85% identity with each other. In some embodiments, the first derivative, the second derivative, and the third derivative of the SARS-CoV-2 spike protein extracellular domain share less than 90% identity with each other. In some embodiments, the first derivative, the second derivative, and the third derivative of the SARS-CoV-2 spike protein extracellular domain share less than 95% identity with each other. In some embodiments, the first derivative, the second derivative, and the third derivative of the SARS-CoV-2 spike protein extracellular domain share less than 98% identity with each other. In some embodiments, the first derivative, the second derivative, and the third derivative of the SARS-CoV-2 spike protein extracellular domain share less than 99% identity with each other. In some embodiments, these three derivatives share 75% to 90% identity with each other. In some embodiments, they share 85% to 90% identity with each other. In some embodiments, they share 90% to 95% identity with each other. In some implementations, the first derivative, the second derivative, and the third derivative of the extracellular domain of the SARS-CoV-2 spike protein share 95% to 99% identity with each other.

[0024] In some embodiments, the first derivative, the second derivative, the third derivative, and the fourth derivative of the SARS-CoV-2 spike protein extracellular domain share less than 85% identity with each other. In some embodiments, these derivatives share less than 90% identity with each other. In some embodiments, they share less than 95% identity with each other. In some embodiments, the first derivative, the second derivative, the third derivative, and the fourth derivative of the SARS-CoV-2 spike protein extracellular domain share less than 98% identity with each other. In some embodiments, these derivatives share less than 99% identity with each other. In some embodiments, they share between 75% and 90% identity with each other. In some implementations, the first derivative, the second derivative, the third derivative, and the fourth derivative of the extracellular domain of the SARS-CoV-2 spike protein share 85% to 90% identity with each other.In some embodiments, the first derivative, the second derivative, the third derivative, and the fourth derivative of the SARS-CoV-2 spike protein extracellular domain share 90% to 95% identity with each other. In some embodiments, the first derivative, the second derivative, the third derivative, and the fourth derivative of the SARS-CoV-2 spike protein extracellular domain share 95% to 99% identity with each other.

[0025] In some embodiments, the extracellular domain of the chimeric F protein is connected via a linker to the transmembrane domain and cytoplasmic domain of the NDV F protein. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the NDV F protein and the cytoplasmic domain comprise the amino acid sequence of SEQ ID NO:42, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:42.

[0026] In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are the transmembrane domain and cytoplasmic domain of the NDV LaSota strain F protein. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are the transmembrane domain and cytoplasmic domain of the NDV Hitchner strain F protein. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein comprise the amino acid sequence of SEQ ID NO:42.

[0027] In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises an amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises an amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39, or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39, or 41. In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises an amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises an amino acid sequence of SEQ ID NO: 6, 18, 22, or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 18, 22, or 39.In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises an amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises an amino acid sequence of SEQ ID NO: 6, 18, 22, or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 18, 22, or 39. In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO:11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:11 or 40.In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises an amino acid sequence of SEQ ID NO:22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22 or 39; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises an amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41. In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises an amino acid sequence of SEQ ID NO:22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22 or 39; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises an amino acid sequence of SEQ ID NO:6 or 18, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:6 or 18.In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0028] In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; The amino acid sequence of NO:22 or 39 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; and (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO:6, 18, 28, or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:6, 18, 28, or 41. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0029] In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO:22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41; The amino acid sequence of NO:22 or 39 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; and (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:6 or 18. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0030] In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; (c) An amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39; and (d) A third recombinant NDV comprising a third transgene that encodes a nucleotide sequence encoding a third chimeric F protein, wherein the third chimeric F protein encodes the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6 or 18; and (d) A fourth recombinant NDV comprising a fourth transgene that encodes a fourth chimeric F protein, wherein the fourth chimeric F protein encodes the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39. The amino acid sequence of NO:28 or 41 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. In some embodiments, the nucleotide sequence encoding the chimeric F protein is codon-optimized.

[0031] In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39, or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39, or 41. In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 22, or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 18, 22, or 39. In some embodiments, this document provides an immunogenic composition comprising (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 22, or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 18, 22, or 39.In some embodiments, this document provides an immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO:11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:11 or 40. In some embodiments, this document provides an immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO:22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22 or 39; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41. In some embodiments, this document provides an immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO:22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22 or 39; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO:6 or 18, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:6 or 18.In some embodiments, this document provides an immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40.

[0032] In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 28, or 41 ...d) an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least The amino acid sequences of NO:6, 18, 28 or 41 have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity. In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO:22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22 or 39; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41. The amino acid sequence of NO:6 or 18 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity.In some embodiments, this document provides a multivalent immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 39. The amino acid sequence of NO:6 or 18 has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence; and (d) a fourth recombinant NDV comprising a fourth chimeric F protein, wherein the fourth chimeric F protein comprises the amino acid sequence of SEQ ID NO:28 or 41, or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:28 or 41.

[0033] In some embodiments, the recombinant NDV described herein is inactivated. In some embodiments, the recombinant NDV described herein is active. In some embodiments, the immunogenic composition described herein further comprises an adjuvant.

[0034] On the other hand, the recombinant NDV and immunogenic compositions described herein are used to induce an immune response, immunize a subject against SARS-CoV-2, and / or prevent COVID-19. In some embodiments, this document provides a method for inducing an immune response against the SARS-CoV-2 spike protein, the method comprising administering the immunogenic composition described herein to a subject. In some embodiments, this document provides a method for preventing COVID-19, the method comprising administering the immunogenic composition described herein to a subject. In some embodiments, this document provides a method for immunizing a subject against SARS-CoV-2, the method comprising administering the immunogenic composition described herein to a subject. In some embodiments, this document provides a method for immunizing a subject against two or more SARS-CoV-2s, the method comprising administering the immunogenic composition described herein to a subject. In some embodiments, this document provides a method for immunizing a subject against one or more SARS-CoV-2s, wherein the one or more SARS-CoV-2s are heterologous to SARS-CoV-2 derived from the extracellular domain of the chimeric F protein contained in the immunogenic composition described herein, the method comprising administering the immunogenic composition to the subject. In some embodiments, this document provides a method for inducing antibodies against one or more SARS-CoV-2 proteins in a subject, wherein the one or more SARS-CoV-2 proteins are heterologous to SARS-CoV-2 derived from the extracellular domain of the chimeric F protein contained in the immunogenic composition described herein, the method comprising administering the immunogenic composition to the subject. In some embodiments, this document provides a method for inducing antibodies that cross-react with one or more SARS-CoV-2 spike proteins in a subject, wherein the one or more SARS-CoV-2 spike proteins are heterologous to SARS-CoV-2 spike proteins derived from the extracellular domain of the immunogenic composition described herein, the method comprising administering the immunogenic composition to the subject. In a specific embodiment, the composition is administered intranasally or intramuscularly to the subject. In a specific embodiment, the subject is a human. In some embodiments, the subject has previously been vaccinated against COVID-19. In some embodiments, at least one booster of the immunogenic composition is administered to the subject.

[0035] On the other hand, this article provides a kit comprising the immunogenic composition described herein.

[0036] 3.1 the term

[0037] As used herein, the terms “about” or “approximately” when used in conjunction with numbers refer to any number within 1%, 5%, or 10% of the cited number, including the cited number.

[0038] As used herein, the term "antibody" refers to a molecule containing an antigen-binding site (e.g., an immunoglobulin). Antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, single-domain antibodies, camel-derived antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fv (sdFv), intracellular antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id and anti-anti-Id antibodies), as well as epitope-binding fragments of any of the above. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0039] As used in this article, the term “older people” refers to people who are 65 years of age or older.

[0040] As used in this article, the term "adult" refers to a person who is 18 years of age or older.

[0041] As used in this article, the term "human child" refers to a person aged 1 to 18 years.

[0042] As used in this article, the term "human infant" refers to a person aged 1 to 3 years.

[0043] As used in this article, the term "human infant" refers to a person from birth to 1 year of age.

[0044] As used herein, the phrase "IFN-deficient system" or "IFN-deficient substrate" refers to a system (e.g., cells, cell lines, and animals such as mice, chickens, turkeys, rabbits, rats, horses, etc.) that does not produce one, two, or more types of IFN, or does not produce any type of IFN, or produces low levels of one, two, or more types of IFN, or produces low levels of any IFN (i.e., under the same conditions, the expression of any IFN is reduced by 5%–10% compared to an IFN-competent system). 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90% or more), unresponsive to one, two or more types of IFN or responding with low efficiency, or unresponsive to any type of IFN, having a delayed response to one, two or more types of IFN, and / or defective activity of antiviral genes induced by one, two or more types of IFN or induced by any type of IFN, or any combination thereof.

[0045] In some embodiments, the nucleotide sequence, nucleic acid sequence, or polynucleotide sequence is isolated. In some embodiments, an "isolated" nucleic acid sequence, nucleotide sequence, or polynucleotide sequence refers to a nucleic acid molecule that is separate from other nucleic acid molecules present in the natural source of that nucleic acid. In other words, an isolated nucleic acid sequence, nucleotide sequence, or polynucleotide sequence may contain heterologous nucleic acids that are not related to it in nature. In other embodiments, an "isolated" nucleic acid sequence, such as a cDNA or RNA sequence, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of nucleic acid sequences, nucleotide sequences, or polynucleotide sequences in which the nucleic acid sequence, nucleotide sequence, or polynucleotide sequence is separate from the cellular components of a cell, which is isolated or recombined from these cells. Therefore, nucleic acid sequences, nucleotide sequences, or polynucleotide sequences that are substantially free of cellular material include preparations of nucleic acid sequences, nucleotide sequences, or polynucleotide sequences having less than about 30%, 20%, 10%, or 5% (by dry weight) of other nucleic acids. The term "substantially free of culture medium" includes preparations of nucleic acid, nucleotide, or polynucleotide sequences in which the culture medium comprises less than about 50%, 20%, 10%, or 5% of the volume of the preparation. The term "substantially free of chemical precursors or other chemicals" includes preparations in which the nucleic acid, nucleotide, or polynucleotide sequence is separated from chemical precursors or other chemicals involved in the synthesis of the nucleic acid, nucleotide, or polynucleotide sequence. In specific embodiments, such preparations of nucleic acid, nucleotide, or polynucleotide sequences, in addition to the nucleic acid, nucleotide, or polynucleotide sequence of interest, have less than about 50%, 30%, 20%, 10%, or 5% (on a dry weight) of chemical precursors or compounds.

[0046] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "subject" refers to an animal. In some embodiments, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a pet (e.g., a dog or cat) or a farm animal (e.g., a horse, pig, or cow). In a particular embodiment, the subject is a human. In some implementations, the mammals (including humans) are 4 to 6 months, 6 to 12 months, 1 to 5 years, 5 to 10 years, 10 to 15 years, 15 to 20 years, 20 to 25 years, 25 to 30 years, 30 to 35 years, 35 to 40 years, 40 to 45 years, 45 to 50 years, 50 to 55 years, 55 to 60 years, 60 to 65 years, 65 to 70 years, 70 to 75 years, 75 to 80 years, 80 to 85 years, 85 to 90 years, 90 to 95 years, or 95 to 100 years old. In specific implementations, the subjects are non-avian animals.

[0047] As used herein, the term “combination” in the context of administering therapy (a) to a subject means the use of more than one therapy. The use of the term “combination” does not restrict the order in which the therapies are administered to the subject. The first therapy may be administered before, simultaneously with, or after the second therapy is administered to the subject.

[0048] As used herein, the terms "SARS-CoV-2 spike protein" and "the spike protein of SARS-CoV-2" include the SARS-CoV-2 spike protein as known to those skilled in the art. Examples of amino acid sequences of the SARS-CoV-2 spike protein and nucleotide sequences encoding the SARS-CoV-2 spike protein can also be found, for example, in GenBank accessions MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, and MT334558. Typical spike proteins contain domains known to those skilled in the art, including an S1 domain, a receptor-binding domain, an S2 domain, a transmembrane domain, and a cytoplasmic domain. See, for example, the descriptions of the SARS-CoV-2 spike protein (especially its structure) in Wrapp et al., 2020, Science 367:1260-1263 and Duan et al., 2020, Front. Immunol., Vol. 11, Paper 576622. The spike protein can be characterized as having a signal peptide, receptor-binding domain, extracellular domain, S1 domain, S2 domain, as well as transmembrane domain and intracellular domain (or cytoplasmic domain).

[0049] As used herein, the terms “SARS-CoV-2 beta variant spike protein” and “SARS-CoV-2 beta variant spike protein” include SARS-CoV-2 beta variant spike proteins known to those skilled in the art. See, for example, GISAID accession numbers EPI_ISL_660610 and EPI_ISL_678626.

[0050] As used herein, the terms “SARS-CoV-2 delta variant spike protein” and “SARS-CoV-2 delta variant spike protein” include SARS-CoV-2 delta variant spike proteins known to those skilled in the art. See, for example, the SARS-CoV-2 delta variants in GISAID accession numbers EPI_ISL_1740580 (hCoV-19 / England / CAMC-151FDF0 / 2021), EPI_ISL_1733902 (hCoV-19 / USA / CA-CDC-FG-021941 / 2021), EPI_ISL_1731755 (hCoV-19 / India / CG-AIIMS-Raipur-L15928 / 2021), and EPI_ISL_4634223hCoV-19 / Spain / CT-LabRefCat-1699309 / 2021. In some implementations, the SARS-CoV-2 delta variant can be found in GISAID accession number EPI_ISL_4634223 (hCoV-19 / Spain / CT-LabRefCat-1699309 / 2021). In one specific implementation, the SARS-CoV-2 delta variant belongs to the B1.617.2 sublineage. In some implementations, the SARS-CoV-2 delta variant belongs to the AY sublineage. In certain embodiments, the SARS-CoV-2 delta variant is AY.4, AY.25, AY.12, AY.3, AY.9, AY.3, AY.9, AY.5, AY.6, AY.20, AY.7.1, AY.23, A Y.14, AY.10, AY.7, AY.13, AY.15, AY.16, AY.19, AY.2, AY.8, AY.11, AY.1, AY.21, AY.22, AY.7.2, AY.5.1 or AY.5.2 pedigree.

[0051] As used herein, the term "W strain" refers to the SARS-CoV-2 strain known to those skilled in the art as the W strain. See, for example, GenBank accession number MN908947.3.

[0052] As used herein, the terms “SARS-CoV-2 gamma variant spike protein” and “SARS-CoV-2 gamma variant spike protein” include SARS-CoV-2 gamma variant spike proteins known to those skilled in the art. See, for example, GISAID accession numbers EPI_ISL_792680 and EPI_ISL_804814.

[0053] As used herein, the terms “SARS-CoV-2μ variant spike protein” and “SARS-CoV-2μ variant spike protein” include SARS-CoV-2μ variant spike proteins known to those skilled in the art. For example, the SARS-CoV-2μ variant spike protein may be a spike protein of the B.1.621 or B.1.621.1 lineage.

[0054] As used herein, the terms “SARS-CoV-2 Omicron variant spike protein” and “SARS-CoV-2 Omicron variant spike protein” include SARS-CoV-2 Omicron variant spike proteins known to those skilled in the art. For example, a SARS-CoV-2 Omicron variant may be a spike protein of the B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, or BA.5 lineage.

[0055] As used herein, the term "therapy" can refer to any regimen, method, agent, or combination thereof that can be used to treat or prevent COVID-19 or for vaccination. In some embodiments, the term "therapy" refers to the recombinant NDV described herein. In other embodiments, the term "therapy" refers to an agent that is not the recombinant NDV described herein. Attached Figure Description

[0056] Figure 1A and Figure 1B The evolution of SARS-CoV-2 and the emergence of variants of concern (VOCs) Figure 1A The phylogenetic tree, comprising 3057 genomes, shows the global evolutionary relationships of the SARS-CoV-2 virus from the ongoing COVID-19 pandemic. Figure 1B The timeline graph shows the global frequency of different SARS-CoV-2 viral clades. The graph is adapted from the website / nextstrain.org / ncov / gisaid / global (accessed on February 5, 2022 via CC-BY(28,29)).

[0057] Figure 2A-2D Design, production, and characterization of the NDV-HXP-S variant vaccine. Figure 2AStructure and design of the NDV-HXP-S construct. A distinct SARS-CoV-2 spike sequence was introduced between the P and M genes of the LaSota L289ANDV strain. The extracellular domain of the spike is linked to the transmembrane domain and cytoplasmic tail (TM / CT) of the NDV F protein. The original polybasic cleavage site was removed by mutating RRAR to A. Stability mutations in HexaPro (F817P, A892P, A899P, A942P, K986P, and V987P) were introduced. This sequence was codon-optimized for mammalian host expression. The original W HXP-S sequence was compared with novel variant constructs: Beta, Gamma, and Delta. Changes relative to the original W HXP-S sequence in the N-terminal domain (NTD), receptor-binding domain (RBD), and spike 2 (S2) are shown. Figure 2B As previously described, the NDV-HXP-S variant was rescued by reverse genetics (Ayllon et al., 2013, J Vis Exp. (80): 50830). Expression plasmids required for NDV viral genome (NP, P, and L) replication and transcription were co-transfected with full-length NDV cDNA. Two or three days later, tissue culture supernatants were inoculated into eight- or nine-day-old specific pathogen-free (SPF) hatched eggs. Antigen identity was confirmed by biochemical methods and sequencing. Genetic stability of the recombinant virus was evaluated through multiple passages in ten-day-old SPF hatched eggs. The NDV-HXP-S vaccine was inactivated with BPL and purified by ultracentrifugation with a sucrose buffer layer (Sun et al., 2021, Nat. Commun. 12: 6197). Figure 2C A comparison of NDV-HXP-SDelta virus with NDV-HXP-SDelta virus possessing the P681R mutation. Spike protein and NDV HN protein were detected by Western blot using anti-spike 2B3E5 mouse monoclonal antibody and anti-HN 8H2 mouse monoclonal antibody, respectively. Figure 2D Protein staining of the NDV-HXP-S variant vaccine, resolved on 4%–20% SDS-PAGE. Viral proteins (L, S0, HN, N, P, and M) were visualized by Coomassie blue staining. The uncut S0 spike protein is highlighted in blue and has an approximate size of 200 kDa.

[0058] Figure 3A-3G NDV-HXP-S Beta and Gamma induce protective antibodies against homologous infections. Figure 3A and Figure 3BStudy and group design. Eight- to ten-week-old female BALB / c mice were vaccinated with either an inactivated NDV-HXP-S variant vaccine or WT NDV (negative control) at a total dose of 1 μg. Two immunizations were administered via intramuscular (IM) on D0 and D21. Mice were treated with Ad5-hACE2 on D44. On D49, mice were challenged with the USA-WA1 / 2020 Beta (B.1.351) or Gamma (P.1) strains, and lungs were harvested and homogenized in 1 mL PBS the day after challenge. Viral titers were measured by plaque assay of Vero E6 cells. Viral titers in lung homogenates after W, Beta, or Gamma challenge (n=5) Figure 3C Viral titers were measured by plaque assays on VeroE6 cells and plotted as GMT in PFU / mL (detection limit equal to 50 PFU / mL; a titer of 25 PFU / mL was specified for negative samples). Error bars represent geometric standard deviation. Geometric mean titers relative to controls are shown in gray. For W spikes (n=10) ( Figure 3D ), Beta spike (n=10)( Figure 3E ), Gamma spike (n=10)( Figure 3F ) and Delta spike protein (n=5) Figure 3G Spike-specific serum IgG levels were measured. Antibodies in booster (D43) serum samples from different immunization regimens were measured by ELISA. GMT AUC plots were plotted. Error bars represent geometric standard deviation. Statistical differences in all plots were analyzed by ordinary one-way ANOVA corrected for Dunnett's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0059] Figures 4A-4D The trivalent and quadrivalent NDV-HXP-S vaccination regimens induce protection against phylogenetically distant SARS-CoV-2 variants. Figure 4A and Figure 4B Study and group design. Eight- to ten-week-old female BALB / c mice were vaccinated with either an inactivated NDV-HXP-S variant vaccine or WT NDV (negative control) at a total dose of 1 μg. Two immunizations were administered via intramuscular (IM) on days 0 and 21. On day 44, mice were treated with Ad5-hACE2. On day 49, mice were challenged with the USA-WA1 / 2020 Delta (B.1.617.2) or Mu (B.1.621) strains, and lungs were harvested and homogenized in 1 mL PBS the day after challenge; titers were measured by plaque assay. Figure 4CViral titers after challenge (n=5). Viral titers were measured by plaque assays in VeroE6 cells (for W strain challenge) and Vero TMPRSS2 cells (for Delta and Mu strain challenge), and plotted as GMT in PFU / mL (limit of detection equal to 50 PFU / mL; a titer of 25 PFU / mL was specified for negative samples). Error bars represent geometric standard deviation. Statistical differences were analyzed by ordinary one-way ANOVA corrected for Dunnett's multiple comparison test. p-values ​​and fold-over titers relative to controls are shown in blue and gray, respectively. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001) Figure 4D Neutralization activity plot. Enhanced pooled sera were tested in a technically reproducible manner for micro-neutralization (MNT) assays against the USA-WA / 2020 strain (WT), Delta (B.1.617.2) variant, Beta (B.1.351) variant, and Omicron (B.1.1.529) variant. GMT serum dilutions (ID50) inhibiting 50% of infection were plotted (limit of detection equal to 10 and assigned to negative samples). Geometric mean fold change is added in blue.

[0060] Figures 5A-5C Trivalent and quadrivalent NDV-HXP-S vaccination regimens induced higher serum antibody titers against phylogenetically distant SARS-CoV-2 variants. Spike specificity against the spike mutant (… Figure 5A ) or RBD specificity ( Figure 5B A heatmap of serum IgG (n=10). Antibody binding for different immunoassays was measured using W, Delta, Alpha, Beta, Gamma, and Omicron spikes or RBD (see [reference needed]). Figures 6A-6C and Figure 6D-6F To view a separate ELISA chart. Figure 5C WS2-specific serum IgG (ten samples pooled, three copies). Antibodies in the enhanced (D43) serum were measured by ELISA. GMTAUC was characterized.

[0061] Figures 6A-6C and Figure 6D-6F Spike and RBD-specific antibody titers after multivalent vaccination. (Compared with...) Figure 5A and Figure 5B Related spike specificity against spike mutants (n=10) Figures 6A-6C ) or RBD specificity ( Figure 6D-6FA graph of serum IgG. Antibody binding was measured using W, Delta, Alpha, Beta, Gamma, and Omicron spikes or RBD for different immunoassays. Antibodies in serum after boosting (D43) were measured by ELISA. GMT AUC is plotted. Error bars represent geometrical SD. Geometric mean fold change is added in blue.

[0062] Figures 7A-7C The trivalent NDV-HXP-S (SARS-CoV-2) live vaccine induced a broad antibody response. Mice were vaccinated intramuscularly with the trivalent NDV-HXP-S vaccine, the monovalent NDV-HXP-S (traditional) vaccine, or wild-type (WT) NDV as a negative control. Mice received a primary dose of the trivalent vaccine, monovalent vaccine, or WT NDV intramuscularly, followed by a booster dose 21 days later. Twenty-one days after the booster, mice were sacrificed to harvest blood and spleens to measure serum IgG levels and spike-specific class-switching memory B cells in the spleen. IgG levels were measured by ELISA, and live CD3+ was detected by flow cytometry. - CD220 + CD19 + IgD - IgM - GL7 - CD38 + APC-S + and PE-S + To examine spike-specific class-switching memory B cells in the spleen ( Figure 7A Serum IgG levels were measured against a group of ancestral and variant RBDs, including ancestral, Beta, Gamma, Delta, BA.1, and BA.2 RBDs. Figure 7B Three weeks after the booster, mouse spleens were harvested and examined using a tetramer-SB cell probe to detect ancestral S-binding or BA.1 S-binding class-switching memory B cells (swMBCs). The frequencies of ancestral S-binding or BA.1 S-binding class-switching memory B cells were plotted. Figure 7C ). Detailed Implementation

[0063] 5.1 Recombinant Newcastle disease virus

[0064] In one aspect, this document provides a transgene comprising a chimeric F protein, wherein the chimeric F protein comprises a SARS-CoV-2 extracellular domain or a derivative thereof, and a transmembrane domain and a cytoplasmic domain of the NDV F protein. This document also provides a recombinant NDV comprising a transgene encoding a chimeric F protein, wherein the chimeric F protein comprises a SARS-CoV-2 extracellular domain or a derivative thereof, and a transmembrane domain and a cytoplasmic domain of the NDV F protein. This document also provides a recombinant NDV comprising a chimeric F protein, wherein the chimeric F protein comprises a SARS-CoV-2 extracellular domain or a derivative thereof, and a transmembrane domain and a cytoplasmic domain of the NDV F protein. The recombinant NDV can be administered as a live or inactivated virus. In another aspect, this document provides the recombinant NDV described herein, which can be used to immunize subjects (e.g., human subjects) against SARS-CoV-2 (e.g., SARS-CoV-2 delta variant, SARS-CoV-2 beta variant, SARS-CoV-2 gamma variant, or W strain). The recombinant NDV can be administered as a live or inactivated virus.

[0065] 5.1.1 NDV

[0066] Newcastle disease virus (NDV) is a member of the genus Avulavirus in the family Paramyxoviridae, and has been shown to infect many avian species (Alexander, DJ (1988). Newcastledisease, Newcastledisease virus--an avian paramyxovirus. Kluwer Academic Publishers: Dordrecht, The Netherlands. pp. 1-22). NDV possesses a negative-sense, single-stranded RNA genome and does not recombine with the host genome or other viruses (Alexander, DJ (1988). Newcastledisease, Newcastledisease virus--an avian paramyxovirus. Kluwer Academic Publishers: Dordrecht, The Netherlands. pp. 1-22). The genomic RNA contains a gene with the sequence 3'-NP-PMF-HN-L-5'. NDV generates two additional proteins, V and W, from the P gene via RNA editing-produced alternative mRNA. The genomic RNA also contains a leader sequence at the 3' end.

[0067] The structural components of a viral particle include a viral envelope, a lipid bilayer derived from the cytoplasmic membrane. A hemagglutinin-neuraminidase (HN) glycoprotein protrudes from the envelope, enabling the virus to simultaneously possess hemagglutinin (e.g., for receptor binding / fusion promotion) and neuraminidase activity. A fusion glycoprotein (F) also interacts with the viral membrane, initially produced as an inactive precursor, followed by post-translational cleavage to produce two disulfide-linked polypeptides. The active F protein participates in NDV penetration into host cells by facilitating the fusion of the viral envelope with the host cell membrane. Matrix proteins (M) are involved in viral assembly and interact with viral membrane and nucleocapsid proteins.

[0068] The major protein subunits of the nucleocapsid are nucleocapsid proteins (NPs), which give the capsid helical symmetry. Associated with the nucleocapsid are the P and L proteins. The phosphoprotein (P) undergoes phosphorylation and is thought to play a regulatory role in transcription, and may also be involved in methylation, phosphorylation, and polyadenylation. The L gene, encoding RNA-dependent RNA polymerase, together with the P protein, is essential for viral RNA synthesis. The L protein accounts for nearly half of the coding capacity of the viral genome; it is the largest viral protein and plays a crucial role in transcription and replication.

[0069] Any NDV type or strain can be used as a "backbone" engineered to include the transgenes described herein, including but not limited to naturally occurring strains, variants or mutants, mutagenic viruses, religands, and / or genetically engineered viruses. Examples of transgenes are provided, for example, in Sections 5.1.2 and 6. In one specific embodiment, the transgenes described herein are incorporated into the genome of an attenuated NDV. In another specific embodiment, the transgenes described herein are incorporated into the genome of the NDV strain LaSota. In yet another embodiment, the transgenes described herein are incorporated into the genome of the NDV strain Hitchner B1. In some embodiments, attenuated strains other than the NDV Hitchner B1 strain are used as a backbone in which nucleotide sequences can be incorporated. The transgene can be incorporated between two transcription units in the NDV genome (e.g., between NDV M and P transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units).

[0070] In one specific embodiment, the NDV engineered to contain the transgene described herein is a naturally occurring strain. Specific examples of NDV strains include, but are not limited to, the Hitchner B1 strain (see, for example, GenBank numbers AF309418 or NC_002617) and the LaSota strain (see, for example, GenBank numbers AY845400, AF07761.1, JF950510.1, and GI number 56799463). In one specific embodiment, the NDV engineered to contain the transgene described herein is the Hitchner B1 strain. In another specific embodiment, the NDV engineered to contain the transgene described herein is the B1 strain identified by GenBank number AF309418 or NC_002617. In one specific embodiment, the nucleotide sequence of the Hitchner B1 genome contains an RNA sequence corresponding to the negative sense strand of the cDNA sequence shown in SEQ ID NO:2. In another specific embodiment, the NDV engineered to contain the transgene described herein is the LaSota strain. In another embodiment, the NDV engineered to contain the transgene described herein is a LaSota strain identified as AY845400, AF07761.1, or JF950510.1. In one specific embodiment, the nucleotide sequence of the LaSota genome comprises an RNA sequence corresponding to the negative sense strand of the cDNA sequence shown in SEQ ID NO:1. In another specific embodiment, the nucleotide sequence of the LaSota genome comprises an RNA sequence corresponding to the negative sense strand of the cDNA sequence shown in SEQ ID NO:3. Those skilled in the art will understand that the NDV genomic RNA sequence is an RNA sequence corresponding to the negative sense strand of the cDNA sequence encoding the NDV genome. Therefore, any procedure for converting nucleotide sequences into their inverse complementary sequences can be used to convert the cDNA sequence encoding the NDV genome into a genomic RNA sequence (see, for example, www.bioinformatics.org / sms / rev_comp.html, www.fr33.net / seqedit.php, and DNAStar). Therefore, the nucleotide sequences provided in Tables 1 through 4 below can be readily converted by those skilled in the art into negative sense RNA sequences for the NDV genome.

[0071] In one specific embodiment, the NDV engineered to contain the transgene described herein contains a genome encoding the NDV F protein, wherein the leucine residue at amino acid position 289 of the NDV F protein is replaced with alanine (as described, for example, by Sergel et al., 2000, Journal of Virology 74:5101-5107). In another specific embodiment, the NDV engineered to contain the transgene described herein contains a genome encoding the NDV F protein, wherein the leucine residue at amino acid position 289 of the NDV F protein (based on the LaSota strain F protein) is replaced with alanine. In another specific embodiment, the NDV engineered to contain the transgene described herein contains a nucleotide sequence encoding the NDV F protein, wherein the leucine corresponding to amino acid position 289 of the LaSota NDV F protein is replaced with alanine. In yet another specific embodiment, the NDV engineered to contain the transgene described herein contains a nucleotide sequence encoding the NDV F protein, wherein the leucine residue at amino acid position 289 of the LaSota NDV F protein is replaced with alanine. In another specific embodiment, the NDV engineered to include the transgene described herein belongs to the LaSota strain (e.g., GenBank accession number AY845400, AF07761.1, or JF950510.1), and the genome of the LaSota strain encodes the NDV F protein, wherein the leucine residue at amino acid position 289 of the NDV F protein is replaced by alanine. In another specific embodiment, the NDV engineered to include the transgene described herein belongs to the LaSota strain (e.g., GenBank accession number AY845400, AF07761.1, or JF950510.1), and the genome of the LaSota strain contains a nucleotide sequence encoding the LaSota NDV F protein, wherein the leucine residue at amino acid position 289 of the NDV F protein (based on the LaSota strain F protein) is replaced by alanine. In another specific embodiment, the NDV engineered to include the transgene described herein belongs to the Hitchner B1 strain (e.g., GenBank accession number AF309418 or NC_002617), and the genome of the Hitchner B1 strain encodes the NDV F protein, wherein the leucine residue at amino acid position 289 of the NDV F protein (based on the LaSota strain F protein) is replaced by alanine.

[0072] In some embodiments, the NDV engineered to include the transgene described herein belongs to the Fuller strain. In some embodiments, the NDV genome engineered to include the transgene described herein belongs to the Ulster strain. In some embodiments, the NDV engineered to include the transgene described herein belongs to the Roakin strain. In some embodiments, the NDV engineered to include the transgene described herein belongs to the Komarov strain. In some embodiments, the NDV engineered to include the transgene described herein belongs to the Roakin strain. In some embodiments, the NDV engineered to include the transgene described herein belongs to the r73T-R116 virus.

[0073] In specific embodiments, the NDV engineered to contain the transgene described herein is non-pathogenic in birds, as assessed by techniques known to those skilled in the art. In some specific embodiments, such as by intracranial injection of the virus into 1-day-old chicks and scoring disease progression and mortality over 8 days, the NDV engineered to contain the transgene described herein is non-pathogenic. In some embodiments, the intracranial pathogenicity index of the NDV engineered to contain the transgene described herein is less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1. In some embodiments, the intracranial pathogenicity index of the NDV engineered to contain the transgene described herein is zero. For a description of this assay, see, for example, OIE Terrestrial Manual 2012, Chapter 2.3.14, entitled “Newcastle Disease (Infection With Newcastle Disease Virus),” which is available at: www.oie.int / fileadmin / Home / eng / Health_standards / tahm / 2.03.14_NEWCASTLE_DIS.pdf Its full text is incorporated into this article by quotation.

[0074] In some implementations, the NDV engineered to include the transgene described herein is a toxic strain that has been genetically engineered so that, as assessed by techniques known to those skilled in the art, it is considered non-pathogenic in birds.

[0075] In a preferred embodiment, the NDV engineered to contain the transgene described herein is nonpathogenic in humans. In a preferred embodiment, the NDV engineered to contain the transgene described herein is nonpathogenic in both humans and poultry. In some embodiments, the NDV engineered to contain the transgene described herein is attenuated such that the NDV retains at least partially infectivity and can replicate in vivo, but produces only low titers, resulting in a nonpathogenic, subclinical level of infection (see, for example, Khattar et al., 2009, J. Virol. 83:7779-7782). Such attenuated NDVs may be particularly suitable for implementations in which the virus is administered to subjects as an immunogen (e.g., a live vaccine). These viruses can be attenuated by any method known in the art. In one specific embodiment, the NDV genome contains sequences necessary for viral infection and replication, such that offspring are produced and the level of infection is subclinical. In some embodiments, the NDV is attenuated by introducing one, two, or more mutations (e.g., amino acid substitutions) into the NDV V protein.

[0076] In one specific embodiment, this document provides a nucleotide sequence comprising: (1) an NDV F transcription unit, (2) an NDV NP transcription unit, (3) an NDV P transcription unit, (4) an NDV M transcription unit, (5) an NDV HN transcription unit, (6) an NDV L transcription unit, and (7) the transgene described herein. In some embodiments, these NDV transcription units are LaSota NDV transcription units. In one specific embodiment, this document provides a nucleotide sequence comprising: (1) an NDV F transcription unit, (2) an NDV NP transcription unit, (3) an NDV P transcription unit, (4) an NDV M transcription unit, (5) an NDV HN transcription unit, (6) an NDV L transcription unit, and (7) the transgene described herein, wherein the NDV F transcription unit encodes an NDV F protein having an amino acid substitution from leucine to alanine at amino acid residue 289 corresponding to amino acid position 289 of the LaSota NDV F protein. In another specific embodiment, this document provides a nucleotide sequence comprising: (1) an NDV F transcription unit, (2) an NDVNP transcription unit, (3) an NDV P transcription unit, (4) an NDV M transcription unit, (5) an NDV HN transcription unit, (6) an NDV L transcription unit, and (7) the transgene described herein, wherein the NDV F transcription unit encodes an NDV F protein having an amino acid substitution from leucine to alanine at amino acid position 289 of the LaSota NDV F protein. In some embodiments, these NDV transcription units are LaSota NDV transcription units. In some embodiments, the nucleotide sequence is part of a vector (e.g., a plasmid, such as those described in the examples below). In a specific embodiment, the nucleotide sequence is isolated.

[0077] In one specific embodiment, this document provides a polynucleotide sequence comprising: (1) a nucleotide sequence encoding NDV F, (2) a nucleotide sequence encoding NDV NP, (3) a nucleotide sequence encoding NDV P, (4) a nucleotide sequence encoding NDV M, (5) a nucleotide sequence encoding NDV HN, (6) a nucleotide sequence encoding NDV L, and (7) the transgene described herein. In another specific embodiment, this document provides a polynucleotide sequence comprising: (1) a nucleotide sequence encoding NDV F, (2) a nucleotide sequence encoding NDV NP, (3) a nucleotide sequence encoding NDV P, (4) a nucleotide sequence encoding NDV M, (5) a nucleotide sequence encoding NDV HN, (6) a nucleotide sequence encoding NDV L, and (7) the transgene described herein, wherein NDV F contains an amino acid substitution from leucine to alanine at amino acid position corresponding to amino acid residue 289 of LaSota NDV F. In another specific embodiment, this document provides a polynucleotide sequence comprising: (1) a nucleotide sequence encoding NDV F, (2) a nucleotide sequence encoding NDV NP, (3) a nucleotide sequence encoding NDV P, (4) a nucleotide sequence encoding NDV M, (5) a nucleotide sequence encoding NDV HN, (6) a nucleotide sequence encoding NDV L, and (7) the transgene described herein, wherein NDV F contains an amino acid substitution from leucine to alanine at amino acid position 289 of LaSotaNDV F. In some embodiments, these NDV proteins are LaSotaNDV proteins. In another specific embodiment, this document provides a polynucleotide sequence comprising a nucleotide sequence of an NDV genome known or described in the art (see, for example, Section 5.1 or the examples below; also see SEQ ID NO: 1, 2 or 3) and the transgene described herein. In some embodiments, the nucleic acid sequence is part of a vector (e.g., a plasmid, such as those described in the examples below). In one specific embodiment, the polynucleotide sequence is isolated.

[0078] In specific embodiments, the polynucleotide sequence, nucleic acid sequence, or nucleotide sequence described herein is the recombinant polynucleotide sequence, recombinant nucleic acid sequence, or recombinant nucleotide sequence described herein. In some embodiments, the polynucleotide sequence, nucleotide sequence, or nucleic acid sequence described herein may be a DNA molecule (e.g., cDNA), an RNA molecule (e.g., mRNA), or a combination of DNA and RNA molecules. In some embodiments, the polynucleotide sequence, nucleotide sequence, or nucleic acid sequence described herein may comprise analogs of DNA or RNA molecules. Such analogs may be produced using, for example, nucleotide analogs, including but not limited to inosine, methylcytosine, pseudouridine, or triphenylmethylated bases. Such analogs may also comprise DNA or RNA molecules containing a modified backbone that imparts beneficial properties to the molecule, such as nuclease resistance or increased ability to cross cell membranes. The polynucleotide sequence, nucleic acid sequence, or nucleotide sequence may be single-stranded, double-stranded, may contain single-stranded and double-stranded portions, and may contain triple-stranded portions. In one specific embodiment, the polynucleotide sequence, nucleotide sequence, or nucleic acid sequence described herein is a negative-sense single-stranded RNA. In another specific embodiment, the polynucleotide sequence, nucleotide sequence, or nucleic acid sequence described herein is a positive-sense single-stranded RNA. In another specific embodiment, the polynucleotide sequence, nucleotide sequence, or nucleic acid sequence described herein is cDNA.

[0079] In some embodiments, this document provides a recombinant NDV comprising the SARS-CoV-2 spike protein or a portion thereof (e.g., the extracellular domain or receptor-binding domain of the SARS-CoV-2 spike protein) or a derivative thereof. In one specific embodiment, this document provides a recombinant NDV comprising a protein containing the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof. In another specific embodiment, this document provides a recombinant NDV comprising a protein containing a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In yet another specific embodiment, this document provides a recombinant NDV comprising a chimeric F protein, wherein the chimeric F protein comprises the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof, as well as the transmembrane domain and cytoplasmic domain of the NDV F protein. In one specific embodiment, this document provides a recombinant NDV comprising a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 spike protein, as well as the transmembrane domain and cytoplasmic domain of the NDV F protein. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the derivative of the SARS-CoV-2 extracellular domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In some embodiments, the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof is encoded by a codon-optimized nucleic acid sequence. In some embodiments, the derivative of the SARS-CoV-2 spike protein is encoded by the nucleotide sequence of SEQ ID NO: 20, 21, 26, 27, 31, 32, 35, or 36. In some embodiments, the derivatives of the SARS-CoV-2 spike protein are encoded by nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the nucleotide sequences of SEQ ID NO: 20, 21, 26, 27, 31, 32, 35, or 36. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein belong to the LaSota or Hitchner strains. In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein comprise SEQ ID NO: 42. In some embodiments, the extracellular domains of the SARS-CoV-2 spike protein and the derivatives of the transmembrane domain and cytoplasmic domain of the NDV F protein are linked via a linker (e.g., a linker described herein, such as SEQ ID NO: 7) or directly linked.

[0080] In some embodiments, this document provides a recombinant NDV comprising a chimeric F protein. In some embodiments, the chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 11, 18, 22, 28, 39, 40, or 41. In some embodiments, the chimeric F protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, 11, 18, 22, 28, 39, 40, or 41. In some embodiments, the chimeric F protein is encoded by a codon-optimized nucleic acid sequence. In some embodiments, the chimeric F protein is encoded by a nucleotide sequence of SEQ ID NO: 5, 19, 25, 33, 34, 37, or 38. In some embodiments, the chimeric F protein is encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the nucleotide sequence of SEQ ID NO: 5, 19, 25, 33, 34, 37, or 38.

[0081] 5.1.2 SARS-CoV-2 variant spike protein / having the extracellular domain of SARS-CoV-2 variant spike protein or other variant spike protein Chimeric F protein of derivatives

[0082] In one specific embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising the SARS-CoV-2 spike protein or a portion thereof (e.g., an extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 spike protein). In another specific embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., an extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein). In yet another specific embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., an extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein). In another specific embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein). In another specific embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising the SARS-CoV-2W strain spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W spike protein).

[0083] In one specific embodiment, a transgene containing a nucleotide sequence encoding a protein (e.g., the NDV LaSota strain) is incorporated into the genome of any NDV type or strain, the protein of which comprises the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. Transgenes encoding the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein) can be inserted into any NDV type or strain (e.g., the NDV LaSota strain). In one specific embodiment, a transgene encoding the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein) is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). In one specific embodiment, the SARS-CoV-2 delta variant belongs to the B1.617.2 sublineage. In some embodiments, the SARS-CoV-2 delta variant belongs to the AY sublineage. See, for example, in Section 3.1, exemplary sequences of the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein) and exemplary nucleic acid sequences encoding the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein). Those skilled in the art will be able to use this sequence information to generate transgenes for incorporation into the genome of any NDV type or strain. Given the degeneracy of the nucleic acid codon, many different polynucleotide sequences can encode the same SARS-CoV-2 delta variant spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein). In one specific embodiment, the transgene encoding the SARS-CoV-2 delta variant spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein) is codon-optimized. For a related discussion of codon optimization, see, for example, Section 5.1.4 below.In some embodiments, the transgene encoding the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein) does not contain the SARS-CoV-2 delta variant spike protein signal peptide. The transgene encoding the SARS-CoV-2 delta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 delta variant spike protein) can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between HN and L transcription units).

[0084] In another specific embodiment, a transgene containing a nucleotide sequence encoding a protein (e.g., the NDV LaSota strain) is incorporated into the genome of any NDV type or strain, the protein of which comprises the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. A transgene encoding the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein) can be inserted into any NDV type or strain (e.g., the NDV LaSota strain). In one specific embodiment, a transgene encoding the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein) is incorporated into the genome of any NDV type or lineage (e.g., the NDV LaSota lineage). In one specific embodiment, the SARS-CoV-2 beta variant belongs to the B.1.351 lineage or its descendants. See, for example, in Section 3.1, exemplary sequences of the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein) and exemplary polynucleotide sequences encoding the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein). Those skilled in the art will be able to use this sequence information to generate transgenes for incorporation into the genome of any NDV type or strain. Given the degeneracy of the nucleic acid codon, many different polynucleotide sequences can encode the same SARS-CoV-2 beta variant spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein). In one specific embodiment, the transgene encoding the SARS-CoV-2 beta variant spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein) is codon-optimized. For a related discussion of codon optimization, see, for example, Section 5.1.4 below.In some embodiments, the transgene encoding the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein) does not contain the SARS-CoV-2 beta variant spike protein signal peptide. The transgene encoding the SARS-CoV-2 beta variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 beta variant spike protein) may be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between HN and L transcription units).

[0085] In another specific embodiment, a transgene containing a nucleotide sequence encoding a protein (e.g., the NDV LaSota strain) is incorporated into the genome of any NDV type or strain, the protein comprising the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein). The transgene encoding the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein) can be inserted into any NDV type or strain (e.g., the NDV LaSota strain). In one specific embodiment, a transgene encoding the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein) is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). In one specific embodiment, the SARS-CoV-2 gamma variant belongs to the P.1 lineage or its descendants, or as described in Section 6. See, for example, Section 3.1, exemplary sequences of the SARS-CoV-2 gamma variant spike protein or portions thereof (e.g., the extracellular domains, S1 domains, S2 domains, or receptor-binding domains of the SARS-CoV-2 gamma variant spike protein) and exemplary polynucleotide sequences encoding the SARS-CoV-2 gamma variant spike protein or portions thereof (e.g., the extracellular domains, S1 domains, S2 domains, or receptor-binding domains of the SARS-CoV-2 gamma variant spike protein). Those skilled in the art will be able to use this sequence information to generate transgenes for incorporation into the genome of any NDV type or strain. Given the degeneracy of the nucleic acid code, many different polynucleotide sequences can encode the same SARS-CoV-2 gamma variant spike protein or portions thereof (e.g., the extracellular domains, S1 domains, S2 domains, or receptor-binding domains of the SARS-CoV-2 gamma variant spike protein). In one specific implementation, the transgene encoding the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein) is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below.In some embodiments, the transgene encoding the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein) does not contain the SARS-CoV-2 gamma variant spike protein signal peptide. The transgene encoding the SARS-CoV-2 gamma variant spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2 gamma variant spike protein) can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between HN and L transcription units).

[0086] In another specific embodiment, a transgene containing a nucleotide sequence encoding a protein (e.g., the NDV LaSota strain) is incorporated into the genome of any NDV type or strain, the protein comprising the SARS-CoV-2W spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W spike protein). The transgene encoding the SARS-CoV-2W strain spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W strain spike protein) can be inserted into any NDV type or strain (e.g., the NDV LaSota strain). In one specific embodiment, a transgene encoding the SARS-CoV-2W strain spike protein or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W strain spike protein) is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). Those skilled in the art will be able to use this sequence information to generate transgenes for incorporation into the genome of any NDV type or strain. Given the degeneracy of the nucleic acid codon, many different polynucleotide sequences can encode the same SARS-CoV-2W strain spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W strain spike protein). In one specific embodiment, the transgene encoding the SARS-CoV-2W strain spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W strain spike protein) is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below. In some embodiments, the transgene encoding the SARS-CoV-2W strain spike protein or portions thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the SARS-CoV-2W strain spike protein) does not contain the SARS-CoV-2W strain spike protein signal peptide. Transgenic material encoding the spike protein of the SARS-CoV-2W strain or a portion thereof (e.g., the extracellular domain, S1 domain, S2 domain, or receptor-binding domain of the spike protein of the SARS-CoV-2W strain) can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between HN and L transcription units).

[0087] In some embodiments, a portion of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises a receptor-binding domain of the SARS-CoV-2 spike protein. In some embodiments, a portion of the SARS-CoV-2 spike protein comprises a receptor-binding domain of the SARS-CoV-2 protein. In some embodiments, a portion of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises a receptor-binding domain of the SARS-CoV-2 spike protein and 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the receptor-binding domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). The 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the receptor-binding domain of the spike protein (or gamma variant), or the 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the receptor-binding domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) and the 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the receptor-binding domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant).In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises a receptor-binding domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the receptor-binding domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) consists of 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the receptor-binding domain, or 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the receptor-binding domain, and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the receptor-binding domain, respectively. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein.

[0088] In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein includes the S1 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein includes the S1 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the S1 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the S1 domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant), or the 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the S1 domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) and the 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the S1 domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant).In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises the S1 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the S1 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The spike protein may be 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the S1 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant), or 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the S1 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the S1 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein.

[0089] In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein includes the S2 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein includes the S2 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the S2 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the S2 domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant), or the 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the S2 domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) and the 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the S2 domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant).In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises the S2 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the S2 domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The spike protein may be 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the S2 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant), or 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the S2 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the S2 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein.

[0090] In some embodiments, a portion of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) comprises the S1 and S2 domains of the SARS-CoV-2 spike protein. In some embodiments, a portion of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) comprises the S1 and S2 domains of the SARS-CoV-2 spike protein and 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the S1 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). The spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) contains 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the S2 domain, or the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) contains 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the S1 domain, or the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) contains 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus of the S2 domain, or the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant).In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises the S1 and S2 domains of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the S1 domain of the SARS-CoV-2 spike protein. The spike protein may contain 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the S2 domain, or 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the S1 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the S2 domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein.

[0091] In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises an extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises an extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus of the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The extracellular domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) contains 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus, or the extracellular domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) contains 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the N-terminus, or the extracellular domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) contains 5, 10, 15, 20, 30, 40, 50, 75 or more amino acid residues at the C-terminus, or the extracellular domain of the spike protein of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant).In some embodiments, a portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein comprises an extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. The spike protein may be 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant), or 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the N-terminus of the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) and 5 to 25, 5 to 50, 25 to 50, 25 to 75, or 50 to 75 amino acid residues at the C-terminus of the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein.

[0092] In some embodiments, a portion of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises 200, 220, 222, 250, 300, 350, 400, or more amino acid residues. In some embodiments, a portion of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or more amino acid residues. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In another specific implementation, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein.

[0093] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a fragment of the full-length SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In some embodiments, the protein further comprises a domain that facilitates the purification, folding, and cleavage of the polypeptide moiety. For example, a His tag His-His-His-His-His-His (SEQ ID NO:44)), a FLAG epitope, or other purification tags can facilitate the purification of the protein provided herein. In some embodiments, the His tag has a sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In some embodiments, the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) has a fragment length of at least 1000, 1025, 1075, 1100, 1125, 1150, 1200, or 1215 amino acid residues. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 W strain spike protein. In some embodiments, SARS-CoV-2 belongs to the B1.617.2 sublineage. In some embodiments, SARS-CoV-2 belongs to the AY sublineage.

[0094] In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a nucleotide sequence of the spike protein of SAR-CoV-2 (e.g., the W strain, delta variant, beta variant, or gamma variant) or a portion thereof (e.g., extracellular domain, S1 domain, S2 domain, or receptor-binding domain) or a fragment thereof. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a nucleotide sequence of the spike protein of SAR-CoV-2 (e.g., the W strain, delta variant, beta variant, or gamma variant) or a portion thereof (e.g., extracellular domain, S1 domain, S2 domain, or receptor-binding domain) or a fragment thereof. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of the spike protein of SARS-CoV-2 (e.g., the W strain, delta variant, beta variant, or gamma variant) or a portion thereof (e.g., extracellular domain, S1 domain, S2 domain, or receptor-binding domain) or a fragment thereof. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 W strain spike protein. Methods / techniques known in the art can be used to determine sequence identity (see, for example, the “Best Fit” or “Gap” program of a sequence analysis software package, version 10; Genetics Computer Group, Inc.). In some embodiments, the protein further comprises one or more polypeptide domains. These polypeptide domains may be located at the C-terminus or N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (SEQ ID NO:44), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein.In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44). In some embodiments, the SARS-CoV-2 spike protein fragment length is at least 250, at least 500, at least 750, at least 1000, at least 1025, at least 1075, at least 1100, at least 1125, at least 1150, at least 1175, at least 1200, or at least 1215 amino acid residues.

[0095] Techniques known to those skilled in the art can be used to determine the percentage of identity between two amino acid sequences or two nucleotide sequences. Typically, to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., vacancies may be introduced into the first amino acid or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. In another embodiment, the percentage of identity is determined over the full length of the amino acid or nucleotide sequence. In some embodiments, the length of the sequence identity comparison may exceed the full length of the two sequences being compared (e.g., the full length of a gene coding sequence or a fragment thereof). In some embodiments, the nucleotide sequence fragment is at least 25, at least 50, at least 75, or at least 100 nucleotides. Similarly, the "sequence identity percentage" of the amino acid sequence on the full length of a protein or a fragment thereof can be readily determined. In some embodiments, the protein fragment contains at least 20, at least 30, at least 40, at least 50 or more consecutive amino acids of the protein. In some embodiments, the protein fragment contains at least 75, at least 100, at least 125, at least 150 or more consecutive amino acids of the protein. In some embodiments, the protein fragment contains at least 175, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, or at least 1250, at least 1300 or more consecutive amino acids of the protein. In some embodiments, the protein fragment comprises 100 to 1300 consecutive amino acids, 100 to 1250 consecutive amino acids, 100 to 1100 consecutive amino acids, 100 to 1000 consecutive amino acids, 500 to 1300 consecutive amino acids, 500 to 1250 consecutive amino acids, 500 to 1100 consecutive amino acids, or 500 to 100 consecutive amino acids. In some embodiments, the protein fragment comprises 50 to 500 consecutive amino acids, 50 to 250 consecutive amino acids, 100 to 500 consecutive amino acids, 200 to 500 consecutive amino acids, 100 to 400 consecutive amino acids, 100 to 300 consecutive amino acids, 200 to 400 consecutive amino acids, 100 to 200 consecutive amino acids, or 200 to 300 consecutive amino acids.

[0096] The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, improved in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm was incorporated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using, for example, a set of NBLAST nucleotide procedure parameters of fraction = 100 and word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using, for example, a set of XBLAST procedure parameters of fraction = 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain vacancy alignments for comparison purposes, GappedBLAST can be used as described by Altschul et al., 1997, Nucleic Acids Res. 25:33893402. Alternatively, PSI BLAST can be used for iterative searches that detect distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. This algorithm was incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residual table, vacancy length penalty 12, and vacancy penalty 4 can be used.

[0097] Similar techniques to those described above can be used to determine the percentage of identity between two sequences, with or without gaps. When calculating the percentage of identity, typically only exact matches are counted.

[0098] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids are substituted by another amino acid (e.g., conserved amino acid substitution). In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the C-terminus are substituted by another amino acid (e.g., conserved amino acid substitution). In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids substituted at the N-terminus by another amino acid (e.g., a conserved amino acid substitution). In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids substituted at the N-terminus by another amino acid (e.g., a conserved amino acid substitution) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids substituted at the C-terminus by another amino acid (e.g., a conserved amino acid substitution). In one embodiment, the N-terminus is the first 100 amino acid residues of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In another embodiment, the C-terminus is the last 100 amino acid residues of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In this embodiment, the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is the mature form of the protein. In other embodiments, the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is the immature form of the protein.In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. Examples of conserved amino acid substitutions include, for example, replacing one class of amino acids with another amino acid of the same class. In a particular embodiment, the conserved substitution does not alter the structure or function of the polypeptide, or both. Amino acid classes may include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformational disruptors (Gly, Pro), and aromatic (Trp, Tyr, Phe). In some embodiments, the protein also comprises one or more polypeptide domains. The one or more polypeptide domains may be located at the C-terminus or N-terminus. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags His-His-His-His-His-His (SEQ ID NO:44), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).

[0099] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) with a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) with a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids from the C-terminus. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or composed of) the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the N-terminus. In one specific embodiment, the N-terminus is the first 100 amino acid residues of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. In one specific embodiment, the C-terminus is the last 100 amino acid residues of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. In a specific embodiment, the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein is the mature form of the protein. In other embodiments, the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is an immature form of the protein. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus or N-terminus. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of the polypeptide moieties.For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).

[0100] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations (e.g., amino acid substitutions, amino acid deletions, amino acid additions, or combinations thereof). In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid deletions and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In some embodiments, the mutation (e.g., amino acid substitution, amino acid deletion, amino acid addition, or a combination thereof) is located at the N-terminus, C-terminus, or both ends. In one specific embodiment, the N-terminus is the first 100 amino acid residues of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the C-terminus is the last 100 amino acid residues of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) is the mature form of the protein. In other embodiments, the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) is the immature form of the protein. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus, N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties.For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).

[0101] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) a receptor-binding domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of the polypeptide moieties. For example, His tags His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one specific embodiment, the protein comprises or is composed of the receptor-binding domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a His tag (e.g., (His)n, where n is 6 (SEQ ID NO: 44)). In some embodiments, the protein comprising (or composed of) the receptor-binding domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike polypeptide / spike protein is a secreted polypeptide. In one specific embodiment, when designing proteins comprising the spike polypeptide / spike protein receptor-binding domain of SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant), care is taken to maintain the stability of the resulting protein. Additionally, in some embodiments, when designing proteins comprising the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof, care is taken to maintain the conformation of the spike protein such that, for example, it can bind to human angiotensin-converting enzyme-2.

[0102] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or consisting of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids are substituted with another amino acid (e.g., a conserved amino acid substitution). In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or consisting of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the C-terminus of the receptor-binding domain are substituted with another amino acid (e.g., a conserved amino acid substitution). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the N-terminus of the receptor-binding domain are replaced by another amino acid (e.g., a conserved amino acid substitution). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) a receptor-binding domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the N-terminus of the receptor-binding domain are substituted with another amino acid (e.g., a conserved amino acid substitution) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the C-terminus of the receptor-binding domain are substituted with another amino acid (e.g., a conserved amino acid substitution). In one specific embodiment, the N-terminus is the first 25 amino acid residues of the receptor-binding domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein. In one specific embodiment, the C-terminus is the last 25 amino acid residues of the receptor-binding domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein.In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. Examples of conserved amino acid substitutions include, for example, replacing one class of amino acids with another amino acid of the same class. In a particular embodiment, the conserved substitution does not alter the structure or function of the polypeptide, or both. Amino acid classes may include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformational disruptors (Gly, Pro), and aromatic (Trp, Tyr, Phe). In some embodiments, the protein also comprises one or more polypeptide domains. The one or more polypeptide domains may be located at the C-terminus, N-terminus, or C-terminus and N-terminus. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).

[0103] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or consisting of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the C-terminus of the receptor-binding domain. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or consisting of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the N-terminus of the receptor-binding domain. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the N-terminus of the receptor-binding domain and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the C-terminus of the receptor-binding domain. In one specific embodiment, the N-terminus is the first 25 amino acid residues of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain. In one specific embodiment, the C-terminus is the last 25 amino acid residues of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein receptor-binding domain. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus or N-terminus. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties.For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).

[0104] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a protein that comprises (or is composed of) the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a protein that comprises (or is composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein that comprises (or is composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), wherein the derivative lacks a multibase cleavage site of the extracellular domain (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with other amino acid residues). In one specific embodiment, amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 are substituted with a single alanine. In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein, with amino acid substitutions for proline at amino acid residues corresponding to amino acid residues 817, 892, 899, 942, 986, and 987. In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein, wherein amino acid residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with a single alanine, and amino acid residues corresponding to amino acid residues 817, 892, 899, 942, 986, and 987 are substituted with proline.In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, eight, or all of the following amino acid modifications: T19R, G142D, delE156, delF157, R158G, L452R, T478K, D614G, P681R, and D950N. In a specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) does not contain the amino acid substitution of P681R. In one specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, or all of the following amino acid modifications: T19R, G142D, delE156, delF157, R158G, L452R, T478K, D614G, and D950N. In another specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having the following amino acid modifications: T19R, G142D, delE156, delF157, R158G, L452R, T478K, D614G, and D950N. In another specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or all of the following amino acid modifications: T19R, G142D, delE156, delF157, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R, and D950N. In another specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) does not contain the amino acid substitution of P681R.In some implementations, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all of the following amino acid modifications: T19R, G142D, delE156, delF157, R158G, A222V, W258L, K417N, L452R, T478K, D614G, and D950N. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having the following amino acid modifications: T19R, G142D, delE156, delF157, R158G, A222V, W258L, K417N, L452R, T478K, D614G, and D950N. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:16 having one, two, three, four, five, six, seven, eight, or all of the following amino acid modifications: T6R, G129D, delE143, delF144, R145G, L439R, T465K, D601G, and D937N. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, eight, nine, or more of the following modifications: L18F, D80A, L246del, L247del, E484K, N501Y, D614G, and A701V. In some implementations, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, eight, nine, or more of the following modifications: L19R, G142D, G157del, G158del, L452R, T478K, D614G, and D950N.In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) comprises the amino acid sequence of SEQ ID NO:12 having one, two, three, four, five, six, seven, eight, nine, or more of the following modifications: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614N, H655Y, and T1027I. In some embodiments, the protein also comprises one or more polypeptide domains. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags His-His-His-His-His-His (SEQ ID NO:44), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one specific embodiment, the protein comprises an extracellular domain of the SARS-CoV-2 spike protein and a His tag (e.g., (His)n, where n is 6 (SEQ ID NO: 44)) or is composed of the extracellular domain and the His tag. In some embodiments, the protein comprising an extracellular domain of the SARS-CoV-2 spike polypeptide or a derivative thereof (or composed of the extracellular domain or a derivative thereof) is a secreted polypeptide. In some embodiments, the protein comprising an extracellular domain of the SARS-CoV-2 spike polypeptide or a derivative thereof comprises one or more trimerizing domains known to those skilled in the art (e.g., T4 fold trimerizing domains) and optional tags (e.g., His tags or Flag tags). In one specific implementation, when designing proteins containing the extracellular domain of the SARS-CoV-2 spike polypeptide or a derivative thereof, care is taken to maintain the stability of the resulting proteins.

[0105] In some embodiments, this document describes transgenic structures comprising a polynucleotide sequence encoding a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the polynucleotide sequence comprises a nucleotide sequence having at least 80%, at least 85%, or at least 90% identity with the nucleotide sequence of SEQ ID NO: 20, 21, 26, 27, 31, 32, 35, or 36. In some embodiments, this document describes transgenic structures comprising a polynucleotide sequence encoding a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the polynucleotide sequence comprises a nucleotide sequence having at least 95%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 20, 21, 26, 27, 31, 32, 35, or 36. In some embodiments, this document describes transgenic structures comprising a polynucleotide sequence encoding a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the polynucleotide sequence comprises a nucleotide sequence of SEQ ID NO: 20, 21, 26, 27, 31, 32, 35, or 36.

[0106] In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein comprising a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein comprising (or composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the derivative comprises the amino acid sequence of GenBank accession number MN908947.3, wherein amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 are substituted with a single alanine, and corresponding to GenB The amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the following positions in ANK accession number MN908947.3 are substituted: 18, 19, 20, 26, 80, 138, 142, 156, 190, 215, 246, 417, 452, 478, 484, 501, 614, 655, 701, 950, and 1027. In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein comprising (or composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the derivative comprises the amino acid sequence of GenBank accession number MN908947.3, wherein (i) amino acid residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with a single alanine, and (ii) amino acid residues corresponding to the following amino acid residues of GenBank accession number MN908947.3 are substituted: F817P A892P, A899P, A942P, K986P, and V987P, and (iii) amino acid variations at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of GenBank accession number MN908947.3: 18, 19, 20, 26, 80, 138, 142, 156, 190, 215, 246, 417, 452, 478, 484, 501, 614, 655, 701, 950, and 1027.

[0107] In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein comprising (or composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the derivative comprises the amino acid sequence of GenBank accession number MN908947.3, wherein (i) amino acid residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with a single alanine, and (ii) amino acid residues corresponding to GenBank accession number MN908947.3 are substituted with a single alanine. Amino acid substitutions of the following amino acid residues in k-accession number MN908947.3: F817P, A892P, A899P, A942P, K986P and V987P, and (iii) corresponding to 1, 2, 3, 4, 5, 6, 7, 8 or all of the following amino acid changes: L18F, D80A, L242del, A243del, L244del, R246I, K417N, E484K, N501Y, D614G and A701V. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 having 1, 2, 3, 4, 5, 6, 7, 8 or all of the following amino acid variations: L18F, D80A, L242del, A243del, L244del, R246I, K417N, E484K, N501Y, D614G and A701V.

[0108] In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein comprising (or composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the derivative comprises GenBank accession number NM908947.3, wherein (i) amino acid residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with a single alanine, and (ii) amino acid residues corresponding to GenBank accession number MN908947.3 are substituted with a single alanine. Amino acid substitutions of the following amino acid residues in catalog number MN908947.3: F817P, A892P, A899P, A942P, K986P and V987P, and (iii) amino acid substitutions corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the following: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614N, H655Y and T1027I. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 having substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the following amino acid substitutions: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614N, H655Y and T1027I.

[0109] In some embodiments, this document describes a transgene comprising a nucleotide sequence encoding a protein comprising (or composed of) a derivative of the extracellular domain of the SARS-CoV-2 spike protein, wherein the derivative comprises GenBank accession number NM908947.3, wherein (i) amino acid residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with a single alanine, (ii) amino acid substitutions corresponding to the following amino acid residues of GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P, and (iii) amino acid changes corresponding to 1, 2, 3, 4, 5, 6, 7, 8, or all of the following: L19R, G142D, G157del, G158del, L452R, T478K, D614G, and D950N. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO:12 having 1, 2, 3, 4, 5, 6, 7, 8 or all of the following amino acid substitutions: L19R, G142D, G157del, G158del, L452R, T478K, D614G and D950N.

[0110] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein extracellular domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids are substituted with another amino acid (e.g., a conserved amino acid substitution). In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein extracellular domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the C-terminus of the extracellular domain are substituted with another amino acid (e.g., a conserved amino acid substitution). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein extracellular domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the N-terminus of the extracellular domain are replaced by another amino acid (e.g., a conserved amino acid substitution). In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein extracellular domain, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the N-terminus of the extracellular domain are substituted by another amino acid (e.g., a conserved amino acid substitution) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the C-terminus of the extracellular domain are substituted by another amino acid (e.g., a conserved amino acid substitution). In one specific embodiment, the C-terminus of the extracellular domain comprises the last 100 amino acid residues. In one specific embodiment, the N-terminus of the extracellular domain comprises the first 100 amino acid residues. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. Examples of conserved amino acid substitutions include, for example, replacing one class of amino acids with another amino acid of the same class.In one particular embodiment, the conserved substitution does not alter the structure or function of the polypeptide, or both. Amino acid classes may include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformational disruptors (Gly, Pro), and aromatic (Trp, Tyr, Phe). In some embodiments, the protein also comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus, N-terminus, or both. In one particular embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO: 44). In some embodiments, the protein containing (or composed of) the extracellular domain of the SARS-CoV-2 delta variant spike protein (e.g., the SARS-CoV-2 delta variant spike polypeptide / protein) is a secreted polypeptide. In some embodiments, the protein containing the extracellular domain of the SARS-CoV-2 delta variant spike protein (e.g., the SARS-CoV-2 delta variant spike polypeptide / protein) comprises one or more trimerizing domains known to those skilled in the art (e.g., the T4 fold trimerizing domain), and an optional tag (e.g., a His tag or a Flag tag).

[0111] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:13. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:13. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding (or consisting of) the amino acid sequence of SEQ ID NO:13. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding (or consisting of) the amino acid sequence of SEQ ID NO:13 minus a signal peptide. For the signal peptide, see SEQ ID NO:15. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:17. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence of SEQ ID NO:17. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:17. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus, N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).In some embodiments, the protein is a secreted polypeptide. In some embodiments, the protein also includes an NDV F protein transmembrane domain and a cytoplasmic domain. In some embodiments, the protein also includes one or more trimerizing domains known to those skilled in the art (e.g., a T4 fold trimerizing domain) and optional tags (e.g., a His tag or a Flag tag).

[0112] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:12. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:12. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:12. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:12 minus a signal peptide. For the signal peptide, see SEQ ID NO:15. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:16. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence of SEQ ID NO:16. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:16. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at a C-terminus, an N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).In some embodiments, the protein is a secreted polypeptide. In some embodiments, the protein also includes an NDV F protein transmembrane domain and a cytoplasmic domain. In some embodiments, the protein also includes one or more trimerizing domains known to those skilled in the art (e.g., a T4 fold trimerizing domain) and optional tags (e.g., a His tag or a Flag tag).

[0113] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:23. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:23. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding (or consisting of) the amino acid sequence of SEQ ID NO:23. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding (or consisting of) the amino acid sequence of SEQ ID NO:23 minus a signal peptide. For the signal peptide, see SEQ ID NO:15. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:24. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence of SEQ ID NO:24. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:24. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at a C-terminus, an N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).In some embodiments, the protein is a secreted polypeptide. In some embodiments, the protein also includes an NDV F protein transmembrane domain and a cytoplasmic domain. In some embodiments, the protein also includes one or more trimerizing domains known to those skilled in the art (e.g., a T4 fold trimerizing domain) and optional tags (e.g., a His tag or a Flag tag).

[0114] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:29. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:29. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding (or consisting of) the amino acid sequence of SEQ ID NO:29 minus a signal peptide. For the signal peptide, see SEQ ID NO:15. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:30. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence of SEQ ID NO:30. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:30. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at a C-terminus, an N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties. For example, His tags (His-His-His-His-His-His (SEQ ID NO:44)), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO:44).In some embodiments, the protein is a secreted polypeptide. In some embodiments, the protein also includes an NDV F protein transmembrane domain and a cytoplasmic domain. In some embodiments, the protein also includes one or more trimerizing domains known to those skilled in the art (e.g., a T4 fold trimerizing domain) and optional tags (e.g., a His tag or a Flag tag).

[0115] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), with amino acid substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and amino acid deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), with amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deleted from the C-terminus. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the N-terminus. In some embodiments, the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein lacks a multibase cleavage site (e.g., amino acid residues 682 to 685 (RRAR) are substituted with a single alanine). In some embodiments, the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein contains the following amino acid substitutions at amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein contains an amino acid substitution of a single alanine at amino acid residues 682 to 685 (RRAR) corresponding to amino acid residues corresponding to GenBank accession number MN908947.3, and contains the following amino acid substitutions at amino acid residues corresponding to amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the C-terminus of the extracellular domain consists of the last 100 amino acid residues. In another specific embodiment, the N-terminus of the extracellular domain consists of the first 100 amino acid residues. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein.In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus, N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of the polypeptide moieties. For example, His tags (SEQ ID NO: 44), FLAG epitopes, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has the sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has the sequence (His)n, where n is 6 (SEQ ID NO: 44). In some embodiments, the protein also comprises a transmembrane domain and a cytoplasmic domain of the NDV F protein. In some embodiments, proteins comprising the extracellular domain of the SARS-CoV-2 spike polypeptide (e.g., the W strain, delta variant, beta variant, or gamma variant) further comprise one or more trimerizing domains known to those skilled in the art (e.g., the T4 fold trimerizing domain), and optional tags (e.g., a His tag or a Flag tag).

[0116] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), with a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant), with a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids from the C-terminus. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the N-terminus. In some embodiments, the extracellular domain of the SARS-CoV-2 delta variant spike protein lacks a multi-base cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with other amino acid residues). In one specific embodiment, amino acid residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, the following amino acid residues corresponding to GenBank accession number MN908947.3 are substituted: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In yet another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus, N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties.For example, a His tag (His-His-His-His-His-His (SEQ ID NO:44)), a FLAG epitope, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has a sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In one embodiment, the His tag has a sequence (His)n, where n is 6 (SEQ ID NO:44). In some embodiments, the protein also comprises a transmembrane domain and a cytoplasmic domain of the NDV F protein. In some embodiments, proteins comprising the extracellular domain of the SARS-CoV-2 spike polypeptide (e.g., the W strain, delta variant, beta variant, or gamma variant) include one or more trimerizing domains known to those skilled in the art (e.g., the T4 fold trimerizing domain) and optional tags (e.g., His tag or Flag tag).

[0117] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations (e.g., amino acid substitutions, amino acid deletions, amino acid additions, or combinations thereof). In yet another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing (or composed of) the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid deletions. In some embodiments, the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lacks a multi-base cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with other amino acid residues). In one specific embodiment, amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, the following amino acid residues corresponding to GenBank accession number MN908947.3 are substituted: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 delta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 beta variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2 gamma variant spike protein. In another specific embodiment, the SARS-CoV-2 spike protein is the SARS-CoV-2W strain spike protein. In some embodiments, the protein further comprises one or more polypeptide domains. These one or more polypeptide domains may be located at the C-terminus, N-terminus, or both. In one specific embodiment, the one or more polypeptide domains are located at the C-terminus. Useful polypeptide domains include those that facilitate the purification, folding, and cleavage of polypeptide moieties.For example, a His tag (SEQ ID NO:44), a FLAG epitope, or other purification tags can facilitate the purification of the proteins provided herein. In some embodiments, the His tag has a sequence (His)n, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater. In some embodiments, the protein also comprises an NDV F protein transmembrane domain and a cytoplasmic domain. In some embodiments, proteins comprising the extracellular domains of the SARS-CoV-2 spike polypeptide (e.g., the W strain, delta variant, beta variant, or gamma variant) include one or more trimerizing domains known to those skilled in the art (e.g., the T4 fold trimerizing domain) and optional tags (e.g., a His tag or a Flag tag).

[0118] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing an extracellular spike protein domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing an extracellular spike protein domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a protein containing an extracellular spike protein domain having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein, wherein the protein comprises an extracellular spike protein domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein, wherein the protein comprises an extracellular spike protein domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein, wherein the protein comprises an extracellular spike protein domain having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein, wherein the protein comprises an extracellular spike protein domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 16, 17, 24, or 30. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein, wherein the protein comprises an extracellular spike protein domain having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 16, 17, 24, or 30.Methods / techniques known in the art can be used to determine sequence identity (see, for example, the “Best Fit” or “Gap” program of the sequence analysis software package, version 10; Genetics Computer Group, Inc.).

[0119] In another embodiment, the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In yet another embodiment, the extracellular domain of the SARS-CoV-2 spike protein or a derivative thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In another embodiment, the SARS-CoV-2 spike protein extracellular domain or a derivative thereof comprises an amino acid sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the nucleotide sequence of SEQ ID NO: 12, 13, 16, 17, 23, 24, 29, or 30. In yet another embodiment, the SARS-CoV-2 spike protein extracellular domain or a derivative thereof provided herein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29, but without a signal sequence. In another embodiment, the extracellular domain of the SARS-CoV-2 delta variant spike protein or a derivative thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29, but without a signal sequence. In yet another embodiment, the extracellular domain of the SARS-CoV-2 delta variant spike protein or a derivative thereof comprises an amino acid sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 12, 13, 23, or 29, but without a signal sequence. Methods / techniques known in the art can be used to determine sequence identity (see, for example, the “Best Fit” or “Gap” program of sequence analysis software packages, version 10; Genetics Computer Group, Inc.).

[0120] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) and the transmembrane and cytoplasmic domains of the NDV F protein as described herein. In a specific embodiment, the chimeric F protein comprises the entire transmembrane and cytoplasmic domains of the NDV F protein. In one specific embodiment, the transmembrane and cytoplasmic domains of the NDV F protein comprise the amino acid sequence of SEQ ID NO:42. In some embodiments, the chimeric F protein comprises more NDV F protein than the entire transmembrane and cytoplasmic domains of the NDV F protein. In some embodiments, the chimeric F protein does not comprise the entire transmembrane and cytoplasmic domains of the NDV F protein. For example, several amino acid residues upstream of the NDV F protein transmembrane (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1-5, 1-10, or 5-15 amino acid residues) may be included in the chimeric F protein and / or several amino acid residues downstream of the NDV F protein cytoplasmic domain (e.g., 1-5, 1-10, or 5-15 amino acid residues) may be included in the chimeric F protein. For example, fewer than a few amino acid residues spanning the entire NDV F protein transmembrane (e.g., 1, 2, 3, 4, 5, or 1-5 amino acid residues) may be included in the chimeric F protein and / or fewer than a few amino acid residues (e.g., 1, 2, 3, 4, 5, or 1-5 amino acid residues) may be included in the chimeric F protein. In specific embodiments, this document describes transgenic proteins comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises the extracellular domain of the SARS-CoV-2 spike protein, the transmembrane domain of the NDV F protein plus or minus 1, 2, 3, 4, or 5 amino acid residues, and the cytoplasmic domain of the NDV F protein plus or minus 1, 2, 3, 4, or 5 amino acid residues. In specific embodiments, the chimeric F protein lacks the entire transmembrane and cytoplasmic domains of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In some embodiments, the chimeric F protein contains 1, 2, or 3 amino acid residues of the transmembrane and / or cytoplasmic domains of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). The extracellular domains, transmembrane domains, and cytoplasmic domains of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) and NDV F protein can be determined using techniques known to those skilled in the art. For example, publicly available information, GenBank, or websites such as the VIPR viral pathogen website ( www.viprbrc.org The extracellular, transmembrane, and cytoplasmic domains of the SARS-CoV-2 spike protein and NDV F protein can be determined using the DTU Bioinformatics website (www.cbs.dtu.dk / services / TMHMM / ) or procedures that can be used to determine transmembrane domains. See, for example, Table 2 below, which illustrates the transmembrane and cytoplasmic domains of the NDV F protein. In specific embodiments, the extracellular domain of the SARS-CoV-2 spike protein is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domains. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some implementations, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may comprise (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). n Where n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are directly fused to the extracellular domain of the SARS-CoV-2 spike protein. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below.

[0121] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises a derivative of the SARS-CoV-2 spike protein extracellular domain (e.g., a derivative of the SARS-CoV-2 spike protein extracellular domain described herein) and the NDV F protein transmembrane domain and cytoplasmic domain. In a specific embodiment, the chimeric F protein comprises the entire NDV F protein transmembrane domain and cytoplasmic domain. In one specific embodiment, the NDV F protein transmembrane domain and cytoplasmic domain comprise the amino acid sequence of SEQ ID NO:42. In some embodiments, the chimeric F protein comprises more NDV F protein than the entire NDV F protein transmembrane domain and cytoplasmic domain. In some embodiments, the chimeric F protein does not comprise the entire NDV F protein transmembrane domain and cytoplasmic domain. For example, several amino acid residues upstream of the NDV F protein transmembrane (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1-5, 1-10, or 5-15 amino acid residues) may be included in the chimeric F protein and / or several amino acid residues downstream of the NDV F protein cytoplasmic domain (e.g., 1-5, 1-10, or 5-15 amino acid residues) may be included in the chimeric F protein. For example, fewer than a few amino acid residues spanning the entire NDV F protein transmembrane (e.g., 1, 2, 3, 4, 5, or 1-5 amino acid residues) may be included in the chimeric F protein and / or fewer than a few amino acid residues (e.g., 1, 2, 3, 4, 5, or 1-5 amino acid residues) may be included in the chimeric F protein. In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., a derivative of the extracellular domain of the SARS-CoV-2 spike protein described herein), the transmembrane domain of the NDV F protein plus or minus 1, 2, 3, 4, or 5 amino acid residues, and the cytoplasmic domain of the NDV F protein plus or minus 1, 2, 3, 4, or 5 amino acid residues. In a specific embodiment, the chimeric F protein does not include the transmembrane domain and cytoplasmic domain of the SARS-CoV-2 spike protein. In some embodiments, the chimeric F protein contains 1, 2, or 3 amino acid residues of the transmembrane domain and / or cytoplasmic domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In a specific implementation, the derivative of the extracellular domain of the SARS-CoV-2 spike protein lacks a multi-base cleavage site (e.g., one, two or more residues of amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 are replaced by other amino acid residues).In one specific embodiment, the derivative comprises amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3, which are substituted with a single alanine. See, for example, Table 2 below, which shows the transmembrane and cytoplasmic domains of the NDV F protein. In some embodiments, the derivative comprises amino acid substitutions for the following amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the derivative comprises amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 substituted with a single alanine, and amino acid substitutions for the following amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In specific embodiments, a derivative of the extracellular domain of the SARS-CoV-2 spike protein is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domains. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may contain (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). nWhere n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are directly fused to a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In a specific embodiment, a transgene comprising a nucleotide sequence encoding a chimeric F protein is described herein, wherein the transgene comprises a negative-sense strand of RNA sequence corresponding to the cDNA sequence of SEQ ID NO: 5 or 43. In another specific embodiment, a transgene comprising a nucleotide sequence encoding a chimeric F protein is described herein, wherein the chimeric F protein comprises the amino acid sequence of SEQ ID NO: 13 or 17 and the transmembrane domain and cytoplasmic domain of the NDV F protein. In a specific embodiment, a transgene comprising a nucleotide sequence encoding a chimeric F protein is described herein, wherein the transgene comprises a negative-sense strand of RNA sequence corresponding to the cDNA sequence of SEQ ID NO: 37 or 38. In another specific embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises the amino acid sequence of SEQ ID NO: 12 or 16 and the transmembrane domain and cytoplasmic domain of the NDV F protein. In another specific embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the transgene comprises a negative-sense RNA sequence corresponding to the cDNA sequence of SEQ ID NO: 19 or 33. In another specific embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises the amino acid sequence of SEQ ID NO: 23 or 24 and the transmembrane domain and cytoplasmic domain of the NDV F protein. In another specific embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the transgene comprises a negative-sense RNA sequence corresponding to the cDNA sequence of SEQ ID NO: 25 or 34. In yet another specific embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises the amino acid sequence of SEQ ID NO: 29 or 30 and the transmembrane domain and cytoplasmic domain of the NDV F protein. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a discussion of codon optimization, see, for example, Section 5.1.4 below. In a preferred embodiment, the transgene comprises a codon-optimized version of the nucleic acid sequence encoding the chimeric F protein.

[0122] In some embodiments, the transgene comprises a codon-optimized version of a nucleic acid sequence encoding a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In one specific embodiment, the transgene described herein comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6 or 18. In another specific embodiment, the transgene described herein comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 11 or 40. In another specific embodiment, the transgene described herein comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 22 or 39. In another specific embodiment, the transgene described herein comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 28 or 41. In another specific embodiment, the transgene described herein comprises a nucleotide sequence of SEQ ID NO: 5 or 43, or an RNA sequence of a negative sense strand corresponding to the cDNA sequence of SEQ ID NO: 5 or 43. In another specific embodiment, the transgene described herein comprises a nucleotide sequence of SEQ ID NO: 37 or 38, or an RNA sequence of a negative sense strand corresponding to the cDNA sequence of SEQ ID NO: 37 or 38. In another specific embodiment, the transgene described herein comprises the nucleotide sequence of SEQ ID NO: 19 or 33, or an RNA sequence of the negative sense strand corresponding to the cDNA sequence of SEQ ID NO: 19 or 33. In another specific embodiment, the transgene described herein comprises the nucleotide sequence of SEQ ID NO: 25 or 34, or an RNA sequence of the negative sense strand corresponding to the cDNA sequence of SEQ ID NO: 25 or 34. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein may be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane domain and cytoplasmic domain of the chimeric F protein of the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the chimeric F protein of the NDV F protein has transmembrane and cytoplasmic domains derived from a different NDV strain than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0123] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein described herein, plus or minus 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid residues at the C-terminus of the extracellular domain, and the transmembrane domain and cytoplasmic domain of the NDV F protein. In other words, the portion of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein of the chimeric F protein does not include the entire SARS-CoV-2 spike protein transmembrane domain and cytoplasmic domain. The extracellular domain, transmembrane domain, and cytoplasmic domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and the NDV F protein can be determined using techniques known to those skilled in the art. For example, publicly available information, GenBank, or websites such as the VIPR viral pathogen website ( www.viprbrc.org The extracellular, transmembrane, and cytoplasmic domains of the SARS-CoV-2 spike protein and NDV F protein can be determined using the DTU Bioinformatics website (www.cbs.dtu.dk / services / TMHMM / ) or procedures that can be used to determine transmembrane domains. See, for example, Table 2 below, which shows the transmembrane and cytoplasmic domains of the NDV F protein (SEQ ID NO:42). In a specific embodiment, the extracellular domain of the SARS-CoV-2 spike protein is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domains. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some implementations, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may comprise (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). nWhere n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are directly fused to the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant). In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcriptional units of the NDV genome. In other embodiments, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0124] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) and the transmembrane and cytoplasmic domains of the NDV F protein, wherein the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid residues with or without the C-terminus of the extracellular domain. In other words, the portion of the SARS-CoV-2 (e.g., the W strain, delta variant, beta variant, or gamma variant) spike protein encoded by the chimeric F protein does not include the entire SARS-CoV-2 spike protein transmembrane and cytoplasmic domains. In specific embodiments, derivatives of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lack a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with other amino acid residues). In specific embodiments, lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin protease. In one specific embodiment, amino acid residues 682 to 685 (RRAR) of amino acid residues corresponding to GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, the derivative contains the following amino acid substitutions at the amino acid residues corresponding to the amino acid residues of GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the derivative contains a single alanine amino acid residue substitution at amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3, and contains the following amino acid substitutions at amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. The extracellular domains, transmembrane domains, and cytoplasmic domains of the SARS-CoV-2 spike protein and NDV F protein can be determined using techniques known to those skilled in the art. For example, publicly available information, GenBank, or websites such as the VIPR viral pathogen website ( www.viprbrc.orgThe extracellular, transmembrane, and cytoplasmic domains of the SARS-CoV-2 spike protein and NDV F protein can be determined using the DTU Bioinformatics website (www.cbs.dtu.dk / services / TMHMM / ) or procedures that can be used to determine transmembrane domains. See, for example, Table 2 below, which illustrates the transmembrane and cytoplasmic domains of the NDV F protein. In a specific embodiment, a derivative of the extracellular domain of the SARS-CoV-2 spike protein is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domains. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some implementations, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may comprise (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). n Where n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are fused directly to a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcriptional units of the NDV genome. In other embodiments, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0125] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of the spike protein are substituted by another amino acid (e.g., conserved amino acid substitution). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the C-terminus of the extracellular domain are replaced by another amino acid (e.g., a conserved amino acid substitution). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane and cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane and cytoplasmic domains of the NDV F protein), wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids at the N-terminus of the extracellular domain are replaced by another amino acid (e.g., a conserved amino acid substitution). In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) and a transmembrane and cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane and cytoplasmic domains of the NDV F protein), wherein the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids substituted at the N-terminus by another amino acid (e.g., a conserved amino acid substitution) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids substituted at the C-terminus by another amino acid (e.g., a conserved amino acid substitution). In some embodiments, the N-terminus is 100 amino acids at the N-terminus of the extracellular domain. In some embodiments, the C-terminus is 100 amino acids at the C-terminus of the extracellular domain.Examples of conserved amino acid substitution include, for example, replacing one class of amino acids with another amino acid of the same class. In a particular embodiment, the conserved substitution does not alter the structure or function of the polypeptide, or both. Amino acid classes may include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformational disruptors (Gly, Pro), and aromatic (Trp, Tyr, Phe). In a particular embodiment, a transgene encoding a chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding a chimeric F protein may be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In one specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcriptional units of the NDV genome. In another specific embodiment, the transmembrane and cytoplasmic domains of the chimeric NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0126] In another embodiment, this document provides a transgenic protein comprising a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) and a transmembrane domain and a cytoplasmic domain of the NDV F protein (or composed of a derivative of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein the derivative of the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids substituted by another amino acid (e.g., conserved amino acid substitution). In a specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein lacks a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted by other amino acid residues). In specific embodiments, lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, the derivative of the SARS-CoV-2 spike protein extracellular domain comprises amino acid residues 682 to 685 (RRAR) corresponding to amino acid residues in GenBank accession number MN908947.3, which are substituted with a single alanine. In some embodiments, the derivative comprises the following amino acid substitutions at the amino acid residues corresponding to the amino acid residues in GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In one specific embodiment, the derivative contains a single alanine amino acid residue substitution at amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3, and contains the following amino acid substitutions at amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In another specific embodiment, the derivative of the SARS-CoV-2 spike protein extracellular domain is fused to the transmembrane domain and cytoplasmic domain of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domain. In some embodiments, the linker is an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids (e.g., a peptide). In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker.For example, the connector may contain (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). n Where n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the linker comprises the sequence GGGGS (SEQ ID NO: 7). In other embodiments, a derivative of the extracellular domain of the SARS-CoV-2 spike protein is fused directly to the transmembrane and cytoplasmic domains of the NDV F protein. Examples of conserved amino acid substitution include, for example, replacing one class of amino acids with another amino acid of the same class. In one specific embodiment, the conserved substitution does not alter the structure or function of the polypeptide, or both. Amino acid classes may include hydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformational disruptors (Gly, Pro), and aromatic (Trp, Tyr, Phe). In one specific embodiment, a transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcription units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0127] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein the extracellular domain of the spike protein has a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the C-terminus. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) and a transmembrane and cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane and cytoplasmic domains of the NDV F protein), wherein the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from its N-terminus. In some embodiments, the N-terminus of the extracellular domain is 100 amino acids. In some embodiments, the C-terminus of the extracellular domain is 100 amino acids. In specific embodiments, the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domain. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may contain (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). nWhere n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In other embodiments, the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is directly fused to the transmembrane and cytoplasmic domains of the NDV F protein. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein of the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the chimeric F protein of the NDV F protein has transmembrane and cytoplasmic domains derived from a different NDV strain than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0128] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising a derivative of the extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of a derivative of the extracellular domain of the spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein), wherein the derivative of the extracellular domain of the spike protein has a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising a derivative of the extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of a derivative of the extracellular domain of the spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein), wherein the derivative of the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the C-terminus. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising a derivative of the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of the NDV F protein (or composed of a derivative of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein the derivative of the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids deleted from the N-terminus. In some embodiments, the derivative of the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein lacks a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) of GenBank accession number MN908947.3 are substituted with other amino acid residues). In specific embodiments, the lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, the derivative contains the following amino acid substitutions at the amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P.In one specific embodiment, the derivative contains a single alanine amino acid residue substitution at amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3, and contains the following amino acid substitutions at amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In another specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) is fused to the transmembrane domain and cytoplasmic domain of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domain. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of length 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may contain (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). n Where n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In other embodiments, a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is directly fused to the transmembrane and cytoplasmic domains of the NDV F protein. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein of the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the chimeric F protein of the NDV F protein has transmembrane and cytoplasmic domains derived from a different NDV strain than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0129] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein), wherein the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations (e.g., amino acid substitutions, amino acid deletions, amino acid additions, or combinations thereof). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising an extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane and cytoplasmic domain of an NDV F protein (or composed of the extracellular domain of the spike protein and the transmembrane and cytoplasmic domains of the NDV F protein), wherein the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid deletions. In a specific embodiment, the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domain. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may contain (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). nWhere n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In other embodiments, the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is directly fused to the transmembrane and cytoplasmic domains of the NDV F protein. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDVLaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein of the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the chimeric F protein of the NDV F protein has transmembrane and cytoplasmic domains derived from a different NDV strain than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0130] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising a derivative of the extracellular domain of a SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of a derivative of the extracellular domain of the spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein), wherein the derivative of the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations (e.g., amino acid substitutions, amino acid deletions, amino acid additions, or combinations thereof). In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising a derivative of the extracellular domain of a SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic domain of an NDV F protein (or composed of a derivative of the extracellular domain of the spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein), wherein the derivative of the extracellular domain of the spike protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid deletions. In some embodiments, derivatives of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) lack a multibase cleavage site (e.g., one, two, or more residues corresponding to amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with other amino acid residues). In specific embodiments, lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin proteases. In one specific embodiment, amino acid residues 682 to 685 (RRAR) in GenBank accession number MN908947.3 are substituted with a single alanine. In some embodiments, the derivative contains the following amino acid substitutions at the amino acid residues corresponding to the amino acid residues in GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P.In one specific embodiment, the derivative contains a single alanine amino acid residue substitution at amino acid residues 682 to 685 (RRAR) corresponding to GenBank accession number MN908947.3, and contains the following amino acid substitutions at amino acid residues corresponding to GenBank accession number MN908947.3: F817P, A892P, A899P, A942P, K986P, and V987P. In another specific embodiment, the derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., W strain, delta variant, beta variant, or gamma variant) is fused to the transmembrane domain and cytoplasmic domain of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domain. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of length 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may contain (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO: 45). In another instance, the connector may contain (G). n Where n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In other embodiments, a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is directly fused to the transmembrane and cytoplasmic domains of the NDV F protein. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDVP and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein of the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the chimeric F protein of the NDV F protein has transmembrane and cytoplasmic domains derived from a different NDV strain than the transcriptional units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0131] In another embodiment, this document describes a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises a derivative of the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and the transmembrane and cytoplasmic domains of the NDV F protein, wherein the derivative comprises amino acid residues corresponding to amino acid residues 817, 892, 899, 942, 986, and 987 of the spike protein found at GenBank accession number MN908947.3, substituted with proline, and wherein the derivative lacks a multibase cleavage site. In a specific embodiment, lack of multibase cleavage means that the multibase site is altered so that it cannot be cleaved by, for example, furin protease. The extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein may lack a multibase cleavage site because amino acid residues 682 to 685 of the multibase cleavage site are substituted with a single alanine. See, for example, Table 2 below, which illustrates the transmembrane and cytoplasmic domains of the NDV F protein. In specific embodiments, a derivative of the extracellular domain of the SARS-CoV-2 spike protein (e.g., the W strain, delta variant, beta variant, or gamma variant) is fused to the transmembrane and cytoplasmic domains of the NDV F protein via a linker (e.g., GGGGS (SEQ ID NO:7)). The linker can be any linker that does not interfere with the folding, function, or both of the extracellular domains. In some embodiments, the linker is an amino acid sequence (e.g., a peptide) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In some embodiments, the linker is a glycine (G) linker or a glycine and serine (GS) linker. For example, the linker may comprise (GGGGS). n The sequence, where n is 1, 2, 3, 4, 5 or more (SEQ ID NO:45). In another instance, the connector may contain (G). nWhere n is 3, 4, 5, 6, 7, 8, or greater. In one specific embodiment, the adapter comprises the sequence GGGGS (SEQ ID NO: 7). In some embodiments, the transmembrane domain and cytoplasmic domain of the NDV F protein are fused directly to a derivative of the extracellular domain of the SARS-CoV-2 spike protein. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units).

[0132] In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:5. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:5 or 43. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:5 or 43. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:5, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:5, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the nucleotide sequence of SEQ ID NO:5, but without a signal sequence. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:5. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:43. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:5 but without a signal sequence. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein, the chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 6 or 18. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein, the chimeric F protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 6 or 18.In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6 or 18. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 6, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 6, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:6, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:6. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:18. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:6 without a signal sequence. Methods / techniques known in the art can be used to determine sequence identity (see, for example, the "Best Fit" or "Gap" program of sequence analysis software packages, version 10; Genetics Computer Group, Inc.). In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In one specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome.In a specific implementation, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific implementation, the NDV genome belongs to the LaSota strain.

[0133] In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:6, wherein the chimeric F protein comprises at least one, at least two, at least three, at least four, or more of the following amino acid modifications: T19R, G142D, delE156, R158G, L452R, T478K, and D950N. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:6. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:18. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:6, wherein the chimeric F protein comprises at least one, at least two, at least three, at least four, or more of the following amino acid modifications: T19R, G142D, delE156, R158G, L452R, T478K, and D950N. In some embodiments, SEQ ID NO:6 lacks a signal sequence. For the signal sequence, see SEQ ID NO:15. Methods / techniques known in the art can be used to determine sequence identity (see, for example, the “Best Fit” or “Gap” program of the sequence analysis software package, version 10; GeneticsComputer Group, Inc.). In one specific embodiment, a transgene encoding a chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV VP and M transcription units, between NDV NP and P transcription units, or between NDV HN and L transcription units). In a specific embodiment, the transmembrane domain and cytoplasmic domain of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome.In a specific implementation, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific implementation, the NDV genome belongs to the LaSota strain.

[0134] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:13, or comprises the amino acid sequence of SEQ ID NO:13 but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:17. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:13, or comprises the amino acid sequence of SEQ ID NO:13 but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the amino acid sequence of SEQ ID NO: 6 or 18. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion of codon optimization, see, for example, Section 5.1.4 below. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above.The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcription units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0135] In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 19 or 33. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 19 or 33. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 19. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 19, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:19, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the nucleotide sequence of SEQ ID NO:19, but without a signal sequence. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:19. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:19 but without a signal sequence. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:33. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:22 or 39. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:22 or 39.In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22 or 39. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:22, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:22, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:22, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:22. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:39. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:22 without a signal sequence. Methods / techniques known in the art can be used to determine sequence identity (see, for example, the “Best Fit” or “Gap” program of sequence analysis software packages, version 10; GeneticsComputer Group, Inc.). In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV VP and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In a specific embodiment, the transmembrane domain and cytoplasmic domain of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome.In a specific implementation, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific implementation, the NDV genome belongs to the LaSota strain.

[0136] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:23, or comprises the amino acid sequence of SEQ ID NO:23 but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:24. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:23, or comprises the amino acid sequence of SEQ ID NO:23 but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the amino acid sequence of SEQ ID NO: 22 or 39. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above.The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcription units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0137] In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:37 or 38. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:37 or 38. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:37 or 38. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:37, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO:37, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the nucleotide sequence of SEQ ID NO:37, but without a signal sequence. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:37. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:37 but without a signal sequence. In another embodiment, this document provides a transgene comprising the nucleotide sequence of SEQ ID NO:38. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 11 or 40. In another embodiment, this document provides a transgenic protein comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 11 or 40.In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11 or 40. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO: 11, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a protein comprising (or consisting of) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 11, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) an amino acid sequence having at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:11, but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:11. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:40. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising (or consisting of) the amino acid sequence of SEQ ID NO:11 without a signal sequence. Methods / techniques known in the art can be used to determine sequence identity (see, for example, the “Best Fit” or “Gap” program of sequence analysis software packages, version 10; GeneticsComputer Group, Inc.). In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above. The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV VP and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In a specific embodiment, the transmembrane domain and cytoplasmic domain of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome.In a specific implementation, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcriptional units of the NDV genome. In one specific implementation, the NDV genome belongs to the LaSota strain.

[0138] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:12, or comprises the amino acid sequence of SEQ ID NO:12 but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:16. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO:12, or comprises the amino acid sequence of SEQ ID NO:12 but without a signal sequence. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and cytoplasmic domain of the NDV F protein, and wherein the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16. In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein, wherein the chimeric F protein comprises (or consists of) the amino acid sequence of SEQ ID NO: 11 or 40. In some embodiments, the transgene encoding the chimeric F protein is codon-optimized. For a related discussion on codon optimization, see, for example, Section 5.1.4 below. In one specific embodiment, the transgene encoding the chimeric F protein is incorporated into the genome of any NDV type or strain (e.g., the NDV LaSota strain). For information on the types and strains of NDV that can be used, see, for example, Section 5.1.1 above.The transgene encoding the chimeric F protein can be incorporated between any two NDV transcription units (e.g., between NDV P and M transcription units, NDV NP and P transcription units, or NDV HN and L transcription units). In a specific embodiment, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from the same NDV strain as the transcription units of the NDV genome. In other embodiments, the transmembrane and cytoplasmic domains of the chimeric F protein in the NDV F protein originate from different NDV strains than the transcription units of the NDV genome. In one specific embodiment, the NDV genome belongs to the LaSota strain.

[0139] In another embodiment, this document provides a transgene comprising a nucleotide sequence encoding a chimeric F protein comprising a derivative of the extracellular domain of the SARS-CoV-2 (e.g., W strain, delta variant, beta variant, or gamma variant) spike protein and a transmembrane domain and a cytoplasmic doma...

Claims

1. An immunogenic composition, comprising: (a) a first recombinant Newcastle disease virus (NDV), wherein the first recombinant NDV comprises a first transgene, the first transgene comprises a nucleotide sequence encoding a first chimeric F protein, and wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprises a nucleotide sequence encoding a second chimeric F protein, and wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; wherein the first derivative and the second derivative are different from each other.

2. The immunogenic composition of claim 1, wherein the first derivative and the second derivative each comprise an amino acid substitution to proline at positions corresponding to positions 817, 892, 899, 942, 986 and 987 of the W strain spike protein, and a RRAR substitution to alanine at positions corresponding to positions 682 to 685 of the W strain spike protein.

3. The immunogenic composition of claim 1 or 2, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

4. The immunogenic composition of claim 1 , 2 or 3, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29 or 30.

5. The immunogenic composition of claim 1 or 2, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

6. The immunogenic composition of claim 1 , 2 or 5, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13, 17, 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, 17, 29 or 30.

7. The immunogenic composition of claim 1 or 2, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

8. The immunogenic composition of claim 1 , 2 or 7, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

9. A multivalent immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, and wherein the first chimeric F protein comprises a first derivative of the extracellular domain of a SARS-CoV-2 spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein; (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprises a nucleotide sequence encoding a second chimeric F protein, and wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; and (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, and wherein the third chimeric F protein comprises a third derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; wherein the first derivative, the second derivative and the third derivative are different from each other.

10. The immunogenic composition of claim 9, wherein the first derivative, the second derivative, and the third derivative each comprise an amino acid substitution to proline at positions corresponding to positions 817, 892, 899, 942, 986, and 987 of the W strain spike protein, and a RRAR substitution to alanine at positions corresponding to positions 682 to 685 of the W strain spike protein.

11. An immunogenic composition according to claim 9 or 10, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

12. The immunogenic composition of claim 9, 10 or 11, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

13. The immunogenic composition of any one of claims 9 to 12, wherein the third derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

14. The immunogenic composition of claim 9 or 10, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

15. The immunogenic composition of claim 14, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

16. The immunogenic composition of claim 14, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

17. The immunogenic composition of any one of claims 14 to 16, wherein the third derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

18. The immunogenic composition of claim 9 or 10, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

19. The immunogenic composition of claim 9 or 10, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

20. The immunogenic composition of claim 18, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

21. The immunogenic composition of claim 19, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

22. The immunogenic composition of claim 20, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

23. The immunogenic composition of any one of claims 9 to 22, comprising a fourth recombinant NDV, wherein the fourth recombinant NDV comprises a fourth transgene, the fourth transgene comprising a nucleotide sequence encoding a fourth chimeric F protein, and wherein the fourth chimeric F protein comprises a fourth derivative of the extracellular domain of the SARS-CoV-2 Spike protein, and a transmembrane domain and a cytoplasmic domain of the NDV F protein, and wherein the fourth derivative is different from the first derivative, the second derivative, and the third derivative.

24. The immunogenic composition of claim 23, wherein the fourth derivative comprises amino acid substitutions to proline at positions corresponding to positions 817, 892, 899, 942, 986 and 987 of the W strain spike protein, and RRAR substitutions to alanine at positions corresponding to positions 682 to 685 of the W strain spike protein.

25. The immunogenic composition of claim 13, further comprising a fourth recombinant NDV, wherein the fourth recombinant NDV comprises a fourth transgene, the fourth transgene comprising a nucleotide sequence encoding a fourth chimeric F protein, and wherein the fourth derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

26. An immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, and wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; and (b) a second recombinant NDV comprising a second chimeric F protein, and wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; wherein the first derivative and the second derivative are different from each other.

27. The immunogenic composition of claim 24, wherein the first derivative and the second derivative each comprise an amino acid substitution to proline at positions corresponding to positions 817, 892, 899, 942, 986 and 987 of the W strain spike protein, and a RRAR substitution to alanine at positions corresponding to positions 682 to 685 of the W strain spike protein.

28. The immunogenic composition of claim 26 or 27, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

29. The immunogenic composition of claim 26, 27 or 28, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, 17, 23, 24, 29 or 30.

30. The immunogenic composition of claim 26 or 27, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

31. The immunogenic composition of claim 26, 27 or 30, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13, 17, 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, 17, 29 or 30.

32. The immunogenic composition of claim 26 or 27, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

33. The immunogenic composition of claim 26, 27 or 32, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

34. A multivalent immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, and wherein the first chimeric F protein comprises a first derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; (b) a second recombinant NDV comprising a second chimeric F protein, and wherein the second chimeric F protein comprises a second derivative of the extracellular domain of the SARS-CoV-2 Spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; and (c) a third recombinant NDV comprising a third chimeric F protein, and wherein the third chimeric F protein comprises a third derivative of the extracellular domain of the SARS-CoV-2 spike protein and the transmembrane domain and the cytoplasmic domain of the NDV F protein; wherein the first derivative, the second derivative and the third derivative are different from each other.

35. The immunogenic composition of claim 34, wherein the first derivative, the second derivative, and the third derivative each comprise an amino acid substitution to proline at positions corresponding to positions 817, 892, 899, 942, 986, and 987 of the W strain spike protein, and a RRAR substitution to alanine at positions corresponding to positions 682 to 685 of the W strain spike protein.

36. The immunogenic composition of claim 34 or 35, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

37. The immunogenic composition of claim 34, 35 or 36, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

38. The immunogenic composition of any one of claims 34 to 37, wherein the third derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

39. The immunogenic composition of claim 34 or 35, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 23 or 24.

40. The immunogenic composition of claim 39, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

41. The immunogenic composition of claim 39, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 16.

42. The immunogenic composition of any one of claims 39 to 41, wherein the third derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

43. The immunogenic composition of claim 34 or 35, wherein the first derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 29 or 30, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 29 or 30.

44. The immunogenic composition of claim 43, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

45. The immunogenic composition of claim 43, wherein the second derivative of the extracellular domain of the SARS-CoV-2 spike protein comprises the amino acid sequence of SEQ ID NO: 13 or 17, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13 or 17.

46. ​​The immunogenic composition of any one of claims 34 to 45, further comprising a fourth recombinant NDV comprising a fourth chimeric F protein, and wherein the fourth chimeric F protein comprises a fourth derivative of the extracellular domain of the SARS-CoV-2 Spike protein and a transmembrane domain and a cytoplasmic domain of the NDV F protein, and wherein the fourth derivative is different from the first derivative, the second derivative, and the third derivative.

47. The immunogenic composition of claim 38, further comprising a fourth recombinant NDV comprising a fourth chimeric F protein, and wherein the fourth chimeric F protein comprises a fourth derivative of the extracellular domain of the SARS-CoV-2 spike protein, the fourth derivative comprising the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 23 or 24.

48. The immunogenic composition of any one of claims 1 to 47, wherein the extracellular domain of the chimeric F protein is connected to the transmembrane domain and the cytoplasmic domain of the NDV F protein via a linker.

49. The immunogenic composition of claim 48, wherein the linker comprises the amino acid sequence of SEQ ID NO:

7.

50. The immunogenic composition of any one of claims 1 to 49, wherein the NDV F protein and cytoplasmic domain comprise the amino acid sequence of SEQ ID NO:

42.

51. An immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 40; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39 or 41.

52. An immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39.

53. An immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18.

54. An immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18; and (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39.

55. A multivalent immunogenic composition comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 40; (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39; and (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6, 18, 28 or 41.

56. A multivalent immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41; (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39; and (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18.

57. A multivalent immunogenic composition, comprising: (a) a first recombinant NDV, wherein the first recombinant NDV comprises a first transgene, the first transgene comprising a nucleotide sequence encoding a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 40; (b) a second recombinant NDV, wherein the second recombinant NDV comprises a second transgene, the second transgene comprising a nucleotide sequence encoding a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39; (c) a third recombinant NDV, wherein the third recombinant NDV comprises a third transgene, the third transgene comprising a nucleotide sequence encoding a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18; and (d) a fourth recombinant NDV, wherein the fourth recombinant NDV comprises a fourth transgene, the fourth transgene comprising a nucleotide sequence encoding a fourth chimeric F protein, wherein the fourth chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41.

58. An immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, and wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 40; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6, 18, 22, 28, 39 or 41.

59. An immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18.

60. An immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39.

61. An immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18; and (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39.

62. A multivalent immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 40; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6, 18, 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6, 18, 28 or 41.

63. A multivalent immunogenic composition, comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39; and (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18.

64. A multivalent immunogenic composition comprising: (a) a first recombinant NDV comprising a first chimeric F protein, wherein the first chimeric F protein comprises the amino acid sequence of SEQ ID NO: 11 or 40, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 40; (b) a second recombinant NDV comprising a second chimeric F protein, wherein the second chimeric F protein comprises the amino acid sequence of SEQ ID NO: 22 or 39, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 22 or 39; (c) a third recombinant NDV comprising a third chimeric F protein, wherein the third chimeric F protein comprises the amino acid sequence of SEQ ID NO: 6 or 18, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 or 18; and (d) a fourth recombinant NDV comprising a fourth chimeric F protein, wherein the fourth chimeric F protein comprises the amino acid sequence of SEQ ID NO: 28 or 41, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 28 or 41.

65. The immunogenic composition of any one of claims 1 to 64, wherein the recombinant NDV is inactivated or live.

66. The immunogenic composition of any one of claims 1 to 65, further comprising an adjuvant.

67. A method for inducing an immune response against the SARS-CoV-2 spike protein, the method comprising administering to a subject the immunogenic composition of any one of claims 1 to 66.

68. A method for preventing COVID-19, the method comprising administering to a subject the immunogenic composition of any one of claims 1 to 66.

69. A method for immunizing a subject against SARS-CoV-2, the method comprising administering to the subject the immunogenic composition of any one of claims 1 to 66.

70. A method for immunizing a subject against two or more SARS-CoV-2, the method comprising administering to the subject the immunogenic composition of any one of claims 1 to 66.

71. A method for immunizing a subject against one or more SARS-CoV-2, wherein the one or more SARS-CoV-2 is heterologous to the SARS-CoV-2 from which the extracellular domain of the chimeric F protein contained in the immunogenic composition is derived, the method comprising administering to the subject the immunogenic composition of any one of claims 1 to 66.

72. A method for inducing antibodies that neutralize one or more SARS-CoV-2 in a subject, wherein the one or more SARS-CoV-2 are heterologous to the SARS-CoV-2 from which the extracellular domain of the chimeric F protein contained in the immunogenic composition is derived, the method comprising administering to the subject the immunogenic composition of any one of claims 1 to 66.

73. A method for inducing antibodies cross-reactive with one or more SARS-CoV-2 spike proteins in a subject, wherein the one or more SARS-CoV-2 spike proteins are heterologous to the SARS-CoV-2 spike protein from which the extracellular domain contained in the immunogenic composition is derived, the method comprising administering to the subject the immunogenic composition of any one of claims 1 to 66.

74. The method of any one of claims 67 to 73, wherein the composition is administered to the subject intranasally or intramuscularly.

75. The method of any one of claims 67 to 74, wherein the subject is a human.

76. A method according to any one of claims 67 to 75, wherein the subject has previously been vaccinated with a COVID-19 vaccine.

77. The method of any one of claims 67 to 76, wherein at least one booster of the immunogenic composition is administered to the subject.

78. A kit comprising the immunogenic composition of any one of claims 1 to 66.

Images