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WO2022103870A1 - Vaccins contre le sars-cov-2 utilisant un virus vivant attenué - Google Patents

Vaccins contre le sars-cov-2 utilisant un virus vivant attenué Download PDF

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WO2022103870A1
WO2022103870A1 PCT/US2021/058829 US2021058829W WO2022103870A1 WO 2022103870 A1 WO2022103870 A1 WO 2022103870A1 US 2021058829 W US2021058829 W US 2021058829W WO 2022103870 A1 WO2022103870 A1 WO 2022103870A1
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sars
virus
live attenuated
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Hiten D. Madhani
Raul Andino
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University of California Berkeley
University of California San Diego UCSD
Chan Zuckerberg Biohub Inc
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University of California Berkeley
University of California San Diego UCSD
Chan Zuckerberg Biohub Inc
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    • C12N2770/20062—Methods of inactivation or attenuation by genetic engineering

Definitions

  • This invention relates to biomedicine and vaccine development.
  • 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.
  • 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).
  • HKU1 Human coronavirus HKU1
  • MERS-CoV Middle East respiratory syndrome- related coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus
  • 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.
  • LAV Live attenuated viruses
  • SARS-CoV-2 vaccine strategies focus on attenuation by codon deoptimization which reduces the replication fitness of the virus by impairing translation.
  • 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.
  • 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.
  • the viral genome comprises inactivating mutation(s) in a sequence(s) encoding one, two, three or more than three SARS-CoV-2 proteins.
  • the SARS-CoV-2 viral genome comprises an inactivating mutation that includes one or more deletion(s) and/or one or more substitution(s).
  • 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.
  • each inactivating mutation(s) is a deletion of at least 10 nucleotides.
  • at least 50% of the length of the sequence encoding the SARS-CoV-2 protein is deleted.
  • 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.
  • 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.
  • the invention provides an immunogenic composition comprising the live attenuated SARS-CoV-2 virus and a pharmaceutically acceptable excipient.
  • 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.
  • the subject is a human.
  • 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.
  • the cDNA sequence is operably linked to a promoter.
  • the promoter is a T7 promoter sequence.
  • 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.
  • 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.
  • Fig. 1A shows a schematic representation of the SARS-CoV-2 genome and protein coding regions.
  • 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.
  • YAC yeast artificial chromosome
  • 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.
  • 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.
  • 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).
  • 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.
  • NLuc Nanoluciferase activity
  • 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.
  • FIG. 5 Schematic of the pY2B_T7-SARS-2-AOrf7eGFP plasmid.
  • the box outlines the BAC regulatory elements inserted in the parental yeast vector.
  • SARS-CoV-2 is a positive-strand, or "sense-strand," RNA virus.
  • 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.
  • 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.
  • 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.
  • 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.
  • the substitution is A>G (WA-1 > HM-l-SARSCoV2) at position 24205.
  • the sequence of HM-l-SARSCoV2 additionally has a non-synonymous substitution in the pplab region.
  • the substitution is G>T at position 3659.
  • 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.
  • human cells e.g., human epithelial cells, such as Calu-3 cells or A549-ACE2 cells
  • Vero cells African green monkey kidney cells
  • a “SARS-CoV-2 variant genome” 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.
  • inactivating mutation 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.
  • the inactivating mutation is a deletion of more than 10 nucleotides as discussed in detail hereinbelow.
  • the inactivating mutation is an inactivating deletion.
  • inactivating deletion 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.
  • 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.
  • 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.
  • a "variant SARS-CoV-2 virus” is a is a SARS-CoV-2 virus that comprises a variant genome.
  • 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.
  • the live attenuated virus is capable of eliciting a protective immune response.
  • the term "corresponding to” is used to describe positions in SARS- CoV-2 variant genome relative to a reference genome.
  • 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).
  • 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.
  • 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.
  • sequence differences e.g., SNPs
  • 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.
  • 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.
  • identity 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.
  • 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.
  • 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.
  • RNA e.g., the positive or negative strand of a wild-type or variant SARS-CoV-2 genome
  • cDNA does not necessarily refer to a polynucleotide produced by reverse transcription from an RNA template.
  • 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.
  • 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.
  • 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.
  • patient refers to a human or non-human mammal to which a vaccine comprising live attenuated virus is administered.
  • immune response refers to a cell-mediated (T-cell) immune response and/or an antibody (B-cell) immune response.
  • 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.
  • immunogenicity effective amount of a vaccine or immunogenic composition is an amount that elicits an immune response towards SARS-CoV-2.
  • 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.
  • “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.
  • 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'.
  • SARS-CoV-2 is an RNA virus that causes COVID-19.
  • 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.
  • the live attenuated virus is capable of eliciting a protective immune response.
  • 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.
  • 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.
  • SARS-CoV-2 structural proteins Spike (S), Nucleocapsid (N), Membrane (M), and Envelope (E), are required to make a complete virus particle.
  • S protein is responsible for receptor binding, membrane fusion, and tissue tropism.
  • SARS-CoV-2 is believed to use the same receptor as SARS-CoV for cell entry: the angiotensin-converting enzyme 2 receptor (ACE2).
  • ACE2 angiotensin-converting enzyme 2 receptor
  • 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).
  • 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.
  • 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.
  • 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
  • SARS-CoV-2 Variant Genomes and Live Attenuated SARS-CoV-2
  • 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.
  • 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.
  • 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.
  • 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.
  • the inactivating mutation is in a sequence encoding a SARS- CoV-2 structural protein.
  • the structural protein is a Spike (S) protein, a Nucleocapsid (N) protein, a Membrane (M) protein, or an Envelope (E) protein.
  • 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.
  • a non-structural protein e.g., nspl, nsp2, nsp3, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsplO, nspll, nspl2, nspl3, nspl4, nspl5, nspl6 protein.
  • 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.
  • a SARS-CoV-2 accessory protein such as an ORF3a, 0RF3b, 0RF6, ORF7a, 0RF7b, 0RF8a, 0RF8b, 0RF9b, or ORFIO protein.
  • multiple genes are deleted by a single contiguous deletion. For example, the deletion of orfN by necessity also removes orf9.
  • 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.
  • 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.
  • the inactivating mutation(s) are in sequences encoding SARS-CoV-2 proteins of the same class.
  • 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.
  • the viral genome comprises inactivating mutation(s) in a sequence(s) encoding three SARS-CoV-2 proteins.
  • the inactivating mutation(s) are in sequences encoding different SARS-CoV-2 proteins.
  • 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.
  • the inactivating mutation(s) are in sequences encoding SARS-CoV-2 proteins of the same class.
  • 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
  • another inactivating mutation may be in a sequence encoding another SARS-CoV-2 non-structural protein, such as an nspl5.
  • the viral genome comprises an inactivating mutation in sequences encoding more than three SARS-CoV-2 proteins.
  • 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.
  • the inactivating mutation is a deletion.
  • the deletion comprises a deletion of the entire sequence encoding a SARS- CoV-2 protein.
  • a deletion may include the sequence from the ATG start codon to the stop codon of a SARS-CoV-2 protein coding sequence.
  • additional upstream untranslated sequences may be deleted and/or additional downstream untranslated sequences may be deleted.
  • 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.
  • 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.
  • a portion of a sequence encoding a SARS-CoV-2 protein may be deleted.
  • 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.
  • 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
  • the sequence may be further modified or optimized to provide or select for various features.
  • 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.
  • 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.
  • the one or more substitutions are in a sequence encoding an nspl4 protein.
  • the substitution is in a region encoding the exoribonuclease (ExoN) of nspl4.
  • the substitution is at positions corresponding to nucleotide positions 18302 to 18303 of SEQ ID NO: 1.
  • the substitution is at positions corresponding to nucleotide positions 18308 to 18309 of SEQ ID NO: 1.
  • the substitution is at positions corresponding to nucleotide positions 18302 to 18303 and 18308 to 18309 of SEQ ID NO: 1.
  • the substitution corresponding to nucleotide positions 18302 to 18303 of SEQ ID NO: 1 is GC>CT.
  • the substitution corresponding to nucleotide positions 18308 to 18309 of SEQ ID NO: 1 is AT>CA.
  • 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.
  • Vaccines of the invention contain live attenuated SARS-CoV-2 variants ("live attenuated virus").
  • 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).
  • ISGs interferon-stimulated genes
  • 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.
  • 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.
  • the virus comprises a reporter gene and the number of cells expressing the reporter gene is representative of the number of infected cells.
  • the viral titer is determined with a plaque assay.
  • 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.
  • PFU plaque-forming units
  • the viral titer may be compared to that of a wild-type SARS-CoV-2 grown under the same conditions.
  • 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.
  • 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.
  • SARS-CoV-2 Spike antigen can be stained for immunofluorescence studies.
  • viral titers e.g., by plaque assay
  • NALT nasal-associated lymphoid tissues
  • cLN cervical lymph nodes
  • trachea trachea
  • lungs spleen and liver
  • 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.
  • 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.
  • 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).
  • animal models e.g., in mice or hamsters that received a dose of the SARS-CoV-2 variant.
  • blood, bronchioalveolar lavage, and nasal swab samples may be analyzed for the presence of serum antigen-specific antibodies using ELISA.
  • the presence of SARS-CoV-2 M, N, and/or S-binding antibodies may be tested.
  • 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.
  • tissue e.g., lung tissue
  • monocytes e.g., monocytes
  • macrophages e.g., T lymphocytes (including CD4 and CD8 subsets)
  • T lymphocytes including CD4 and CD8 subsets
  • B lymphocytes including plasmablasts
  • cytokine response are examined using Luminex assays.
  • 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.
  • 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.
  • a virus such as a coronavirus (e.g., SARS-CoV-2)
  • SARS-CoV-2 coronavirus
  • 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.
  • 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.
  • the virus e.g., SARS-CoV-2
  • blood samples from animals administered with a SARS-CoV-2 variant can be collected at their endpoints and used for neutralization assays.
  • 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).
  • the expression level of interferon-stimulated genes 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.
  • 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.
  • 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.
  • the YAC is a shuttle vector that can propagate in a yeast species (e.g., Streptomyces) and a bacterial species (e.g., E. coli).
  • a full-length SARS-CoV-2 cDNA is generated using transformation- associated recombination (TAR) methods.
  • TAR transformation- associated recombination
  • Example 2 transformation- associated recombination
  • 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.
  • Fig. IB shows the general workflow of TAR cloning.
  • genome reconstruction in yeast e.g., Saccharomyces cerevisiae
  • yeast e.g., Saccharomyces cerevisiae
  • the TAR cDNA fragments are assembled by homologous recombination in yeast to generate a YAC containing the full-length SARS-CoV-2 sequence.
  • 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.
  • SARS-CoV-2 cDNA with inactivating mutations can be produced by providing cDNA fragments with the desired mutations for assembly by transformation-associated recombination.
  • 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.
  • 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.
  • modifications are made using CRISPR-Cas technology, e.g., as described in Example 3.
  • TAR-mediated assembly and/or modification e.g., CRISPR- mediated gene editing
  • a viral genome is carried out using a yeast-bacterial shuttle vector.
  • 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.
  • pY2B YAC-to-BAC plasmid
  • 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.
  • 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.
  • SopA, SopB, SopC genes encoding plasmid partitioning proteins
  • incC incompatibility region, e.g., derived from the bacterial F plasmid
  • RepE repplication initiation site
  • 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.
  • pBeloBACll GenBank Accession no. U51113
  • U51113 New England Biolabs
  • oriV high-copy origin of replication
  • SEQ ID NO:13 An exemplary regulated bacterial origin of replication is found in SEQ ID NO:13.
  • 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).
  • the pY2B includes one or more yeast selectable markers (e.g., neoR, natR, hygR, TRP1, URA3, HIS3, LYS2, LEU 2, TRP1, MET15).
  • yeast selectable markers e.g., neoR, natR, hygR, TRP1, URA3, HIS3, LYS2, LEU 2, TRP1, MET15.
  • 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 )).
  • auxotrophic markers e.g., ampicillin resistance (AmpR); chloramphenicol resistance (Cm R )).
  • Cm R chloramphenicol resistance
  • Fig. 5 illustrates a pY2B structure (shown with the viral genome).
  • 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).
  • 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.
  • 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.
  • 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.
  • the pY2B RNA encodes a reporter (usually a reporter protein), that allows expression of the encoded polypeptide to be monitored.
  • a reporter usually a reporter protein
  • 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.
  • 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.
  • the reporter gene is inserted in place of Orf7a, e.g., as described in Example 2.
  • 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.
  • 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.
  • a promoter e.g., T7, T3, and SP6 RNA promoters and compatible RNA polymerases
  • a promoter is selected for expression of viral particles in mammalian cells transfected with a vector comprising a SARS-Covid-2 variant genome.
  • nucleic acid constructs that include the nucleic acid sequences provided herein.
  • 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.
  • the nucleic acid construct may comprise a T7 RNA polymerase promoter sequence that allows in-vitro transcription by T7 polymerase.
  • 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.
  • vectors including nucleic acids or nucleic acid constructs according to the embodiments of the present invention can include necessary regulatory elements (as described above) that direct and regulate transcription of the nucleic acid sequences included in the vector.
  • a T7 promoter is used to express the pY2B cargo (e.g., viral genome cDNA).
  • pY2B cargo e.g., viral genome cDNA
  • An exemplary construct is shown in Fig. 5.
  • 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.
  • the cytomegalovirus (CMV) promoter is commonly used to express heterologous genes in vivo (e.g., in mammalian cells) or in vitro.
  • CMV cytomegalovirus
  • 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.
  • HDV hepatitis delta virus
  • Rz hepatitis delta virus
  • 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.
  • RNA-dependent RNA polymerase makes copies of itself using the viral RNA-dependent RNA polymerase.
  • 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.
  • tracrRNA transactivating crisp RNA
  • crRNA crisp RNA
  • Some CRISPR systems e.g., CRISPR Casl2a/Cpfl
  • 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.
  • sgRNA chimeric single guide RNA
  • 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).
  • 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.
  • Cas proteins and their amino acid sequence are well known in the art.
  • 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.
  • 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.
  • PAM protospacer adjacent motif
  • Cas9 generally requires the PAM motif NGG for activity.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • in-vitro transcription is used to prepare viral RNA from the SARS-CoV-2 variant cDNA isolated from yeast.
  • 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).
  • the cell is a Vero E6 cell.
  • the cell is a BHK-21 cell.
  • the nucleic acid as described herein e.g., in the form of RNA
  • 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.
  • aspects of the invention relate to an isolated cell comprising the nucleic acid, or the live attenuated SARS-CoV-2 virus described herein.
  • the isolated cell is a mammalian cell.
  • 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).
  • BHK Baby Hamster Kidney fibroblast
  • Vero cell e.g., a Vero E6 cell
  • 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.
  • 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.
  • pneumonia became mild with focal lesion areas at seven days post - infection, suggesting a non-lethal and self-limiting infection course.
  • 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.
  • 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.
  • 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.
  • interferon-stimulated genes 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.
  • 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.
  • 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
  • SARS-CoV-2 variants 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.
  • 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).
  • 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.
  • proinflammatory cytokines e.g., TNF, IL-1, IL-6
  • Th2 cytokines e.g., IL-4, IL-13
  • chemokines e.g., IL-4, IL-13
  • 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.”
  • 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.
  • solutions e.g., a sterile injectable solution
  • 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.
  • 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
  • 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.
  • 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.
  • the immunogenic composition is administered by injection, such as intravenous injection.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • IFU virus infectious units
  • the composition can be administered in one or more dose administrations daily, for one or several days, including a prime-boost paradigm.
  • the composition may be administered at one point in time, followed by second administration from 2 weeks to 2 years later.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • WT wild-type
  • innate immunity pathways capable of restricting virus replication of those variants with defects modulating of the host antiviral defense.
  • DESeq2 may be used to analyze transcriptomics.
  • 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.
  • 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.
  • 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.
  • DNA from each strain can be prepared by in-vitro transcription.
  • 200 ng of plasmid DNA can be reproducibly purified from yeast.
  • T7 RNA polymerase 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).
  • This example illustrates one approach of generating a YAC-to-BAC shuttle vector (pY2B) using a YAC vector as the backbone.
  • 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.
  • 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.
  • 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).
  • 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.
  • SEB buffer IM Sorbitol, 0.1M EDTA, 0.1% R-mercaptoethanol
  • 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).
  • EDS buffer 0.05M EDTA, 2% SDS, 2.5pM NaOH
  • 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.
  • 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.
  • a Gene Pulser II Biorad
  • 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.
  • 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.
  • 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.
  • RecBCD Exonuclease V
  • 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.
  • NEB Sall-HF enzyme
  • 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.

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Abstract

La divulgation concerne des vaccins préparés à l'aide d'un virus vivant atténué. La divulgation concerne notamment le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) vivant atténué comprenant un génome viral qui comprend une ou plusieurs mutations d'inactivation dans une séquence codant pour une protéine du SARS-CoV-2. La divulgation concerne également des procédés de fabrication et d'utilisation du SARS-CoV-2 vivant atténué en tant que vaccin pour provoquer une réponse immunitaire dirigée contre le SARS-CoV-2 afin d'empêcher l'apparition de la COVID-19 ou d'autres maladies à coronavirus.
PCT/US2021/058829 2020-11-10 2021-11-10 Vaccins contre le sars-cov-2 utilisant un virus vivant attenué Ceased WO2022103870A1 (fr)

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IT202200015231A1 (it) * 2022-07-20 2024-01-20 Bioinnova S R L S Microalghe esprimenti prodotti biologicamente attivi
WO2025012671A1 (fr) * 2023-07-07 2025-01-16 Institute National De La Sante Et De La Recherche Medicale (Inserm) Séquences 5'utr du sars-cov-2 et leur liaison à des séquences de codage

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WO2006136448A2 (fr) * 2005-06-24 2006-12-28 Consejo Superior De Investigaciones Cientificas Sras attenue: utilisation comme vaccin

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IT202200015231A1 (it) * 2022-07-20 2024-01-20 Bioinnova S R L S Microalghe esprimenti prodotti biologicamente attivi
WO2024018036A1 (fr) * 2022-07-20 2024-01-25 Bioinnova S.R.L.S. Microalgues exprimant des produits biologiquement actifs
WO2025012671A1 (fr) * 2023-07-07 2025-01-16 Institute National De La Sante Et De La Recherche Medicale (Inserm) Séquences 5'utr du sars-cov-2 et leur liaison à des séquences de codage

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