CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional application no. 63/111,929, filed November 10, 2020, the entire content of which is incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to biomedicine and vaccine development.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0003] This invention was made with Government support under Grant Number R01AI100272-09S1, awarded by the National Institutes of Health. The Government has certain rights in the invention.

SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 9, 2021, is named 103182-1268901-005810WO_SL.txt and is 188,093 bytes in size.

BACKGROUND OF THE INVENTION

[0005] Coronavirus are enveloped, single-stranded RNA viruses from the viral family of Coronaviridae. Coronavirus causes diseases in mammals and birds. Coronavirus hosts include bats, pigs, dogs, cats, mice, rats, cows, rabbits, chickens and turkeys. In humans, coronaviruses cause mild to severe respiratory tract infections. See Fehr A.R., Perlman S. (2015) "Coronaviruses: An Overview of Their Replication and Pathogenesis" in: Maier H., Bickerton E., Britton P. (eds) Coronaviruses. Methods in Molecular Biology, vol 1282. Humana Press, New York, NY. https://doi.org/10.1007/978-l-4939-2438-7_l, incorporated herein by reference, for a review. Some examples of human coronaviruses are: Human coronavirus 229E (HCoV-229E); Human coronavirus OC43 (HCoV-OC43); Severe acute respiratory syndrome coronavirus (SARS-CoV); Human coronavirus NL63 (HCoV-NL63, New Haven coronavirus); Human coronavirus HKU1 (HCoV-HKUl); Middle East respiratory syndrome- related coronavirus (MERS-CoV), also known as novel coronavirus 2012 and HCoV-EMC; and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0006] The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has spread throughout the world and is causing massive sustained morbidity, mortality, and societal disruption. The development of an effective vaccine is critical to contain the spread of this virus. In addition, suitable animal models for checking the efficacy and toxicity of vaccines as well as appropriate systems for studying virus-host protein-protein interactions are needed to facilitate research and development of SARS-CoV-2 vaccines and therapies.

[0007] There are multiple efforts to develop SARS-CoV-2 vaccines with the most advanced vaccines being developed on pre-existing platforms. Protein or nucleic acid-based vaccines can be developed relatively rapidly and can be safe, but are often ineffective. Other vaccines in development are either inactivated or vector-based, and most strategies focus on a single antigen, the viral Spike protein or a fragment of it (Funk et al., 2020, "A Snapshot of the Global Race for Vaccines Targeting SARS-CoV-2 and the COVID-19 Pandemic," Front. Pharmacol., 11, 937). However, T-cell responses may be required to protect against SARS-CoV-2, as has been shown to be the case for other coronaviruses in small-animal models (Sariol and Perlman (2020), "Lessons for COVID-19 immunity from other coronavirus infections," Immunity, 18; 53(2): 248-263). Further, these types of vaccines will be expensive and difficult to produce in sufficient quantities for global immunization. Inactivated vaccines may be prepared with less effort, but it has been shown that they not abolish viral replication post-challenge even after three immunizations of nonhuman primates (Gao et al., 2020, "Development of an inactivated vaccine candidate for SARS-CoV-2," Science; 369 (6499):77-81). Human immunization will likely involve one immunization (due to compliance challenges), and thus, this type of vaccine response is unlikely to prevent transmission or be effective in the elderly. [0008] Live attenuated viruses (LAV) in general have been shown to be highly effective for preventing viral diseases and interrupting the chain of transmission (Vetter et al., 2018, "Understanding modern-day vaccines: what you need to know," Ann Med; 50(2): 110-20). Current SARS-CoV-2 vaccine strategies focus on attenuation by codon deoptimization which reduces the replication fitness of the virus by impairing translation. However, these and similar strategies failed to produce an immunogenic polio vaccine, because the codon- deoptimized virus reduces overall viral protein production (Van Damme et al., 2019, "The safety and immunogenicity of two novel live attenuated monovalent (serotype 2) oral poliovirus vaccines in healthy adults: a double-blind, single-centre phase 1 study," Lancet 394 (10193): 148-58). Furthermore, because the deoptimized virus still contains the full suite of viral proteins, it may still recombine or evolve to produce pathology.

BRIEF SUMMARY OF THE INVENTION

[0009] Aspects of the present invention relate to a live attenuated SARS-CoV-2 for use in preparation of a vaccine against COVID-19 or other SARS-CoV-2 illnesses. The live attenuated SARS-CoV-2 disclosed herein is a SARS-CoV-2 variant that differs from wild-type SARS-CoV-2 by virtue of inactivating mutation(s) (e.g., deletions) in the coding sequences of one or more viral proteins. In one aspect, the invention provides a live attenuated SARS-CoV-2 virus comprising a viral genome, where the viral genome comprises inactivating mutation(s) in a sequence(s) encoding one or more SARS-CoV-2 proteins. In some embodiments, the viral genome comprises inactivating mutation(s) in a sequence(s) encoding one, two, three or more than three SARS-CoV-2 proteins.

[0010] In some embodiments, the SARS-CoV-2 viral genome comprises an inactivating mutation that includes one or more deletion(s) and/or one or more substitution(s). In some embodiments, the inactivating mutation(s) comprises an inactivating deletion in at least one sequence encoding a SARS-CoV-2 protein and comprises an inactivating substitution in at least one sequence encoding a SARS-CoV-2 protein. In some embodiments, each inactivating mutation(s) is a deletion of at least 10 nucleotides. In some embodiments, at least 50% of the length of the sequence encoding the SARS-CoV-2 protein is deleted. In some embodiments, the deletion(s) is within a region(s) corresponding to nucleotide positions of SEQ ID NO: 1 selected from a region listed in Table 1.

[0011] In some aspects, at least one of, optionally all of, the SARS-CoV-2 protein(s) comprising inactivating mutation(s) is a SARS-CoV-2 structural protein, a SARS-CoV-2 non- structural protein, or a SARS-CoV-2 accessory protein.

[0012] In some aspects, the invention provides an immunogenic composition comprising the live attenuated SARS-CoV-2 virus and a pharmaceutically acceptable excipient. [0013] The invention further includes a method for eliciting an immune response against a SARS-CoV-2 virus in a subject, comprising administering an immunogenically effective amount of the live attenuated SARS-CoV-2 virus or the immunogenic composition to the subject. In some aspects, the subject is a human.

[0014] In one aspect the invention provides a construct comprising (i) a yeast-bacterial shuttle vector, referred to as "pY2B," comprising (a) a yeast autonomously replicating sequence [ARS], and (b) a yeast centromere [CEN] sequence; (ii) a regulated bacterial origin of replication comprising repE, sopA, sopB, sopC, incC, and ori2; and, (iii) a cDNA sequence encoding a SARS-CoV-2 genome, wherein the SARS-CoV-2 genome comprises at least one inactivating mutation. In some embodiments the cDNA sequence is operably linked to a promoter. In some embodiments the promoter is a T7 promoter sequence. In some embodiments the promoter is a promoter active in mammalian cells. In some embodiments the promoter is a CMV promoter. In an aspect the disclosure provides a yeast cell comprising the SARS-CoV-2 genome having at least one inactivating mutation and/or the pY2B containing the SARS-CoV-2 genome. In an aspect the disclosure provides a mammalian cell comprising the SARS-CoV-2 genome having at least one inactivating mutation and/or the pY2B containing the SARS-CoV-2 genome . In one aspect a mammalian cell that produces a live attenuated SARS-CoV-2 virus is disclosed.

[0015] Aspects of the invention further relate to the use of the live attenuated SARS-CoV-2 virus or the immunogenic composition for the preparation of a vaccine for eliciting an immune response towards a SARS-CoV-2 virus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1A shows a schematic representation of the SARS-CoV-2 genome and protein coding regions.

[0017] Fig. IB shows the general workflow of transformation-associated recombination (TAR) cloning. In-yeast genome reconstruction requires the delivery of overlapping cDNA fragments of the viral genome and a TAR vector in yeast. Transformed cDNA fragments are assembled by homologous recombination in yeast to generate a yeast artificial chromosome (YAC) that comprises the full-length SARS-CoV-2 cDNA sequence.

[0018] Fig. 2 shows a scheme illustrating the introduction of deletions into the SARS-CoV-2 cDNA using CRISPR-Cas technology. The PCR-product sizes (bottom) are shown for exemplary SARS-CoV-2 variant cDNAs comprising a deletion in the sequence encoding an 0RF6 or S protein, confirming the presence of the deletion.

[0019] Fig. 3A shows a schematic representation of the SARS CoV-2 genome from engineered viruses. The genomes schematized represent: infectious clone SARS CoV-2 WT(icSARS CoV-2), icSARS CoV-2 GFP (where Orf 7a has been replaced by GFP), and icSARS replicon (NLuc Rep), in which the entire structural protein coding region has been replaced with nanoLuciferase.

[0020] Fig. 3B shows gel images illustrating PCR results of viral RNAs produced from molecular clones of SARS-CoV-2 generated with the genomic platform. RNA was electroporated into BHK cells (lanes 1-5) or Vero cells (lanes 6-8). Viral replication was assessed by RT-PCR and confirmed by primers that amplify subgenomic M RNA also containing the leader sequence (lanes 1 to 8). As a positive control, viral replication in SARS- CoV-2 infected Vero cells was assessed by RT-PCR (lane 7).

[0021] Fig. 3C shows bar graphs illustrating replication of SARS-CoV-2 Replicon-Nluc in Vero cells. Nanoluciferase (NLuc) activity was measured 24h and 48h post electroporation of the engineered SARS-CoV2 replicon RNA together with SARS-CoV2 N mRNA in Vero cells. As a negative control, Vero cells were transfected only with SARS-CoV2 N mRNA.

[0022] Fig. 4 shows gel images of diagnostic PCR results demonstrating production of SARS- CoV-2 variants. Genomic DNA was extracted from yeast strains following identification of positives produced CRISPR-Cas9 based editing by colony PCR. Top panels: primers flanking a deletion. The first lane is the wild-type control. Subsequent lanes are independently generated variants. The bottom panels show a lack of a signal using primer pairs internal to the deletion endpoints. The first lane in each set shows a positive control using DNA extracted from yeast harboring the wild-type SARS-CoV-2 cDNA clone.

[0023] Fig. 5. Schematic of the pY2B_T7-SARS-2-AOrf7eGFP plasmid. The box outlines the BAC regulatory elements inserted in the parental yeast vector.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions, Terminology, and Conventions

[0024] SARS-CoV-2 is a positive-strand, or "sense-strand," RNA virus. As used herein, "SARS- CoV-2" may be used to refer to the virus (i.e., viral particle) or to the viral genome, as will be understood from context. [0025] A "SARS-CoV-2 strain" refers to a certain genetic subtype, or isolate of SARS-CoV-2. A SARS-CoV-2 strain may be a naturally occurring isolate (e.g., WA-1) or a recombinantly generated SARS-CoV-2 variant as described herein.

[0026] As used herein, the term "viral genome" refers to a wild-type or variant genomic RNA of a wild-type or attenuated SARS virus (in particular a SARS-CoV-2 virus), and the nucleotide sequence of the viral RNA. Additionally, as will be apparent from context, "viral genome" may refer to the DNA equivalent of the RNA sense strand (i.e., in which uracil in RNA is replaced by thymine in DNA) and to the complement of the DNA or RNA sequences.

[0027] A "wild-type SARS-CoV-2 genome" may be naturally occurring or recombinant and is a genome that encodes the proteins encoded in SEQ ID NO: 2. The nucleotide sequence of a wild-type SARS-CoV-2 genome may differ from strain to strain or isolate to isolate. Examples of wild-type SARS-CoV-2 genomes include the WA-1 genome (Genbank Accession No. MN985325.1) [SEQ ID NO: 1], the Wuhan-Hu-1 genome (Genbank Accession No. MN 908947.3), and HM-l-SARSCoV2. HM-l-SARSCoV2 has the same sequence has WA-1 (SEQ ID NO: 1) except for a synonymous substitution in the S coding sequence. In some cases, the substitution is A>G (WA-1 > HM-l-SARSCoV2) at position 24205. In some cases, the sequence of HM-l-SARSCoV2 additionally has a non-synonymous substitution in the pplab region. In some cases, the substitution is G>T at position 3659.

[0028] A "wild-type SARS-CoV-2 virus" is a SARS-CoV-2 virus that comprises a wild-type genome, is able to replicate in human cells (e.g., human epithelial cells, such as Calu-3 cells or A549-ACE2 cells) and/or Vero cells (African green monkey kidney cells), and is pathogenic.

[0029] In contrast to a "wild-type SARS-CoV-2 genome" a "SARS-CoV-2 variant genome," does not encode active forms of all of the proteins encoded in SEQ ID NO: 1, and has inactivating mutations in one or more protein coding regions. In a variant genome, at least one wild-type protein a not expressed, or is expressed in a form that does not have the activity or function of the wild-type protein.

[0030] The term "inactivating mutation," as used herein refers to a mutation in a nucleotide sequence of a virus genome, that interferes with the function or expression of the SARS-CoV- 2 protein normally encoded in the nucleotide sequence. In some embodiments, the inactivating mutation is a deletion of more than 10 nucleotides as discussed in detail hereinbelow. In some embodiments, the inactivating mutation is an inactivating deletion. The term "inactivating deletion" as used herein refers to a deletion in a nucleotide sequence of a virus genome, that interferes with the function or expression of the SARS-CoV-2 protein normally encoded in the nucleotide sequence. In some embodiments, the inactivating mutation is a nucleotide substitution, or a combination of multiple contiguous or noncontiguous substitutions. In some embodiments, the inactivating mutation is an inactivating substitution. The term "inactivating substitution" as used herein refers to a substitution in a nucleotide sequence of a virus genome, that interferes with the function or expression of the SARS-CoV-2 protein normally encoded in the nucleotide sequence. In some embodiments, the inactivating mutation is an insertion or deletion of one or more nucleotides that causes a reading frame shift mutation that when translated produces an inactive (e.g., truncated) protein.

[0031] A "variant SARS-CoV-2 virus" is a is a SARS-CoV-2 virus that comprises a variant genome.

[0032] As used herein, the term "live attenuated" refers to a virus that is (i) capable of replication, (ii) capable of eliciting a immune response, and (iii) which is not pathogenic (e.g., does not infect human cells and/or does not cause COVID-19. In preferred embodiments, the live attenuated virus is capable of eliciting a protective immune response.

[0033] As used herein, the term "corresponding to" is used to describe positions in SARS- CoV-2 variant genome relative to a reference genome. Unless otherwise specified, the reference genome is a wild-type SARS CoV-2 genome, which may be SEQ ID NO: 1 (the genome of the WA-1 strain of SARS-CoV-2 (SARS-CoV-2/human/USA/WA-CDC-WAl/2020), Genbank Accession no. MN985325.1; See Section 18, below). To identify a sequence in, or absent (deleted) from a SARS CoV-2 variant genome, the variant genome or subregion thereof is aligned with the reference sequence and the nucleotide numbering of the reference sequence is applied to the variant genome sequence. For example, in SEQ ID NO:1, orf3a extends from base 25393 to base 26220. See Table 5. A variant genome in which the entire orf3a sequence is deleted can be described a having a deletion of the region corresponding to base 25393 to base 26220 of the reference sequence. Similarly, in a genome in which orf3a is not deleted but which has sequence differences (e.g., SNPs) relative to a reference sequence, the two orf3a sequences "correspond" to each other, not withstanding the differences in sequence.

[0034] Two nucleotide or amino acid sequences can be aligned by art known methods. In general, sequence alignment is performed to determine sequence identity or to identify corresponding regions or nucleotide positions between sequences. The sequences are aligned for maximum correspondence over a comparison window or designated region. A comparison window includes reference to a segment of any one of the number of contiguous positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has from 10 to 600 residues, e.g., about 50 to about 200 residues, or more usually about 100 to about 150 residues, in which two sequences with the same number of contiguous positions may be compared after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0035] The terms "identity" or "identical" in the context of two or more amino acid or nucleic acid sequences, refer to two or more sequences that are the same ("identical") or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 90% identity, preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over the entire sequence of a specified region. Two polynucleotides or two polypeptides may have less that 100% identity (due to natural polymorphism or artificial variation) but are recognized as variants of the same nucleic acid or polypeptide sequence. For example, two polypeptides with 95% sequence identity can be recognized as the same (e.g., both S proteins). Sequence identity can be used to describe relationships with any polynucleotide or polypeptide sequence referred to in this disclosure. Two polynucleotides or two polypeptides may be described as substantially identical or having substantial identity when they have at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 90% identity, preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

[0036] As used herein, the term "cDNA" refers to a DNA that has the sequence corresponding to a specified RNA (e.g., the positive or negative strand of a wild-type or variant SARS-CoV-2 genome), except for the substitution of thymine in DNA for uracil in RNA. Thus, the term cDNA does not necessarily refer to a polynucleotide produced by reverse transcription from an RNA template.

[0037] As used herein, the terms "pathogenic" or "pathogenicity" have their normal meanings in the art and refer to the potential of a virus to infect human cells and cause disease in a subject. The pathogenicity of a coronavirus may be assessed using methods well-known in the art. Typically, the pathogenicity of a SARS-CoV-2 is determined by assaying disease associated symptoms in a subject, for example fever, cough, and shortness of breath. The terms "reduced pathogenicity" or "non-pathogenic" describe a SARS-CoV-2 that is less pathogenic than (i.e., pathogenicity is decreased or diminished compared to) a wild-type SARS-CoV-2 such as CoV-2-WA-l.

[0038] The term "patient" or "subject" refers to a human or non-human mammal to which a vaccine comprising live attenuated virus is administered.

[0039] The term "immune response" refers to a cell-mediated (T-cell) immune response and/or an antibody (B-cell) immune response.

[0040] The terms "immunogenic composition," or "vaccine" are used interchangeably and refer to a composition that elicits an immune response in a subject, especially a human. An immunogenic composition or vaccine can be used prophylactically to prevent COVID-19 or other coronavirus diseases.

[0041] The term "immunogenicity effective amount" of a vaccine or immunogenic composition is an amount that elicits an immune response towards SARS-CoV-2.

[0042] As used herein, "administering" or "administration of" a vaccine refer to (i) the act of physically delivering a substance as it exists outside the body (e.g., by injection or inhalation) as well as (ii) instructing that a vaccine should be administered by, for example, writing a prescription for vaccination or directing medical professionals to vaccinate a subject. [0043] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" are used interchangeably and refer to a substance or compound that aids or facilitates preparation, storage, administration, delivery, effectiveness, absorption by a subject, or any other feature of the composition for its intended use or purpose. Such pharmaceutically acceptable carrier is not biologically or otherwise undesirable and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the subject or interacting in a deleterious manner with the other components of the pharmaceutical composition.

[0044] It will be understood that the viral genome or portions thereof can be defined by reference to either the sense or the antisense strand sequence. Unless otherwise indicated, nucleotide sequences are presented 5' to 3'.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

2. Introduction

[0046] SARS-CoV-2 is an RNA virus that causes COVID-19. In one aspect, the present invention relates to a live attenuated SARS-CoV-2 for use in preparation of a vaccine against COVID-19 or other SARS-CoV-2 illnesses. The live attenuated SARS-CoV-2 disclosed herein is a SARS-CoV-2 variant that differs from wild-type SARS-CoV-2 by virtue of inactivating mutation(s) (e.g., deletions) in the coding sequences of one or more viral proteins. A live attenuated SARS-CoV-2 variant of the invention is (i) capable of replication, (ii) capable of eliciting a immune response, and (iii) not pathogenic (e.g., does not infect human cells or does not cause COVID-19. In preferred embodiments, the live attenuated virus is capable of eliciting a protective immune response.

2.1 Proteins and Protein Coding Sequences

[0047] The RNA genome of SARS-CoV-2 virus is about 29.8 kb to 30 kb in length and encodes at least 29 proteins ("SARS-CoV-2 proteins") including four structural proteins, at least 16 predicted non-structural proteins, and several accessory proteins. See, e.g., Finkel et al. (2020), "The coding capacity of SARS-CoV-2," bioRxiv, 2020.05.07.082909. See Fig. 1A. In general, the genome of SARS-CoV-2 is arranged in the order 5'-leader - UTR - replicase - S - E - IVI - N - 3'UTR - poly (A) tail with accessory genes interspersed within the structural genes S, E, M, N. See Michel, CJ. et al.., 2020, "Characterization of accessory genes in coronavirus genomes," Virol J 17, 131, https://doi.org/10.1186/sl2985-020-01402-l. Table 1, below, shows nucleotide positions (with reference to SEQ ID NO: 1) defining the putative boundaries of coding sequence for SARS-CoV-2 proteins.

2.1.1 Structural Proteins

[0048] The SARS-CoV-2 structural proteins, Spike (S), Nucleocapsid (N), Membrane (M), and Envelope (E), are required to make a complete virus particle. The S protein is responsible for receptor binding, membrane fusion, and tissue tropism. The SARS-CoV-2 is believed to use the same receptor as SARS-CoV for cell entry: the angiotensin-converting enzyme 2 receptor (ACE2). Work in SARS-CoV-l suggests that the E protein might be nonessential for viral replication (Graham et al., 2012, "A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease," Nat Med, 18 (12): 1820-6; Siu et al., 2014, "Suppression of innate antiviral response by severe acute respiratory syndrome coronavirus M protein is mediated through the first transmembrane domain, "Cell Mol Immunol, 11 (2): 141-9).

2.1.2 Non-Structural Proteins

[0049] The genome of SARS-CoV-2 encodes at least 16 predicted non-structural proteins (e.g., Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, an Nspl6). The 16 non-structural proteins are produced after viral entry from two large precursor proteins (ORFla and ORFlb). Work in SARS-CoV and other coronaviruses suggests that Nsp2, Nsp6, and Nspl5 might be nonessential for viral replication in cells (Graham et al., 2006, "The nsp2 proteins of mouse hepatitis virus and SARS coronavirus are dispensable for viral replication," Adv Exp Med Biol; 581: 67-72; Dediego et al., 2008, "Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice," Virology, 376(2): 379-89; Narayanan et al. (2008), "Severe acute respiratory syndrome coronavirus nspl suppresses host gene expression, including that of type I interferon, in infected cells," J Virol, 82(9): 4471-9; Graham et al. (2012), "A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease," Nat Med; 18 (12): 1820-6; Siu et al. (2014), "Suppression of innate antiviral response by severe acute respiratory syndrome coronavirus M protein is mediated through the first transmembrane domain, "Cell Mol Immunol, 11 (2): 141-9; Jimenez-Guardeno et al. (2015), "Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine," PLoS Pathog, 11(10): el005215.; Castano- Rodriguez et al. (2018), "Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis," mBio, 9 (3); Menachery et al. (2018), "Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines," J Virol, 92 (17); Finkel et al. (2020), "The coding capacity of SARS-CoV-2," bioRxiv, 2020.05.07.082909). Nspl3 is a bifunctional RNA/NTP triphosphatase (TPase) and helicase; nspl4, is a bifunctional 3'->5' mismatch exonuclease, and mRNA cap guanine-N7 methyltransferase, and nspl6 is a cap ribose 2'-0 methyltransferase; that, in conjunction with nsplO, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, See Viswanathan et al., "Structural basis of RNA cap modification by SARS-CoV-2". Nat Commun 11, 3718 (2020). doi.org/10.1038/s41467-020-17496-8.

2.1.3 Accessory Proteins

[0050] The genome of SARS-CoV-2 encodes several accessory proteins, including ORF3a, 0RF3b, 0RF6, ORF7a, 0RF7b, ORF8a, 0RF8b, 0RF9b, and ORFIO. See e.g., Finkel et al. (2020), "The coding capacity of SARS-CoV-2," bioRxiv, 2020.05.07.082909. The SARS-CoV-2 accessory proteins are likely to contribute to pathogenesis. Work in SARS-CoV and other coronaviruses suggests that at least six of the accessory proteins, ORF3a, 0RF6, ORF7a, 0RF8 and 0RF9 might be nonessential for viral replication in cells

[0051] Table 1. Deletion Boundary Positions By Protein

*including stop codon

3. SARS-CoV-2 Variant Genomes and Live Attenuated SARS-CoV-2

[0052] Aspects of the invention relate to a live attenuated SARS-CoV-2 having a viral genome that comprises an inactivating mutation in one or multiple protein encoding sequences, where the inactivating mutation prevents expression of a functional protein. The inventors have generated a series of SARS-CoV-2 variants comprising an inactivating mutation in one or more sequences encoding a SARS-CoV-2 protein. See Table 2 and Table 3. Certain inactivating mutation(s) and combinations of inactivating mutations will result in SARS-CoV-2 variants that show robust replication but are nonpathogenic, and, thus, can be used as live attenuated virus vaccines against SARS-CoV-2.

[0053] Accordingly, the present disclosure provides methods and reagents for eliciting an immune response towards a SARS-CoV-2 in a subject in need thereof by delivering live attenuated SARS-CoV-2 to the subject. In one approach, the methods and reagents described herein are used to provide immunoprotection against infections elicited by coronaviruses (e.g., SARS-CoV-2), i.e., to prevent occurrence or recurrence of COVID-19 or other coronavirus diseases.

3.1 Exemplary Deletions and Point Mutations (Single Nucleotide Deletions, Substitutions, or Insertions)

[0054] Work on SARS-CoV-l showed that nonessential functions in essential viral proteins can be inactivated by small deletions or point mutations. Importantly, in some cases, this can enable generation of nonpathogenic strains. These include the C-terminus of Nspl (Jimenez- Guardeno et al. (2015), "Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine," PLoS Pathog, 11(10): el005215), which has involved in host translation shutdown by blocking the ribosome mRNA tunnel (Schubert et al. (2020), "SARS-CoV-2 Nspl binds ribosomal mRNA channel to inhibit translation:, bioRxiv 2020: 2020.07.07.191676), the enzymatic activity of the proofreading exonuclease of Nspl4, both of which were incorporated into nonpathogenic SARS live attenuated vaccine candidates (Graham et al. (2006), "The nsp2 proteins of mouse hepatitis virus and SARS coronavirus are dispensable for viral replication," Adv Exp Med Biol; 581: 67- 72; Castano-Rod riguez et al. (2018), "Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis," mBio, 9 (3)). Thus, in addition to deletions in ORFs as described above, we will create mutations in Nspl, Nspl4, and other proteins, that inactivate these nonessential functions.

[0055] Finally, we will generate a mutation that deletes a short N-terminal region of the M protein implicated in interferon antagonism (Siu et al. (2014), "Suppression of innate antiviral response by severe acute respiratory syndrome coronavirus M protein is mediated through the first transmembrane domain," Cell Mol Immunol, 11 (2): 141-9).

[0056] In some embodiments, the inactivating mutation is in a sequence encoding a SARS- CoV-2 structural protein. In some aspects, the structural protein is a Spike (S) protein, a Nucleocapsid (N) protein, a Membrane (M) protein, or an Envelope (E) protein. In some embodiments, inactivating mutation is in a sequence encoding a non-structural protein, e.g., nspl, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsplO, nspll, nspl2, nspl3, nspl4, nspl5, nspl6 protein. In some embodiments, the inactivating mutation is in a sequence encoding a SARS-CoV-2 accessory protein, such as an ORF3a, 0RF3b, 0RF6, ORF7a, 0RF7b, 0RF8a, 0RF8b, 0RF9b, or ORFIO protein. In some cased, multiple genes are deleted by a single contiguous deletion. For example, the deletion of orfN by necessity also removes orf9.

3.1.1 Inactivating mutations in at least two genes

[0057] In some embodiments, the viral genome of the live attenuated SARS-CoV-2 may comprise inactivating mutations in two or more viral proteins. Accordingly, in some embodiments, the viral genome comprises inactivating mutation(s) in a sequence(s) encoding two SARS-CoV-2 proteins. In some embodiments, the inactivating mutation(s) are in sequences encoding different SARS-CoV-2 proteins. In some embodiments, the inactivating mutation(s) are in sequences encoding different classes of SARS-CoV-2 proteins. For example, one inactivating mutation may be in a sequence encoding a SARS-CoV-2 non-structural protein and another inactivating mutation may be in a sequence encoding a SARS-CoV-2 accessory protein. In another embodiment, the inactivating mutation(s) are in sequences encoding SARS-CoV-2 proteins of the same class. For example, one inactivating mutation may be in a sequence encoding a SARS-CoV-2 non-structural protein, such as an nsp 2, and another inactivating mutation may be in a sequence encoding another SARS-CoV-2 non-structural protein, such as an nsp6.

3.1.2 Inactivating mutations (e.g., deletions) in three or more genes

[0058] In some embodiments, the viral genome comprises inactivating mutation(s) in a sequence(s) encoding three SARS-CoV-2 proteins. In some embodiments, the inactivating mutation(s) are in sequences encoding different SARS-CoV-2 proteins. In some embodiments, the inactivating mutation(s) are in sequences encoding different classes of SARS-CoV-2 proteins. For example, one inactivating mutation may be in a sequence encoding a SARS-CoV- 2 non-structural protein, another inactivating mutation may be in a sequence encoding a SARS-CoV-2 accessory protein, and another inactivating mutation may be in a sequence encoding a SARS-CoV-2 structural protein. In another embodiment, the inactivating mutation(s) are in sequences encoding SARS-CoV-2 proteins of the same class. For example, one inactivating mutation may be in a sequence encoding a SARS-CoV-2 non-structural protein, such as an nsp 2, another inactivating mutation may be in a sequence encoding another SARS-CoV-2 non-structural protein, such as an nsp6, and another inactivating mutation may be in a sequence encoding another SARS-CoV-2 non-structural protein, such as an nspl5. [0059] In some embodiments, the viral genome comprises an inactivating mutation in sequences encoding more than three SARS-CoV-2 proteins. In some embodiments, the viral genome comprises an inactivating mutation in sequences encoding four SARS-CoV-2 proteins. In some embodiments, the viral genome comprises an inactivating mutation in sequences encoding five SARS-CoV-2 proteins. In some embodiments, the viral genome comprises an inactivating mutation in sequences encoding six SARS-CoV-2 proteins.

3.1.3 Extent of deletion

[0060] In some embodiments, the inactivating mutation is a deletion. In some embodiments, the deletion comprises a deletion of the entire sequence encoding a SARS- CoV-2 protein. For example, a deletion may include the sequence from the ATG start codon to the stop codon of a SARS-CoV-2 protein coding sequence. In some cases additional upstream untranslated sequences may be deleted and/or additional downstream untranslated sequences may be deleted.

[0061] In some embodiments, the deletion comprises a deletion of at least 10 nucleotides. In some embodiments, the deletion is at least 10, at least 25, at least 50, at least 100, at least 200, or at least 500 nucleotides in length.

[0062] In some embodiments, the viral genome of the live attenuated SARS-CoV-2 of the present invention comprises a deletion(s) in a within a region(s) corresponding to nucleotide positions of SEQ ID NO: 1 selected from a region listed in Table 1. Table 1 lists the nucleotide position boundaries for viral proteins. Shown are nucleotide positions corresponding to SEQ ID NO: 1 and the SARS-CoV-2 protein encoded in the deleted region.

[0063] In other embodiments, a portion of a sequence encoding a SARS-CoV-2 protein may be deleted. In some aspects, the extent of deletion is in the range from 10% to 100% of a sequence encoding a SARS-CoV-2 protein. For example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of a sequence encoding a SARS-CoV-2 protein may be deleted.

3.1.4 Exemplary deletions

[0064] As described in the Examples and shown in Table 2, below, a series of SARS-CoV-2 variants were produced. In some embodiments, the viral genome of the SARS-CoV-2 has inactivating mutation(s) in one, or in at least one, of the genes selected from the genes listed in Table 2, below. In some embodiments, the viral genome of the SARS-CoV-2 has inactivating mutation(s) in two genes selected from the combination of genes listed in Table 3, below. [0065] Table 2. SARS-CoV-2 variants with inactivating mutations in one gene [0066] Table 3. SARS-CoV-2 variants with inactivating mutations in two genes

[0067] In some approaches, the sequence may be further modified or optimized to provide or select for various features. Thus, in addition to deletions described above, the genome of the live attenuated virus may comprise other deletions and/or mutations including, additions, substitutions, or combinations thereof. The mutations may also include, replacing a sequence of the SARS-CoV-2 genome with an analogous sequence of the viral genome of a different species, of a different subgroup, or of a different variant.

3.1.5 Exemplary Substitutions

[0068] In some embodiments, the live attenuated SARS-CoV-2 comprises a viral genome comprising one or more substitutions in a sequence(s) encoding one, two, three or more than three SARS-CoV-2 proteins. In some embodiments, the one or more substitutions are in a sequence encoding an nspl4 protein. In some embodiments, the substitution is in a region encoding the exoribonuclease (ExoN) of nspl4. In some embodiments, the substitution is at positions corresponding to nucleotide positions 18302 to 18303 of SEQ ID NO: 1. In some embodiments, the substitution is at positions corresponding to nucleotide positions 18308 to 18309 of SEQ ID NO: 1. In some embodiments, the substitution is at positions corresponding to nucleotide positions 18302 to 18303 and 18308 to 18309 of SEQ ID NO: 1. In some embodiments, the substitution corresponding to nucleotide positions 18302 to 18303 of SEQ ID NO: 1 is GC>CT. In some embodiments, the substitution corresponding to nucleotide positions 18308 to 18309 of SEQ ID NO: 1 is AT>CA.

3.2 Properties of Live Attenuated Virus

[0069] A live attenuated SARS-CoV-2 virus strain must preserve functions that enable sufficient viral replication in vivo to induce protective immunity, but limit functions that result in disease. Inactivating mutations should be non-revertable, and thus, we favor a deletion strategy over a point-mutation strategy and a combination of attenuating genetic lesions.

[0070] Vaccines of the invention contain live attenuated SARS-CoV-2 variants ("live attenuated virus"). Properties of the live attenuated virus include (i) capable of replication, (ii) capable of eliciting a immune response, preferably a protective immune response, in a mammal (e.g., mouse) or in a human subject, (iii) which is not pathogenic (e.g., does not infect human cells or does not cause COVID-19). Additional desirable properties of a live attenuated virus are (iv) infection with the virus does not result in suppression of specific host defense pathways; (v) infection with the attenuated virus triggers an increase in expression of interferon-stimulated genes (ISGs) that is greater than the level of expression see with infection with wild-type virus. See Blanco-Melo D et al. (2020), "Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19," Cell, 181(5): 1036-45 e9).

3.2.1 Replication

[0071] The live attenuated SARS-CoV-2 of the invention is able to replicate in human cells (e.g., human epithelial cells, such as Calu-3 cells (ATCC Number HTB-55) or A549 cells (ATCC Number CCL-185) that have been modified to express ACE2 (A549-ACE2) cells, and/or Vero cells. Vero cells are derived from African green monkey kidney cells, and are used as a cell line for virus production. Non-limiting examples of Vero cells are Vero E6 cells (or Vero E Cl 008; ATCC Number CRL-1586), Vero cells (ATCC Number CCL-81), and Vero 76 cells (ATCC Number CRL-1587). Replication capability and rate can be determined by any standard technique known in the art. The rate of replication is represented by the growth rate of the virus and can be determined by plotting the viral titer over the time post infection. In certain embodiments, a suspension containing the virus is incubated with cells (e.g., Vero cells) that are susceptible to infection by the virus, and subsequently, the number of infected cells is determined. In certain embodiments, the virus comprises a reporter gene and the number of cells expressing the reporter gene is representative of the number of infected cells. In a specific embodiment, the viral titer is determined with a plaque assay. To perform a plaque assay, dilutions of a virus stock are prepared and a monolayer of cells that are susceptible to infection are infected with the virus. Plaques produced by infected cells can then be counted and the titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter.

[0072] To determine if a SARS-CoV-2 variant is able to replicate, the viral titer may be compared to that of a wild-type SARS-CoV-2 grown under the same conditions. In certain aspects, a SARS-CoV-2 variant is identified as being replication-competent if the viral titer (in PFU/ml) of the SARS-CoV-2 variant is at least 10% of the viral titer of the wild-type SARS-CoV- 2 grown under the same conditions, preferably at least 20%, sometimes at least 30%, sometimes at least 50%, sometimes at least 75%, sometimes at least 90% or sometimes at least 95% of the viral titer of the wild-type SARS-CoV-2 grown under the same conditions.

3.2.2 Non-Pathogenicity

[0073] Pathogenicity of a SARS-CoV-2 variant may be determined in animal studies (e.g., in mice or hamsters). For example, intranasal administration of a SARS-CoV-2 variant can be performed in mice (e.g., transgenic mice expressing human ACE2) or hamsters followed by examinations and measurements that determine the pathogenicity of the variant. In certain embodiments, pathogenicity is determined through pulmonary function measurements by whole-body plethysmography. In some cases, mice can be euthanized and lungs may be examined for lung hemorrhage. In some cases, examination may include sectioning of the lungs followed by staining of lung sections. In some cases, SARS-CoV-2 Spike antigen can be stained for immunofluorescence studies. In some approaches, viral titers (e.g., by plaque assay) in the nasal-associated lymphoid tissues (NALT; mouse equivalent of tonsils), cervical lymph nodes (cLN), trachea, lungs, spleen and liver are measured over time. In certain cases, pathogenicity can be determined through examination of virus spread in tissue. For example, tissues from infected mice and hamsters may be collected, homogenized, and used for plaque assay to determine virus titer. SARS-CoV-2 variants that replicate transiently in the upper respiratory tract but show limited or no replication in the lungs or other target tissues, including brain, heart and kidneys can be identified as non-pathogenic.

3.2.3 Immune Response

[0074] An immunogenic composition may have one or more of the following effects upon administration to a subject: production of antibodies by B-cells; and/or the activation of suppressor, cytotoxic, or helper T-cells and/or T-cells directed specifically to an antigen protein present in the immunogenic composition. Various aspects of an immune response elicited by an immunogenic compositions can be evaluated using standard assays to determine whether a SARS-CoV-2 variant elicits an immune response.

[0075] In some approaches, the presence of antibodies is measured is animal models (e.g., in mice or hamsters that received a dose of the SARS-CoV-2 variant). In some approaches, blood, bronchioalveolar lavage, and nasal swab samples may be analyzed for the presence of serum antigen-specific antibodies using ELISA. For example, the presence of SARS-CoV-2 M, N, and/or S-binding antibodies may be tested.

[0076] In some approaches, cellular response may be examined by measuring the levels of circulating and/or tissue (e.g., lung tissue) neutrophils, monocytes, macrophages, T lymphocytes (including CD4 and CD8 subsets), and/or B lymphocytes (including plasmablasts) by flow cytometry using murine subset stains or hamsters-specific reagents. See e.g., Rees et al. (2017), "Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules", Vet Immunol Immunopathol, 183: 40-4.

[0077] In some approaches, serum cytokine response are examined using Luminex assays. For example, cytokine response may be examined in blood, bronchiolar lavage fluid, lung and associated lymphoid tissue (NALT and cLNs), or using nasal swab samples. Cytokines of interest include type I and III interferons, proinflammatory cytokines (including TNF, IL-lb, IL- 18, IL-6), type I cytokines (IFN-y and IL-12) type II cytokines (IL-4, IL-13), a type III cytokine (ILI A), a modulatory cytokine (IL-10), and chemokines.

3.2.4 Protective Immune Response

[0078] Immune response elicited in the subject may serve to neutralize infectivity of a virus, such as a coronavirus (e.g., SARS-CoV-2), and/or mediate antibody-complement, or antibody dependent cell cytotoxicity to provide protection against viral infection to an immunized subject. [0079] The ability of a SARS-CoV-2 variant to elicit an antibody response may be evaluated in various types of virus neutralization tests or assays. Virus neutralization assays are known and widely used for a variety of viruses. The dilution of a serum that provides 50% or more reduction of infectivity is referred to as the neutralization titer (Niedrig et al. (2008) Clin. Vaccine Immunol. 15, 177). In some approaches, the level of infectivity of a virus may be monitored in a standardized target cell culture, and the reduction in infectivity of the virus (e.g., SARS-CoV-2) may be evaluated after incubation with the tested serum. For example, blood samples from animals administered with a SARS-CoV-2 variant can be collected at their endpoints and used for neutralization assays.

[0080] In some approaches, the expression level of certain proteins involved in host defense pathways can be used to determine whether a SARS-CoV-2 variant provides protection against infection. For example, RNA-seq and/or RT-qPCR may be used to examine the transcriptional profile of Vero, hACE2-A549 and Calu3 cells at different time points after infection with a SARS-CoV-2 variant (as described in e.g., Blanco-Melo D et al. (2020), "Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19," Cell, 181(5): 1036-45 e9). In some approaches, the expression level of interferon-stimulated genes (ISGs) can be used to determine whether a SARS-CoV-2 variant provides protection against infection. Wild-type SARS-CoV-2 typically triggers a muted interferon response upon infection. Thus, an increase in expression of ISGs that is greater than the level of expression seen with infection with wild-type SARS-CoV-2 may indicate that the SARS-CoV-2 variant has protective properties.

4. Producing SARS-CoV-2 Variants

[0081] The live attenuated SARS-CoV-2 of the present invention may be produced using various art-known methods. In some embodiments, methods described herein are used.

4.1 Producing SARS-CoV-2 Variant Genomes

[0082] A live attenuated SARS-CoV-2 genome of the present invention may be prepared using yeast artificial chromosome (YAC) expressed in a yeast species (e.g., Saccharomyces cerevisiae). Yeast artificial chromosome (YAC) vectors are well known. See, for example, Arnak et al., 2012, "Yeast Artificial Chromosomes" in: eLS. John Wiley & Sons, Ltd: Chichester; DOklO.1002/9780470015902. a0000379.pub3; Gnugge and Rudolf, 2017, "Saccharomyces cerevisiae shuttle vectors," Yeast 34:205-221; Zeng et al., 2001, "Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast," Genomics 77:27-34; Bajpai B., 2013, "High Capacity Vectors," Advances in Biotechnology ppl- 10. doi:10.1007/978-81-322-1554-7_l; Burke et al., 1987, "Cloning of large segments of DNA into yeast by means of artificial chromosome vectors," Science 236:806-812; Kouprina et al., 2003, "Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes," Microbiol Rev 5:649 DO 10.1016/50168-6445(03)00070-6. In some embodiments the YAC is a shuttle vector that can propagate in a yeast species (e.g., Streptomyces) and a bacterial species (e.g., E. coli).

4.2 Transformation-Associated Recombination (TAR)

[0083] In one approach, a full-length SARS-CoV-2 cDNA is generated using transformation- associated recombination (TAR) methods. TAR is described in Example 2, below. Also see, e.g., Kouprina and Larionov, 2016, "Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology," Chromosoma 125(4): 621-632; Kouprina and Larionov, 2008, "Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae," Nat. Protoc. 3, 371-377; Kouprina and Larionov, 2006, "TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution," PNAS, 7 (10), 805-812; Kouprina and Larionov, 2003, "Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes," FEMS Microbiol Rev. 27(5):629-49. Also see U.S. Pat. No. 6,391,642.

[0084] Fig. IB shows the general workflow of TAR cloning. In brief, genome reconstruction in yeast (e.g., Saccharomyces cerevisiae) requires the delivery of overlapping cDNA fragments of the viral genome and a TAR vector in yeast. The TAR cDNA fragments are assembled by homologous recombination in yeast to generate a YAC containing the full-length SARS-CoV-2 sequence. In some approaches the full-length SARS-CoV-2 may have a nucleic acid sequence set forth in SEQ ID NO: 1 or a substantially identical variant thereof.

[0085] SARS-CoV-2 cDNA with inactivating mutations can be produced by providing cDNA fragments with the desired mutations for assembly by transformation-associated recombination. For example, TAR cloning may be used to introduce only certain sequences of the viral genome and exclude sequences that are not desired to be included in the viral genome of the SARS-CoV-2 variant without the preparation of a full-length SARS-CoV-2 cDNA as an intermediate product. The generated cDNA encoding the viral genome of a SARS-CoV-2 variant can be isolated to prepare viral RNA, which may then be introduced into suitable cells for virus production.

[0086] Alternatively, YACs comprising a wild-type or variant genome can prepared using TAR or other approaches and then modified to introduce one or more inactivating mutations in a sequence encoding a SARS-CoV-2 protein. In one approach, modifications are made using CRISPR-Cas technology, e.g., as described in Example 3.

4.3 Y2B Shuttle Vectors

[0087] In one approach, TAR-mediated assembly and/or modification (e.g., CRISPR- mediated gene editing) of a viral genome is carried out using a yeast-bacterial shuttle vector. In one approach a YAC shuttle vector comprising a bacterial low copy number replication system, referred to herein as a "pY2B" vector, is used. pY2B vectors are described in more detail hereinbelow.

[0088] Although large DNA constructs, such as the 30 KB SARS-CoV-2 genome cDNA and variants thereof, can be cloned and stably maintained in Saccharomyces cerevisiae as conventional yeast artificial chromosome (YAC), the extraction of YAC constructs from yeast generally suffers from very low yield. In contrast, plasmid production in E. coli can yield high quantity of DNA. However, E. coli plasmids containing large fragments of exogenous and potentially toxic DNA are usually heavily mutated in this process. To achieve high yield of plasmid DNA preparation, we have engineered a YAC-to-BAC plasmid (hereafter referred to as pY2B), where regulatory genes from a Bacterial Artificial Chromosome (BAC) are inserted into a Yeast Artificial Chromosome shuttle vector to allow cloning in yeast , amplification in E. coli at low copy number while avoiding deleterious mutation of the plasmid, and expression of the genome. The pY2B vector is adapted to for expression of large viral cDNA, such as SARS- CoV-2 variant constructs of the invention.

[0089] In one embodiment the pY2B comprises YAC components such as an autonomously replicating sequence [ARS], and a yeast centromere [CEN] sequence (e.g., CEN4 or CEN6) along with a bacterial origin of replication (e.g., ori2), a regulated bacterial origin of replication that keeps the plasmid copy number in bacteria around 1, and an expression system for producing viral RNA in bacteria, e.g., E. coli. The regulated bacterial origin of replication system generally includes comprising E. coli SopA, SopB, SopC (genes encoding plasmid partitioning proteins), incC (incompatibility region, e.g., derived from the bacterial F plasmid), and RepE (replication initiation site), or SopA, SopB, SopC, incC, and RepE variants or homologs that function in E. coli or other host bacteria. See, Imber et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7132-7136; Disque-Kochem et al., 1986, Mol. Gen. Genet. 202, 132-135; and Komori et al., 1999, EMBOJ. 18, 4597-4607. Suitable homologs or variants of SopA, SopB, SopC, incC, and RepE can be used. See, for example, Chen et al., 2004, "Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43" Gene 337:189- 98.

[0090] One suitable regulated bacterial origin of replication is found in pBeloBACll (GenBank Accession no. U51113), which is available from New England Biolabs (Ipswich, MA). Generally the pY2B vector does not contain a yeast 2 micron origin. Generally the Y2B vector does not contain oriV (high-copy origin of replication). An exemplary regulated bacterial origin of replication is found in SEQ ID NO:13.

[0091] An important feature of the pY2B vector is an expression system for expressing the viral genome, as discussed in greater detail below. Expression may be in vitro or in cells (e.g., mammalian cells).

[0092] Usually the pY2B includes one or more yeast selectable markers (e.g., neoR, natR, hygR, TRP1, URA3, HIS3, LYS2, LEU 2, TRP1, MET15).

[0093] Usually the pY2B includes one or more bacterial selectable markers, including auxotrophic markers or drug resistance markers (e.g., ampicillin resistance (AmpR); chloramphenicol resistance (Cm ^R )).

[0094] Fig. 5 illustrates a pY2B structure (shown with the viral genome). In one embodiment the pY2B vector has the sequence shown in SEQ ID NO:12 (minus the portion of SEQ ID NO.:12 encoding the viral genome and eGFP reporter). In some embodiments the pY2B has a sequence with substantial identity to SEQ ID NO:12 (e.g., at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 90% identity, preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) along with feature characteristic of a pY2B as described herein.

4.3.1 pY2B Cargo

[0095] The pY2B cargo is generally a viral genome, especially a cDNA encoding the genome of an RNA virus, in particular a SARS virus, such as SARS-CoV-2 virus. In one aspect, the invention provides a pY2B shuttle vector comprising a SARS-CoV-2 variant genome disclosed herein. An exemplary pY2B is pY2B_T7-SARS-2-AOrf7eGFP plasmid. pY2B_T7-SARS-2- A0rf7eGFP includes the pY2B components as well as A SARS-CoV-2 genome modified by replacement of Orf7a with an enhanced green fluorscent protein reporter.

[0096] In some cases the pY2B RNA encodes a reporter (usually a reporter protein), that allows expression of the encoded polypeptide to be monitored. Exemplary reporters function in yeast and/or bactieria. Exemplary reporters include green fluorescent protein (GFP), enhanced green fluorscent protein (eGFP), luciferase, nanoluciferase, and others known in the art.

[0097] In one approach the reporter gene is inserted into the SARS viral genome in place of a nonessential protein such as Nsp2, Nsp6, Nspl5, ORF3a, 0RF6, ORF7a, 0RF8 and 0RF9. In one approach the reporter gene is inserted in place of Orf7a, e.g., as described in Example 2.

4.3.2 Expression System

[0098] In some versions, the pY2B for use in the present invention includes an expression system functional in E. coli or other host bacteria. The expression system comprises a promoter operably linked to the viral genome (cDNA) sequence such that it is under control of a promoter. The term "promoter" describes the combination of the promoter (RNA polymerase binding site) and operators. The promoter may function in vivo or in a cell-free system. Any number of promoters may be used depending on the needs and preferences of the practitioner. Promoters for controlling RNA in vitro transcription can be any promoter for any DNA dependent RNA polymerase, for example. Generally a promoter (e.g., T7, T3, and SP6 RNA promoters and compatible RNA polymerases) is selected for in vitro expression for producing viral RNA.

[0099] In some cases a promoter is selected for expression of viral particles in mammalian cells transfected with a vector comprising a SARS-Covid-2 variant genome.

[0100] Provided in this disclosure are nucleic acid constructs that include the nucleic acid sequences provided herein. For example, a nucleic acid construct can be a recombinant DNA nucleic acid sequence comprising a promoter operably linked to a nucleic acid encoding the viral genome of the live attenuated SARS-CoV-2 of the present invention. A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. A promoter is a region or a sequence located upstream and/or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription, e.g., directing or driving expression of a nucleic acid sequence encoding the live attenuated SARS-CoV-2 in a cell or organism of interest. In some embodiments, the nucleic acid construct may comprise a T7 RNA polymerase promoter sequence that allows in-vitro transcription by T7 polymerase. For preparing nucleic acid constructs according to the embodiments of the present invention, nucleic acids may be manipulated to provide for the nucleic acid sequence to be in the proper orientation and proper reading frame. A nucleic acid according to the embodiments of the present invention can be included in an expression cassette for expression of a live attenuated SARS-CoV-2 in a cell or an organism of interest. An expression cassette can include various regulatory regions or sequences, such as transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, initiation codons, or termination signals. Also included among the embodiments of the present invention are vectors including nucleic acids or nucleic acid constructs according to the embodiments of the present invention. Such vectors can include necessary regulatory elements (as described above) that direct and regulate transcription of the nucleic acid sequences included in the vector.

4.3.2.1 T7 Promoter

[0101] In one approach a T7 promoter is used to express the pY2B cargo (e.g., viral genome cDNA). An exemplary construct is shown in Fig. 5. For example, transcription may be initiated by T7 polymerase that binds to the T7 RNA polymerase promoter included in the cDNA upstream of the SARS-CoV-2 genome sequence.

4.3.2.2 CMV promoter

[0102] The cytomegalovirus (CMV) promoter is commonly used to express heterologous genes in vivo (e.g., in mammalian cells) or in vitro. In one approach the CMV promoter is placed upstream of SARS-CoV-2 5'UTR, and a hepatitis delta virus (HDV) ribozyme (Rz) is inserted just downstream of SARS-CoV-2 3'UTR followed by a bovine growth hormone termination and polyadenylation signal. After delivery of the pY2B into host cells, the CMV promoter initiates the production of viral RNA from the nuclei of transfected cells by cellular RNA polymerase II. Using this construct eliminates the need for in vitro transcription. See Ye at al., 2020, "Rescue of SARS-CoV-2 from a single bacterial artificial chromosome," mBio 11(5) doi.org/10.1128/mBio.02168-20. Other promoters active in mammalian cells can be used in place of the CMV promoter including, without limitation, the RPE65 promoter, BEST1 promoter, CBA promoter, and VMD2 promoter. In pY2B constructs in which the pY2B is transfected into mammalian cells, a bacterial promoter or heterologous promoter (e.g., T7 promoter) is not required and may be omitted. It will be understood that within the host cell the virus makes copies of itself using the viral RNA-dependent RNA polymerase. Some of the RNA produced serve as mRNAs while the full length plus strand is what is packaged into virus particles.

4.4 Gene editing to create SARS-CoV-2 variants

4.4.1 CRISPR-Cas systems

[0103] In some approaches, CRISPR technology is used to introduce inactivating mutation(s) into the SARS-CoV-2 cDNA in yeast. The CRISPR technology is a gene-editing method that makes use of the CRISPR/CAS system. The "CRISPR/Cas" system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR/Cas systems include type I, II, and III sub-types. Wild-type type II CRISPR/Cas systems use the RNA- mediated nuclease, for example, Cas9, in complex with guide and activating RNA to recognize and cleave foreign nucleic acid. CRISPR systems usually include transactivating crisp RNA (tracrRNA), which binds the Cas endonuclease, and crisp RNA (crRNA), which binds to the DNA target sequence. Some CRISPR systems (e.g., CRISPR Casl2a/Cpfl) require only crRNA. CRISPR/Cas system in research and biomedical applications typically use a chimeric single guide RNA ("sgRNA"), which is a crRNA-tracrRNA fusion that binds both the Cas endonuclease and the DNA target sequence.

[0104] In some approaches, a SARS-CoV-2 variant cDNA is generated using a CRISPR/Cas system, where the system comprises a Cas protein and a guide RNA (e.g., an sgRNA). In addition, a donor polynucleotide that serves as a homology-directed repair (HDR) template harboring a mutation of interest is co-delivered with the Cas protein and a guide RNA. The cut genomic DNA is repaired by homologous recombination using the donor polynucleotide, resulting in a change in the genomic sequence from the wild-type to the SARS-CoV-2 variant. The HDR template sequence generally requires a certain amount of overlap (homology) on each side of the cut site.

[0105] Cas proteins and their amino acid sequence are well known in the art. Non-limiting examples of Cas proteins that can be used include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (or Csnl/Csxl2), CaslO, Casl2, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. The amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9; Sampson et al., Nature. 2013 May 9;497(7448):254-7; and Jinek, et al., Science. 2012 Aug 17;337(6096):816-21; Pickar-Oliver and Gersbach (2019), Nat. Rev. Mol. Cell Biol., 20: 490-507.

4.4.2 Guide RNAs and homology-directed repair (HDR) templates

[0106] Methods for designing sgRNAs that target a specified target sequence are well known in the art. See e.g., Mohr et al (2016). "CRISPR guide RNA design for research applications", The FEBS Journal. 283 (17): 3232-8; Doench et al. (2016), Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9", Nat. Biotechnol. 34:184-191; Horlbeck et al. (2016), "Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation, eLive. 5, el9760 (2016); Cui et al., "Review of CRISPR/Cas9 sgRNA Design Tools. Interdiscip. Sci. 2018, 10:455-465; and Kiani et al. (2015), "Cas9 gRNA engineering for genome editing, activation and repression", Nat Methods 2015;12:1051-4. The most commonly used sgRNA's are approximately 100 nucleotides in length. The programmable guiding sequence or targeting sequence comprises approximately 20 nucleotides at or near the 5' end of the sgRNA. The CRISPR Cas9 system can be targeted towards any genomic region complementary to that guiding sequence. In some cases, the sgRNA may be complementary to the target sequence in the viral genome. The degree of complementarity or identity between an sgRNA and its target sequence may be 100%, or less than 100, e.g., at least 95%, at least 90%, at least 85%, or at least 80%. The sgRNA may bind to a target sequence that is contiguous with a protospacer adjacent motif (PAM) recognized by the Cas protein. For example, Cas9 generally requires the PAM motif NGG for activity. Thus, in some systems, certain target sequences will be preferred based on the proximity of the target sequence to a PAM, while some Cas proteins (e.g., variants of Cas9) have flexible PAM requirements (see Karvekis et al., 2019, "PAM recognition by miniature CRISPR-Casl4 triggers programmable double-stranded DNA cleavage." bioRxiv.; Legut et al., 2020, "High-Throughput Screens of PAM-Flexible Cas9", Cell Reports 30:2859-2868), and other Cas proteins are PAM-independent (e.g., Casl4al). Exemplary PAMs are described, e.g., in Zhao et al. (2017), CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif. Int J Biol Sci; 13(12):1470-1478.

4.4.3 Delivery of CRISPR-Cas elements to yeast cells

[0107] Methods for the delivery of CRISPR-Cas elements into a cell are well known in the art. The guide RNA and the Cas protein may be delivered in DNA form, e.g., in a suitable vector that can be introduced into a yeast cell. Generally, DNA encoding the gRNA is cloned into a vector downstream of a promoter for expression. The sgRNA and Cas may be expressed from the same vector of the system or from different vectors.

5. Production of virus

[0108] Production of viral stock comprising any of the SARS-CoV-2 variants described herein may be performed using methods known in the art. See, e.g., Thao et al., 2020, "Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform,". bioRxiv, 2020.02.21.959817); https://www.nature.com/articles/s41586-020-2294-9. For general methods for genetic and recombinant engineering, and transfection techniques see e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Graham et al., Virol., 52:456 (1973); Davis et al., Basic Methods in Molecular Biology, Elsevier, (1986); and Chu et al., Gene 13:197 (1981).

5.1 Growth of SARS-CoV-2 in VeroE6 cells and rescue of infectious virus

[0109] [0121] SARS-CoV-2 variant viruses can be rescued by art known means, e.g., co-electroporation in BHK-21 cells with RNA encoding the viral N protein with viral RNA produced by in-vitro transcription of plasmid isolated from yeast (described in e.g., Thao et al., 2020, "Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform, "bioRxiv, 2020.02.21.959817; and Leem et al., 2008, "Purification of circular YACs from yeast cells for DNA sequencing," Genome, 51:155-8). The cells can be cocultured with Vero E6 cells as described (Thao et al., 2020, supra). We will incubate cells until a total cytopathic effect (CEP) is observed. Some variants may present fitness defects so CPE could be delayed.

5.2 Nucleic acids, vectors, and cells

[0110] Aspects of the invention relate to a nucleic acid comprising a sequence encoding the viral genome of the live attenuated SARS-CoV-2 as described above. The nucleic acids according to the embodiments of the present invention can be DNA or RNA. Nucleic acids described in the present disclosure can be used for producing the live attenuated SARS-CoV- 2 to be used as immunogenic compositions, or vaccines, against a coronaviruses (e.g., SARS- CoV-2).

[0111] The invention further includes a yeast cell comprising the nucleic acid described herein. Aspects of the invention also relate to a yeast cell comprising the nucleic acid or the vector as described herein.

[0112] The invention further includes a yeast cell comprising the nucleic acid described herein. Aspects of the invention also relate to a yeast cell comprising the nucleic acid or the vector as described herein.

[0113] In one example illustrating a process of generating a live attenuated SARS-CoV-2 virus as described in the present disclosure, in-vitro transcription is used to prepare viral RNA from the SARS-CoV-2 variant cDNA isolated from yeast. For example, transcription may be initiated by T7 polymerase that binds to the T7 RNA polymerase promoter included in the cDNA upstream of the SARS-CoV-2 genome sequence. Viral RNA is then introduced into suitable cells for virus production. Any suitable method can be used to introduce the viral RNA into a cell. In some embodiments, viral RNA is introduced into a cell by electroporation. Suitable cells forvirus production and propagation include mammalian cell, such as Vero cells, Baby Hamster Kidney fibroblast (BHK). In some embodiment, the cell is a Vero E6 cell. In some embodiment, the cell is a BHK-21 cell. In some embodiments, the nucleic acid as described herein (e.g., in the form of RNA) may be electroporated into BHK-21 cells and co-cultured with Vero E6 cells. The cultures are fed with medium capable of supporting growth of the cells. The cells are maintained in culture for several days until the desired virus titer is achieved. Virus can be harvested from these cultures by collecting the supernatants and re-feeding the cells. Viral particles can be recovered and purified using well known methods.

[0114] Accordingly, aspects of the invention relate to an isolated cell comprising the nucleic acid, or the live attenuated SARS-CoV-2 virus described herein. In some embodiments, the isolated cell is a mammalian cell. In certain aspects, the mammalian cell is a Baby Hamster Kidney fibroblast (BHK) cell (e.g., a BHK-21 cell) or a Vero cell (e.g., a Vero E6 cell).

6. Evaluation of attenuated virus

[0115] Virus with variant genomes can be evaluated for use as an attenuated viral vaccine.

Such evaluation may include one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) of the assays described below. In some assessments, transgenic mice expressing the human ACE2 receptor are used. These mice support SARS-CoV-2 infection and can be used for the study of drugs and vaccines (Bao et al., 2020, "The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice," Nature; Jiang et al., 2020, "Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin- Converting Enzyme 2," Cell, 182(1): 50-8 e8). hACE2 transgenic mice infected with SARS CoV2 demonstrate robust virus replication, weight loss, infiltration of lymphocytes and monocytes in alveolar interstitium and accumulation of macrophages in alveolar cavities 3-5 days postinfection. However, pneumonia became mild with focal lesion areas at seven days post - infection, suggesting a non-lethal and self-limiting infection course. As alternative, nontransgenic small -animal model, golden Syrian hamsters are used (Imai et al., 2020, "Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development," Proc Natl Acad Sci U S A, 117(28): 16587-95; Sia et al. (2020), "Pathogenesis and transmission of SARS-CoV-2 in golden hamsters," Nature) producing significant pulmonary disease which is ultimately self-limiting.

6.1 Viral replication ability of SARS-CoV-2 variants in hACE2-A549 and Calu3 cells

[0116] Given than the goal is to produce attenuated virus that replicate robustly in humans, virus with modified genomes can be characterized in human cells to determine if the inactivating mutations introduced in the viral genome affect replication fitness in hACE2-A549 and Calu3 cells. Virus replication kinetics can be examined and compare replication phenotype of SARS-CoV-2 variants with WT SARS-CoV-2 reference strain. These experiments identify variants that replicate in simple systems despite carrying one or more inactivating mutations in conserved viral proteins.

6.2 Assessing antiviral interferon responses elicited by SARS-CoV-2 variants

[0117] Host cell responses to infection with each of the viable variants, can be assessed using bulk and transcriptomics (RNA-seq using timepoints and methods described, see e.g., Blanco-Melo D et al., 2020, "Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19," Cell, 181(5): 1036-45 e9). Transcriptional dynamics may be monitored in Vero, hACE2-A549 and Calu3 cells at 0, 6, 12 and 24 h post-infection. Using these different cell types, it is possible to elucidate host responses to different variants, which will help us to better understand the role of each deleted gene in controlling host antiviral responses. In particular, this analysis will reveal whether deletion of a given gene results in an increase expression of interferon-stimulated genes (ISGs). This is important as WT SARS-CoV-2 triggers a muted interferon response upon infection (Blanco-Melo D et al., 2020, "Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19," Cell, 181(5): 1036-45 e9). Viral variants that replicate well in Vero cells and other cell lines but trigger the strongest interferon response (as determined by ISG induction) will be prioritized for animal experiments.

6.3 Virulence and viral replication ability of SARS-CoV-2 variants in hACE2 transgenic mice and hamsters

[0118] The replication ability and virulence of candidate live-attenuated SARS-CoV-2 viruses can be determined after intranasal inoculation in animal models, including hACE2 transgenic mice and hamsters.

[0119] To test SARS-CoV-2 variants for virulence, studies are conducted in hACE2- transgenic mice. Similar numbers (N = 9-10/sex/group) of 23-24-week-old male and female mice will be assigned to each infection group. Mice will be anaesthetized with and intranasally infected with either 5xl0 ⁴ or 5xl0 ⁵ pfu SARS-CoV-2 in 50 pl of virus collection medium. To monitor morbidity, mice and hamsters will be weighed daily out to 6 dpi. Animals that lose >20% of their starting weight will be humanely sacrificed by CO2 inhalation and cervical dislocation.

[0120] To elucidate the magnitude of SARS-CoV-2 variant replication and test for pathology, 12 9-12-week-old female animals are infected with 5xl0 ⁴ in 50 pl of virus collection medium as above. A subset of each cohort can be randomly assigned for pulmonary function measurements by whole-body plethysmography daily. On several dpi, animals will be euthanized, lungs will be visually scored for lung hemorrhage, and the inferior right lobe will be frozen for viral titration by plaque assay as described. The large left lobe will be placed in 10% buffered formalin and stored at 4°C for subsequent histological sectioning and analysis. Lung sectioning and hematoxylin and eosin staining will be performed. Also, SARS-CoV-2 Spike antigen will be stained for immunofluorescence studies. We will measure viral titers (by plaque assay) in the nasal-associated lymphoid tissues (NALT; mouse equivalent of tonsils), cervical lymph nodes (cLN), trachea, lungs, spleen and liver over time. Based on preliminary data, we expect to detect viral particles in lymphoid tissues (cLN, NALT) and lungs. A comparison of females and males revealed more severe weight loss in males, suggestive of delayed viral clearance. 6.4 Assessment of tissue distribution and shedding of SARS-CoV-2 variants in hACE2 transgenic mice and hamsters

[0121] As SARS-CoV-2 replicates in the lower respiratory track (LRT) in hamsters, it is important to test if SARS-CoV-2 variants abolish viral ability to establish a productive infection in the LTR. The level of virus shedding into feces and saliva will be also examined, as these are the main routes of dispersion of vaccine strains. Reduction of spread should increase the safety of these vaccine candidates. Tissues from infected mice and hamsters will be collected, homogenized, and used for plaque assay to determine virus titer.

6.5 Examination of antibody and cytokine responses to SARS-CoV-2 variants in hACE2 transgenic mice and hamsters

[0122] The ability of SARS-CoV-2 variants to induce cellular and antibody responses can be examined. Blood samples from immunized mice and hamsters will be collected at Day 21 after infection and used for neutralization assay to determine antibody against SARS-CoV-2. Briefly, serum samples collected from immunized animals will inactivated at 56 degrees C for 0.5 h and serially diluted with cell culture medium in two-fold steps. Diluted sera will be mixed with a virus suspension of 100 TCID50 in 96-well plates at a ratio of 1:1, followed by 2 h of incubation at 36.5 degrees C in 5% CO2. 1-2 x 10 ⁴ Vero cells will be added to the serum-virus mixture, and the plates will be incubated for 5 days at 36.5 degrees C. CPEs of each well will be recorded under microscopes, and the neutralizing titer calculated by the dilution number of 50% protective condition.

[0123] Samples will be analyzed for SARS-CoV-2-specific antibodies with ELISA, including M- , N- and S-binding antibodies. Samples will also be isotyped for IgG and IgA such reagents are available for hamsters, see e.g., Rees et al. (2017), "Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules", Vet Immunol Immunopathol, 183: 40-4). We will also examine cytokine responses, including type I and III interferons, proinflammatory cytokines (e.g., TNF, IL-1, IL-6), Th2 cytokines (e.g., IL-4, IL-13), and chemokines.

6.6 Genetic stability of SARS-CoV-2 variants in hACE2 transgenic mice and hamsters

[0124] Genetic stability of potential SARS CoV2 vaccine strains will be examined with sequencing analysis. Mice and hamsters will be infected with SARS CoV2 vaccine candidates, and viruses recovered from tissues of infected mice will be used for subsequent infections. The process will be repeated 5 times, and all recovered viruses will be analyzed by sequencing analysis to determine if mutations have been accumulated during the adaptation experiment. We will also determine if virus isolated 5 dpi have an increase in virulence, using the assays described above.

6.7 Protective potential of candidate SARS-CoV-2 vaccines in K18-hACE2 transgenic mice

[0125] Once the optimal vaccine candidate is identified, we will compare the replication and spread of WT SARS-CoV-2 in vaccinated and naive animals. Intranasal challenge of K18- hACE2 transgenic mice with SARS-CoV-2 causes rapid virus spread to the alveoli and brain with up-regulation of pro-inflammatory cytokines and chemokines. At 3-5 dpi, K18-hACE2 mice begin to lose weight, and all die within 7 days due to brain infection. Thus, this model poses a rigorous challenge for vaccine efficacy.

7. Administration of Immunogenic Compositions ("Vaccination")

[0126] Also provided herein are immunogenic compositions comprising the live attenuated SARS-CoV-2 virus, the nucleic acid, the vector, or the isolated cell of the invention. The immunogenic compositions described herein can be delivered to subjects by any suitable route or a combination of different routes. Immunogenic compositions according to the embodiments of the present invention can be also referred to as "vaccines." In some embodiments, the immunogenic composition comprising the live attenuated SARS-CoV-2 virus, the nucleic acid, the vector, or the isolated cell as described herein further comprises a pharmaceutically acceptable excipient or carrier.

[0127] In some approaches, solutions (e.g., a sterile injectable solution) can be prepared with the live attenuated SARS-CoV-2 virus, the nucleic acid, the vector, or the isolated cell in the required amount and an excipient suitable for injection into a subject, e.g., a human subject. For example, a suitable carrier may be buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels, stabilizing agents, adjuvants, diluents, or surfactants. For injection, the excipient will typically be a liquid. Exemplary pharmaceutically acceptable excipients include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline, or a combination thereof. The pH of the liquid excipient is generally about 5 to about 8 or from about 7 to 7.5. An excipient may include a pH controlling buffer. 7.1 Routes of Administration

[0128] Aspects of the invention include methods of administering the live attenuated SARS- CoV-2 virus of the present disclosure for inducing an immune response towards a SARS-CoV- 2 virus in a subject. In some approaches, the administration includes administering the live attenuated SARS-CoV-2. Administration is not limited to a particular site or method. Any suitable route of administration or combination of different routes can be used, including, but not limited to, parenteral administration (e.g., intravenous, intramuscular, subcutaneous, or intradermal injection), nebulization/inhalation, oral administration (e.g., in the form of a tablet or capsule), or by installation via bronchoscopy.

[0129] In some approaches, the immunogenic composition is administered by injection, such as intravenous injection. In some approaches, the immunogenic composition is administered by oral inhalation, nasal inhalation, or intranasal mucosal administration. Administration of the immunogenic compositions described in the present disclosure by inhalant can be through the nose or mouth via delivery by spraying or droplet mechanism, for example, in the form of an aerosol. The composition may be administered alone or with an adjuvant as described above.

7.2 Dosage and effective amounts

[0130] Dosage values may depend on the nature of the product. It is to be understood that for any particular subject, specific dosage regimens can be adjusted over time and in course of a vaccine treatment according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Accordingly, dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0131] The amount of immunogenic compositing administered will be an "immunologically effective amount," i.e., an amount that is effective, at dosages and for periods of time necessary, to achieve a desired result. A desired result would include eliciting an immune response against a coronavirus (e.g., SARS-CoV-2).

[0132] A immunologically effective amount may vary according to factors such as age, sex, medical condition, and weight of the subject, or or whether other drugs are included in the regimen. Dosage regimens may be adjusted to provide the optimum response and can be adjusted by a medical professional in the event of any contraindications. An immunologically effective amount is also one in which any toxic or detrimental effects of the live attenuated virus are outweighed by the therapeutically beneficial effects.

[0133] For example, a suitable dosage may comprise at least 10 virus infectious units (IFU), at least 50 IFU, at least 10 ² IFU, at least 5 x 10 ² IFU, at least 10 ³ IFU, at least 5 x 10 ³ IFU, at least 10 ⁴ IFU, at least 5 x 10 ⁴ IFU, at least 10 ⁵ IFU, at least 5 x 10 ⁵ IFU, at least 10 ⁶ IFU, at least 5 x 10 ⁶ IFU, at least 10 ⁷ IFU, at least 5 x 10 ⁷ IFU, at least 5 x 10 ⁸ IFU, or at least 10 ⁸ IFU of the live attenuated SARS-CoV-2.

[0134] The composition can be administered in one or more dose administrations daily, for one or several days, including a prime-boost paradigm. For example, the composition may be administered at one point in time, followed by second administration from 2 weeks to 2 years later. In some embodiments, between 1 and 10, e.g., between 2 and 8, or between 4 and 6 doses may be administered over a 1 year period. Booster vaccinations may be given periodically thereafter.

7.3 Subjects and therapeutic uses

[0135] Subjects who are candidates for a vaccine treatment with the live attenuated SARS- CoV-2 virus as described herein include healthy individuals without higher risk for a SARS- CoV-2 infection than the general public. In some instances, the subject can have an elevated risk of developing a coronavirus infection such that they are predisposed to contracting an infection, or may be predisposed to developing a serious form of coronavirus disease, such as COVID-19 (for example, persons over 65, persons with asthma or other chronic respiratory disease, young children, pregnant women, individuals with a hereditary predisposition, individuals with a compromised immune system may be predisposed to developing a serious form of COVID-19). A "subject" herein refers to any single animal, including, for example, a mammal, such as a human, a non-human primate, or a mouse.

[0136] The immunogenic compositions can be used before the subject is infected with SARS-CoV-2 or any other coronavirus to prevent disease. Administration of the composition may be performed prior to the appearance of signs or symptoms of a SARS-CoV-2 or coronavirus disease. Thus, provided herein are methods and compositions for preventing development of COVID-19 or any other coronavirus disease. The immunogenic compositions can also be used in subjects with a current coronavirus infection, having one or more symptoms of the infection. A subject with a coronavirus infection may have been diagnosed with coronavirus infection based on the symptoms or the results of diagnostic test. The disease can be diagnosed using criteria generally accepted in the art. For example, viral infection can be diagnosed by the measurement of viral titer in a biological sample e.g., a nostril swab or mucosal sample) from the subject.

8. Other Coronaviruses

[0137] The compositions and methods described herein may also be used to prepare vaccines against other (known or as yet unknown) coronaviruses, such as SARS-CoV, MERS- CoV, HCoV-HKUl, HCoV-229E, HCoV-OC43, and HCoV-NL63. Generally, coronaviruses share the same genome organization. Accordingly, deletions and substitutions that result in live attenuated variants of SARS-CoV-2 may be introduced to sequences encoding corresponding proteins to produce live attenuated variants of other viruses that replicate but are non- pathogenic.

[0138] Additionally, administration of the live attenuated SARS-CoV-2 virus of the present disclosure may reduce the incidence of an infection caused by other coronaviruses. Exemplary coronaviruses include SARS-CoV, MERS-CoV, HCoV-HKUl, HCoV-229E, HCoV-OC43, and HCoV-NL63.

9. Examples

9.1 Example 1. Approach and Methods

[0139] The experiments below test the replication characteristics of SARS-CoV-2 vaccine candidates in cell culture and their safety and immunogenicity in susceptible small-animal models. Without intending to be bound by a particular mechanism, we hypothesize that the ability to suppress the diverse host antiviral mechanisms enables SARS-CoV-2 to cause disease. Therefore, our approach is to inactivate viral factors that inhibit the host defense mechanisms while retaining the ability to replicate well enough to induce an immune response. A panel of SARS-CoV-2 variants with systematic deletions in each of the accessory ORFs and mutations in genes/functions implicated in suppression of innate immunity can be generated. We hypothesize that some of these SARS-CoV-2 variants will not produce disease but elicit a robust immune response. We will identify which of these variants pose these phenotypic characteristics through the following overall approaches.

[0140] Variants can be screened in cell lines that support replication of viruses defective in suppressing host antiviral pathways. SARS-CoV-2 variants with individual or combined inactivating mutations will be characterized for (1) virus replication and (2) suppression of specific host defense pathways. The abilities of these variants with those of wild-type (WT) virus to replicate in primary cells in culture can be compared, with innate immunity pathways capable of restricting virus replication of those variants with defects modulating of the host antiviral defense. For those variants that replicate well in cell culture, their pathogenicity, tissue distribution, immunogenicity after infection of a susceptible mouse and hamster model of COVID-19 are assessed. DESeq2 may be used to analyze transcriptomics. For all other assays, data will be analyzed by plotting, computation of means and standard deviations, and the Mann-Whitney U test will be used to determine the probability of the data given the null hypothesis except for animal survival analysis that will be analyzed using Cox proportional hazards regression test. In some cases, at least three biological replicates are performed for all cell line and animal experiments, except for survival analysis which will use 10 animals to maximize power to detect virulence of putative attenuated strains.

9.2 Example 2. Generating full-length SARS-CoV-2 cDNA in yeast

[0141] Generation of recombinant coronaviruses has been laborious and time consuming due to their large genome size (26-34 kb). Furthermore, for several coronaviruses, cloned DNAs are unstable in E. coli. \Ne, therefore, generated recombinant coronaviruses based on "transformation-associated recombination" (TAR) in yeast (Thao et al., 2020, "Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform,". bioRxiv, 2020.02.21.959817). We assembled a full-length SARS-CoV-2 cDNA from 13 cDNA fragments of the viral genome into the yeast CEN-ARS vector pRS313 (HIS3; Fig. IB) to produce a yeast artificial chromosome (YAC). See e.g., Sikorski and Hieter, 1989, "A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae," Genetics, 122 (1): 19-27. To enhance circular YAC purification, we employed a cir- 0 strain lacking the endogenous 2 micron plasmid. Sequencing of the full-length SARS-CoV-2 cDNA demonstrates no amino acid changes relative to the original Wuhan (Wu-1) strains of SARS-CoV-2. Two synonymous SNPs are shared with the earliest Washington State isolate (WA-1), and an additional synonymous SNP in the S coding sequence appears to be a PCR error (useful for identifying the recombinant virus). We named this yeast strain stock YM3986. We now routinely obtain 0.2 pg of DNA from IL of yeast. This enables reliable virus rescue by electroporation with the N protein RNA into both BHK-21 and Vero E6 cells. We have also used TAR to replace the accessory ORF7a with GFP or a nanoluciferase and rescued virus (Fig.

3A, Fig. 3B, and Fig. 3C).

9.3 Example 3. CRISPR-Cas9 mutagenesis of SARS-CoV-2 cDNA in yeast

[0142] To enable rapid and sequential mutagenesis of the SARS-CoV-2 cDNA in YM3986, we implemented CRISPR-Cas9 technology in yeast (Fig. 2). In our approach, a CEN-ARS URA3- marked plasmid encoding Cas9 driven from the PGK1 promoter and a sgRNA expression cassette driven by the SNR52 promoter are co-transformed with a donor oligonucleotide that serves as a homology-directed repair (HDR) template harboring a mutation of interest (Anand et al., 2017, "Rad51-mediated double-strand break repair and mismatch correction of divergent substrates," Nature, 544(7650): 377-80). After identifying colonies harboring the desired change (efficiency ranges are typically 70-100%), we used media containing 5- fluoroorotic acid (Boeke et al., 1984, "A positive selection for mutants lacking orotidine-5'- phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance," Mol Gen Genet; 197(2): 345-6) to select for loss of the Cas9-sgRNA plasmid to eliminate the editor (Fig. 2). We designed and cloned sgRNAs along with HDR templates to delete each annotated ORF to test the essentiality of each. While many can be predicted from prior studies of other coronaviruses, we nonetheless generated deletion alleles of each, which could be useful for complementation studies. For the proteins encoded in the la and lab polyproteins, deletions were constructed so that they preserved sequences required for proteolytic processing and do not impact ribosomal frameshifting signals. For other ORFs, deletions were designed to avoid mutation of overlapping ORFs. Fig. 2 (bottom) and Fig. 4 show gel images illustrating PCR-product sizes for exemplary SARS-CoV-2 variant cDNAs comprising a deletion in SARS- CoV-2 protein coding sequences, confirming the presence of deletions.

[0143] The sgRNA sequences and repair oligonucleotides to be used are shown in Table 4. HDR oligonucleotides for selections correspond to the 40 bp upstream and downstream of the deletion endpoints. In each case, care in the design was taken maintain proteolytic cleavage sites (Nspl, 2, 6, 15) and to avoid overlapping essential ORFs. The ORF3a deletion removes putative 0RF3b, which is not thought to be expressed (Finkel et al., 2020, "The coding capacity of SARS-CoV-2," bioRxiv, 2020.05.07.082909), and the ORF7a deletion leaves 0RF7b intact and vice-versa. sgRNAs were selected using CHOPCHOP to avoid off-target effects. For the Nspl4 double point mutation, the repair template introduces synonymous changes in the PAM to prevent Cas9 cutting after introduction of the mutations. sgRNA- encoding oligonucleotides will be cloned into pJ H2972 and confirmed by Sanger sequencing. pJH2972 derivates will be cotransformed into YM3986 (yeast strain ha rboring pRS313-CoV- 2), and transformants will be selected on SC -His -Ura media. Transformants will be colony- purified and tested for mutations by colony PCR and gel electrophoresis for delete alleles or Sanger sequencing for point mutations. Positives will be streaked onto 5FOA-His media to select for loss of the Cas9 plasmid. Strains will be frozen in 20% glycerol. A small sample of each will be used for a crude DNA prep for PCR amplification and Sanger sequencing of the entire length of each clone.

[0144] Table 4. sgRNAs targeting SARS-CoV-2 proteins

9.4 Example 4. Preparation of SARS-CoV-2 variant RNA

[0145] By using the modified yeast plasmid preparation procedure above, DNA from each strain can be prepared by in-vitro transcription. We found that 200 ng of plasmid DNA can be reproducibly purified from yeast. After in-vitro transcription with T7 RNA polymerase, this typically yields 300 ng of full-length capped SARS-CoV-2 (as determined by RT-qPCR with primers at the 3' end of the genome).

9.5 Example 5. Generation of a Yeast-to-Bacteria Shuttle Vector (pY2B)

[0146] This example illustrates one approach of generating a YAC-to-BAC shuttle vector (pY2B) using a YAC vector as the backbone.

[0147] A DNA insert comprising open reading frames SARS-Cov-2 Orfab, S, M and E was incorporated into the pY2B vector by transformation-associated-recombination (TAR) in 5. cerevisiae generally as described in Example 2. The resulting plasmid contains the insert and is capable of replicating the genome of SARS-CoV-2 and variants thereof, as well as other large DNA molecules in a bacterial host, under the control of a low copy number origin of replication.

[0148] The Orf7a accessory gene (AOrf7-eGFP) of the full length SARS-CoV-2 was substituted with an eGFP reporter coding sequence and was cloned into a pRS313 YAC plasmid by Transformation-Associated-Recombination in 5. cerevisiae. The resulting plasmid was named p313-T7-SARS-2-AOrf7-eGFP.

[0149] A 4426 base pair long fragment, containing the regulatory genes repE, sopA, sopB and sopC) and sequence (incC), and the origin of replication (ori2), was obtained from a BAC, e.g., the pBeloBacllloxP2272 plasmid (Addgene 60342) using unique restriction enzymes and standard molecular biology approaches. The BAC fragment was cloned into the aforementioned p313-T7-SARS-2-AOrf7-eGFP in lieu of its high copy number origin of replication, to confer to the new plasmid low copy number replication when produced in bacteria. The resulting plasmid was named pY2B-T7-SARS-2-AOrf7-eGFP (see Fig. 5 and SEQ ID NO:13).

9.6 Example 6. Plasmid DNA preparation from yeast

[0150] Plasmid DNA was prepared from a 5mL overnight culture of 5. cerevisae transformed with pY2B-T7-SARS-2-AOrf7-eGFP and recovered using standard protocol for genomic DNA prep. Briefly, after centrifugation of the culture are 13,000 rpm for 30 sec, the cell pellet was resuspended in 400 pL SEB buffer (IM Sorbitol, 0.1M EDTA, 0.1% R-mercaptoethanol) with lpL zymolase 20T (30mg/mL) and incubated for 30 to 60 minutes at 37°C and then spun at 13,000 rpm for 30 sec. After removal of the supernatant, the pellet was resuspended in EDS buffer (0.05M EDTA, 2% SDS, 2.5pM NaOH) and incubated at 65°C for 10 minutes. After addition of 200pL 10M ammonium acetate, the sample was placed on ice for 30 minutes then spun at 13,000 rpm for 10 minutes. DNA in the supernatant was precipitated by addition of 1 volume of isopropanol and finally resuspended in 300pL TE buffer (lOmM Tris pH 8, ImM EDTA) and incubated for 30 minutes at 37°C with lpL RNaseA (lOmg/mL). Finally, the DNA prep was cleaned up by proteinase K treatment (50pg final, lh at 55°C), extracted with PhenokChloroform, precipitated with Ethanol and resuspended in lOpL lOmM Tris pH 8.

9.7 Example 7. Amplification of YAC-to-BAC plasmid in E. coli

Transformation in DHIOfi E. coli

[0151] lpL of DNA plasmid extracted from yeast was electroporated into 25pL DH10R E. coli (NEB 10-beta electrocompetent E. coli, catalog #C3020K) on a Gene Pulser II (Biorad) with a 0.1cm cuvette and Biorad, using the following conditions: 2.0 kV, 200Q, and 25 pF. Immediately after electroporation, cells were recovered in 250pL NEB outgrowth media and allow to recover at 30°C for 30 minutes on an orbital shaker (at 210rpm). 200pL of cells were plated on a LB Ampicillin (LBA) plate and incubated overnight at 30°C.

[0152] The following day, 3 colonies were picked and used to start three 3mL cultures in LBA (4-5 hours at 30°C, 210rpm). The culture were then pooled into a 45mL LBA culture and grown overnight under the same conditions. Purification of low copy pY2B plasmid

[0153] DNA plasmid prep from the E. coli culture was performed using ZR BAG DNA Miniprep kit (Zymo research) according to the manufacturer's instructions with the following modifications. Buffers volumes were adjusted to the culture volume as followed: 1.8mL of Pl and P2 buffer, and 3.6mL P3 buffer. After lysis and neutralization, the supernatant was divided into 4 columns from the kit, processed according to the manufacturer's instructions. DNA plasmid was eluted from each column with lOpL elution buffer. All four eluates were then pooled.

Removal of bacterial DNA

[0154] Bacterial DNA was removed by adding 5pL lOx NEB4 buffer, 5pL lOmM ATP and 3pL Exonuclease V (RecBCD) (NEB) to the low copy plasmid prep. The mixture was incubated for 1 hour at 37°C, heat inactivated for 30 minutes at 70°C and extracted with phenokchloroform, followed by precipitation with ethanol.

9.8 Example 8. Production of SARS-CoV-2-AOrf7-eGFP mRNA

Plasmid linearization

[0155] Before in vitro transcription, the pY2B-T7-SARS-2-AOrf7-eGFP was linearized by overnight digestion with Sall-HF enzyme (NEB) to cut the unique Sall site, located just after the poly A tail at the end of SARS-CoV-2 genome.

[0156] The next day, the linearized plasmid was extracted with phenokchloroform and precipitated with ethanol and resuspended in 8pL RNase and DNase-free water.

In vitro transcription

[0157] Viral RNA was produced by in vitro transcription from the pY2B-T7-SARS-CoV-2 using the HiScribe T7 ARCA mRNA kit (with tailing) (NEB), according the manufacturer's instruction with one modification: in vitro transcription was performed at 30°C for 4h. The final viral mRNA product was resuspended in 6pL lOmM Tris pH 7, O.lmM EDTA.

9.9 Example 9. Production of SARS-CoV-2-AOrf7-eGFP mRNA virus

[0158] All subsequent work, including in vivo and in vitro experiments with infectious SARS- CoV-2, was performed under an appropriate biosafety level 2 (BSL2) laboratory.

Transfection of viral mRNA into mammalian cell

[0159] Transfection of viral mRNA was performed using a Neon electroporation system (ThermoFisher Scientific) according to manufacturer's instructions, in BHK-N cells, engineered to expressed SARS-CoV-1 nucleocapsid under doxycycline treatment, that were obtained from Dr. Volker Thiel and used previously to rescue SARS-CoV-2 infectious clones (Thi Nhu Thao et al., 2020, "Rapid reconstruction of SARS-CoV-2 using a synthetic genomic platform." Nature. 582: 561-565.)

[0160] 1.5pL SARS-CoV-2-AOrf7-eGFP mRNA from in vitro transcription was mixed with 13.5pL BHK-N cells (at 10e5 cells in lOpL buffer R) and electroporated using a lOpL Neon tip with the following settings: 1200V, 20 seconds, 2 pulses.

[0161] Immediately after electroporation, cells were transferred in a 6 well plate to 4xl0e5 Vero cells overexpressing human TMPRSS-2 and ACE-2 per well, in 2mL DMEM supplemented with 5% FBS, non essential amino acid (MEM NEAA, Gibco), Sodium pyruvate (Gibco), doxycycline (5pg/mL) and without antibiotics. After 3 days at 37°C, cultures with transfected cells showed signs of cytopathic effect. After 4 days at 37°C, the cell culture supernatant was collected, as the original viral rescue preparation (P0) and stored at -80°C.

[0162] Infectious viruses in the P0 stock was later confirmed by plaque assay in Vero E6 cells. The P0 stock was then amplified by adding 250pL of P0 stock to 4xl0e5 naive Vero cells overexpressing human TMPRSS-2 and ACE-2 in a 6 well plate as described above. After 5 days at 37°C, all cells in the P0 infected well were dead, whereas cells in untreated wells were healthy and confluent. The new cell culture supernatant was collected, labelled as Pl stock and stored at -80°C. 200pL of P0 and Pl stocks were extracted with 3 volumes of TRI reagent (Thermofisher) followed by RNA purification using Monarch Total RNA Miniprep kit (NEB). 4pL of the lOOpL eluate were used for each RT-qPCR reaction to quantify the viral RNA copy number of SARS-CoV-2 N and E mRNAs and genomic Orflb RNA (2 probes Pl and P2), using a serial dilution of commercial available SARS-CoV-2 genome standard (IDT). All probes were obtained from IDT. Amplification of the viral stock was confirmed by plaque assay and RT- qPCR, showing a 1000 fold increase of viral RNA between P0 and Pl stocks.

10. Table 5: Sequence Listing and Annotation

[0163] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.