WO2023111725A1 - Sars-cov-2 vaccines - Google Patents

Sars-cov-2 vaccines Download PDF

Info

Publication number
WO2023111725A1
WO2023111725A1 PCT/IB2022/061202 IB2022061202W WO2023111725A1 WO 2023111725 A1 WO2023111725 A1 WO 2023111725A1 IB 2022061202 W IB2022061202 W IB 2022061202W WO 2023111725 A1 WO2023111725 A1 WO 2023111725A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
mutation
acid residue
protein
sars
Prior art date
Application number
PCT/IB2022/061202
Other languages
French (fr)
Inventor
Jaroslaw JURASZEK
Adriaan Hugo DE WILDE
Original Assignee
Janssen Pharmaceuticals, Inc.
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 Pharmaceuticals, Inc. filed Critical Janssen Pharmaceuticals, Inc.
Publication of WO2023111725A1 publication Critical patent/WO2023111725A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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 invention relates to the fields of virology and medicine.
  • the invention relates to vaccines for the prevention of disease induced by a SARS-CoV-2 virus.
  • SARS-CoV-2 is a coronavirus that was first discovered late 2019 in the Wuhan region in China, originally referred to as Wuhan-Hu-1.
  • SARS-CoV-2 is a beta-coronavirus, like MERS-CoV and SARS-CoV, all of which have their origin in bats.
  • the name of this disease caused by the virus is coronavirus disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases.
  • SARS-CoV-2 has strong genetic similarity to bat coronaviruses, from which it likely originated, although an intermediate reservoir host such as a pangolin is thought to be involved. From a taxonomic perspective SARS-CoV-2 is classified as a strain of the severe acute respiratory syndrome (SARS)-related coronavirus species. Coronaviruses are enveloped RNA viruses.
  • the major surface protein is the large, trimeric spike glycoprotein (S) that mediates binding to host cell receptors as well as fusion of viral and host cell membranes.
  • the S protein is composed of an N-terminal SI subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively.
  • SI NTD N-terminal domain
  • SI RBD receptor-binding domain
  • SARS-CoV-2 makes use of its SI RBD to bind to human angiotensin-converting enzyme 2 (ACE2).
  • ACE2 angiotensin-converting enzyme 2
  • the rapid expansion of the CO VID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health priority. Since the novel SARS-CoV-2 virus was first observed in humans in late 2019, over 270 million people have been infected and more than 5 million have died as a result of COVID-19.
  • SARS-CoV-2 and coronaviruses more generally, lack effective treatment, leading to a large unmet medical need.
  • mRNA vaccines and vector-based vaccines such as Ad26.CoV2.S.
  • VOC Variants of Concern
  • VOI Variants of Interest
  • the B.1.1.529 variant was reported to WHO from South Africa on 24 November 2021. In recent weeks, infections have increased steeply, coinciding with the detection of B.1.1.529 variant. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant, as compared to other VOCs. The number of cases of this variant appears to be increasing in almost all provinces in South Africa. This variant has been detected at faster rates than previous surges in infection, suggesting that this variant may have a growth advantage.
  • the present invention relates to a recombinant nucleic acid encoding a coronavirus S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a
  • the recombinant nucleic acid encodes a SARS-CoV-2 S protein, comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 32, or 33 mutations selected from the group consisting of a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375
  • the present invention relates to a recombinant nucleic acid encoding a stabilized SARS-CoV-2 S protein, said stabilized SARS CoV-2 protein comprising an amino acid sequence of SEQ ID NO: 2.
  • the invention in another aspect relates to a recombinant coronavirus S protein comprising the amino acid sequence of SEQ ID NO 2, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof.
  • the invention relates to vectors comprising the nucleic acids as described herein.
  • the vector is a recombinant human adenovirus of serotype 26.
  • the invention relates to compositions comprising such nucleic acids, proteins, and/or vectors.
  • the invention relates to methods for vaccinating a subject against COVID-19, caused by SARS CoV-2 and/or a variant thereof, the method comprising administering to the subject a composition according to the invention.
  • the invention relates to an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a SARS- CoV-2 S protein or fragment thereof.
  • the invention in another aspect, relates to methods for making a vaccine against COVID-19, said methods comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus in a pharmaceutically acceptable composition.
  • the recombinant human adenovirus of this aspect may be any of the adenoviruses described herein.
  • the invention in another aspect, relates to an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof.
  • the adenovirus may also be any of the adenoviruses as described in the embodiments above.
  • FIG. 1 Comparison of CV3-25 and ACE2-Fc antibody binding after expression by Ad26.COV2.S and Ad26.COV2.S.529.
  • Antibody binding affinity (Kd) values are shown, each point represents the mean value of a duplicate measurement.
  • FIG. 2 SARS-COV-2 Delta and Omicron spike neutralizing antibodies induced by Ad26.COV2.S.529.
  • psVNA pseudotyped virus neutralization assay
  • Horizontal red bars and values per immunization group represent geometric mean (GMT) titers. Dashed horizontal lines represent the lower limit of detection (LLOD), of a 1 :20 dilution, the lowest dilution measured in the assay. Samples with no measurable titer were set at an LLOD of 20. Pairwise comparisons were performed by a t-test, Tobit Z-test or Mann-Whitney test. Significance is shown compared to the Ad26. Empty -immunized group, unless depicted otherwise. **, P ⁇ 0.05; ***, P ⁇ 0.001.
  • nucleic acid, protein and/or adenovirus implicates that it has been modified by the hand of man, e.g., in case of an adenovector it may have altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e., it is not a naturally occurring wild type adenovirus.
  • the Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. All of these genera contain pathogenic viruses that can infect a wide variety of animals, including birds, cats, dogs, cows, bats, and humans. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses that cause respiratory disease in humans are members of the genus Betacoronavirus. These include SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKUl, and the currently circulating SARS-CoV-2.
  • S protein viral spike protein
  • SARS-CoV-2 can cause severe respiratory disease in humans.
  • a safe and effective SARS-CoV-2 vaccine is required to end the COVID-19 pandemic.
  • SARS CoV-2 refers to the SARS CoV-2 isolate that was originally identified in Wuhan (also referred to as the Wuhan-Hu-1).
  • a variant as used herein refers to a SARS-CoV-2 variant virus comprising one or more mutations in the SARS CoV-2 spike (S) protein, as compared to the S protein of the Wuhan-Hu-1 virus strain, including but not limited to the B.l, Bl.1.7, B.1.351, Pl, B.1.427, B.1.429 and B.l.1.529.
  • VOC Variant of Interest
  • VOC Variant of Concern
  • VOHC Variant of High Consequence
  • SARS-CoV-2 typically have one or more mutations in the SARS CoV-2 spike (S) protein.
  • the viral S protein binds to angiotensin-converting enzyme 2 (ACE2), which is the entry receptor utilized by SARS-CoV-2.
  • ACE2 is a type I transmembrane metallocarboxypeptidase with homology to ACE, an enzyme long-known to be a key player in the Renin-Angiotensin system (RAS) and a target for the treatment of hypertension.
  • the spike (S) protein of coronaviruses is a major surface protein and target for neutralizing antibodies in infected patients (Lester et al., Access Microbiology 2019; 1) and is therefore considered a potential protective antigen for vaccine design.
  • S protein spike protein of coronaviruses
  • Several emerging variants having mutations in the S protein indeed have shown decreased susceptibility to neutralization by vaccine induced immunity, most notably the B.1.351 variant, although the overall impact on vaccine efficacy remains to be determined.
  • the present invention provides a recombinant nucleic acid encoding a coronavirus S protein, in particular as SARS-CoV-2 S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417
  • the recombinant nucleic acid encodes a SARS-CoV-2 S protein, comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 32, or 33 mutations selected from the group consisting of: a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position
  • the nucleic acid encodes a coronavirus S protein comprising the amino acid sequence of SEQ ID NO: 2, or a fragment thereof.
  • nucleic acids can encode the same polypeptide or protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acids, to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleotide 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 include introns.
  • the nucleic acid is codon-optimized for expression in human cells.
  • the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 3, or a fragment thereof.
  • the invention further provides a recombinant coronavirus S protein, in particular a SARS-CoV-2 S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the
  • the a SARS-CoV-2 S protein comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 32, or 33 mutations selected from the group consisting of: a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue
  • the SARS-CoV-2 S protein comprises the amino acid sequence of SEQ ID NO: 2, or a fragment thereof.
  • the S protein may or may not comprise the signal peptide (or leader sequence).
  • the signal peptide may comprise the amino acids 1- 13 of SEQ ID NO: 1.
  • the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 2.
  • the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 2 without the signal peptide.
  • fragment refers to a protein or (poly)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 the SARS- CoV-2 S protein, in particular the full-length sequence of a SARS-CoV-2 S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein does not need to be full length nor have all its wild type functions, and that fragments of the protein (i.e., without signal peptide, or the ectodomain (without the transmembrane and cytoplasmic regions)) 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 SARS-CoV-2 S protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the SARS-CoV-2 S protein.
  • the person skilled in the art will also appreciate that changes can be made to a protein, e.g., by amino acid substitutions, deletions, and/or additions, using routine molecular biology procedures. Generally, conservative amino acid substitutions may be applied without loss of function or immunogenicity of a polypeptide.
  • the present invention further provides vector comprising a nucleic acid sequence according to the invention.
  • the vector is an adenovirus (or adenoviral vector).
  • An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus.
  • adenovirus 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).
  • bovine adenovirus e.g., bovine adenovirus 3, BAdV3
  • CAdV2 canine adenovirus
  • porcine adenovirus e.g., PAdV3 or 5
  • simian adenovirus which includes a monkey adenovirus and an ape a
  • 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., the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26.
  • the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.
  • Human adenoviruses are preferred according to certain aspects of the invention.
  • a recombinant adenovirus according to the invention thus 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 preexisting 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., in US6083716; WO 2005/071093; WO 2010/086189; WO 2010085984).
  • 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). In certain embodiments, 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 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.
  • characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like.
  • 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 can be double-stranded or single-stranded.
  • the adenovirus vectors useful in the invention are preferably 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-CoV-2 S protein, or fragment thereof (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.
  • the vector is 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 synthetic SARS CoV- 2 S antigens is cloned into the El and/or the E3 region of the adenoviral genome.
  • the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.
  • CMV cytomegalovirus
  • TetO tetracycline operator
  • a “promoter” is a nucleic acid sequence enabling the initiation of the transcription of a gene sequence in a messenger RNA, such transcription being initiated with the binding of an RNA polymerase on or nearby the promoter.
  • the promoter is a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif.
  • TetO motif can be referred to a “regulatory sequence” or “regulatory element,” which as used herein refers to a segment of nucleic acid, typically, but not limited to DNA, that modulates the transcription of the nucleic acid sequence to which it is operatively linked, and, thus, acts as a transcriptional modulator.
  • a regulatory sequence often comprises nucleic acid sequences that are transcription binding domains that are recognized by the nucleic acid-binding domains of transcriptional proteins and/or transcription factors, enhancers, or repressors, etc.
  • a repressor sequence can be bound by a repressor protein that can decrease or prevent the expression of the transgene in a production cell line that expresses the repressor protein.
  • a regulatory sequence can include one or more tetracycline operator (TetO) motifs/sequences, such that expression is inhibited in the presence of the tetracycline repressor protein (TetR).
  • the TetR protein In the absence of tetracycline, the TetR protein is able to bind to the TetO sites and to repress transcription of a transgene (e.g., SARS CoV-2 S antigen) operably linked to the TetO motifs/sequences.
  • a transgene e.g., SARS CoV-2 S antigen
  • the nucleic acid encoding the SARS-CoV-2 S protein when present in the adenoviral vector, is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif, such that the expression of the SARS CoV-2 S protein is inhibited in recombinant adenoviruses that are produced in the producer cell line in which the TetR protein is expressed. Expression will not be inhibited when the recombinant adenoviral vector is introduced into a subject or into cells that do not express the TetR protein.
  • CMV cytomegalovirus
  • TetO tetracycline operator
  • the term “repressor” refers to molecules (e.g., proteins) having the capability to inhibit, interfere, retard, and/or repress the production of a heterologous protein product of a recombinant expression vector (e.g., an adenoviral vector).
  • the repressor can inhibit expression by interfering with a binding site at an appropriate location along the expression vector, such as in an expression cassette (e.g., a TetR can bind the TetO motif in the CMV promoter). Repression of vector transgene expression during vector propagation can prevent transgene instability and can increase yields of vectors having the transgene during production.
  • a nucleic acid is “operably linked” when it is placed into a structural or functional relationship with another nucleic acid sequence.
  • one segment of DNA can be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding sequence so as to facilitate transcription of the coding sequence; a ribosome binding site that is positioned relative to a coding sequence so as to facilitate translation; or a pre-sequence or secretory leader that is positioned relative to a coding sequence so as to facilitate expression of a pre-protein (e.g., a pre-protein that participates in the secretion of the encoded polypeptide).
  • a pre-protein e.g., a pre-protein that participates in the secretion of the encoded polypeptide
  • the operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them.
  • Enhancers for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.
  • the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 4, preferably the CMV promoter consists of SEQ ID NO: 4.
  • the vector according to the invention comprises a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 3. In certain preferred embodiments, the vector according to the invention comprises a nucleic acid consisting of SEQ ID NO: 3.
  • compositions in particular pharmaceutical compositions, comprising a nucleic acid, a protein, and/or vector according to the invention.
  • the invention may employ pharmaceutical compositions comprising the nucleic acid, a protein, and/or vector 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.
  • the purified nucleic acid, a protein, and/or vector preferably is formulated and administered as a sterile solution although it is also 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, preferably in the range of pH 5.0 to 7.5.
  • nucleic acid, a protein, and/or vector typically is in a solution having a suitable pharmaceutically acceptable buffer, and the solution may also contain a salt.
  • stabilizing agent may be present, such as albumin.
  • detergent is added.
  • nucleic acid, a protein, and/or vector may be formulated into an injectable preparation. These formulations contain effective amounts of nucleic acid, a protein, and/or vector, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
  • adenovirus may be stored in the buffer that is also used for the Adenovirus World Standard (Hoganson et al, Development of a stable adenoviral vector formulation, Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol.
  • Another useful formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgC12, 25 mM NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v.
  • many other buffers can be used, and several examples of suitable formulations for the storage and for pharmaceutical administration of purified (adeno)virus preparations can for instance be found in European patent no.
  • a composition according to the invention comprises a(n) (adeno) vector according to the invention in combination with a further active component.
  • Such further active components may comprise one or more SARS-CoV-2 protein antigens, e.g., a SARS-CoV-2 protein according to the invention, or any other SARS-CoV-2 protein antigen, or additional vectors comprising nucleic acid encoding similar or alternative SARS- CoV-2 antigens.
  • Such vectors again may be non-adenoviral or adenoviral, of which the latter can be of any serotype.
  • a composition according to the invention comprises at least a first vector according to the invention and at least a second vector comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 5, or a fragment thereof.
  • the first and second vector comprise a recombinant human adenovirus of serotype 26.
  • compositions may or may not comprise one or more adjuvants.
  • adjuvants are known in the art to further increase the immune response to an applied antigenic determinant, and pharmaceutical compositions comprising adenovirus and suitable adjuvants are for instance disclosed in WO 2007/110409, incorporated by reference herein.
  • the terms “adjuvant” and “immune stimulant” are used interchangeably 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 adenovirus vectors of the invention.
  • suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-emulsion 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.
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O- deacylated MPL
  • CpG-motif containing oligonucleotides such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like
  • vector-encoded adjuvant e.g., by using heterologous nucleic acid that encodes a fusion of the oligomerization domain of C4-binding protein (C4bp) to the antigen of interest (e.g., Solabomi et al, 2008, Infect Immun. 76: 3817- 23).
  • C4bp C4-binding protein
  • compositions do not comprise adjuvants.
  • the present invention further provides compositions for use as a vaccine against COVID-19 caused by SARS- CoV-2 Wuhan-Hu, or a variant thereof, such as the B.1.1.529 variant, comprising a nucleic acid, a protein, and/or vector according to the invention.
  • the term “vaccine” refers to a (pharmaceutical) composition containing an active component effective to induce a therapeutic degree of immunity in a subject against a certain pathogen or disease.
  • the vaccine preferably comprises an effective amount of a recombinant adenovirus of serotype 26 that encodes a SARS CoV-2 S protein, in particular a SARS CoV-2 protein that comprises the amino acid sequence of SEQ ID NO: 2, or an antigenic fragment thereof, which results in an immune response, preferably a protective immune response, against the S protein of SARS CoV-2, or a variant thereof, such as the B 1.351 variant.
  • the vaccine comprises a recombinant human adenovirus of serotype 26 that comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the vaccine comprises a recombinant human adenovirus of serotype 26 that comprises a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 3.
  • the “vaccine” 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 other components that induce an immune response, such as but not limited to a second adenoviral vector encoding a different SARS-CoV-2 protein, or a SARS-CoV-2 protein as such.
  • the vaccine compositions of the invention may be used in a method of preventing serious lower respiratory tract disease leading to hospitalization, and/or the decrease the frequency of complications such as pneumonia and bronchiolitis, and/or death due to infection with SARS-CoV-2, or a variant thereof, including, but not limited to, the B.1.1.529 variant.
  • the vaccine may also be used in so-called Post-exposure prophylaxis (PEP), i.e., for preventing illness after potential or documented exposure to the coronavirus and/or for reducing the risk of secondary spread of infection.
  • PEP Post-exposure prophyl
  • the invention thus also provides a method for vaccinating a subject against COVID- 19, caused by SARS CoV-2 (Wuhan-Hu-1), or a variant thereof, said method comprising administering to the subject a vaccine as described herein.
  • the vaccine is administered to a naive (or seronegatieve) subject, preferably a subject that has no circulating antibodies against SARS-CoV-2 or a variant thereof.
  • a naive (or seronegatieve) subject preferably a subject that has no circulating antibodies against SARS-CoV-2 or a variant thereof.
  • the subject has not been vaccinated against COVID-19 and has not been infected with SARS CoV-2 virus (Wuhan-Hu-1), or a variant thereof, prior to the administration of the vaccine.
  • the vaccine is administered to a subject that has been vaccinated at least once against COVID-19 prior to administration of the vaccine.
  • the subject may have been vaccinated using any available vaccine, including, but not limited to, mRNA vaccines such as BNT162b2 and mRNA- 1273, vector-based vaccines, such as AZDI 222 and Ad26.COV2.S, or protein vaccines, such as NVX-CoV2373.
  • the subject was vaccinated using a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding a SARS-CoV-2 S protein that comprises the amino acid sequence of SEQ ID NO: 5, or a fragment thereof (also referred to as Ad26.COV2.S).
  • the vaccine according to the present invention is administered to the subject between 6 and 12 months after the previous vaccination.
  • the total dose of the adenovirus provided to a subject preferably is between IxlO 8 vp and 2xlO n vp, for instance between 3xl0 8 and IxlO 11 vp per administration.
  • the total dose of the adenovirus provided to the subject ranges from 1 x IO 10 vp to 1 x 10 11 vp per dose.
  • the adenovirus is administered at a total dose of 5 x IO 10 vp per administration.
  • the vaccine comprises a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding the SARS CoV-2 S protein of SEQ ID NO: 2 at a dose of 2.5 x IO 10 vp and a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding a SARS-CoV-2 S protein that comprises the amino acid sequence of SEQ ID NO: 5 at a dose of 2.5 x IO 10 vp.
  • the total dose of adenovirus per administration of the vaccine thus is 5 x IO 10 vp.
  • Administration of adenovirus compositions can be performed using standard routes of administration.
  • Non-limiting embodiments include parenteral administration, such as by injection, e.g., intramuscular, intradermal, subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. It is particularly preferred according to the present invention to administer the vaccine intramuscularly, such as into the deltoid muscle of the arm, or vastus lateralis muscle of the thigh.
  • the subject is a human subject.
  • the subject can be of any age, e.g., from about 1 month to 100 years old, e.g., from about 2 months to about 80 years old, e.g., from about 1 month to about 3 years old, from about 3 years to about 50 years old, from about 50 years to about 75 years old, etc.
  • the subject is a human from 2 years of age, preferably a human from 12 years of age, more preferably a human from 18 years of age.
  • the vaccine is administered to the subject more than once, e.g., once a year.
  • the method of vaccination consists of a single administration of the composition or vaccine to the subject. It is also possible to provide one or more booster administrations of the vaccine of the invention. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a moment between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases sometimes referred to as ‘priming vaccination’). In certain embodiments, the vaccine is administered every two, three, four or five years.
  • the invention further provides a method for inducing binding antibodies to the S protein of a of SARS-CoV-2 variant, including but not limited to the B.1.1.529 variant, and to the SARS CoV-2 Wuhan-Hu-1 S protein, in a subject in need thereof, as measured e.g., by ELISA, comprising administering to the subject a vaccine as described herein.
  • the amount (titer) of binding antibodies against SARS-CoV-2 Wuhan-Hu-1 is non-inferior to the amount of binding antibodies against the variant.
  • the amount (titer) of binding antibodies against SARS-CoV-2 Wuhan-Hui is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amount of binding antibodies against the variant, e.g., the B.1.1.529 variant.
  • the invention also provides a method for inducing antibodies capable of neutralizing a SARS-CoV-2 variant, including but not limited to the B.1.1.529 variant, and SARS CoV-2 Wuhan-Hu 1, in a subject in need thereof, as measured, e.g., by wtVNA or psVNA, comprising administering to the subject a vaccine as described herein.
  • the neutralizing antibody response against SARS CoV-2 Wuhan-Hu-1 is non-inferior to the neutralizing antibody response against the variant, such as B.1.1.529.
  • the neutralizing antibody response to Wuhan-Hu-1 is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the neutralizing antibody response against the variant.
  • non-inferiority (NI) of the vaccine of the current invention means that it is not inferior to an existing one (such as Ad26.CoV2.S), i.e., that it is either equally effective or better (e.g., with a NI margin of 0.67).
  • the vaccine of the current invention is not inferior to an existing one (such as Ad26.CoV2.S), i.e., is either equally effective or better, with a NI margin of 0.67, in their respective matched virus neutralization assays.
  • Ad26.CoV2.S an existing one
  • NI margin 0.67
  • the invention also provides a method for inducing a specific T cell response against a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, and against SARS CoV-2 Wuhan-Hu-1, in a subject in need thereof, as assessed, e.g., by flow cytometry after SARS-CoV2 S protein peptide stimulation of peripheral blood mononuclear cells (PBMCs) and intracellular staining, comprising administering to the subject a vaccine as described herein.
  • PBMCs peripheral blood mononuclear cells
  • the T cell response against SARS-CoV-2 is similar (non-inferior) to the T cell response against the variant.
  • the T cell response to SARS-CoV-2 is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cell response to the variant.
  • the invention also provides a method for reducing infection and/or replication of a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant and of SARS CoV- 2 Wuhan-Hu-1, in, e.g., the nasal tract and lungs of, a subject, comprising administering to the subject a vaccine as described herein.
  • This will reduce adverse effects resulting from infection by SARS-CoV2 (Wuhan-Hu-1), or a variant thereof, in a subject, and thus contribute to protection of the subject against such adverse effects.
  • adverse effects of infection may be essentially prevented, i.e., reduced to such low levels that they are not clinically relevant.
  • the invention also provides a method for prevention of molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, comprising administering to the subject a vaccine as described herein,
  • the invention also provides a method for prevention of molecularly confirmed, moderate to severe/critical COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, comprising administering to the subject a vaccine as described herein.
  • the invention also provides a method for preventing or reducing the occurrence of pneumonia linked to any molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, when compared to a placebo or a different COVID-19 vaccine, such as, but not limited to Ad26.COV2.S.
  • the invention also provides a method for preventing or reducing the occurrence of hospitalization linked to any molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, when compared to a placebo or a different vaccine, such as, but not limited to Ad26.COV2.S.
  • the invention also provides a method for preventing or decreasing death linked to molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, when compared to placebo, or a different vaccine.
  • the effects of the vaccine occur between 14 and 28 days after vaccination.
  • the invention further provides an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising a nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof comprising the nucleotide sequence of SEQ ID NO: 3.
  • the invention further provides methods for making a vaccine COVID-19, comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-COV-2 S protein or fragment thereof, as described herein, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition.
  • adenoviral particle comprising a SARS-Co-V-2 S protein as described herein.
  • the methods comprise (a) contacting a host cell of the invention with an adenoviral vector of the invention and (b) growing the host cell under conditions wherein the adenoviral particle comprising the SARS-CoV-2 antigen is propagated.
  • Recombinant adenovirus can be prepared and propagated in host cells, according to well-known methods, which entail cell culture of the host cells that are infected with the adenovirus.
  • 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.
  • a host cell (sometimes also referred to in the art and herein as “packaging cell” or “complementing cell” or “producer cell”) that can be used can be any host cell wherein a desired adenovirus can be propagated.
  • a host cell line is typically used to produce sufficient amounts of adenovirus vectors of the invention.
  • a host cell is a cell that comprises those genes that have been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, PER.C6®, 911, 293, and
  • the host cell further comprises a nucleotide sequence encoding a tetracycline repressor (TetR) protein.
  • the nucleotide sequence encoding the TetR protein can, for example, be integrated in the genome of the host cell.
  • the nucleotide sequence encoding the TetR protein can be integrated in chromosome 1.
  • the host cell line can, for example, be a PER.C6® cell.
  • the invention further provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV2 S protein or fragment thereof.
  • SARS-CoV-2 S severe acute respiratory syndrome coronavirus 2 Spike
  • SARS-CoV-2 S severe acute respiratory syndrome coronavirus 2 Spike
  • the gene was cloned in house in the expression cassette under transcriptional control of a human cytomegalovirus (CMV.TetO) promoter and the SV-40 polyA sequence.
  • CMV.TetO human cytomegalovirus
  • the plasmid DNA was subjected to a DNA cleaning process and DNA sequence analysis prior to Ad26 vector generation.
  • Ad26.COV2.S.529 The recombinant Ad26 vector, Ad26.COV2.S.529, is replication-incompetent due to deletions in El (A E1A/E1B).
  • the El deletion renders the vector replication-incompetent in non-complementing cells such as normal human cells.
  • Ad5 El complementing cell lines like HEK293, PER.C6, PER.C6 TetR and HER96 cells the virus can be propagated.
  • the E3 gene has been removed (AE3) to create sufficient space in the viral genome for insertion of foreign antigens, and the Ad26 E4 orf6 has been exchanged by the Ad5 homologue to allow efficient production of replication-incompetent Ad26 vectors in Ad5 El complementing cell lines.
  • a single genome plasmid is used to generate the Ad26 vector on PER.C6 TetR cells (Research Cell Bank II (RCB II).
  • RBC II Research Cell Bank II
  • these suspension cells were cultured in DMEM without geneticin, supplemented with 10% FBS (y-irradiated, complying with EMA/CHMP/BWP/457920/2012 rev 1) in PLL coated plates.
  • Cells were transfected with the linearized plasmid using the agent Lipofectamine 2000CDTM.
  • Single plaques were isolated by 1 round of plaque purification on monolayers of PER.C6 TetR cells covered with an agarose overlay (sea plaque agarose).
  • Plaques were amplified on PER.C6 TetR cells grown in DMEM supplemented with 10% y-irradiated FBS. The final steps were performed in suspension cultures. Multiple plaques were tested for integrity and identity of the adenovirus genome and correct expression of the antigen and one plaque was selected for manufacturing.
  • Virus seed stocks derived from a single plaque, are used to infect PER.C6 TetR cells (Research Cell Bank II (RCB II) cultivated in DMEM without geneticin, supplemented with 10% FBS (y-irradiated, complying with EMA/CHMP/BWP/457920/2012 rev 1) in order to manufacture the Ad26.COV2.S.529 pre-master virus seed (preMVS).
  • RBC II Research Cell Bank II
  • FBS y-irradiated, complying with EMA/CHMP/BWP/457920/2012 rev 1
  • FBS y-irradiated, complying with EMA/CHMP/BWP/457920/2012 rev 1
  • preMVS pre-master virus seed
  • Cell material is harvested by centrifugation and used for purification of the recombinant adenovirus. Purification is performed using 2 successive rounds of cesium chloride (CsCl) density centrifugation. Dialysis is performed to remove excess CsCl and
  • the combined dilution factor associated with plasmid and Ad26 vector purification i.e., single clone selection, 1 round of plaque purification and 2 ultracentrifugation purification steps) is calculated to be at least 10 27 .
  • the purified virus suspension is tested for quantity, infectivity, identity, and adventitious agents.
  • DS batches are produced from the Master Virus Seed (MVS). All raw materials are chemically defined and of non-animal (derived) or non-human origin.
  • Ad26.COV2.S.529 (also known as VAC31518, JNJ-87918883) is a monovalent vaccine composed of a recombinant, replication-incompetent adenovirus type 26 (Ad26) vector, constructed to encode the Spike (S) protein derived from a SARS-CoV-2 clinical isolate (B.1.1.529 lineage, Omicron variant), with an inactivated furin cleavage site between the SI and S2 protein subunits and substitution of 2 prolines to enhance prefusion conformation.
  • Ad26 a monovalent vaccine composed of a recombinant, replication-incompetent adenovirus type 26 (Ad26) vector, constructed to encode the Spike (S) protein derived from a SARS-CoV-2 clinical isolate (B.1.1.529 lineage, Omicron variant), with an inactivated furin cleavage site between the SI and S2 protein subunits and substitution of 2 prolines to enhance prefusion conformation.
  • mice will be used to compare immunogenicity of Ad26.COV2.S.529 with that of the current vaccine Ad26.COV2.S by an Omicron virus neutralization assay (VNA).
  • VNA Omicron virus neutralization assay
  • the Pfizer-BioNTech COVID-19 vaccine, BNT162b2 is a lipid nanoparticle- formulated, nucleoside-modified RNA vaccine encoding a prefusion stabilized, membrane- anchored SARS CoV-2 full-length spike protein.
  • Ad26.COV2.S vaccine directed against the original SARS- CoV-2 strain is associated with demonstrated clinical protective efficacy against COVID-19.
  • This study will assess the reactogenicity, safety, and immunogenicity of a booster dose of Ad26.COV2.S or Ad26.COV2.S.529 in adults >18 years of age, who have previously received primary vaccination with Ad26.COV2.S, booster vaccination with Ad26.COV2.S or booster vaccination with Pfizer mRNA-based vaccine BNT162b2.
  • the purpose of the study is to demonstrate that the humoral neutralizing immune responses elicited by a booster vaccination with Ad26.COV2.S.529 against the Omicron variant, given after a single dose of Ad26.COV2.S (Cohort 1), is non-inferior (NI) to the responses elicited by primary vaccination with the initially authorized vaccine (Ad26.COV2.S) against the original strain (Primary Objective la), which is associated with demonstrated clinical protective efficacy against COVID-19 .
  • NI non-inferior
  • a heterologous regimen will also be tested (Cohort 3), where the NI of neutralizing antibody responses against the Omicron variant induced by Ad26.COV2.S.529 in participants who received a 2-dose Pfizer BNT162b2 primary series and Pfizer BNT162b2 booster, compared to neutralizing antibody responses against the standard strain induced by the 2-dose primary regimen with Pfizer BNT162b2 (Primary Objective 1c).
  • the objectives la, lb, 1c will examine the NI of the neutralizing antibody levels against Omicron induced by a booster with Ad26.COV2.S.529 compared to those responses by primary regimens against the standard strain at neutralizing antibody levels and seroresponse rates where protection has been shown. It will also show the superiority of a final boost with Ad26.COV2.S.529 vs Ad26.COV2.S when given after a primary series, and a primary series followed by a boost, which will reflect the majority of individuals that have received COVID-19 vaccines.
  • Reactogenicity data will be collected for 28 days following COV2015 study booster vaccination.
  • Safety data (SAEs/AESIs/MAAEs) will be collected for 360 days after CO V2015 study booster vaccination.
  • a Responders are defined as 4-fold rise from pre-vaccination titers or 4 fold rise above the lower limit of quantitation if pre-vaccination titer is below the level of quantification. Further information on responder definitions will be described in the Statistical Analysis Plan.
  • b Alpha is determined using an hierarchical testing approach as outlined in the Statistical Considerations.
  • PBMC Blood Collection Schedule
  • Subset 2 Subset 2 Subset 2 Subset 2 Subset 2 Cohort 1 Participant only received Ad26.COV2.S (5x 1010 vp) primary vaccination (ie, single dose).
  • Cohort 2 Participant received Ad26.COV2.S (5x 1010 vp) primary vaccination followed by an initial boost with Ad26.COV2.S (5x 1010 vp) which occurred >2 months57 days after primary vaccination.
  • Cohort 3 Participant completed primary vaccination with a 2-dose regimen of BNT162b2 vaccine (Pfizer) and a booster dose of BNT162b2 which occurred >4 months after primary vaccination.
  • Ad26.COV2.S vaccine is a replication-incompetent human adenovirus type 26 (Ad26) vector encoding a stabilized pre-fusion SARS-CoV-2 spike protein based on the Wuhan-Hu-1 isolate.
  • Ad26.COV2.S was 73% efficacious at preventing severe-critical COVID- 192
  • the Ad26.COV2.S COVID-19 vaccine was granted emergency use authorization in US and (conditional) marketing authorization in the European Union and in more than 50 other countries.
  • Ad26.COV2.S.529 induces robust Omicron neutralizing antibody titers in naive mice and in hamsters with pre-existing SARS-CoV-2 spike protein immunity.
  • Ad26 vector-based vaccines were generated as described previously using the AdVac system (Bos et al. NPJ Vaccines 5, 91 (2020)).
  • Ad26NCOV006 and Ad26.COV2.S encode a SARS-COV-2 spike protein sequence based on SARS-CoV-2 Wuhan-Hu-1 spike (GenBank accession number MN908947), while Ad26.COV2.S.529 encode a SARS-COV-2 spike protein sequence based on SARS-CoV-2 Omicron; BA. l spike (GISAID accession number EPI ISL 6913991).
  • Spike protein encoded by Ad26.COV2.S and Ad26.COV2.S.529 was stabilized in the prefusion conformation by R682S and R685G that abolish the furin cleavage site and by the proline substitutions K986P and V987P.
  • the negative control vector Ad26.Empty which does not contain a transgene, was used a control.
  • A549 cells were seeded at 2.9 * 10 4 cells/well in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) in a flat-bottomed 96-well microtiter plate (Corning). The plate was incubated overnight at 37 °C in 10% CO2. After 24 h, cells were transduced with Ad26.COV2.S or Ad26.COV2.S.529 at a dose of 2000 infectious units [IU]/cell and the plate was incubated for 48 h at 37 °C in 5% CO2. Two days post transduction, cells were washed four times with PBS and subsequently fixed with 4% formaldehyde in PBS.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS heat-inactivated fetal bovine serum
  • CV3-25 was produced at ImmunoPreci se according to Jennewein et al.21 and ACE2-Fc was made according to Liu et al22. After 30 to 60 minutes of incubation, cells were washed four times with 0.05% Tween-20 in PBS. Next, cells were incubated with mouse HRP-conjugated antihuman IgG Fc (Jackson, 1 : 8000) for 40 min at RT. The plate was washed four times with 0.05% Tween-20 in PBS. 3,3',5,5'-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma) was added and after 20 minutes the reaction was terminated by adding Stop Reagent for TMB Substrate (Sigma).
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • mice Female Syrian golden hamsters (Mesocricetus auratus), strain RjHan:aura, aged 9-11 weeks at the start of the study, were purchased from Janvier Labs, France. Hamsters were immunized via the intramuscular route with 100 pl (50 pl per hindleg) vaccine under general anesthesia with isoflurane. Blood samples were collected via the retro-orbital route under anesthesia as described above.
  • mice 8-10 weeks at the start of study were provided by Charles River Laboratories, Germany. Mice were immunized via the intramuscular route with 100 pl (50 pl per hindleg) vaccine under general anesthesia with isoflurane. Blood samples were collected via the submandibular bleeding route. Blood from all animal experiments was processed for serum isolation.
  • HlV Human Immunodeficiency Virus
  • SARS-CoV-2 spike variants by psVNA.
  • Human Immunodeficiency Virus (HlV)-based lentiviruses, pseudotyped with SARS-CoV-2 spike protein (based on Wuhan-Hu- 1; GenBank accession number MN908947) were generated as described previously (Solforosi et al., J. Exp. Med. 218, e20202756 (2021); Jongeneelen et al., 2021.07.01.450707 https://www.biorxiv.Org/content/10. l 101/2021.07.01.450707vl (2021) doi: 10.1101/2021.07.01.450707). Substitutions and deletions in the spike protein open reading frame for the variant B.1.617.2 and BA.1 (GISAID accession number
  • EPI ISL 6913991 were introduced using standard molecular biology techniques and confirmed by sequencing.
  • Assays were performed on Hek293T target cells stably expressing the human angiotensinconverting enzyme 2 (ACE2) and human transmembrane serine protease 2 (TMPRSS2) genes (VectorBuilder, Cat. CL0015). The cells were seeded in white half-density area 96- well tissue culture plates (Perkin Elmer) at a density of 1.5E+04 cells/well.
  • ACE2 angiotensinconverting enzyme 2
  • TMPRSS2 human transmembrane serine protease 2
  • Luciferase activity was measured 40h after transduction by adding an equal volume of NeoLite substrate (Perkin Elmer) to the wells according to the manufacturer’s protocol, followed by read out of RLU on the EnSight Multimode Plate Reader (Perkin Elmer).
  • SARS-CoV-2 neutralizing titers were calculated in R using a four-parameter curve fit as the sample dilution at which a 50% reduction (N50) of luciferase readout was observed compared with luciferase readout in the absence of serum (High Control). The starting serum sample dilution of 20 was fixed as the limit of detection (LLOD).
  • Ad26.COV2.S.529 vector spike expression and antigenicity were characterized in vitro and compared to Ad26.COV2.S. Spike protein expression was evaluated after transduction of A549 cells using a quantitative cell-based ELISA with CV3-25 and ACE2-Fc.
  • CV3-25 is an antibody that binds to the stem region of the SARS-CoV-2 spike S221, a region which is conserved between the Wuhan-1 -Hu and BA.l spike protein.
  • CV3-25-binding to the spike protein expressed after transduction of A549 cells with Ad26.COV2.S or Ad26.COV2.S.529 was comparable (Figure 1A).
  • Ad26.COV2.S.529 induces Omicron neutralizing antibodies in naive mice and hamsters with pre-existing immunity.
  • Naive mice were immunized with 10 8 , 10 9 , IO 10 vp of Ad26.COV2.S, Ad26.COV2.S.529 or IO 10 vp Ad26.Empty mock control vector.
  • SARS-CoV-2 Delta (B.1.617.2) and Omicron (BA. l) spike neutralization titers were evaluated using a psVNA assay in sera collected 4 weeks after immunization.
  • a single immunization with Ad26.COV2.S.529 induced dose-dependent Omicron spike neutralizing antibodies that were significantly higher than after vaccination with Ad26.COV2.S at all doses tested (Figure 2A).
  • Ad26.COV2.S.529 As an increasing part of the population acquired pre-existing immunity either by infection or vaccination, we also evaluated the immunogenicity of Ad26.COV2.S.529 in hamsters with pre-existing immunity to a Wuhan-Hu-1 SARS-CoV-2 spike protein. Hamsters were first immunized with 10 7 vp Ad26NCOV006 , which encodes the ancestral SARS-CoV- 2 Wuhan-Hu- 1 spike protein. The mock control group was immunized with 10 7 vp of Ad26. empty. Six weeks later, the hamsters received a vaccination with 10 10 vp of Ad26.COV2.S, Ad26.COV2.S.529 or Ad26.Empty.
  • SARS-CoV-2 Delta and Omicron spike neutralization titers were evaluated using a psVNA assay in sera collected 4 weeks after vaccination. Vaccination of pre-immune hamsters with Ad26.COV2.S and Ad26.COV2.S.529 resulted in comparable Delta spike neutralizing antibody titers that were significantly higher than in control animals ( Figure 2B). While Omicron spike neutralizing antibody titers were undetectable in 7 out of 8 Ad26.COV2.S-vaccinated hamsters, vaccination with Ad26.COV2.S.529 induced robust Omicron spike neutralizing antibodies.
  • Ad26 vector encoding an Omicron (BA. l) spike protein was generated (Ad26.COV2.S.529).
  • Ad26.COV2.S.529 is similarly prefusion stabilized as the current COVID-19 vaccine Ad26.COV2.S encoding Wuhan-Hu-1 spike and it was verified that spike expression was comparable to Ad26.COV2.S. Immunogenicity of Ad26.COV2.S.529 was then evaluated in naive mice and SARS-CoV-2 Wuhan-Hu-1 spike pre-immunized hamsters.
  • Ad26.COV2.S.529 elicited robust neutralizing antibodies against SARS-CoV-2 Omicron (BA.1) but not to SARS-CoV-2 Delta in naive mice while the opposite was observed for Ad26.COV2.S.
  • Ad26.COV2.S.529 vaccination resulted in robust increases in neutralizing antibody titers against both SARS-CoV-2 Omicron (BA.l) and Delta, while Ad26.COV2.S vaccination only increased neutralizing antibody titers against the Delta variant.
  • Our data imply that Ad26.COV2.S.529
  • TAA CMVdell34 including 2x TO; SEO ID NO: 4

Abstract

The present invention relates to isolated nucleic and/or recombinant nucleic acid encoding a coronavirus S protein, and to the coronavirus S proteins, as well as to the use of the nucleic acids and/or proteins thereof in vaccines.

Description

SARS-CoV-2 Vaccines
Introduction
The invention relates to the fields of virology and medicine. In particular, the invention relates to vaccines for the prevention of disease induced by a SARS-CoV-2 virus.
Background
SARS-CoV-2 is a coronavirus that was first discovered late 2019 in the Wuhan region in China, originally referred to as Wuhan-Hu-1. SARS-CoV-2 is a beta-coronavirus, like MERS-CoV and SARS-CoV, all of which have their origin in bats. The name of this disease caused by the virus is coronavirus disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases.
As indicated above, SARS-CoV-2 has strong genetic similarity to bat coronaviruses, from which it likely originated, although an intermediate reservoir host such as a pangolin is thought to be involved. From a taxonomic perspective SARS-CoV-2 is classified as a strain of the severe acute respiratory syndrome (SARS)-related coronavirus species. Coronaviruses are enveloped RNA viruses. The major surface protein is the large, trimeric spike glycoprotein (S) that mediates binding to host cell receptors as well as fusion of viral and host cell membranes. The S protein is composed of an N-terminal SI subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively. Recent cryo- EM reconstructions of the CoV trimeric S structures of alpha-, beta-, and deltacoronaviruses revealed that the SI subunit comprises two distinct domains: an N-terminal domain (SI NTD) and a receptor-binding domain (SI RBD). SARS-CoV-2 makes use of its SI RBD to bind to human angiotensin-converting enzyme 2 (ACE2). The rapid expansion of the CO VID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health priority. Since the novel SARS-CoV-2 virus was first observed in humans in late 2019, over 270 million people have been infected and more than 5 million have died as a result of COVID-19.
SARS-CoV-2, and coronaviruses more generally, lack effective treatment, leading to a large unmet medical need. Several vaccines have recently come to market, including mRNA vaccines and vector-based vaccines such as Ad26.CoV2.S.
All viruses, including SARS-CoV-2, change over time. Most changes have little to no impact on the virus’ properties. However, some changes may affect the virus’s properties, such as how easily it spreads, the associated disease severity, or the performance of vaccines, therapeutic medicines, diagnostic tools, or other public health and social measures. The World Health Organization (WHO) and the European Centre for Disease prevention and Control (ECDC) publish weekly overviews of so-called Variants of Concern (VOC) and Variants of Interest (VOI). These variants are carefully monitored and mapped due to their (potential) high-risk characteristics and degree of spread. The emergence and rapid spread of some variants of SARS-CoV-2 has raised important questions about how these variants may impact both natural and vaccine-elicited immunity. For example, the B.1.1.7 variant, initially identified in the UK, has demonstrated enhanced transmissibility, while the B.1.351 variant, initially identified in South Africa, exhibits partial evasion of antibody responses.
More recently, the B.1.1.529 variant was reported to WHO from South Africa on 24 November 2021. In recent weeks, infections have increased steeply, coinciding with the detection of B.1.1.529 variant. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant, as compared to other VOCs. The number of cases of this variant appears to be increasing in almost all provinces in South Africa. This variant has been detected at faster rates than previous surges in infection, suggesting that this variant may have a growth advantage.
Concerns exists that the current COVID-19 vaccines (herein referred to as prototype vaccines) will provide reduced protection against the current variants or further (circulating or future) variants (Rambaut et al., Virological. 2020: https://virological.org/t/preliminary- genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defmed-by-a-novel- set-of-spike-mutations/563; Tegally et al., MedRxiv. 2020. https://www.medrxiv.org/content/10.1101/2020.12.21.20248640vl). For example, data suggest that the B.1.351 variant is not neutralized by some monoclonal antibodies directed to the SARS-CoV-2 spike protein and is resistant to neutralization by plasma from individuals previously infected with SARS-CoV-2 (Wibmer et al., Nat Med (2021) https://doi.org/10.1038/s41591-021-01285-x.).
There is thus still an urgent need for novel vaccines that can be used to prevent coronavirus induced respiratory disease caused by SARS-CoV-2 (Wuhan-hu-1) and variants derived therefrom.
Summary of the Invention
In a first aspect, the present invention relates to a recombinant nucleic acid encoding a coronavirus S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1.
In certain embodiments, the recombinant nucleic acid encodes a SARS-CoV-2 S protein, comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 32, or 33 mutations selected from the group consisting of a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1.
In a preferred embodiment, the present invention relates to a recombinant nucleic acid encoding a stabilized SARS-CoV-2 S protein, said stabilized SARS CoV-2 protein comprising an amino acid sequence of SEQ ID NO: 2.
In another aspect the invention relates to a recombinant coronavirus S protein comprising the amino acid sequence of SEQ ID NO 2, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof.
In yet another aspect, the invention relates to vectors comprising the nucleic acids as described herein. In certain embodiments, the vector is a recombinant human adenovirus of serotype 26.
In another aspect, the invention relates to compositions comprising such nucleic acids, proteins, and/or vectors.
In another aspect, the invention relates to methods for vaccinating a subject against COVID-19, caused by SARS CoV-2 and/or a variant thereof, the method comprising administering to the subject a composition according to the invention.
In another aspect, the invention relates to an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a SARS- CoV-2 S protein or fragment thereof.
In another aspect, the invention relates to methods for making a vaccine against COVID-19, said methods comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus in a pharmaceutically acceptable composition. The recombinant human adenovirus of this aspect may be any of the adenoviruses described herein.
In another aspect, the invention relates to an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof. The adenovirus may also be any of the adenoviruses as described in the embodiments above.
Brief description of the drawings
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings.
FIG. 1: Comparison of CV3-25 and ACE2-Fc antibody binding after expression by Ad26.COV2.S and Ad26.COV2.S.529. A. CV3-25 or B. ACE2-Fc fusion protein binding to the spike protein in A549 cells after transduction with 2000 infectious units/cell Ad26.COV2.S.529 (grey line) or Ad26.COV2.S (black line). Antibody binding affinity (Kd) values are shown, each point represents the mean value of a duplicate measurement.
FIG. 2: SARS-COV-2 Delta and Omicron spike neutralizing antibodies induced by Ad26.COV2.S.529. A. Mice were immunized with 108, 109 or IO10 viral particles (vp) Ad26.COV2.S or Ad26.COV2.S.529 (both n=8); IO10 vp Ad26.Empty (n=5). Serum was collected 4 weeks after immunization to measure neutralizing titers against pseudotyped viruses expressing SARS-CoV-2 B.1.617.2 (Delta) or BA.l (Omicron) spike in a pseudotyped virus neutralization assay (psVNA). B. Hamsters with pre-existing immunity were generated by vaccination with 107 viral particles (vp) of Ad26NCOV006 (Ad26 vector encoding Wuhan-Hu-1 spike) or Ad26.Empty. 6 weeks later hamsters were immunized with IO10 Ad26.COV2.S, Ad26.COV2.S.529 (both n=8) or Ad26.Empty (n=4). Serum was collected 4 weeks after this last immunization for psVNA analysis. Neutralizing antibody titers are expressed as the dilution giving a 50% reduction (N50) in the normalized luciferase readout (normalization relative to control wells without any serum added). Horizontal red bars and values per immunization group represent geometric mean (GMT) titers. Dashed horizontal lines represent the lower limit of detection (LLOD), of a 1 :20 dilution, the lowest dilution measured in the assay. Samples with no measurable titer were set at an LLOD of 20. Pairwise comparisons were performed by a t-test, Tobit Z-test or Mann-Whitney test. Significance is shown compared to the Ad26. Empty -immunized group, unless depicted otherwise. **, P<0.05; ***, P<0.001.
Detailed Description of the Invention
The term “recombinant” for a nucleic acid, protein and/or adenovirus, as used herein implicates that it has been modified by the hand of man, e.g., in case of an adenovector it may have altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e., it is not a naturally occurring wild type adenovirus.
Nucleotide sequences herein are provided from 5’ to 3’ direction, as custom in the art.
The Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. All of these genera contain pathogenic viruses that can infect a wide variety of animals, including birds, cats, dogs, cows, bats, and humans. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses that cause respiratory disease in humans are members of the genus Betacoronavirus. These include SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKUl, and the currently circulating SARS-CoV-2.
As described above, SARS-CoV-2 can cause severe respiratory disease in humans. A safe and effective SARS-CoV-2 vaccine is required to end the COVID-19 pandemic.
As used herein SARS CoV-2 refers to the SARS CoV-2 isolate that was originally identified in Wuhan (also referred to as the Wuhan-Hu-1).
A variant as used herein refers to a SARS-CoV-2 variant virus comprising one or more mutations in the SARS CoV-2 spike (S) protein, as compared to the S protein of the Wuhan-Hu-1 virus strain, including but not limited to the B.l, Bl.1.7, B.1.351, Pl, B.1.427, B.1.429 and B.l.1.529.
It is well known that viruses constantly change through mutation, and new variants of a virus are expected to occur over time. Sometimes new variants emerge and disappear. Other times, new variants emerge and persist. Multiple variants of the virus that causes CO VID-19 have already been identified globally during this pandemic. Scientists are continuously monitoring changes in the virus, including changes to the spike protein on the surface of the virus. These studies, including genetic analyses of the virus, are helping scientists understand how changes to the virus might affect how it spreads and what happens to people who are infected with it. In collaboration with a SARS-CoV-2 Interagency Group (SIG), CDC established 3 classifications for the SARS-CoV-2 variants being monitored: Variant of Interest (VOI), Variant of Concern (VOC), and Variant of High Consequence (VOHC). There are currently several VOCs identified, including:
B.1.1.7: This variant was initially detected in the UK.
B.1.351 : This variant was initially detected in South Africa in December 2020.
P.1 : This variant was initially identified in travelers from Brazil, who were tested during routine screening at an airport in Japan, in early January. B.1.427 and B.1.429: These two variants were first identified in California in
February 2021 and were classified as VOCs in March 2021.
B.1.1.529, first identified in November 2021.
At least some of these variants seem to spread more easily and quickly than other variants, which may lead to more cases of CO VID-19. An increase in the number of cases will put more strain on health care resources, lead to more hospitalizations, and potentially more deaths.
The emerging variants of SARS-CoV-2 typically have one or more mutations in the SARS CoV-2 spike (S) protein. The viral S protein binds to angiotensin-converting enzyme 2 (ACE2), which is the entry receptor utilized by SARS-CoV-2. ACE2 is a type I transmembrane metallocarboxypeptidase with homology to ACE, an enzyme long-known to be a key player in the Renin-Angiotensin system (RAS) and a target for the treatment of hypertension. The spike (S) protein of coronaviruses is a major surface protein and target for neutralizing antibodies in infected patients (Lester et al., Access Microbiology 2019; 1) and is therefore considered a potential protective antigen for vaccine design. Several emerging variants having mutations in the S protein indeed have shown decreased susceptibility to neutralization by vaccine induced immunity, most notably the B.1.351 variant, although the overall impact on vaccine efficacy remains to be determined.
The present invention provides a recombinant nucleic acid encoding a coronavirus S protein, in particular as SARS-CoV-2 S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1.
In certain embodiments, the recombinant nucleic acid encodes a SARS-CoV-2 S protein, comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 32, or 33 mutations selected from the group consisting of: a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1.
In a preferred embodiment, the nucleic acid encodes a coronavirus S protein comprising the amino acid sequence of SEQ ID NO: 2, or a fragment thereof.
It is understood by a skilled person that numerous different nucleic acids can encode the same polypeptide or protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acids, to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleotide 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 include introns.
In certain embodiments, the nucleic acid is codon-optimized for expression in human cells.
In a preferred embodiment, the nucleic acid comprises a nucleotide sequence of SEQ ID NO: 3, or a fragment thereof.
The invention further provides a recombinant coronavirus S protein, in particular a SARS-CoV-2 S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1.
In certain embodiments, the a SARS-CoV-2 S protein, comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 32, or 33 mutations selected from the group consisting of: a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1.
In a preferred embodiment, the SARS-CoV-2 S protein comprises the amino acid sequence of SEQ ID NO: 2, or a fragment thereof. The S protein may or may not comprise the signal peptide (or leader sequence). The signal peptide may comprise the amino acids 1- 13 of SEQ ID NO: 1. In certain embodiments, the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 2. In certain embodiments, the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 2 without the signal peptide.
The term “fragment” as used herein refers to a protein or (poly)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 the SARS- CoV-2 S protein, in particular the full-length sequence of a SARS-CoV-2 S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein does not need to be full length nor have all its wild type functions, and that fragments of the protein (i.e., without signal peptide, or the ectodomain (without the transmembrane and cytoplasmic regions)) 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 SARS-CoV-2 S protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the SARS-CoV-2 S protein.
The person skilled in the art will also appreciate that changes can be made to a protein, e.g., by amino acid substitutions, deletions, and/or additions, using routine molecular biology procedures. Generally, conservative amino acid substitutions may be applied without loss of function or immunogenicity of a polypeptide. The present invention further provides vector comprising a nucleic acid sequence according to the invention.
In certain embodiments of the invention, the vector is an adenovirus (or adenoviral 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). Preferably, 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). As used herein, a human adenovirus is meant if referred to as Ad without indication of species, e.g., the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.
Human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, a recombinant adenovirus according to the invention thus is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low preexisting 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., in US6083716; WO 2005/071093; WO 2010/086189; WO 2010085984). Hence, in other embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g., a chimpanzee adenovirus. In certain embodiments, 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. In certain embodiments, 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). In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see, e.g., WO 2019/086466). In certain embodiments, 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).
In a preferred embodiment of the invention, the adenoviral vectors comprise capsid proteins from rare serotypes, e.g., including Ad26. In the typical embodiment, 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. As used herein 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. In certain embodiments, the capsid protein is an entire capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber are of Ad26.
One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable properties from different serotypes can be produced.
Thus, in some embodiments, 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)
In certain preferred embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e., the vector is rAd26). In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the El region of the genome. For adenoviruses being derived from non-group C adenovirus, such as Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the El genes of Ad5, such as for example HEK-293 cells, PER.C6 cells, and the like (see, e.g., WO 03/104467). However, such adenoviruses will not be capable of replicating in non-complementing cells that do not express the El genes of Ad5.
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.
Typically, 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). Thus, 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.
As described above, the adenovirus vectors useful in the invention are preferably replication deficient. In these embodiments, 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-CoV-2 S protein, or fragment thereof (usually linked to a promoter) within the region. In some embodiments, 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. For 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.
In a preferred embodiment of the invention, the vector is 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. Typically, the nucleic acid sequence encoding the synthetic SARS CoV- 2 S antigens is cloned into the El and/or the E3 region of the adenoviral genome.
In certain embodiments, the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. This allows for the cost-effective, large-scale manufacturing of adenoviral particles comprising the SARS CoV-2 S protein insert. Without intending to be limited by theory, it is believed that the SARS CoV-2 S protein leads to lower levels of adenoviral particle production. The addition of the TetO motif to the CMV promoter allows for higher levels of adenoviral particle production.
As used herein, a “promoter” is a nucleic acid sequence enabling the initiation of the transcription of a gene sequence in a messenger RNA, such transcription being initiated with the binding of an RNA polymerase on or nearby the promoter.
As defined above, in certain embodiments, the promoter is a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif. The TetO motif can be referred to a “regulatory sequence” or “regulatory element,” which as used herein refers to a segment of nucleic acid, typically, but not limited to DNA, that modulates the transcription of the nucleic acid sequence to which it is operatively linked, and, thus, acts as a transcriptional modulator. A regulatory sequence often comprises nucleic acid sequences that are transcription binding domains that are recognized by the nucleic acid-binding domains of transcriptional proteins and/or transcription factors, enhancers, or repressors, etc. For example, it is possible to operably couple a repressor sequence to the promoter, which repressor sequence can be bound by a repressor protein that can decrease or prevent the expression of the transgene in a production cell line that expresses the repressor protein. This can improve genetic stability and/or expression levels of the nucleic acid molecule upon passaging and/or when this is produced at high quantities in the production cell line. Such systems have been described in the art. A regulatory sequence can include one or more tetracycline operator (TetO) motifs/sequences, such that expression is inhibited in the presence of the tetracycline repressor protein (TetR). In the absence of tetracycline, the TetR protein is able to bind to the TetO sites and to repress transcription of a transgene (e.g., SARS CoV-2 S antigen) operably linked to the TetO motifs/sequences. In certain embodiments, the nucleic acid encoding the SARS-CoV-2 S protein, when present in the adenoviral vector, is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif, such that the expression of the SARS CoV-2 S protein is inhibited in recombinant adenoviruses that are produced in the producer cell line in which the TetR protein is expressed. Expression will not be inhibited when the recombinant adenoviral vector is introduced into a subject or into cells that do not express the TetR protein. The invention, however, is not limited to use of a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif.
As used herein, the term “repressor” refers to molecules (e.g., proteins) having the capability to inhibit, interfere, retard, and/or repress the production of a heterologous protein product of a recombinant expression vector (e.g., an adenoviral vector). The repressor can inhibit expression by interfering with a binding site at an appropriate location along the expression vector, such as in an expression cassette (e.g., a TetR can bind the TetO motif in the CMV promoter). Repression of vector transgene expression during vector propagation can prevent transgene instability and can increase yields of vectors having the transgene during production.
A nucleic acid is “operably linked” when it is placed into a structural or functional relationship with another nucleic acid sequence. For example, one segment of DNA can be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding sequence so as to facilitate transcription of the coding sequence; a ribosome binding site that is positioned relative to a coding sequence so as to facilitate translation; or a pre-sequence or secretory leader that is positioned relative to a coding sequence so as to facilitate expression of a pre-protein (e.g., a pre-protein that participates in the secretion of the encoded polypeptide). In other examples, the operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.
In certain embodiments, the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 4, preferably the CMV promoter consists of SEQ ID NO: 4.
In certain preferred embodiments, the vector according to the invention comprises a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 3. In certain preferred embodiments, the vector according to the invention comprises a nucleic acid consisting of SEQ ID NO: 3.
The invention further provides compositions, in particular pharmaceutical compositions, comprising a nucleic acid, a protein, and/or vector according to the invention. For administering to humans, the invention may employ pharmaceutical compositions comprising the nucleic acid, a protein, and/or vector and a pharmaceutically acceptable carrier or excipient. In the present context, the term “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. Such 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. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The purified nucleic acid, a protein, and/or vector preferably is formulated and administered as a sterile solution although it is also 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, preferably in the range of pH 5.0 to 7.5. The nucleic acid, a protein, and/or vector typically is in a solution having a suitable pharmaceutically acceptable buffer, and the solution may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, nucleic acid, a protein, and/or vector may be formulated into an injectable preparation. These formulations contain effective amounts of nucleic acid, a protein, and/or vector, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
For instance, adenovirus may be stored in the buffer that is also used for the Adenovirus World Standard (Hoganson et al, Development of a stable adenoviral vector formulation, Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol. Another useful formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgC12, 25 mM NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v. Obviously, many other buffers can be used, and several examples of suitable formulations for the storage and for pharmaceutical administration of purified (adeno)virus preparations can for instance be found in European patent no. 0853660, US patent 6,225,289 and in international patent applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.
In certain embodiments, a composition according to the invention comprises a(n) (adeno) vector according to the invention in combination with a further active component. Such further active components may comprise one or more SARS-CoV-2 protein antigens, e.g., a SARS-CoV-2 protein according to the invention, or any other SARS-CoV-2 protein antigen, or additional vectors comprising nucleic acid encoding similar or alternative SARS- CoV-2 antigens. Such vectors again may be non-adenoviral or adenoviral, of which the latter can be of any serotype.
In certain embodiments, a composition according to the invention comprises at least a first vector according to the invention and at least a second vector comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 5, or a fragment thereof.
In preferred embodiments, the first and second vector comprise a recombinant human adenovirus of serotype 26.
The (pharmaceutical) compositions may or may not comprise one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant, and pharmaceutical compositions comprising adenovirus and suitable adjuvants are for instance disclosed in WO 2007/110409, incorporated by reference herein. The terms “adjuvant” and “immune stimulant” are used interchangeably and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the adenovirus vectors of the invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-emulsion 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. It is also possible to use vector-encoded adjuvant, e.g., by using heterologous nucleic acid that encodes a fusion of the oligomerization domain of C4-binding protein (C4bp) to the antigen of interest (e.g., Solabomi et al, 2008, Infect Immun. 76: 3817- 23).
In a preferred embodiment, the compositions do not comprise adjuvants.
The present invention further provides compositions for use as a vaccine against COVID-19 caused by SARS- CoV-2 Wuhan-Hu, or a variant thereof, such as the B.1.1.529 variant, comprising a nucleic acid, a protein, and/or vector according to the invention. The term “vaccine” refers to a (pharmaceutical) composition containing an active component effective to induce a therapeutic degree of immunity in a subject against a certain pathogen or disease. According to the present invention, the vaccine preferably comprises an effective amount of a recombinant adenovirus of serotype 26 that encodes a SARS CoV-2 S protein, in particular a SARS CoV-2 protein that comprises the amino acid sequence of SEQ ID NO: 2, or an antigenic fragment thereof, which results in an immune response, preferably a protective immune response, against the S protein of SARS CoV-2, or a variant thereof, such as the B 1.351 variant.
In certain embodiments, the vaccine comprises a recombinant human adenovirus of serotype 26 that comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the vaccine comprises a recombinant human adenovirus of serotype 26 that comprises a nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 3.
The “vaccine” according to the invention 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 other components that induce an immune response, such as but not limited to a second adenoviral vector encoding a different SARS-CoV-2 protein, or a SARS-CoV-2 protein as such. The vaccine compositions of the invention may be used in a method of preventing serious lower respiratory tract disease leading to hospitalization, and/or the decrease the frequency of complications such as pneumonia and bronchiolitis, and/or death due to infection with SARS-CoV-2, or a variant thereof, including, but not limited to, the B.1.1.529 variant. The vaccine may also be used in so-called Post-exposure prophylaxis (PEP), i.e., for preventing illness after potential or documented exposure to the coronavirus and/or for reducing the risk of secondary spread of infection.
The invention thus also provides a method for vaccinating a subject against COVID- 19, caused by SARS CoV-2 (Wuhan-Hu-1), or a variant thereof, said method comprising administering to the subject a vaccine as described herein.
In certain embodiments, the vaccine is administered to a naive (or seronegatieve) subject, preferably a subject that has no circulating antibodies against SARS-CoV-2 or a variant thereof. Typically, the subject has not been vaccinated against COVID-19 and has not been infected with SARS CoV-2 virus (Wuhan-Hu-1), or a variant thereof, prior to the administration of the vaccine.
In certain embodiments, the vaccine is administered to a subject that has been vaccinated at least once against COVID-19 prior to administration of the vaccine. The subject may have been vaccinated using any available vaccine, including, but not limited to, mRNA vaccines such as BNT162b2 and mRNA- 1273, vector-based vaccines, such as AZDI 222 and Ad26.COV2.S, or protein vaccines, such as NVX-CoV2373. In a preferred embodiment, the subject was vaccinated using a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding a SARS-CoV-2 S protein that comprises the amino acid sequence of SEQ ID NO: 5, or a fragment thereof (also referred to as Ad26.COV2.S). Preferably, the vaccine according to the present invention is administered to the subject between 6 and 12 months after the previous vaccination. The total dose of the adenovirus provided to a subject preferably is between IxlO8 vp and 2xlOn vp, for instance between 3xl08 and IxlO11 vp per administration.
In a preferred embodiment, the total dose of the adenovirus provided to the subject ranges from 1 x IO10 vp to 1 x 1011 vp per dose. Preferably, the adenovirus is administered at a total dose of 5 x IO10 vp per administration.
In certain embodiments, the vaccine comprises a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding the SARS CoV-2 S protein of SEQ ID NO: 2 at a dose of 2.5 x IO10 vp and a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding a SARS-CoV-2 S protein that comprises the amino acid sequence of SEQ ID NO: 5 at a dose of 2.5 x IO10 vp. When administered the total dose of adenovirus per administration of the vaccine thus is 5 x IO10 vp. Administration of adenovirus compositions can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as by injection, e.g., intramuscular, intradermal, subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. It is particularly preferred according to the present invention to administer the vaccine intramuscularly, such as into the deltoid muscle of the arm, or vastus lateralis muscle of the thigh.
Preferably, the subject is a human subject. The subject can be of any age, e.g., from about 1 month to 100 years old, e.g., from about 2 months to about 80 years old, e.g., from about 1 month to about 3 years old, from about 3 years to about 50 years old, from about 50 years to about 75 years old, etc. In certain embodiments, the subject is a human from 2 years of age, preferably a human from 12 years of age, more preferably a human from 18 years of age.
In certain embodiments, the vaccine is administered to the subject more than once, e.g., once a year. In certain embodiments, the method of vaccination consists of a single administration of the composition or vaccine to the subject. It is also possible to provide one or more booster administrations of the vaccine of the invention. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject at a moment between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases sometimes referred to as ‘priming vaccination’). In certain embodiments, the vaccine is administered every two, three, four or five years.
The invention further provides a method for inducing binding antibodies to the S protein of a of SARS-CoV-2 variant, including but not limited to the B.1.1.529 variant, and to the SARS CoV-2 Wuhan-Hu-1 S protein, in a subject in need thereof, as measured e.g., by ELISA, comprising administering to the subject a vaccine as described herein. Preferably, the amount (titer) of binding antibodies against SARS-CoV-2 Wuhan-Hu-1 is non-inferior to the amount of binding antibodies against the variant. In certain embodiments, the amount (titer) of binding antibodies against SARS-CoV-2 Wuhan-Hui, as measured by ELISA, is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amount of binding antibodies against the variant, e.g., the B.1.1.529 variant.
The invention also provides a method for inducing antibodies capable of neutralizing a SARS-CoV-2 variant, including but not limited to the B.1.1.529 variant, and SARS CoV-2 Wuhan-Hu 1, in a subject in need thereof, as measured, e.g., by wtVNA or psVNA, comprising administering to the subject a vaccine as described herein. Preferably, the neutralizing antibody response against SARS CoV-2 Wuhan-Hu-1 is non-inferior to the neutralizing antibody response against the variant, such as B.1.1.529. In certain embodiments, the neutralizing antibody response to Wuhan-Hu-1 is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the neutralizing antibody response against the variant. According to the present invention, non-inferiority (NI) of the vaccine of the current invention means that it is not inferior to an existing one (such as Ad26.CoV2.S), i.e., that it is either equally effective or better (e.g., with a NI margin of 0.67).
In certain embodiments, the vaccine of the current invention is not inferior to an existing one (such as Ad26.CoV2.S), i.e., is either equally effective or better, with a NI margin of 0.67, in their respective matched virus neutralization assays.
The invention also provides a method for inducing a specific T cell response against a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, and against SARS CoV-2 Wuhan-Hu-1, in a subject in need thereof, as assessed, e.g., by flow cytometry after SARS-CoV2 S protein peptide stimulation of peripheral blood mononuclear cells (PBMCs) and intracellular staining, comprising administering to the subject a vaccine as described herein. Preferably, the T cell response against SARS-CoV-2 is similar (non-inferior) to the T cell response against the variant. In certain embodiments, the T cell response to SARS-CoV-2 is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cell response to the variant.
The invention also provides a method for reducing infection and/or replication of a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant and of SARS CoV- 2 Wuhan-Hu-1, in, e.g., the nasal tract and lungs of, a subject, comprising administering to the subject a vaccine as described herein. This will reduce adverse effects resulting from infection by SARS-CoV2 (Wuhan-Hu-1), or a variant thereof, in a subject, and thus contribute to protection of the subject against such adverse effects. In certain embodiments, adverse effects of infection may be essentially prevented, i.e., reduced to such low levels that they are not clinically relevant.
The invention also provides a method for prevention of molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, comprising administering to the subject a vaccine as described herein,
The invention also provides a method for prevention of molecularly confirmed, moderate to severe/critical COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, comprising administering to the subject a vaccine as described herein.
The invention also provides a method for preventing or reducing the occurrence of pneumonia linked to any molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, when compared to a placebo or a different COVID-19 vaccine, such as, but not limited to Ad26.COV2.S.
The invention also provides a method for preventing or reducing the occurrence of hospitalization linked to any molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, when compared to a placebo or a different vaccine, such as, but not limited to Ad26.COV2.S.
The invention also provides a method for preventing or decreasing death linked to molecularly confirmed COVID-19, caused by a SARS-CoV-2 variant, including, but not limited to, the B.1.1.529 variant, or SARS-CoV-2 Wuhan-Hu-1, when compared to placebo, or a different vaccine.
In certain embodiments the effects of the vaccine occur between 14 and 28 days after vaccination.
The invention further provides an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising a nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof comprising the nucleotide sequence of SEQ ID NO: 3. The invention further provides methods for making a vaccine COVID-19, comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-COV-2 S protein or fragment thereof, as described herein, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition.
Also provided herein are methods of producing an adenoviral particle comprising a SARS-Co-V-2 S protein as described herein. The methods comprise (a) contacting a host cell of the invention with an adenoviral vector of the invention and (b) growing the host cell under conditions wherein the adenoviral particle comprising the SARS-CoV-2 antigen is propagated. Recombinant adenovirus can be prepared and propagated in host cells, according to well-known methods, which entail cell culture of the host cells that are infected with the adenovirus. 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. Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable (see, e.g., WO 2010/060719, and WO 2011/098592, both incorporated by reference herein, which describe suitable methods for obtaining and purifying large amounts of recombinant adenoviruses).
A host cell (sometimes also referred to in the art and herein as “packaging cell” or “complementing cell” or “producer cell”) that can be used can be any host cell wherein a desired adenovirus can be propagated. A host cell line is typically used to produce sufficient amounts of adenovirus vectors of the invention. A host cell is a cell that comprises those genes that have been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, PER.C6®, 911, 293, and
El A549.
In certain embodiments, the host cell further comprises a nucleotide sequence encoding a tetracycline repressor (TetR) protein. The nucleotide sequence encoding the TetR protein can, for example, be integrated in the genome of the host cell. By way of an example, the nucleotide sequence encoding the TetR protein can be integrated in chromosome 1. The host cell line can, for example, be a PER.C6® cell.
The invention further provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV2 S protein or fragment thereof.
The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.
EXAMPLES
Example 1. Viral Sources and Recombinant Vector Generation - DNA Preparation
After generation of the severe acute respiratory syndrome coronavirus 2 Spike (SARS-CoV-2 S) gene, encoding the SARS-CoV-2 S protein of SEQ ID NO: 2, the gene was cloned in house in the expression cassette under transcriptional control of a human cytomegalovirus (CMV.TetO) promoter and the SV-40 polyA sequence. The plasmid DNA was subjected to a DNA cleaning process and DNA sequence analysis prior to Ad26 vector generation.
The recombinant Ad26 vector, Ad26.COV2.S.529, is replication-incompetent due to deletions in El (A E1A/E1B). The El deletion renders the vector replication-incompetent in non-complementing cells such as normal human cells. In Ad5 El complementing cell lines like HEK293, PER.C6, PER.C6 TetR and HER96 cells the virus can be propagated. In addition, the E3 gene has been removed (AE3) to create sufficient space in the viral genome for insertion of foreign antigens, and the Ad26 E4 orf6 has been exchanged by the Ad5 homologue to allow efficient production of replication-incompetent Ad26 vectors in Ad5 El complementing cell lines.
A single genome plasmid is used to generate the Ad26 vector on PER.C6 TetR cells (Research Cell Bank II (RCB II). In order to perform a plaque purification these suspension cells were cultured in DMEM without geneticin, supplemented with 10% FBS (y-irradiated, complying with EMA/CHMP/BWP/457920/2012 rev 1) in PLL coated plates. Cells were transfected with the linearized plasmid using the agent Lipofectamine 2000CD™. Single plaques were isolated by 1 round of plaque purification on monolayers of PER.C6 TetR cells covered with an agarose overlay (sea plaque agarose). Plaques were amplified on PER.C6 TetR cells grown in DMEM supplemented with 10% y-irradiated FBS. The final steps were performed in suspension cultures. Multiple plaques were tested for integrity and identity of the adenovirus genome and correct expression of the antigen and one plaque was selected for manufacturing.
Virus seed stocks, derived from a single plaque, are used to infect PER.C6 TetR cells (Research Cell Bank II (RCB II) cultivated in DMEM without geneticin, supplemented with 10% FBS (y-irradiated, complying with EMA/CHMP/BWP/457920/2012 rev 1) in order to manufacture the Ad26.COV2.S.529 pre-master virus seed (preMVS). Cell material is harvested by centrifugation and used for purification of the recombinant adenovirus. Purification is performed using 2 successive rounds of cesium chloride (CsCl) density centrifugation. Dialysis is performed to remove excess CsCl and to prepare the suspension in the required formulation buffer. The combined dilution factor associated with plasmid and Ad26 vector purification (i.e., single clone selection, 1 round of plaque purification and 2 ultracentrifugation purification steps) is calculated to be at least 1027. The purified virus suspension is tested for quantity, infectivity, identity, and adventitious agents.
DS batches are produced from the Master Virus Seed (MVS). All raw materials are chemically defined and of non-animal (derived) or non-human origin.
Example 2. A Randomized, Double-blind, Phase 2 Study to Evaluate the Immunogenicity,
Reactogenicity and Safety of Ad26.COV2.S.529 Administered as Booster Vaccination in
Adults 18 Years of Age and Older Who Have Previously Received Primary or Booster
Vaccination with Ad26.COV2.S or Primary and Booster Vaccination with BNT162b2
Ad26.COV2.S.529 (also known as VAC31518, JNJ-87918883) is a monovalent vaccine composed of a recombinant, replication-incompetent adenovirus type 26 (Ad26) vector, constructed to encode the Spike (S) protein derived from a SARS-CoV-2 clinical isolate (B.1.1.529 lineage, Omicron variant), with an inactivated furin cleavage site between the SI and S2 protein subunits and substitution of 2 prolines to enhance prefusion conformation.
Immunogenicity of the SARS-CoV-2 Omicron Spike (S) encoding vaccine Ad26.COV2.S.529 will be confirmed in small animal models prior to dosing in humans.
Mice will be used to compare immunogenicity of Ad26.COV2.S.529 with that of the current vaccine Ad26.COV2.S by an Omicron virus neutralization assay (VNA). To assess the immune responses induced by these vaccines in the context of pre-existing immunity against the parental SARS-CoV-2 strain, Syrian hamsters that have been pre-immunized with a low dose of Ad26 encoding wild-type S from the parental Wuhan virus strain will receive booster doses of Ad26.COV2.S.529 or Ad26.COV2.S. The resulting immune responses will be analyzed by Omicron VNA and by VNA assessing neutralizing antibody titers directed against the parental strain. The Pfizer-BioNTech COVID-19 vaccine, BNT162b2, is a lipid nanoparticle- formulated, nucleoside-modified RNA vaccine encoding a prefusion stabilized, membrane- anchored SARS CoV-2 full-length spike protein.
The currently authorized Ad26.COV2.S vaccine directed against the original SARS- CoV-2 strain is associated with demonstrated clinical protective efficacy against COVID-19. This study will assess the reactogenicity, safety, and immunogenicity of a booster dose of Ad26.COV2.S or Ad26.COV2.S.529 in adults >18 years of age, who have previously received primary vaccination with Ad26.COV2.S, booster vaccination with Ad26.COV2.S or booster vaccination with Pfizer mRNA-based vaccine BNT162b2.
The purpose of the study is to demonstrate that the humoral neutralizing immune responses elicited by a booster vaccination with Ad26.COV2.S.529 against the Omicron variant, given after a single dose of Ad26.COV2.S (Cohort 1), is non-inferior (NI) to the responses elicited by primary vaccination with the initially authorized vaccine (Ad26.COV2.S) against the original strain (Primary Objective la), which is associated with demonstrated clinical protective efficacy against COVID-19 . In a similar manner, the NI of neutralizing antibody responses to the Omicron variant induced by Ad26.COV2.S.529 when given after a booster (2nd injection) of Ad26.COV2.S (Cohort 2) compared to neutralizing responses elicited by primary vaccination with the initially authorized vaccine (Ad26.COV2.S) against the original strain will be demonstrated (Primary Objective lb). A heterologous regimen will also be tested (Cohort 3), where the NI of neutralizing antibody responses against the Omicron variant induced by Ad26.COV2.S.529 in participants who received a 2-dose Pfizer BNT162b2 primary series and Pfizer BNT162b2 booster, compared to neutralizing antibody responses against the standard strain induced by the 2-dose primary regimen with Pfizer BNT162b2 (Primary Objective 1c). The superiority of neutralizing antibody responses against the Omicron variant induced by the final booster with Ad26.COV2.S.529 vs those induced by the final booster with Ad26.COV2.S when each is given after a primary series with Ad26.COV2.S (Cohort 1, Primary Objective Id) when given after a primary series + boost of Ad26.COV2.S (Cohort 2 Objective le) and when given after a primary series and boost with Pfizer BNT162b2 (Cohort 3, Primary Objective le ). The objectives la, lb, 1c will examine the NI of the neutralizing antibody levels against Omicron induced by a booster with Ad26.COV2.S.529 compared to those responses by primary regimens against the standard strain at neutralizing antibody levels and seroresponse rates where protection has been shown. It will also show the superiority of a final boost with Ad26.COV2.S.529 vs Ad26.COV2.S when given after a primary series, and a primary series followed by a boost, which will reflect the majority of individuals that have received COVID-19 vaccines.
Reactogenicity data will be collected for 28 days following COV2015 study booster vaccination. Safety data (SAEs/AESIs/MAAEs) will be collected for 360 days after CO V2015 study booster vaccination.
Objectives and Endpoints (Protocol Section 3)
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
* As defined by the Centers for Disease Control and Prevention (CDC Aug 2021 c) at the time of the analysis a Responders are defined as 4-fold rise from pre-vaccination titers or 4 fold rise above the lower limit of quantitation if pre-vaccination titer is below the level of quantification. Further information on responder definitions will be described in the Statistical Analysis Plan. b Alpha is determined using an hierarchical testing approach as outlined in the Statistical Considerations.
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000056_0002
Blood Collection Schedule (Whole Blood) for Humoral Immunogenicity Assessments
Figure imgf000057_0001
a. Selected timepoints include samples for PAXgene analysis
Blood Collection Schedule (PBMC) for Cellular Immunogenicity Assessments
Cohort Group Day 1 Day 29 Day71 Day 181 Day 361
Subset 2 Subset 2 Subset 2 Subset 2 Subset 2
1
N = 25 N = 25 N = 25 N = 25 N = 25
1
Subset 2 Subset 2 Subset 2 Subset 2 Subset 2
2
N = 75 N = 75 N = 75 N = 75 N = 75
Subset 2 Subset 2 Subset 2 Subset 2 Subset 2
3 N = 25 N = 25 N = 25 N = 250 N = 25
2
Subset 2 Subset 2 Subset 2 Subset 2 Subset 2
4
N = 75 N = 75 N = 75 N = 75 N = 75
Subset 2 Subset 2 Subset 2 Subset 2 Subset 2
5
3 N = 25 N = 25 N = 25 N = 25 N = 25
6 Subset 2 Subset 2 Subset 2 Subset 2 Subset 2
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Cohort 1 : Participant only received Ad26.COV2.S (5x 1010 vp) primary vaccination (ie, single dose).
Cohort 2: Participant received Ad26.COV2.S (5x 1010 vp) primary vaccination followed by an initial boost with Ad26.COV2.S (5x 1010 vp) which occurred >2 months57 days after primary vaccination.
Cohort 3: Participant completed primary vaccination with a 2-dose regimen of BNT162b2 vaccine (Pfizer) and a booster dose of BNT162b2 which occurred >4 months after primary vaccination.
Example 3. Immunogenicity of SARS-CoV-2 Omicron vaccine in naive mice and SARS-CoV- 2 spike immune hamsters
In response to the SARS-CoV-2 (COVID-19) pandemic, multiple mostly spike based vaccines were rapidly and successfully developed. These authorized vaccines showed high efficacy against COVID-19 and have been deployed worldwide. The Ad26.COV2.S vaccine is a replication-incompetent human adenovirus type 26 (Ad26) vector encoding a stabilized pre-fusion SARS-CoV-2 spike protein based on the Wuhan-Hu-1 isolate. A phase 3 trial demonstrated that Ad26.COV2.S was 73% efficacious at preventing severe-critical COVID- 192 The Ad26.COV2.S COVID-19 vaccine was granted emergency use authorization in US and (conditional) marketing authorization in the European Union and in more than 50 other countries.
Several rapidly spreading SARS-CoV-2 variants have evolved since the initial introduction of the virus into humans. The Beta (B.1.351) and Delta (B.1.617.2) variants of concern (VOC) were initially thought to potentially evade vaccine elicited immunity, however SARS-CoV-2 vaccine efficacy was largely maintained. The Delta VOC obtained virtually worldwide dominance until it was in turn replaced by the Omicron variant, BA.1 (formerly named B.1.1.529) after its first reporting in November 2021 and instantly declared a VOC due to its rapid spread and the unparalleled number of mutations in the spike protein. BA.1 carries 15 mutations in its receptor binding domain (RBD), which is an immunodominant target for neutralizing antibodies, raising the possibility of reduced effectiveness of both vaccines and (therapeutic) monoclonal antibodies targeting this region.
An Omicron (BA.l) based vaccine candidate using the Ad26 vaccine platform (Ad26.COV2.S.529) was generated to assess whether the immunogenicity against the Omicron variant could be further improved. Here, it is shown that Ad26.COV2.S.529 induces robust Omicron neutralizing antibody titers in naive mice and in hamsters with pre-existing SARS-CoV-2 spike protein immunity.
Materials and methods
Vaccines and challenge stocks
Replication-incompetent El/E3-deleted adenovirus serotype 26 (Ad26) vector-based vaccines were generated as described previously using the AdVac system (Bos et al. NPJ Vaccines 5, 91 (2020)). Ad26NCOV006 and Ad26.COV2.S encode a SARS-COV-2 spike protein sequence based on SARS-CoV-2 Wuhan-Hu-1 spike (GenBank accession number MN908947), while Ad26.COV2.S.529 encode a SARS-COV-2 spike protein sequence based on SARS-CoV-2 Omicron; BA. l spike (GISAID accession number EPI ISL 6913991). Spike protein encoded by Ad26.COV2.S and Ad26.COV2.S.529 was stabilized in the prefusion conformation by R682S and R685G that abolish the furin cleavage site and by the proline substitutions K986P and V987P. The negative control vector Ad26.Empty, which does not contain a transgene, was used a control. Cell-based ELISA
A549 cells were seeded at 2.9 * 104 cells/well in Dulbecco's Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) in a flat-bottomed 96-well microtiter plate (Corning). The plate was incubated overnight at 37 °C in 10% CO2. After 24 h, cells were transduced with Ad26.COV2.S or Ad26.COV2.S.529 at a dose of 2000 infectious units [IU]/cell and the plate was incubated for 48 h at 37 °C in 5% CO2. Two days post transduction, cells were washed four times with PBS and subsequently fixed with 4% formaldehyde in PBS. After a 20-minute incubation at room temperature (RT), wells were washed four times with 0.05% Tween-20 in PBS. Cells were permeabilized by incubation with 1% Elugent (Merck) for 15-20 minutes, after which the plates were washed four times with 0.05% Tween-20 in PBS. Next, Casein blocking buffer (Thermo Scientific) was added per well and the plate was incubated for 60 to 90 minutes at 37 °C in 10% CO2. Plates were washed four times with 0.05% Tween-20 in PBS, after which two-fold diluted CV3-25 antibody or ACE2-Fc fusion protein (0.007 to 15 pg/ml) was added per well. CV3-25 was produced at ImmunoPreci se according to Jennewein et al.21 and ACE2-Fc was made according to Liu et al22. After 30 to 60 minutes of incubation, cells were washed four times with 0.05% Tween-20 in PBS. Next, cells were incubated with mouse HRP-conjugated antihuman IgG Fc (Jackson, 1 : 8000) for 40 min at RT. The plate was washed four times with 0.05% Tween-20 in PBS. 3,3',5,5'-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma) was added and after 20 minutes the reaction was terminated by adding Stop Reagent for TMB Substrate (Sigma). Signal was measured at 450 nm (signal) and at 630nm (for background subtraction) using a Biotek Synergy Neo reader. Data was analyzed using GraphPad Prism 9, using the “Specific binding with Hill slope” module to calculate the antibody binding affinity. Animal studies
Animal experiments were approved by the Central Authority for Scientific Procedures on Animals (Centrale Commissie Dierproeven) and conducted in accordance with the European guidelines (EU directive on animal testing 86/609ZEEC) and local Dutch legislation on animal experiments.
Female Syrian golden hamsters (Mesocricetus auratus), strain RjHan:aura, aged 9-11 weeks at the start of the study, were purchased from Janvier Labs, France. Hamsters were immunized via the intramuscular route with 100 pl (50 pl per hindleg) vaccine under general anesthesia with isoflurane. Blood samples were collected via the retro-orbital route under anesthesia as described above.
BALB/c mice aged 8-10 weeks at the start of study were provided by Charles River Laboratories, Germany. Mice were immunized via the intramuscular route with 100 pl (50 pl per hindleg) vaccine under general anesthesia with isoflurane. Blood samples were collected via the submandibular bleeding route. Blood from all animal experiments was processed for serum isolation.
Recombinant lentivirus-based
Figure imgf000064_0001
virus neutralization assay
Figure imgf000064_0002
Neutralizing antibody titers were measured against several SARS-CoV-2 spike variants by psVNA. Human Immunodeficiency Virus (HlV)-based lentiviruses, pseudotyped with SARS-CoV-2 spike protein (based on Wuhan-Hu- 1; GenBank accession number MN908947) were generated as described previously (Solforosi et al., J. Exp. Med. 218, e20202756 (2021); Jongeneelen et al., 2021.07.01.450707 https://www.biorxiv.Org/content/10. l 101/2021.07.01.450707vl (2021) doi: 10.1101/2021.07.01.450707). Substitutions and deletions in the spike protein open reading frame for the variant B.1.617.2 and BA.1 (GISAID accession number
EPI ISL 6913991 were introduced using standard molecular biology techniques and confirmed by sequencing.
Assays were performed on Hek293T target cells stably expressing the human angiotensinconverting enzyme 2 (ACE2) and human transmembrane serine protease 2 (TMPRSS2) genes (VectorBuilder, Cat. CL0015). The cells were seeded in white half-density area 96- well tissue culture plates (Perkin Elmer) at a density of 1.5E+04 cells/well.
Two-fold serial dilutions were prepared from heat-inactivated serum samples serum samples in phenol red free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% FBS and 1% PenStrep. The serially diluted serum samples were incubated at room temperature with an equal volume of pseudovirus particles with titers of approximately 1E+05 Relative Luminescence Units (RLU) luciferase activity. After one hour incubation, the serum-particle mixture was inoculated onto Hek293T.ACE2.TMPRSS2 cells. Luciferase activity was measured 40h after transduction by adding an equal volume of NeoLite substrate (Perkin Elmer) to the wells according to the manufacturer’s protocol, followed by read out of RLU on the EnSight Multimode Plate Reader (Perkin Elmer). SARS-CoV-2 neutralizing titers were calculated in R using a four-parameter curve fit as the sample dilution at which a 50% reduction (N50) of luciferase readout was observed compared with luciferase readout in the absence of serum (High Control). The starting serum sample dilution of 20 was fixed as the limit of detection (LLOD).
Statistical analysis
Statistical comparisons were performed in SAS 9.4 using a paired-sample t-test from an ANOVA. If titers were censored at LLOD, then a Tobit z-test from a Tobit ANOVA was used instead. If a vaccine-dose had more than 50% censored measurements, the non- parametric Mann-Whitney U-test was used instead. No adjustments for multiple comparisons were done.
Results
In-vitro characterization of spike expression by Ad26.COV2.S.529
Ad26.COV2.S.529 vector spike expression and antigenicity were characterized in vitro and compared to Ad26.COV2.S. Spike protein expression was evaluated after transduction of A549 cells using a quantitative cell-based ELISA with CV3-25 and ACE2-Fc. CV3-25 is an antibody that binds to the stem region of the SARS-CoV-2 spike S221, a region which is conserved between the Wuhan-1 -Hu and BA.l spike protein. Here, we show that CV3-25-binding to the spike protein expressed after transduction of A549 cells with Ad26.COV2.S or Ad26.COV2.S.529 was comparable (Figure 1A). Similarly, ACE2-Fc fusion protein binding to both spike proteins was comparable despite the fact that the ACE2 binding sites in the spike RBD region are not conserved between the spike proteins expressed by Ad26.COV2.S and Ad26.COV2.S.529 (Figure IB).
Ad26.COV2.S.529 induces Omicron neutralizing antibodies in naive mice and hamsters with pre-existing immunity.
Naive mice were immunized with 108, 109, IO10 vp of Ad26.COV2.S, Ad26.COV2.S.529 or IO10 vp Ad26.Empty mock control vector. SARS-CoV-2 Delta (B.1.617.2) and Omicron (BA. l) spike neutralization titers were evaluated using a psVNA assay in sera collected 4 weeks after immunization. A single immunization with Ad26.COV2.S.529 induced dose-dependent Omicron spike neutralizing antibodies that were significantly higher than after vaccination with Ad26.COV2.S at all doses tested (Figure 2A). In contrast, Omicron spike neutralizing titers 4 weeks after vaccination with Ad26.COV2.S were comparable to animals vaccinated with Ad26.Empty. While Ad26.COV2.S induced robust dose-dependent Delta spike neutralizing antibody titers, Delta titers in animals vaccinated with 108 and 109 vp Ad26.COV2.S.529 were in the same range as after vaccination with Ad26. Empty (Figure 2A). Only the highest dose of Ad26.COV2.S.529 tested (IO10 vp) induced statistically significantly Delta spike neutralization titers compared with Ad26.Empty, albeit at lower levels compared to 1010 vp Ad26.COV2.S.
As an increasing part of the population acquired pre-existing immunity either by infection or vaccination, we also evaluated the immunogenicity of Ad26.COV2.S.529 in hamsters with pre-existing immunity to a Wuhan-Hu-1 SARS-CoV-2 spike protein. Hamsters were first immunized with 107 vp Ad26NCOV006 , which encodes the ancestral SARS-CoV- 2 Wuhan-Hu- 1 spike protein. The mock control group was immunized with 107 vp of Ad26. empty. Six weeks later, the hamsters received a vaccination with 1010 vp of Ad26.COV2.S, Ad26.COV2.S.529 or Ad26.Empty. SARS-CoV-2 Delta and Omicron spike neutralization titers were evaluated using a psVNA assay in sera collected 4 weeks after vaccination. Vaccination of pre-immune hamsters with Ad26.COV2.S and Ad26.COV2.S.529 resulted in comparable Delta spike neutralizing antibody titers that were significantly higher than in control animals (Figure 2B). While Omicron spike neutralizing antibody titers were undetectable in 7 out of 8 Ad26.COV2.S-vaccinated hamsters, vaccination with Ad26.COV2.S.529 induced robust Omicron spike neutralizing antibodies.
Conclusion
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant sparked concern due to its fast spread and the unprecedented number of mutations in the spike protein that enables it to partially evade spike-based COVID-19 vaccine-induced humoral immunity. In anticipation of a potential need for an Omicron spike-based vaccine, a Ad26 vector encoding an Omicron (BA. l) spike protein was generated (Ad26.COV2.S.529). Ad26.COV2.S.529 is similarly prefusion stabilized as the current COVID-19 vaccine Ad26.COV2.S encoding Wuhan-Hu-1 spike and it was verified that spike expression was comparable to Ad26.COV2.S. Immunogenicity of Ad26.COV2.S.529 was then evaluated in naive mice and SARS-CoV-2 Wuhan-Hu-1 spike pre-immunized hamsters.
Ad26.COV2.S.529 elicited robust neutralizing antibodies against SARS-CoV-2 Omicron (BA.1) but not to SARS-CoV-2 Delta in naive mice while the opposite was observed for Ad26.COV2.S. In pre-immune hamsters, Ad26.COV2.S.529 vaccination resulted in robust increases in neutralizing antibody titers against both SARS-CoV-2 Omicron (BA.l) and Delta, while Ad26.COV2.S vaccination only increased neutralizing antibody titers against the Delta variant. Our data imply that Ad26.COV2.S.529
Sequences full length S protein (underline
Figure imgf000069_0001
double underline TM and
Figure imgf000069_0002
domain)
Figure imgf000069_0003
FSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS
LLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS
QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF S ALEPL VDLP
IGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA
VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRF
ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRG
DEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN
LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTD
AVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPT
WRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVAS
QSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC
SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILP
DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLT
DEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI
ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDIL
SRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSK
RVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGV
FVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL
DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYI KWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG
VKLHYT
SEQ ID NO: 2 Ad26.COV2.S.529 (Omicron variant) transgene protein sequence
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPF
FSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLI
VNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD
LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGF S ALEPL VDLPIGINIT
RFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCA
LDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVY
AWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR
QIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFE
RDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPA
TVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRD PQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVY
STGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHSRAGSVASQSIIAY TMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL
QYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKP SKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPPLLTDEMI
AQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQ FNS AIGKIQDSLS ST ASALGKLQD VVNHNAQALNTLVKQLS SKFGAIS S VLNDIF SRL DPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDF
CGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSN GTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYF
KNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW YIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLH
YT**
SEQ ID NO: 3 Ad26.COV2.S.529 (Omicron variant) transgene nucleotide sequence
ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAATGCGTGAACCTGA
CCACAAGAACCCAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAGGCGTGT
ACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTT
CCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGTGATCTCCGGCACCAATGGC
ACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCA
GCATCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACA
GCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAG
TGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACCACAAGAACAACAAGA
GCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTTG
AATACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCA
AGAACCTGCGCGAGTTCGTGTTCAAGAACATCGACGGCTACTTCAAGATCTACA
GCAAGCACACCCCTATCATCGTCAGAGAGCCCGAGGATCTGCCTCAGGGCTTCTC
TGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAG
ACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGA
TGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTTC
TGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGG
ATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCA
TCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCC
CAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCC
TCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCC
GTGCTGTACAACCTGGCCCCCTTCTTCACCTTCAAGTGCTACGGCGTGTCCCCTAC CAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGG
GGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACTGGCAACATCGCCGACTAC
AACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAAC
AAGCTGGACTCCAAAGTCAGCGGCAACTACAATTACCTGTACCGGCTGTTCCGG
AAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCC
GGCAACAAGCCTTGTAACGGCGTGGCCGGCTTCAACTGCTACTTCCCACTGAGAT
CCTACAGCTTTAGACCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGG
TGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAG
CACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGAAGGG
CACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGG
CCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAAT
CCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACC
AACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTG
CCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCG
GCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAATACGTGA
ACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGCATCTGTGCCAGCTACCA
GACACAGACAAAGAGCCACAGCAGAGCCGGATCTGTGGCCAGCCAGAGCATCAT
TGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCT
ATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGT
CCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCG
AGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAGAGAG
CCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCC
AAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTT
CAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGA
CCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGC GATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAAGG
GACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATC
TGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCTGGCGCCGC
TCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTG
ACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGC
GCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAA
GCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCA
GCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCTTCAGCAGA
CTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACCGGAAGGCTG
CAGTCCCTGCAGACCTACGTTACCCAGCAGCTGATCAGAGCCGCCGAGATTAGA
GCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCA
AGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGC
CCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAAT
TTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAG
GCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGA
GCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTG
ATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCT
TCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACC
TGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCG
ACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAG
AACTGGGAAAATACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCT
TTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGAC
CAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTC
GACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGA
TAA CMVdell34 including 2x TO; SEO ID NO: 4
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTG
ATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT
TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCA
ACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGG
TAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAG
AGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAG
ATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACC
GATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAA
>COR200007 SEO ID NO: 5
Figure imgf000074_0001
FFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQ
SLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS
QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF S ALEPL VDLP
IGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDA
VDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRF
ASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRG DEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSN
LKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTD
AVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPT
WRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPSRAGSVAS
QSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTEC
SNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILP
DPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLT
DEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLI
ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDIL
SRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKR
VDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF
VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELD
KYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK
WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGV KLHYT
>COR200007 SEQ ID NO: 6
ATGTTCGTGTTTCTGGTACTGCTCCCCCTCGTCTCCAGTCAATGCGTGAACCTGAC
CACAAGAACCCAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAGGCGTGTA
CTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTTC
CTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCA
ATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTT
TGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACT
GGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCAT CAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTACTATCAC
AAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAA
CAACTGCACCTTTGAATACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAG
CAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTCAAGAACATCGACGGCTAC
TTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAGG
GCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCG
GTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAG
CAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAG
AACCTTTCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTG
TGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAA
AAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTG
CGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCA
GATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCG
ACTACTCCGTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGT
GTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTC
GTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACTGGCAAGATC
GCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGA
ACAGCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGC
TGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTA
TCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCA
CTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAG
TGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAA
GAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCT
GACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCA
GTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACT GGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCT
GGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGACGTGAACTGTACC
GAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACT
CCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGC
ACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGCATCTGTGCCA
GCTACCAGACACAGACAAACAGCCCCAGCAGAGCCGGATCTGTGGCCAGCCAGA
GCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAA
CAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTG
CCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATT
CCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAA
TAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTT
CGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGGACTTCGGCGGCTTC
AATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCG
AGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGT
ATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTT
TAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTAC
ACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCTGGC
GCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCG
GAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCA
ACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGG
GAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAGGCACTGAACACCCTGGTCA
AGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAG
CAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACCGGAAG
GCTGCAGTCCCTGCAGACCTACGTTACCCAGCAGCTGATCAGAGCCGCCGAGATT
AGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAG AGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAG
TCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGA
AGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAG
AGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTC TACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACG
TCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGA
CAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGT
GGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGA
GATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCT GCAAGAACTGGGAAAATACGAGCAGTACATCAAGTGGCCTTGGTACATCTGGCT
GGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGC
ATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCA
AGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACA
CA

Claims

Claims A recombinant nucleic acid encoding a coronavirus S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid
78 residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1. The nucleic acid according to claim 1, wherein the coronavirus S protein comprises an amino acid sequence of SEQ ID NO: 2, or a fragment thereof. The nucleic acid according to claim 1 or 2, which is codon optimized for expression in human cells. The nucleic acid according to claim 1, 2 or 3, comprising a nucleotide sequence of SEQ ID NO: 3, or a fragment thereof. A recombinant coronavirus S protein, or a fragment thereof, said S protein comprising an amino acid sequence comprising a mutation of the amino acid residue A at position 67 into V, a deletion of the amino acid residues at position 69 and 70, a mutation of the amino acid residue G at position 95 into I, a mutation of the amino acid residue G at position 142 into D, a deletion of the amino acid residues 143-145, a deletion of the amino acid residue at position 211, a mutation of the amino acid residue L at position 212 into I, an insertion of the amino acid sequence EPE at position 214, a mutation of the amino acid residue G at position 339 into D, a mutation of the amino acid residue S at position 371 into L, a mutation of the amino acid residue S at position 373 into P, a mutation of the amino acid residue S at position 375 into F, a mutation of the amino acid residue K at position 417 into N, a mutation of the amino acid residue N at position 440 into K, a mutation of the amino acid residue G at position 446 into S, a mutation of the
79 amino acid residue S at position 477 into N, a mutation of the amino acid residue T at position 478 into K, a mutation of the amino acid residue E at position 484 into A, a mutation of the amino acid residue Q at position 493 into R, a mutation of the amino acid residue G at position 496 into S, a mutation of the amino acid residue Q at position 498 into R, a mutation of the amino acid residue N at position 501 into Y, a mutation of the amino acid residue Y at position 505 into H, a mutation of the amino acid residue T at position 547 into K, a mutation of the amino acid residue D at position 614 into G, a mutation of the amino acid residue H at position 655 into Y, a mutation of the amino acid residue N at position 679 into K, a mutation of the amino acid residue P at position 681 into H, a mutation of the amino acid residue R at position 682 into S, a mutation of the amino acid residue R at position 685 into G, a mutation of the amino acid residue N at position 764 into K, a mutation of the amino acid residue D at position 796 into Y, a mutation of the amino acid residue N at position 856 into K, a mutation of the amino acid residue Q at position 954 into H, a mutation of the amino acid residue N at position 969 into K, a mutation of the amino acid residue L at position 981 into F, a mutation of the amino acid residue K at position 986 into P and a mutation of the amino acid residue V at position 987 into P, wherein the numbering of amino acid position is according to the numbering of amino acid positions in SEQ ID NO: 1. The protein according to claim 5, comprising an amino acid sequence of SEQ ID NO: 2, or a fragment thereof. A vector comprising a nucleic acid according to any one of the claims 1-4. A vector comprising a nucleic acid encoding a protein according to claim 5 or 6. The vector according to claim 7 or 8, wherein the vector is a recombinant human adenoviral vector.
80
10. The vector according to any one of the claims 7-9, wherein the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.
11. The vector according to claim 10, wherein the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 4.
12. The vector according to any one of the claims 7-11, comprising a nucleic acid comprising a nucleic acid sequence of SEQ ID NO: 3.
13. The vector according to any one of the claims 9-12, wherein the recombinant human adenovirus has a deletion in the El region, a deletion in the E3 region, or a deletion in both the El and the E3 region of the adenoviral genome.
14. The vector to any one of the claims 7-13, wherein the vector is a recombinant human adenovirus of serotype 26.
15. A composition comprising a nucleic acid according to claim 1-4, a protein according to claim 5 or 6, and/or vector according to any one of claims 7-14.
16. A composition comprising a first vector according to any one of the claims 7-14 and a second vector comprising a nucleic acid encoding a coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 5, or a fragment thereof.
17. The composition according to claim 16, wherein the first and second vector comprise a recombinant human adenovirus of serotype 26.
18. The composition according to claim 15, 16 or 17 for use as a vaccine against COVID-19, caused by a SARS CoV-2 variant, or SARS CoV-2 Wuhan-Hu-1.
19. A method for vaccinating a subject against COVID-19, caused by SARS CoV-2 and/or a SARS-CoV-2 variant, said method comprising administering to the subject a composition according to any of the claims 15-18.
81
20. The method according to claim 19, wherein the composition is administered to a subject that has been vaccinated against CO VID-19 prior to administration of the composition.
21. The method according to claim 20, wherein the composition is administered to the subject between 6 and 12 months after the previous vaccination. 22. The method according to any one of the claims 19-21, consisting of a single administration of the composition to the subject.
23. The method according to any one of the claims 19-21, consisting of a yearly administration of the composition to the subject.
24. The method according to any of the claims 19-23, wherein the recombinant human adenovirus of serotype 26 is administered at a dose of 5 x IO10 vp per administration.
25. An isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a SARS-CoV2 S protein or fragment thereof comprising the nucleotide sequence of SEQ ID NO: 3.
82
PCT/IB2022/061202 2021-12-14 2022-11-21 Sars-cov-2 vaccines WO2023111725A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202163265379P 2021-12-14 2021-12-14
US63/265,379 2021-12-14
US202263266381P 2022-01-04 2022-01-04
US63/266,381 2022-01-04
EP22158884 2022-02-25
EP22158884.1 2022-02-25

Publications (1)

Publication Number Publication Date
WO2023111725A1 true WO2023111725A1 (en) 2023-06-22

Family

ID=84363765

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2022/061202 WO2023111725A1 (en) 2021-12-14 2022-11-21 Sars-cov-2 vaccines

Country Status (1)

Country Link
WO (1) WO2023111725A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990003184A1 (en) 1988-09-30 1990-04-05 Bror Morein Matrix with immunomodulating activity
WO1990014837A1 (en) 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
WO1996011711A1 (en) 1994-10-12 1996-04-25 Iscotec Ab Saponin preparations and use thereof in iscoms
EP0853660A1 (en) 1996-07-16 1998-07-22 Transgene S.A. Method for preserving infectious recombinant viruses, aqueous viral suspension and use as medicine
WO1999012568A1 (en) 1997-09-05 1999-03-18 Merck & Co., Inc. Stabilizers containing recombinant human serum albumin for live virus vaccines
WO1999041416A2 (en) 1998-02-17 1999-08-19 Schering Corporation Compositions comprising viruses and methods for concentrating virus preparations
WO2000029024A1 (en) 1998-11-16 2000-05-25 Introgen Therapeutics, Inc. Formulation of adenovirus for gene therapy
US6083716A (en) 1996-09-06 2000-07-04 The Trustees Of The University Of Pennsylvania Chimpanzee adenovirus vectors
US6225289B1 (en) 1998-12-10 2001-05-01 Genvec, Inc. Methods and compositions for preserving adenoviral vectors
WO2001066137A1 (en) 2000-03-07 2001-09-13 Merck & Co., Inc. Adenovirus formulations
WO2003049763A1 (en) 2001-12-12 2003-06-19 Fh Faulding & Co Limited Composition for the preservation of viruses
WO2003061708A1 (en) 2002-01-18 2003-07-31 Schering Aktiengesellschaft Stabilized formulations of adenovirus
WO2003078592A2 (en) 2002-03-15 2003-09-25 Cell Genesys, Inc. Method for the purification, production and formulation of oncolytic adenoviruses
WO2003104467A1 (en) 2002-04-25 2003-12-18 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
WO2004004762A1 (en) 2002-07-05 2004-01-15 Isconova Ab Iscom preparation and use thereof
WO2005002620A1 (en) 2003-07-07 2005-01-13 Isconova Ab Quil a fraction with low toxicity and use thereof
WO2005071093A2 (en) 2004-01-23 2005-08-04 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Chimpanzee adenovirus vaccine carriers
WO2006040330A2 (en) 2004-10-13 2006-04-20 Crucell Holland B.V. Improved adenoviral vectors and uses thereof
WO2007104792A2 (en) 2006-03-16 2007-09-20 Crucell Holland B.V. Recombinant adenoviruses based on serotype 26 and 48, and use thereof
WO2007110409A1 (en) 2006-03-27 2007-10-04 Crucell Holland B.V. Compositions comprising a recombinant adenovirus and an adjuvant
WO2010060719A1 (en) 2008-11-03 2010-06-03 Crucell Holland B.V. Method for the production of adenoviral vectors
WO2010086189A2 (en) 2009-02-02 2010-08-05 Okairòs Ag, Switzerland Simian adenovirus nucleic acid- and amino acid-sequences, vectors containing same, and uses thereof
WO2010085984A1 (en) 2009-02-02 2010-08-05 Okairos Ag Simian adenovirus nucleic acid- and amino acid-sequences, vectors containing same, and uses thereof
WO2011098592A1 (en) 2010-02-15 2011-08-18 Crucell Holland B.V. Method for the production of ad26 adenoviral vectors
WO2012082918A1 (en) 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 Adenovirus serotype 26 and serotype 35 filovirus vaccines
WO2012172277A1 (en) 2011-05-25 2012-12-20 Isis Innovation Limited Simian adenovirus and hybrid adenoviral vectors
WO2018215766A1 (en) 2017-05-26 2018-11-29 Oxford University Innovation Limited Compositions and methods for inducing an immune response
WO2019086461A1 (en) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adenovirus vectors and uses thereof
WO2019086456A1 (en) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
WO2019086466A1 (en) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
US20190351048A1 (en) * 2016-12-23 2019-11-21 Curevac Ag Mers coronavirus vaccine

Patent Citations (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
WO1990003184A1 (en) 1988-09-30 1990-04-05 Bror Morein Matrix with immunomodulating activity
WO1990014837A1 (en) 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
WO1996011711A1 (en) 1994-10-12 1996-04-25 Iscotec Ab Saponin preparations and use thereof in iscoms
EP0853660A1 (en) 1996-07-16 1998-07-22 Transgene S.A. Method for preserving infectious recombinant viruses, aqueous viral suspension and use as medicine
US6083716A (en) 1996-09-06 2000-07-04 The Trustees Of The University Of Pennsylvania Chimpanzee adenovirus vectors
WO1999012568A1 (en) 1997-09-05 1999-03-18 Merck & Co., Inc. Stabilizers containing recombinant human serum albumin for live virus vaccines
WO1999041416A2 (en) 1998-02-17 1999-08-19 Schering Corporation Compositions comprising viruses and methods for concentrating virus preparations
WO2000029024A1 (en) 1998-11-16 2000-05-25 Introgen Therapeutics, Inc. Formulation of adenovirus for gene therapy
US6225289B1 (en) 1998-12-10 2001-05-01 Genvec, Inc. Methods and compositions for preserving adenoviral vectors
WO2001066137A1 (en) 2000-03-07 2001-09-13 Merck & Co., Inc. Adenovirus formulations
WO2003049763A1 (en) 2001-12-12 2003-06-19 Fh Faulding & Co Limited Composition for the preservation of viruses
WO2003061708A1 (en) 2002-01-18 2003-07-31 Schering Aktiengesellschaft Stabilized formulations of adenovirus
WO2003078592A2 (en) 2002-03-15 2003-09-25 Cell Genesys, Inc. Method for the purification, production and formulation of oncolytic adenoviruses
WO2003104467A1 (en) 2002-04-25 2003-12-18 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
WO2004004762A1 (en) 2002-07-05 2004-01-15 Isconova Ab Iscom preparation and use thereof
WO2005002620A1 (en) 2003-07-07 2005-01-13 Isconova Ab Quil a fraction with low toxicity and use thereof
WO2005071093A2 (en) 2004-01-23 2005-08-04 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Chimpanzee adenovirus vaccine carriers
WO2006040330A2 (en) 2004-10-13 2006-04-20 Crucell Holland B.V. Improved adenoviral vectors and uses thereof
WO2007104792A2 (en) 2006-03-16 2007-09-20 Crucell Holland B.V. Recombinant adenoviruses based on serotype 26 and 48, and use thereof
WO2007110409A1 (en) 2006-03-27 2007-10-04 Crucell Holland B.V. Compositions comprising a recombinant adenovirus and an adjuvant
WO2010060719A1 (en) 2008-11-03 2010-06-03 Crucell Holland B.V. Method for the production of adenoviral vectors
WO2010085984A1 (en) 2009-02-02 2010-08-05 Okairos Ag Simian adenovirus nucleic acid- and amino acid-sequences, vectors containing same, and uses thereof
WO2010086189A2 (en) 2009-02-02 2010-08-05 Okairòs Ag, Switzerland Simian adenovirus nucleic acid- and amino acid-sequences, vectors containing same, and uses thereof
WO2011098592A1 (en) 2010-02-15 2011-08-18 Crucell Holland B.V. Method for the production of ad26 adenoviral vectors
WO2012082918A1 (en) 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 Adenovirus serotype 26 and serotype 35 filovirus vaccines
WO2012172277A1 (en) 2011-05-25 2012-12-20 Isis Innovation Limited Simian adenovirus and hybrid adenoviral vectors
US20190351048A1 (en) * 2016-12-23 2019-11-21 Curevac Ag Mers coronavirus vaccine
WO2018215766A1 (en) 2017-05-26 2018-11-29 Oxford University Innovation Limited Compositions and methods for inducing an immune response
WO2019086461A1 (en) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adenovirus vectors and uses thereof
WO2019086456A1 (en) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof
WO2019086466A1 (en) 2017-10-31 2019-05-09 Janssen Vaccines & Prevention B.V. Adenovirus and uses thereof

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. MN908947
"Pharmaceutical Formulation Development of Peptides and Protein", 2000, PHARMACEUTICAL PRESS
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
ABBINK ET AL., VIROL, vol. 81, no. 9, 2007, pages 4654 - 63
ABBINK, J VIROL, vol. 81, no. 9, 2007, pages 4654 - 63
BOS ET AL., NPJ VACCINES, vol. 5, 2020, pages 91
BOS RINKE ET AL: "Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses", vol. 5, no. 1, 1 December 2020 (2020-12-01), XP055920608, Retrieved from the Internet <URL:https://www.nature.com/articles/s41541-020-00243-x.pdf> DOI: 10.1038/s41541-020-00243-x *
CALLAWAY EWEN: "Heavily mutated Omicron variant puts scientists on alert", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 600, no. 7887, 25 November 2021 (2021-11-25), pages 21, XP037632537, ISSN: 0028-0836, [retrieved on 20211125], DOI: 10.1038/D41586-021-03552-W *
GAGNE MATTHEW ET AL: "mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron", BIORXIV, 4 February 2022 (2022-02-04), XP055944343, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2022.02.03.479037v1.full.pdf> [retrieved on 20220719], DOI: 10.1101/2022.02.03.479037 *
HOGANSON ET AL.: "Development of a stable adenoviral vector formulation", BIOPROCESSING, March 2002 (2002-03-01), pages 43 - 48
HOWARD D ET AL: "Severe acute respiratory syndrome coronavirus 2 surface glycoprotein, EMBL accession no. UHS83992", 3 January 2022 (2022-01-03), XP055944347, Retrieved from the Internet <URL:https://www.ebi.ac.uk/ena/browser/api/embl/UHS83992.1?lineLimit=1000> [retrieved on 20220719] *
KUMAR SURESH ET AL: "Omicron and Delta Variant of SARS-CoV-2: A Comparative Computational Study of Spike protein", BIORXIV, 3 December 2021 (2021-12-03), XP093019898, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.12.02.470946v1.full.pdf> [retrieved on 20230201], DOI: 10.1101/2021.12.02.470946 *
LESTER ET AL., ACCESS MICROBIOLOGY, 2019, pages 1
RAMBAUT ET AL., VIROLOGICAL, 2020, Retrieved from the Internet <URL:https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563>
SOLABOMI ET AL., INFECT IMMUN., vol. 76, 2008, pages 3817 - 23
SOLFOROSI ET AL., J. EXP. MED., vol. 218, 2021, pages e20202756
SWART MAARTEN ET AL: "Immunogenicity of an Ad26-based SARS-CoV-2 Omicron Vaccine in Naïve Mice and SARS-CoV-2 Spike Pre-immune Hamsters", BIORXIV, 4 March 2022 (2022-03-04), pages 1 - 14, XP055944339, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2022.03.04.482636v1.full.pdf> [retrieved on 20220719], DOI: 10.1101/2022.03.04.482636 *
TEGALLY ET AL., MEDRXIV, 2020, Retrieved from the Internet <URL:https://www.medrxiv.org/content/10.1101/2020.12.21.20248640vl>
WIBMER ET AL., NAT MED, 2021, Retrieved from the Internet <URL:https://doi.org/10.1038/s41591-021-01285-x>
YING BAOLING ET AL: "Boosting with Omicron-matched or historical mRNA vaccines increases neutralizing antibody responses and protection against B.1.1.529 infection in mice", BIORXIV, 9 February 2022 (2022-02-09), XP055944341, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2022.02.07.479419v1.full.pdf> [retrieved on 20220719], DOI: 10.1101/2022.02.07.479419 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Similar Documents

Publication Publication Date Title
US11911462B2 (en) Nucleic acid vaccine against the SARS-CoV-2 coronavirus
US11801297B2 (en) Vaccine against RSV
JP7229239B2 (en) Adenovirus vector and use thereof
US20220133878A1 (en) Method for the safe induction of immunity against rsv
KR102023791B1 (en) Vaccine against rsv
WO2018215766A1 (en) Compositions and methods for inducing an immune response
JP2003180385A (en) Composition and method for vaccination against coronavirus
US20220016234A1 (en) Anti covid-19 therapies using nucleocapsid and spike proteins
Khanam et al. Induction of neutralizing antibodies and T cell responses by dengue virus type 2 envelope domain III encoded by plasmid and adenoviral vectors
US20230233661A1 (en) Vaccine combination against repiratory syncytial virus infection
WO2023111725A1 (en) Sars-cov-2 vaccines
JP2021500880A (en) Saladenovirus vector with two expression cassettes
JP2023543387A (en) Novel coronavirus recombinant spike protein, a polynucleotide encoding the same, a vector comprising the polynucleotide, and a vaccine for preventing or treating coronavirus infection, comprising the vector
US11964013B2 (en) Nucleic acid vaccine against the SARS-CoV-2 coronavirus
WO2023020939A1 (en) Sars-cov-2 vaccines
US11607449B2 (en) Synthetic plasmid DNA vaccine expressing a codon-optimized SARS-COV-2 spike protein
US11857620B2 (en) Method of inducing immunity against SARS-CoV-2 using spike (s) and nucleocapsid (N)-ETSD immunogens delivered by a replication-defective adenovirus
JP2024503482A (en) Replication-competent adenovirus type 4 SARS-COV-2 vaccines and their use
WO2021178844A1 (en) Zika and flavivirus immunogenic compositions and their use
Solforosi et al. ongeneelen, M
WO2022175479A1 (en) Vaccine combinations against respiratory syncytial virus strain a and b infections
WO2023026182A1 (en) Sars-cov-2 vaccines
WO2018146257A1 (en) Hepatitis e virus vaccine
NZ785676A (en) Vaccine against rsv
Lobanova Development of novel vaccine candidates for measles

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: 22813746

Country of ref document: EP

Kind code of ref document: A1