WO2021155323A1 - Compositions and methods for preventing and treating coronavirus infection-sars-cov-2 vaccines - Google Patents

Compositions and methods for preventing and treating coronavirus infection-sars-cov-2 vaccines Download PDF

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Publication number
WO2021155323A1
WO2021155323A1 PCT/US2021/015946 US2021015946W WO2021155323A1 WO 2021155323 A1 WO2021155323 A1 WO 2021155323A1 US 2021015946 W US2021015946 W US 2021015946W WO 2021155323 A1 WO2021155323 A1 WO 2021155323A1
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Prior art keywords
protein
seq
sars
nucleic acid
cov
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PCT/US2021/015946
Other languages
French (fr)
Inventor
Dan H. Barouch
Johannes Petrus Maria LANGEDIJK
Lucy RUTTEN
Mark Johannes Gerandus BAKKERS
Rinke Bos
Frank Wegmann
David Adrianus Theodorus Maria ZUIJDGEEST
An Vandebosch
Mathieu Claude Michel LE GARS
Jerald C. Sadoff
Original Assignee
Beth Israel Deaconess Medical Center, Inc.
Janssen Pharmaceuticals, Inc.
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Priority claimed from CA3101131A external-priority patent/CA3101131A1/en
Application filed by Beth Israel Deaconess Medical Center, Inc., Janssen Pharmaceuticals, Inc. filed Critical Beth Israel Deaconess Medical Center, Inc.
Priority to JP2022546341A priority Critical patent/JP2023512519A/en
Priority to KR1020227030114A priority patent/KR20220134622A/en
Priority to CN202180025856.2A priority patent/CN116096732A/en
Priority to BR112022014808A priority patent/BR112022014808A2/en
Priority to EP21747489.9A priority patent/EP4097122A1/en
Priority to US17/759,803 priority patent/US20230075527A1/en
Publication of WO2021155323A1 publication Critical patent/WO2021155323A1/en

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    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61K2039/53DNA (RNA) vaccination
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    • C07K2317/00Immunoglobulins specific features
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Definitions

  • the invention relates to the fields of virology and medicine.
  • the invention relates to vaccines for the prevention of disease induced by SARS-CoV-2.
  • SARS-CoV-2 is a coronavirus that was first discovered late 2019 in the Wuhan region in China.
  • SARS-CoV-2 is a beta-coronavirus, like MERS-CoV and SARS-CoV, all of which have their origin in bats.
  • MERS-CoV and SARS-CoV beta-coronavirus
  • the name of this disease caused by the virus is coronavirus disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases.
  • SARS-CoV-2 has strong genetic similarity to bat coronaviruses, from which it likely originated, although an intermediate reservoir host such as a pangolin is thought to be involved. From a taxonomic perspective SARS-CoV-2 is classified as a strain of the severe acute respiratory syndrome (SARS)-related coronavirus species.
  • SARS severe acute respiratory syndrome
  • Coronaviruses are enveloped RNA viruses.
  • the major surface protein is the large, trimeric spike glycoprotein (S) that mediates binding to host cell receptors as well as fusion of viral and host cell membranes.
  • S protein is composed of an N-terminal S1 subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively.
  • Recent cryo-EM reconstructions of the CoV trimeric S structures of alpha-, beta-, and deltacoronaviruses revealed that the S1 subunit comprises two distinct domains: an N-terminal domain (S1 NTD) and a receptor-binding domain (S1 RBD).
  • SARS-CoV-2 makes use of its S1 RBD to bind to human angiotensin-converting enzyme 2 (ACE2).
  • ACE2 human angiotensin-converting enzyme 2
  • Wuhan coronavirus (2019-nCoV; also referred to as SARS-CoV-2) is a coronavirus that is responsible for an unprecedented current epidemic in China. 2019-nCoV is known to cause respiratory symptoms and fever, which may result in death. The World Health Organization declared the 2019-nCoV outbreak a Public Health Emergency of International Concern on January 30, 2020 and has confirmed over 11,000 cases in 16 countries. While the rapid development of a safe and effective 2019-nCoV vaccine is a global health priority, very little is currently known about 2019-nCoV immunology and mechanisms of immune protection.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a 2019-NCOV Spike (S) protein (also referred to as SARS-CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEC ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEC ID NO: 90).
  • S 2019-NCOV Spike
  • the isolated nucleic acid molecule encodes a 2019-NCOV Spike (S) protein that comprises the following further modification to the full-length amino acid sequence of SEC ID NO: 29: c. deletion of the signal sequence.
  • S 2019-NCOV Spike
  • the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (SEQ ID NO: 219) promoter, preferably the CMV immediate early promoter.
  • the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.
  • the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 219.
  • the CMV promotor consists of the nucleotide sequence of SEQ ID NO: 219.
  • the present invention thus relates to isolated and/or recombinant nucleic acids encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acids comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 211- 218, or fragments thereof.
  • the present invention relates to an isolated and/or recombinant nucleic acid encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acid comprising a nucleotide of SEQ ID NO: 211, or fragments thereof.
  • the invention provides an isolated 2019-NCOV Spike (S) protein (also referred to as SARS- CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
  • S 2019-NCOV Spike
  • the isolated 2019-NCOV Spike (S) protein comprises the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
  • the invention relates to isolated and/or recombinant coronavirus S proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 205-210, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof.
  • the invention relates to an isolated and/or recombinant coronavirus S proteins comprising an amino acid sequence of SEQ ID NO: 205, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof.
  • the invention relates to vectors comprising such nucleic acids.
  • the vector is a recombinant human adenovirus of serotype 26.
  • the invention relates to compositions and vaccines comprising such nucleic acids, proteins and/or vectors.
  • the invention relates to methods for vaccinating a subject against COVID-19, the method comprising administering to the subject a vaccine or composition according to the invention.
  • the invention relates to an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof.
  • the invention in another aspect, relates to methods for making a vaccine against COVID-19, comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus in a pharmaceutically acceptable composition.
  • the recombinant human adenovirus of this aspect may be any of the adenoviruses described herein.
  • the invention in another aspect, relates to an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof.
  • the adenovirus may also be any of the adenoviruses as described in the embodiments above.
  • the invention also relates to a composition for use in prevention of molecularly confirmed, moderate to severe/critical COVID-19 in a subject in need thereof, comprising administering to the subject a composition or immunogenic composition of the invention as described herein, wherein the composition is administered at a dose of 5x10 10 vp per dose in a one dose regimen (i.e. a single dose).
  • the present invention features optimized and/or non-naturally occurring coronavirus (e.g., 2019- nCoV) nucleic acid molecules and polypeptides for the generation of DNA or RNA vaccines, antibodies, and immunogenic compositions and their use in methods of preventing, reducing and/or treating a coronavirus (e.g., 2019-nCoV) infection in a subject (e.g., a mammalian subject (e.g., a human)).
  • a coronavirus e.g., 2019-nCoV
  • One aspect of the invention features an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 1-84.
  • a) the polypeptide is capable of eliciting an immune response in a subject; or b) the polypeptide has at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to, or the polypeptide sequence of, any one of SEQ ID NOs: 1-84.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 56.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 51.
  • nucleic acid molecule comprising a nucleotide sequence having at least 85% sequence identity to all or a portion of any one of SEQ ID NOs: 93-181 , 190-195, and 199-204, or a complementary sequence thereof.
  • the nucleic acid molecule has at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to, or the nucleotide sequence of, any one of SEQ ID NOs: 93-181, 190-195, and 199-204.
  • the nucleic acid molecule is capable of eliciting an immune response in a subject.
  • the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 195.
  • the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 143.
  • the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 204.
  • the nucleic acid molecule has the nucleotide sequence of nucleotides 19-3837 of SEQ ID NO: 204.
  • polypeptide comprising an amino acid sequence having at least 85% sequence identity to all or a portion of any one of SEQ ID NOs: 1-84.
  • said polypeptide has at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to, or the amino acid sequence of, any one of SEQ ID NOs: 1-84.
  • the polypeptide, or a portion thereof is capable of eliciting an immune response in a subject.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 28.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 51.
  • the vector is replication-defective (e.g., lacking an E1, E3, and/or E4 region).
  • the vector is a mammalian, bacterial, or viral vector.
  • the vector is an expression vector.
  • 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.
  • the vector is an adenovirus.
  • the adenovirus is selected from the group consisting of Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52, Ad59, and Pan9.
  • the Ad52 is a rhesus Ad52 or the Ad59 is a rhesus Ad59.
  • the adenovirus is Ad26.
  • the adenovirus is an Ad26 vector that comprises a nucleic acid molecule with nucleotides 19-3837 of SEQ ID NO: 204 or all of the nucleotides of SEQ ID NO: 204.
  • the adenovirus is an Ad26 vector that comprises a nucleic acid molecule encoding a polypeptide with at least 85% or more (e.g., 90%, 95%, 99%, or 100%) sequence identity to the polypeptide of SEQ ID NOL 51.
  • the antibody is generated by immunizing a mammal with the nucleic acid, the polypeptide, or the vector.
  • the mammal is a human, cow, goat, mouse, or rabbit.
  • the antibody is humanized.
  • the antibody is an IgG.
  • the antibody is a bis-Fab, Fv, Fab, Fab’-SH, F(ab’)2, a diabody, a linear antibody, or a scFV.
  • Another aspect features a method of producing an anti-2019-Wuhan coronavirus (2019-nCoV) antibody, comprising administering an amount of the nucleic acid molecule, the polypeptide, and/or the vector to a subject sufficient to elicit the production of neutralizing anti-2019-nCoV antisera after administration to said subject.
  • 2019-nCoV anti-2019-Wuhan coronavirus
  • Another aspect features an isolated anti-2019-nCoV antibody produced by any of the abovementioned methods.
  • the antibody binds to an epitope within any one of SEQ ID NOs: 1-84.
  • compositions comprising the nucleic acid molecule, the polypeptide, the vectors or the antibody.
  • the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • the composition further comprises an adjuvant or an immunostimulatory agent.
  • an immunogenic composition comprising the nucleic acid molecule, the polypeptide, the vector, or the antibody.
  • the immunogenic composition is a vaccine.
  • the immunogenic composition is capable of treating or reducing the risk of a coronavirus infection (e.g., a 2019-nCoV infection) in a subject in need thereof.
  • the immunogenic composition elicits production of neutralizing anti-2019-nCoV antisera after administration to said subject.
  • the subject is a mammal.
  • the mammal is a human.
  • the human has an underlying health condition.
  • the underlying health condition is hypertension, diabetes, or cardiovascular disease.
  • Another aspect features a method of identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection comprising determining whether the subject has a protective level of an anti-coronavirus antibody (such as an anti-Spike antibody) in a sample from the subject, wherein preferably 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; (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.
  • an anti-coronavirus antibody such as an anti-Spike antibody
  • the protective level of an anti-coronavirus antibody is a level sufficient to prevent or reduce the development of severe disease.
  • the method further comprises administering an effective amount of the composition or the immunogenic composition to the subject having less than a protective level of the anti-coronavirus antibody.
  • the method further comprises identifying a subclass and/or an effector function of the anti-coronavirus antibody (e.g., the anti- Spike antibody).
  • the subclass is IgM, IgA, lgG1, lgG2, lgG3, or FcgR2A.
  • 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.
  • the sample is a bodily fluid from the subject, wherein preferably the bodily fluid is blood.
  • the coronavirus is 2019-nCoV.
  • Another aspect features 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 composition or the immunogenic composition to said subject.
  • the method further comprises measuring an anti-coronavirus antibody (e.g., an anti-Spike antibody) level in the subject.
  • the anti-coronavirus antibody level in the subject is measured before and/or after the administration.
  • 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.
  • the anti-coronavirus antibody level of the subject is below a protective level and wherein the method further comprises re-administering the composition or the immunogenic composition or administering a different anti-coronavirus composition to the subject.
  • the protective level is a level sufficient to reduce symptoms or duration of a coronavirus-mediated disease.
  • the protective level is a level sufficient to prevent or reduce the development of severe disease (e.g., which can be characterized by weight loss (e.g., a weight reduction of at least about 5% (e.g., at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 20%, at least about 25% or more) relative to the subject’s initial weight pre-infection), the development of pneumonia and/or respiratory failure, and/or increased risk of death).
  • severe disease e.g., which can be characterized by weight loss (e.g., a weight reduction of at least about 5% (e.g., at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 20%, at least about 25% or more) relative to the subject’s initial weight pre-infection)
  • weight loss e.g., a weight reduction of at least about 5% (e.g., at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at
  • 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; (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.
  • the coronavirus is 2019-nCoV.
  • the method further includes measuring the coronavirus (e.g., 2019-nCoV) viral load in a sample from the subject.
  • the sample is a bronchoalveolar lavage (BAL) or a nasal swab (NS).
  • the sample is a bodily fluid (e.g., blood, e.g., whole blood or plasma) from the subject.
  • the sample is a tissue sample (e.g., a respiratory tract tissue sample) from the subject.
  • viral load is a detectible nucleic acid (e.g., subgenomic mRNA) level or a detectible protein (e.g., nucleocapsid protein (N)) level.
  • the detectible nucleic acid e.g., subgenomic mRNA
  • the detectible protein is determined by an immunoassay (e.g., an immunohistochemical (IHC) assay or a lateral flow immunoassay).
  • a detectable viral load indicates that the subject is susceptible to disease (e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease).
  • a viral load of greater than 3.85 logio sgmRNA copies/mL in BAL or 3.78 logio sgmRNA copies/mL in NS indicates that the subject is susceptible to disease (e.g., a 2019- nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease).
  • a viral load of greater than 3.85 logio sgmRNA copies/mL in BAL or 3.78 logio 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 logio sgmRNA copies/g of tissue indicates that the subject is susceptible to severe COVID-19 disease.
  • a viral load of greater than about 3% SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to disease.
  • a viral load of greater than about 5% SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to severe COVID-19 disease.
  • coronavirus (e.g., 2019- nCoV) viral load is measured one or more times over about 1, 2, 3, 4, 5, or 6 days or 1 , 2, 3, 4,
  • a subject determined to be susceptible to disease e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease
  • a subject determined to be susceptible to disease is administered the composition or the immunogenic composition of the present disclosure alone or in combination with an additional therapeutic agent.
  • Another aspect features a method of reducing a coronavirus-mediated activity (e.g., 2019- nCoV-mediated activity) in a subject infected with a 2019-nCoV, comprising administering a therapeutically effective amount of the composition or the immunogenic composition to said subject.
  • the therapeutically effective amount is sufficient to produce a log serum anti-Spike antibody titer greater than 2 in a subject, as measured by an ELISA assay.
  • the therapeutically effective amount is between 15 pg and 300 pg of the composition or the immunogenic composition.
  • the activity is viral titer, viral spread, infection, or cell fusion.
  • the viral titer is decreased after administration of the composition or the immunogenic composition. 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, the administering occurs prior to exposure to the coronavirus. In some embodiments, the administering occurs at least 1 hour prior to exposure to said coronavirus. In some embodiments, the administering occurs at least 1 week, 1 month, or a year prior to exposure to said coronavirus. In some embodiments, the administering occurs post-exposure to the coronavirus.
  • the administering occurs at least 15 minutes post-exposure to said coronavirus. In some embodiments, the administering occurs at least 1 hour, 1 day, 1- week, post-exposure to said coronavirus. In some embodiments, the subject is administered at least one dose of the nucleic acid molecule, polypeptide, vector, composition, immunogenic composition, and antibody. In some embodiments, the subject is administered at least two doses. In some embodiments, the nucleic acid molecule, polypeptide, vector, composition, or immunogenic composition is administered to said subject as a prime, a boost, or as a prime boost.
  • the nucleic acid molecule, polypeptide, vector, composition, immunogenic composition, or antibody 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, intraperi cardially, 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.
  • the nucleic acid molecule, polypeptide, vector, composition, immunogenic composition, or antibody is administered intramuscularly.
  • the subject is a mammal.
  • the mammal is a human.
  • the human has an underlying health condition.
  • the underlying health condition is hypertension, diabetes, or cardiovascular disease.
  • the method promotes an immune response in said subject.
  • the immune response is a humoral immune response.
  • the humoral immune response is an IgG response.
  • Another aspect 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, comprising a therapeutically effective amount of the composition or the immunogenic composition.
  • compositions for use in reducing a coronavirus-mediated activity e.g., 2019-nCoV-mediated activity
  • a coronavirus-mediated activity e.g., 2019-nCoV-mediated activity
  • a subject infected with a 2019-nCoV comprising a therapeutically effective amount of the composition or the immunogenic composition.
  • Another aspect features a method of manufacturing an immunogenic composition for treating or reducing the risk of a coronavirus (e.g., 2019-nCoV) infection in a subject in need thereof, said method comprising the steps of: (a) admixing at least one of the nucleic acid molecule, the polypeptide, the vector, the composition, and the antibody with a pharmaceutically acceptable carrier, excipient, or diluent to form the immunogenic composition; and (b) placing the immunogenic composition in a container.
  • a coronavirus e.g., 2019-nCoV
  • kits comprising: (a) a first container comprising at least one of the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, and the antibody; (b) instructions for use thereof; and optionally (c) a second container comprising a pharmaceutically acceptable carrier, excipient, or diluent.
  • the first container further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • the kit optionally includes an adjuvant and/or an immunostimulatory agent.
  • kits comprising: one or more reagents for determining the presence of an anti-coronavirus antibody (such as an anti-Spike antibody) in a sample (e.g., a blood sample) from a subject and instructions for identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection.
  • an anti-coronavirus antibody such as an anti-Spike antibody
  • the kit further comprises reagents for identifying a subclass and/or an effector function of the anti-coronavirus antibody.
  • the kit further comprises standards or samples for comparison.
  • FIG. 1 is a diagram showing Spike protein immunogens.
  • Annotated domains of 2019-nCoV Spike (SEQ ID NO: 1) including the S1 (SEQ ID NO: 4), S2 (amino acids 665-1191 of SEQ ID NO: 1), TM (SEQ ID NO: 86), and CT (SEQ ID NO: 85) domains.
  • Full-length Spike (SEQ ID NO: 1), SdCT (SEQ ID NO: 2), S.Ecto (SEQ ID NO: 3), S1-foldon (SEQ ID NO: 9), RBD-foldon (SEQ ID NO: 10), and S.Ecto-PP-foldon (SEQ ID NO: 22) protein immunogens are labeled.
  • White boxes indicate foldon domain and the double intersecting lines in S.Ecto-PP-foldon indicate the approximate position of two stabilizing mutations (proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 1).
  • FIG. 2 is a western blot showing the recognition of recombinant 2019-nCoV proteins by polyclonal anti-SARS antiserum.
  • Cell lysates (left panel) and supernatants (right panel) from cells transfected with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), and SS-S.Ecto-dF-PP-foldon (lane 4) were probed using polyclonal anti-SARS antiserum.
  • Numbered black lines to the left of each blot indicate approximate molecular weight in kDa and numbers at the top of each blot indicate lane number.
  • FIG. 3 is a graph showing the recognition of full-length Spike by antibodies produced in 2019-nCoV vaccinated mice. Serum was collected from mice 4-weeks post-vacci nation with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), SS-S1-foldon (lane 4), SS-RBD-foldon (lane 5), and SS-S.Ecto-dF-PP-foldon (lane 6) and used in an ELISA with full- length Spike (SEQ ID NO: 1). Gray bars represent mean ELISA titer.
  • FIG. 4 is a graph showing the recognition of S.dTM.PP by antibodies produced in 2019- nCoV vaccinated mice. Serum was collected from mice 4-weeks post-vaccination with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), SS-S1-foldon (lane 4), SS- RBD-foldon (lane 5), and SS-S.Ecto-dF-PP-foldon (lane 6) and used in an ELISA with full-length ectodomain S.Ecto-PP (SEQ ID NO: 19). Gray bars represent mean ELISA titer.
  • FIG. 5 is a graph showing the neutralizing activity of antibodies produced in 2019-nCoV vaccinated mice. Serum was collected from mice 4-weeks post-vaccination with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), SS-S1-foldon (lane 4), SS- RBD-foldon (lane 5), and SS-S.Ecto-dF-PP-foldon (lane 6) and used in an in vitro 2019-nCoV Spike pseudovirus neutralization assay. Gray bars represent mean IC50 titer.
  • FIGS. 6A-6E are graphs showing humoral immune responses in vaccinated rhesus macaques. Humoral immune responses were assessed following immunization by (FIG. 6A) binding antibody ELISA, (FIG. 6B) pseudovirus neutralization assays, and (FIG. 6C) live virus neutralization assays. (FIG. 6D) Comparison of pseudovirus neutralization titers in vaccinated macaques (all animals and SS-Spike / SS-SdCT groups), a cohort of 9 convalescent macaques, and a cohort of 27 convalescent humans from Boston who had recovered from 2019-nCoV infection. (FIG.
  • FIGS. 7A-7B are graphs showing cellular immune responses in vaccinated rhesus macaques.
  • Cellular immune responses were assessed following immunization by (FIG. 7 A) IFN-g ELISPOT assays and (FIG. 7B) multiparameter intracellular cytokine staining assays in response to pooled S peptides. Red bars reflect mean responses.
  • FIGS. 8A-8D are graphs showing viral loads in 2019-nCoV challenged rhesus macaques.
  • FIGS. 9A-9C are graphs showing immune correlates of protection. Correlations of (FIG.
  • FIG. 9A pseudovirus NAb titers and
  • FIG. 9B live NAb titers prior to challenge with log peak sgmRNA copies/ L in BAL or log peak sgmRNA copies/swab in nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 9C The heat map (top panel) shows the Spearman and Pearson correlations between antibody features and log peak sgmRNA copies/mL in BAL (*q ⁇ 0.05, **q ⁇ 0.01, ***q ⁇ 0.001, with q-values obtained by Benjamini- Hochberg correction for multiple testing).
  • the bar graph (bottom left panel) shows the rank of the Pearson correlation of the most predictive combination or individual antibody features defined by recursive feature elimination for partial least square regression (PLSR) and random forest (RF) regression.
  • the correlation heatmap (bottom right panel) represents pairwise Pearson correlations between features across all animals.
  • FIG. 10 is a graph showing correlation of pseudovirus and live virus NAb assays in vaccinated macaques. Red line reflects the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 11 is a graph showing viral RNA following 2019-nCoV challenge in sham controls in BAL and nasal swabs. Red lines reflect median viral loads.
  • FIG. 12 is a graph showing viral RNA following 2019-nCoV challenge in vaccinated animals in BAL. Red lines reflect median viral loads.
  • FIG. 13 is a graph showing viral RNA following 2019-nCoV challenge in vaccinated animals in nasal swabs. Red lines reflect median viral loads.
  • FIG. 14 is a graph showing Peak viral RNA following 2019-nCoV challenge in vaccinated animals in BAL and nasal swabs. Red lines reflect median viral loads. P-values indicate two- sided Wilcoxon rank-sum tests.
  • FIG. 15 is a graph showing correlations of log ELISA titers prior to challenge with log sgmRNA in BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
  • FIG. 16 is a graph showing correlations of log ELISPOT responses prior to challenge with log sgmRNA in BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
  • FIG. 17 is a graph showing correlations of log CD4+ ICS responses prior to challenge with log sgmRNA in BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
  • FIG. 18 is a graph showing correlations of log CD8+ ICS responses prior to challenge with log sgmRNA copies/mL in BAL and log sgmRNA copies/swab in nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 19 is a graph showing anamnestic ELISA responses following challenge. Responses on day 0 and day 14 following challenge are shown. Red lines reflect median responses.
  • FIG. 20 is a graph showing anamnestic pseudovirus NAb responses following challenge.
  • FIG. 21 is a graph showing anamnestic live virus NAb responses following challenge.
  • FIG. 22 is a graph showing anamnestic ELISPOT responses following challenge.
  • FIGS. 23A-23C are graphs showing viral loads in 2019-nCoV challenged rhesus macaques.
  • FIG. 23A Log viral RNA copies/mL (limit 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) at multiple timepoints following challenge.
  • FIG. 23B LogTM viral RNA copies/swab and
  • FIG. 23C log sgmRNA copies/swab (limit 50 copies/swab) were assessed in nasal swabs (NS) at multiple timepoints following challenge. Red horizontal bars reflect median viral loads.
  • FIG. 24A-24F are graphs showing immune responses in 2019-nCoV challenged rhesus macaques.
  • Humoral immune responses were assessed following challenge by (FIG. 24A) binding antibody ELISA, (FIG. 24B) pseudovirus neutralization assays, (FIG. 24C) live virus neutralization assays, and (FIG. 24D) systems serology profiles including antibody subclasses and effector functions to receptor binding domain (RBD), soluble spike (S) ectodomain, and nucleocapsid (N) proteins on day 35.
  • RBD receptor binding domain
  • S soluble spike
  • N nucleocapsid
  • ADCD Antibody-dependent complement deposition
  • ADCP antibody-dependent cellular phagocytosis
  • ADNP antibody-dependent neutrophil phagocytosis
  • NK CD107a antibody-dependent NK cell degranulation
  • NK Ml P1 b cytokine secretion
  • FIG. 24E IFN-g ELISPOT assays
  • FIG. 24F multiparameter intracellular cytokine staining assays in response to pooled S peptides. Red and horizontal bars reflect mean responses.
  • FIGS. 25A-25L is a set of micrographs showing that 2019-nCoV induces acute viral interstitial pneumonia.
  • FIGS. 25A-25F H&E sections of fixed lung tissue from 2019-nCoV infected rhesus macaques 2 days following challenge showing (FIG. 25A) interstitial edema and regional lung consolidation, (FIG. 25B) intra-alveolar edema and infiltrates of neutrophils, (FIGS. 25C-25D) bronchiolar epithelial sloughing and necrosis, (FIG. 25E) bronchiolar epithelial syncytial cell formation, and (FIG. 25F) hyaline membranes within alveolar septa. (FIGS. 25A-25F) H&E sections of fixed lung tissue from 2019-nCoV infected rhesus macaques 2 days following challenge showing (FIG. 25A) interstitial edema and regional lung consolidation, (FIG. 25B) intra
  • FIG. 25G- 25H IHC for SARS nucleocapsid showing virus infected cells within interstitial spaces including (FIG. 25G) a viral syncytial cell within the lumen and (FIG. 25H) virus infected alveolar lining cells.
  • FIG. 25I Inflammatory infiltrate showing multiple cells containing 2019-nCoV RNA by RNASCOPE® in situ hybridization.
  • FIGGS. 25J-25L bronchial respiratory epithelium showing (FIG. 25J) inflammation within the submucosa and transmigration of inflammatory cells into the ciliated columnar respiratory epithelium of a bronchus,
  • FIG. 25K 2019-nCoV RNA
  • FIG. 25K 2019-nCoV RNA
  • FIGS. 26A-26K is a set of micrographs showing that 2019-nCoV infects alveolar epithelial cells in rhesus macaques.
  • FIG. 26A Whole slide image of a lung with DAPI staining for cell nuclei, regions of nuclear consolidation (arrows), and foci of viral replication (box).
  • FIG. 26B Higher magnification images of inset box showing
  • FIG. 26C SARS nucleocapsid positive cells (green) and DAPI for cell nuclei (blue).
  • FIG. 26D Bright-field IHC for SARS nucleocapsid from corresponding lung region depicted in (FIG. 26B and FIG. 26C).
  • FIGS. 27A-27F are graphs showing viral loads following 2019-nCoV re-challenge in rhesus macaques.
  • Three naive animals were included as a positive control in the re-challenge experiment. (FIG.
  • FIG. 27A Logio viral RNA copies/mL (limit 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) at multiple timepoints following re-challenge. One of the naive animals could not be lavaged.
  • FIG. 27B Comparison of viral RNA in BAL following primary challenge and re challenge.
  • FIG. 27C Logio viral RNA copies/mL and
  • FIG. 27E logio sgmRNA copies/swab (limit 50 copies/mL) were assessed in nasal swabs (NS) at multiple timepoints following re challenge.
  • FIG. 28 is a series of graphs showing anamnestic immune responses following 2019- nCoV re-challenge in rhesus macaques.
  • Virus-specific binding antibody ELISAs, pseudovirus neutralization assays, live virus neutralization assays, and IFN-g ELISPOT assays are depicted prior to and 7 days following 2019-nCoV re-challenge.
  • Red lines reflect mean responses.
  • P-values reflect two-sided Mann-Whitney tests.
  • FIG. 29 is a series of graphs showing plasma viral loads in 2019-nCoV challenged rhesus macaques. Groups 1-3 are described in FIG. 23. Logio viral RNA copies/mL (limit 50 copies/mL) were assessed in plasma at multiple timepoints following challenge. Red horizontal bars reflect median viral loads.
  • FIG. 30 is a series of graphs showing clinical scores of 2019-nCoV challenged rhesus macaques. Groups 1-3 are described in FIG. 23. Semi-quantitative clinical scoring of animals based on appearance, dyspnea, recumbency, appetite, and responsiveness respiratory distress at multiple timepoints following challenge. Red horizontal bars reflect median clinical scores.
  • FIG. 31 is a graph showing hematology in 2019-nCoV challenged rhesus macaques.
  • FIG. 32 is a graph showing tissue viral loads in 2019-nCoV challenged rhesus macaques.
  • FIGS. 33A-33J are micrographs showing that 2019-nCoV replication induces polymorphonuclear and mononuclear inflammatory infiltrates associated with type 1 interferon responses.
  • FIG. 33A and FIG. 33F Detection of 2019-nCoV RNA
  • FIG. 33B and FIG. 33G IHC for myeloperoxidase (MPO)
  • FIG. 33C and FIG. 33H double staining for CD4 (brown) and macrophages (CD68 and CD163, red
  • FIG. 33D and FIG. 33I CD8 T lymphocytes
  • FIG. 33E and FIG. 33J MX1 (type 1 interferon response gene) in (FIG. 33A- 33E) a 2019-nCoV infected rhesus macaque on day 2 following challenge and (FIG. 33F-33G) an uninfected rhesus macaque.
  • Scale bars 100 microns.
  • FIGS. 34A-34B is a graph showing quantitative analysis of inflammatory infiltrates in lung tissue.
  • FIG. 34A Lung alveoli polymorphonuclear (PMN) cell infiltration and
  • FIG. 34B extent of MX1 staining in 2019-nCoV infected versus uninfected rhesus macaques.
  • P values reflect 2- sided Mann-Whitney tests.
  • FIGS. 35A-35J are micrographs showing inflammatory infiltrates within regions of lung consolidation in 2019-nCoV acute infection.
  • FIG. 35B Bronchus associated lymphoid tissue (BALT) and
  • FIG. 35C and FIG. 35D bronchiolar epithelium within ROI 1 showing bronchiolar necrosis with (FIG. 35C) migration of CD3+ T lymphocytes into the bronchiole lumen and
  • MPO myeloperoxidase
  • FIG. 35F CD16 positivity (red) in consolidated tissue (DNA, gray) with (FIG. 35E, FIG. 35G, and FIG. 35I) low 2019-nCoV positivity in ROI 1 as compared to (FIG. 35F, FIG. 35H, and FIG. 35J) high 2019-nCoV positivity in ROI 3.
  • FIG. 35G SARS-N (green) and pan-CK (epithelium, red) staining in (FIG. 35G) ROI 1 versus (FIG. 35H) ROI 3.
  • FIG. 36 is a graph showing plaque assays of BAL and nasal swabs following 2019-nCoV primary challenge and re-challenge. Peak plaque-forming units (PFU) per ml for BAL or per swab for nasal swabs from days 1-7 following primary challenge or re-challenge are shown.
  • PFU Peak plaque-forming units
  • Red horizontal bars reflect median PFU titers.
  • FIG. 37 is a graph showing clinical scores of 2019-nCoV re-challenged rhesus macaques.
  • Groups 1-3 are described in FIG. 23. Semi-quantitative clinical scoring of animals based on appearance, dyspnea, recumbency, appetite, and responsiveness respiratory distress at multiple timepoints following challenge.
  • FIG. 38A is a diagram showing the construction of Ad26 vectors. Seven Ad26 vectors were produced expressing SARS-CoV-2 S protein variants: (i) tPA leader sequence with full- length S (tPA.S (SEQ ID NO: 1, with a tPA leader sequence fused to the N-terminus)), (ii) tPA leader sequence with full-length S with mutation of the furin cleavage site and two proline stabilizing mutations (tPA.S.
  • PP (SEQ ID NO: 23, with a tPA leader sequence fused to the N- terminus)), (iii) wildtype leader sequence with native full-length S (S (SEQ ID NO: 29)), (iv) wildtype leader sequence with S with deletion of the cytoplasmic tail (S.dCT (SS-SdCT) (SEQ ID NO: 30)), (v) tandem tPA and wildtype leader sequences with full-length S (tPA.WT.S (SEQ ID NO: 29, with a tPA leader sequence fused to the N-terminus)), (vi) wildtype leader sequence with S with deletion of the transmembrane region and cytoplasmic tail, reflecting the soluble ectodomain, with mutation of the furin cleavage site, proline stabilizing mutations, and a foldon trimerization domain (S.dTM.PP (SS-S.Ecto-dF-PP-foldon) (SEQ ID NO: 56)), and (vii) wildtype leader sequence with full-length S
  • FIG. 38B is a western blot showing the recognition of SARS-CoV-2 S variatns by polyclonal anti-SARS antibody.
  • FIGS. 39A-39D are graphs showing humoral immune responses in vaccinated rhesus macaques. Humoral immune responses were assessed at weeks 0, 2, and 4 by (FIG. 39A) RBD-specific binding antibody ELISA, (FIG. 39B) pseudovirus neutralization assays, and (FIG. 39C) live virus neutralization assays. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation. (FIG. 39D) S- and RBD-specific antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent monocyte cellular phagocytosis (ADCP), antibody- dependent complement deposition (ADCD), and antibody-dependent NK cell activation (ADNKA) at week 4 are shown as radar plots.
  • ADNP S- and RBD-specific antibody-dependent neutrophil phagocytosis
  • ADCP antibody-dependent monocyte cellular phagocytosis
  • ADCD antibody-dependent complement deposition
  • ADNKA antibody-dependent NK cell activation
  • the size and color intensity of the wedges indicate the median of the feature for the corresponding group (blue depicts antibody functions, red depicts antibody isotype/subclass/FcyR binding).
  • the principal component analysis (PCA) plot shows the multivariate antibody profiles across groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses shows the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution.
  • FIGS. 40A-40B are graphs showing cellular immune responses in vaccinated rhesus macaques.
  • Cellular immune responses were assessed at week 4 following immunization by (FIG. 40A) IFN-Y ELISPOT assays and (FIG. 40B) IFN-Y+CD4+ and IFN-Y+CD8+ T cell intracellular cytokine staining assays in response to pooled S peptides. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
  • FIGS. 41A-41D are graphs showing viral loads in rhesus macaques following SARS-CoV- 2 challenge. Rhesus macaques were challenged by the intranasal and intratracheal routes with 1.2x10 8 VP (1.1x10 4 PFU) SARS-CoV-2.
  • FIGS. 41A-41B Log sgmRNA copies/mL (limit of quantification 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) in sham controls and in vaccinated animals following challenge.
  • FIG. 42 is a graph showing summary of peak viral loads following SARS-CoV-2 challenge.
  • Peak viral loads in BAL and NS following challenge Peak viral loads occurred variably on day 1-4 following challenge. Red lines reflect median viral loads. P-values indicate two-sided Mann-Whitney tests (*P ⁇ 0.05, **P ⁇ 0.001, ***P ⁇ 0.0001).
  • the two groups were compared by two-sided Mann-Whitney tests, and stars indicate the Benjamini-Hochberg corrected q-values (*q ⁇ 0.05).
  • the dot plots show differences in the features that best discriminated completely protected and partially protected animals, including NAb titers, S-specific antibody-dependent NK cell activation (ADNKA), and antibody-dependent monocyte cellular phagocytosis (ADCP).
  • P-values indicate two-sided Mann-Whitney tests.
  • the bar plot shows the cross-validated area under the receiver operator characteristics curves using the features indicated on the x-axis in a logistic regression model. The top three 1 -feature and 2-feature models are shown. Error bars indicate the mean and standard deviation for 100 repetitions of 10-fold cross-validation.
  • FIGS. 44A-44B are graphs showing immune responses following SARS-CoV-2 challenge.
  • FIG. 44A Pseudovirus NAb titers prior to challenge and on day 14 following challenge
  • FIG. 44B IFN-Y+CD8+ and IFN-y+CD4+ T cell responses by intracellular cytokine staining assays in response to pooled spike (S1, S2), nucleocapsid (NCAP), and non-structural proteins (N6, N7a, N8) peptides on day 14 following challenge in sham controls and in Ad26-S.PP (Ad26 SS-Spike-dF-PP) vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
  • FIG. 45 is a pair of graphs showing correlation of pseudovirus NAb titers and ELISA or live virus NAb assays in vaccinated macaques. Red line reflects the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
  • FIG. 46 is a graph showing a comparison of pseudovirus NAb titers in vaccinated macaques and convalescent macaques and humans. Comparison of pseudovirus NAb in macaques vaccinated with Ad26-S.PP (Ad26 SS-Spike-dF-PP) with previously reported cohorts of convalescent macaques and convalescent humans who had recovered from SARS-CoV-2 infection.
  • FIG. 47 is a pair of graphs showing humoral immune responses in BAL in vaccinated rhesus macaques. S-specific IgG and IgA at week 4 in BAL by ELISA in sham controls and in Ad26-S.PP (Ad26 SS-Spike-dF-PP) vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
  • FIG. 48 is a series of graphs showing humoral immune responses in vaccinated rhesus macaques.
  • S- and RBD-specific antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent monocyte cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent NK cell activation (ADNKA) are shown. Red bars reflect median responses.
  • FIG. 49 is a pair of graphs showing cellular immune responses in vaccinated rhesus macaques. IFN-y+, IL-2+, IL-4+, and IL-10+ CD4+ T cell intracellular cytokine staining assays in response to pooled S peptides in Ad26-S.dTM.PP (Ad26 SS-S.Ecto-dF-PP-foldon) and Ad26- S.PP (Ad26 SS-Spike-dF-PP) vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
  • FIG. 50 is a pair of graphs showing ELISA correlates of protection. Correlations of binding ELISA titers at week 2 and week 4 with log peak sgmRNA copies/swab in NS following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 51 is a pair of graphs showing pseudovirus NAb correlates of protection.
  • FIG. 52 is a pair of graphs showing live virus NAb correlates of protection. Correlations of live virus NAb titers at week 2 and week 4 with log peak sgmRNA copies/swab in NS following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 53 is a series of graphs showing antibody correlates of protection.
  • the dot plots show differences in the features between completely protected and partially protected animals. P-values indicate two-sided Mann-Whitney tests.
  • FIG. 54 is a series of graphs showing NAb titers following SARS-CoV-2 challenge.
  • Pseudovirus NAb titers prior to challenge and on day 14 following challenge in vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
  • FIGS. 55A-55C are graphs showing clinical disease following SARS-CoV-2 infection in hamsters.
  • FIG. 55A Median percent weight change following challenge. The numbers reflect the number of animals at each timepoint. In the high-dose group, 4 animals were necropsied on day 2, 4 animals were necropsied on day 4, 4 animals met euthanization criteria on day 6, and 2 animals met euthanization criteria on day 7.
  • FIGS. 55A-55C are graphs showing clinical disease following SARS-CoV-2 infection in hamsters.
  • FIGS. 56A-56L are a series of graphs showing pathologic features of high-dose SARS- CoV-2 infection in hamsters.
  • FIG. 56A Necrosis and inflammation (arrow) in nasal turbinate, H&E (d2).
  • FIG. 56A Necrosis and inflammation (arrow) in nasal turbinate, H&E (d2).
  • FIG. 56B Bronchiolar epithelial necrosis with cellular debris and degenerative neutrophils in lumen (arrow) and transmigration of inflammatory cells in vessel wall (arrowhead), H&E (d2).
  • FIG. 56C Interstitial pneumonia, hemorrhage, and consolidation of lung parenchyma, H&E (d2).
  • FIG. 56D Nasal turbinate epithelium shows strong positivity for SARS-CoV-N by IHC (d2).
  • FIG. 56E Bronchiolar epithelium and luminal cellular debris show strong positivity for SARS-CoV-N by IHC (d2).
  • FIG. 56F Pneumocytes and alveolar septa show multifocal strong positivity for SARS-CoV-N by IHC (d2).
  • FIG. 56G Diffuse vRNA staining by RNASCOPE® within pulmonary interstitium (arrow, interstitial pneumonia) and within bronchiolar epithelium (arrowhead; d2).
  • FIG. 56H Diffuse vRNA staining by RNASCOPE® within pulmonary interstitium (d4).
  • FIG. 56I lba-1 IHC (macrophages) within pulmonary interstitium (d7).
  • FIG. 56J CD3+ T lymphocytes within pulmonary interstitium, CD3 IHC (d4).
  • MPO neurotrophil myeloperoxidase
  • IHC interstitial neutrophils
  • FIG. 56L Interferon-inducible gene, MX1, IHC shows strong and diffuse positivity throughout the lung (d4).
  • FIGS. 57A-57F are a series of graphs showing longitudinal quantitative image analysis of viral replication and associated inflammation in lungs.
  • FIG. 57A Percent lung area positive for anti-sense SARS-CoV-2 viral RNA (vRNA) by RNASCOPE® ISH.
  • FIG. 57B Percentage of total cells positive for SARS-CoV-N protein (nuclear or cytoplasmic) by IHC.
  • FIG. 57C lba-1 positive cells per unit area by IHC.
  • FIG. 57D CD3 positive cells per unit area.
  • FIG. 57E MPO positive cells per unit area.
  • FIG. 57F Percentage of MX1 positive lung tissue as a proportion of total lung area.
  • ISH in situ hybridization
  • IHC immunohistochemistry
  • SARS- N SARS-CoV nucleocapsid
  • MPO myeloperoxidase
  • MX1 myxovirus protein 1 (a type 1 interferon inducible gene). Each dot represents one animal.
  • FIGS. 58A-580 are a series of graphs showing lung viral dynamics and ACE2 receptor expression patterns.
  • Hamsters were necropsied before (SARS-CoV-2 Negative) or after high- dose SARS-CoV-2 challenge on day 2 (D2), day 4 (D4), day 7 (D7), and day 14 (D14) following challenge.
  • Serial sections of lung tissue were stained for vRNA anti-sense RNASCOPE® (FIGS. 58A-58E), for vRNA sense RNASCOPE® (FIGS. 58F-58J), and ACE2 IHC (FIGS. 58K- 580).
  • Anti-sense RNASCOPE® used a sense probe; sense RNASCOPE® used an anti-sense probe.
  • FIGS. 59A-59L are a series of graphs showing extrapulmonary pathology.
  • FIG. 59A Anti- sense SARS-CoV-2 viral RNA (vRNA) in brainstem on day 2 following challenge.
  • FIG. 59B Higher magnification showing cytoplasmic vRNA staining in neurons in the absence of inflammation and pathology.
  • FIG. 59C Anti-sense SARS-CoV-2 vRNA staining in the lamina intestinal villus on day 2 following challenge.
  • FIG. 59D Higher magnification showing cytoplasmic and nuclear vRNA staining in an individual mononuclear cell in the absence of inflammation and tissue pathology.
  • FIG. 59A Anti- sense SARS-CoV-2 viral RNA (vRNA) in brainstem on day 2 following challenge.
  • FIG. 59B Higher magnification showing cytoplasmic vRNA staining in neurons in the absence of inflammation and pathology.
  • FIG. 59C Anti-sense SARS-CoV-2 v
  • FIG. 59E Anti-sense SARS-CoV-2 vRNA staining within the myocardium and along the epicardial surface of the heart on day 4 following challenge.
  • FIG. 59F Higher magnification showing staining of inflammatory mononuclear cell infiltrates consistent with focal myocarditis.
  • FIG. 59G Pulmonary vessel showing endothelialitis day 4 (d4) following challenge.
  • FIG. 59H Pulmonary vessel showing CD3+ T lymphocyte staining by IHC adhered to endothelium and within vessel wall, d4 following challenge.
  • FIG. 59I Pulmonary vessel showing lba-1+ staining by IHC of macrophages along endothelium and perivascularly, d4.
  • FIG. 59J Pulmonary vessel showing minimal vascular staining for SARS-CoV-N by IHC, d4.
  • FIGS. 60A-60F are a series of graphs showing SARS-CoV-2 in blood mononuclear cells and bone marrow.
  • FIG. 60A-60C SARS-CoV-2 anti-sense vRNA staining within mononuclear cells within lung thrombus on day 2 following challenge.
  • FIGS. 61A-61F are graphs showing humoral immune responses in vaccinated hamsters.
  • FIG. 61 A SARS-CoV-2 spike (S) immunogens with (i) deletion of the transmembrane region and cytoplasmic tail reflecting the soluble ectodomain with a foldon trimerization domain (S.dTM.PP) or (ii) full-length S (S.PP), both with mutation of the furin cleavage site and two proline stabilizing mutations.
  • Red X depicts furin cleavage site mutation
  • red vertical lines depict proline mutations
  • open square depicts foldon trimerization domain.
  • S1 and S2 represent the first and second domain of the S protein
  • TM depicts the transmembrane region
  • CT depicts the cytoplasmic domain.
  • FIG. 61 E Principal component analysis (PCA) plot showing the multivariate antibody profiles across vaccination groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses show the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution.
  • FIG. 61 F The heat map shows the differences in the means of z-scored features between vaccine groups S.PP and S.dTM.PP. The two groups were compared by two-sided Mann-Whitney tests and stars indicate the Benjamini-Hochberg corrected q-values (*q ⁇ 0.05, ** q ⁇ 0.01, *** q ⁇ 0.001).
  • FIGS. 62A-62C are graphs showing the correlation of antibody titers and survival curves.
  • FIG. 62A Correlations of binding ELISA titers and pseudovirus NAb titers at week 2 and week 4. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 62B Survival curve for the vaccine study. P values indicate two-sided Fisher’s exact tests. N denotes number of animals in each group.
  • FIG. 62C Combined analysis of the two hamster studies involving all animals that received the 5x10 5 TCID 5 o challenge dose and were followed longitudinally. P values indicate two-sided Fisher’s exact tests. N denotes number of animals in each group.
  • FIGS. 63A-63D are graphs showing clinical disease in hamsters following high-dose SARS-CoV-2 challenge.
  • FIG. 63A Median percent weight change following challenge.
  • FIG. 63B Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 4.
  • FIG. 63A Median percent weight change following challenge.
  • FIG. 63B Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 4.
  • FIG. 63C Maximal weight loss in the combined Ad26-S.d
  • FIGS. 64A-64B are graphs showing antibody correlates of clinical protection. Correlations of (FIG. 64A) binding ELISA titers and (FIG. 64B) pseudovirus NAb titers at week 2 and week 4 with maximum percent weight loss following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
  • FIGS. 66A-66D are graphs showing antibody correlates of protection. Correlations of (FIGS. 66A, 66C) binding ELISA titers and (FIGS. 66B, 66D) pseudovirus NAb titers at week 2 and week 4 with logio RNA copies per gram (FIGS. 66A, 66B) lung and (FIGS. 66C, 66D) nasal turbinate tissue in the animals that were necropsied on day 4. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
  • FIGS. 67A-67B are graphs showing antibody correlates of protection and anamnestic responses.
  • FIG. 67B ELISA and NAb responses in surviving hamsters on day 14 following SARS-CoV-2 challenge.
  • FIGS. 68A-68S is a series of graphs showing Ad26 vaccination protects against SARS- CoV-2 pathology. Histopathological H&E evaluation of (FIGS. 68A-68E, 68K-68N) sham controls and (FIGS. 68F-J, 680-R) Ad26-S.PP vaccinated hamsters shows in sham controls (FIG. 68A) severe consolidation of lung parenchyma and infiltrates of inflammatory cells, (FIG. 68B) bronchiolar epithelial syncytia and necrosis, (FIG. 68C) SARS-CoV-N positive (IHC) bronchiolar epithelial cells, (FIG.
  • FIG. 69 Schematic representation of several designs of SARS-CoV-2 S constructs (S1 light grey, S2 dark grey). In the bottom designs, the wildtype (wt) signal peptide was changed to the tPA signal peptide.
  • Vertical line between S1 and S2 indicates the mutation in the furin cleavage site. Dotted line indicates the double proline mutations at position 986 and 987. Delta ERRS indicates deletion of the C-terminal residues that contain the endoplasmic reticulum retention signal.
  • FIG. 70 Detection of expression of SARS-CoV-2 S with several SARS-CoV-1 specific antibodies on the cell surface after transfection with several different S variants based on luminescence intensities using a cell-based ELISA. Data are represented as mean ⁇ SEM.
  • FIG. 71 Detection of expression of SARS-CoV-2 S with several SARS-CoV-1 specific antibodies and an antibody specific for a high-mannose patch on HIV gp120 (2G12), on the cell surface after transfection with several different S variants using flow cytometry. Data are represented as mean ⁇ SEM.
  • FIG. 72 Serum SARS-CoV-2 Spike-specific antibody titers on day 19 by ELISA.
  • ULOQ upper limit of detection
  • LLOD lower limit of detection.
  • Statistical analysis One-way ANOVA with Bonferroni multiple comparison correction; n.s., not significant (p>0.05).
  • FIG. 73 Effect of different signal peptides on protein expression. Detection of expression of SARS-CoV-2 S constructs with different signal peptides with a ACE2-antibody construct and SARS-CoV-1 specific antibody (CR3022) on the cell surface based on luminescence intensities using cell-based ELISA. Data are represented as mean ⁇ SEM.
  • FIG. 74 Cloning the SARS-CoV-2 coding sequence in linearized target vector. Two DNA fragments coding parts of SARS-CoV-2 spike protein were assembled into their target vector.
  • FIG. 75 Introduction of expression cassette coding the SARS-CoV-2 sequence into the E1 region of the Ad26 vector.
  • FIGs. 76A-76B S protein binding antibody titers as measured by ELISA.
  • FIG. 76A week 2;
  • FIG. 76B week 4.
  • the median response per group is indicated with a horizontal line.
  • the dotted line indicates the LLOD.
  • Ad26NCOV030 Ad26COVS1
  • FIGs. 77A-77B Neutralizing antibody titers as measured by wtVNA determining the cytopathic effect (CPE) of virus isolate Leidenl (L-001) on Vero E6 cells.
  • CPE cytopathic effect
  • FIG. 77 A week 2;
  • FIG. 77B week 4.
  • the median response per group is indicated with a horizontal line.
  • the dotted line indicates the LLOD. Animals with a response at or below the LLOD are shown as open symbols.
  • wtVNA wild-type virus neutralization assay.
  • Ad26NCOV030 Ad26COVS1
  • Ad26NCOV006, and Ad26NCOV028 were performed with a z-test for Tobit ANOVA with Bonferroni correction for multiple comparisons. **: p ⁇ 0.01.
  • FIG. 78 SARS-CoV-2 S Protein Specific Cellular Response in Mice; The sum of SFU from stimulation with peptide pools 1 and 2 is shown. IFN-y production by splenocytes was measured by ELISpot after 18 hours of peptide stimulation. The dotted line indicates the LLOD of 200 IFN- Y SFU/10 6 unstimulated splenocytes incubated with medium and DMSO. Animals with a response at or below the LLOD are shown as open symbols.
  • FIGs. 79A-79C Cytokine concentration in cell supernatant was determined by a Multiplex ELISA based analysis, and ratios were calculated.
  • FIG. 79A Ratio IFNY/IL-4
  • FIG. 79B Ratio IFNY/IL-5
  • FIG. 79C Ratio IFNY/IL-10. The median ratio per group is indicated with a horizontal line.
  • Ad26COVS1 Ad26NCOV030
  • adjuvanted S protein calculated by Mann-Whitney U-testwith 2-fold Bonferroni correction. ***: p ⁇ 0.001.
  • FIGs. 80A-80C S protein binding lgG1 and lgG2a titers were measured by ELISA.
  • FIG. 80A lgG1;
  • FIG. 80B lgG2a.
  • the median response per group is indicated with a horizontal line. Dotted lines indicate the LLOD and ULOD of the assays. Animals with a response at or below the LLOD or at or above the ULOD are shown as open symbols.
  • FIG. 80C The ratio of lgG2a/lgG1 titers was calculated.
  • the median response per group is indicated with a horizontal line.
  • the dotted line indicates an lgG2a/lgG1 ratio of 1.
  • Ad26COVS1 Ad26NCOV030
  • Ad26NCOV030 Ad26NCOV030
  • FIGs. 81A-81B S protein binding antibody titers as measured by ELISA.
  • FIG. 81 B Neutralizing antibody titers as measured by wtVNA determining the cytopathic effect (CPE) of virus isolate Leidenl (L-001) on Vero E6 cells. The median response per group is indicated with a horizontal line. The lower dotted line indicates the LLOD of 1.699 logio for ELISA and 5.7 for wtVNA. Animals with a response at or below the LLOD are shown as open symbols. Across- dose comparisons were performed with a t-test from ANOVA (ELISA) and with a Cochran- Mantel-Haenszel test (wtVNA), with Bonferroni adjustment for multiple comparisons.
  • CPE cytopathic effect
  • FIG. 82 IFN-y production by PBMC measured after 18 hours of peptide stimulation by ELISpot. The sum of SFU from stimulation with peptide pools 1 and 2 is shown. The dotted line indicates the LOB of 4.5 IFN-g SFU/10 6 PBMC, calculated as the 95th percentile of SFU from unstimulated cells incubated with medium and DMSO. Animals with a response at or below the LOB are shown as open symbols. Across-dose comparisons were performed with a t-test from ANOVA, with Bonferroni correction for multiple comparisons.
  • FIGs. 83A-83B (FIG. 83A) S protein binding antibody titers as measured by ELISA.
  • FIG. 84 Replication competent virus (TCID 5 o per gram lung) measured by plaque assay. The median viral load per group is indicated with a horizontal line. Animals with a response at or below the LLOD are shown as open symbols. Comparisons were performed between the Ad26. Empty group and the Ad26COVS1 (Ad26NCOV030) and Ad26NCOV006 groups by Mann-Whitney U-test. No corrections for multiple comparisons were made. **: p ⁇ 0.01.
  • FIG. 85 Body weight as measured prior to challenge on Day relative to challenge [C]0 and 4 days post challenge on Day C4. The relative change in body weight is expressed as the body weight % relative to Day CO. The median change in body weight per group is indicated with a horizontal line. Comparisons were performed between the Ad26. Empty group and the Ad26COVS1 (Ad26NCOV030) and Ad26NCOV006 groups by t-test from ANOVA. No corrections for multiple comparisons were made. ***: p ⁇ 0.001 ; **: p ⁇ 0.01; *: p ⁇ 0.05.
  • FIGs. 86A-86D S protein binding antibody titers as measured by ELISA.
  • FIG. 86B Neutralizing antibody titers as measured by ppVNA.
  • FIG. 86C Neutralizing antibody titers as measured by wtVNA using the Seattle Washington isolate, designed to express luciferase and GFP, incubated on Vero E6 cells. The titers were measured via Nano-Glo Luciferase Assay System.
  • FIG. 86D IFN-y production of SARS-CoV-2 peptide stimulated PBMC as measured by ELISpot. The sum of SFU from stimulation with peptide pools 1 and 2 is shown. The dotted line indicates the LOB of 50 IFN-g SFU/10 6 PBMC. Animals with a response at or below the LOB are shown as open symbols. The median response per group is indicated with a horizontal line.
  • ppVNA pseudotyped virus neutralization assay
  • SFU spot forming units
  • vp virus particles
  • wtVNA wild-type virus neutralization assay.
  • FIG. 87 Western blot analyses for expression from Ad26 vectors encoding tPA.S (lane 1), tPA.S.PP (lane 2), S (lane 3), S.dCT (lane 4), tPA.WT.S (lane 5), S.dTM.PP (lane 6), and S.PP (lane 7) in cell lysates using an anti-SARS polyclonal antibody.
  • FIGs. 88A-88B Humoral immune responses in vaccinated rhesus macaques. Humoral immune responses were assessed at weeks 0, 2, and 4 by (FIG. 88A) RBD-specific binding antibody ELISA, and (FIG. 88B) pseudovirus neutralization assays. Red (solid) bars reflect median responses.
  • FIGs. 89A-89B Cellular immune responses in vaccinated rhesus macaques. Cellular immune responses were assessed at week 4 following immunization by (FIG. 89A) IFN-g ELISPOT assays and (FIG. 89B) IFN-y+CD4+ and IFN-y+CD8+ T cell intracellular cytokine staining assays in response to pooled S peptides. Red (solid) bars reflect median responses.
  • FIGs. 90A-90D Viral loads in rhesus macaques following SARS-CoV-2 challenge. Rhesus macaques were challenged by the intranasal and intratracheal route with 1.2x10 8 VP (1.1x10 4 PFU) SARS-CoV-2.
  • FIG. 90A, FIG. 90B Logio sgmRNA copies/ml (limit 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) in sham controls and in vaccinated animals following challenge.
  • FIG. 90C, FIG. 90D Logio sgmRNA copies/swab (limit 50 copies/swab) were assessed in nasal swabs (NS) in sham controls and in vaccinated animals following challenge. Red lines reflect median values.
  • FIG. 91 _Summary of peak viral loads following SARS-CoV-2 challenge. Peak viral loads in BAL and NS following challenge. Peak viral loads occurred variably on day 1-4 following challenge. Red lines reflect median viral loads. P-values indicate two-sided Mann-Whitney tests (*P ⁇ 0.05, ** P ⁇ 0.001, *** P ⁇ 0.0001).
  • FIG. 92 Immune responses following SARS-CoV-2 challenge. Pseudovirus NAb titers prior to challenge and on day 14 following challenge. Red bars reflect median responses.
  • FIG. 93 Comparison of pseudovirus NAb titers in vaccinated macaques and convalescent macaques and humans. Comparison of pseudovirus NAb in macaques vaccinated with Ad26.S.PP with previously reported cohorts of convalescent macaques and convalescent humans who had recovered from SARS-CoV-2 infection.
  • FIG. 94 Humoral immune responses in BAL in vaccinated rhesus macaques. S-specific IgG and IgA at week 4 in BAL by ELISA in sham controls and in Ad26.S.PP vaccinated animals.
  • FIG. 95 Cellular immune responses in vaccinated rhesus macaques. IFN-Y+, IL-2+, IL-4+, and IL-10+ CD4+ T cell intracellular cytokine staining assays in response to pooled S peptides in Ad26.S.dTM.PP and Ad26.S.PP vaccinated animals. Red bars reflect median responses.
  • FIGs. 96A-96C Median percent weight change following challenge. The numbers reflect the number of animals at each timepoint. In the high-dose group, 4 animals were necropsied on day 2, 4 animals were necropsied on day 4, 4 animals met euthanization criteria on day 6, and 2 animals met euthanization criteria on day 7.
  • FIG. 96B Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 2 and day 4.
  • FIGs. 97A-97L Pathologic features of high-dose SARS-CoV-2 infection in hamsters.
  • FIG. 97A Necrosis and inflammation (arrow) in nasal turbinate, H&E (d2).
  • FIG. 97B Bronchiolar epithelial necrosis with cellular debris and degenerative neutrophils in lumen (arrow) and transmigration of inflammatory cells in vessel wall (arrowhead), H&E (d2).
  • FIG. 97C Interstitial pneumonia, hemorrhage, and consolidation of lung parenchyma, H&E (d2).
  • FIG. 97D Nasal turbinate epithelium shows strong positivity for SARS-CoV-N by IHC (d2).
  • FIG. 97E Pathologic features of high-dose SARS-CoV-2 infection in hamsters.
  • FIG. 97A Necrosis and inflammation (arrow) in nasal turbinate, H&E (d2).
  • FIG. 97B Bronchiolar epithelial nec
  • Bronchiolar epithelium and luminal cellular debris show strong positivity for SARS-CoV-N by IHC (d2).
  • FIG. 97F Pneumocytes and alveolar septa show multifocal strong positivity for SARS-CoV-N by IHC (d2).
  • FIG. 97G Diffuse vRNA staining by RNAscope within pulmonary interstitium (arrow, interstitial pneumonia) and within bronchiolar epithelium (arrowhead; d2).
  • FIG. 97H Diffuse vRNA staining by RNAscope within pulmonary interstitium (d4).
  • FIG. 97I lba-1 IHC (macrophages) within pulmonary interstitium (d7).
  • FIG. 97J CD3+ T lymphocytes within pulmonary interstitium, CD3 IHC (d4).
  • FIG. 97K MPO (neutrophil myeloperoxidase) IHC indicating presence of interstitial neutrophils (d7).
  • FIG. 97L Interferon-inducible gene, MX1,
  • FIGs. 98A-98F Humoral immune responses in vaccinated hamsters.
  • SARS-CoV-2 spike (S) immunogens with (i) deletion of the transmembrane region and cytoplasmic tail reflecting the soluble ectodomain with a foldon trimerization domain (S.dTM.PP) or (ii) full-length S (S.PP), both with mutation of the furin cleavage site and two proline stabilizing mutations.
  • Red X depicts furin cleavage site mutation
  • red vertical lines depict proline mutations
  • open square depicts foldon trimerization domain.
  • S1 and S2 represent the first and second domain of the S protein
  • TM depicts the transmembrane region
  • CT depicts the cytoplasmic domain.
  • the size and color intensity of the wedges indicate the median of the feature for the corresponding group (antibody subclass, red; FcyR binding, blue; ADCD, green).
  • FIG. 98E Principal component analysis (PCA) plot showing the multivariate antibody profiles across vaccination groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses show the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution.
  • FIG. 98F The heat map shows the differences in the means of z-scored features between vaccine groups S.PP and S.dTM.PP. The two groups were compared by two-sided Mann-Whitney tests and stars indicate the Benjamini-Hochberg corrected q-values (*q ⁇ 0.05, ** q ⁇ 0.01, *** q ⁇ 0.001).
  • FIGs. 99A-99C Clinical disease in hamsters following high-dose SARS-CoV-2 challenge.
  • FIG. 99A Median percent weight change following challenge.
  • FIG. 99B Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 4.
  • P values indicate two-sided Mann Whitney tests. N reflects all animals that were followed for weight loss and were not necropsied on day 4.
  • FIGs. 100A-100F Longitudinal quantitative image analysis of viral replication and associated inflammation in lungs.
  • FIG. 100A Percent lung area positive for anti-sense SARS-CoV-2 viral RNA (vRNA) by RNAscope ISH.
  • FIG. 100B Percentage of total cells positive for SARS-CoV-N protein (nuclear or cytoplasmic) by IHC.
  • FIG. 100C lba-1 positive cells per unit area by IHC.
  • FIG. 100D CD3 positive cells per unit area.
  • FIG. 100E MPO positive cells per unit area.
  • FIG. 100F Percentage of MX1 positive lung tissue as a proportion of total lung area.
  • ISH in situ hybridization
  • IHC immunohistochemistry
  • SARS-N SARS-CoV nucleocapsid
  • MPO myeloperoxidase
  • MX myxovirus protein 1 (a type 1 interferon inducible gene). Each dot represents one animal.
  • FIG. 101 Participants were enrolled concurrently at Belgian and US sites. Participants were randomized in parallel in a 1:1:1 :1 :1 ratio to one of five vaccination groups to receive one or two IM injections of Ad26.COV2.S at two dose levels of either 5x10 10 vp or 1x10 11 vp, or placebo. For each cohort, in the absence of clinically significant findings 24 hours after the first vaccination was administered to five sentinel participants (one per dose level and one placebo), another ten participants were vaccinated across all groups. Safety data up to day 28 were then reviewed by an internal data review committee before the remaining participants were randomized.
  • FIGs. 102A-102C Flow charts for cohort 1a (FIG. 102A), cohort 1b (FIG. 102B) and cohort 3 (FIG. 102C).
  • FIGs. 103A-103D Humoral and cellular immune responses.
  • FIG. 103A Log geometric mean titers (GMTs - as illustrated by the horizontal bars and the numbers below each timepoint) of serum SARS-CoV-2 binding antibodies, measured by ELISA (ELISA Units per mL [EU/mL]), at baseline and 29 days post vaccination, among all participants, according to schedule in cohort 1a and 3. Dotted lines indicate the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the assay, error bars indicate 95% confidence interval (Cl). For values below the LLOQ, LLOQ/2 values were plotted.
  • LLOQ lower limit of quantification
  • UEOQ upper limit of quantification
  • Th1 IFNy and/or IL-2, not IL-4, IL-5 and IL-13
  • Th2 IL-4 and/or IL-5 and/or IL-13 and CD40L
  • ICS intracellular cytokine staining
  • FIG. 104 Graphical representation of VNA responses against SARS-CoV-2 (geometric mean titers [GMTs] with corresponding 95% Cls) over time.
  • FIG. 105 depicts the effect of age on the neutralizing antibody response by Day 29.
  • FIG. 106 depicts the neutralizing antibody response and responder rates in the study cohorts.
  • FIG. 107 depicts reverse cumulative distribution curves for the 5 c 10 10 vp vaccine group and the 1x10 11 vp vaccine group.
  • FIG. 108 graphic representation of SARS-CoV-2 S protein binding antibody responses as measured by ELISA.
  • FIG. 109 depicts the effect of age on the binding antibody response at Day 29.
  • FIG. 110 graphic representation of SARS-CoV-2 S protein binding antibody responses as measured by ELISA.
  • FIG. 111 depicts reverse cumulative distribution curves for the 5 c 10 10 vp vaccine group and the 1x10 11 vp vaccine group.
  • FIG. 112 shows that wtVNA titers highly correlated with ELISA titers at both Day 15 and Day 29, with Spearman Correlation coefficients of 0.734 and 0.72; respectively.
  • FIG. 113 shows the percentage of CD4+ T cells expressing IFNy and/or IL-2 (Th1), and not Th2 cytokines, and expressing IL-4 and/or IL-5/IL-13 and CD40L (Th2).
  • FIG. 114 shows the combined regimen profile.
  • FIG. 115 shows the descriptive statistics for CD8+ T cells producing IFNy and/or IL-2 in response to SARS-CoV-2 S peptide stimulation.
  • FIG. 116 shows the combined regimen profile.
  • FIG. 117A-117C SARS-CoV-2-specific humoral immune responses to 1- and 2-dose Ad26.COV2.S vaccine regimes in adult rhesus macaques.
  • Spike (S) protein binding antibody levels were measured over time with a qualified ELISA for human samples, using a trimeric, soluble stabilized S protein produced in mammalian cells as coating antigen. Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The geometric mean titers (GMT) of binding antibody responses per group is indicated with the red line. The dotted lines indicate the lower limit of detection (LLOD) and lower limit of quantification (LLOQ).
  • LLOD lower limit of detection
  • LLOQ lower limit of quantification
  • S protein neutralizing antibody levels were measured over time with a qualified psVNA for human samples, using pseudotyped virus particles made from a modified Vesicular Stomatitis Virus (VSVAG) backbone and bear the S glycoprotein of SARS-CoV- 2.
  • Neutralizing antibody responses are measured as the reciprocal of the sample dilution where 50% neutralization is achieved (IC50) ⁇ Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The GMT of neutralizing antibody responses per group is indicated with the red line.
  • the dotted lines indicate the LLOD and LLOQ.
  • FIG. 117C Correlation between S-specific binding antibody levels and neutralizing antibody titers per animal for all groups and timepoints except the sham control group and week 0 (baseline). The dotted lines indicate the LLOD for each assay.
  • FIG. 118A-118C SARS-CoV-2-specific humoral and cellular immune responses after vaccination of aged rhesus macaques.
  • Spike (S) protein binding antibody levels were measured over time with a qualified ELISA for human samples, using a trimeric, soluble stabilized S protein produced in mammalian cells as coating antigen. Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The geometric mean titer (GMT) of binding antibody responses per group is indicated with the red line. The dotted lines indicate the lower limit of detection (LLOD) and lower limit of quantification (LLOQ).
  • LLOD lower limit of detection
  • LLOQ lower limit of quantification
  • SARS-Cov-2 neutralization antibody titers over time, as measured by wtVNA. Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The GMT per group is indicated with the red line. The dotted line indicates the LLOD.
  • FIG. 118C Correlation between S-specific binding antibody levels and neutralizing antibody titers per animal for all groups and timepoints except the sham control group and week 0. The dotted lines indicate the LLOD for each assay.
  • FIG. 119A-119C SARS-CoV-2-specific cellular immune responses after vaccination of aged rhesus macaques.
  • FIG. 119A Spike (S) protein-specific T cell responses as measured with an IFN-y/IL-4 Double-color ELISpot at indicated timepoints. The geometric mean titer (GMT) response per group is indicated with a horizontal line. Samples with background subtracted counts below or equal to 0 were set a 10 for visualization purposes, indicated by the dotted line.
  • FIG. 119B Spike (S) protein-specific T cell responses as measured by intracellular cytokine staining at indicated timepoints. Frequency of CD4+CD69+ T cell expressing cytokines.
  • the geometric mean titer (GMT) response per group is indicated with a horizontal line. The dotted line indicates the technical threshold. Open symbols denote samples at technical threshold.
  • FIG. 119C ratio of CD4+CD69+ T cells expressing Th1 (IFN-y or IL-2) or Th2 (IL-4, IL-5, IL-13) cytokines.
  • the geometric mean titer (GMT) response per group is indicated with a horizontal line. Open symbols denote values were either cells expressing Th1, Th2 or any cytokine were at the technical threshold. The dotted horizontal line is set at a ratio of 1 for visualization purposes.
  • FIG. 120 Spike (S) protein-specific T cell responses as measured by intracellular cytokine staining at indicated timepoints. Frequency of CD8+CD69+ T cell expressing cytokines. The geometric mean titer (GMT) response per group is indicated with a horizontal line. The dotted line indicates the technical threshold. Open symbols denote samples at technical threshold.
  • FIGs. 121A-121D Log geometric mean titers (GMTs - as illustrated by the horizontal bars and the numbers below each timepoint) of SARS-CoV-2 Spike protein binding antibodies in serum as measured by ELISA (ELISA Units per mL [EU/mL]), at baseline and at Day 29 post vaccination, among all participants, and Day 57 and Day 71 for those available for cohort 1a, according to schedule in cohort 1a (18-55 years old) (FIG. 121A) and cohort 3 (>65 years old) (FIG. 121 B). Dotted lines indicate the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the assay, error bars indicate 95% confidence interval (Cl). For values below the LLOQ, LLOQ/2 values were plotted.
  • LLOQ lower limit of quantification
  • UEOQ upper limit of quantification
  • FIGs. 122A-122F Log GMTs of serum SARS-CoV-2 neutralizing antibodies (wtVNA), measured by 80% neutralization assay.
  • FIG. 123 Correlation plot between Ad26 and SARS-CoV-2 neutralizing antibody titers: baseline Ad26 VNA baseline vs. wtVNA day 29, and Ad26 VNA at day 57 vs. wtVNA at day 71
  • FIGs. 124A-124C Expression of Th1 (IFN-y and/or IL-2, and not IL-4, IL-5 and IL-13) (FIG. 124A), and Th2 (IL-4 and/or IL-5 and/or IL-13 and CD40L) (FIG. 124B) cytokines by CD4+ T cells was measured by intracellular cytokine staining (ICS).
  • Th1 IFN-y and/or IL-2, and not IL-4, IL-5 and IL-13
  • Th2 IL-4 and/or IL-5 and/or IL-13 and CD40L
  • FIG. 124C Expression of IFN-g and/or IL-2 cytokines by CD8+ T cells was measured by ICS. Median (as illustrated by the horizontal bars and the numbers below each timepoint) and individual ICS responses to SARS-CoV-2 S protein peptide pool in peripheral blood mononuclear cells, at baseline and 15 days post vaccination, among a subset of participants from cohort 1a (18-55 years old) and cohort 3 (>65 years old), according to schedule, are given. The Y-axis denotes the percentage of CD8+ T cells positive for IFN-g and/or IL-2 cytokines. Dotted line indicates the LLOQ. Values below the LLOQ were plotted as LLOQ/2.
  • FIGs. 125A-125B Relative productivity of Ad26NCOV030 (JNJ 78436735) and Ad26NCOV028 purified material in SPER.C6 and PER.C6 TetR cells.
  • SPER.C6 cells B) SPER.C6 TetR cells. Cells were transduced in shaker flasks with purified material of the Ad26 vector (70 or 300 VP/cell). Samples were taken at 0, 1, 2, 3, and 4 days post infection, and vector particle concentration was measured by VP-qPCR. Standard control (Ad26.ZEBOV) and low control (26ZIK001). VP, viral particles; qPCR, quantitative polymerase chain reaction; TetR, tetracycline repressor.
  • FIGs. 126A-126E Titration of SARS-CoV-2 challenge dose and characterization of histopathology in Syrian hamsters.
  • Daily throat swabs were taken, and 2, 3, 4, and 7 days p.i., 3 hamsters per group were sacrificed and nose and lung tissue collected for virological analysis and histopathology.
  • Replication competent viral load in (FIG. 126A) lung tissue (FIG.
  • FIG. 126B nose tissue, and (FIG. 126C) throat swabs, was determined by TCID 5 o assay on Vero E6 cells. LLOD was calculated per animal per gram or milliliter of tissue, and animals with a response at or below the LLOD are shown as open symbols.
  • FIG. 126D Lung tissue was analyzed and scored for presence and severity of alveolitis, alveolar damage, alveolar edema, alveolar hemorrhage, type II pneumocyte hyperplasia, bronchitis, bronchiolitis, peribronchial and perivascular cuffing. Sum of scores are presented as sum of LRT disease parameters (potential range: 0 - 24).
  • FIGs. 127A-127C SARS-CoV-2 neutralizing antibody response elicited by 1- and 2-dose Ad26.COV2.S vaccine regimes in Syrian hamsters.
  • SARS-CoV-2 neutralization titers were measured 4 weeks after dose 1 and (FIG.
  • FIGs. 128A-128C SARS-CoV-2 spike protein binding antibody response, and ratio with neutralizing antibody responses, elicited by 1- and 2-dose Ad26 vaccine regimes in Syrian hamsters.
  • Syrian hamsters were immunized with either 10 9 or 10 10 VP of Ad26-based vaccine candidates Ad26.S, Ad26.dTM.PP or Ad26.COV2.S, or with 10 10 vp of an Ad26 vector without gene insert as control (Ad26. empty).
  • Ad26. empty Ad26. empty
  • the ratio of SARS-CoV-2 neutralizing antibodies over binding antibodies was calculated by dividing antibody titers as measured by wtVNA, by antibody titers as measured by ELISA. Animals with neutralization titers at or below the LLOD are displayed as open symbols. Median responses per group are indicated with horizontal lines.
  • LLOD Lower Limit of Detection
  • ELISA Enzyme-linked Immunosorbent assay
  • wtVNA wild-type virus neutralization assay
  • VP virus particles
  • FIGs. 129A-129C SARS-CoV-2 spike protein binding antibody response and neutralizing antibody response elicited by a 2-dose Ad26 vaccine regime in New Zealand White rabbits.
  • New Zealand white rabbits were intramuscularly immunized with a 2-dose regimen of 5x10 9 vp or 5x10 10 vp Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or saline (FIG. 129A). Serum was sampled prior to immunization (day -3) and days 14, 21 , 35, 42, 56, 63, 70, 84 and at sacrifice (days 99- 101, depicted in the graph as day 100).
  • FIG. 129B SARS-CoV-2 spike protein-specific antibody binding titers were measured by ELISA.
  • FIG. 129C Neutralization titers were measured on days 14, 35, 56, 70 and at sacrifice.
  • ELISA Enzyme-linked Immunosorbent assay
  • wtVNA wild-type virus neutralization assay
  • LLOQ Lower Limit of Qualification
  • ULOQ Upper Limit of Qualification
  • LLOD Lower Limit of Detection
  • VP virus particles.
  • FIGs. 130A-130F Protection against SARS-CoV-2 viral replication in Syrian hamsters immunized with Ad26-based vaccine candidates.
  • Syrian hamsters were intramuscularly immunized with a 1-dose regimen and a 2-dose regimen of Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or Ad26. empty (Ad26 vector not encoding any SARS-CoV-2 antigens).
  • FIGs. 130A, 130B Right lung tissue and (FIGs. 130C, 130D) right nasal turbinates were harvested at the end of the 4-day inoculation phase for viral load analysis. Replication competent virus was measured by TCID50 assay.
  • FIGs. 130E, 130F Throat swab samples were taken daily after inoculation, and viral load area under the curve during the four-day follow up was calculated as TCIDso/ml x day.
  • the median viral load per group is indicated with a horizontal line.
  • FIGs. 131A-131D Protection against SARS-CoV-2 IHC and histopathology in lung tissue of Syrian hamsters immunized with Ad26-based vaccine candidates.
  • Syrian hamsters were intramuscularly immunized with a 1-dose regimen and a 2-dose regimen of Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or Ad26. empty (Ad26 vector not encoding any SARS-CoV-2 antigens).
  • the hamsters received intranasal inoculation with 10 2 TCID50 SARS-CoV-2 strain BetaCoV/Munich/BavPat1/20204 weeks post-dose 1 (week 4), or 4 weeks post-dose 2 (week 8).
  • FIGs. 131 A and 131 B Presence of SARS-CoV-2 NP was determined by immunohistochemical staining.
  • FIGs. 132A-132D Protection against SARS-CoV-2 IHC and histopathology in nasal tissue of Syrian hamsters immunized with Ad26-based vaccine candidates.
  • Syrian hamsters were intramuscularly immunized with a 1-dose regimen and a 2-dose regimen of Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or Ad26. empty (Ad26 vector not encoding any SARS-CoV-2 antigens).
  • the hamsters received intranasal inoculation with 10 2 TCID50 SARS-CoV-2 strain BetaCoV/Munich/BavPat1/20204 weeks post-dose 1 (week 4), or 4 weeks post-dose 2 (week 8).
  • FIGs. 132A and 132B Presence of SARS-CoV-NP was determined by immunohistochemical staining.
  • FIGs. 132C and 132D Nasal tissue was scored for severity of rhinitis.
  • FIGs. 133A-133D Dose responsiveness of Ad26.COV2.S on immunogenicity and lung viral load in hamsters.
  • SARS-CoV-2 Spike protein-specific antibody binding titers FIG. 133A
  • SARS-CoV-2 neutralizing antibodies FIG. 133B
  • Ad26.lrr Ad26 vector not encoding any SARS-CoV-2 antigens
  • LLOD lower limit of detection
  • N number of animals
  • TCIDso/g 50% tissue culture infective dose per gram tissue
  • VP virus particles
  • NP Nucleocapsid protein.
  • N number of animals
  • VP virus particles
  • FIGs. 135A-135D Binding and neutralizing antibodies correlate with protection. Protection per vaccine construct was defined as a viral load below 10 2 TCIDso/g in lung tissue, irrespective of vaccine regimen and dose level (see Fig 129A and B, and Fig 133C).
  • Hamsters were inoculated with 10 2 TCID 50 SARS- CoV-2, and four days later sacrificed for virological analysis of lung tissue.
  • FIG. 135A Prior to virus inoculation serum samples were analyzed for (FIG. 135A) antibody binding titers and (FIG. 135B) virus neutralizing antibodies. Median antibody responses per group is indicated with horizontal lines. Dotted lines indicate the LLOD.
  • FIGs. 136A-136C Rapid induction of binding and neutralizing antibodies following Ad26.COV2.S vaccination.
  • FIG. 136A S-specific binding antibodies by ELISA
  • FIG. 136B RBD-specific binding antibodies by ELISA
  • FIG. 136C pseudovirus neutralizing antibodies (psVNA) on day 1 and day 8 in recipients of the high dose (HD) and low dose (LD) Ad26.COV2.S or placebo (PL).
  • Red bars reflect geometric mean titers (GMT).
  • P values reflect two-sided Mann-Whitney tests.
  • FIGs. 137A-137B Kinetics and magnitude of binding and neutralizing antibodies following Ad26.COV2.S vaccination.
  • A S- and RBD-specific binding antibodies by ELISA and
  • B SARS-CoV-2 pseudovirus neutralizing antibody (psVNA) and Ad26 virus neutralizing antibody (Ad26 VNA) responses following Ad26.COV2.S vaccination.
  • LD low dose
  • HD high dose
  • PL placebo.
  • Red bars reflect geometric mean titers (GMT). Dotted lines reflect lower limits of quantitation.
  • FIG. 138 Correlations of humoral immune responses. Correlations of logi 0 S-specific ELISA titers, logio RBD-specific ELISA titers, and Iog10 neutralizing antibody (NAb) titers on day 29. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIGs. 139A-139B Antibody cross-reactivity following Ad26.COV2.S vaccination. Electrochemiluminescence assay (Meso Scale Discovery SARS-CoV-2 IgG Panel 2; K15369U- 2) assessing binding antibody responses to the S proteins from (FIG. 139A) SARS-CoV-2 and SARS-CoV-1 as well as (FIG. 139B) CoV-229E, CoV-HKLH, CoV-NL63, and CoV-OC43 following Ad26.COV2.S vaccination. LD, low dose; HD, high dose; PL, placebo. Red bars reflect geometric mean responses. Dotted lines reflect lower limits of quantitation. FIGs.
  • 140A-140B Kinetics and magnitude of cellular immune responses following Ad26.COV2.S vaccination.
  • FIG. 140A IFN-g and IL-4 ELISPOT responses
  • FIG. 140B IFN-g central memory CD27+ CD45RA- CD4+ and CD8+ T cell responses by ICS assays following Ad26.COV2.S vaccination.
  • ICS assays were performed in a subset of participants with sufficient peripheral blood mononuclear cells (PBMCs) on day 71/85.
  • PBMCs peripheral blood mononuclear cells
  • SFC spot-forming cells
  • LD low dose
  • HD high dose
  • PL placebo. Red bars reflect geometric mean responses. Dotted lines reflect lower limits of quantitation.
  • FIG. 141 Correlations of cellular and humoral immune responses. Correlations of logio ELISPOT responses with logio S-specific ELISA titers, logio RBD-specific ELISA titers, and Iog10 neutralizing antibody (NAb) titers on day 29. P and R values reflect two-sided Spearman rank-correlation tests.
  • FIG. 142 Cumulative Incidence of Molecularly Confirmed Moderate to Severe/Critical COVID- 19 Cases with Onset at Least 1 Day after Vaccination, Full Analysis Set.
  • FIG. 143 Cumulative Incidence of Molecularly Confirmed Moderate to Severe/Critical COVID- 19 Cases with Onset at Least 1 Day After Vaccination Until Day 29 by Serostatus; Full Analysis Set.
  • FIG. 144 Cumulative Incidence of Molecularly Confirmed Severe/Critical COVID-19 Cases with Onset at Least 1 Day after Vaccination, Full Analysis Set.
  • FIG. 145 Force of infection. Placebo COVID-19 incidence rate in different countries.
  • 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 2019-nCoV immunogen) into a eukaryotic cell, such that the polynucleotide is subsequently expressed.
  • a polynucleotide of interest e.g., a polynucleotide encoding a 2019-nCoV immunogen
  • the adenovirus is Ad26.
  • 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.
  • 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 Wuhan coronavirus (2019-nCoV) vaccine)) to a subject.
  • a pharmaceutical composition e.g., an immunogenic composition (e.g., a vaccine (e.g., a Wuhan coronavirus (2019-nCoV) vaccine)
  • a pharmaceutical composition e.g., an immunogenic composition (e.g., a vaccine (e.g., a Wuhan coronavirus (2019-nCoV) vaccine)
  • 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 preferred 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).
  • 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 (l), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • codon 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.
  • codon also refers to base triplets in a DNA strand.
  • convalescent refers to subjects who have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV).
  • 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., 2019-nCoV).
  • a cohort of convalescent humans will share common characteristics with a subject having a risk of 2019-nCoV infection or suspected of being susceptible to a coronavirus infection.
  • samples from convalescent humans will be obtained at least 7 days after documented recovery (e.g., determined with a negative nasal swab).
  • an 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).
  • an ectodomain includes the S1 domain (e.g., SEQ ID NO: 4) and RBD (e.g., SEQ ID NO: 5) of a Spike polypeptide.
  • 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 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.
  • exogenous polynucleotide sometimes referred to as a "transgene”
  • 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 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.
  • 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.
  • the gene product is from a virus (e.g., a 2019-nCoV) and may include, for example, any one or more of the viral proteins, or fragments thereof, described herein.
  • 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., 2019-nCoV).
  • 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.
  • 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.
  • immunogen any polypeptide that can induce an immune response in a subject upon administration.
  • 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)).
  • immunogenic composition is defined as material used to provoke an immune response and may confer immunity after administration of the immunogenic composition to a subject.
  • 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).
  • isolated is meant separated, recovered, or purified from a component of its natural environment.
  • a nucleic acid molecule or polypeptide may be isolated from a component of its natural environment by 1% (2%, 3%, 4%, 5%, 6%, 7%, 8% 9% 10%, 20%,
  • composition any composition that contains a therapeutically or biologically active agent, such as an immunogenic composition or vaccine (e.g., a 2019-nCoV nucleic acid molecule, vector, and/or polypeptide), preferably including 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., 2019-nCoV infection) or reduces or ameliorates one or more symptoms of the disease (e.g., 2019-nCoV viral titer, viral spread, infection, and/or cell fusion)).
  • a therapeutically or biologically active agent such as an immunogenic composition or vaccine (e.g., a 2019-nCoV nucleic acid molecule, vector, and/or polypeptide), preferably including 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., 2019-nCo
  • compositions include vaccines
  • 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 (21 st 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.
  • linking 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.
  • 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.
  • 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).
  • a nucleic acid is “operably linked” when it is placed into a structural or functional relationship with another nucleic acid sequence.
  • one segment of DNA 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).
  • a pre-protein e.g., a pre-protein that participates in the secretion of the encoded polypeptide
  • the operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them.
  • Enhancers for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.
  • 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).
  • 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
  • the optimized viral polypeptide may correspond to a “parent” viral gene sequence; alternatively, the 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.
  • the optimized polypeptide is a Spike polypeptide, which has been further altered to include a leader/signal sequence (e.g., a Spike signal sequence or a tPA signal sequence) for maximal protein expression, a factor Xa site, a foldon trimerization domain (see, e.g., SEQ ID NO: 87), and/or linker or spacer (e.g., SEQ ID NOs: 88 or 89) 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:91)).
  • an optimized viral polypeptide is 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.
  • 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.
  • optimal 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.
  • 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.
  • a pharmaceutically acceptable carrier is physiological saline.
  • 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).
  • 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.
  • 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,
  • 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,
  • 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 (SEQ ID NO: 121).
  • 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: 143 and 204).
  • 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,
  • a fragment of a nucleic acid molecule may include at least 20,
  • 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, or more consecutive nucleotides of the polynucleotide SS-S1-foldon (SEQ ID NO: 129).
  • 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, or more consecutive nucleotides of the polynucleotide SS-RBD-foldon (SEQ ID NO: 130).
  • 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, or more consecutive nucleotides of the polynucleotide SS-S.Ecto-dF-foldon (SEQ ID NO: 136 or 193).
  • a fragment of a nucleic acid molecule may include at least 5, 6, 7, 8, 9, 10, 20, 30,
  • a fragment of a nucleic acid molecule 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,
  • a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100,
  • 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 polypeptide SS-SdCT (SEQ ID NO: 30).
  • 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 polypeptide SS-Spike-dF-PP (SEQ ID NO: 51).
  • 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 polypeptide SS-S.Ecto (SEQ ID NO: 31).
  • a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200,
  • 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 polypeptide SS-RBD-foldon (SEQ ID NO: 38).
  • 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, or more consecutive amino acids of polypeptide SS-S.Ecto-dF-foldon (SEQ ID NO: 44). 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, or more consecutive amino acids of polypeptide S.Ecto-PP-foldon (SEQ ID NO: 50).
  • 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, 525, 550, 575, 600, 625, 650, 675, or more consecutive amino acids of polypeptide SS-S.Ecto-dF-PP-foldon (SEQ ID NO: 56).
  • 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, 525, 550, 575, 600, 625, 650, 675, or more consecutive amino acids of polypeptide prM-Env with JEV Stem/TM (SEQ ID NO: 27).
  • administration of a fragment of a polynucleotide (e.g., SEQ ID NOs: 93-181, 190-195, and 199-204) and/or a polypeptide (e.g., SEQ ID NOs: 1-84, e.g., SEQ ID NO: 51) to a subject may illicit an immune response in the subject.
  • a fragment of a polynucleotide e.g., SEQ ID NOs: 93-181, 190-195, and 199-204
  • a polypeptide e.g., SEQ ID NOs: 1-84, e.g., SEQ ID NO: 51
  • 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.
  • promoters 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 2019-nCoV)) or protein targets in a subject to which the pharmaceutical composition (e.g., an immunogenic composition or vaccine) has been administered.
  • a humoral response e.g., the production of antibodies
  • a cellular response e.g., the activation of T cells, macrophages, neutrophils, and/or natural killer cells
  • infective agents e.g., a virus (e.g., a 2019-nCoV)
  • protein targets in a subject to which the pharmaceutical composition e.g., an immunogenic composition or vaccine
  • 2019-nCoV-mediated disease used interchangeably with “Coronavirus disease 2019 (COVID-19)" herein, as well as grammatical variants thereof, 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 2019-nCoV (SARS-CoV-2) infection 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.
  • 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.
  • 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)
  • An amount or level of a marker that is above the protective level indicates protection from coronavirus infection (e.g., a 2019-nCoV infection) or disease (e.g., a 2019-nCoV-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 2019-nCoV-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).
  • the protective level is an anti-coronavirus 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.
  • the protective level is an anti- coronavirus antibody titer of at least about 100 as measured using the pseudovirus neutralization assay described herein.
  • 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.
  • 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.
  • 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.
  • signal peptide 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: 92) at the N-terminus of a polypeptide that directs a polypeptide towards the secretory pathway (e.g., the extracellular space).
  • the signal peptide is typically cleaved during secretion of the polypeptide.
  • the signal sequence may direct the polypeptide to an intracellular compartment or organelle, e.g., the Golgi apparatus.
  • a 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.
  • a signal peptide can be one that is, for example, substantially identical to the amino acid sequence of SEC ID NO: 92.
  • 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 K D of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a K D 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 K D of greater than 100 nM (e.g., greater than 500 nm, 1 mM, 100 mM, 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.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate.
  • 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. coli 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 2019-nCoV infection or having a 2019-nCoV 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.
  • a subject to be treated according to the present invention 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 2019-nCoV, for example, due to travel to an area where 2019-nCoV infection is prevalent).
  • humans with underlying health conditions e.g., hypertension, diabetes, or cardiovascular disease
  • a coronavirus e.g., 2019-nCoV.
  • the methods of treating a human subject with a composition are, therefore, particularly useful in treating, reducing, and/or preventing a 2019- nCoV infection in humans with underlying health conditions.
  • 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.
  • an exogenous nucleic acid molecule e.g., DNA, such as an expression vector
  • electroporation e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection, and the like.
  • 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.
  • “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 anti-inflammatory 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.
  • 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.
  • vector is meant a DNA construct that includes one or more polynucleotides, or fragments thereof, such as from a viral species, such as a 2019-nCoV 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.
  • plasmid 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
  • 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”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • 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.
  • virus 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.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors (e.g., see PCT publication no. WO 2006/002203), alphavirus vectors and the like.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes (WO 95/27071). Ads 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 invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a 2019-NCOV Spike (S) protein (also referred to as SARS-CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
  • S 2019-NCOV Spike
  • SEQ ID NO: 29 provides the amino acid sequence of the full-length 2019-NCOV Spike (S) protein; see also NCBI Reference Sequence: YP_009724390.1.
  • the stabilising mutations are from the original amino acid in this sequence to proline.
  • the original amino acid at position 986 is lysine (lys, K) and at position 987 is valine (val, V) as shown in SEQ ID NO: 29.
  • the furin cleavage site within the 2019-NCOV Spike (S) protein comprises, or has, the amino acid sequence RARR (SEQ ID NO: 90).
  • Suitable mutations may comprise mutation to SRAG (SEQ ID NO: 225) or GGSG (SEQ ID NO: 91).
  • the SRAG mutation is achieved by introducing a R682S and a R685G mutation into the amino acid sequence.
  • the GGSG mutation is achieved by introducing a R682G, a R683G, a A684S and a R685G mutation into the amino acid sequence.
  • a preferred nucleic acid molecule encodes a full-length 2019-NCOV Spike (S) protein with the stabilising mutations and mutations to the furin cleavage site as the only modifications.
  • the isolated nucleic acid molecule encodes a 2019-NCOV Spike (S) protein that comprises the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
  • the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter, preferably the CMV immediate early promoter.
  • CMV cytomegalovirus
  • the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.
  • the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 219.
  • the CMV promotor consists of the nucleotide sequence of SEQ ID NO: 219.
  • the invention also provides an isolated 2019-NCOV Spike (S) protein (also referred to as SARS-CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
  • S 2019-NCOV Spike
  • the isolated 2019-NCOV Spike (S) protein comprises the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
  • nucleic acid, protein and/or adenovirus implicates that it has been modified by the hand of man, e.g. in case of an adenovector it has altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e. it is not a naturally occurring wild type adenovirus.
  • the Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. All of these genera contain pathogenic viruses that can infect a wide variety of animals, including birds, cats, dogs, cows, bats, and humans. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses that cause respiratory disease in humans are members of the genus Betacoronavirus. These include SARS-CoV-1, SARS-CoV-2 and MERS, OC43 and HKU1.
  • SARS-CoV-2 can cause severe respiratory disease in humans.
  • a safe and effective SARS-CoV-2 vaccine may be required to end the COVID-19 pandemic.
  • SARS CoV-2 viral spike (S) protein binds to angiotensin-converting enzyme 2 (ACE2), which is the entry receptor utilized by SARS-CoV-2.
  • ACE2 is a type I transmembrane metallocarboxypeptidase with homology to ACE, an enzyme long-known to be a key player in the Renin-Angiotensin system (RAS) and a target for the treatment of hypertension. It is expressed in, inter alia, vascular endothelial cells, the renal tubular epithelium, and in Leydig cells in the testes.
  • PCR analysis revealed that ACE-2 is also expressed in the lung, kidney, and gastrointestinal tract, tissues shown to harbor SARS-CoV-2.
  • the spike (S) protein of coronaviruses is a major surface protein and target for neutralizing antibodies in infected patients (Lester et al., Access Microbiology 2019; 1) and is therefore considered a potential protective antigen for vaccine design.
  • S protein of the SARS-CoV-2 virus several antigen constructs based on the S protein of the SARS-CoV-2 virus were designed. It was surprisingly found that the nucleic acid of the invention (i.e. SEQ ID NO: 211) was superior in immunogenicity when expressed and that adenovectors containing this nucleic acid could be manufactured in high yields.
  • Ad26 vector containing a nucleic acid encoding the SARS CoV- 2 S protein of SEQ ID NO: 205 induced robust neutralizing antibody responses and provided complete protection in bronchoalveolar lavage and or near-complete protection in nasal swabs following SARS-CoV-2 challenge. In addition, as shown in the Examples, it showed a robust single-shot vaccine protection against SARS-CoV-2 in nonhuman primates.
  • the present invention thus provides isolated and/or recombinant nucleic acids encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acids comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 211- 218, or fragments thereof.