[go: up one dir, main page]

WO2024061759A1 - Protéines s de coronavirus stabilisées - Google Patents

Protéines s de coronavirus stabilisées Download PDF

Info

Publication number
WO2024061759A1
WO2024061759A1 PCT/EP2023/075414 EP2023075414W WO2024061759A1 WO 2024061759 A1 WO2024061759 A1 WO 2024061759A1 EP 2023075414 W EP2023075414 W EP 2023075414W WO 2024061759 A1 WO2024061759 A1 WO 2024061759A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
hcov
amino acid
proteins
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2023/075414
Other languages
English (en)
Inventor
Johannes Petrus Maria Langedijk
Mark Johannes Gerardus BAKKERS
Jaroslaw JURASZEK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Vaccines and Prevention BV
Original Assignee
Janssen Vaccines and Prevention BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Janssen Vaccines and Prevention BV filed Critical Janssen Vaccines and Prevention BV
Publication of WO2024061759A1 publication Critical patent/WO2024061759A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of medicine.
  • the invention in particular, relates to stabilized recombinant prefusion Coronavirus spike (S) proteins, in particular to NL63 S proteins, to nucleic acid molecules encoding said NL63 S proteins, and uses thereof, e.g. in vaccines.
  • S Coronavirus spike
  • Coronaviruses are a family of enveloped, single-stranded positive-sense RNA viruses belonging to the order Nidovirales, which can infect a broad range of mammalian and avian species, causing respiratory or enteric diseases. Coronaviruses possess large, trimeric spike glycoproteins that mediate binding to host cell receptors as well as fusion of viral and host cell membranes.
  • the Coronavirus family contains the genera Alphacoronavirus (a-CoV), Betacoronavirus (0-CoV), Gammacoronavirus (y-CoV), and Deltacoronavirus (5-CoV). The host range of these viruses is primarily determined by the viral spike protein (S) protein.
  • Coronaviruses that can infect humans are found both in the genus a-CoV, namely NL63 and 229E, and in the genus p-CoV, namely SARS-CoV, MERS-CoV, HCoV- OC43,HCoV-HKUl, and SARS-CoV-2.
  • the currently identified human coronaviruses have been shown to originate from previous zoonotic transmissions, with rodents and bats likely serving as the source of most a-CoVs and 0-CoVs, while birds are the main reservoir of y- CoVs and 5-CoVs (Su et al. (2016), Trends Microbiol, 24:490-502).
  • S proteins are comprised of a S 1 and S2 subunit and form homotrimers on the viral membrane.
  • the aminoterminal SI subunit contains the N-terminal domain (NTD) and receptor binding domain (RBD) which is responsible for host-receptor attachment.
  • NTD N-terminal domain
  • RBD receptor binding domain
  • SARS-CoV-2, SARS-CoV, and HCoV-NL63 S bind to human angiotensin-converting enzyme 2 (ACE2) via their receptorbinding domain (RBD) (Hoffmann et al. (2020), Cell, 181 :271-280; Wrapp et al.
  • the carboxy-terminal S2 subunit contains the fusion machinery and is required for fusion of the viral and host membranes (Heald- Sargent and Gallagher (2012), Viruses, 4:557-580).
  • Coronavirus S proteins are class I fusion proteins that are inherently metastable. Their metastable nature is a prerequisite for their ability to undergo extensive conformational changes from a labile prefusion to a stable post-fusion conformation, a pivotal event that drives membrane fusion.
  • the prefusion conformation of S as present on the infectious viral particle, contains the epitopes for neutralizing antibodies and thus holds most promise as a vaccine immunogen (Chen et al. (2020), Hum Vaccin Immunother, 16: 1239-1242; Liu et al. (2020), Nature, 584:450-456; Yuan et al. (2020), Science, 368;630-633; Brouwer et al.
  • the HR1 region in the S2 subunit consists of several discontinuous a-helices in the prefusion structure, which rearrange to a single extended a-helix in the postfusion conformation (Walls et al. (2017), Proc Natl Acad Sci USA, 17: 11157-11162).
  • the 2P substitution prevents this reorganization and consequently stabilizes the S protein in the prefusion conformation (Hsieh et al. (2020), Science, 369:150-1505; Juraszek et al.
  • CoV NL63 currently exists.
  • the present invention aims at providing means for obtaining a stable prefusion NL63 S protein for use in vaccinating against HCoV-NL63.
  • FIGURE 1 Domain organization of NL63 spike protein
  • A Schematic representation of the conserved elements of the NL63 spike (S) protein in both the full-length membrane bound protein (‘full-length’, top panel) and in the mature, soluble ectodomain (‘ectodomain’, bottom panel).
  • the N-terminal domain is preceded by a signal peptide sequence (SP) that is cleaved off during protein maturation (indicated by arrow).
  • SP signal peptide sequence
  • S protein domains include N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 protease cleavage site (S1/S2), S2’ protease cleavage site (S2’) (indicated by arrows), heptadrepeat region 1 (HR1), heptad-repeat region 2 (HR2), transmembrane-domain (TMD), cytosolic tail (CT).
  • NTD N-terminal domain
  • RBD receptor-binding domain
  • S1/S2 protease cleavage site S2’ protease cleavage site (S2’) (indicated by arrows)
  • HR1 heptadrepeat region 1
  • HR2 heptad-repeat region 2
  • TMD transmembrane-domain
  • CT cytosolic tail
  • GCN4 trimerization domain GCN4 trimerization domain
  • FIGURE 2 Expression profile of NL63 S proteins with proline substitutions in the «13al4 loop of the HR1 region in crude cell supernatant.
  • FIGURE 3 Expression profile of NL63 S proteins with proline substitutions in the hinge loop region at the C-terminus of HR1 in crude cell supernatant.
  • FIGURE 4 Expression profile in crude supernatant and characterization of purified NL63 S proteins with proline substitutions A996P and S1052P.
  • HCV-NL63 Human coronavirus NL63
  • HCV-NL63 Human coronavirus NL63
  • the virus is found primarily in young children, the elderly, and immunocompromised patients with acute respiratory illness. It also has a seasonal association in temperate climates. A study performed in Amsterdam estimated the presence of HCoV-NL63 in approximately 4.7% of common respiratory illnesses. Viruses closely related to NL63 are found in palm civets and bats, which thus form natural reservoirs.
  • the spike protein (S) of HCoV-NL63 is involved in fusion of the viral membrane with a host cell membrane, which is required for infection.
  • HCoV-NL63 S RNA is translated into a 1356 amino acid precursor protein, which contains a signal peptide sequence at the N-terminus (e.g. amino acid residues 1-15 of SEQ ID NO: 1, predicted by SignalP 6.0) which is removed by a signal peptidase in the endoplasmic reticulum.
  • S protein typically involves cleavage by host proteases at the boundary between the SI and S2 subunits (S1/S2) in a subset of coronaviruses (including HCov-NL63), and at a conserved site upstream of the fusion peptide (S2’) in all known coronaviruses.
  • the prefusion conformation of S contains the epitopes for neutralizing antibodies and thus holds most promise as a vaccine immunogen (Chen et al. (2020), Hum Vaccin Immunother, 16: 1239-1242; Liu et al. (2020), Nature, 584:450-456; Yuan et al. (2020), Science, 368;630-633; Brouwer et al. (2020), Science, 369;643-650; Hsieh et al. (2020), Science, 369: 150-1505; Robbiani et al. (2020), Nature, 584:437-442.). Since class I proteins are metastable proteins, increasing the stability of the prefusion conformation is desirable for use of fusion proteins as vaccine components.
  • stabilization of prefusion S is desired for a soluble, subunit-based vaccine, which requires truncation of the viral fusion protein by deletion of the transmembrane (TM) and the cytoplasmic region.
  • the remaining ectodomain of the fusion protein is considerably more labile due to removal of the membrane anchor and will either not form as a trimeric protein or even more readily refold into the post-fusion end-state.
  • stabilization of fusion proteins generally increases the expression level of the protein, because less protein will be misfolded and more protein will successfully transport through the secretory pathway. For manufacturability purposes, high S protein expression and stable S trimeric conformation is critical for protein-based vaccines.
  • the present invention provides stabilized HCoV-NL63 S proteins, or fragments thereof, comprising a proline substitution in the al3al4 (amino acid residues 992-1001) loop and/or in the hinge loop region (amino acid residues 1049-1054) of HR1, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.
  • the proteins comprise a proline substitution in the al3al4 (amino acid residues 992-1001) loop and in the hinge loop region (amino acid residues 1049- 1054) ofHRl.
  • the proline is introduced in the al3al4 loop at position 993, 996, 997, 998, and/or 1000, wherein the numbering of the amino acid positions in said NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.
  • the proline in the al3al4 loop is introduced at position 996.
  • the proline is introduced in the hinge loop region at position 1051, 1052, 1053, and/or 1054, wherein the numbering of the amino acid positions in said
  • NL63 S protein is according to the numbering of the amino acid positions in SEQ ID NO: 1.
  • the proline in the hinge loop is introduced at position
  • the protein according to the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3-8 and 12-16, or a fragment thereof. In a preferred embodiment, the protein according to the invention comprises an amino acid sequence of SEQ ID NO: 16, or a fragment thereof.
  • the numbering of the positions of the amino acid residues is according to the numbering of the amino acid residues in the amino acid sequence of SEQ ID NO: 1. According to the invention it has been demonstrated that the presence of the specific amino acids at the indicated positions increases the stability of the S proteins in the prefusion conformation and/or increases trimer yields.
  • the specific amino acids may be already present in the amino acid sequence of the S protein or may be introduced by substitution (mutation) of a naturally occurring amino acid residue at that position into the specific amino acid residue according to the invention.
  • the proteins comprise one or more mutations in their amino acid sequence as compared to the amino acid sequence of a wild type S protein.
  • the wording ‘the amino acid at position 996” thus refers to the amino acid residue that is at position 996 in SEQ ID NO: 1.
  • the multimeric HCoV-NL63 S proteins are trimeric, i.e. comprise three monomers comprising identical amino acid sequences.
  • An amino acid residue according to the invention can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as e.g. D-amino acids (the D- enantiomers of amino acids with a chiral center), or any variants that are not naturally found in proteins, such as e.g. norleucine.
  • the standard amino acids can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups. These properties are important for protein structure and protein-protein interactions.
  • amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that induces turns of the polypeptide backbone, and glycine that is more flexible than other amino acids.
  • Table 1 shows the abbreviations and properties of the standard amino acids.
  • fragment refers to a peptide that has an amino-terminal and/or carboxy-terminal and/or internal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence of a HCoV-NL63 S protein, for example, the full-length sequence of a HCoV-NL63 S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein does not have to be full length nor have all its wild type functions, and fragments of the protein are equally useful.
  • a fragment according to the invention is an immunologically active fragment, and typically comprises at least 15 amino acids, or at least 30 amino acids of the HCoV-NL63 S protein.
  • the fragment comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the HCoV-NL63 S protein.
  • the fragment is the HCoV-NL63 S ectodomain.
  • the proteins according to the invention are soluble proteins, i.e. S protein ectodomains.
  • the S proteins comprise a truncated S2 domain.
  • a “truncated” S2 domain refers to a S2 domain that is not a full length S2 domain, i.e. wherein either N-terminally or C-terminally one or more amino acid residues have been deleted.
  • at least the transmembrane domain and cytoplasmic domain have been deleted to permit expression as a soluble ectodomain, corresponding to the amino acids 1-1283 (or 16-1283 without signal peptide) of SEQ ID NO:
  • a heterologous trimerization domain such as a GCN4 trimerization domain
  • a heterologous trimerization domain may be fused to the C-terminus of the HCoV-NL63 S protein ectodomain (Walls et al. (2016) Nat Struct Mol Biol, 23:899-905).
  • the transmembrane region has been replaced by a heterologous trimerization domain.
  • the heterologous trimerization domain is GCN4 domain comprising the amino acid sequence of SEQ ID NO: 2.
  • the stabilized S proteins do not comprise a heterologous trimerization domain.
  • the soluble HCoV-NL63 S proteins do not comprise a heterologous trimerization domain.
  • pre-fusion HCoV-NL63 S proteins, or fragments thereof, according to the invention are trimeric and stable, i.e. do not readily change into the post-fusion conformation upon processing of the proteins, such as e.g. upon purification, freeze-thaw cycles, and/or storage etc.
  • the proteins according to the invention may comprise a signal peptide, also referred to as signal sequence or leader peptide, corresponding to amino acids 1-15 of SEQ ID NO: 1 (as predicted by SignalP v6.0).
  • Signal peptides are short (typically 5-30 amino acids long) peptides present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.
  • the proteins according to the invention do not comprise a signal peptide.
  • the proteins according to the invention may comprise a linker and C-terminal tag (C- tag), such as GSGEPEA (SEQ ID NO: 17). In certain embodiments, the proteins do not comprise a C-tag.
  • nucleic acid molecule refers to a polymeric form of nucleotides (i.e. polynucleotides) and includes both DNA (e.g. cDNA, genomic DNA) and RNA (e.g. mRNA, modified RNA, circular mRNA), and synthetic forms and mixed polymers of the above.
  • the nucleic acid molecules encoding the proteins according to the invention have been codon-optimized for expression in mammalian cells, preferably human cells, or insect cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells).
  • a sequence is considered codon-optimized if at least one non-preferred codon as compared to a wild type sequence is replaced by a codon that is more preferred.
  • a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon.
  • the frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon.
  • nucleic acid sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may or may not include introns.
  • Nucleic acid sequences can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt, GenScript, Invitrogen, Eurofins).
  • the invention also provides vectors comprising a nucleic acid molecule as described above.
  • a nucleic acid molecule according to the invention thus is part of a vector.
  • Such vectors can easily be manipulated by methods well known to the person skilled in the art and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells.
  • many vectors can be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome.
  • the vector used can be any vector that is suitable for cloning DNA and that can be used for transcription of a nucleic acid of interest.
  • the vector is an adenovirus vector.
  • An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g. CAdV2), a porcine adenovirus (e.g.
  • PAdV3 or 5 or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus).
  • the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd).
  • a human adenovirus is meant if referred to as Ad without indication of species, e.g.
  • Ad26 means the same as HAdV26, which is human adenovirus serotype 26.
  • rAd means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.
  • a recombinant adenovirus according to the invention is based upon a human adenovirus.
  • the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc.
  • an adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials.
  • Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g. US6083716; WO 2005/071093; WO 2010/086189; WO 2010085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al,
  • the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus.
  • the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P.
  • the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see e.g. WO 2012/172277), or ChAdOx 2 (see e.g. WO 2018/215766).
  • the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see e.g. WO 2019/086466).
  • the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see e.g. WO 2019/086456), or BZ1 (see e.g. WO 2019/086466).
  • the adenovirus vector is a replication deficient recombinant viral vector, such as rAd26, rAd35, rAd48, rAd5HVR48, etc.
  • the adenoviral vectors comprise capsid proteins from rare serotypes, e.g. including Ad26.
  • the vector is an rAd26 virus.
  • An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining the serotype and/or tropism of a particular adenovirus.
  • Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins.
  • a “capsid protein” for a particular adenovirus such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein.
  • the capsid protein is an entire capsid protein of Ad26.
  • the hexon, penton and fiber are of Ad26.
  • a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of a first serotype with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like. See for example WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also e.g.
  • WO 2019/086461 for chimeric adenoviruses Ad26HVRPtrl, Ad26HVRPtrl2, and Ad26HVRPtrl3, that include an Ad26 virus backbone having partial capsid proteins of Ptrl, Ptrl2, and Ptrl3, respectively)
  • the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26).
  • the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome.
  • non-group C adenovirus such as Ad26 or Ad35
  • rAd26 vectors The preparation of recombinant adenoviral vectors is well known in the art. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Examples of vectors useful for the invention for instance include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.
  • a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector).
  • a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector).
  • the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention.
  • the nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically.
  • the DNA can be double-stranded or single-stranded.
  • the adenovirus vectors useful in the invention are typically replication deficient.
  • the virus is rendered replication deficient by deletion or inactivation of regions critical to replication of the virus, such as the El region.
  • the regions can be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding the SARS-CoV2 S protein (usually linked to a promoter) within the region.
  • the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions.
  • E2- and/or E4-mutated adenoviruses generally E2- and/or E4-complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication.
  • a packaging cell line is typically used to produce sufficient amounts of adenovirus vectors for use in the invention.
  • a packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell.
  • Suitable packaging cell lines for adenoviruses with a deletion in the El region include, for example, PER.C6, 911, 293, and El A549.
  • the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the El region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p. 4654-63, which is incorporated herein by reference.
  • the nucleic acid sequence encoding the stabilized SARS-CoV2 S protein is cloned into the El and/or the E3 region of the adenoviral genome.
  • Host cells comprising the nucleic acid molecules encoding the pre-fusion HCoV- NL63 S proteins also form part of the invention.
  • the pre-fusion HCoV-NL63 S proteins may be produced through recombinant DNA technology involving expression of the molecules in host cells, e.g. Chinese hamster ovary (CHO) cells, tumor cell lines, BHK cells, human cell lines such as HEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like, or transgenic animals or plants.
  • the cells are from a multicellular organism, in certain embodiments they are of vertebrate or invertebrate origin.
  • the cells are mammalian cells, such as human cells, or insect cells.
  • the production of a recombinant proteins, such the pre-fusion HCoV-NL63 S proteins of the invention, in a host cell comprises the introduction of a heterologous nucleic acid molecule encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid molecule and allowing expression of the protein in said cell.
  • the nucleic acid molecule encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like.
  • enhancer(s) promoter
  • polyadenylation signal and the like.
  • Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed.
  • Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here the pre-fusion HCoV- NL63 S proteins.
  • the suitable medium may or may not contain serum.
  • a “heterologous nucleic acid molecule” (also referred to herein as ‘transgene’) is a nucleic acid molecule that is not naturally present in the host cell. It is introduced into for instance a vector by standard molecular biology techniques.
  • a transgene is generally operably linked to expression control sequences. This can for instance be done by placing the nucleic acid encoding the transgene(s) under the control of a promoter. Further regulatory sequences may be added.
  • Many promoters can be used for expression of a transgene(s), and are known to the skilled person, e.g. these may comprise viral, mammalian, synthetic promoters, and the like.
  • a non-limiting example of a suitable promoter for obtaining expression in eukaryotic cells is a CMV-promoter (US 5,385,839), e.g. the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter.
  • a polyadenylation signal for example the bovine growth hormone poly A signal (US 5,122,458), may be present behind the transgene(s).
  • several widely used expression vectors are available in the art and from commercial sources, e.g.
  • pcDNA and pEF vector series of Invitrogen pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to recombinantly express the protein of interest, or to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like.
  • the cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.
  • adherent cell culture e.g. cells attached to the surface of a culture vessel or to microcarriers
  • suspension culture Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up.
  • continuous processes based on perfusion principles are becoming more common and are also suitable.
  • Suitable culture media are also well known to the skilled person and can generally be obtained from commercial sources in large quantities, or custom-made according to standard protocols. Culturing can be done for instance in dishes, roller bottles or in bioreactors, using batch, fed-batch, continuous systems and the like. Suitable conditions for culturing cells are known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and R.I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition (W
  • the invention further provides compositions comprising a pre-fusion HCoV-NL63 S protein and/or a nucleic acid molecule, and/or a vector, as described above.
  • the invention also provides compositions comprising a nucleic acid molecule and/or a vector, encoding such pre-fusion HCoV-NL63 S protein.
  • the invention further provides immunogenic compositions comprising a pre-fusion HCoV-NL63 S protein, and/or a nucleic acid molecule, and/or a vector, as described above.
  • the invention also provides the use of a stabilized prefusion HCoV-NL63 S protein, a nucleic acid molecule, and/or a vector, according to the invention, for inducing an immune response against a HCoV-NL63 S protein in a subject. Further provided are methods for inducing an immune response against HCoV-NL63 S protein in a subject, comprising administering to the subject a pre-fusion HCoV-NL63 S protein, and/or a nucleic acid molecule, and/or a vector according to the invention.
  • pre-fusion HCoV-NL63 S proteins, nucleic acid molecules, and/or vectors, according to the invention for use in inducing an immune response against HCoV-NL63 S protein in a subject. Further provided is the use of the pre-fusion HCoV-NL63 S proteins, and/or nucleic acid molecules, and/or vectors according to the invention for the manufacture of a medicament for use in inducing an immune response against HCoV-NL63 S protein in a subject.
  • the nucleic acid molecule is DNA and/or an RNA molecule.
  • the pre-fusion HCoV-NL63 S proteins, nucleic acid molecules, or vectors of the invention may be used for prevention (prophylaxis, including post-exposure prophylaxis) of HCoV-NL63 infections.
  • the prevention may be targeted at patient groups that are susceptible for and/or at risk of HCoV-NL63 infection or have been diagnosed with a HCoV-NL63 infection.
  • target groups include, but are not limited to e.g., the elderly (e.g. > 50 years old, > 60 years old, and preferably > 65 years old), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.
  • the target population comprises human subjects from 2 months of age.
  • the pre-fusion HCoV-NL63 S proteins, nucleic acid molecules and/or vectors according to the invention may be used e.g. in stand-alone treatment and/or prophylaxis of a disease or condition caused by HCoV-NL63, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.
  • the invention further provides methods for preventing and/or treating HCoV-NL63 infection in a subject utilizing the pre-fusion HCoV-NL63 S proteins, nucleic acid molecules and/or vectors according to the invention.
  • a method for preventing and/or treating HCoV-NL63 infection in a subject comprises administering to a subject in need thereof an effective amount of a pre-fusion-SARS CoV-2 S protein, nucleic acid molecule and/or a vector, as described above.
  • a therapeutically effective amount refers to an amount of a protein, nucleic acid molecule or vector, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by HCoV-NL63.
  • Prevention encompasses inhibiting or reducing the spread of HCoV-NL63 or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by SARS CoV-2.
  • Amelioration as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of HCoV-NL63 infection.
  • the invention may employ pharmaceutical compositions comprising a pre-fusion HCoV-NL63 S protein, a nucleic acid molecule and/or a vector as described herein, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered.
  • pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L.
  • the CoV S proteins, or nucleic acid molecules preferably are formulated and administered as a sterile solution although it may also be possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers.
  • the pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5.
  • the CoV S proteins typically are in a solution having a suitable pharmaceutically acceptable buffer, and the composition may also contain a salt.
  • stabilizing agent may be present, such as albumin.
  • detergent is added.
  • the CoV S proteins may be formulated into an injectable preparation.
  • a composition according to the invention further comprises one or more adjuvants.
  • Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant.
  • the terms “adjuvant” and “immune stimulant' are used interchangeably herein and are defined as one or more substances that cause stimulation of the immune system.
  • an adjuvant is used to enhance an immune response to the HCoV-NL63 S proteins of the invention.
  • suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oilemulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see e.g.
  • WO 90/14837 saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g.
  • compositions of the invention comprise aluminium as an adjuvant, e.g. in the form of aluminium hydroxide, aluminium phosphate, aluminium potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminium content per dose.
  • compositions do not comprise adjuvants.
  • the pre-fusion HCoV-NL63 S proteins may also be administered in combination with or conjugated to nanoparticles, such as e.g. polymers, liposomes, virosomes, virus-like particles.
  • nanoparticles such as e.g. polymers, liposomes, virosomes, virus-like particles.
  • the HCoV-NL63 S proteins may be combined with or encapsidated in or conjugated to the nanoparticles with or without adjuvant. Encapsulation within liposomes is described, e.g. in US 4,235,877. Conjugation to macromolecules is disclosed, for example in
  • the HCoV-NL63 S proteins may be fused to a self-assembling protein domain (e.g. I53_dn5 trimerization domains ) that can self-assemble into 2-component particles by addition of pentamers (Boyoglu-Bamum, S. et al., Nature 592, 623-628, (2021)).
  • a self-assembling protein domain e.g. I53_dn5 trimerization domains
  • pentamers Boyoglu-Bamum, S. et al., Nature 592, 623-628, (2021).
  • the invention provides methods for making a vaccine against a HCoV-NL63 virus, comprising providing a composition according to the invention and formulating it into a pharmaceutically acceptable composition.
  • the term "vaccine” refers to an agent or composition containing an active component effective to induce a certain degree of immunity in a subject against a certain pathogen or disease, which will result in at least a decrease (up to complete absence) of the severity, duration or other manifestation of symptoms associated with infection by the pathogen or the disease.
  • the vaccine comprises an effective amount of a pre-fusion HCoV-NL63 S protein and/or a nucleic acid molecule encoding a pre-fusion HCoV-NL63 S protein, and/or a vector comprising said nucleic acid molecule, which results in an immune response against the S protein of HCoV-NL63.
  • vaccine refers to that it is a pharmaceutical composition, and thus typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients. In certain embodiments it may be a combination vaccine that further comprises additional components that induce an immune response against HCoV-NL63, e.g. against other antigenic proteins of HCoV-NL63, or may comprise different forms of the same antigenic component.
  • a combination product may also comprise immunogenic components against other infectious agents, e.g.
  • the administration of the additional active components may for instance be done by separate, e.g. concurrent administration, or in a prime-boost setting, or by administering combination products of the vaccines of the invention and the additional active components.
  • the HCoV-NL63 S proteins may also be used to isolate monoclonal antibodies from a biological sample, e.g. a biological sample (such as blood, plasma, or cells) obtained from an immunized animal or infected human.
  • a biological sample such as blood, plasma, or cells
  • the invention thus also relates to the use of the HCoV- NL63 protein as bait for isolating monoclonal antibodies.
  • HCoV-NL63 S proteins of the invention in methods of screening for candidate HCoV-NL63 antiviral agents, including but not limited to antibodies against HCoV-NL63.
  • the proteins of the invention may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the protein of the invention.
  • the invention thus also relates to an in vitro diagnostic method for detecting the presence of an ongoing or past HCoV-NL63 infection in a subject said method comprising the steps of a) contacting a biological sample obtained from said subject with a protein according to the invention; and b) detecting the presence of antibody -protein complexes.
  • the invention is further illustrated in the following example.
  • the example does not limit the invention in any way. It merely serves to clarify the invention.
  • Plasmids encoding NL63 S protein ectodomain in which the transmembrane and cytoplasmic tail were replaced with a GCN4 trimerization domain and a C-tag were synthesized and codon-optimized at Genscript.
  • Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37°C and 10% CO2. The culture supernatant was collected, and cells and cellular debris were removed by centrifugation for 10 minutes at 600 g, followed by sterile filtration using a 0.22 pm filter.
  • the cell culture supernatants of the different NL63 S variants were analyzed by quantitative Octet (qOctet) using biolayer interferometry with biotinylated anti-C-tag antibody immobilized to streptavidin sensors.
  • Streptavidin-conjugated sensors (ForteBio) were hydrated in kinetic buffer (ForteBio) for 10 minutes prior to nanobody loading.
  • Biotinylated C-tag nanobody was loaded onto the corresponding sensors at 5 pg/mL for 30 minutes. Sensors were blocked for 10 minutes in supernatant of mock-transfected cells in 96-well black flat-bottom microplates (Corning).
  • Proline screening at positions 1048-1054 demonstrated an increase in NL63 S expression by introducing a proline substitution at positions 1051, 1052, 1053, and 1054, but not at positions 1048, 1049, and 1050 (Figure 3B).
  • NL63 S trimer was purified from sterile-filtered crude cell culture supernatant using a two-step purification protocol including CaptureSelectTM C-tagXL affinity column (Thermofisher Scientific), followed by size-exclusion chromatography using a Superose 6 10/300 GL column (Cytiva).
  • the trimeric fraction was pooled and further characterized by analytical SEC using an ultra- high-performance liquid chromatography system (Vanquish, Thermofisher Scientific). Protein was loaded onto an SRT-10C SEC-500 15 cm column with the corresponding guard column (Sepax Technologies) in 150 mM sodium phosphate, 50 mM NaCl, pH 7.0 buffer.
  • NL63 S variants were trimeric proteins of the expected molecular weight, with a glycan mass of approximately 26%, and with comparable retention time and hydrodynamic radius (Figure 4D).
  • the melting temperature (Tm50) of purified NL63 S trimers was determined by Differential Scanning Fluorimetry (DSF). To this end, the fluorescent emission of Sypro Orange Dye (Thermofisher Scientific) added to NL63 S protein in solution was monitored. The measurement was performed with a starting temperature of 25 °C and a final temperature of 95 °C (54 °C increase per hour).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des protéines S de HCoV-NL63 stabilisées, ou des fragments associés, des séquences d'acides nucléiques codant pour de telles protéines, ainsi que des utilisations associées.
PCT/EP2023/075414 2022-09-23 2023-09-15 Protéines s de coronavirus stabilisées Ceased WO2024061759A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22197356.3 2022-09-23
EP22197356 2022-09-23

Publications (1)

Publication Number Publication Date
WO2024061759A1 true WO2024061759A1 (fr) 2024-03-28

Family

ID=83438778

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/075414 Ceased WO2024061759A1 (fr) 2022-09-23 2023-09-15 Protéines s de coronavirus stabilisées

Country Status (1)

Country Link
WO (1) WO2024061759A1 (fr)

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235877A (en) 1979-06-27 1980-11-25 Merck & Co., Inc. Liposome particle containing viral or bacterial antigenic subunit
US4372945A (en) 1979-11-13 1983-02-08 Likhite Vilas V Antigen compounds
US4474757A (en) 1981-01-13 1984-10-02 Yeda Research & Development Co., Ltd. Synthetic vaccine and process for producing same
WO1990003184A1 (fr) 1988-09-30 1990-04-05 Bror Morein Matrice a activite immunomodulatrice
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US5122458A (en) 1984-08-24 1992-06-16 The Upjohn Company Use of a bgh gdna polyadenylation signal in expression of non-bgh polypeptides in higher eukaryotic cells
US5385839A (en) 1985-01-30 1995-01-31 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter regulatory DNA sequence
WO1996009378A1 (fr) 1994-09-19 1996-03-28 The General Hospital Corporation Surexpression de proteines mammaliennes et virales
WO1996011711A1 (fr) 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
US6083716A (en) 1996-09-06 2000-07-04 The Trustees Of The University Of Pennsylvania Chimpanzee adenovirus vectors
WO2003104467A1 (fr) 2002-04-25 2003-12-18 Crucell Holland B.V. Moyens et procede de production de vecteurs d'adenovirus
WO2004004762A1 (fr) 2002-07-05 2004-01-15 Isconova Ab Preparation d'iscom et son utilisation
WO2005002620A1 (fr) 2003-07-07 2005-01-13 Isconova Ab Fraction de quil a a faible toxicite, utilisation de cette derniere
WO2005071093A2 (fr) 2004-01-23 2005-08-04 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Porteurs de vaccin adenoviral de chimpanze
WO2006040330A2 (fr) 2004-10-13 2006-04-20 Crucell Holland B.V. Vecteurs adenoviraux ameliores et leurs utilisations
WO2007104792A2 (fr) 2006-03-16 2007-09-20 Crucell Holland B.V. Adénovirus recombinés basés sur les sérotypes 26 et 48 et utilisation de ceux-ci
WO2010086189A2 (fr) 2009-02-02 2010-08-05 Okairòs Ag, Switzerland Séquences d'acide aminé et d'acide nucléique d'adénovirus simien, vecteurs les contenant, et utilisations afférentes
WO2010085984A1 (fr) 2009-02-02 2010-08-05 Okairos Ag Séquences d'acide nucléique et d'acides aminés d'adénovirus simiens, vecteurs les contenant et leurs utilisations
WO2012082918A1 (fr) 2010-12-14 2012-06-21 The Goverment Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vaccins contre filovirus d'adénovirus de sérotype 26 et sérotype 35
WO2012172277A1 (fr) 2011-05-25 2012-12-20 Isis Innovation Limited Adénovirus simien et vecteurs adénoviraux hybrides
WO2017037196A1 (fr) 2015-09-02 2017-03-09 Janssen Vaccines & Prevention B.V. Protéines de fusion de classe i virales stabilisées
WO2018081318A1 (fr) * 2016-10-25 2018-05-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines de spicule de coronavirus de préfusion et utilisation associée
WO2018215766A1 (fr) 2017-05-26 2018-11-29 Oxford University Innovation Limited Compositions et procédés d'induction d'une réponse immunitaire
WO2019086461A1 (fr) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Vecteurs d'adénovirus et leurs utilisations
WO2019086466A1 (fr) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adénovirus et utilisations associées
WO2019086456A1 (fr) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adénovirus et utilisations associées
WO2021178971A1 (fr) * 2020-03-06 2021-09-10 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Vaccins contre le sras-cov-2 et d'autres coronavirus
WO2022228827A1 (fr) * 2021-04-26 2022-11-03 Institut Pasteur Anticorps monoclonaux neutralisants humains contre le sras-cov-2 et leurs utilisations
WO2023283651A1 (fr) * 2021-07-09 2023-01-12 Modernatx, Inc. Vaccins pan-coronavirus humains
WO2023047349A1 (fr) * 2021-09-24 2023-03-30 Janssen Pharmaceuticals, Inc. Protéines de fusion protéines de spicule de coronavirus stabilisées
WO2023086961A1 (fr) * 2021-11-12 2023-05-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Spike du sars-cov-2 fusionnée à un antigène de surface de l'hépatite b

Patent Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235877A (en) 1979-06-27 1980-11-25 Merck & Co., Inc. Liposome particle containing viral or bacterial antigenic subunit
US4372945A (en) 1979-11-13 1983-02-08 Likhite Vilas V Antigen compounds
US4474757A (en) 1981-01-13 1984-10-02 Yeda Research & Development Co., Ltd. Synthetic vaccine and process for producing same
US5122458A (en) 1984-08-24 1992-06-16 The Upjohn Company Use of a bgh gdna polyadenylation signal in expression of non-bgh polypeptides in higher eukaryotic cells
US5385839A (en) 1985-01-30 1995-01-31 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter regulatory DNA sequence
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
WO1990003184A1 (fr) 1988-09-30 1990-04-05 Bror Morein Matrice a activite immunomodulatrice
WO1990014837A1 (fr) 1989-05-25 1990-12-13 Chiron Corporation Composition d'adjuvant comprenant une emulsion de gouttelettes d'huile d'une taille inferieure au micron
WO1996009378A1 (fr) 1994-09-19 1996-03-28 The General Hospital Corporation Surexpression de proteines mammaliennes et virales
WO1996011711A1 (fr) 1994-10-12 1996-04-25 Iscotec Ab Preparations a base de saponine et leur utilisation dans des complexes immunostimulants
US6083716A (en) 1996-09-06 2000-07-04 The Trustees Of The University Of Pennsylvania Chimpanzee adenovirus vectors
WO2003104467A1 (fr) 2002-04-25 2003-12-18 Crucell Holland B.V. Moyens et procede de production de vecteurs d'adenovirus
WO2004004762A1 (fr) 2002-07-05 2004-01-15 Isconova Ab Preparation d'iscom et son utilisation
WO2005002620A1 (fr) 2003-07-07 2005-01-13 Isconova Ab Fraction de quil a a faible toxicite, utilisation de cette derniere
WO2005071093A2 (fr) 2004-01-23 2005-08-04 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Porteurs de vaccin adenoviral de chimpanze
WO2006040330A2 (fr) 2004-10-13 2006-04-20 Crucell Holland B.V. Vecteurs adenoviraux ameliores et leurs utilisations
WO2007104792A2 (fr) 2006-03-16 2007-09-20 Crucell Holland B.V. Adénovirus recombinés basés sur les sérotypes 26 et 48 et utilisation de ceux-ci
WO2010086189A2 (fr) 2009-02-02 2010-08-05 Okairòs Ag, Switzerland Séquences d'acide aminé et d'acide nucléique d'adénovirus simien, vecteurs les contenant, et utilisations afférentes
WO2010085984A1 (fr) 2009-02-02 2010-08-05 Okairos Ag Séquences d'acide nucléique et d'acides aminés d'adénovirus simiens, vecteurs les contenant et leurs utilisations
WO2012082918A1 (fr) 2010-12-14 2012-06-21 The Goverment Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vaccins contre filovirus d'adénovirus de sérotype 26 et sérotype 35
WO2012172277A1 (fr) 2011-05-25 2012-12-20 Isis Innovation Limited Adénovirus simien et vecteurs adénoviraux hybrides
WO2017037196A1 (fr) 2015-09-02 2017-03-09 Janssen Vaccines & Prevention B.V. Protéines de fusion de classe i virales stabilisées
WO2018081318A1 (fr) * 2016-10-25 2018-05-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines de spicule de coronavirus de préfusion et utilisation associée
WO2018215766A1 (fr) 2017-05-26 2018-11-29 Oxford University Innovation Limited Compositions et procédés d'induction d'une réponse immunitaire
WO2019086461A1 (fr) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Vecteurs d'adénovirus et leurs utilisations
WO2019086466A1 (fr) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adénovirus et utilisations associées
WO2019086456A1 (fr) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adénovirus et utilisations associées
WO2021178971A1 (fr) * 2020-03-06 2021-09-10 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Vaccins contre le sras-cov-2 et d'autres coronavirus
WO2022228827A1 (fr) * 2021-04-26 2022-11-03 Institut Pasteur Anticorps monoclonaux neutralisants humains contre le sras-cov-2 et leurs utilisations
WO2023283651A1 (fr) * 2021-07-09 2023-01-12 Modernatx, Inc. Vaccins pan-coronavirus humains
WO2023047349A1 (fr) * 2021-09-24 2023-03-30 Janssen Pharmaceuticals, Inc. Protéines de fusion protéines de spicule de coronavirus stabilisées
WO2023086961A1 (fr) * 2021-11-12 2023-05-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Spike du sars-cov-2 fusionnée à un antigène de surface de l'hépatite b

Non-Patent Citations (39)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. EF 153474
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
"Tissue Culture", 1973, ACADEMIC PRESS
ABBINK ET AL., VIROL, vol. 81, no. 9, 2007, pages 4654 - 63
ABBINK, J VIROL, vol. 81, no. 9, 2007, pages 4654 - 63
BANGARIMITTAL, VACCINE, vol. 24, 2006, pages 849 - 62
BOYOGLU-BARNUM, S ET AL., NATURE, vol. 592, 2021, pages 623 - 628
BROUWER ET AL., SCIENCE, vol. 369, 2020, pages 1260 - 1505
BURTONWALKER, CELL HOST MICROBE, vol. 27, 2020, pages 695 - 698
CHEN ET AL., HUM VACCIN IMMUNOTHER, vol. 16, 2020, pages 1239 - 1242
CHIU ET AL., CLIN INFECT DIS, vol. 40, 2005, pages 1721 - 1729
COHEN ET AL., J GEN VIROL, vol. 83, 2002, pages 151 - 55
DATABASE UniProt [online] 7 June 2017 (2017-06-07), "Full=Spike glycoprotein; OS Human coronavirus NL63 (HCoV-NL63)", XP093029275, retrieved from EBI accession no. UNIPROT:A0A1V0E0W0 Database accession no. A0A1V0E0W0 *
FARINA ET AL., J VIROL, vol. 75, 2001, pages 11603 - 13
GRAHAM, ANNU REV MED, vol. 70, 2019, pages 91 - 104
HASTIE, SCIENCE, vol. 356, 2017, pages 923 - 928
HAVENGA ET AL., J GEN VIROL, vol. 87, 2006, pages 2135 - 43
HEALD-SARGENTGALLAGHER, VIRUSES, vol. 4, 2012, pages 557 - 580
HOFFMANN ET AL., CELL, vol. 181, 2020, pages 271 - 280
HOFFMANN, PROC NATL ACAD SCI USA, vol. 102, 2005, pages 7988 - 7993
HSIEH CHING-LIN ET AL: "Structure-based design of prefusion-stabilized SARS-CoV-2 spikes", vol. 369, no. 6510, 18 September 2020 (2020-09-18), US, pages 1501 - 1505, XP055780339, ISSN: 0036-8075, Retrieved from the Internet <URL:https://science.sciencemag.org/content/369/6510/1501.full.pdf> DOI: 10.1126/science.abd0826 *
HSIEH CHING-LIN ET AL: "Supplementary Materials for: Structure-based design of prefusion-stabilized SARS-CoV-2 spikes", SCIENCE, vol. 369, no. 6510, 18 September 2020 (2020-09-18), US, pages 1501 - 1505, XP093030004, ISSN: 0036-8075, Retrieved from the Internet <URL:https://www.science.org/doi/suppl/10.1126/science.abd0826/suppl_file/abd0826_hsieh_sm.pdf> DOI: 10.1126/science.abd0826 *
JURASZEK ET AL., NAT COMMUN, vol. 12, 2021, pages 244
JURASZEK JAREK ET AL: "Abstract", vol. 12, no. 1, 1 January 2021 (2021-01-01), XP055827254, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-020-20321-x.pdf> DOI: 10.1038/s41467-020-20321-x *
JURASZEK JAREK ET AL: "Supplementary Figures of : Stabilizing the Closed SARS-CoV-2 Spike Trimer", NATURE COMMUNICATIONS, 11 January 2021 (2021-01-11), XP093030022, Retrieved from the Internet <URL:https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-020-20321-x/MediaObjects/41467_2020_20321_MOESM1_ESM.pdf> [retrieved on 20230308] *
KISTLERBEDFORD, ELIFE, vol. 10, 2021, pages e64509
KOBINGER ET AL., VIROLOGY, vol. 346, 2006, pages 394 - 401
KRARUP ET AL., NAT COMM, vol. 6, 2015, pages 8143
LASAROERTL, MOL THER, vol. 17, 2009, pages 1333 - 39
PALLESEN ET AL., PROC NATL ACAD SCI USA, vol. 114, 2017, pages E7348 - E73 57
R.I. FRESHNEY: "Pharmaceutical Formulation Development of Peptides and Proteins", 2000, PHARMACEUTICAL PRESS
ROBBIANI ET AL., NATURE, vol. 584, 2020, pages 437 - 442
RUTTEN ET AL., CELL REP, vol. 30, 2020, pages 4540 - 4550
SU ET AL., TRENDS MICROBIOL, vol. 24, 2016, pages 490 - 502
TATSIS ET AL., MOLECULAR THERAPY, vol. 15, 2007, pages 608 - 17
TORTORICIVEESLER, ADV VIRUS RES, vol. 105, 2019, pages 93 - 116
VAN DER HOEK ET AL., FEMS MICROBIOL REV, vol. 30, 2006, pages 760 - 773
WALLS ET AL., NAT STRUCT MOL BIOL, vol. 23, 2016, pages 899 - 905
ZHU ET AL., RESPIR RES, vol. 21, 2020, pages 224

Similar Documents

Publication Publication Date Title
US20240197859A1 (en) Stabilized Pre-Fusion PIV3 F Proteins
US20240189416A1 (en) Stabilized coronavirus spike protein fusion proteins
EP4448802A1 (fr) Protéines de fusion hmpv pré-hybrides stabilisées
WO2024175579A1 (fr) Protéines de fusion de vrs trimères stabilisées sans domaine de trimérisation hétérologue
US20230302119A1 (en) Stabilized Corona Virus Spike Protein Fusion Proteins
WO2023047349A1 (fr) Protéines de fusion protéines de spicule de coronavirus stabilisées
US20240132548A1 (en) Stabilized pre-fusion rsv fb antigens
WO2024061759A1 (fr) Protéines s de coronavirus stabilisées
EP4598939A1 (fr) Protéines f du piv3 sous forme pré-fusion stabilisées
CA3269073A1 (fr) Protéines f du piv3 sous forme pré-fusion stabilisées
WO2023047348A1 (fr) Protéines de fusion de protéine de spicule de coronavirus stabilisées
EP4590690A1 (fr) Protéines f du piv1 humain en pré-fusion
CN116745408A (zh) 稳定的冠状病毒刺突蛋白融合蛋白

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23772223

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23772223

Country of ref document: EP

Kind code of ref document: A1