WO2024050482A2 - Compositions and methods for treating coronavirus infection - Google Patents

Compositions and methods for treating coronavirus infection Download PDF

Info

Publication number
WO2024050482A2
WO2024050482A2 PCT/US2023/073246 US2023073246W WO2024050482A2 WO 2024050482 A2 WO2024050482 A2 WO 2024050482A2 US 2023073246 W US2023073246 W US 2023073246W WO 2024050482 A2 WO2024050482 A2 WO 2024050482A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nucleic acid
variant
polypeptide
acid molecule
Prior art date
Application number
PCT/US2023/073246
Other languages
French (fr)
Other versions
WO2024050482A3 (en
Inventor
Dan H. Barouch
Bette T. Korber
Original Assignee
Beth Israel Deaconess Medical Center, Inc.
Triad National Security, Llc
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 Beth Israel Deaconess Medical Center, Inc., Triad National Security, Llc filed Critical Beth Israel Deaconess Medical Center, Inc.
Publication of WO2024050482A2 publication Critical patent/WO2024050482A2/en
Publication of WO2024050482A3 publication Critical patent/WO2024050482A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Disclosed are immunogenic compositions and vaccines containing rationally designed coronavirus Spike proteins and polynucleotides encoding the same that can be administered to treat or inhibit a coronavirus infection. The compositions and vaccines can also be used to produce anti-coronavirus antibodies (e.g., broadly neutralizing anti-coronavirus antibodies), which can also be used for prophylactic or therapeutic purposes in the treatment of a coronavirus infection.

Description

COMPOSITIONS AND METHODS FOR TREATING CORONAVIRUS INFECTION
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on August 28, 2023, is named “01948-285WO2_Sequence_Listing_8_28_23” and is 262,144 bytes in size.
BACKGROUND
Wuhan coronavirus (2019-nCoV; also referred to as SARS-CoV-2) is a coronavirus that is responsible for a worldwide pandemic. SARS-CoV-2 and subsequently arising lineage variants thereof are known to cause respiratory symptoms and fever, which may result in death.
The World Health Organization declared the SARS-CoV-2 or a variant thereof outbreak a Public Health Emergency of International Concern on January 30, 2020 and, to date, there are over 560,000,000 reported cases in over 200 countries. Over the course of the pandemic, various SARS-CoV-2 lineages carrying different mutations in the Spike protein have developed throughout the world, such as, for example, the Omicron variant (B.1 .1 .529) of SARS-CoV-2, which was first reported on November 24, 2021 , as well as Omicron subvariants, such as BA.4 and BA.5. Some of these lineages may be less susceptible to neutralization by antibodies produced by the original SARS-CoV-2 strain, as well as currently available vaccines directed against this strain. Accordingly, there is an unmet need in the field for therapy of SARS-CoV-2 and variants thereof, including a vaccine with enhanced breadth of antibody response (e.g., to multiple different Spike variants).
SUMMARY OF THE DISCLOSURE
Epigraph (EG) designed immunogens (e.g., EGO, EG1 , EG2, and EG3) can be used to produce a vaccine composition for producing neutralizing antibodies against currently existing SARS-CoV-2 and its lineage variants, as well as newly arising variants.
A first aspect of the disclosure features an isolated nucleic acid molecule with a nucleotide sequence that encodes a polypeptide having an amino acid sequence of any one or more of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a variant thereof (e.g., a variant polypeptide with an amino acid sequence with at least 85% sequence identity (e.g., at least 90%, 95%, 97%, and 99% sequence identity) to SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107).
In some embodiments, the polypeptide is capable of eliciting an immune response in a subject.
In some embodiments, the nucleic acid molecule includes the nucleotide sequence of any one or more of SEQ ID NOs: 42, 43, 47, 52, and 57-60, or a variant thereof or a complementary sequence thereof.
In some embodiments, the nucleic acid molecule encodes the polypeptide having the amino acid sequence of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or each of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or said variant thereof. In some embodiments, the nucleotide sequence encodes the polypeptide having the amino acid sequence of any one or more of SEQ ID NOs: 87-90 and 103-107 or said variant thereof.
In some embodiments, the nucleic acid molecule includes the nucleotide sequence of any one or more of SEQ ID NOs: 57-60 or a variant thereof or a complementary sequence thereof.
In some embodiments, the nucleic acid molecule encodes the polypeptide having the amino acid sequence of two, three, four, five, six, seven, eight, or each of SEQ ID NOs: 87-90 and 103-107 or said variant thereof.
In some embodiments, the nucleotide sequence encodes the polypeptide having an amino acid sequence of any one or more of SEQ ID NOs: 72, 73, 77, 82, and 97-102 or said variant thereof.
In some embodiments, the nucleic acid molecule includes the nucleotide sequence of any one or more of SEQ ID NOs: 42, 43, 47, and 52 or a variant thereof or a complementary sequence thereof.
In some embodiments, nucleic acid molecule encodes the polypeptide having the amino acid sequence of two, three, or each of SEQ ID NOs: 72, 73, 77, 82, and 97-102 or said variant thereof.
A second aspect of the disclosure features an isolated polypeptide having an amino acid sequence of any one of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a portion or variant thereof (e.g., a variant polypeptide with an amino acid sequence with at least 85% sequence identity (e.g., at least 90%, 95%, 97%, and 99% sequence identity) to SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107).
In some embodiments, the polypeptide is encoded by any one of the nucleic acid molecules of the first aspect.
In some embodiments, the polypeptide, or portion thereof, is capable of eliciting an immune response in a subject.
In some embodiments, the polypeptide has the amino acid sequence of any one of SEQ ID NOs: 87-90 and 103-107 or said variant thereof.
In some embodiments, the polypeptide has the amino acid sequence of any one of SEQ ID NOs: 72, 73, 77, 82, and 97-102 or said variant thereof.
A third aspect of the disclosure features an isolated vector including any one or more of the nucleic acid molecules of the first aspect.
In some embodiments, the nucleic acid molecule includes a nucleotide sequence of any one or more of SEQ ID NOs: 57-60 or a variant thereof.
In some embodiments, the nucleic acid molecule encodes a polypeptide having the amino acid sequence of two or three, four, five, six, seven, eight, or each, of SEQ ID NOs: 87-90 and 103-107 or a variant thereof.
In some embodiments, the vector is replication-defective.
In some embodiments, the vector is a mammalian, bacterial, or viral vector.
In some embodiments, the vector is an expression vector.
In some embodiments, the viral vector is a virus selected from the group including a retrovirus, adenovirus, adeno-associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox.
In some embodiments, the vector is an adenovirus. In some embodiments, the adenovirus is selected from the group including Ad26, Ad52, Ad59, Ad2, Ad5, Ad11 , Ad12, Ad24, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, and Pan9, wherein optionally the adenovirus is Ad26 or Ad52.
In some embodiments, the Ad52 is a rhesus Ad52 or the Ad59 is a rhesus Ad59.
A fourth aspect of the disclosure features a composition including any one or more of the nucleic acid molecules of the first aspect, any one or more of the polypeptides of the second aspect, or any one or more of the vectors of the third aspect.
In some embodiments, the composition includes any two or more of the nucleic acid molecules of the first aspect, wherein optionally the nucleic acid molecule of the composition encodes any two or more of the polypeptides of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a variant thereof (e.g., a variant polypeptide with an amino acid sequence with at least 85% sequence identity (e.g., at least 90%, 95%, 97%, and 99% sequence identity) to SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107). In some embodiments, the composition includes any two or more of the polypeptides of the second aspect, wherein optionally the composition includes any two or more of the polypeptides of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a portion or variant thereof.
In some embodiments, the composition includes any three or more of the nucleic acid molecules of the first aspect, wherein optionally the nucleic acid molecule of the composition encodes any three or more of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a variant thereof. In some embodiments, the composition includes any three or more of the polypeptides of the second aspect, wherein optionally the composition includes any three or more of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a portion or variant thereof (e.g., a variant polypeptide with an amino acid sequence with at least 85% sequence identity (e.g., at least 90%, 95%, 97%, and 99% sequence identity) to SEQ ID NOs: 87-90 and 103-107).
In some embodiments, the composition includes any four or more of the nucleic acid molecules of the first aspect, wherein optionally the nucleic acid molecule of the composition encodes any four or more of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a variant thereof (e.g., a variant polypeptide with an amino acid sequence with at least 85% sequence identity (e.g., at least 90%, 95%, 97%, and 99% sequence identity) to SEQ ID NOs: 87-90 and 103-107). In some embodiments, the composition includes any four or more of the polypeptides of the second aspect, wherein optionally the composition includes each of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a portion or variant thereof.
In some embodiments, the composition further includes a pharmaceutically acceptable carrier, excipient, or diluent.
In some embodiments, the composition further includes an adjuvant or an immunostimulatory agent.
In some embodiments, the composition is a vaccine.
In some embodiments, the vaccine is a monovalent or a polyvalent vaccine.
In some embodiments, the composition is capable of treating or reducing the risk of a coronavirus infection, such as, for example, infection by a 2019-nCoV virus or a variant thereof, in a subject in need thereof.
In some embodiments, the composition elicits production of neutralizing anti-2019-nCoV antisera in said subject. In some embodiments, the subject is infected with a lineage of 2019-nCoV.
In some embodiments, the lineage of 2019-nCoV is one or more of B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1.1.28, B.1.351 , or A23.1.
In some embodiments, the lineage of 2019-nCoV is B.1 .1 .529.
In some embodiments, the subject is a mammal.
In some embodiments, the mammal is a human.
In some embodiments, the human has an underlying health condition.
In some embodiments, the underlying health condition is hypertension, diabetes, or cardiovascular disease.
In some embodiments, the composition further includes any one or more of the following: (a) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 72 or a variant thereof; (b) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 73 or a variant thereof; (c) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 77 or a variant thereof; (d) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 82 or a variant thereof; (e) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 97 or a variant thereof; (f) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 98 or a variant thereof; (g) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 99 or a variant thereof; (h) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 100 or a variant thereof; (i) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 101 or a variant thereof; and (j) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 102 or a variant thereof, wherein optionally the composition includes a single nucleic acid molecule. The variant polypeptide of this paragraph has an amino acid sequence with at 85% sequence identity (e.g., at least 90%, 95%, 97%, and 99% sequence identity) to any of SEQ ID NOs: 72, 73, 77, 92, and 97-102.
In some embodiments, the nucleic acid molecule of the composition includes any two or more of the following: (a) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 72 or said variant thereof; (b) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 73 or said variant thereof; (c) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 77 or said variant thereof; (d) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 82 or said variant thereof; (e) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 97 or a variant thereof; (f) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 98 or a variant thereof; (g) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 99 or a variant thereof; (h) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 100 or a variant thereof; (i) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 101 or a variant thereof; and (j) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 102 or a variant thereof.
In some embodiments, the nucleic acid molecule of the composition includes any three or more of the following: (a) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 72 or said variant thereof; (b) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 73 or said variant thereof; (c) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 77 or said variant thereof; (d) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 82 or said variant thereof; (e) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 97 or a variant thereof; (f) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 98 or a variant thereof; (g) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 99 or a variant thereof; (h) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 100 or a variant thereof; (i) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 101 or a variant thereof; and (j) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 102 or a variant thereof.
In some embodiments, the nucleic acid molecules of the composition include the nucleotide sequence of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 47, and/or SEQ ID NO: 52.
A fifth aspect of the disclosure features a method of producing an antibody, the method including administering any one of the nucleic acid molecules of the first aspect, any one of the polypeptides of the second aspect, any one of the vectors of the third aspect, or any one of the compositions of the fourth aspect to a subject to elicit production of neutralizing antisera in said subject.
A sixth aspect of the disclosure features a method of identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection, the method including the steps of: (a) determining whether the subject has a protective level of a broadly neutralizing anti-coronavirus antibody (bNAb) against two or more lineages of coronavirus (such as an anti-Spike antibody) in a sample from the subject, wherein optionally the protective level is: (i) a level that is at or above a titer of at least about 70, as determined using a pseudovirus neutralization assay; or (ii) a level that is at or above a titer of at least about 25, as determined using a live virus neutralization assay; or (iii) a level that is at least 80% of a median level of an anti-coronavirus antibody in a cohort of convalescent humans, as determined by a pseudovirus neutralization assay or live virus neutralization assay; and (b) administering an effective amount of any one of the nucleic acid molecules of the first aspect, any one of the polypeptides of the second aspect, any one of the vectors of the third aspect, or any one of the compositions of the fourth aspect to the subject having less than a protective level of the bNAb.
In some embodiments, the method further includes identifying a subclass and/or an effector function of the bNAb.
In some embodiments, the subclass is (a) IgM, IgA, IgG 1 , lgG2, lgG3, or FcgR2A; and/or (b) the effector function is antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation.
In some embodiments, the sample is a bodily fluid from the subject, wherein optionally the bodily fluid is blood.
In some embodiments, the coronavirus is 2019-nCoV.
In some embodiments, the two or more lineages of coronavirus are selected from the group including B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , and A23.1 . A seventh aspect of the disclosure features a method of treating or reducing the risk of a coronavirus infection in a subject in need thereof, the method including administering a therapeutically effective amount of any one of the nucleic acid molecules of the first aspect, any one of the polypeptides of the second aspect, any one of the vectors of the third aspect, or the any one of the compositions of the fourth aspect to said subject.
In some embodiments, the method includes administering: (a) an Ad26 or Ad52 vector including the nucleic acid molecule; and/or (b) an Ad26 or Ad52 vector including a nucleic acid molecule that encodes the polypeptide.
In some embodiments, the method further includes measuring an anti-coronavirus antibody (e.g., an anti-Spike antibody) level in the subject.
In some embodiments, the anti-coronavirus antibody level in the subject is measured before and/or after administration of the composition.
In some embodiments, the anti-coronavirus antibody level in the subject is measured one or more times over about 1 , 2, 3, 4, 5, or 6 days, 1 , 2, 3, 4, 5, 6, or 7 weeks, 2, 3, 4, 5, or 6 months, 1 , 2, 3, 4, or 5 years after administration.
In some embodiments, the anti-coronavirus antibody level of the subject is below a protective level and wherein the method further includes re-administering any one of the compositions of the fourth aspect to said subject or administering a different anti-coronavirus composition to the subject.
In some embodiments, the protective level is a level sufficient to reduce symptoms or duration of a coronavirus-mediated disease.
In some embodiments, the protective level is: (a) a level that is at or above a titer of at least about 70, as determined using a pseudovirus neutralization assay; or (b) a level that is at or above a titer of at least about 25, as determined using a live virus neutralization assay; or (c) a level that is at least 80% of a median level of an anti-coronavirus antibody in a cohort of convalescent humans, as determined by a pseudovirus neutralization assay or live virus neutralization assay.
In some embodiments, the coronavirus infection is infection by 2019-nCoV, wherein optionally said 2019-nCoV is of the lineage B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , or A23.1 . In some embodiments, said 2019-nCoV is an Omicron variant. In some embodiments, the Omicron variant is BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6.
An eighth aspect of the disclosure features a method of reducing a coronavirus-mediated activity in a subject infected with a 2019-nCoV or a variant thereof, the method including administering a therapeutically effective amount of any one of the nucleic acid molecules of the first aspect, any one of the polypeptides of the second aspect, any one of the vectors of the third aspect, or any one of the compositions of the fourth aspect to said subject.
In some embodiments, the method includes administering: (a) an Ad26 or Ad52 vector including the nucleic acid molecule; and/or (b) an Ad26 or Ad52 vector including a nucleic acid molecule that encodes the polypeptide, wherein optionally the therapeutically effective amount of the composition or the immunogenic composition is sufficient to produce a log serum anti-Spike antibody titer greater than 2 in a subject, as measured by an ELISA assay.
In some embodiments, the therapeutically effective amount is between 15 pg and 300 pg of the one or more of the compositions of the fourth aspect. In some embodiments, said activity is viral titer, viral spread, infection, or cell fusion.
In some embodiments, said viral titer is decreased after administration of the one or more compositions of the fourth aspect.
In some embodiments, the viral titer is decreased by 25% or more.
In some embodiments, the viral titer is decreased by 50% or more.
In some embodiments, the viral titer is decreased by 75% or more.
In some embodiments, the coronavirus is undetectable after said administration.
In some embodiments, said administering occurs prior to exposure to the coronavirus.
In some embodiments, said administering occurs at least 1 hour prior to exposure to said coronavirus.
In some embodiments, said administering occurs at least 1 week, 1 month, or a year prior to exposure to said coronavirus.
In some embodiments, said administering occurs post-exposure to the coronavirus.
In some embodiments, said administering occurs at least 15 minutes post-exposure to said coronavirus.
In some embodiments, said administering occurs at least 1 hour, 1 day, or 1 week post-exposure to said coronavirus.
In some embodiments, said subject is administered at least one dose of the one or more compositions of the fourth aspect.
In some embodiments, said subject is administered at least two doses of the one or more compositions of the fourth aspect.
In some embodiments, the composition is administered to said subject as a prime, a boost, or as a prime-boost.
In some embodiments, the composition is administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivelly, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in creams, or in lipid compositions.
In some embodiments, the subject is a mammal.
In some embodiments, the mammal is a human.
In some embodiments, the human has an underlying health condition.
In some embodiments, the underlying health condition is hypertension, diabetes, or cardiovascular disease.
In some embodiments, the method promotes an immune response in said subject.
In some embodiments, the immune response is a humoral immune response.
In some embodiments, the humoral immune response is an IgG response.
A ninth aspect of the disclosure features a composition for use in treating or reducing the risk of a coronavirus infection, such as a 2019-nCoV infection, in a subject in need thereof, including a therapeutically effective amount of any one of the nucleic acid molecules of the first aspect, any one of the polypeptides of the second aspect, any one of the vectors of the third aspect, or any one of the compositions of the fourth aspect.
A tenth aspect of the disclosure features a composition for use in reducing a coronavirus- mediated activity in a subject infected with a 2019-nCoV or a variant thereof, including a therapeutically effective amount of any one of the nucleic acid molecules of the first aspect, any one of the polypeptides of the second aspect, any one of the vectors of the third aspect, or any one of the compositions of the fourth aspect.
In some embodiments, the composition for use includes: (a) an Ad26 vector including the nucleic acid molecule of the composition or the immunogenic composition; and/or (b) an Ad26 vector including a nucleic acid molecule that encodes the polypeptide of the composition or the immunogenic composition.
An eleventh aspect of the disclosure features a method of manufacturing an immunogenic composition for treating or reducing the risk of a coronavirus infection in a subject in need thereof, said method including the steps of: (a) admixing at least one of the nucleic acid molecules of the first aspect, at least one of the polypeptides of the second aspect, at least one of the vectors of the third aspect, and at least one of the compositions of the fourth aspect with a pharmaceutically acceptable carrier, excipient, or diluent to form the immunogenic composition; and (b) placing the immunogenic composition in a container.
A twelfth aspect of the disclosure features a kit including: (a) a first container including at least one said nucleic acid molecule of the first aspect, at least one said polypeptide of the second aspect, at least one said vector of the third aspect, and/or at least one said composition of the fourth aspect; (b) instructions for use thereof; and optionally (c) a second container including a pharmaceutically acceptable carrier, excipient, or diluent.
In some embodiments, the first container further includes a pharmaceutically acceptable carrier, excipient, or diluent.
In some embodiments, the kit optionally includes an adjuvant and/or an immunostimulatory agent.
In other embodiments of any of the foregoing aspects, the nucleic acid molecules, vectors, compositions, immunogenic compositions, and methods may have, or further include, the nucleotide sequence of any one or more of SEQ ID NOs: 44-46, 48-51 , 53-56, and 61 -66, or a variant thereof (e.g., a variant with 80% or greater sequence identity thereto) or a complementary sequence thereof.
In other embodiments of any of the foregoing aspects, the polypeptides, compositions, immunogenic compositions, and methods may have, or further include, the polypeptide sequence of any one or more of SEQ ID NOs: 74-76, 78-81 , 83-86, and 91 -107 or a variant thereof (e.g., a variant with 80% or greater sequence identity thereto).
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations. FIG. 1 shows a multiple sequence alignment (MSA) of Epigraph (EG) designed immunogen (e.g., EGO, EG1 , EG2, and EG3) protein sequences (e.g., SEQ ID NOs: 87 to 90, respectively). Shaded residues indicate mutations, relative to SEQ ID NO: 21 or 23.
FIG. 2 shows theoretical coverage of 1 , 2, 3, 4, and 5 Spike Epigraph immunogens (“EP-1 ” refers to a single Spike epigraph sequence, whereas “EP-5” refers to five Spike epigraph sequences). Percent missed epitopes is shown. Four Spike Epigraph immunogens provide high level coverage of SARS-CoV- 2 sequences, including coverage for Omicron variants (e.g., BA.4 and BA.5).
FIG. 3 shows theoretical coverage of 1 , 2, 3, 4, and 5 Nucleocapsid Epigraph immunogens (“EP- 1 ” refers to a single Nucleocapsid epigraph sequence, whereas “EP-5” refers to five Nucleocapsid epigraph sequences). Percent missed epitopes is shown. Two Nucleocapsid Epigraph immunogens provide high level coverage of SARS-CoV-2 sequences.
FIG. 4 shows theoretical coverage of 1 , 2, 3, 4, and 5 Membrane Epigraph immunogens (“EP-1 ” refers to a single Membrane epigraph sequence, whereas “EP-5” refers to five Membrane epigraph sequences). Percent missed epitopes is shown. One Membrane Epigraph immunogen provides high level coverage of SARS-CoV-2 sequences.
FIG. 5 shows theoretical coverage of 1 , 2, 3, 4, and 5 Conserved Epigraph immunogens (“EP-1 ” refers to a single Conserved epigraph sequence, whereas “EP-5” refers to five Conserved epigraph sequences). Percent missed epitopes is shown. One Conserved Epigraph immunogen provides high level coverage of SARS-CoV-2 sequences.
DEFINITIONS
As used herein, the term “about” means +/- 10% of the recited value.
The terms “adenovirus vector” and “adenoviral vector” are used interchangeably and refer to a genetically-engineered adenovirus that is designed to insert a polynucleotide of interest (e.g., a polynucleotide encoding a SARS-CoV-2 immunogen described herein) into a eukaryotic cell, such that the polynucleotide is subsequently expressed. Examples of adenoviruses that can be used as a viral vector include those having, or derived from, the serotypes Ad2, Ad5, Ad11 , Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), Ad59 (e.g., RhAd59), and Pan9 (also known as AdC68); these vectors can be derived from, for example, human, chimpanzee, or rhesus adenoviruses. In some embodiments, the adenovirus is Ad26.
The term “adjuvant” refers to a pharmacological or immunological agent that modifies the effect of other agents (e.g., vaccines) while having few if any direct effects when given by itself. They are often included in vaccines to enhance the recipient’s immune response to a supplied antigen while keeping the injected foreign material at a minimum.
As used herein, by “administering” is meant a method of giving a dosage of a pharmaceutical composition (e.g., an immunogenic composition (e.g., a vaccine (e.g., a monovalent or a polyvalent coronavirus vaccine (SARS-CoV-2 or a variant thereof)), such as those described herein) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions. The method of administration can vary depending on various factors (e.g., the components of the composition being administered, and the severity of the condition being treated).
The terms “antibody” and “immunoglobulin (lg)” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments. An antibody typically comprises both “light chains” and “heavy chains.” The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, lg D, lg E, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG 1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, E, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
By "broadly neutralizing antibody" or "bnAb," with respect to coronavirus (e.g., SARS-CoV-2 or a variant thereof), is meant an antibody that recognizes a specific antigen (e.g., Spike (e.g., the NTD and/or RBD of a spike protein, such as the EGO, EG1 , EG2, EG3, EG5, EG6, EG7, EG8, and EG9, MEMO, MEM5, MEM6, CRO, CR5, NULO, NUL1 , NUL5, NUL6, and/or NUL7, immunogens described herein)) and inhibits the effect(s) of the antigen of at least 2, 3, 4. 5, 6, 7, 8, 9 or more different strains of SARS-CoV-2 or a variant thereof, the strains belonging to the same or different clades, in the host subject (e.g., human). As used herein, the antibody can be a single antibody or a plurality of antibodies.
As used herein, the term “clade” refers to related coronaviruses classified according to their degree of genetic similarity. A clade generally refers to a distinctive branch in a phylogenetic tree. In certain exemplary embodiments, a composition described herein (e.g., a nucleic acid molecule, polypeptide, vector, and/or antibody composition described herein, such as a monovalent or polyvalent vaccine composition as described herein) can be used to elicit an immune response (e.g., the generation of neutralizing anti-coronavirus antisera) against two, three, four, five, six, seven, eight, nine, ten or more clades. In some embodiments, the two or more clades (e.g., also referred to herein as “lineages”) of coronavirus are selected from the group consisting of B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , A23.1 B.1 .617.1 , B.1 .617.2, B.1 .427, B.1 .525, B.1 .526, P.1 , P.2, P.3, C.36, C.37, B.1 .1 .519, B.1 .526.1 , B.1 .526.2, R.1 , B.1 .258.17, B.1 .575, B.1 .214.2, A.2.5.2, AT.1 , B.1 .1 .523, and B.1 .620. The SARS-CoV-2 may be an Alpha, Beta, Gamma, Delta, or Omicron variant. For example, the Omicron variant can be BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6. Other clades are described at cov-lineages.org/lineage_list.html, incorporated herein by reference.
The term “codon” as used herein refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule, or coding strand of DNA, that specifies a particular amino acid or a starting or stopping signal for translation. The term codon also refers to base triplets in a DNA strand. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
As used herein, the terms "conservative mutation," "conservative substitution," and "conservative amino acid substitution" refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1 below.
Table 1. Representative physicochemical properties of naturally occurring amino acids
Figure imgf000012_0001
From this table it is appreciated that the conservative amino acid families include (i) G, A, V, L and I; (ii) D and E; (Hi) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. A conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g., a substitution of Ser for Thr or Lys for Arg). Table 1 also presents the one letter code for amino acids, which is used herein to designate an amino acid mutation at a particular residue position (e.g., “L18F” refers to the substitution of leucine for phenylalanine at residue position 18 of a polypeptide sequence (e.g., the sequence of a coronavirus Spike protein)).
The term “convalescent” as used herein refers to subjects who have recovered or are recovering from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof). A “cohort of convalescent humans” refers to a group of humans that share common characteristics (e.g., sex, age, weight, medical history, race, ethnicity, or environment) and have recovered or are recovering from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof). A cohort of convalescent humans may share common characteristics with a subject having a risk of coronavirus (e.g., SARS-CoV-2 or variant thereof) infection or suspected of being susceptible to a coronavirus infection. As an example, samples from convalescent humans can be obtained at least 7 days after documented recovery (e.g., determined with a negative nasal swab).
The terms “ectodomain” and “extracellular domain” refer to the portion of a coronavirus Spike polypeptide that extends beyond the transmembrane domain into the extracellular space. The ectodomain mediates binding of a Spike polypeptide to one or more coronavirus receptors (e.g., ACE2). For instance, an ectodomain includes the S1 domain (e.g., SEQ ID NO: 27) and RBD (e.g., SEQ ID NO: 28) of a Spike polypeptide (e.g., SEQ ID NO: 21 ).
A “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
"Gene delivery," "gene transfer," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of techniques such as, for example, vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides).
The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are capable of mediating transfer of genes to mammalian cells.
By “gene product” is meant to include mRNAs or other nucleic acids (e.g., microRNAs) transcribed from a gene, as well as polypeptides translated from those mRNAs. In some embodiments, the gene product is from a virus (e.g., a SARS-CoV-2 or variant thereof) and may include, for example, any one or more of the viral proteins, or fragments thereof, and/or immunogens (e.g., EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and EG9) described herein.
By “heterologous nucleic acid molecule” is meant a nucleotide sequence that may encode proteins derived or obtained from pathogenic organisms, such as viruses, which may be incorporated into a polynucleotide or vector. Heterologous nucleic acids may also encode synthetic or artificial proteins, such as immunogenic epitopes, constructed to induce immunity. An example of a heterologous nucleic acid molecule is one that encodes one or more immunogenic peptides or polypeptides derived from a coronavirus (e.g., SARS-CoV-2 or variant thereof). The heterologous nucleic acid molecule is one that is not normally associated with the other nucleic acid molecules found in the polynucleotide or vector into which the heterologous nucleic acid molecule is incorporated.
The term “host cell,” refers to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Host cells include cells within the body of a subject (e.g., a mammalian subject (e.g., a human)) into which an exogenous nucleic acid has been introduced. Host cells may also include “producer cells,” such as those described in Kovesdi and Hedley, Viruses, 2(8): 1681 -1703 (e.g., HEK293, 911 , HeLa-E1 , VL2-20, 293-E4, E2T), herein incorporated by reference, which allow for production of a vector (e.g., an adenovirus),
By “immunogen” is meant any polypeptide that can induce an immune response in a subject upon administration. In some embodiments, the immunogen is encoded by a nucleic acid molecule that may be incorporated into, for example, a polynucleotide or vector, for subsequent expression of the immunogen (e.g., a gene product of interest, or fragment thereof (e.g., a polypeptide)). Examples of immunogens include, e.g., EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and EG9, and variants thereof (e.g., those with at least 85% or more sequence identity to the amino acid sequence of these immunogens), described herein.
The term “immunogenic composition” as used herein, is defined as material used to provoke an immune response and may confer immunity after administration of the immunogenic composition to a subject.
The term “immunostimulatory agent” refers to substances (e.g., drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. An immunostimulatory agent includes a cytokine (e.g., the granulocyte macrophage colony-stimulating factor) and interferon (e.g., IFN-a and/or IFN-y).
By “isolated” is meant separated, recovered, or purified from a component of its natural environment. For example, a nucleic acid molecule or polypeptide may be isolated from a component of its natural environment by 1% (e.g., by 2%, 3%, 4%, 5%, 6%, 7%, 8% 9% 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or 90%) or more.
By “pharmaceutical composition” is meant any composition that contains a therapeutically or biologically active agent, such as an immunogenic composition or vaccine (e.g., a nucleic acid molecule encoding a protein of SARS-CoV-2 or a variant thereof (e.g., a Spike protein), a vector containing the nucleic acid molecule, and/or a polypeptide encoded by the nucleic acid molecule, such as those described herein), that, in particular, includes a nucleotide sequence encoding an antigenic gene product of interest, or fragment thereof, that is suitable for administration to a subject and that treats or prevents a disease (e.g., infection by SARS-CoV-2 or a variant thereof) or reduces or ameliorates one or more symptoms of the disease (e.g., viral titer, viral spread, infection, and/or cell fusion caused by SARS-CoV-2 or a variant thereof)). For the purposes of this disclosure, pharmaceutical compositions include vaccines (e.g., monovalent vaccines and polyvalent vaccines), and pharmaceutical compositions suitable for delivering a therapeutic or biologically active agent can include, for example, tablets, gelcaps, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. Any of these formulations can be prepared by well-known and accepted methods of art. See, for example, Remington: The Science and Practice of Pharmacy (21st ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.
The terms “linked” or “links” or “link” as used herein are meant to refer to the covalent joining of two amino acid sequences or two nucleic acid sequences together through peptide or phosphodiester bonds, respectively, such joining can include any number of additional amino acid or nucleic acid sequences between the two amino acid sequences or nucleic acid sequences that are being joined.
As used herein, the term “mutation” refers to a change in the nucleotide sequence of a gene or a change in the polypeptide sequence of a protein. Mutations in a gene or protein may occur naturally as a result of, for example, errors in DNA replication, DNA repair, irradiation, and exposure to carcinogens or mutations may be induced as a result of administration of a transgene expressing a mutant gene. Mutations may result from single or multiple nucleotide insertions, deletions, or substitutions. The nomenclature for describing mutations and sequence variations uses the format “reference sequence code,” wherein the reference sequence may be “A” or “del,” designating a deletion, or may contain reference to the substitutions occurring. The nomenclature for describing mutations resulting from amino acid substitutions uses the format “AnB,” where “A” designates the amino acid found in the wild type variant of the protein, “n” designates the number of the amino acid within the peptide chain, and “B” designates the new amino acid that resulted from the substitution. For example, the SARS-CoV-2 spike protein EG1 (SEQ ID NO: 88) described herein contains a substitution described as N501 Y, which corresponds to a change in the protein at amino acid residue #501 , in which an asparagine is substituted for a tyrosine. The mutations described herein appear in the context of a Spike protein of a coronavirus (e.g., SARS-CoV-2, for example corresponding to the amino acid sequence of SEQ ID NO: 21 ). Thus, references herein to mutations made “relative to the amino acid sequence of SEQ ID NO: 21 ”, or made “relative to SEQ ID NO: 23”, indicate that the Spike protein containing one or more of the indicated mutations is in the context of a Spike (S) protein of SARS-CoV-2 or a variant thereof with the referenced sequence (e.g., the reference sequence serves as the backbone for the Spike protein with the indicated mutation(s).
“Nucleic acid molecule” or “polynucleotide,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label.
A "nucleic acid vaccine" refers to a vaccine that includes a heterologous nucleic acid molecule under the control of a promoter for expression in a subject. The heterologous nucleic acid molecule can be incorporated into an expression vector, such as a plasmid. A “DNA vaccine” refers to a vaccine in which the nucleic acid is DNA. An “RNA vaccine” refers to a vaccine in which the nucleic acid is RNA (e.g., an mRNA). As used herein, a “monovalent vaccine” refers to a vaccine which contains a single strain of a single antigen (e.g., a Spike protein, or a nucleic acid molecule encoding the Spike protein, from SARS-CoV-2 or a variant thereof, such as one of EGO, EG1 , EG2, and EG3, and variants thereof), whereas a “polyvalent vaccine” refers to a vaccine containing more than one antigen (e.g., multiple different Spike proteins, or nucleic acid molecules encoding multiple different Spike proteins from SARS- CoV-2 or a variant thereof, such as two or more of EGO, EG1 , EG2, and EG3, and variants thereof). A monovalent or polyvalent vaccine as described herein may contain one or more Spike proteins, or nucleic acid molecules encoding one or more Spike proteins, from a SARS-CoV-2 that is an Alpha, Beta, Gamma, Delta, or Omicron variant, such as a variant of the lineage B.1 .1 .529 (e.g., BA.1 , BA.2, BA.3, BA.4, and BA.5), B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , A23.1 , B.1 .617.1 , B.1 .617.2, B.1 .427, B.1 .525, B.1.526, P.1 , P.2, P.3, C.36, C.37, B.1.1.519, B.1 .526.1 , B.1 .526.2, R.1 , B.1 .258.17, B.1.575, B.1.214.2, A.2.5.2, AT.1 , B.1 .1 .523, and/or B.1 .620 (e.g., a Spike protein encoded by the nucleic acid molecule of any one of SEQ ID NOs: 57-60). By way of example, the Omicron variant can be BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6. The vaccine may also include one or more of the Membrane, Nucleocapsid, and Conserved Region epigraph sequences described herein (either as a protein sequence or as a nucleic acid molecule encoding the protein). A monovalent or polyvalent vaccine as described herein can be used to induce an immune response (e.g., a neutralizing antibody response) against one or more different coronaviruses.
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 may 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.
By “optimized” is meant an immunogenic polypeptide that is not a naturally-occurring peptide, polypeptide, or protein, such as a non-naturally occurring viral polypeptide (e.g., a Spike polypeptide). Optimized viral polypeptide sequences are initially generated by modifying the amino acid sequence of one or more naturally-occurring viral gene products (e.g., peptides, polypeptides, and proteins) to increase the breadth, intensity, depth, or longevity of the antiviral immune response (e.g., cellular or humoral immune responses) generated upon immunization (e.g., when incorporated into a composition, e.g., vaccine) of a subject (e.g., a human). Thus, an optimized viral polypeptide may correspond to a “parent” viral gene sequence; alternatively, an optimized viral polypeptide may not correspond to a specific “parent” viral gene sequence but may correspond to analogous sequences from various strains or quasi-species of a virus. Modifications to the viral gene sequence that can be included in an optimized viral polypeptide include amino acid additions, substitutions, and deletions. In one embodiment, an optimized polypeptide is a Spike polypeptide from a coronavirus (e.g., from SARS-CoV-2 or a variant thereof), which has been further altered to include a leader/signal sequence (e.g., a Spike signal sequence (SS) or a tPA signal sequence or other leader/signal sequence) for maximal protein expression, a factor Xa site, a foldon trimerization domain (see, e.g., SEQ ID NO: 32) or other trimerization domain known in the art, and/or linker or spacer (e.g., SEQ ID NOs: 33 or 34) sequences. An optimized polypeptide may, but need not, also include a cleavage site mutation(s) (e.g., a furin cleavage site mutation (e.g., SEQ ID NO: 36)). Methods of generating an optimized viral polypeptide are described in, e.g., Fisher et al. “Polyvalent Vaccine for Optimal Coverage of Potential T-Cell Epitopes in Global HIV-1 Variants,” Nat. Med. 13(1 ):100-106 (2007) and International Patent Application Publication WO 2007/024941 , herein incorporated by reference. Once the optimized viral polypeptide sequence is generated, the corresponding polypeptide can be produced or administered by standard techniques (e.g., recombinant viral vectors, such as the adenoviral vectors disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, herein incorporated by reference) and optionally assembled to form a stabilized polypeptide trimer.
The terms “optimized codon” and “codon optimized” as used herein refer to a codon sequence that has been modified to match codon frequencies in a target (e.g., a subject) or host organism, but that does not alter the amino acid sequence of the original translated protein.
By “pharmaceutically acceptable diluent, excipient, carrier, or adjuvant” is meant a diluent, excipient, carrier, or adjuvant that is physiologically acceptable to the subject while retaining the therapeutic properties of the pharmaceutical composition with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable diluents, excipients, carriers, or adjuvants and their formulations are known to one skilled in the art (see, e.g., U.S. Pub. No. 2012/0076812).
By “portion” or “fragment” is meant a part of a whole. A portion may comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the entire length of a polynucleotide or polypeptide sequence region. For polynucleotides, for example, a portion may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 or more contiguous nucleotides of a reference polynucleotide molecule. For polypeptides, for example, a portion may include at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 or more continuous amino acids of a reference polypeptide molecule.
In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more consecutive nucleotides of the polynucleotide SS-Spike-dF-PP (SEQ ID NOs: 5-6, 57-60, and 62- 65) or one or more of the polynucleotides encoding a polypeptide of SEQ ID NOs: 21 -22, 87-90, 92-95, and 103-107.
In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of SEQ ID NO: 21 . In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or more consecutive amino acids of a EGO polypeptide (SEQ ID NO: 87 or 92). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or more consecutive amino acids of a EG1 polypeptide (SEQ ID NO: 88 or 93). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG2 polypeptide (SEQ ID NO: 89 or 94). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG3 polypeptide (SEQ ID NO: 90 or 95). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG5 polypeptide (SEQ ID NO: 103). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG6 polypeptide (SEQ ID NO: 104). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG7 polypeptide (SEQ ID NO: 105). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG8 polypeptide (SEQ ID NO: 106). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of a EG9 polypeptide (SEQ ID NO: 107).
In some instances, administration of a fragment of a polynucleotide (e.g., a portion of one or more of SEQ ID NOs: 5-6, 57-60, and 62-65) and/or a polypeptide (e.g., a portion of one or more of SEQ ID NOs: 21 , 23, 87-90, 92-95, and 103-107) to a subject may elicit an immune response in the subject.
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.
By “promotes an immune response” is meant eliciting a humoral response (e.g., the production of antibodies) or a cellular response (e.g., the activation of T cells, macrophages, neutrophils, and/or natural killer cells) directed against, for example, one or more infective agents (e.g., a virus (e.g., a SARS-CoV-2 or a variant thereof)) or protein targets in a subject to which the pharmaceutical composition (e.g., an immunogenic composition or vaccine) has been administered.
The term "SARS-CoV-2 or a variant thereof-mediated disease" is used interchangeably with the terms "Coronavirus disease 2019 (COVID-19)" and “SARS-CoV-2” herein, as well as grammatical variants thereof, and refers to any pathology or sequelae known in the art to be caused by (alone or in association with other mediators), exacerbated by, or associated with SARS-CoV-2 or a variant thereof infection, including infection by a variant of SARS-CoV-2, such as those described herein or others that are later arising, or exposure in the subject having the disease. Disease can be acute (e.g., fever) or chronic (e.g., chronic fatigue), mild (e.g., hair loss) or severe (e.g., organ failure), and early-onset (e.g., 2- 14 days post-infection) or late-onset (e.g., 2 weeks post-infection). Non-limiting examples of severe disease include pneumonia, acute respiratory distress syndrome (ARDS), acute respiratory failure, pulmonary edema, organ failure, or death. Non-limiting examples of symptoms include weight loss, fever, cough, difficulty breathing, fatigue, headache, loss of taste or smell, hair loss, rash, sore throat, nausea, and diarrhea. Symptoms can be mild or severe (e.g., weight loss of greater than about 5% within a week and high fever) and temporary or permanent. In some embodiments, the SARS-CoV-2 is an Alpha, Beta, Gamma, Delta, or Omicron variant, such as a variant of the lineage B.1 .1 .529 (e.g., BA.1 , BA.2, BA.3, BA.4, and BA.5), B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , A23.1 , B.1 .617.1 , B.1 .617.2, B.1 .427, B.1 .525, B.1.526, P.1 , P.2, P.3, C.36, C.37, B.1.1.519, B.1 .526.1 , B.1 .526.2, R.1 , B.1 .258.17, B.1.575, B.1.214.2, A.2.5.2, AT.1 , B.1 .1 .523, or B.1 .620, or other known or subsequently arising lineage (see, e.g., cov- lineages.org/lineage_list.html, which is incorporated herein by reference). For example, the Omicron variant can be BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6.
A “protective level” refers to an amount or level of a marker (e.g., an antibody, a cell (e.g., an immune cell, e.g., a T cell, a B cell, an NK cell, or a neutrophil)) that is indicative of partial or complete protection from coronavirus infection or disease. An amount or level of a marker that is above the protective level indicates protection from coronavirus infection (e.g., a SARS-CoV-2 or a variant thereof infection) or disease (e.g., a SARS-CoV-2 or a variant thereof-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). An amount or level of a marker that is below the protective level indicates susceptibility to coronavirus infection or disease (e.g., a SARS-CoV-2 or a variant thereof-mediated disease, e.g., COVID-19, e.g., severe clinical disease). The marker may be a single measure (e.g., neutralizing antibody level) or the marker may be a combination of multiple measures (e.g., neutralizing antibody level and RBD-specific lgG2 level). In some instances, the protective level is an anticoronavirus antibody titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, an anti-coronavirus antibody titer of at least about 25 as measured using the live virus neutralization assay described herein, or an anti-coronavirus antibody titer that is above a level of at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. In some instances, the protective level is an anti-coronavirus antibody titer of at least about 100 as measured using the pseudovirus neutralization assay described herein.
As used herein, the term “sample” is a composition that is obtained or derived from a subject that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. A sample may be solid tissue as from a fresh, frozen, and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid. The sample may also be primary or cultured cells or cell lines. The sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, wax, nutrients, antibiotics, or the like.
By “sequence identity” or “sequence similarity” is meant that the identity or similarity, respectively, between two or more amino acid sequences, or two or more nucleotide sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of “percentage (%) identity,” in which a higher percentage indicates greater identity shared between the sequences. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similarity shared between the sequences. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Sequence identity/similarity can be determined across all or a defined portion of the two or more sequences compared.
By “signal peptide” or “signal sequence (SS)” is meant a short peptide (e.g., 5-30 amino acids in length, such as 17 amino acids in length, e.g., SEQ ID NO: 29) at the N-terminus of a polypeptide that directs a polypeptide towards the secretory pathway (e.g., the extracellular space). The signal peptide/signal sequence is typically cleaved during secretion of the polypeptide. The signal peptide/signal sequence may direct the polypeptide to an intracellular compartment or organelle, e.g., the Golgi apparatus. A signal peptide/signal sequence may be identified by homology, or biological activity, to a peptide with the known function of targeting a polypeptide to a particular region of the cell. One of ordinary skill in the art can identify a signal peptide/signal sequence by using readily available software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs). A signal peptide/signal sequence can be one that is, for example, substantially identical to the amino acid sequence of SEQ ID NO: 29. By “substantially identical” is meant that the signal peptide/signal sequence can have at least 80% or more (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 29.
As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nm, 1 pM, 100 pM, 500 pM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
As used herein, the term “stabilized polypeptide trimer” or “stabilized trimer” refers, but is not limited to, an oligomer that includes a protein and/or polypeptide sequence that increases the stability (e.g., via the presence of one or more oligomerization domains) of the trimeric structure (e.g., reduces dissociation of a trimer into monomeric units). The stabilized polypeptide trimer, for example, may be a homotrimer. An “oligomerization domain” refers, but is not limited to, a polypeptide sequence that can be used to increase the stability of an oligomeric envelope protein such as, e.g., to increase the stability of a Spike trimer. Oligomerization domains can be used to increase the stability of homooligomeric polypeptides as well as heterooligomeric polypeptides. Oligomerization domains are well known in the art, and include “trimerization domains.” A trimerization domain refers to an oligomerization domain that stabilizes trimeric polypeptides (e.g., trimers consisting of one or more of the Spike polypeptides). Examples of trimerization domains include, but are not limited to, the T4-fibritin “foldon” trimerization domain; the coiled-coil trimerization domain derived from GCN4 (Yang et al. (2002) J. Virol. 76:4634); and the catalytic subunit of E. co// aspartate transcarbamoylase as a trimer tag (Chen et al. (2004) J. Virol. 78:4508).
A “subject” is a vertebrate, such as a mammal (e.g., a primate and a human, in particular a human with underlying health conditions (e.g., hypertension, diabetes, or cardiovascular disease)). Mammals also include, but are not limited to, farm animals (such as cows), sport animals (e.g., horses), pets (such as cats, and dogs), mice, rats, bats, civets, and raccoon dogs. A subject to be treated according to the methods described herein (e.g., a subject in need of protection from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof) or having a coronavirus infection may be one who has been diagnosed by a medical practitioner as having such a need or infection. Diagnosis may be performed by any suitable means. A subject in whom the development of an infection is being prevented may or may not have received such a diagnosis. One skilled in the art will understand that a subject to be treated according to the disclosure may have been subjected to standard tests or may have been identified, without examination, as one with a suspected infection or at high risk of infection due to the presence of one or more risk factors (e.g., exposure to a coronavirus (e.g., SARS-CoV-2 or a variant thereof), for example, due to travel to an area where coronavirus infection is prevalent). Additionally, humans with underlying health conditions (e.g., hypertension, diabetes, or cardiovascular disease) are identified as subjects at high risk of infection with a coronavirus (e.g., SARS-CoV-2 or a variant thereof). The methods of treating a human subject with a composition are, therefore, particularly useful in treating, reducing, and/or preventing a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof) in humans with underlying health conditions.
As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of an exogenous nucleic acid molecule (e.g., DNA, such as an expression vector) into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection, and the like.
As used herein, the term “transformation” refers to any of a wide variety of techniques commonly used for the introduction of a virus (e.g., an adenovirus) into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection, and the like. Transformation also includes the use of a vector (e.g., an adenoviral vector), such as a vector described herein, to infect cells of a subject (e.g., a mammal, such as a human) being therapeutically-treated with the vector.
As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms (e.g., fever, joint pain, rash, conjunctivitis, muscle pain, headache, retro-orbital pain, edema, lymphadenopathy, malaise, asthenia, sore throat, cough, nausea, vomiting, diarrhea, and hematospermia) or conditions (Zammarchi et al., J. Clin. Virol. 63:32-5, 2015; Waddell et al., PLoS One 11 (5): e0156376, 2016); diminishment of the extent of disease, disorder, or condition; stabilization (e.g., not worsening) of a state of disease, disorder, or condition; prevention of spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or the time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. A treatment can include one or more therapeutic agents, such as one or more of the compositions described herein and/or one or more additional therapeutic agents. Additional therapeutic agents can include agents that stimulate (e.g., interferons) or inhibit (e.g., an antiinflammatory agent, such as corticosteroids, e.g., dexamethasone) the immune response. A treatment can include one or more therapeutic interventions, such as surgery or prone positioning.
The term “vaccine” as used herein, is defined as material used to provoke an immune response and that confers immunity for a period of time after administration of the vaccine to a subject.
By “vector” is meant a DNA construct that includes one or more polynucleotides, or fragments thereof, such as from a viral species, such as SARS-CoV-2 species. The vector can be used to infect cells of a subject, which results in the translation of the polynucleotides of the vector into a protein product. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the disclosure, “plasmid” and “vector” may, at times, be used interchangeably as the plasmid is the most commonly used form of vector. Other vectors include, e.g., viral vectors, such as adenoviral vectors (e.g., an Ad26 vector), in particular, those described herein.
The term “virus,” as used herein, is defined as an infectious agent that is unable to grow or reproduce outside a host cell and that infects mammals (e.g., humans).
A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (e.g., see PCT publication no. WO 2006/002203), alphaviral vectors and the like.
In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad (e.g., Ad26 and Ad52)) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Adenoviruses are a relatively well characterized, homogenous group of viruses, including over 50 serotypes (WO 95/27071 ). Adenoviruses are easy to grow and do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed (WO 95/00655 and WO 95/11984). Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo. To optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
The term “Spike” or “S”, as used herein, refers to a Spike sequence that does not contain a signal sequence (e.g., SEQ ID NOs: 1 , 4, 20, 22, and variants thereof). The term “SS”, as used herein, refers to a Spike signal sequence (e.g., SEQ ID NOs: 12, 29, and variants thereof). Therefore, as an example, the term “SS-Spike” would refer to a Spike sequence that contains a signal sequence (e.g., SEQ ID NOs: 2, 3, 5, 21 , or 23). The term “dF”, as used herein, refers to a mutated (i.e. , dead) furin cleavage site (e.g., SEQ ID NO: 18 residing within a Spike polynucleotide sequence being mutated into, e.g., SEQ ID: 19 ; or SEQ ID NO: 35 residing within a Spike polypeptide sequence being mutated into, e.g., SEQ ID NO: 36). The term “PP”, as used herein, refers to proline-stabilizing mutations (e.g., proline substitutions corresponding to amino acids K969 and V970 of full-length Spike (e.g., SEQ ID NO: 20)). As an example, the term “SS-Spike-dF-PP”, as used herein, refers to a Spike sequence that contains a signal sequence, a mutated furin cleavage site, and proline stabilizing mutations (e.g., SEQ ID NO: 23 or 5). The term “SdCT”, as used herein, refers to a Spike sequence that does not contain a signal sequence and a cytoplasmic region (e.g., SEQ ID NO: 7 or 24). As an example, the term “SS-SdCT” refers to a Spike sequence that contains a signal sequence and does not contain a cytoplasmic region (e.g., SEQ ID NO: 8 or 25). The term “S.Ecto”, as used herein, refers to a Spike ectodomain sequence that does not contain a signal sequence (e.g., SEQ ID NO: 9 or 26). The term “foldon” refers to a T4-fibritin foldon trimerization domain (e.g., SEQ ID NO: 15 or 32). As an example, the term “SS-S.Ecto-dF-PP-foldon”, refers to a Spike ectodomain sequence containing a signal sequence, a mutated furin cleavage site, proline stabilizing mutations, and a T4-fibritin foldon trimerization domain. The term “S1 ”, as used herein, refers to the S1 domain of Spike (e.g., residues 18-682 of SEQ ID NO: 21 or 23). The term “transmembrane domain” refers to a hydrophobic region of a protein that can be inserted or traverse a lipid membrane (e.g., SEQ ID NO: 13 or 30). Other features and advantages will be apparent from the following Detailed Description, the drawings, and the claims.
DETAILED DESCRIPTION
Described herein are Epigraph (EG) designed immunogens directed against Wuhan coronavirus (SARS-CoV-2) and, in particular, variants and subvariants thereof (e.g., Omicron), which can be used to elicit protective and therapeutic immune responses (e.g., humoral responses and/or cellular responses) against infection by the coronavirus infection (e.g., infection by SARS-CoV-2 or a variant thereof) when administered to a subject (e.g., a human subject). The use of Epigraph, which is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population, to design immunogens is described in Theiler et al. (Statistics in Medicine 37:181 , 2018) and Theiler et al. (Scientific Reports 6:33987, 2016), both of which are incorporated herein by reference. The compositions described herein can be prepared for administration to a subject (e.g., a human subject) and can include a protein of SARS-CoV-2, such as the spike (S) protein (e.g., an EGO, EG1 , EG2, EG3, and EG4 polypeptide of SEQ ID NOs: 87-91 , respectively; a spike-modified EGO, EG1 , EG2, EG3, and EG4 polypeptide of SEQ ID NOs: 92-96, respectively; a pan-sarbecovirus S protein (e.g., EG5, EG6, EG7, EG8, and EG9 polypeptide of SEQ ID NOs: 103-107); or any portion or variant polypeptide thereof with at least 85%, at least 90%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity thereto). These compositions can also include one or more of a SARS-CoV-2 nucleomembrane (MEM) protein (e.g., a MEMO, MEM1 , MEM2, MEM3, and MEM4 polypeptide of SEQ ID NO: 82-86, one or more of a pan-sarbecovirus MEM protein (e.g., MEM5 and MEM6), or any portion or variant polypeptide with at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity thereto); one or more of a SARS-CoV-2 nucleocapsid (NUL) protein (e.g., a NULO, NUL1 , NUL2, NUL3, NUL4 polypeptide of SEQ ID NO: 72-76, respectively), a pan-sarbecovirus NUL protein (e.g., NUL5, NUL6, and NUL7 polypeptide of SEQ ID NOs: 97-99), or any portion or variant polypeptide with at least 85%, at least 90%, at least 94%, at least 95%, at least 99% or more sequence identity thereto); and one or more of a SARS-CoV-2 conserved region (CR) protein (e.g., a CR0, CR1 , CR2, CR3, or CR4 polypeptide of SEQ ID NO: 77-81 ), a pan-sarbecovirus CR protein (e.g., a CR5 polypeptide of SEQ ID NO: 102), or any portion or variant polypeptide with at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity thereto). See Table 2 below for a summary of the S, MEM, NUL, CR, and other nucleotide and proteins sequences described herein (e.g., SEQ ID NOs: 1 -107).
Table 2. Summary of Nucleotide and Protein Sequences
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Spike = WT Spike without signal sequence; SdCT = WT Spike without signal sequence and without cytoplasmic region; S.Ecto = WT Spike ectodomain without signal sequence; S1 = WT Spike S1 domain without signal sequence; RBD = WT Spike receptor binding domain without signal sequence; SS- = WT Spike signal sequence; -dF = mutated "dead" furin cleavage site; -PP = proline stabilizing mutations (proline substitutions corresponding to amino acids K969 and V970 of full-length Spike); -foldon = foldon trimerization domain with linker; K.H. Kozak- = Kpnl-Hindlll-Kozak; -S2.N.E = -Stop2-Nhel-EcoRI; EG = Epigraph.
While the nucleotide sequences described here, such as those in in Table 2 (e.g., SEQ ID NO: 1 - 19 and 37-66), are provided as a DNA sequence (e.g., consisting of A, T, C, and G), the present disclosure contemplates the nucleotide sequences described here, such as those in in Table 2 (e.g., SEQ ID NO: 1 -19 and 37-66), as an RNA sequence (e.g., consisting of an A, U, C, and G) as well. For example, to generate an RNA sequence of any one of SEQ ID NOs: 1 -19 and 37-66, one may replace all thymine (“T”) nucleotides with uracil (“U”) nucleotides. Therefore, the nucleic acid molecules, vectors, and compositions described below may contain a DNA or RNA nucleotide sequence of any one or more of SEQ ID NOs: 1 -19 and 37-66.
A composition of the disclosure can also include one or more of the SARS-CoV-2 MEM protein (e.g., a MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, and MEM6 polypeptide of SEQ ID NO: 82-86, 100, and 101 , or any portion or variant polypeptide with at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity thereto), one or more of the SARS-CoV-2 NUL protein (e.g., a NULO, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and NUL7 polypeptide of SEQ ID NOs: 72-76 and 97-99, respectively, or any portion or variant polypeptide with at least 85%, at least 90%, at least 94%, at least 95%, at least 99% or more sequence identity thereto), and one or more of the SARS-CoV-2 CR protein (e.g., a CR0, CR1 , CR2, CR3, CR4, and CR5 polypeptide of SEQ ID NO: 77-81 and 102, or any portion or variant polypeptide with at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity thereto). The MEM, NUL, and CR proteins can be administered alone or in combination with one or more of the Spike proteins described herein (e.g., EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and EG9, or a portion or variant thereof). A composition of the disclosure also includes a vector (e.g., an expression vector, such as a plasmid, or a viral vector, such as an adenovirus (e.g., Ad26 or Ad52), poxvirus, adeno-associated virus, retroviral, or other viral vector, or naked or encapsulated DNA) containing a nucleic acid sequence that encodes a SARS-CoV-2 polypeptide (e.g., a S, MEM, CR, and/or NUL protein) or a variant thereof (e.g., a nucleic acid molecule with the sequence of any one of SEQ ID NOs: 42-66, or a nucleic acid molecule with at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity thereto). The composition may contain a vector(s) that encodes only one or more S proteins, such as those described herein (e.g., an EGO, EG1 , EG2, EG3, and/or EG4 polypeptide, or a portion or variant thereof, as described herein). The composition may be one that contains a vector(s) that encodes only MEM, CR, and/or NUL polypeptides, such as those described herein. The composition may also contain a vector(s) that encodes a S, MEM, CR, and/or NUL polypeptides, as described herein.
The generation of DNA and RNA vaccines expressing a S, MEM, CR, and/or NUL protein of SARS-CoV-2, or a variant thereof, are described herein. The DNA and RNA vaccines can be generated by incorporating a polynucleotide (e.g., all or a fragment of any one or more of SEQ ID NOs: 42-66, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto) encoding S, MEM, CR, NUL, or a portion thereof (e.g., all or a fragment of any one or more of SEQ ID NOs: 72-107), or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto), or a complement sequence thereof, into a mammalian expression vector (e.g., pcDNA3.1 + ; Invitrogen, CA, USA) or a viral vector (e.g., an adenoviral vector) to generate the vaccine. The polynucleotide may also be one that encodes the polypeptide of any one of SEQ ID NOs: 97-107 or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), or a complement sequence thereof.
Generation of recombinant viral vectors (e.g., adenoviral vectors, such as Ad26 and Ad52 viral vectors) expressing a modified S, MEM, CR and/or NUL protein(s) of SARS-CoV-2 or a portion or variant thereof are also described. A viral vector encoding a modified S, MEM, CR, and/or NUL protein(s) of SARS-CoV-2 or a portion or variant thereof can be generated by incorporating a polynucleotide (e.g., all or a fragment of any one or more of SEQ ID NOs: 42-66, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto) encoding S, MEM, CR, and/or NUL, or a portion thereof (e.g., SEQ ID NOs: 72-107), or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto (e.g., all or a fragment of any one or more of SEQ ID NOs: 72-107) into a viral vector (e.g., an Ad26 or Ad52 viral vector).
Anti-coronavirus antibodies (e.g., antibodies against a modified S, MEM, CR, or NUL protein of SARS-CoV-2 or a variant thereof, such as those described herein (e.g., anti-Spike antibodies, anti-Spike neutralizing antibodies, or broadly neutralizing anti-Spike antibodies) present in a sample from a subject (e.g., a human subject) can be used to detect and/or monitor a protective antibody response in the subject. The anti-coronavirus antibodies (e.g., anti-Spike antibodies, anti-Spike neutralizing antibodies, or broadly neutralizing anti-Spike antibodies) may be measured in a short timeframe (e.g., between 1 day post-administration and 8-weeks post-administration) or a longer timeframe (e.g., between 2 month post- administration and 15 years post-administration) after administration of a therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein).
The nucleic acid molecules, polypeptides, vectors, monovalent vaccines, polyvalent vaccines, compositions, and antibodies described herein can also be used in methods of treating and/or inhibiting a SARS-CoV-2 infection in a subject (e.g., a human).
COMPOSITIONS AND METHODS
Nucleic Acid Molecules
Nucleic acid molecules of the disclosure include Epigraph (EG) spike (S) polynucleotide sequences (e.g., EG0-EG9), membrane (MEM, e.g., MEM0-MEM6), conserved region (CR, e.g., CRO- CR5), and nucleocapsid (NUL, e.g., NUL0-NUL7) polynucleotide sequences. The EG, MEM, CR, and NUL polynucleotide sequence may be in the form of a DNA polynucleotide (e.g., SEQ ID NOs: 1 -19 and 37-66) or an RNA polynucleotide (e.g., by replacing all thymine (“T”) nucleotides with uracil (“U”) nucleotides in SEQ ID NOs: 1 -19 and 37-66.
The nucleic acid molecules of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, and/or EG4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 57-66. The nucleic acid molecules of the disclosure may contain one or more NUL (e.g., NULO, NUL1 , NUL2, NUL3, and/or NUL4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 42-46. The nucleic acid molecules of the disclosure may contain one or more CR (e.g., CR0, CR1 , CR2, CR3, and/or CR4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 47-51 . The nucleic acid molecules of the disclosure may contain one or more MEM (e.g., MEMO, MEM1 , MEM2, MEM3, and/or MEM4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 52-56. The nucleic acid molecules of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, and/or EG4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 57-66, as well as one or more NUL (e.g., NULO, NUL1 , NUL2, NUL3, and/or NUL4), CR (e.g., CR0, CR1 , CR2, CR3, and/or CR4), and/or MEM (e.g., MEMO, MEM1 , MEM2, MEM3, and/or MEM4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 42-56. The nucleic acid molecules of the disclosure may also encode an EG, MEM, NUL, or CR polypeptide with an amino acid sequence of any one of SEQ ID NOs: 97-107 or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), or a complement sequence thereof.
The nucleic acid molecules of SARS-CoV-2 spike Epigraph (EG) immunogen EGO (e.g., SEQ ID NOs: 57 and 62 or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto), EG1 (e.g., SEQ ID NOs: 58 and 63 or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto), EG2 (e.g., SEQ ID NOs: 59 and 64 or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto), and EG3 (e.g., SEQ ID NOs: 60 and
65 or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more sequence identity thereto) were designed using methods developed in earlier studies (e.g., Theiler et al., Statistics in Medicine 37:181 (2018); Theiler et al., Scientific Reports 6:33987 (2016), herein incorporated by reference) of emerging variants around the world of the Wuhan coronavirus (SARS-CoV-2 or a variant thereof) relative to the wildtype SARS-CoV-2 S protein (e.g., SEQ ID NO: 2) and existing optimizations thereof (e.g., SEQ ID NO: 5, e.g., see U.S. Patent Application number 63/066,147, incorporated by reference herein; see also PCT/US2021/015946, incorporated by reference herein).
The nucleic acid molecules described herein encode an S protein of SARS-CoV-2 or a variant thereof that has been modified to include one or more resistance mutations and includes, for example, the EGO polypeptide, which includes the mutations: T19R, G142D, T478K, D614G, and P681 R, relative to the amino acid sequence of SEQ ID NO: 21 or 23; the EG1 polypeptide, which includes the mutations: L5F, V36F, L54F, A67V, A69-70, D80Y, T95I, S112L, V126A, A156-157, R158G, G181V, R190S V213G, A222V, I233V, A241-243, D253G, A262S, P272L, V289I, T299I, V308L, T323I, G339D, R346K, R357K, S371 F, S373P, S375F, T376A, T385I, D405N, R408S, K417N, T430I, N440K, L452R, S477N, T478K, E484A, Q493R, Q498R, N501 Y, Y505H, A520S, T547K, K558N, A570D, E583D, T604I, Q613H, D614G, V622F, P631 S, S640F, H655Y, I670V, N679K, P681 H, A694V, S704L, T716I, T732A, T747I, N764K, E780Q, D796Y, P809S, L822F, A831V, A845S, N856K, I870V, A879V, F888L, A899S, I909V, S929T, S939F, D950N, N969K, L981 F, T1006I, T1027I, L1049I, L1063F, A1078S, A1087S, V1104L, D1118H, D1127G, L1141 F, D1153Y, P1162S, V1176F, K1191 N, E1202Q, G1219V, V1228L, M1237I, S1252F, and V1264L relative to the amino acid sequence of SEQ ID NO: 21 or 23; the EG2 polypeptide, which includes the mutations: V3G, T19I, A24-26, A27S, V36F, H49Y, A69-70, D80A, S98F, D1 11 N, V127F, G142D, A143-145, E154K, N164K, L176F, L189F, D198G, N211 I, L212V, 213-214insRE, V213P, R214E, D228H, T240I, P251 L, G261V, R273S, T284I, T307I, V320I, P330S, G339D, N354K, S371 L, S373P, S375F, P384L, D405N, K417T, D427N, N440K, G446S, L455F, Q493R, G496S, Q498R, N501Y, A522V, T547I, N556K, T572I, L585F, Q628K, F643L, H655Y, N658S, A672V, S691 F, A701V, T719I, M731 I, M740V, G769V, T778I, Q787H, D796H, P812S, L822F, I834V, I850L, T859N, A879S, A892V, I909V, L922F, D936Y, Q954H, N969K, S982A, T1009I, A1020S, H1058Y, Q1071 H, 11081V, R1091 H, H1101Y, I1114T, G1124V, V1133I, E1150D, G1167V, V1177I, E1207D, G1219C, M1229I, T1238I, C1247F, and D1259Y, all relative to the amino acid sequence of SEQ ID NO: 21 or 23; and the EG3 polypeptide, which includes the mutations: V6F, L18F, T20N, V36F, Q52R, D80G, T95I, K97E, L118F, V127F, A145, M153T, N164T, Q173H, G184S, V193L, A211 , L212I, S221 L, R237S, T250I, T259I, E281 Q, T299I, E309Q, V327I, R346K, K356R, V367L, V382L, Q414R, D427V, N439K, L452Q, T478K, E484A, F486V, N501Y, A522S, N540S, A570S, E583Q, T604I, Q613H, A626S, A647S, E661 D, A672V, P681 H, A688V, A706V, T723I, S735A, T747I, V772I, T791 I, P812L, L822F, I834T, A846V, T859I, A871 S, T883I, A892S, I909V, S929I, L938F, D950H, T961 M, D979E, T1006I, A1020V, M1050I, L1063F, N1074S, H1083Y, H1101 D, T1117I, 11132V, S1147L, P1162L, 11179V, Q1208H, 11221 T, L1234I, C1243F, S1252P, and P1263L, all relative to the amino acid sequence of SEQ ID NO: 21 or 23. The nucleic acid molecules may also encode a S protein of SARS-CoV-2 or a variant thereof containing further modifications to one or more regions. For example, the full-length (SEQ ID NO: 2) Spike with a deletion of the cytoplasmic region (SEQ ID NO: 7), the ectodomain (SEQ ID NO: 9), S1 (SEQ ID NO: 10), and the receptor binding domain (SEQ ID NO: 11 ). The nucleic acid molecules may also feature additional modifications to regions of S, including deletion of or inclusion of a signal sequence (e.g., SEQ ID NO: 12), one or more stabilizing mutations (e.g., proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 20), a mutation that inactivates a furin cleavage site (e.g., SEQ ID NO: 19), introduction of a trimerization domain (e.g., a foldon trimerization domain, e.g., SEQ ID NO: 15), introduction of linker or spacer sequences (e.g., SEQ ID NOs: 16 and 17), and combinations thereof.
The nucleic acid molecules may also encode an S protein of SARS-CoV-2 or a variant thereof containing further modifications to one or more regions. For example, the nucleic acid molecule may encode a S protein, as defined herein, with a deletion of the cytoplasmic region (e.g., SEQ ID NO: 7), with a deletion of the cytoplasmic (e.g., SEQ ID NO: 14) and transmembrane (e.g., SEQ ID NO: 13) domains, leaving only S protein ectodomain (e.g., SEQ ID NO: 9), a deletion of the S1 domain (e.g., a deletion of SEQ ID NO: 10, or variant thereof, within a S polynucleotide (e.g., SEQ ID NO: 2), or variant thereof)), a deletion of the S2 region such that only the S1 region of S remains, or a S protein with a deletion of the receptor binding domain (e.g., a deletion of SEQ ID NO: 11 , or variant thereof, within a S polynucleotide (e.g., SEQ ID NO: 2), or variant thereof).
The nucleic acid molecules may also feature additional modifications to regions of S protein, including deletion of or inclusion of a signal sequence (e.g., SEQ ID NO: 12), one or more stabilizing mutations (e.g., proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 20), a mutation that inactivates a furin cleavage site (e.g., SEQ ID NO: 19), introduction of a trimerization domain (e.g., a foldon trimerization domain, e.g., SEQ ID NO: 15), introduction of linker or spacer sequences (e.g., SEQ ID NOs: 16 and 17), and combinations thereof. The nucleic acid molecule may also contain a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to at least 500 contiguous amino acids within positions 18-1208 (e.g., positions 50-1100, 100-1000, 200- 900, and 300-800) of any one of SEQ ID NOs: 21 and 23 or a complementary sequence thereof, in which the polypeptide has at least one of the following mutations: V3G, L5F, V6F, 213-214insRE, L18F, T19I, T19R, T20N, A24-26, A27S, V36F, H49Y, Q52R, L54F, A67V, A69-70, D80A, D80G, D80Y, T95I, K97E, S98F, V126A, V127F, G142D, A143-145, A145, M153T, E154K, A156-157, R158G, N164K, N164T, Q173H, L176F, G181V, G184S, L189F, R190S, V193L, D198G, A211 , N211 I, L212I, L212V, V213G, V213P, R214E, S221 L, A222V, D228H, I233V, R237S, T240I, A241 -243, T250I, P251 L, D253G, T259I, G261V, A262S, P272L, R273S, E281 Q, T284I, V289I, T299I, T307I, V308L, E309Q, V320I, T323I, V327I, P330S, G339D, R346K, N354K, K356R, R357K, V367L, S371 F, S371 L, S373P, S375F, T376A, V382L, P384L, T385I, D405N, R408S, Q414R, K417N, K417T, D427N, D427V, T430I, N439K, N440K, G446S, L452Q, L452R, L455F, S477N, T478K, E484A, F486V, Q493R, G496S, Q498R, N501 Y, Y505H, A520S, A522S, A522V, N540S, T547I, T547K, N556K, K558N, A570D, A570S, T572I, E583D, E583Q, L585F, T604I, Q613H, D614G, V622F, A626S, Q628K, P631 S, S640F, F643L, A647S, H655Y, N658S, E661 D, I670V, A672V, N679K, A688V, S691 F, A694V, A701V, S704L, A706V, T716I, T719I, T723I, M731 I, T732A, S735A, M740V, T747I, N764K, G769V, V772I, T778I, E780Q, Q787H, T791 I, D796H, D796Y, P809S, P812L, P812S, L822F, A831V, I834T, I834V, A845S, A846V, I850L, N856K, T859I, T859N, I870V, A871 S, A879S, A879V, T883I, F888L, A892S, A892V, A899S, I909V, L922F, S929I, S929T, D936Y, L938F, S939F, D950H, D950N, Q954H, T961 M, N969K, D979E, L981 F, S982A, T1006I, T1009I, A1020S, A1020V, T1027I, L1049I, H1058Y, M1050I, L1063F, A1078S, N1074S, Q1071 H, A1087S, H1083Y, 11081 V, R1091 H, H1101 D, H1101Y, V1104L, D1118H, D111 N, I1114T, T1117I, D1127G, G1124V, S112L, 11132V, V1133I, L1141 F, S1147L, D1153Y, E1150D, G1167V, P1162L, P1162S, 11179V, V1176F, V1177I, L118F, K1191 N, E1202Q, E1207D, Q1208H, G1219C, G1219V, I1221T, M1229I, V1228L, L1234I, M1237I, T1238I, C1243F, C1247F, D1259Y, S1252F, S1252P, P1263L, and V1264L (or any mutations shown in Figure 1 ) relative to the amino acid sequence of SEQ ID NO: 21 or 23.
In some embodiments, the nucleic acid molecule contains a nucleotide sequence that encodes a coronavirus Spike polypeptide containing two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty- three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty- two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty- two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty-two, sixty- three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty-nine, seventy, seventy-one, seventy- two, seventy-three, seventy-four, seventy-five, seventy-six, seventy-seven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, eighty-eight, eighty-nine, ninety, ninety-one, ninety-two, ninety-three, ninety-four, ninety-five, ninety-six, ninety-seven, ninety-eight, ninety-nine, one hundred, one hundred one, one hundred two, or one hundred three of the mutations in the context of a polypeptide molecule with a polypeptide sequence of SEQ ID NO: 21 or 23, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. In particular, the Spike polypeptide contains five to one hundred three of the following mutations: V3G, L5F, V6F, 213-214insRE, L18F, T19I, T19R, T20N, A24-26, A27S, V36F, H49Y, Q52R, L54F, A67V, A69-70, D80A, D80G, D80Y, T95I, K97E, S98F, V126A, V127F, G142D, A143-145, A145, M153T, E154K, A156-157, R158G, N164K, N164T, Q173H, L176F, G181V, G184S, L189F, R190S, V193L, D198G, A211 , N2111, L212I, L212V, V213G, V213P, R214E, S221 L, A222V, D228H, I233V, R237S, T240I, A241 -243, T250I, P251 L, D253G, T259I, G261V, A262S, P272L, R273S, E281 Q, T284I, V289I, T299I, T307I, V308L, E309Q, V320I, T323I, V327I, P330S, G339D, R346K, N354K, K356R, R357K, V367L, S371 F, S371 L, S373P, S375F, T376A, V382L, P384L, T385I, D405N, R408S, Q414R, K417N, K417T, D427N, D427V, T430I, N439K, N440K, G446S, L452Q, L452R, L455F, S477N, T478K, E484A, F486V, Q493R, G496S, Q498R, N501Y, Y505H, A520S, A522S, A522V, N540S, T547I, T547K, N556K, K558N, A570D, A570S, T572I, E583D, E583Q, L585F, T604I, Q613H, D614G, V622F, A626S, Q628K, P631 S, S640F, F643L, A647S, H655Y, N658S, E661 D, I670V, A672V, N679K, A688V, S691 F, A694V, A701V, S704L, A706V, T716I, T719I, T723I, M731 I, T732A, S735A, M740V, T747I, N764K, G769V, V772I, T778I, E780Q, Q787H, T791 I, D796H, D796Y, P809S, P812L, P812S, L822F, A831V, I834T, I834V, A845S, A846V, I850L, N856K, T859I, T859N, I870V, A871 S, A879S, A879V, T883I, F888L, A892S, A892V, A899S, I909V, L922F, S929I, S929T, D936Y, L938F, S939F, D950H, D950N, Q954H, T961 M, N969K, D979E, L981 F, S982A, T1006I, T1009I, A1020S, A1020V, T1027I, L1049I, H1058Y, M1050I, L1063F, A1078S, N1074S, Q1071 H, A1087S, H1083Y, 11081 V, R1091 H, H1101 D, H1101Y, V1104L, D1118H, D111 N, I1114T, T1117I, D1127G, G1 124V, S1 12L, 11 132V, V1 133I, L1 141 F, S1 147L, D1 153Y, E1 150D, G1 167V, P1 162L, P1 162S, 11 179V, V1 176F, V1 177I, L1 18F, K1 191 N, E1202Q, E1207D, Q1208H, G1219C, G1219V, I1221 T, M1229I, V1228L, L1234I, M1237I, T1238I, C1243F, C1247F, D1259Y, S1252F, S1252P, P1263L, and V1264L relative to the amino acid sequence of SEQ ID NO: 21 or 23.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 spike polypeptide with one or more of the mutations: T19R, G142D, T478K, D614G, and P681 R relative to the amino acid sequence of SEQ ID NO: 21 or 23. The polypeptide may contain two, three, four, or five of the mutations: T19R, G142D, T478K, D614G, and P681 R, relative to the amino acid sequence of SEQ ID NO: 21 or 23. In particular, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with each of the mutations: T19R, G142D, T478K, D614G, and P681 R, relative to the amino acid sequence of SEQ ID NO: 21 or 23. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 87, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 92, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. The nucleic acid molecule may have the nucleic acid sequence of SEQ ID NOs: 57 or 62, or a variant thereof with at least 85% sequence identity thereto, such as a variant that encodes a polypeptide that contains each of the indicated mutations.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 spike polypeptide with one or more of the mutations: L5F, V36F, L54F, A67V, A69-70, D80Y, T95I, S1 12L, V126A, A156-157, R158G, G181V, R190S V213G, A222V, I233V, A241 -243, D253G, A262S, P272L, V289I, T299I, V308L, T323I, G339D, R346K, R357K, S371 F, S373P, S375F, T376A, T385I, D405N, R408S, K417N, T430I, N440K, L452R, S477N, T478K, E484A, Q493R, Q498R, N501 Y, Y505H, A520S, T547K, K558N, A570D, E583D, T604I, Q613H, D614G, V622F, P631 S, S640F, H655Y, I670V, N679K, P681 H, A694V, S704L, T716I, T732A, T747I, N764K, E780Q, D796Y, P809S, L822F, A831 V, A845S, N856K, I870V, A879V, F888L, A899S, I909V, S929T, S939F, D950N, N969K, L981 F, T1006I, T1027I, L1049I, L1063F, A1078S, A1087S, V1104L, D1 1 18H, D1 127G, L1 141 F, D1 153Y, P1 162S, V1 176F, K1 191 N, E1202Q, G1219V, V1228L, M1237I, S1252F, and V1264L, relative to the amino acid sequence of SEQ ID NO: 21 or 23. The polypeptide may contain two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirtyseven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, fortyseven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fiftyeight, fifty-nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixtyeight, sixty-nine, seventy, seventy-one, seventy-two, seventy-three, seventy-four, seventy-five, seventy- six, seventy-seven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, eighty-eight, eighty-nine, ninety, ninety-one, ninety-two, ninety-three, ninety-four, ninety-five, ninety-six, ninety-seven, ninety-eight, ninety-nine, one hundred, one hundred one, one hundred two, or one hundred three of the mutations: L5F, V36F, L54F, A67V, A69-70, D80Y, T95I, S112L, V126A, A156-157, R158G, G181V, R190S V213G, A222V, I233V, A241-243, D253G, A262S, P272L, V289I, T299I, V308L, T323I, G339D, R346K, R357K, S371 F, S373P, S375F, T376A, T385!, D405N, R408S, K417N, T430I, N440K, L452R, S477N, T478K, E484A, Q493R, Q498R, N501 Y, Y505H, A520S, T547K, K558N, A570D, E583D, T604I, Q613H, D614G, V622F, P631 S, S640F, H655Y, I670V, N679K, P681 H, A694V, S704L, T716I, T732A, T747I, N764K, E780Q, D796Y, P809S, L822F, A831V, A845S, N856K, I870V, A879V, F888L, A899S, I909V, S929T, S939F, D950N, N969K, L981 F, T1006I, T1027I, L1049I, L1063F, A1078S, A1087S, V1104L, D1118H, D1127G, L1141 F, D1153Y, P1162S, V1176F, K1191 N, E1202Q, G1219V, V1228L, M1237I, S1252F, and V1264L, relative to the amino acid sequence of SEQ ID NO: 21 or 23. In particular, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with each of the mutations: L5F, V36F, L54F, A67V, A69-70, D80Y, T95!, S112L, V126A, Al 56-157, R158G, G181V, R190S V213G, A222V, I233V, A241-243, D253G, A262S, P272L, V289I, T299I, V308L, T323I, G339D, R346K, R357K, S371 F, S373P, S375F, T376A, T385I, D405N, R408S, K417N, T430I, N440K, L452R, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, A520S, T547K, K558N, A570D, E583D, T604S, Q613H, D614G, V622F, P631 S, S640F, H655Y, 1670V, N679K, P681 H, A694V, S704L, T716I, T732A, T747I, N764K, E780Q, D796Y, P809S, L822F, A831V, A845S, N856K, I870V, A879V, F888L, A899S, I909V, S929T, S939F, D950N, N969K, L981 F, T1006I, T1027I, L1049I, L1063F, A1078S, A1087S, V1104L, D1118H, D1127G, L1141 F, D1153Y, P1162S, V1176F, K1191 N, E1202Q, G1219V, V1228L, M1237I, S1252F, and V1264L, relative to the amino acid sequence of SEQ ID NO: 21 or 23. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 88, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 93, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. The nucleic acid molecule may have the nucleic acid sequence of SEQ ID NOs: 58 or 63, or a variant thereof with at least 85% sequence identity thereto, such as a variant that encodes a polypeptide that contains each of the indicated mutations.
A nucleic acid molecule of the disclosure may also contain a nucleotide sequence that encodes a SARS-CoV-2 spike polypeptide with one or more of the mutations: V3G, T19I, A24-26, A27S, V36F, H49Y, A69-70, D80A, S98F, D111 N, V127F, G142D, A143-145, E154K, N164K, L176F, L189F, D198G, N211 I, L212V, 213-214insRE, V213P, R214E, D228H, T240I, P251 L, G261V, R273S, T284I, T307I, V320I, P330S, G339D, N354K, S371 L, S373P, S375F, P384L, D405N, K417T, D427N, N440K, G446S, L.455F, Q493R, G496S, Q498R, N501 Y, A522V, T547I, N556K, T572I, L585F, Q628K, F643L, H655Y, N658S, A672V, S691 F, A701V, T719I, M731 I, M740V, G769V, T778I, Q787H, D796H, P812S, L822F, I834V, I850L, T859N, A879S, A892V, I909V, L922F, D936Y, Q954H, N969K, S982A, T1009I, A1020S, H1058Y, Q1071 H, 11081V, R1091 H, H1101Y, I1114T, G1124V, V1133I, E1150D, G1167V, V1177I, E1207D, G1219C, M1229I, T1238I, C1247F, and D1259Y, relative to the amino acid sequence of SEQ ID NO: 21 or 23. The polypeptide may contain two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty- one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty- one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty-nine, seventy, seventy-one, seventy-two, seventy-three, seventy-four, seventy-five, seventy-six, seventy-seven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, eighty-eight, eighty-nine, ninety, ninety-one, ninety-two, ninety-three, ninety-four, ninety-five, ninety-six, ninety-seven, ninety-eight, or ninety-nine of the mutations: V3G, T19I, A24-26, A 70, D80A, S98F, D111 N, V127F, G142D, A143-145, E154K, N164K, L176F, L18 L212V, 213-214insRE, V213P, R214E, D228H, T240I, P251 L, G261 V, R273S, T P330S, G339D, N354K, S371 L, S373P, S375F, P384L, D405N, K417T, D427N, Q493R, G496S, Q498R, N501 Y, A522V, T547I, N556K, T572I, L585F, Q628K, F A672V, S691 F, A701V, T719I, M731 I, M740V, G769V, T778I, Q787H, D796H, P I850L, T859N, A879S, A892V, I909V, L922F, D936Y, Q954H, N969K, S982A, T1
Figure imgf000034_0001
Q1071 H, 11081V, R1091 H, H1101Y, I1114T, G1124V, V1133I, E1150D, G1167V, V1177I, E1207D, G1219C, M1229I, T1238I, C1247F, and D1259Y, relative to the amino acid sequence of SEQ ID NO: 21 or 23. In particular, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with each of the mutations: V3G, T19I, A24-26, A27S, V36F, H49Y, A69-70, D80A, S98F, D111 N, V127F, G142D, A143-145, E154K, N164K, L176F, L189F, D198G, N211 I, L212V, 213-214insRE, V213P, R214E, D228H, T240I, P251 L, G261 V, R273S, T284I, T307I, V320I, P330S, G339D, N354K, S371 L, S373P, S375F, P384L, D405N, K417T, D427N, N440K, G446S, L455F, Q493R, G496S, Q498R, N501Y, A522V, T547I, N556K, T572I, L585F, Q628K, F643L, H655Y, N658S, A672V, S691 F, A701 V, T719I, M731 I, M740V, G769V, T778I, Q787H, D796H, P812S, L822F, I834V, I850L, T859N, A879S, A892V, I909V, L922F, D936Y, Q954H, N969K, S982A, T1009I, A1020S, H1058Y, Q1071 H, 11081V, R1091 H, H1101Y, I1114T, G1124V, V1133I, E1150D, G1167V, V1177I, E1207D, G1219C, M1229I, T1238I, C1247F, and D1259Y, relative to the amino acid sequence of SEQ ID NO: 21 or 23. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide has the amino acid sequence of SEQ ID NO: 89, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide has the amino acid sequence of SEQ ID NO: 94, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. The nucleic acid molecule may have the nucleic acid sequence of SEQ ID NOs: 59 or 64, or a variant thereof with at least 85% sequence identity thereto, such as a variant that encodes a polypeptide that contains each of the indicated mutations.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 spike polypeptide with one or more of the mutations: V6F, L18F, T20N, V36F, Q52R, D80G, T95I, K97E, L118F, V127F, A145, M153T, N164T, Q173H, G184S, V193L, A211 , L.212I, S221 L, R237S, T250I, T259I, E281 Q, T299I, E309Q, V327I, R346K, K356R, V367L, V382L, Q414R, D427V, N439K, L.452Q, T478K, E484A, F486V, N501Y, A522S, N540S, A570S, E583Q, T604I, Q613H, A626S, A647S, E661 D, A672V, P681 H, A688V, A706V, T723I, S735A, T747I, V772I, T791 I, P812L, L822F, I834T, A846V, T859I, A871 S, T883I, A892S, I909V, S929I, L938F, D950H, T961 M, D979E, T1006I, A1020V, M1050I, L1063F, N1074S, H1083Y, H1101 D, T1117I, 11132V, S1147L, P1162L, 11179V, Q1208H, 11221 T, L1234I, C1243F, S1252P, and P1263L relative to the amino acid sequence of SEQ ID NO: 21 or 23. The polypeptide may contain two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirtyeight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, fortyeight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty- nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty- nine, seventy, seventy-one, seventy-two, seventy-three, seventy-four, seventy-five, seventy-six, seventyseven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, or eighty-eight of the mutations: V6F, L18F, T20N, V36F, Q52R, D80G, T95I, K97E, L118F, V127F, A145, M153T, N164T, Q173H, G184S, V193L, A211 , L212I, S221 L, R237S, T250I, T259I, E281 Q, T299I, E309Q, V327I, R346K, K356R, V367L, V382L, Q414R, D427V, N439K, L452Q, T478K, E484A, F486V, N501Y, A522S, N540S, A570S, E583Q, T604I, Q613H, A626S, A647S, E661 D, A672V, P681 H, A688V, A706V, T723I, S735A, T747I, V772I, T791 I, P812L, L822F, I834T, A846V, T859I, A871 S, T883I, A892S, I909V, S929I, L938F, D950H, T961 M, D979E, T1006I, A1020V, M1050I, L1063F, N1074S, H1083Y, H1101 D, T1117I, 11132V, S1147L, P1162L, 11179V, Q1208H, 11221 T, L1234I, C1243F, S1252P, and P1263L, relative to the amino acid sequence of SEQ ID NO: 21 or 23. In particular, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with each of the mutations: V6F, L18F, T20N, V36F, Q52R, D80G, T95I, K97E, L118F, V127F, A145, M153T, N164T, Q173H, G184S, V193L, A211 , L212I, S221 L, R237S, T250I, T259I, E281 Q, T299I, E309Q, V327I, R346K, K356R, V367L, V382L, Q414R, D427V, N439K, L452Q, T478K, E484A, F486V, N501Y, A522S, N540S, A570S, E583Q, T604I, Q613H, A626S, A647S, E661 D, A672V, P681 H, A688V, A706V, T723I, S735A, T747I, V772I, T791 I, P812L, L822F, I834T, A846V, T859I, A871 S, T883I, A892S, I909V, S929I, L938F, D950H, T961 M, D979E, T1006I, A1020V, M1050I, L1063F, N1074S, H1083Y, H1101 D, T1117I, 11132V, S1147L, P1162L, 11179V, Q1208H, 11221 T, L1234I, C1243F, S1252P, and P1263L, relative to the amino acid sequence of SEQ ID NO: 21 or 23. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 90, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. For example, the nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 95, or a variant thereof with at least 85% sequence identity thereto, such as a variant that contains each of the indicated mutations. The nucleic acid molecule may have the nucleic acid sequence of SEQ ID NOs: 60 or 65, or a variant thereof with at least 85% sequence identity thereto, such as a variant that encodes a polypeptide that contains each of the indicated mutations.
In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having 100% sequence identity to amino acids 18-1208 of SEQ ID NO: 87 or SEQ ID NO: 92. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 18-1201 of SEQ ID NO: 88 or SEQ ID NO: 93. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 18-1200 of SEQ ID NO: 89 or SEQ ID NO: 94. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 18-1204 of SEQ ID NO: 90 or SEQ ID NO: 95.
In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 103. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 104. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 105. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 106. In some embodiments, an isolated nucleic acid molecule may contain a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 107.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 membrane (MEM) polypeptide having at least 85% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200) of SEQ ID NO: 82 (e.g., MEMO) or a complementary sequence thereof. In some embodiments, the nucleotide sequence has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of SEQ ID NO: 52, or a complementary sequence thereof. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject. In some embodiments, the nucleic acid molecule has the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the nucleic acid molecule has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 52 but does not encode the sequence of SEQ ID NO: 82.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 membrane (MEM) polypeptide having at least 85% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200) of SEQ ID NO: 100 (e.g., MEM5) or SEQ ID NO: 101 (e.g., MEM6), or a complementary sequence thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of SEQ ID NO: 100 (e.g., MEM5), or a complementary sequence thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of SEQ ID NO: 101 (e.g., MEM6), or a complementary sequence thereof. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject. A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV2 nucleocapsid (NUL) polypeptide having at least 85% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200) of any one of SEQ ID NOs: 72-73 (e.g., NULO-2, respectively) or a complementary sequence thereof. In some embodiments, the nucleotide sequence has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of any one of SEQ ID NOs: 42-43, or a complementary sequence thereof. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject. In some embodiments, the nucleic acid molecule has the nucleic acid sequence of any one of SEQ ID NOs: 42-43. In some embodiments, the nucleic acid molecule has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of any one of SEQ ID NOs: 42-43, but does not encode the sequence of SEQ ID NO: 72 or 73
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV2 nucleocapsid (NUL) polypeptide having at least 85% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200) of any one of SEQ ID NOs: 97 (e.g., NUL5, respectively), or a complementary sequence thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of any one of SEQ ID NOs: 98 (e.g., NUL6), or a complementary sequence thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of any one of SEQ ID NOs: 99 (e.g., NUL7), or a complementary sequence thereof. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 conserved region (CR) polypeptide having at least 85% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200) of SEQ ID NO: 77 (e.g., CR0) or a complementary sequence thereof. In some embodiments, the nucleotide sequence has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of SEQ ID NO: 47, or a complementary sequence thereof. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject. In some embodiments, the nucleic acid molecule has the nucleic acid sequence of SEQ ID NO: 47. In some embodiments, the nucleic acid molecule has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 47 but does not encode the sequence of SEQ ID NO: 77.
A nucleic acid molecule of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 conserved region (CR) polypeptide having at least 85% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200) of SEQ ID NO: 102 (e.g., CR5) or a complementary sequence thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to all or a portion of SEQ ID NO: 102 (e.g., CR5), or a complementary sequence thereof. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject.
The nucleic acid molecules of the disclosure may contain a spike, membrane, or nucleocapsid nucleotide sequence that has been modified relative to a wild-type or natural variant of SARS-CoV-2 for improved expression in host cells (e.g., mammalian (e.g., human) host cells). Optimization can include the addition of a leader sequence, restriction site, and/or a Kozak sequence.
The nucleic acid molecules may be further modified, such as by codon optimization, for expression in a targeted mammalian subject (e.g., human or a non-human animal for vaccine production).
The nucleic acid molecules may also be inserted into expression vectors, such as a plasmid, or a viral vector, such as an adenovirus (e.g., Ad26 vector), poxvirus, adeno-associated virus, retroviral, or other viral vector, or prepared as naked or encapsulated DNA and incorporated into compositions.
Polypeptides
The polypeptides of the disclosure have been rationally designed using information from naturally occurring spike (S), membrane (MEM), conserved region (CR), and nucleocapsid (NUL) polypeptides from SARS-CoV-2 lineage variants.
The polypeptides of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, and/or EG4) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 87-96. The polypeptides of the disclosure may contain one or more NUL (e.g., NULO, NUL1 , NUL2, NUL3, and/or NUL4) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 72-76. The polypeptides of the disclosure may contain one or more CR (e.g., CR0, CR1 , CR2, CR3, and/or CR4) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 77-81 . The polypeptides of the disclosure may contain one or more MEM (e.g., MEMO, MEM1 , MEM2, MEM3, and/or MEM4) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 82-86.
The polypeptides of the disclosure may contain or further contain one or more EG (e.g., EG5, EG6, EG7, EG8 and/or EG9) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 103-107. The polypeptides of the disclosure may contain or further contain one or more NUL (e.g., NUL5, NUL6, and/or NUL7) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 97-99. The polypeptides of the disclosure may contain a CR5 amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 102. The polypeptides of the disclosure may contain one or more MEM (e.g., MEM5 and/or MEM6) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 100-101 .
The S polypeptides of the disclosure are modified S proteins containing one or more mutations relative to the Wuhan spike protein (e.g., relative to SEQ ID NO: 21 ) and existing optimizations thereof (e.g., SEQ ID NO: 23) that were designed based on a study of emerging variants of the Wuhan coronavirus around the world. The polypeptides described herein have been modified to include one or more resistance mutations identified in one or more of these lineage variants.
The polypeptides disclosed herein may be less than the full-length S protein (i.e., they may contain one or more regions of the S protein from SARS-CoV-2 or a variant thereof, such as the NTD and/or RBD). The polypeptides may also include, in addition to one or more of the resistance mutations described herein, most or all of the regions of the S protein, such that the S protein of the disclosure is capable of forming into a structurally and/or functionally stable S protein.
The S protein of the disclosure may have the amino acid sequence of any one of SEQ ID NOs: 87-90, 92-95, and 103-107 or a variant thereof with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to all or a portion of any one of SEQ ID NOs: 87-90, 92-95, and 103-107. The polypeptides may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 or more continuous or non-continuous amino acids of any one of SEQ ID NOs: 87-90, 92-95, and 103- 107.
Polypeptides of the disclosure may also include a deletion of or an inclusion of a signal sequence (e.g., SEQ ID NO: 29), stabilizing mutations (e.g., proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 20), mutations to a furin cleavage site (e.g., SEQ ID NO: 36), introduction of a trimerization domain (e.g., a foldon trimerization domain, e.g., SEQ ID NO: 32, or other trimerization domain known in the art), introduction of linker or spacer sequences (e.g., SEQ ID NOs: 33 and 34), and combinations thereof. A signal sequence and each of these modifications are present in the S protein of SEQ ID NO: 23, and any of SEQ ID NOs: 87-90 and 92-95 could be similarly modified using the sequence of SEQ ID NO: 23 as a guide (e.g., by aligning the sequences of SEQ ID NO: 87-90 and 92-95 with SEQ ID NO: 23 and making the equivalent modifications found in SEQ ID NO: 23 at the corresponding sequence of SEQ ID NOs: 87-90 and 92-95.
In some embodiments, an isolated polypeptide may contain an amino acid sequence having 100% sequence identity to amino acids 18-1208 of SEQ ID NO: 87 or SEQ ID NO: 92. In some embodiments, an isolated polypeptide may contain an amino acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 18-1201 of SEQ ID NO: 88 or SEQ ID NO: 93. In some embodiments, an isolated polypeptide may contain an amino acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 18-1200 of SEQ ID NO: 89 or SEQ ID NO: 94. In some embodiments, an isolated polypeptide may contain an amino acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to amino acids 18-1204 of SEQ ID NO: 90 or SEQ ID NO: 95.
An isolated polypeptide of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 membrane polypeptide. In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, SEQ ID NO: 82 (e.g., MEMO). In some embodiments, the polypeptide, or a portion or fragment thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 82. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 82, but is not the sequence of SEQ ID NO: 82.
An isolated polypeptide of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 membrane polypeptide. In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, SEQ ID NO: 100 (e.g., MEM5). In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, SEQ ID NO: 101 (e.g., MEM6). In some embodiments, the polypeptide, or a portion or fragment thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 100. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 101 . In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 100, but is not the sequence of SEQ ID NO: 100. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 101 , but is not the sequence of SEQ ID NO: 101 .
An isolated polypeptide of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV2 nucleocapsid polypeptide. In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, any one of SEQ ID NOs: 72-73 (e.g., NULO-2, respectively). In some embodiments, the polypeptide, or a portion or fragment thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 72. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 73. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of any one of SEQ ID NOs: 72 or 73, but is not the sequence of SEQ ID NOs: 72 or 73.
An isolated polypeptide of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV2 nucleocapsid polypeptide. In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, any one of SEQ ID NOs: 97-99 (e.g., NUL5-7, respectively). In some embodiments, the polypeptide, or a portion or fragment thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 97. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 98. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 99. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 97, but is not the sequence of SEQ ID NO: 97. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 98, but is not the sequence of SEQ ID NO: 98. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 99, but is not the sequence of SEQ ID NO: 99.
An isolated polypeptide of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 conserved region polypeptide. In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, SEQ ID NO: 77 (e.g., CR0). In some embodiments, the polypeptide, or a portion or fragment thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 77. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 77, but is not the sequence of SEQ ID NO: 77.
An isolated polypeptide of the disclosure may also be one that contains a nucleotide sequence that encodes a SARS-CoV-2 conserved region polypeptide. In some embodiments, the polypeptide has at least 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to at least 100 contiguous amino acids (e.g., positions 1 -100, 1 -200, and 100-200), or the amino acid sequence of, SEQ ID NO: 102 (e.g., CR5). In some embodiments, the polypeptide, or a portion or fragment thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 102. In some embodiments, the polypeptide has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to all or a portion of SEQ ID NO: 102, but is not the sequence of SEQ ID NO: 102.
The polypeptides of the disclosure may be produced (e.g., recombinant methods) and may be purified during the production (e.g., isolated from other components, such as components with which the polypeptides are natively associated). The purified polypeptides may then be incorporated into a composition of the disclosure (e.g., an immunogenic composition or a vaccine composition).
Vectors
The disclosure also features recombinant vectors (e.g., an Ad26 or Ad52 viral vector) including any one or more of the S, MEM, CR, and/or NUL polynucleotides described above.
The vectors of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, and/or EG4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 57-66. The vectors of the disclosure may contain one or more NUL (e.g., NUL0, NUL1 , NUL2, NUL3, and/or NUL4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 42-46. The vectors of the disclosure may contain one or more CR (e.g., CR0, CR1 , CR2, CR3, and/or CR4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 47-51 . The vectors of the disclosure may contain one or more MEM (e.g., MEMO, MEM1 , MEM2, MEM3, and/or MEM4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 52-56. The vectors of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, and/or EG4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 57-66, as well as one or more NUL (e.g., NULO, NUL1 , NUL2, NUL3, and/or NUL4), CR (e.g., CRO, CR1 , CR2, CR3, and/or CR4), and/or MEM (e.g., MEMO, MEM1 , MEM2, MEM3, and/or MEM4) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 42-56.
The vectors can be used to deliver a nucleic acid (e.g., RNA or DNA) expressing an immunogen (e.g., one or more of SEQ ID NOs: 87-90, 92-95, and 103-107 or variants thereof, having at least 85-99% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto), and include mammalian, viral, and bacterial expression vectors. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 57-60 and 62-65, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of any one of SEQ ID NOs: 87-90 and 92-95, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 57 or 62, or a variant thereof with up to 85% sequence identity thereto), expressing an immunogen with the amino acid sequence of SEQ ID NO: 87 or 92, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 58 or 63, or a variant thereof with up to 85% sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 88 or 93, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 59 or 64, or a variant thereof with up to 85% sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 89 or 94, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 60 or 65, or a variant thereof with up to 85% sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 90 or 95, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The mammalian, viral, and bacterial vectors can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 57-60 and 62-65 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, as well as complementary sequences thereof.
The vectors can be used to deliver a nucleic acid (e.g., RNA or DNA) having a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 103. The vectors can be used to deliver a nucleic acid (e.g., RNA or DNA) having a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 104. The vectors can be used to deliver a nucleic acid (e.g., RNA or DNA) having a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 105. The vectors can be used to deliver a nucleic acid (e.g., RNA or DNA) having a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 106. The vectors can be used to deliver a nucleic acid (e.g., RNA or DNA) having a nucleotide sequence that encodes a polypeptide having 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 107.
If desired, one or more of the vectors can also include a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 6, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, that encodes an immunogen with the amino acid sequence of SEQ ID NO: 23, or a variant thereof with up to 85% sequence identity thereto, or a complementary sequence thereof.
The vectors of the disclosure may contain a nucleic acid molecule with different combinations of different nucleic acid molecules described herein (e.g., combinations of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more nucleic acid molecules described herein). The vectors of the disclosure may contain a nucleic acid molecule with combinations of different nucleic acid molecules (e.g., a vector may contain a nucleic acid molecule with the nucleotide sequence of SEQ ID NOs: 57 and 58, SEQ ID NOs: 57 and 59, SEQ ID NOs: 57 and 60, SEQ ID NOs: 58 and 59, SEQ ID NOs 58 and 60, SEQ ID NOs 59 and 60, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 58, 59, and 60, SEQ ID NOs: 57, 59, and 60, SEQ ID NOs: 57-60, SEQ ID NOs: 62 and 63, SEQ ID NOs: 62 and 64, SEQ ID NOs: 62 and 65, SEQ ID NOs: 63 and 64, SEQ ID NOs: 63 and 65, SEQ ID NOs 64 and 65, SEQ ID NOs: 62, 63, and 64, SEQ ID NOs: 63, 64, and 65, SEQ ID NOs: 62, 64, and 65, or SEQ ID NOs: 62-65, or variants thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto). The nucleic acid molecule of the vector may encode two or more immunogens, such as immunogens with the amino acid sequences of SEQ ID NOs: 57 and 58, SEQ ID NOs: 57 and 59, SEQ ID NOs: 57 and 60, SEQ ID NOs: 58 and 59, SEQ ID NOs 58 and 60, SEQ ID NOs 59 and 60, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 58, 59, and 60, SEQ ID NOs: 57, 59, and 60, SEQ ID NOs: 57-60, SEQ ID NOs: 62 and 63, SEQ ID NOs: 62 and 64, SEQ ID NOs: 62 and 65, SEQ ID NOs: 63 and 64, SEQ ID NOs: 63 and 65, SEQ ID NOs 64 and 65, SEQ ID NOs: 62, 63, and 64, SEQ ID NOs: 63, 64, and 65, SEQ ID NOs: 62, 64, and 65, or SEQ ID NOs: 62-65, or variants thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The vector may also include a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 6, or a variant thereof with up to 85% sequence identity thereto (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%), that encodes an immunogen with the amino acid sequence of SEQ ID NO: 23, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto.
The vectors of the disclosure may contain a nucleic acid molecule that encodes two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more immunogens, such as immunogens with the amino acid sequences of SEQ ID NOs: 97-107. For example, the vector can encode two immunogens with the amino acid sequences of: SEQ ID NO: 103 and 104; SEQ ID NO: 103 and 105; SEQ ID NO: 103 and 106; or SEQ ID NO: 103 and 107. In another example, the vector can encode three immunogens with the amino acid sequences of: SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 103, 104, 106; SEQ ID NOs: 103, 104, and 107; SEQ ID NOs: 103, 105, and 106; SEQ ID NOs: 103, 105, and 107; SEQ ID NOs: 103, 106, and 107; SEQ ID NOs: 104, 105, and 106; SEQ ID NOs: 104, 105, and 107; or SEQ ID NOs: 105, 106, and 107. In yet another example, the vector can encode four immunogens with the amino acid sequences of any four of SEQ ID NOs: 103-107. In yet another example, the vector can encode five immunogens with the amino acid sequences of SEQ ID NOs: 103- 107. The vectors of the disclosure may be a mammalian, viral, and bacterial and, further, can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 2-6, 57-60 and 62-65 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, as well as complementary sequences thereof.
The recombinant vectors of the disclosure can be used to deliver any one or more nucleic acids expressing a SARS-CoV-2 S immunogen (e.g., one or more of SEQ ID NOs: 21 , 23, 87-90, and/or 92-95, or variants thereof with at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto), and include mammalian, viral, and bacterial expression vectors. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of one or more of SEQ ID NOs: 57- 60 and 62-65, or a variant thereof with up to 85% sequence identity thereto) expressing an immunogen with the amino acid sequence of any one of SEQ ID NOs: 87-90 and 92-95, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 57 or 62, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), expressing an immunogen with the amino acid sequence of SEQ ID NO: 87 or 92, or a variant thereof with up to 85% sequence identity thereto (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 58 or 63, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 88 or 93, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 59 or 64, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 89 or 94, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 60 or 65, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 90 or 95, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The mammalian, viral, and bacterial vectors can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 2-6, 57-60 and 62-65 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, as well as complementary sequences thereof.
The recombinant vectors of the disclosure can be used to deliver any one or more nucleic acids expressing a SARS-CoV-2 MEM immunogen (e.g., one or more of SEQ ID NOs: 82, 100, and 101 or variants thereof with at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto), a SARS- CoV-2 CR immunogen (e.g., SEQ ID NO: 77 or SEQ ID NO: 102), and/or a SARS-CoV-2 NUL immunogen (e.g., one or more of SEQ ID NOs: 72-73 and 97-99, or variants thereof with at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto). The recombinant vectors of the disclosure can include mammalian, viral, and bacterial expression vectors. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of one or more of SEQ ID NOs: 42, 43, 47, 52, or a variant thereof with up to 85% sequence identity thereto) expressing an immunogen with the amino acid sequence of any one of SEQ ID NOs: 72, 73, 77, and 82 , or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 42, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), expressing an immunogen with the amino acid sequence of SEQ ID NO: 72 (e.g., NUL0), or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 43, or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 73 (e.g., NUL1 ) or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 47 or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 77 (e.g., CR0), or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 51 , or a variant thereof with up to 85% (e.g., 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) expressing an immunogen with the amino acid sequence of SEQ ID NO: 82 (e.g., MEMO), or a variant thereof with up to 85% sequence identity thereto. The mammalian, viral, and bacterial vectors can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 42, 43, 47, and 52 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, as well as complementary sequences thereof.
The vectors may be, for example, plasmids, artificial chromosomes (e.g., BAG, PAC, YAC), and virus or phage vectors, and may optionally include a promoter, enhancer, or regulator for the expression of the polynucleotide. The vectors may also contain one or more selectable marker genes, for example an ampicillin, neomycin, and/or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example, for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell, e.g., for the production of protein encoded by the vector. The vectors may also be adapted to be used in vivo, for example in a method of DNA vaccination, RNA vaccination, or gene therapy.
Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals, such as cadmium, and the p-actin promoter. A viral promoter, which can be obtained from the genome of a virus, such as, for example, polyoma virus, fowlpox virus, adenovirus (A), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), and human papillomavirus (HPV), may also be used. These promoters are well known and readily available in the art.
A particular promoter element is the CMV immediate early promoter. In some embodiments, the expression plasmid is pcDNA3.1 + (Invitrogen, CA, USA). In some embodiments, the expression vector is a viral vector, such as a vector derived from adenovirus or poxvirus.
Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors that can be used to deliver a nucleic acid expressing an immunogen (e.g., an immunogen with the amino acid sequence of one or more of SEQ ID NOs: 21 , 23, 87-90, 92-95, and 97-107 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto) include a retrovirus, adenovirus (e.g., Ad2, Ad5, Ad11 , Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., a RhAd52), Ad59 (e.g., a RhAd59), and Pan9 (also known as AdC68)), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding immunogens (e.g., polypeptides) include Norwalk virus, togavirus, coronavirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). For example, the vector can be Ad26. These adenovirus vectors can be derived from, for example, human, chimpanzee, or rhesus adenoviruses. Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent. No. 5,801 ,030); incorporated herein in its entirety by reference. The nucleic acid material (e.g., including a nucleic acid molecule) of the viral vector may be encapsulated, e.g., in a lipid membrane or by structural proteins (e.g., capsid proteins), that may include one or more viral polypeptides (e.g., a glycoprotein). The viral vector can be used to infect cells of a subject, which, in turn, promotes the translation of the heterologous gene(s) of the viral vector into the immunogens. For example, a viral vector can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 2, 3, 5, 6, 57-60, and 62-65, or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, and complements thereof.
Adenoviral vectors disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, each incorporated by reference herein, are particularly useful as vectors. These adenoviral vectors can encode and/or deliver one or more of the immunogens described herein (e.g., one or more of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9 immunogens, the MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, and/or MEM6 immunogens, the CR0, CR1 , CR2, CR3, CR4, and/or CR5 immunogens, and/or one or more of the NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7 immunogens, or any combination of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, EG9 MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, MEM6, CR0, CR1 , CR2, CR3, CR4, CR5, NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7 immunogens, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) to treat a subject having a pathological condition associated with a viral infection (e.g., a SARS-CoV-2 infection or a lineage variant thereof). In some embodiments, one or more recombinant adenovirus vectors can be administered to the subject in order to express more than one type of immunogen (e.g., one or more of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9 immunogens, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto). In some embodiments, a recombinant adenovirus vector can be modified to change the hexon HVR domains (e.g., replace one or more HVRs with those of a different serotype). Besides adenoviral vectors, other viral vectors and techniques are known in the art that can be used to facilitate delivery and/or expression of one or more of the immunogens in a subject (e.g., a human). These viruses include poxviruses (e.g., vaccinia virus and modified vaccinia virus Ankara (MV A); see, e.g., U.S. Patent Nos. 4,603,112 and 5,762,938, each incorporated by reference herein), herpesviruses, togaviruses (e.g., Venezuelan Equine Encephalitis virus; see, e.g., U.S. Patent No. 5,643,576, incorporated by reference herein), picornaviruses (e.g., poliovirus; see, e.g., U.S. Patent No. 5,639,649, incorporated by reference herein), baculoviruses, and others described by Wattanapitayakul and Bauer (Biomed. Pharmacother. 54:487 (2000), incorporated by reference herein).
Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide into the host genome, although such recombination is not preferred. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. The replication-defective adenoviral vector may contain a deletion in or of one or more of the E1 , E3, and/or E4 regions. Alternatively, the adenoviral vector may contain one or more of the E1 , E3, and/or E4 regions and may be replication-competent.
Vectors capable of driving expression in insect cells (for example baculovirus vectors), in human cells, in yeast or in bacteria may be employed in order to produce quantities of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, EG9, MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, MEM6, CRO, CR1 , CR2, CR3, CR4, CR5, NULO, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7 immunogens, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, encoded by the polynucleotides of the disclosure, for example, for use as a subunit vaccine or in an immunoassay.
In some embodiments, the vector is an expression vector. In some embodiments, the viral vector is a virus selected from the group consisting of a retrovirus, adenovirus, adeno-associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox. In some embodiments, the vector is an adenovirus. In some embodiments, the adenovirus is selected from the group consisting of Ad26, Ad52, Ad59, Ad2, Ad5, Ad11 , Ad12, Ad24, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, and Pan9. For example, the adenovirus is Ad26. In some embodiments, the adenovirus is Ad52, such as a rhesus Ad52, or the adenovirus is Ad59, such as a rhesus Ad59. In some embodiments, the vector is a replication-defective vector. In some embodiments, the replication-defective vector is a viral vector (e.g., an adenoviral vector) that contains a deletion in or of one or more of the E1 , E3, and/or E4 regions. In other embodiments, the viral vector (e.g., an adenoviral vector) includes one or more of the E1 , E3, and/or E4 regions and is replication-competent.
Antibodies
Anti-SARS-CoV-2 antibodies of the disclosure are capable of specifically binding to a S protein of SARS-CoV-2 or a variant thereof or a Spike polypeptide of a lineage variant thereof (such as the EGO EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9 immunogens described herein), and are capable of inhibiting a SARS-CoV-2-mediated activity (e.g., viral spread, infection, and or cell fusion) in a subject (e.g., a human). The result of such binding may be, for example, a reduction in viral titer (e.g., viral load), by about 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more, after administration of an antibody to a subject infected with SARS-CoV-2 or a variant thereof. The anti-SARS-CoV-2 antibodies may selectively bind to an epitope comprising all, or a portion of, the NTD or RBD region of a S protein of SARS-CoV-2 or a variant thereof.
The antibodies of the disclosure may bind to one or more EG proteins (e.g., EGO, EG 1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 87-96 and 103-107. The antibodies of the disclosure may bind to one or more NUL proteins (e.g., NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 72-76 and 97-99. The antibodies of the disclosure may bind to one or more CR proteins (e.g., CR0, CR1 , CR2, CR3, CR4, and/or CR5) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 77-81 and 102. The antibodies of the disclosure may bind to one or more MEM proteins (e.g., MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5 and/or MEM6) amino acid sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 82-86, 100, and 101 .
In particular, the anti-SARS-CoV-2 antibodies may selectively bind to an epitope containing all, or a portion of, any one of SEQ ID NOs: 87-90, 92-95, and 103-107. In some embodiments, the anti-SARS- CoV-2 antibodies may bind to an epitope of a S protein of SARS-CoV-2 or a variant thereof that contains one or more of the mutations V3G, L5F, V6F, 213-214insRE, L18F, T19I, T19R, T20N, A24-26, A27S, V36F, H49Y, Q52R, L54F, A67V, A69-70, D80A, D80G, D80Y, T95I, K97E, S98F, V126A, V127F, G142D, A143-145, A145, M153T, E154K, A156-157, R158G, N164K, N164T, Q173H, L176F, G181V, G184S, L189F, R190S, V193L, D198G, A211 , N2111, L212I, L212V, V213G, V213P, R214E, S221 L, A222V, D228H, I233V, R237S, T240I, A241 -243, T250I, P251 L, D253G, T259I, G261 V, A262S, P272L, R273S, E281 Q, T284I, V289I, T299I, T307I, V308L, E309Q, V320I, T323I, V327I, P330S, G339D, R346K, N354K, K356R, R357K, V367L, S371 F, S371 L, S373P, S375F, T376A, V382L, P384L, T385I, D405N, R408S, Q414R, K417N, K417T, D427N, D427V, T430I, N439K, N440K, G446S, L452Q, L452R, L455F, S477N, T478K, E484A, F486V, Q493R, G496S, Q498R, N501 Y, Y505H, A520S, A522S, A522V, N540S, T547I, T547K, N556K, K558N, A570D, A570S, T572I, E583D, E583Q, L585F, T604I, Q613H, D614G, V622F, A626S, Q628K, P631 S, S640F, F643L, A647S, H655Y, N658S, E661 D, I670V, A672V, N679K, A688V, S691 F, A694V, A701 V, S704L, A706V, T716I, T719I, T723I, M731 I, T732A, S735A, M740V, T747I, N764K, G769V, V772I, T778I, E780Q, Q787H, T7911, D796H, D796Y, P809S, P812L, P812S, L822F, A831V, I834T, I834V, A845S, A846V, I850L, N856K, T859I, T859N, I870V, A871 S, A879S, A879V, T883I, F888L, A892S, A892V, A899S, I909V, L922F, S929I, S929T, D936Y, L938F, S939F, D950H, D950N, Q954H, T961 M, N969K, D979E, L981 F, S982A, T1006I, T1009I, A1020S, A1020V, T1027I, L1049I, H1058Y, M1050I, L1063F, A1078S, N1074S, Q1071 H, A1087S, H1083Y, 11081V, R1091 H, H1101 D, H1101Y, V1104L, D1118H, D111 N, I1114T, T1117I, D1127G, G1124V, S112L, 11132V, V1133I, L1141 F, S1147L, D1153Y, E1150D, G1167V, P1162L, P1162S, 11179V, V1176F, V1177I, L118F, K1191 N, E1202Q, E1207D, Q1208H, G1219C, G1219V, I1221T, M1229I, V1228L, L1234I, M1237I, T1238I, C1243F, C1247F, D1259Y, S1252F, S1252P, P1263L, and V1264L relative to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 21 .
For example, an antibody of the disclosure may neutralize one or more of the B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , A23.1 , BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , and XBC.1 .6 lineages of SARS-CoV-2. In particular, an antibody may bind to an epitope containing all, or a portion of, SEQ ID NOs: 87-90, 92-95, and 103-107, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, such as an epitope within the NTD or RBD region of the Spike protein of SEQ ID NOs: 87-90, 92- 95, and 103-107. For example, antibodies may bind to an epitope of a coronavirus spike protein containing one or more of the mutations V3G, L5F, V6F, 213-214insRE, L18F, T19I, T19R, T20N, A24- 26, A27S, V36F, H49Y, Q52R, L54F, A67V, A69-70, D80A, D80G, D80Y, T95I, K97E, S98F, V126A, V127F, G142D, A143-145, A145, M153T, E154K, A156-157, R158G, N164K, N164T, Q173H, L176F, G181V, G184S, L189F, R190S, V193L, D198G, A211 , N2111, L212I, L212V, V213G, V213P, R214E, S221 L, A222V, D228H, I233V, R237S, T240I, A241 -243, T250I, P251 L, D253G, T259I, G261V, A262S, P272L, R273S, E281 Q, T284I, V289I, T299I, T307I, V308L, E309Q, V320I, T323I, V327I, P330S, G339D, R346K, N354K, K356R, R357K, V367L, S371 F, S371 L, S373P, S375F, T376A, V382L, P384L, T385I, D405N, R408S, Q414R, K417N, K417T, D427N, D427V, T430I, N439K, N440K, G446S, L452Q, L452R, L455F, S477N, T478K, E484A, F486V, Q493R, G496S, Q498R, N501Y, Y505H, A520S, A522S, A522V, N540S, T547I, T547K, N556K, K558N, A570D, A570S, T572I, E583D, E583Q, L585F, T604I, Q613H, D614G, V622F, A626S, Q628K, P631 S, S640F, F643L, A647S, H655Y, N658S, E661 D, I670V, A672V, N679K, A688V, S691 F, A694V, A701V, S704L, A706V, T716I, T719I, T723I, M731 I, T732A, S735A, M740V, T747I, N764K, G769V, V772I, T778I, E780Q, Q787H, T791 I, D796H, D796Y, P809S, P812L, P812S, L822F, A831V, I834T, I834V, A845S, A846V, I850L, N856K, T859I, T859N, I870V, A871 S, A879S, A879V, T883I, F888L, A892S, A892V, A899S, I909V, L922F, S929I, S929T, D936Y, L938F, S939F, D950H, D950N, Q954H, T961 M, N969K, D979E, L981 F, S982A, T1006I, T1009I, A1020S, A1020V, T1027I, L1049I, H1058Y, M1050I, L1063F, A1078S, N1074S, Q1071 H, A1087S, H1083Y, 11081 V, R1091 H, H1101 D, H1101Y, V1104L, D1118H, D111 N, I1114T, T1117I, D1127G, G1124V, S112L, 11132V, V1133I, L1141 F, S1147L, D1153Y, E1150D, G1167V, P1162L, P1162S, 11179V, V1176F, V1177I, L118F, K1191 N, E1202Q, E1207D, Q1208H, G1219C, G1219V, I1221T, M1229I, V1228L, L1234I, M1237I, T1238I, C1243F, C1247F, D1259Y, S1252F, S1252P, P1263L, and V1264L, relative to the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 21 . The antibodies may neutralize one coronavirus, such as a coronavirus of lineage B.1 .1 .529. The antibodies may neutralize one coronavirus, such as a coronavirus of lineage B.1 .1 .7. The antibodies may also neutralize more than one coronavirus, such as coronaviruses of lineages B.1 .1 .7 and B.1 .429. The antibodies can therefore be used to inhibit or treat a coronavirus infection (e.g., infection by SARS-CoV-2 or a variant thereof). The antibodies may also neutralize one or more Omicron variants selected from can be BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6. Due to the broad neutralizing abilities of the immunogens described here, the antibodies may neutralize virtually any SARS-CoV2 variant or any emerging variant thereof.
The antibodies may be specifically generated by using one or more of the EGO, EG1 , EG2, and/or EG3 immunogens of SEQ ID NOs 87-90, 92-95, and 103-107, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. Furthermore, the anti-SARS-CoV-2 antibodies may selectively bind to an epitope containing all, or a portion of, any one of SEQ ID NOs: 87-90, any one of SEQ ID NOs: 92-95, or any one of SEQ ID NOs: 103-107. In some embodiments, the anti-SARS-CoV-2 antibodies may bind to an epitope of a S protein of SARS-CoV-2 or a variant thereof that contains one or more of the mutations V3G, L5F, V6F, 213-214insRE, L18F, T19I, T19R, T20N, A24-26, A27S, V36F, H49Y, Q52R, L54F, A67V, A69-70, D80A, D80G, D80Y, T95I, K97E, S98F, V126A, V127F, G142D, A143-145, A145, M153T, E154K, A156-157, R158G, N164K, N164T, Q173H, L176F, G181V, G184S, L189F, R190S, V193L, D198G, A211 , N211 I, L212I, L212V, V213G, V213P, R214E, S221 L, A222V, D228H, I233V, R237S, T240I, A241 -243, T250I, P251 L, D253G, T259I, G261V, A262S, P272L, R273S, E281 Q, T284I, V289I, T299I, T307I, V308L, E309Q, V320I, T323I, V327I, P330S, G339D, R346K, N354K, K356R, R357K, V367L, S371 F, S371 L, S373P, S375F, T376A, V382L, P384L, T385I, D405N, R408S, Q414R, K417N, K417T, D427N, D427V, T430I, N439K, N440K, G446S, L452Q, L452R, L455F, S477N, T478K, E484A, F486V, Q493R, G496S, Q498R, N501Y, Y505H, A520S, A522S, A522V, N540S, T547I, T547K, N556K, K558N, A570D, A570S, T572I, E583D, E583Q, L585F, T604I, Q613H, D614G, V622F, A626S, Q628K, P631 S, S640F, F643L, A647S, H655Y, N658S, E661 D, I670V, A672V, N679K, A688V, S691 F, A694V, A701V, S704L, A706V, T716I, T719I, T723I, M731 I, T732A, S735A, M740V, T747I, N764K, G769V, V772I, T778I, E780Q, Q787H, T791 I, D796H, D796Y, P809S, P812L, P812S, L822F, A831V, I834T, I834V, A845S, A846V, I850L, N856K, T859I, T859N, I870V, A871 S, A879S, A879V, T883I, F888L, A892S, A892V, A899S, I909V, L922F, S929I, S929T, D936Y, L938F, S939F, D950H, D950N, Q954H, T961 M, N969K, D979E, L981 F, S982A, T1006I, T1009I, A1020S, A1020V, T1027I, L1049I, H1058Y, M1050I, L1063F, A1078S, N1074S, Q1071 H, A1087S, H1083Y, 11081V, R1091 H, H1101 D, H1101Y, V1104L, D1118H, D111 N, I1114T, T1117I, D1127G, G1124V, S112L, 11132V, V1133I, L1141 F, S1147L, D1153Y, E1150D, G1167V, P1162L, P1162S, 11179V, V1176F, V1177I, L118F, K1191 N, E1202Q, E1207D, Q1208H, G1219C, G1219V, 11221 T, M1229I, V1228L, L1234I, M1237I, T1238I, C1243F, C1247F, D1259Y, S1252F, S1252P, P1263L, and V1264L, relative to the amino acid sequence of SEQ ID NO: 21 or 23 (e.g., the antibodies may bind an epitope of a S protein that includes one or more of these mutations). The specific binding of an antibody or antigen-binding fragment thereof to S protein of SARS-CoV-2 or a variant thereof can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody needed to achieve half-maximal inhibition of viral spread (e.g., viral titer) in vitro (IC50) and the equilibrium constant (KD) of the antibody-SARS-CoV-2 polyprotein complex dissociation. The equilibrium constant, KD, that describes the interaction of SARS-CoV-2 polyprotein with an antibody is the chemical equilibrium constant for the dissociation reaction of a SARS-CoV-2 polyprotein-antibody complex into solvent-separated SARS-CoV-2 polyprotein and antibody molecules that do not interact with one another.
In some embodiments, anti-SARS-CoV-2 antibodies of the disclosure may specifically bind to a MEM, CR, and/or NUL protein of SARS-CoV-2 or a variant thereof or polypeptide of a lineage variant thereof (such as the MEMO, MEM5, MEM6, CR0, CR5, NUL0, NUL1 , NUL5, NUL6, and NUL7 immunogens described herein), and are capable of inhibiting a SARS-CoV-2-mediated activity (e.g., viral spread, infection, and or cell fusion) in a subject (e.g., a human). The result of such binding may be, for example, a reduction in viral titer (e.g., viral load), by about 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more, after administration of an antibody to a subject infected with SARS-CoV-2 or a variant thereof. The MEM antibodies may be specifically generated by using the MEMO immunogens of SEQ ID NO: 82, the MEM5 immunogen of SEQ ID NO: 100, or the MEM6 immunogen of SEQ ID NO: 101 , or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The NUL antibodies may be specifically generated by using one or more of the NUL0 immunogen of SEQ ID NO: 72, the NUL1 immunogen of SEQ ID NO: 73, the NUL5 immunogen of SEQ ID NO: 97, the NUL6 immunogen of SEQ ID NO: 98, and the NUL7 immunogen of SEQ ID NO 99, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The CR antibodies may be specifically generated by using the CR0 immunogens of SEQ ID NO: 77, the CR5 immunogen of SEQ ID NO: 102, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The anti-SARS-CoV-2 MEM antibodies may selectively bind to an epitope comprising all, or a portion of, SEQ ID NO: 82, SEQ ID NO: 100, or SEQ ID NO: 101 . The anti-SARS- CoV-2 CR antibodies may selectively bind to an epitope comprising all, or a portion of, SEQ ID NO: 77 or SEQ ID NO: 102. The anti-SARS-CoV-2 NUL antibodies may selectively bind to an epitope comprising all, or a portion of, any one of SEQ ID NOs: 72-73, and 97-99. The MEM, CR, and NUL antibodies may neutralize one coronavirus, such as a coronavirus of lineage B.1 .1 .529. The antibodies can therefore be used to inhibit or treat a coronavirus infection (e.g., infection by SARS-CoV-2 or a variant thereof). In some embodiments, the antibodies generated with the immunogens described herein (e.g., SEQ ID NOs: 97-107) can be pan-sarbecovirus antibodies that treat any SARS-CoV2 infection.
Antibodies are those that specifically bind to a SARS-CoV-2 or a variant thereof polyprotein (e.g., the S, MEM, or NUL region of SARS-CoV-2 or a variant thereof) with a KD value of less than 1 pM (e.g., 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In certain cases, antibodies are those that specifically bind to a SARS-CoV-2 or a variant thereof polyprotein with a KD value of less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM,
850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM,
730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM,
610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM,
490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM, 390 pM, 380 pM,
370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM,
250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM,
130 pM, 120 pM, 1 10 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).
Antibodies of the disclosure can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the KD or ICso of a SARS-CoV-2 or a variant thereof antibody include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays, among others. ELISA represents a particularly useful method for analyzing antibody activity, as such assays typically require minimal concentrations of antibodies. A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a SARS-CoV-2 or a variant thereof antibody). Antibodies are capable of binding SARS-CoV-2 or a variant thereof and epitopes derived thereof, such as epitopes containing one or more residues of any one of SEQ ID NOs: 21 , 23, 87-90, 92-95, and 103-107, as well as isolated peptides derived from SARS- CoV-2 or a variant thereof that structurally pre-organize various residues in a manner that may simulate the conformation of these amino acids in the native protein. For instance, antibodies may bind peptides containing the amino acid sequence of any one of SEQ ID NOs: 21 , 23, 87-90, 92-95, and 103-107, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids of any one of SEQ ID NOs: 21 , 23, 87-90, 92-95, and 103-107. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigen-antibody complex bound to an HRP- conjugated secondary antibody.
Antibodies include those that are generated by immunizing a host (e.g., a mammalian host, such as a human) with the polypeptides of SEQ ID NOs: 21 , 23, 87-90, 92-95, and 103-107. The antibodies can be prepared recombinantly and, if necessary, humanized, for subsequent administration to a human recipient if the host in which the antibodies against a modified S protein of SARS-CoV-2 or a variant thereof are generated is not a human.
Compositions
Compositions of the disclosure may include Epigraph (EG) spike (S) polynucleotide (e.g., RNA or DNA) sequences (e.g., EG0-EG9), membrane (MEM, e.g., MEM0-MEM6), conserved region (CR, e.g., CR0-CR5), and nucleocapsid (NUL, e.g., NUL0-NUL7) polynucleotide sequences.
The compositions of the disclosure may contain one or more EG (e.g., EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 57-66. The compositions of the disclosure may contain one or more NUL (e.g., NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 42-46. The compositions of the disclosure may contain one or more CR (e.g., CR0, CR1 , CR2, CR3, CR4 and/or CR5) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 47-51 . The compositions of the disclosure may contain one or more MEM (e.g., MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, and/or MEM6) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 52-56. The compositions of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 57-66, as well as one or more NUL (e.g., NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7), CR (e.g., CR0, CR1 , CR2, CR3, CR4, and/or CR5), and/or MEM (e.g., MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, and/or MEM6) nucleotide sequences having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 42-56.
Compositions of the disclosure may include Epigraph (EG) spike (S) amino acid sequences (e.g., EG0-EG9), membrane (MEM, e.g., MEM0-MEM6), conserved region (CR, e.g., CR0-CR5), and nucleocapsid (NUL, e.g., NUL0-NUL7) amino acid sequences.
The compositions of the disclosure may contain one or more EG (e.g., EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9) proteins containing an amino acid sequence having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 87-96 and 103-107. The compositions of the disclosure may contain one or more NUL (e.g., NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7) proteins containing an amino acid sequence having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 72-76 and 97-99. The compositions of the disclosure may contain one or more CR (e.g., CR0, CR1 , CR2, CR3, CR4, and/or CR5) proteins containing an amino acid sequence up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 77-81 and 102. The compositions of the disclosure may contain one or more MEM (e.g., MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, and/or MEM6) proteins containing an amino acid sequence having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 82-86, 100, and 101 . The compositions of the disclosure may contain one or more EG (e.g., EGO, EG 1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and/or EG9) proteins containing an amino acid sequence having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 87-96 and 103-107, as well as one or more NUL (e.g., NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and/or NUL7), CR (e.g., CR0, CR1 , CR2, CR3, CR4, and/or CR5), and/or MEM (e.g., MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, and/or MEM6) proteins containing an amino acid sequence having up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 72-86 and 97- 102.
Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of one or more of the EGO, EG1 , EG2, and EG3 immunogens of SEQ ID NOs: 87-90 (or SEQ ID NOs: 92-95), respectively, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The DNA or RNA vector may include all or a fragment of the nucleic acid molecule of any one of SEQ ID NOs: 57-60 and 62-65, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs: 57-60 and 62-65, or a complement thereof. The composition may be an immunogenic composition, which is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera).
Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of one or more of the EG5, EG6, EG7, EG8, and EG9 immunogens of SEQ ID NOs: 103-107, respectively, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The DNA or RNA vector may include all or a fragment of a nucleic acid molecule encoding the polypeptide of any one of SEQ ID NOs: 103-107, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The composition may be an immunogenic composition that is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera).
Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of the MEMO immunogen of SEQ ID NO: 82, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The DNA or RNA vector may include all or a fragment of the nucleic acid molecule of SEQ ID NO: 52, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 52, or a complement thereof. The composition may be an immunogenic composition that is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera).
Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of the MEM5 and/or MEM6 immunogen of SEQ ID NO: 100 and SEQ ID NO: 101 , respectively, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The composition may be an immunogenic composition, which is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera). Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of one or more of the NUL0 and NUL1 immunogens of SEQ ID NOs: 72-73, respectively, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The DNA or RNA vector may include all or a fragment of the nucleic acid molecule of any one of SEQ ID NOs: 42-43, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs: 42-43, or a complement thereof. The composition may be an immunogenic composition, which is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera).
Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of one or more of the NUL5-7 immunogens of SEQ ID NOs: 97-99, respectively, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The composition may be an immunogenic composition, which is capable of eliciting the production of anticoronavirus antisera (e.g., neutralizing antisera). Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of the CRO immunogen of SEQ ID NO: 77, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The DNA or RNA vector may include all or a fragment of the nucleic acid molecule of SEQ ID NO: 47, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 47, or a complement thereof. The composition may be an immunogenic composition, which is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera).
Compositions of the disclosure may further include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, corresponding to all or a fragment of the CR5 immunogen of SEQ ID NO: 102, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, as described herein. The composition may be an immunogenic composition, which is capable of eliciting the production of anti-coronavirus antisera (e.g., neutralizing antisera).
The DNA or RNA vector of the disclosure may also contain a nucleic acid molecule with all or a fragment of the nucleic acid sequence of any one of SEQ ID NOs: 1 -6, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto.
A composition of the disclosure can include an immunogenic polypeptide with the amino acid sequence of all or a fragment of any one or more of SEQ ID NOs: 87-90 and 92-95, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The immunogenic polypeptide composition of the disclosure may further include an immunogenic polypeptide with the amino acid sequence of all or a fragment of any one or more of SEQ ID NOs: 72, 73, 77, and 82, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The composition may be an immunogenic composition, which is capable of eliciting the production of anticoronavirus antisera (e.g., neutralizing antisera).
A composition of the disclosure may also contain an anti-coronavirus antibody (e.g., an anti-Spike antibody or a broadly neutralizing anti-Spike antibody) capable of binding SARS-CoV-2 or a variant thereof and epitopes derived thereof, such as epitopes containing one or more of residues of any one of SEQ ID NOs: 87-90, 92-95, and 103-107. For example, a composition can include an antibody capable of binding epitopes within the NTD or RBD region of the polypeptide of any one or more of SEQ ID NOs: 87-90, 92-95, and 103-107. The antibody may be generated by immunization of a host (e.g., a mammal) with a polypeptide of any one of SEQ ID NOs: 87-90, 92-95, and 103-107. For example, an antibody may be generated by immunization of a host with two or more polypeptides having the amino acid sequence of SEQ ID NOs: 87-90, 92-95, and 103-107, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or one or more nucleic acid molecules encoding the polypeptides. For example, the antibodies may be generated by administering a vector containing a nucleic acid molecule encoding combinations of different immunogens (e.g., the vector may contain a nucleic acid molecule with the nucleotide sequence of SEQ ID NOs: 57 and 58, SEQ ID NOs: 57 and 59, SEQ ID NOs: 57 and 60, SEQ ID NOs: 58 and 59, SEQ ID NOs 58 and 60, SEQ ID NOs 59 and 60, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 58, 59, and 60, SEQ ID NOs: 57, 59, and 60, SEQ ID NOs: 57-60, SEQ ID NOs: 62 and 63, SEQ ID NOs: 62 and 64, SEQ ID NOs: 62 and 65, SEQ ID NOs: 63 and 64, SEQ ID NOs: 63 and 65, SEQ ID NOs 64 and 65, SEQ ID NOs: 62, 63, and 64, SEQ ID NOs: 63, 64, and 65, SEQ ID NOs: 62, 64, and 65, or SEQ ID NOs 62-65 or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto). The nucleic acid molecule of the vector may encode two or more immunogens, such as immunogens with the amino acid sequences of SEQ ID NOs: 57 and 58, SEQ ID NOs: 57 and 59, SEQ ID NOs: 57 and 60, SEQ ID NOs: 58 and 59, SEQ ID NOs 58 and 60, SEQ ID NOs 59 and 60, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 58, 59, and 60, SEQ ID NOs: 57, 59, and 60, SEQ ID NOs: 57-60, SEQ ID NOs: 62 and 63, SEQ ID NOs: 62 and 64, SEQ ID NOs: 62 and 65, SEQ ID NOs: 63 and 64, SEQ ID NOs: 63 and 65, SEQ ID NOs 64 and 65, SEQ ID NOs: 62, 63, and 64, SEQ ID NOs: 63, 64, and 65, SEQ ID NOs: 62, 64, and 65, or SEQ ID NOs 62-65, or variants thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto.
The antibody of the composition may be generated by immunization of a host with two or more polypeptides having the amino acid sequence of SEQ ID NOs: 103-107, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or one or more nucleic acid molecules encoding the polypeptides. For example, the composition of the disclosure can contain a nucleic acid molecule that encodes two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more immunogens, such as immunogens with the amino acid sequences of SEQ ID NOs: 97-107. For example, the composition of the disclosure can contain a nucleic acid molecule that encodes two immunogens with the amino acid sequences of: SEQ ID NO: 103 and 104; SEQ ID NO: 103 and 105; SEQ ID NO: 103 and 106; or SEQ ID NO: 103 and 107. In another example, the composition of the disclosure can contain a nucleic acid molecule that encodes three immunogens with the amino acid sequences of: SEQ ID NOs: 103, 104, and 105; SEQ ID NOs: 103, 104, 106; SEQ ID NOs: 103, 104, and 107; SEQ ID NOs: 103, 105, and 106; SEQ ID NOs: 103, 105, and 107; SEQ ID NOs: 103, 106, and 107; SEQ ID NOs: 104, 105, and 106; SEQ ID NOs: 104, 105, and 107; or SEQ ID NOs: 105, 106, and 107. In yet another example, the composition of the disclosure can contain a nucleic acid molecule that encodes four immunogens with the amino acid sequences of any four of SEQ ID NOs: 103-107. In yet another example, the composition of the disclosure can contain a nucleic acid molecule that encodes five immunogens with the amino acid sequences of SEQ ID NOs: 103-107. The nucleic acid molecule in the compositions described herein may be in a vector, such as a mammalian, viral, and bacterial vector.
The vector may also include a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 6, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, that encodes an immunogen with the amino acid sequence of SEQ ID NO: 23, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. Antibodies generated by administering the vector to a mammal (to elicit the production of the antibodies) can be used to purified or otherwise prepared and used to manufacture a composition of the disclosure.
A composition of the disclosure may contain, or further contain, an anti-MEM antibody, anti-CR antibody, and/or anti-NUL antibody capable of binding SARS-CoV-2 or a variant thereof and epitopes derived thereof, such as epitopes containing one or more of residues of any one of SEQ ID NOs: 72, 73, 77,, 82, and 97-102. The antibody may be generated by immunization of a host (e.g., a mammal) with a polypeptide of any one of SEQ ID NOs: 72, 73, 77, 82, and 97-102. For example, an antibody may be generated by immunization of a host with two or more polypeptides having the amino acid sequence of SEQ ID NOs: 72, 73, 77, 82, and 97-102, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or one or more nucleic acid molecules encoding the polypeptides. For example, the antibodies may be generated by administering a vector containing a nucleic acid molecule encoding combinations of different immunogens. The vector may include a nucleic acid molecule containing one or more of the nucleotide sequences of SEQ ID NO: 42, 43, 47, and 52, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, that encodes one or more immunogens with the amino acid sequence of SEQ ID NOs: 72, 73, 77, and 82, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. Antibodies generated by administering the vector to a mammal (to elicit the production of the antibodies) can be purified or otherwise prepared and used to manufacture a composition of the disclosure.
Compositions of the disclosure designed to contain or express immunogens having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one or more of SEQ ID NOs: 103-107 may be used as a pan-sarbecovirus neutralizer, such as a vaccine.
A composition of the disclosure may contain a viral vector (e.g., an adenovirus vector or a poxvirus vector) containing a nucleic acid molecule(s) of the disclosure. Recombinant adenoviruses offer several significant advantages for use as vectors for the expression of, for example, one or more of the immunogens (e.g., SARS-CoV-2 or a variant thereof polypeptides). The viruses can be prepared to high titer, can infect non-replicating cells, and can confer high-efficiency transduction of target cells ex vivo following contact with a target cell population. Furthermore, adenoviruses do not integrate their DNA into the host genome. Thus, their use as expression vectors has a reduced risk of inducing spontaneous proliferative disorders. In animal models, adenoviral vectors have generally been found to mediate high- level expression for approximately one week. The duration of transgene expression (expression of a nucleic acid molecule) can be prolonged by using cell or tissue-specific promoters. Other improvements in the molecular engineering of the adenovirus vector itself have produced more sustained transgene expression and less inflammation. This is seen with so-called “second generation” vectors harboring specific mutations in additional early adenoviral genes and “gutless” vectors in which virtually all the viral genes are deleted utilizing a Cre-Lox strategy (Engelhardt et al., Proc. Natl. Acad. Sci. USA 91 :6196 (1994) and Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731 (1996), each herein incorporated by reference). Therapeutic formulations of the compositions are prepared for administration to a subject (e.g., a human) using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Therapeutic formulations of the compositions are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable carriers, include saline, or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™, or PEG.
Optionally, the formulation contains a pharmaceutically acceptable salt, such as sodium chloride, and may be present in the formulation at about physiological concentrations. Optionally, the formulations can contain a pharmaceutically acceptable preservative. The preservative concentration may range from about 0.1 to about 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts, such as benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Optionally, the formulations can include a pharmaceutically acceptable surfactant at a concentration of about 0.005 to about 0.02%.
Optionally, the compositions may be formulated to include for co-administration, or sequential administration with, an adjuvant and/or an immunostimulatory agent, (e.g., a protein), such as receptor molecules, nucleic acids, immunogenic proteins, pharmaceuticals, chemotherapy agents, and accessory cytokines. For example, interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11 ), interleukin-12 (IL-12), interleukin-13 (IL- 13), lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin B, Type I interferon, Type II interferon, transforming growth factor-p (TGF-p), lymphotoxin migration inhibition factor, granulocytemacrophage colony-stimulating factor (CSF), monocyte-macrophage CSF, granulocyte CSF, vascular epithelial growth factor (VEGF), angiogenin, transforming growth factor (TGF-a), heat shock proteins (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factors, nucleotides, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof, may be included in formulations of, or for co-administration with, the compositions.
The pharmaceutical compositions can be administered in a therapeutically effective amount that provides an immunogenic and/or protective effect against an infective agent (e.g., a SARS-CoV-2 or a variant thereof). In some embodiments, a composition containing a nucleic acid molecule, polypeptide, vector, and/or antibodies may be formulated for administration at a dose of at least 1 -1 ,000 pg (e.g., at least 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 pg or more). In some embodiments, the subject is administered two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) doses of the composition. The dose may be in a volume of 0.2 mL to 1 .0 mL or up to 1 L (e.g., if prepared as an infusion). In some embodiments, a composition containing a nucleic acid molecule, vector, polypeptide, and/or antibodies is administered at a dose of 50 pg.
In some embodiments, the subject is administered a single type of a pharmaceutical composition described herein (e.g., a pharmaceutical composition containing either an EGO immunogen (e.g., SEQ ID NO: 87 or 92), or a nucleic acid molecule (e.g., a vector) encoding an EGO immunogen (e.g., SEQ ID NO: 57 or 62); a pharmaceutical composition containing an EG1 immunogen (e.g., SEQ ID NO: 88 or 93), or a nucleic acid molecule (e.g., a vector) encoding an EG1 immunogen (e.g., SEQ ID NO: 58 or 63); a pharmaceutical composition containing an EG2 immunogen (e.g., SEQ ID NO: 89 or 94), or a nucleic acid molecule (e.g., a vector) encoding an EG2 immunogen (e.g., SEQ ID NO: 59 or 64); or a pharmaceutical composition containing an EG3 immunogen (e.g., SEQ ID NO: 90 or 95), or a nucleic acid molecule (e.g., a vector) encoding an EG3 immunogen (e.g., SEQ ID NO: 60 or 65)).
In another embodiment, the subject is administered a single type of a pharmaceutical composition described herein (e.g., a pharmaceutical composition containing either an EG5 immunogen (e.g., SEQ ID NO: 103), or a nucleic acid molecule (e.g., a vector) encoding an EG5 immunogen; a pharmaceutical composition containing an EG6 immunogen (e.g., SEQ ID NO: 104), or a nucleic acid molecule (e.g., a vector) encoding an EG6 immunogen; a pharmaceutical composition containing an EG7 immunogen (e.g., SEQ ID NO: 105), or a nucleic acid molecule (e.g., a vector) encoding an EG7 immunogen; a pharmaceutical composition containing an EG8 immunogen (e.g., SEQ ID NO: 106), or a nucleic acid molecule (e.g., a vector) encoding an EG8 immunogen; or a pharmaceutical composition containing an EG9 immunogen (e.g., SEQ ID NO: 107), or a nucleic acid molecule (e.g., a vector) encoding an EG9 immunogen).
In some embodiments, the subject is administered two different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing an EG1 immunogen (e.g., SEQ ID NO: 87 or 92), or a nucleic acid molecule encoding an EG1 immunogen (e.g., SEQ ID NO: 57 or 62)), and a pharmaceutical composition containing an EG2 immunogen (e.g., SEQ ID NO: 88 or 93), or a nucleic acid molecule (e.g., a vector) encoding an EG2 immunogen (e.g., SEQ ID NO: 58 or 63)).
In another embodiment, the subject is administered two different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing either an EG5 immunogen (e.g., SEQ ID NO: 103), or a nucleic acid molecule (e.g., a vector) encoding an EG5 immunogen; and a pharmaceutical composition containing an EG6 immunogen (e.g., SEQ ID NO: 104), or a nucleic acid molecule (e.g., a vector) encoding an EG6 immunogen).
In some embodiments, the subject is administered three different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing an EG1 immunogen (e.g., SEQ ID NO: 87 or 92), or a nucleic acid molecule (e.g., a vector) encoding an EG1 immunogen (e.g., SEQ ID NO: 57 or 62), a pharmaceutical composition containing an EG2 immunogen (e.g., SEQ ID NO: 88 or 93), or a nucleic acid molecule (e.g., a vector) encoding an EG2 immunogen (e.g., SEQ ID NO: 58 or 63), and a pharmaceutical composition containing an EG3 immunogen (e.g., SEQ ID NO: 89 or 94), or a nucleic acid molecule (e.g., a vector) encoding an EG3 immunogen (e.g., SEQ ID NO: 59 or 64)).
In another embodiment, the subject is administered three different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing either an EG5 immunogen (e.g., SEQ ID NO: 103), or a nucleic acid molecule (e.g., a vector) encoding an EG5 immunogen; a pharmaceutical composition containing an EG6 immunogen (e.g., SEQ ID NO: 104), or a nucleic acid molecule (e.g., a vector) encoding an EG6 immunogen; and a pharmaceutical composition containing an EG7 immunogen (e.g., SEQ ID NO: 105), or a nucleic acid molecule (e.g., a vector) encoding an EG7 immunogen).
In some embodiments, the subject is administered four different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing an EGO immunogen (e.g., SEQ ID NO: 87 or 92), or a nucleic acid molecule (e.g., a vector) encoding an EGO immunogen (e.g., SEQ ID NO: 57 or 62), a pharmaceutical composition containing EG1 immunogen (e.g., SEQ ID NO: 88 or 93), or a nucleic acid molecule (e.g., a vector) encoding an EG1 immunogen (e.g., SEQ ID NO: 58 or 63), a pharmaceutical composition containing an EG2 immunogen (e.g., SEQ ID NO: 89 or 94), or a nucleic acid molecule (e.g., a vector) encoding an EG2 immunogen (e.g., SEQ ID NO: 59 or 64), and a pharmaceutical composition containing an EG3 immunogen (e.g., SEQ ID NO: 90 or 95), or a nucleic acid molecule (e.g., a vector) encoding an EG3 immunogen (e.g., SEQ ID NO: 60 or 65).
In another embodiment, the subject is administered four different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing either an EG5 immunogen (e.g., SEQ ID NO: 103), or a nucleic acid molecule (e.g., a vector) encoding an EG5 immunogen; a pharmaceutical composition containing an EG6 immunogen (e.g., SEQ ID NO: 104), or a nucleic acid molecule (e.g., a vector) encoding an EG6 immunogen; a pharmaceutical composition containing an EG7 immunogen (e.g., SEQ ID NO: 105), or a nucleic acid molecule (e.g., a vector) encoding an EG7 immunogen; and a pharmaceutical composition containing an EG8 immunogen (e.g., SEQ ID NO: 106), or a nucleic acid molecule (e.g., a vector) encoding an EG8 immunogen).
In some embodiments, the subject is administered five different types of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition containing either an EG5 immunogen (e.g., SEQ ID NO: 103), or a nucleic acid molecule (e.g., a vector) encoding an EG5 immunogen; a pharmaceutical composition containing an EG6 immunogen (e.g., SEQ ID NO: 104), or a nucleic acid molecule (e.g., a vector) encoding an EG6 immunogen; a pharmaceutical composition containing an EG7 immunogen (e.g., SEQ ID NO: 105), or a nucleic acid molecule (e.g., a vector) encoding an EG7 immunogen; a pharmaceutical composition containing an EG8 immunogen (e.g., SEQ ID NO: 106), or a nucleic acid molecule (e.g., a vector) encoding an EG8 immunogen; and a pharmaceutical composition containing an EG9 immunogen (e.g., SEQ ID NO: 107), or a nucleic acid molecule (e.g., a vector) encoding an EG9 immunogen).
In any of these examples, the pharmaceutical compositions described may further include one or more of the following: a pharmaceutical composition containing a MEMO immunogen (e.g., SEQ ID NO: 82), or a nucleic acid molecule (e.g., a vector) encoding an MEM immunogen (e.g., SEQ ID NO: 52); a pharmaceutical composition containing a MEM5 immunogen (e.g., SEQ ID NO: 100), or a nucleic acid molecule (e.g., a vector) encoding an MEM5 immunogen; a pharmaceutical composition containing a MEM6 immunogen (e.g., SEQ ID NO: 101 ), or a nucleic acid molecule (e.g., a vector) encoding an MEM6 immunogen; a pharmaceutical composition containing a CR0 immunogen (e.g., SEQ ID NO: 77), or a nucleic acid molecule (e.g., a vector) encoding an CR0 immunogen (e.g., SEQ ID NO: 47); a pharmaceutical composition containing a CR5 immunogen (e.g., SEQ ID NO: 102), or a nucleic acid molecule (e.g., a vector) encoding an CR0 immunogen; a pharmaceutical composition containing a NUL0 immunogen (e.g., SEQ ID NO: 72), or a nucleic acid molecule (e.g., a vector) encoding an NUL1 immunogen (e.g., SEQ ID NO: 42); a pharmaceutical composition containing a NUL1 immunogen (e.g., SEQ ID NO: 73), or a nucleic acid molecule (e.g., a vector) encoding a NUL1 immunogen (e.g., SEQ ID NO: 43); a pharmaceutical composition containing a NUL5 immunogen (e.g., SEQ ID NO: 97), or a nucleic acid molecule (e.g., a vector) encoding an NUL5 immunogen; a pharmaceutical composition containing a NUL6 immunogen (e.g., SEQ ID NO: 98), or a nucleic acid molecule (e.g., a vector) encoding an NUL6 immunogen; and a pharmaceutical composition containing a NUL7 immunogen (e.g., SEQ ID NO: 999), or a nucleic acid molecule (e.g., a vector) encoding an NUL7 immunogen.
In any of these examples, the subject may also be administered an amount of a pharmaceutical composition containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5, nucleotides 19-3837 of SEQ ID NO: 6, the nucleotide sequence of SEQ ID NO: 6, the nucleotide of SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 3, a polypeptide containing the amino acid sequence of SEQ ID NO: 23, a polypeptide having at least 85% sequence identity to amino acids 18- 1208 of SEQ ID NO: 23, or a polypeptide containing the amino acid sequence of SEQ ID NO: 21 , or at least 85% sequence identity thereof.
The compositions utilized in the methods described herein can be formulated, for example, for administration intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions.
Pharmaceutical compositions according to the disclosure described herein may be formulated to release the composition immediately upon administration (e.g., targeted delivery) or at any predetermined time period after administration using controlled or extended release formulations. Administration of the pharmaceutical composition in controlled or extended release formulations is useful where the composition, either alone or in combination, has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, Tl, is defined as the ratio of median lethal dose (LDso) to median effective dose (EDso)); (ii) a narrow absorption window at the site of release (e.g., the gastro-intestinal tract); or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain a therapeutic level.
Many strategies can be pursued to obtain controlled or extended release in which the rate of release outweighs the rate of metabolism of the pharmaceutical composition. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Suitable formulations are known to those of skill in the art. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes. The compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation may be administered in powder form or combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11 , such as between 5 and 9 or between 6 and 8, and, in particular between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of an immunogenic composition (e.g., a vaccine as described herein or an anti-SARS-CoV-2 or a variant thereof antibody described herein) and, if desired, one or more immunomodulatory agents, such as in a sealed package of tablets or capsules, or in a suitable dry powder inhaler (DPI) capable of administering one or more doses.
Methods of Treatment Using a Composition of the Disclosure
One or more of the pharmaceutical compositions described herein (e.g., an immunogenic composition and/or an anti-SARS-CoV-2 or a variant thereof antibody-containing composition) can be used to treat a subject (e.g., a human) at risk of exposure to a coronavirus (e.g., SARS-CoV-2 or a variant thereof), a subject susceptible to a coronavirus (e.g., SARS-CoV-2 or a variant thereof), or to treat a subject infected with a coronavirus (e.g., SARS-CoV-2 or a variant thereof). In particular, the compositions can be used to treat (pre- or post-exposure) infection by a SARS-CoV-2 or a variant thereof. In some embodiments, the treatment can induce a protective level of anti-coronavirus antibodies (e.g., antibodies against a modified S, MEM, and/or NUL protein of SARS-CoV-2 or a variant thereof (e.g., one or more of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, EG9, MEMO, MEM1 , MEM2, MEM3, MEM4, MEM5, MEM6, CRO, CR1 , CR2, CR3, CR4, CR5, NULO, NUL1 , NUL2, NUL3, NUL4, NUL5, and NUL6 immunogens), e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies, e.g., broadly neutralizing anti-Spike antibodies, e.g., anti-membrane antibodies, e.g., anti-membrane neutralizing antibodies, e.g., broadly neutralizing anti-membrane antibodies, e.g., anti-nucleocapsid antibodies, e.g., anti-nucleocapsid neutralizing antibodies, e.g., broadly neutralizing anti-nucleocapsid antibodies). In some embodiments, the protective level is a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is above a level of at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. In some embodiments, treatment with a composition may reduce a SARS-CoV-2 or a variant thereof-mediated activity in a subject, such as viral titer, viral spread, infection, and or cell fusion. In some embodiments, SARS-CoV-2 or a variant thereof-mediated activity is viral load in the respiratory tract (e.g., the upper respiratory tract and/or the lower respiratory tract). In some embodiments, SARS-CoV-2 or a variant thereof-mediated activity is viral load in the lung, nares, and/or trachea. In some embodiments, the SARS-CoV-2 or a variant thereof viral load is decreased by about 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, 99.99%, or more). In some embodiments, SARS-CoV-2 or a variant thereof titer in a treated subject infected with SARS-CoV-2 or a variant thereof is decreased by at least about 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 95%, 99%, 99.9%, 99.99%, or more) after administration of a composition (e.g., vaccine) to the subject.
The compositions (e.g., any of the compositions described herein) can be used to induce an immune response (e.g., a humoral and/or cellular immune response) in a subject (e.g., a human subject). The immune response induced may be different (e.g., different in the specificity, robustness, or durability) depending on the composition or combination of compositions administered. For example, a composition can induce an antibody response with different antibody types (e.g., different proportions of IgM, IgA, IgG 1 , lgG2, lgG3, or FcgR2A.1 ) or different functional characteristics (e.g., ability to induce antibodydependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation (IFN-Y secretion, CD107a degranulation, and MIP-1 p expression)). Compositions described herein (e.g., SS-Spike and SS-SdCT) may induce an ADCD response that can be monitored (e.g., to assess therapeutic efficacy). Compositions described herein (e.g., SS-RBD-foldon and SS-S.Ecto-dF-PP-foldon) may induce an antibody-dependent NK cell activation response that can be monitored (e.g., to assess therapeutic efficacy). Compositions may also induce cellular responses with different characteristics (e.g., Th1 , Th2, or Th17 responses). Compositions described herein (e.g., SS-Spike, SS-SdCT, and SS- S.Ecto-dF-PP-foldon) may induce an S-specific CD4+ or CD8+ T cell response that can be monitored (e.g., to assess therapeutic efficacy).
The vectors (e.g., mammalian, bacterial, or viral (e.g., Ad26) derived expression vectors) can be used to deliver a nucleic acid expressing an immunogen (e.g., one or more of SEQ ID NOs: 87-90, 92-95, and 103-107 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto) to a subject in a method of inhibiting and/or treating a SARS-CoV-2 or a variant thereof infection or infection by a lineage variant thereof. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of one or more of SEQ ID NOs: 57-60 and 62-65, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) encoding an immunogen with the amino acid sequence of one or more SEQ ID NOs: 87-90, 92-95, and 103-107. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of one or more of SEQ ID NOs: 57- 60 and 62-65, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto) that encodes an immunogen with the amino acid sequence of all or a fragment of any one of SEQ ID NOs: 87-90, 92-95, and 103-107, or a variant thereof with up to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The vectors (e.g., mammalian, bacterial, or viral derived expression vectors) can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 57-60 and 62-65 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, and complements thereof.
In particular, adenoviral vectors (e.g., vectors derived from Ad2, Ad5, Ad11 , Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (RhAd52), Ad59 (RhAd59), and Pan9 (also known as AdC68)) disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, each incorporated by reference herein, are particularly useful as vectors in methods of delivering an immunogen to a subject. For example, the vector can be Ad26. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent. No. 5,801 ,030); incorporated herein, in its entirety, by reference.
Useful gene therapy methods for the delivery of immunogens to a subject in need thereof include those described in PCT publication no. WO 2006/060641 , U.S. Patent No. US 7,179,903, and PCT publication no. WO 2001/036620, which described the use of, for example, an adenovirus vector (e.g., vectors derived from Ad2, Ad5, Ad11 , Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (RhAd52), Ad59 (RhAd59), and Pan9 (also known as AdC68)) for therapeutic protein delivery.
One or more of the compositions (e.g., pharmaceutical compositions (e.g., immunogenic compositions and antibodies against a modified S protein of SARS-CoV-2 or a variant thereof)) described herein can be used in the treatment of a subject with or at risk of exposure to a coronavirus (SARS-CoV-2 or a lineage variant thereof). The treatment can include administration of one or more of the compositions described herein, either alone or with one or more additional therapeutic agents (e.g., proinflammatory (e.g., interferons) or anti-inflammatory agents (e.g., corticosteroids, e.g., dexamethasone)) and/or one or more therapeutic interventions (e.g., surgery and prone positioning). The therapeutic agents and/or interventions can be administered sequentially (e.g., administration of one or more of any of the compositions described herein before disease or at an early stage of disease (e.g., within a week of symptom onset), then administration of an additional therapeutic agent (e.g., an antiinflammatory agent (e.g., a corticosteroid, e.g., dexamethasone) at a later stage of disease (e.g., after a week of symptom onset))) or simultaneously (e.g., administration of one or more of any of the compositions described herein and/or one or more additional therapeutic agents). Additional therapeutic agents can include corticosteroids (e.g., glucocorticoids (e.g., dexamethasone, prednisone, and hydrocortisone)), interferons (e.g., interferon beta), deoxycholic acid, colony stimulating factors (e.g., G- CSF and GM-CSF), and non-steroidal anti-inflammatory drugs (e.g., aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as sulindac, indomethacin, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib). Other agents that can be administered in combination with the compositions described herein include remdesivir, chloroquine, hydroxychloroquine, baricitinib, lopinavir/ritonavir, umifenovir, favipiravir, tocilizumab, and ribavirin.
Administration
The pharmaceutical compositions can be administered to a subject (e.g., a human) pre- or postexposure to an infective agent (e.g., a coronavirus, such as SARS-CoV-2 or a variant thereof) to treat, prevent, ameliorate, inhibit the progression of, or reduce the severity of one or more symptoms of infection (e.g., a coronavirus infection, such as a SARS-CoV-2 or a variant thereof infection). For example, the compositions can be administered to a subject having a SARS-CoV-2 or a variant thereof infection. Examples of symptoms of diseases caused by a viral infection, such as SARS-CoV-2 or a variant thereof, that can be treated using the compositions include, for example, fever, pneumonia, respiratory failure, weight loss, joint pain, rash, conjunctivitis, muscle pain, headache, retro-orbital pain, edema, lymphadenopathy, malaise, asthenia, sore throat, cough, nausea, vomiting, diarrhea, and hematospermia. These symptoms, and their resolution during treatment, may be measured by, for example, a physician during a physical examination or by other tests and methods known in the art. A pharmaceutical composition described herein can be administered to a subject (e.g., a human) pre- or post-exposure to an infective agent (e.g., a coronavirus, such as SARS-CoV-2 or a variant thereof) to reduce or prevent the risk of mortality caused by the infective agent.
The method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated). Formulations suitable for oral or nasal administration may consist of liquid solutions, such as an effective amount of the composition dissolved in a diluent (e.g., water, saline, or PEG-400), capsules, sachets, tablets, or gels, each containing a predetermined amount of the chimeric Ad5 vector composition. The pharmaceutical composition may also be an aerosol formulation for inhalation, for example, to the bronchial passageways. Aerosol formulations may be mixed with pressurized, pharmaceutically acceptable propellants (e.g., dichlorodifluoromethane, propane, or nitrogen). In particular, administration by inhalation can be accomplished by using, for example, an aerosol containing sorbitan trioleate or oleic acid, for example, together with trichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethane, or any other biologically compatible propellant gas.
Immunogenicity of the composition may be significantly improved if it is co-administered with an immunostimulatory agent and/or adjuvant. Suitable adjuvants well-known to those skilled in the art include, for example, aluminum phosphate, aluminum hydroxide, QS21 , Quil A (and derivatives and components thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of an amino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof.
The compositions may be administered to provide pre-exposure prophylaxis or after a subject has been diagnosed as having a viral infection (e.g., SARS-CoV-2 or a variant thereof infection) or a subject exposed to an infective agent, such as a virus (e.g., a coronavirus infection, such as a SARS-CoV-2 or a variant thereof). The composition may be administered, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months pre-exposure to a SARS-CoV-2 or a variant thereof, or may be administered to the subject 15-30 minutes or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48, or 72 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, 3, 4, 6, or 9 months, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years or post-exposure to a coronavirus (e.g., SARS-CoV-2 or a variant thereof).
When treating viral infection (e.g., a SARS-CoV-2 or a variant thereof infection), the compositions may be administered to the subject either before the occurrence of symptoms or a definitive diagnosis or after diagnosis or symptoms become evident. For example, the composition may be administered, for example, immediately after diagnosis or the clinical recognition of symptoms or 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days after diagnosis or detection of symptoms.
One or more doses (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) of an immunogenic composition or anti-SARS-CoV-2 or a variant thereof antibody-containing composition may be administered to a subject in need thereof. In some embodiments, a subject is administered at least one dose. In some embodiments, a subject is administered at least two doses. In some embodiments, doses are administered on the same day. In some embodiments, doses are administered on different days. In some embodiments, an immunogenic composition is administered to a subject in need thereof as a prime, a boost, or as a prime-boost. In some embodiments, the boost is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 days, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years after the prime of a prime-boost regimen. In other embodiments, multiple boost doses are administered, in which each boost does is administered at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 days, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years apart.
One or more doses of any of the compositions described herein (e.g., any of the immunogenic compositions described herein) may be administered with one or more additional therapeutic agents either sequentially or simultaneously.
Dosages
The dose of the compositions or the number of treatments using the compositions may be increased or decreased based on the severity of, occurrence of, or progression of, the disease in the subject (e.g., based on the severity of one or more symptoms of, e.g., viral infection).
The pharmaceutical compositions can be administered in a therapeutically effective amount that provides an immunogenic and/or protective effect against an infective agent (e.g., a SARS-CoV-2 or a variant thereof). In some embodiments, a composition containing a nucleic acid molecule, polypeptide, vector, and/or antibodies may be administered in a dose of at least 1 pg to 100 mg (e.g., at least 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 225 pg, 250 pg, 275 pg, 300 pg, 325 pg, 350 pg, 375 pg, 400 pg, 425 pg, 450 pg, 475 pg, 500 pg, 525 pg, 550 pg, 575 pg, 600 pg, 625 pg, 650 pg, 675 pg, 700 pg, 725 pg, 750 pg, 775 pg, 800 pg, 825 pg, 850 pg, 875 pg, 900 pg, 925 pg, 950 pg, 975 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6mg, 7mg, 8mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or more). In some embodiments, a composition containing a nucleic acid molecule, vector, and/or antibody is administered at a dose of about 50 pg (e.g., a dose between about 25 pg and about 75 pg). In some embodiments, a composition containing a nucleic acid molecule, vector, and/or antibody is administered at a dose of about 5 mg (e.g., a dose of about 1 mg to about 10 mg).
In some instances, administration of an effective amount of a composition of the disclosure (e.g., an immunogen, such as a protein having all or a fragment of the amino acid sequence of one or more of SEQ ID NOs: 87-90, 92-95, and 103-107 or a nucleic acid molecule (e.g., a vector, such as a viral vector) encoding the immunogen, which can be administered alone or in combination with a protein having the amino acid sequence of SEQ ID NO: 21 or 23) induces a protective level (e.g., above a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, above a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is above a level of at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein) of anti- coronavirus antibodies (e.g., antibodies against a modified S, MEM, CR, and/or NUL protein of SARS- CoV-2 or a variant thereof (e.g., one or more of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, EG9, MEMO, MEM5, MEM6, NULO, NUL1 , NUL5, NUL6, and NUL7 immunogens), e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies, e.g., broadly neutralizing anti-Spike antibodies, e.g., anti-membrane antibodies, e.g., anti-membrane neutralizing antibodies, e.g., broadly neutralizing antimembrane antibodies, e.g., anti-nucleocapsid antibodies, e.g., anti-nucleocapsid neutralizing antibodies, e.g., broadly neutralizing anti-nucleocapsid antibodies). In some instances, the protective level is a titer of at least about 70 (e.g., at least about 80, at least about 100, or at least about 120) as measured using the pseudovirus neutralization assay described herein. In some instances, the protective level is a titer of at least about 100, as measured using the pseudovirus neutralization assay described herein. In some instances, administration of an effective amount of a composition results in a protective level of anticoronavirus antibodies (e.g., antibodies against a modified S protein of SARS-CoV-2 or a variant thereof , e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies, e.g., broadly neutralizing anti-Spike antibodies) that are maintained for at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 years or more.
In some instances, administration of an effective amount of a composition (e.g., an immunogen, such as a protein having all or a fragment of the amino acid sequence of one or more of SEQ ID NOs: 87- 90 and 92-95 or a nucleic acid molecule (e.g., a vector, such as a viral vector) encoding the immunogen, which can be administered alone or in combination with a protein having the amino acid sequence of SEQ ID NO: 21 or 23) reduces SARS-CoV-2 or a variant thereof serum viral loads determined from a subject having a SARS-CoV-2 or a variant thereof infection by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to viral loads determined from the patient prior to administration of an effective amount of a composition. In some instances, administration of an effective amount of a composition reduces serum viral loads to an undetectable level compared to viral loads determined from the patient prior to administration of an effective amount of a composition. In some instances, administration of an effective amount of a composition results in a reduced and/or undetectable serum viral load that may be maintained for at least about 1 , 2, 3, 4, 5, 6, 7 days; 1 , 2, 3, 4, weeks; 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months; or 1 year or more.
The dosage administered may depend on the subject to be treated (e.g., the age, body weight, capacity of the immune system, and general health of the subject being treated), the form of administration (e.g., as a solid or liquid), the manner of administration (e.g., by injection, inhalation, or dry powder propellant), and the cells targeted (e.g., epithelial cells, such as blood vessel epithelial cells, nasal epithelial cells, or pulmonary epithelial cells). The composition may be administered in an amount that provides a sufficient level of the antigenic or therapeutic gene product, or fragment thereof (e.g., a level of an antigenic gene product that elicits an immune response without undue adverse physiological effects in the host caused by the antigenic gene product).
The method of delivery, for example a DNA or RNA vaccine, may also determine the dose amount. In some cases, dosage administered by injections by intravenous (i.v.) or intramuscular (i.m.) route may require variable amounts of a DNA or RNA vaccine, for example from 10 pg-1 mg. However, administration using a gene gun may require a dose of a DNA or RNA vaccine between 0.2 pg and 20 pg (e.g., 0.2, 0.1 , 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 pg). In some instances, the use of a gene gun to deliver a dose of a DNA or RNA vaccine may require only ng quantities of DNA or RNA, for example between 10 ng and 200 ng (e.g., 10, 12, 13, 14, 15, 16, 17, 18, 19, 20.30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 ng).
In other embodiments, the delivery vector is a virus (e.g., an Ad26 virus) and the subject can be administered at least about 1 x103 viral particles (VP)/dose or between 1 x101 and 1 x1020 VP/dose (e.g., 1 x101 , 1 x102, 1 x103, 1 x104, 1 x105, 1 x106, 1 x107, 1 x10s, 1 x109, 1 x1010, 1 x1011 , 1 x1012, 1 x1013, 1 x1014, 1 x1015, 1 x1016, 1 x1017, 1 x1018, 1 x1019, or 1 x1020 VP/dose). For example, the subject can be administered about 1 x106 to about 1 x1014 VP/dose (e.g., about 1 x107, about 1 x108, about 1 x109, about 1 x1010, about 1 x1011 , about 1 x1012, about 1 x1013, about 1 x1014, or about 1 x1015 VP/dose). For example, the subject can be administered about 1 x1011 , about 1 x1012, about 1 x1013, or about 1 x1014 VP/dose.
In addition, single or multiple administrations of the compositions of the disclosure may be given (pre- or post-exposure and/or pre- or post-diagnosis) to a subject (e.g., one administration or administration two or more times). For example, subjects who are particularly susceptible to, for example, viral infection (e.g., a SARS-CoV-2 or a variant thereof infection) may require multiple treatments to establish and/or maintain protection against the virus. Levels of induced immunity provided by the pharmaceutical compositions described herein can be monitored by, for example, measuring amounts of neutralizing secretory and serum antibodies. The dosages may then be adjusted or repeated as necessary to trigger the desired level of immune response. For example, the immune response triggered by a single administration (prime) of a composition may not sufficiently potent and/or persistent to provide effective protection. Accordingly, in some embodiments, repeated administration (boost), such that a prime boost regimen is established, can significantly enhance humoral and cellular responses to the antigen of the composition.
Alternatively, the efficacy of treatment can be determined by monitoring the level of the antigenic or therapeutic gene product, or fragment thereof, expressed in a subject (e.g., a human) following administration of the compositions. For example, the blood or lymph of a subject can be tested for antigenic or therapeutic gene product, or fragment thereof, using, for example, standard assays known in the art.
In some instances, efficacy of treatment can be determined by monitoring a change in the serum viral load from a sample from the subject obtained prior to and after administration of an effective amount of a composition (e.g., an immunogen, such as a protein having all or a fragment of the amino acid sequence of one or more of SEQ ID NOs: 87-90, 92-95, and 103-107, or a nucleic acid molecule (e.g., a vector, such as a viral vector) encoding the immunogen, which can be administered alone or in combination with a protein having the amino acid sequence of SEQ ID NO: 23). A reduction in serum viral load of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to viral load determined from the subject prior to administration of an effective amount of a composition may indicate that the subject is receiving benefit from the treatment. If a viral load does not decrease by at least about 10%, 20%, 30%, or more after administration of a composition, the dosage of the composition to be administered may be increased. For example, by increasing the pg or mg amount of a DNA vaccine (e.g., a DNA vaccine containing a nucleic acid molecule with a nucleic acid sequence of one or more of SEQ ID NOs: 21 , 23, 57-60 and 62-65, which can be administered alone or in combination with any one of the nucleic acid molecules with the sequence of SEQ ID NO: 2-6, 42, 43, 47, and 52) administered to the subject or by increasing the number of viral particles (VP) of an adenovirus vector-based vaccine (e.g., an adenovirus vector-based vaccine containing the nucleic acid sequence of one or more of SEQ ID NOs: 21 , 23, 57-60 and 62-65, which can be administered alone or in combination with a nucleic acid molecule with the sequence of SEQ ID NO: 2-6, 42, 43, 47, and 52).
A single dose of a composition may achieve protection, pre-exposure or pre-diagnosis. In addition, a single dose administered post-exposure or post-diagnosis can function as a treatment according to the disclosure.
A single dose of a composition can also be used to achieve therapy in subjects being treated for an infection (e.g., a coronavirus infection, such as a SARS-CoV-2 or a variant thereof infection). Multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more doses) can also be administered to these subjects, if deemed to be therapeutically beneficial.
Methods of Diagnosing and Predicting Susceptibility to Coronavirus Infection
Diagnostic Methods
Provided herein are methods for identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection. The method includes measuring the level or amount of a broadly neutralizing anti-coronavirus antibody (bNAb) against two or more lineages of coronavirus (such as an anti-Spike antibody) in a sample (e.g., a whole blood sample, e.g., a serum or plasma sample) from the subject. In some embodiments, the lineage is an Alpha, Beta, Gamma, Delta, or Omicron (e.g., BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6) variant. In other embodiments, the two or more lineages of coronavirus are selected from the group consisting of B.1 .1 .529 (e.g., BA.1 , BA.2, BA.3, BA.4, and BA.5), B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , A23.1 , B.1 .617.1 , B.1.617.2, B.1.427, B.1.525, B.1.526, P.1 , P.2, P.3, C.36, C.37, B.1.1.519, B.1 .526.1 , B.1 .526.2, R.1 , B.1.258.17, B.1.575, B.1.214.2, A.2.5.2, AT.1 , B.1 .1 .523, B.1.620, BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6, or other known or subsequently arising lineage (see, e.g., cov-lineages.org/lineage_list.html, which is incorporated herein by reference). In some embodiments, the coronavirus is SARS-CoV-2 or a variant thereof. In some embodiments, the subject is determined to be susceptible to the coronavirus infection if the broadly neutralizing anti-coronavirus antibody (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti-nucleocapsid antibody) amount or level is below a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or below 80% of a median level of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof)) as determined by a pseudovirus neutralization assay or live virus neutralization assay) and determined to not be susceptible to the coronavirus infection if the broadly neutralizing anticoronavirus antibody (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti-nucleocapsid antibody) level is above a protective level. In some embodiments, the protective level is a broadly neutralizing anti-coronavirus antibody titer (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti- nucleocapsid antibody titer) of at least about 70 (e.g., about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 110, 115, 120, 125, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350 or more) as determined in a pseudovirus neutralization assay. In some embodiments, the protective level is a broadly neutralizing anti-coronavirus antibody titer (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti- nucleocapsid antibody titer) of at least about 83 as determined in a pseudovirus neutralization assay. In some embodiments, the protective level is a broadly neutralizing anti-coronavirus antibody titer (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti-nucleocapsid antibody titer) of at least about 25 (e.g., about 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 110, 115, 120, 125, 130, 140, 150, 175, 200 or more) as determined in a live virus neutralization assay. In some embodiments, the protective level is a broadly neutralizing anti-coronavirus antibody titer (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti-nucleocapsid antibody titer) of at least about 35 as determined in a live virus neutralization assay In some embodiments, the protective level is a broadly neutralizing anti-coronavirus antibody titer (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti- nucleocapsid antibody titer) that is at least about 60% (e.g., about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 110%, about 120%) of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. In some embodiments, the protective level is a broadly neutralizing anti-coronavirus antibody titer (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti-nucleocapsid antibody titer) that is at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. A subject determined to be susceptible to the coronavirus infection (a subject with a broadly neutralizing anticoronavirus antibody (e.g., a broadly neutralizing anti-Spike, anti-membrane, or anti-nucleocapsid antibody) amount or level is below a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or below 80% of a median level of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof)) as determined by a pseudovirus neutralization assay or live virus neutralization assay)) can be administered a therapy (e.g., administered any of the compositions described herein), such as an effective amount of one or more of the pharmaceutical compositions (e.g., immunogenic compositions and antibodies against a modified S, MEM, CR, and/or NUL protein of SARS-CoV-2 or a variant thereof) described herein. A subject may be re-administered a therapy until the subject is determined to not be susceptible to the coronavirus infection (e.g., until the subject has a level of a broadly neutralizing anti-coronavirus antibody (e.g., a broadly neutralizing anti-Spike antibody, such as an antibody that specifically binds to one or more of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, EG9, MEMO, MEM5, MEM6, NUL0, NUL1 , NUL2, NUL3, NUL4, NUL5, NUL6, and NUL7 immunogens) that is above a protective level (e.g., a level above a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, above a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is at a level that is at least 80% of a median level (such as at or above a median level) of a broadly neutralizing anticoronavirus antibody of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof)) as determined by a pseudovirus neutralization assay or live virus neutralization assay)). The method may also involve determining whether the anti-Spike antibody is an RBD-specific antibody. The method may also involve determining whether the anti-Spike antibody is an S1 -specific antibody. The method may also involve determining whether the anti-Spike antibody is an S2-specific antibody. The method may also involve identifying the subclass (e.g., IgM, IgA, IgG 1 , lgG2, lgG3, or FcgR2A.1 ) and/or effector function (e.g., antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation (IFN-Y secretion, CD107a degranulation, and MIP-1 p expression)) of the anti-coronavirus antibody. The method may further include administering one or more of the pharmaceutical compositions (e.g., one of the immunogenic compositions and/or a composition containing an antibody against a modified S (e.g., one or more of the EGO, EG1 , EG2, EG3, EG4, EG5, EG6, EG7, EG8, and EG9 immunogens), MEM (e.g., one or more of the MEMO, MEM5, and MEM6 immunogens), CR (e.g., one or more of the CRO and CR5 immunogen) and/or NUL (e.g., one or more of the NULO, NUL1 , NUL5, NUL6, and NUL7 immunogens) protein of SARS-CoV-2 or a variant thereof) described herein to a subject determined to be in need of further therapy.
The method may include measuring the coronavirus (e.g., SARS-CoV-2 or a variant thereof) viral load in a sample from the subject. In some embodiments, the sample is a bronchoalveolar lavage (BAL) or a nasal swab (NS). In some embodiments, the sample is a bodily fluid (e.g., blood, e.g., whole blood or plasma) from the subject. In some embodiments, the sample is a tissue sample (e.g., a respiratory tract tissue sample) from the subject. In some embodiments, viral load is a detectible nucleic acid (e.g., subgenomic mRNA) level or a detectible protein (e.g., nucleocapsid protein (NUL)) level. In some embodiments, the detectible nucleic acid (e.g., subgenomic mRNA) is determined by RNA-seq, RT- qPCR, qPCR, multiplex qPCR or RT-qPCR, LAMP, microarray analysis, or hybridization (e.g., ISH (e.g., FISH)). In some embodiments, the detectible protein (e.g., nucleocapsid protein (NUL)) is determined by an immunoassay (e.g., an immunohistochemical (IHC) assay or a lateral flow immunoassay). In some embodiments, a detectable viral load indicates that the subject is susceptible to disease (e.g., a SARS- CoV-2 or a variant thereof-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than at least about 3.5 log sgmRNA copies/mL (e.g., about 3.75 logw sgmRNA copies/mL, about 3.8 logio sgmRNA copies/mL, about 3.9 logio sgmRNA copies/mL, about 4.0 log sgmRNA copies/mL, about 4.25 logw sgmRNA copies/mL, about 4.5 logw sgmRNA copies/mL, about 4.75 logw sgmRNA copies/mL, about 5.0 logw sgmRNA copies/mL, about 5.5 logw sgmRNA copies/mL, about 6.0 logw sgmRNA copies/mL, about 6.5 logw sgmRNA copies/mL, about 7.0 logw sgmRNA copies/mL, about 7.5 logw sgmRNA copies/mL, about 8.0 logw sgmRNA copies/mL, about 8.5 logw sgmRNA copies/mL, about 9 logw sgmRNA copies/mL, about 10 logw sgmRNA copies/mL, about 11 logw sgmRNA copies/mL, about 12 log w sgmRNA copies/mL, about 13 logw sgmRNA copies/mL or more). In some embodiments, a viral load of greater than 3.85 logw sgmRNA copies/mL in BAL or 3.78 logw sgmRNA copies/mL in NS indicates that the subject is susceptible to disease (e.g., a SARS-CoV-2 or a variant thereof-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than 3.85 log sgmRNA copies/mL in BAL or 3.78 log 1 o sgmRNA copies/mL in NS indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 2.0 logw sgmRNA copies/g (e.g., about 2.0 logw sgmRNA copies/g, about 2.5 logw sgmRNA copies/g, about 3.0 logw sgmRNA copies/g, about 3.5 logw sgmRNA copies/g, about 4.0 logw sgmRNA copies/g, about 4.25 logw sgmRNA copies/g, about 4.5 logw sgmRNA copies/g, about 4.75 logw sgmRNA copies/g, about 5.0 logw sgmRNA copies/g, about 5.5 logw sgmRNA copies/g, about 6.0 logw sgmRNA copies/g, about 6.5 logw sgmRNA copies/g, about 7.0 logw sgmRNA copies/g, about 7.5 logw sgmRNA copies/g, about 8.0 logw sgmRNA copies/g, about 8.5 logw sgmRNA copies/g, about 9 logw sgmRNA copies/g, about 10 logw sgmRNA copies/g, about 11 logw sgmRNA copies/g, about 12 logw sgmRNA copies/g, about 13 logw sgmRNA copies/g or more) of tissue indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 8.0 logw sgmRNA copies/g in lung tissue, about 7.0 logw sgmRNA copies/g in nares tissue, about 6.0 logw sgmRNA copies/g in trachea tissue, about 5.5 logw sgmRNA copies/g in heart tissue, or about 2.0 logw sgmRNA copies/g in Gl, spleen, liver, kidney, or brain tissue indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 3% (e.g., about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) SARS- CoV-2 vRNA staining by ISH indicates that the subject is susceptible to disease (e.g., a SARS-CoV-2 or a variant thereof-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than about 5% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 5% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, coronavirus (e.g., SARS- CoV-2 or a variant thereof) viral load is measured one or more times over about 1 , 2, 3, 4, 5, or 6 days or 1 , 2, 3, 4, 5, 6, or 7 weeks post-infection.
Monitoring Responsiveness
Provided herein are methods for monitoring an anti-coronavirus immune response of a subject to a therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein) for treating or inhibiting or reducing the risk of a coronavirus infection. The method includes measuring the level or amount of an anti-coronavirus antibody (e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody or a broadly neutralizing anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody) in the subject. In some embodiments, the coronavirus is SARS- CoV-2 or a variant thereof. In some embodiments, the anti-coronavirus antibody (e.g., an anti-Spike, antimembrane, anti-conserved region, or anti-nucleocapsid antibody) is a neutralizing antibody. The anticoronavirus antibody (e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody, e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid neutralizing antibody, e.g., a broadly neutralizing anti-Spike, anti-membrane, anti-conserved region, or anti- nucleocapsid antibody) may be measured in a short timeframe (e.g., in order to measure the robustness of the antibody response) or a longer timeframe (e.g., in order to measure the durability of the antibody response) after administration of a therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein). In some embodiments, the anti-coronavirus antibody (e.g., an antiSpike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody or a broadly neutralizing antiSpike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody) is measured about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 days, about 5, 6, 7, 8, 9, 10, 1 1 , or 12 weeks, about 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 months, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years after the subject is administered the therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein).
The subject is determined to be responsive to the therapeutic composition if the anti-coronavirus antibody (e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody or a broadly neutralizing anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody) detected in the subject (e.g., in the subject’s blood) is above a protective level (e.g., above a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, above a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is at a level that is at least 80% of a median level (such as at or above a median level) of an anti-coronavirus antibody of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., SARS-CoV-2 or a variant thereof)) as determined by a pseudovirus neutralization assay or live virus neutralization assay). Alternatively, the subject is determined to be non- responsive to the therapeutic composition if the anti-coronavirus antibody (e.g., an anti-Spike, antimembrane, anti-conserved region, or anti-nucleocapsid antibody or a broadly neutralizing anti-Spike, antimembrane, anti-conserved region, or anti-nucleocapsid antibody) detected in the subject is below a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is at a level that is below 80% of a median level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay). A protective level of an anti-coronavirus antibody (e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid neutralizing antibody) corresponds to a titer of at least about 70 (e.g., about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 1 10, 1 15, 120, 125, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350 or more) as determined in a pseudovirus neutralization assay (e.g., the pseudovirus neutralization assay described herein). In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid neutralizing antibody titer) of at least about 25 (e.g., about 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 1 10, 1 15, 120, 125, 130, 140, 150, 175, 200 or more) as determined in a live virus neutralization assay (e.g., the pseudovirus neutralization assay described herein). In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike, antimembrane, anti-conserved region, or anti-nucleocapsid neutralizing antibody titer) that is at least about 60% (e.g., about 61 %, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71 %, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 110%, about 120%) of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein.
If, over time, an anti-coronavirus antibody (e.g., an anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody or a broadly neutralizing anti-Spike, anti-membrane, anti-conserved region, or anti-nucleocapsid antibody) titer in the subject (e.g., in the blood of a subject) falls below or fails to reach a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or below 80% of a median level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay described herein), the subject may be administered or may be re-administered a coronavirus vaccine composition (e.g., one or more of the therapeutic or immunogenic compositions described herein) alone or in combination with an additional therapeutic agent, such as one or more of the additional therapeutic agents described herein. Administration of a composition of the disclosure to a subject in need thereof can be performed one or more times (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more times) over one or more days (e.g., 1 , 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 weeks), months (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months), or years (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years), or over the life of the subject, as needed to maintain a protective level of an anti-coronavirus antibody in the subject, thereby protecting the subject against coronavirus infection (e.g., infection by SARS-CoV-2 or a variant thereof).
The method may include measuring the coronavirus (e.g., SARS-CoV-2 or a variant thereof) viral load in a sample from the subject. In some embodiments, the coronavirus is SARS-CoV-2 or a variant thereof. In some embodiments, the viral load is measured about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 30, 36, 42, or 48 hours or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 days post-infection. In some embodiments, the sample is a bronchoalveolar lavage (BAL) or a nasal swab (NS). In some embodiments, the sample is a bodily fluid (e.g., blood, e.g., whole blood or plasma) from the subject. In some embodiments, the sample is a tissue sample (e.g., a respiratory tract tissue sample) from the subject. In some embodiments, viral load is a detectible nucleic acid (e.g., subgenomic mRNA) level or a detectible protein (e.g., nucleocapsid protein (NUL)) level. In some embodiments, the detectible nucleic acid (e.g., subgenomic mRNA) is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT- qPCR, LAMP, microarray analysis, or hybridization (e.g., ISH (e.g., FISH)). In some embodiments, the detectible protein (e.g., nucleocapsid protein (NUL)) is determined by an immunoassay (e.g., an immunohistochemical (IHC) assay or a lateral flow immunoassay). The subject is determined to be responsive to the therapeutic composition if the viral load is below a pre-assigned level. In some embodiments, the pre-assigned level is less than about 3.5 logw sgmRNA copies/mL BAL or NS or less than about 5.0 logw sgmRNA copies/g of tissue (e.g., lung, nares, trachea, heart, Gl, spleen, liver, kidney, or brain tissue). In some embodiments, the subject is determined to be responsive to the therapeutic composition if the viral load decreases in the subject. If the subject is not determined to be responsive as determined by viral load, then the subject may be administered or may be re-administered a coronavirus vaccine composition (e.g., one or more of the therapeutic or immunogenic compositions described herein) alone or in combination with an additional therapeutic agent, such as one or more of the additional therapeutic agents described herein.
Administration of a composition of the disclosure to a subject in need thereof can be performed one or more times (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more times) over one or more days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 days) as needed to reduce the viral load.
EXAMPLES
The following examples are to illustrate the invention. They are not meant to limit the invention in any way.
Example 1. Administration of EGO-3 nucleic acid vaccines to a human subject
A human subject can be administered a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) composition of this disclosure pre- or post-exposure to SARS-CoV-2 or a variant thereof according to the methods described herein. The human subject may be identified as being at high risk for infection, such as an individual who has or will be traveling to a region where infection of SARS-CoV-2 or a variant thereof is prevalent, or may be identified as presenting with symptoms consistent with an infection of SARS-CoV-2 or a variant thereof. The nucleic acid vaccine administered may include, e.g., the nucleic acid sequence of EGO (e.g., SEQ ID NOs: 57 or 62, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof), EG1 (e.g., SEQ ID NOs: 58 or 63, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof), EG2 (e.g., SEQ ID NOs: 59 or 64, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof), and/or EG3 (e.g., SEQ ID NOs: 60 or 65, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof), or any combination of EGO-3.
For example, a human identified as having a risk of infection of SARS-CoV-2 or a variant thereof and may be administered a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) containing a nucleic acid molecule encoding a modified Spike (S) protein of SARS-CoV-2 or a variant thereof (e.g., a nucleic acid molecule of any one or more of SEQ ID NOs: 57-60 and 62-65, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof), e.g., in an adenoviral vector (e.g., Ad26) at a dose of between 10 pg and 10 mg. The nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) may contain the nucleic acid sequence of one or more of SEQ ID NOs: 57-60 and 62-65.
The subject may also be administered a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) containing a nucleic acid molecule encoding a 2019-nCoV (Wuhan/WIV04/2019) nucleic acid (e.g., a nucleic acid molecule with the nucleic acid sequence of one or more of SEQ ID NOs: 2-6). The subject may also be administered, either separately or in combination with one of the other nucleic acid vaccines described in this example, a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) containing a nucleic acid molecule encoding a modified membrane (MEM), a conserved region (CR) and/or nucleocapsid (NUL) protein of SARS-CoV-2 or a variant thereof (e.g., a nucleic acid molecule of any one or more of SEQ ID NOs: 42, 43, 47, 52, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof), e.g., in an adenoviral vector (e.g., Ad26) at a dose of between 10 pg and 10 mg.
The nucleic acid vaccine may be one that contains one or more of the MEM, CR, and NUL nucleic acid sequences in combination with one or more of the EGO, EG1 , EG2, and EG3 nucleic acid sequences (e.g., as a polyvalent vaccine).
The subject can then be monitored for presentation of symptoms of 2019-nCoV infection, the resolution of symptoms, and/or the production of antibodies against the modified S, MEM, or NUL protein of SARS-CoV-2 or a variant thereof. If necessary, a second dose or additional doses of the nucleic acid vaccine(s) can be administered.
Example 2. Administration of EG0-4 immunogenic S proteins of SARS-CoV-2 polypeptide to a human subject
A human subject can be administered an immunogenic composition (e.g., containing a modified S protein of a coronavirus) of this disclosure pre- or post-exposure to SARS-CoV-2 or a variant thereof according to the methods described herein. The human subject may be identified as being at high risk for infection, such as an individual who has or will be traveling to a region where infection of SARS-CoV-2 or a variant thereof is prevalent, or may be identified as presenting with symptoms consistent with an infection of SARS-CoV-2 or a variant thereof.
For example, a human with an underlying health condition (e.g., one or more of hypertension, diabetes, and cardiovascular disease) may be identified as having a risk of infection of SARS-CoV-2 or a variant thereof and may be administered a modified S protein of SARS-CoV-2 or a variant thereof as an immunogen (e.g., a Spike protein of any one of SEQ ID NOs: 87-90 and 92-95, or a variant thereof with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., at a dose of between 10 pg and 10 mg. The immunogen is one or more polypeptides encoded by SEQ ID NOs: 57-60 and 62-65. The subject may also be administered a 2019-nCoV Spike protein as an immunogen (e.g., a Spike protein having the sequence of SEQ ID NO: 21 or 23, or a variant thereof with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., at a dose of between 10 pg and 10 mg. The subject may also be administered an immunogen containing a modified membrane (MEM), conserved region (CR), and/or nucleocapsid (NUL) protein of SARS-CoV-2 or a variant thereof (e.g., a polypeptide of any one or more of SEQ ID NOs: 42, 43, 47, 52, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., in an adenoviral vector (e.g., Ad26) at a dose of between 10 pg and 10 mg.
The subject can then be monitored for presentation of symptoms of infection of SARS-CoV-2 or a variant thereof, the resolution of symptoms, and/or the production of antibodies against the S, MEM, CR, or NUL protein of SARS-CoV-2 or a variant thereof. If necessary, a second dose or additional doses of the immunogen(s) can be administered.
Example 3. Administration of antibodies against EGO-3 S proteins of SARS-CoV-2 or a variant thereof to a human subject at risk of, or presenting symptoms of, infection by SARS-CoV-2 or a variant thereof
A human subject infected with SARS-CoV-2 or a variant thereof or identified as having a risk of infection by SARS-CoV-2 or a variant thereof (e.g., a subject that has traveled to a region where infection of SARS-CoV-2 or a variant thereof is prevalent) can be administered an antibody against a modified S protein of SARS-CoV-2 or a variant thereof that binds an epitope within the amino acid sequence of any one of SEQ ID NOs: 87-90 and 92-95, such as the NTD and/or RBD region of SEQ ID NOs:87-90 and 92- 95, and, in particular, an epitope containing one or more of the EGO, EG1 , EG2, and EG3 mutations. For example, the antibody may have been generated against one or more of the polypeptides of SEQ ID NOs: 87-90 and 92-95. The antibody composition can be administered to the subject at a dose of the antibody of between 1 -1 ,000 mg. In addition, administration can optionally include 1 -1000 mg of an antibody against a modified membrane (MEM), conserved region (CR), and/or nucleocapsid (NUL) protein of SARS-CoV-2 or a variant thereof that binds to an epitope with the amino acid sequence of any one of SEQ ID NOs: 42, 43, 47, 52, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The antibody composition may be administered to the subject as a prophylactic therapy, e.g., prior to or post-exposure to a SARS-CoV-2 or a variant thereof. The subject can then be monitored for presentation of symptoms of infection of SARS-CoV-2 or a variant thereof or the resolution of symptoms. If necessary, a second dose or subsequent doses of the antibody composition can be administered to the subject.
Example 4. Administration of EG5-9 nucleic acid vaccines to a human subject
A human subject can be administered a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) composition of this disclosure pre- or post-exposure to SARS-CoV-2 or a variant thereof according to the methods described herein. The human subject may be identified as being at high risk for infection, such as an individual who has or will be traveling to a region where infection of SARS-CoV-2 or a variant thereof is prevalent, or may be identified as presenting with symptoms consistent with an infection of SARS-CoV-2 or a variant thereof. The nucleic acid vaccine administered may include, e.g., the nucleic acid sequence of EG5 (e.g., SEQ ID NO: 103, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof); EG6 (e.g., SEQ ID NO: 104, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof); EG7 (e.g., SEQ ID NO: 105, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof); EG8 (e.g., SEQ ID NO: 106, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof); and/or EG9 (e.g., SEQ ID NO: 107, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, or a complementary sequence thereof); or any combination of EG5-9.
For example, a human identified as having a risk of infection of SARS-CoV-2 or a variant thereof and may be administered a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) containing a nucleic acid molecule encoding a modified Spike (S) protein of SARS-CoV-2 or a variant thereof (e.g., a nucleic acid molecule encoding the polypeptide of any one or more of SEQ ID NOs: 103-107, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto, e.g., in an adenoviral vector (e.g., Ad26) at a dose of between 10 pg and 10 mg.
The subject may also be administered a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) containing a nucleic acid molecule encoding a 2019-nCoV (Wuhan/WIV04/2019) nucleic acid (e.g., a nucleic acid molecule with the nucleic acid sequence of one or more of SEQ ID NOs: 2-6).
The subject may also be administered, either separately or in combination with one of the other nucleic acid vaccines described in this example, a nucleic acid vaccine (e.g., a DNA vaccine or an RNA vaccine) containing a nucleic acid molecule encoding a modified membrane (MEM), a conserved region (CR) and/or nucleocapsid (NUL) protein of SARS-CoV-2 or a variant thereof (e.g., a nucleic acid molecule encoding the polypeptide of any one or more of SEQ ID NOs: 97-102, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., in an adenoviral vector (e.g., Ad26) at a dose of between 10 pg and 10 mg.
The nucleic acid vaccine may be one that contains one or more of the MEM, CR, and NUL nucleic acid sequences in combination with one or more of the EG5, EG6, EG7, EG8, and EG9 nucleic acid sequences (e.g., as a polyvalent vaccine).
The subject can then be monitored for presentation of symptoms of 2019-nCoV infection, the resolution of symptoms, and/or the production of antibodies against the modified S, MEM, or NUL protein of SARS-CoV-2 or a variant thereof. If necessary, a second dose or additional doses of the nucleic acid vaccine(s) can be administered.
Example 5. Administration of EG5-9 immunogenic S proteins of SARS-CoV-2 polypeptide to a human subject
A human subject can be administered an immunogenic composition (e.g., containing a modified S protein of a coronavirus) of this disclosure pre- or post-exposure to SARS-CoV-2 or a variant thereof according to the methods described herein. The human subject may be identified as being at high risk for infection, such as an individual who has or will be traveling to a region where infection of SARS-CoV-2 or a variant thereof is prevalent, or may be identified as presenting with symptoms consistent with an infection of SARS-CoV-2 or a variant thereof.
For example, a human with an underlying health condition (e.g., one or more of hypertension, diabetes, and cardiovascular disease) may be identified as having a risk of infection of SARS-CoV-2 or a variant thereof and may be administered a modified S protein of SARS-CoV-2 or a variant thereof as an immunogen (e.g., a Spike protein of any one of SEQ ID NOs: 103-107, or a variant thereof with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., at a dose of between 10 pg and 10 mg. The subject may also be administered a 2019-nCoV Spike protein as an immunogen (e.g., a Spike protein having the sequence of SEQ ID NO: 21 or 23, or a variant thereof with at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., at a dose of between 10 pg and 10 mg. The subject may also be administered an immunogen containing a modified membrane (MEM), conserved region (CR), and/or nucleocapsid (NUL) protein of SARS-CoV-2 or a variant thereof (e.g., a polypeptide of any one or more of SEQ ID NOs: 97-102, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto), e.g., in an adenoviral vector (e.g., Ad26) at a dose of between 10 pg and 10 mg.
The subject can then be monitored for presentation of symptoms of infection of SARS-CoV-2 or a variant thereof, the resolution of symptoms, and/or the production of antibodies against the S, MEM, CR, or NUL protein of SARS-CoV-2 or a variant thereof. If necessary, a second dose or additional doses of the immunogen(s) can be administered.
Example 6. Administration of antibodies against EG5-9 S proteins of SARS-CoV-2 or a variant thereof to a human subject at risk of, or presenting symptoms of, infection by SARS-CoV-2 or a variant thereof
A human subject infected with SARS-CoV-2 or a variant thereof or identified as having a risk of infection by SARS-CoV-2 or a variant thereof (e.g., a subject that has traveled to a region where infection of SARS-CoV-2 or a variant thereof is prevalent) can be administered an antibody against a modified S protein of SARS-CoV-2 or a variant thereof that binds an epitope within the amino acid sequence of any one of SEQ ID NOs: 103-107, such as the NTD and/or RBD region of SEQ ID NOs: 103-107, and, in particular, an epitope containing one or more of the EG5, EG6, EG7, EG8, and EG9 mutations. For example, the antibody may have been generated against one or more of the polypeptides of SEQ ID NOs: 103-107. The antibody composition can be administered to the subject at a dose of the antibody of between 1 -1 ,000 mg. In addition, administration can optionally include 1 -1000 mg of an antibody against a modified membrane (MEM), conserved region (CR), and/or nucleocapsid (NUL) protein of SARS-CoV-2 or a variant thereof that binds to an epitope with the amino acid sequence of any one of SEQ ID NOs: 97- 102, or a variant thereof with 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The antibody composition may be administered to the subject as a prophylactic therapy, e.g., prior to or post-exposure to a SARS-CoV-2 or a variant thereof. The subject can then be monitored for presentation of symptoms of infection of SARS-CoV-2 or a variant thereof or the resolution of symptoms. If necessary, a second dose or subsequent doses of the antibody composition can be administered to the subject.
Other Embodiments
All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

1 . An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence of any one or more of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a variant thereof.
2. The nucleic acid molecule of claim 1 , wherein the polypeptide is capable of eliciting an immune response in a subject.
3. The nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule comprises the nucleotide sequence of any one or more of SEQ ID NOs: 42, 43, 47, 52, and 57-60, or a variant thereof or a complementary sequence thereof.
4. The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule encodes the polypeptide comprising the amino acid sequence of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or each of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or said variant thereof.
5. The nucleic acid molecule of any one of claims 1 -4, wherein the nucleotide sequence encodes the polypeptide having the amino acid sequence of any one or more of SEQ ID NOs: 87-90 and 103-107 or said variant thereof.
6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of any one or more of SEQ ID NOs: 57-60 or a variant thereof or a complementary sequence thereof.
7. The nucleic acid molecule of claim 5 or 6, wherein the nucleic acid molecule encodes the polypeptide comprising the amino acid sequence of two, three, four, five, six, seven, eight, or each of SEQ ID NOs: 87-90 and 103-107 or said variant thereof.
8. The nucleic acid molecule of any one of claims 1 -4, wherein the nucleotide sequence encodes the polypeptide having an amino acid sequence of any one or more of SEQ ID NOs: 72, 73, 77, 82, and 97- 102 or said variant thereof.
9. The nucleic acid molecule of claim 8, wherein the nucleic acid molecule comprises the nucleotide sequence of any one or more of SEQ ID NOs: 42, 43, 47, and 52 or a variant thereof or a complementary sequence thereof.
10. The nucleic acid molecule of claim 8 or 9, wherein the nucleic acid molecule encodes the polypeptide comprising the amino acid sequence of two, three, four, five, six, seven, eight, nine, or each of SEQ ID NOs: 72, 73, 77, 82, and 97-102 or said variant thereof.
11 . An isolated polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a portion or variant thereof.
12. The polypeptide of claim 11 , wherein the polypeptide is encoded by the nucleic acid molecule of any one of claims 1 -10 or a variant thereof.
13. The polypeptide of claim 11 or 12, wherein the polypeptide, or portion thereof, is capable of eliciting an immune response in a subject.
14. The polypeptide of any one of claims 11 -13, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 87-90 and 103-107 or said variant thereof.
15. The polypeptide of any one of claims 11 -13, wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 72, 73, 77, 82, and 97-102 or said variant thereof.
16. An isolated vector comprising the nucleic acid molecule of any one of claims 1 -10 or a variant thereof.
17. The isolated vector of claim 16, wherein the nucleic acid molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 57-60 or a variant thereof.
18. The isolated vector of claim 16 or 17, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of two or three, four, five, six, seven, eight, or each, of SEQ ID NOs: 87-90 and 103-107 or a variant thereof.
19. The isolated vector of any one of claims 16-18, wherein the vector is replication-defective.
20. The isolated vector of any one of claims 16-19, wherein the vector is a mammalian, bacterial, or viral vector.
21 . The isolated vector of claim 20, wherein the vector is an expression vector.
22. The isolated vector of claim 20, wherein the viral vector is a virus selected from the group consisting of a retrovirus, adenovirus, adeno-associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox.
23. The isolated vector of claim 22, wherein the vector is an adenovirus.
24. The isolated vector of claim 23, wherein the adenovirus is selected from the group consisting of Ad26, Ad52, Ad59, Ad2, Ad5, Ad11 , Ad12, Ad24, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, and Pan9, wherein optionally the adenovirus is Ad26 or Ad52.
25. The isolated vector of claim 24, wherein the Ad52 is a rhesus Ad52 or the Ad59 is a rhesus Ad59.
26. A composition comprising the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 11 -15, or the vector of any one of claims 16-25.
27. The composition of claim 26, comprising any two or more of the nucleic acid molecules of any one of claims 1 -10, wherein optionally the nucleic acid molecule of the composition encodes any two or more of the polypeptides of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a variant thereof, or any two or more of the polypeptides of any one of claims 11 -15, wherein optionally the composition comprises any two or more of the polypeptides of SEQ ID NOs: 72, 73, 77, 82, 87-90, and 97-107 or a portion or variant thereof.
28. The composition of claim 26, comprising any three or more of the nucleic acid molecules of any one of claims 1 -10, wherein optionally the nucleic acid molecule of the composition encodes any three or more of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a variant thereof, or any three or more of the polypeptides of any one of claims 11 -15, wherein optionally the composition comprises any three or more of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a portion or variant thereof.
29. The composition of claim 26, comprising any four or more of the nucleic acid molecules of any one of claims 1 -10, wherein optionally the nucleic acid molecule of the composition encodes any four or more of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a variant thereof, or any four or more of the polypeptides of any one of claims 11 -15, wherein optionally the composition comprises each of the polypeptides of SEQ ID NOs: 87-90 and 103-107 or a portion or variant thereof.
30. The composition of any one of claims 26-29, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.
31 . The composition of claim 30, further comprising an adjuvant or an immunostimulatory agent.
32. The composition of any one of claims 26-31 , wherein the composition is a vaccine.
33. The composition of claim 32, wherein the vaccine is a monovalent or a polyvalent vaccine.
34. The composition of claim 32 or 33, wherein the composition is capable of treating or reducing the risk of a coronavirus infection, such as, for example, infection by a 2019-nCoV virus or a variant thereof, in a subject in need thereof.
35. The composition of any one of claims 26-34, wherein said composition elicits production of neutralizing anti-2019-nCoV antisera in said subject.
36. The composition of claim 35, wherein the subject is infected with a lineage of 2019-nCoV.
37. The composition of claim 36, wherein the lineage of 2019-nCoV is an Alpha, Beta, Gamma, Delta, or Omicron variant, such as, e.g., a variant of the lineage B.1 .1 .529 (e.g., BA.1 , BA.2, BA.3, BA.4, and BA.5), B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , A23.1 , P.1 , B.1 .617.2, BA.1 , BA.2, BA.4/5, BQ.1 .1 , XBB.1 , XBB.1 .5, EG.5, EG.5.1 , FL.1 .5.1 , or XBC.1 .6.
38. The composition of claim 37, wherein the lineage of 2019-nCoV is B.1 .1 .529.
39. The composition of any one of claims 26-38, wherein the subject is a mammal.
40. The composition of claim 39, wherein the mammal is a human.
41 . The composition of claim 40, wherein the human has an underlying health condition.
42. The composition of claim 41 , wherein the underlying health condition is hypertension, diabetes, or cardiovascular disease.
43. The composition of any one of claims 26-42, further comprising any one or more of the following:
(a) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
72 or a variant thereof;
(b) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
73 or a variant thereof;
(c) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 77 or a variant thereof;
(d) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 82 or a variant thereof;
(e) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
97 or a variant thereof;
(f) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
98 or a variant thereof;
(g) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
99 or a variant thereof;
(h) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
100 or a variant thereof;
(i) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
101 or a variant thereof; and (j) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 102 or a variant thereof, wherein optionally the composition comprises a single nucleic acid molecule.
44. The composition of claim 43, wherein the nucleic acid molecule of the composition comprises any two or more of the following:
(a) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
72 or said variant thereof;
(b) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
73 or said variant thereof;
(c) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 77 or said variant thereof;
(d) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 82 or said variant thereof;
(e) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
97 or a variant thereof;
(f) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
98 or a variant thereof;
(g) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
99 or a variant thereof;
(h) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
100 or a variant thereof;
(i) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
101 or a variant thereof; and
(j) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
102 or a variant thereof,
45. The composition of claim 44, wherein the nucleic acid molecule of the composition comprises any three or more of the following:
(a) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
72 or said variant thereof;
(b) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
73 or said variant thereof;
(c) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
77 or said variant thereof; and
(d) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO: 82 or said variant thereof;
(e) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
97 or a variant thereof;
(f) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
98 or a variant thereof; (g) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
99 or a variant thereof;
(h) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
100 or a variant thereof;
(i) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
101 or a variant thereof; and
(j) a nucleic acid molecule that encodes a polypeptide with the amino acid sequence of SEQ ID NO:
102 or a variant thereof,
46. The composition of any one of claims 43-45, wherein:
(a) the nucleic acid molecule of (a) has the nucleotide sequence of SEQ ID NO: 42;
(b) the nucleic acid molecule of (b) has the nucleotide sequence of SEQ ID NO: 43;
(c) the nucleic acid molecule of (c) has the nucleotide sequence of SEQ ID NO: 47; and/or
(d) the nucleic acid molecule of (d) has the nucleotide sequence of SEQ ID NO: 52.
47. A method of producing an antibody comprising administering the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 11-15, the vector of any one of claims 16-25, or the composition of any one of claims 26-46 to a subject to elicit production of neutralizing antisera in said subject.
48. A method of identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection comprising:
(a) determining whether the subject has a protective level of a broadly neutralizing anti-coronavirus antibody (bNAb) against two or more lineages of coronavirus (such as an anti-Spike antibody) in a sample from the subject, wherein optionally the protective level is:
(i) a level that is at or above a titer of at least about 70, as determined using a pseudovirus neutralization assay; or
(ii) a level that is at or above a titer of at least about 25, as determined using a live virus neutralization assay; or
(iii) a level that is at least 80% of a median level of an anti-coronavirus antibody in a cohort of convalescent humans, as determined by a pseudovirus neutralization assay or live virus neutralization assay; and
(b) administering an effective amount of the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 11 -15, the vector of any one of claims 16-25, or the composition of any one of claims 26-46 to the subject having less than a protective level of the bNAb.
49. The method of claim 48, wherein the method further comprises identifying a subclass and/or an effector function of the bNAb.
50. The method of claim 49, wherein:
(a) the subclass is IgM, IgA, IgG 1 , lgG2, lgG3, or FcgR2A; and/or (b) the effector function is antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation.
51 . The method of any one of claims 48-50, wherein the sample is a bodily fluid from the subject, wherein optionally the bodily fluid is blood.
52. The method of any one of claims 48-51 , wherein the coronavirus is 2019-nCoV.
53. The method of any one of claims 48-52, wherein the two or more lineages of coronavirus are selected from the group consisting of B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , and A23.1 .
54. A method of treating or reducing the risk of a coronavirus infection in a subject in need thereof, comprising administering a therapeutically effective amount of the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 11-15, the vector of any one of claims 16-25, or the composition of any one of claims 26-46 to said subject.
55. The method of claim 54, comprising administering:
(a) an Ad26 or Ad52 vector comprising the nucleic acid molecule; and/or
(b) an Ad26 or Ad52 vector comprising a nucleic acid molecule that encodes the polypeptide.
56. The method of claim 54 or 55, further comprising measuring an anti-coronavirus antibody (e.g., an anti-Spike antibody) level in the subject.
57. The method of claim 56, wherein the anti-coronavirus antibody level in the subject is measured before and/or after administration of the composition.
58. The method of claim 57, wherein the anti-coronavirus antibody level in the subject is measured one or more times over about 1 , 2, 3, 4, 5, or 6 days, 1 , 2, 3, 4, 5, 6, or 7 weeks, 2, 3, 4, 5, or 6 months, 1 , 2, 3, 4, or 5 years after administration.
59. The method of any one of claims 54-58, wherein the anti-coronavirus antibody level of the subject is below a protective level and wherein the method further comprises re-administering the composition of any one of claims 26-40 to said subject or administering a different anti-coronavirus composition to the subject.
60. The method of claim 59, wherein the protective level is a level sufficient to reduce symptoms or duration of a coronavirus-mediated disease.
61 . The method of claim 59 or 60, wherein the protective level is: (a) a level that is at or above a titer of at least about 70, as determined using a pseudovirus neutralization assay; or
(b) a level that is at or above a titer of at least about 25, as determined using a live virus neutralization assay; or
(c) a level that is at least 80% of a median level of an anti-coronavirus antibody in a cohort of convalescent humans, as determined by a pseudovirus neutralization assay or live virus neutralization assay.
62. The method of any one of claims 54-61 , wherein the coronavirus infection is infection by 2019-nCoV, wherein optionally said 2019-nCoV is of the lineage B.1 .1 .529, B.1 .1 .7, B.1 .429, B.1 .1 .28, B.1 .351 , or A23.1 .
63. A method of reducing a coronavirus-mediated activity in a subject infected with a 2019-nCoV or a variant thereof, comprising administering a therapeutically effective amount of the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 1 1 -15, the vector of any one of claims 16- 25, or the composition of any one of claims 26-46 to said subject.
64. The method of claim 63, comprising administering:
(a) an Ad26 or Ad52 vector comprising the nucleic acid molecule; and/or
(b) an Ad26 or Ad52 vector comprising a nucleic acid molecule that encodes the polypeptide, wherein optionally the therapeutically effective amount of the composition or the immunogenic composition is sufficient to produce a log serum anti-Spike antibody titer greater than 2 in a subject, as measured by an ELISA assay.
65. The method of claim 63 or 64, wherein the therapeutically effective amount is between 15 pg and 300 pg of the one or more of compositions of any one of claims 26-46.
66. The method of any one of claims 63-65, wherein said activity is viral titer, viral spread, infection, or cell fusion.
67. The method of claim 66, wherein said viral titer is decreased after administration of the one or more compositions of any one of claims 26-46.
68. The method of claim 67, wherein the viral titer is decreased by 25% or more.
69. The method of claim 68, wherein the viral titer is decreased by 50% or more.
70. The method of claim 69, wherein the viral titer is decreased by 75% or more.
71 . The method of claim 70, wherein the coronavirus is undetectable after said administration.
72. The method of any one of claims 63-71 , wherein said administering occurs prior to exposure to the coronavirus.
73. The method of claim 72, wherein said administering occurs at least 1 hour prior to exposure to said coronavirus.
74. The method of claim 73, wherein said administering occurs at least 1 week, 1 month, or a year prior to exposure to said coronavirus.
75. The method of any one of claims 63-71 , wherein said administering occurs post-exposure to the coronavirus.
76. The method of claim 75, wherein said administering occurs at least 15 minutes post-exposure to said coronavirus.
77. The method of claim 76 wherein said administering occurs at least 1 hour, 1 day, or 1 week postexposure to said coronavirus.
78. The method of any one of claims 63-77, wherein said subject is administered at least one dose of the one or more compositions.
79. The method of claim 78, wherein said subject is administered at least two doses of the one or more compositions.
80. The method of claim 79, wherein the composition is administered to said subject as a prime, a boost, or as a prime-boost.
81 . The method of any one of claims 63-80, wherein the composition is administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivelly, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in creams, or in lipid compositions.
82. The method of any one of claims 63-81 , wherein the subject is a mammal.
83. The method of claim 82, wherein the mammal is a human.
84. The method of claim 83, wherein the human has an underlying health condition.
85. The method of claim 84, wherein the underlying health condition is hypertension, diabetes, or cardiovascular disease.
86. The method of any one of claims 63-85, wherein the method promotes an immune response in said subject.
87. The method of claim 86, wherein the immune response is a humoral immune response.
88. The method of claim 87, wherein the humoral immune response is an IgG response.
89. A composition for use in treating or reducing the risk of a coronavirus infection, such as a 2019-nCoV infection, in a subject in need thereof, comprising a therapeutically effective amount of the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 1 1 -15, the vector of any one of claims 16-25, or the composition of any one of claims 26-46.
90. A composition for use in reducing a coronavirus-mediated activity in a subject infected with a 2019- nCoV or a variant thereof, comprising a therapeutically effective amount of the nucleic acid molecule of any one of claims 1 -10, the polypeptide of any one of claims 1 1 -15, the vector of any one of claims 16-25, or the composition of any one of claims 26-46.
91 . The composition for use according to claim 89 or 90, wherein said composition comprises:
(a) an Ad26 vector comprising the nucleic acid molecule of the composition or the immunogenic composition; and/or
(b) an Ad26 vector comprising a nucleic acid molecule that encodes the polypeptide of the composition or the immunogenic composition.
92. A method of manufacturing an immunogenic composition for treating or reducing the risk of a coronavirus infection in a subject in need thereof, said method comprising the steps of:
(a) admixing at least one of the nucleic acid molecules of any one of claims 1 -10, at least one of the polypeptides of any one of claims 1 1 -15, the vector of any one of claims 16-25, and at least one of the compositions of any one of claims 26-46 with a pharmaceutically acceptable carrier, excipient, or diluent to form the immunogenic composition; and
(b) placing the immunogenic composition in a container.
93. A kit comprising:
(a) a first container comprising at least one said nucleic acid molecule of any one of claims 1 -4, at least one said polypeptide of any one of claims 1 1 -15, at least one said vector of any one of claims 16-25, and/or at least one said composition of any one of claims 26-46;
(b) instructions for use thereof; and optionally
(c) a second container comprising a pharmaceutically acceptable carrier, excipient, or diluent.
94. The kit of claim 93, wherein the first container further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
95. A kit of claim 93 or 94, wherein the kit optionally includes an adjuvant and/or an immunostimulatory agent.
PCT/US2023/073246 2022-08-31 2023-08-31 Compositions and methods for treating coronavirus infection WO2024050482A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263374174P 2022-08-31 2022-08-31
US63/374,174 2022-08-31

Publications (2)

Publication Number Publication Date
WO2024050482A2 true WO2024050482A2 (en) 2024-03-07
WO2024050482A3 WO2024050482A3 (en) 2024-05-02

Family

ID=90098765

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/073246 WO2024050482A2 (en) 2022-08-31 2023-08-31 Compositions and methods for treating coronavirus infection

Country Status (1)

Country Link
WO (1) WO2024050482A2 (en)

Also Published As

Publication number Publication date
WO2024050482A3 (en) 2024-05-02

Similar Documents

Publication Publication Date Title
US20190144506A1 (en) Compositions and methods for preventing and treating zika virus infection
US20190055290A1 (en) Stabilized human immunodeficiency virus (hiv) envelope (env) trimer vaccines and methods of using same
KR101971808B1 (en) Methods and Compositions for Inducing Protective Immunity Against Human Immunodeficiency Virus Infection
JP5123343B2 (en) HIVCON: HIV immunogen and use thereof
US20220396810A1 (en) Vaccines and uses thereof to induce an immune response to sars-cov2
US8101739B2 (en) Recombinant expression vectors comprising a human codon-optimized marburg virus (MARV) angola glycoprotein gene insert and method of immunization employing said vector
JP2022101658A (en) HIV vaccination and immunotherapy
US11896657B2 (en) Replication-deficient modified vaccinia Ankara (MVA) expressing Marburg virus glycoprotein (GP) and matrix protein (VP40)
EA038402B1 (en) Adenovirus polynucleotides and polypeptides
US20220160853A1 (en) Cancer vaccine compositions and methods for use thereof
BR112014015390B1 (en) MODIFIED MATRIX PROTEIN (M) OF A VESICULAR STOMATITIS VIRUS, RECOMBINANT VESICULAR STOMATITIS VIRUS, VACCINE COMPRISING THE SAME, KIT AND USE OF A VACCINE
WO2022197720A2 (en) Compositions and methods for treating coronavirus infection
EA035504B1 (en) Methods and compositions for inducing protective immunity against filovirus infection
US20220275346A1 (en) Hantavirus antigenic composition
JP2023529124A (en) Coronavirus vaccine constructs and methods of making and using them
WO2024050482A2 (en) Compositions and methods for treating coronavirus infection
WO2023036191A1 (en) Vaccine for novel coronavirus sars-cov-2 delta variant and application
JP5636284B2 (en) Treatment before or after exposure to filovirus or arenavirus infection
Shimada et al. Effect of therapeutic immunization using Ad5/35 and MVA vectors on SIV infection of rhesus monkeys undergoing antiretroviral therapy
Liu et al. Construction and evaluation of DNA vaccine encoding Ebola virus glycoprotein fused with lysosome-associated membrane protein
RU2290204C1 (en) Recombinant fused protein, preparation for immunotherapy based on thereof and method for immunotherapy of relapsing larynx papillomatosis
RU2811791C1 (en) Expression vector based on human adenovirus 19 serotype and method of its application
US20240156940A1 (en) Replication-deficient modified vaccinia ankara (mva) expressing marburg virus glycoprotein (gp) and matrix protein (vp40)
WO2021249011A1 (en) Hiv vaccine compositions, methods, and uses thereof
CN115770291A (en) Novel coronavirus SARS-CoV-2 vaccine booster immunity and its application

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: 23861573

Country of ref document: EP

Kind code of ref document: A2