EP4118210A1 - Traitement du covid-19 et procédés correspondants - Google Patents

Traitement du covid-19 et procédés correspondants

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Publication number
EP4118210A1
EP4118210A1 EP21768699.7A EP21768699A EP4118210A1 EP 4118210 A1 EP4118210 A1 EP 4118210A1 EP 21768699 A EP21768699 A EP 21768699A EP 4118210 A1 EP4118210 A1 EP 4118210A1
Authority
EP
European Patent Office
Prior art keywords
etsd
sars
protein
recombinant
had5
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21768699.7A
Other languages
German (de)
English (en)
Other versions
EP4118210A4 (fr
Inventor
Patrick Soon-Shiong
Peter Sieling
Annie SHIN
Leonard SENDER
Jeffrey SAFRIT
Adrian RICE
Shahrooz Rabizadeh
Kayvan Niazi
Lise GEISSERT
Elizabeth GABITZSCH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immunitybio Inc
Original Assignee
Immunitybio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/883,263 external-priority patent/US11684668B2/en
Application filed by Immunitybio Inc filed Critical Immunitybio Inc
Priority claimed from PCT/US2021/021737 external-priority patent/WO2021183665A1/fr
Publication of EP4118210A1 publication Critical patent/EP4118210A1/fr
Publication of EP4118210A4 publication Critical patent/EP4118210A4/fr
Pending legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/6031—Proteins
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20071—Demonstrated in vivo effect
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the field of the present disclosure is vaccine compositions and methods to generate immunity against coronaviruses, and particularly as it relates to SARS-CoV-2.
  • a recombinant modified nucleocapsid protein and/or a modified spike protein of a coronavirus induces immunity against the coronavirus in a subject.
  • the recombinant proteins are encoded in a recombinant nucleic acid that can be delivered in a lipid formulation, as part of a recombinant virus, and/or as part of a recombinant yeast or yeast lysate.
  • a recombinant nucleic acid that comprises a first portion encoding a severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein (N) fused to an endosomal targeting sequence (N-ETSD), wherein the first portion is functionally coupled to one or more regulatory elements that enable N-ETSD expression, and a second portion encoding a SARS virus spike protein (S), wherein the second portion is each functionally coupled to one or more regulatory elements that enable S expression.
  • SARS severe acute respiratory syndrome
  • N-ETSD endosomal targeting sequence
  • the SARS virus is SARS-CoV-2
  • the endosomal targeting sequence of the N-ETSD is encoded at a 5’-end and/or a 3’-end of the first portion.
  • the second portion encodes S optimized for surface expression.
  • the first and second portions can be arranged in a bicistronic sequence.
  • the N-ETSD may have an amino acid sequence that has at least 90% identity to amino acid sequence SEQ ID NO: 1.
  • the first portion may, in certain embodiments, have nucleotide sequence SEQ ID NO:2.
  • the S protein or S-fusion protein may have an amino acid sequence that has at least 90% identity to amino acid sequence SEQ ID NO:3 (e.g., SEQ ID NO:3) or at least 90% identity to SEQ ID NO:4 (e.g., SEQ ID NO:4).
  • the second portion may, in certain embodiments, have nucleotide sequence SEQ ID NO:5 or SEQ ID NO:6.
  • the recombinant nucleic acid may further comprise a third portion that encodes a co-stimulatory molecule or an immune stimulatory cytokine.
  • the recombinant nucleic acid may also be integrated into a viral or yeast expression vector (e.g., an adenoviral expression vector having an El gene region deletion and an E2b gene region deletion, and/or a yeast expression vector for Saccharomyces cerevisiae ).
  • the nucleic acid is a deoxyribonucleic acid (DNA).
  • a recombinant replication defective adenovirus is described herin that comprises an El gene deletion, an E2b gene deletion, and a recombinant nucleic acid that includes a first portion encoding a SARS coronavirus N-ETSD.
  • the first portion is functionally coupled to one or more regulatory elements that enable N-ETSD expression.
  • the adenovirus also includes a second portion encoding a SARS S protein.
  • the second portion is each functionally coupled to one or more regulatory elements that enable S expression.
  • the adenovirus may further comprise a third portion encoding a co-stimulatory molecule or an immune stimulatory cytokine, and/or the recombinant adenovirus may have an E3 and/or E4 gene region deletion.
  • a recombinant yeast comprises a recombinant nucleic acid that includes a first portion encoding a SARS coronavirus N-ETSD, wherein the first portion is functionally coupled to one or more regulatory elements that enable N- ETSD expression, and a second portion encoding a SARS S protein, wherein the second portion is each functionally coupled to one or more regulatory elements that enable S expression.
  • the yeast may be S. cerevisiae. In certain embodiments, the yeast may be lysed.
  • a vaccine composition that includes the recombinant nucleic acids presented herein, and the recombinant nucleic acid may be encapsulated in a lipid nanoparticle.
  • a vaccine composition comprises aragonite particles admixed with a recombinant replication defective adenovirus as presented herein, wherein the recombinant replication defective adenovirus is lyophilized.
  • the aragonite particles have an average particle size between 100 nm and 1 mm.
  • FIG.l schematically illustrates various crystalline forms of calcium carbonate.
  • FIG.2 shows three photographs (right to left: 1, 2, 3) of bivalent human adenovirus serotype 5 COVID-Spike and Nucleocapsid antigen vaccine (hAD5-COVID-S/N) in a non-coated aragonite capsule (Sample #6) in 0.1 M hydrochloric acid (HCL) with observed wrinkling, swelling, or a hole in capsule as indicated: 1): 2 minutes post HCL acid exposure; 2): 2 hours post HCL acid exposure; and 3) 2 hours post HCL acid exposure and dried.
  • HCL hydrochloric acid
  • FIG.3 shows three photographs (right to left: 1, 2, 3) ofhAD5-COVID-S/N in a non-coated aragonite capsule (Samples #7 or #8) in 0.1 M HCL with observed swelling, twisting, or a hole in capsule as indicated: 1): Sample #7 at 2 hours post HCL acid exposure; 2): Sample #8 at 2 hours post HCL acid exposure; 3) At 2 hours post HCL acid exposure and dried.
  • FIG.4 shows two photographs (right to left: 1, 2) of hAD5-COVID-S/N in a non-coated lactose capsule (Samples #3 or #4) in 0.1 M HCL with observed swelling of capsule as indicated: 1): Sample #3 at 2 hours post HCL acid exposure; 2): Sample #4 at 2 hours post HCL acid exposure.
  • FIG.5 shows two photographs (right to left: 1, 2) of hAD5-COVID-S/N in a coated aragonite capsule (Samples #1 or #5) in 0.1 M HCL with observed swelling of capsule as indicated: 1): Sample #1 at 2 hours post HCL acid exposure; 2): Sample #5 at 2 hours post HCL acid exposure.
  • FIG.6 shows Infectious Units per gram (IFU/g) (y-axis) for indicated hAD5-COVID-S/N Capsule Type, as indicated.
  • FIG.7 shows the percentage (%) of Virus Recovery (y-axis) for each hAD5-COVID-S/N Capsule Type as indicated.
  • FIG.8 shows IFU/g for each hAD5-COVID-S/N Capsule Type and corresponding pH as indicated.
  • FIG.9 shows the percentage (%) of Virus Recovery for each hAD5-COVID-S/N Capsule Type, as indicated.
  • FIG.10 shows Percent Virus Recovered for each hAD5-COVID-S/N Capsule Type as indicated, with acid treatment indicated for those with shading.
  • FIG.ll shows IFU/g for each hAD5-COVID-S/N Capsule Type as indicated, with acid treatment indicated for those with shading.
  • FIG.12 depicts a conceptual illustration of an ideal vaccine that will elicit durable and effective immunity across multiple pathways.
  • FIG.13 depicts results of an exemplary expression experiment where SARS-CoV2 N is overexpressed in S. cerevisiae.
  • FIG.14 shows ELISA results detecting IgG seroreactivity against SARS-CoV-2 spike in sera samples drawn from immunized macaques.
  • FIG.15 depicts serum inhibiting SARS-CoV-2 infectivity.
  • Panel A shows sera from vaccinated Group 1 macaques inhibiting SARS-CoV-2 infectivity in vitro.
  • Panel B shows sera from vaccinated Group 2 macaques. The dotted line indicates 20% inhibition.
  • FIG.16 depicts non-human primate (NHP) nasopharyngeal viral load over time.
  • Panel A shows viral load (qPCR) in nasal swabs from macaques following SC+SC+oral vaccination.
  • Panel B shows viral load (qPCR) in nasal swabs following SC+oral+oral vaccination.
  • FIG.17 depicts the viral load in NHP over time in the lungs.
  • Panel A shows viral load (qPCR) in BAL from Group 1 macaques following SC+SC+oral vaccination
  • Panel B shows viral load (qPCR) in BAL from Group 1 macaques following SC+oral+oral vaccination.
  • FIG.18 depicts IgG & IgM seroreactivity against SARS-CoV-2 spike in sera samples from human patients immunized with various experimental anti- SARS-CoV-2 vaccines.
  • FIG.19 shows ELISpot results from Thl N-responsive patients.
  • FIG.20 shows ELISpot results from patient 4 (N-unresponsive) and patient 10 (weakly Thl N-responsive).
  • FIG.21 depicts exemplary results for humoral responses and neutralizing capability of sera from hAd5 S-Fusion + N-ETSD vaccinated NHP.
  • Anti-spike IgG levels (ELISA; OD 450nm) are shown for (A) individual SC Oral-Oral NHP along with the (B) geometric mean and (C) inhibition in the surrogate assay.
  • SC-SC-Oral NHP (D) individual anti-S IgG, (E) geometric mean, and (F) inhibition in the surrogate assay. Inhibition above 20% (dashed line) with a sera dilution of 1:30 is correlated with neutralization of SARS-CoV-2 infection.
  • NHP received vaccination on Days 0, 14, & 28 (black arrows).
  • FIG.22 depicts viral load (gRNA) in nasal passages and lung of SC-Oral-Oral and SC-SC- Oral vaccinated NHP post-challenge.
  • A Individual viral gRNA (RT qPCR) and (B) the geometric mean for nasal swab samples; and (C) gRNA and (D) the geometric mean for bronchoalveolar lavage (BAL) samples from SC-Oral-Oral NHP.
  • E Individual gRNA and (F) the geometric mean for nasal swab samples; and (G) gRNA and (H) the geometric means for BAL samples from SC- SC-Oral NHP.
  • SARS-CoV-2 challenge was on Day 56 (black arrows).
  • the level of detection (LOD; dashed line) was 54 gene copies/mL (GC/mL) for gRNA and 119 GC/mL for sgRNA. For values below the LOD, half the LOD value (or 27 GC/mL for gRNA and 59 GC/mL for sgRNA) was used for graphing of individual values and calculation of the geometric mean.
  • FIG.23 depicts viral replication (sgRNA) in nasal passages and lung in SC-Oral-Oral and SC-SC-Oral vaccinated NHP post-challenge.
  • A Individual viral sgRNA (RT qPCR) and (B) the geometric mean for nasal swab samples; and (C) sgRNA and (D) the geometric mean for bronchoalveolar lavage (BAL) samples from SC-Oral-Oral NHP.
  • E Individual sgRNA and (F) the geometric mean for nasal swab samples; and (G) sgRNA and (H) the geometric means for BAL samples from SC-SC-Oral NHP.
  • SARS-CoV-2 challenge was on Day 56 (black arrows).
  • the LOD (dashed line) was 54 GC/mL for gRNA and 119 GC/mL for sgRNA. For values below the LOD, half the LOD value was used for graphing of individual values and calculation of the geometric mean.
  • FIG.24 depicts T-cell responses to vaccination and neutralization capability of sera post- SARS-CoV-2 challenge.
  • IFN-g Interferon-g
  • B interleukin-4
  • IL-4 interleukin-4
  • S spike
  • N nucleocapsid
  • C the ratio IFN-y/IL-4 ratio are shown. Ratios of ‘infinity’ due to undetectable IL-4 are represented as open circles. Cells pulsed with PMA-ionomycin were used as positive controls. Data graphed with mean and SEM.
  • MN50 (dilution factor which SARS-CoV-2 infection of Vero E6 cells is inhibited by 50%) throughout the course of the study is shown; an unpaired, two-tailed Student’s t-test was used to compare MN50 for vaccinated and placebo NHP on Day 70.
  • Nasal gRNA (E) and sgRNA (F) on Days 57, 63, & 70, as well as lung gRNA (G) and sgRNA on Days 57 & 63 are presented.
  • FIG.25 schematically depicts the hAd5 platform and the hAd5 S-Fusion + N-ETSD construct.
  • Panel A shows the human adenovirus serotype 5 vaccine platform with El, E2b, and E3 regions deleted (*). The vaccine construct is inserted in the El regions (arrow).
  • Panel B shows the dual-antigen vaccine comprises both S-Fusion and N-ETSD under control of cytomegalovirus (CMV) promoters and with C-terminal SV40 poly-A sequences delivered by the hAd5 [E1-, E2b- , E3-] platform.
  • CMV cytomegalovirus
  • FIG.26 depicts exemplary constructs for cloning into an adenovirus.
  • FIG.27 is a western blot showing in vitro construct expression and detection of S and N.
  • FIG.28 depicts additional exemplary constructs for cloning into an adenovirus.
  • FIG.29 depicts antibody response to N with a Thl phenotype. Humoral Immune Responses THI VS TH2 associated isotype analysis is shown.
  • FIG.30 depicts cell mediated immunity (CMI) response to N focus phenotype - IFN-g and IL-2 ELISpot.
  • CMI cell mediated immunity
  • FIG.31 depicts enhanced cell surface expression of RBD with S Fusion and with S Fusion+N combination constructs compared to S-WT.
  • FIG.32 depicts antigen recognition by recovered COVID-19 patient plasma.
  • Antigens include RBD-ETSD and fusion S / N-ETSD constructs.
  • FIG.33 depicts the SARS-CoV-2 virus, spike, the hAd5 [E1-, E2b-, E3-] vector and vaccine candidate constructs
  • Trimeric S protein is displayed on the viral surface; the N protein is associated with the viral RNA.
  • RBD is within the SI region, followed by other functional regions, the transmembrane domain (TM) and the C-terminus (CT), which is within the virus
  • TM transmembrane domain
  • CT C-terminus
  • the second-generation human adenovirus serotype 5 (hAd5) vector used has the El, E2b, and E3 regions deleted.
  • FIG.34 depicts HEK293T transfection with hAd5 S-Fusion + ETSD, resulting in enhanced RBD surface expression.
  • Flow cytometric analysis of an anti-RBD antibody with construct- transfected cells reveals no detectable RBD surface expression in either S-WT or (b) S-WT + N- ETSD transfected cells.
  • Surface RBD expression was high for S RBD- ETSD and S RBD-ETSD + N-ETSD (c, d). Expression was low in (e) S-Fusion transfected cells.
  • Cell surface expression of the RBD was high in (1) S-Fusion + N-ETSD transfected cells, particularly at day 1 and 2.
  • No expression was detected the N-ETSD negative control.
  • Y-axis scale is normalized to mode (NM).
  • FIG.35 depicts immunoblot analysis of S expression.
  • Cell surface RBD expression with (a) hAd5 S-WT, S-Fusion, and (c) S-Fusion + N-ETSD in HEK 293T cells shows high correlation with (d) expression of S in immunoblots of HEK 293T cell lysates probed using anti-full length (S2) antibody.
  • Y-axis scale is normalized to mode (NM).
  • FIG.36 depicts binding of recombinant ACE2-Fc HEK293T cell-surface expressed RBD after transfection confirms native protein folding.
  • Flow cytometric analysis of binding between recombinant ACE2- Fc, with which the spike RBD interacts in vivo to initiate infection, and cell- surface antigens expressed after transfection of HEK293T cells with (a) hAd5 S-WT, (b) hAd5 S- Fusion, (c) hAd5 S-Fusion + N-ETSD, (d) hAd5 S RBD-ETSD, or (e) hAd5 S RBD-ETSD + N- ETSD constructs reveals the highest binding is seen for both ACE-Fc and an anti-RBD specific antibody (f-j) after transfection with the bivalent S-Fusion + N-ETSD. Both S RBD-ETSD- containing constructs also showed binding.
  • Y-axis scale is normalized to mode (NM).
  • FIG.37 depicts N expressed from hAd5 N-ETSD is localized to the endosomal/lysosomal compartment.
  • N infected with N-ETSD, (a) N (red) co-localizes with the endosomal marker CD71 (b) as indicated by the arrow in (c).
  • N-ETSD also co- localizes with the lysosomal marker Lampl, whereas (e) N wild type (N-WT) does not, showing instead difluse cytoplasmic distribution.
  • FIG.38 depicts ICS detection of cytokine-expressing splenocytes fromhAd5 S-Fusion + N-
  • FIG.39 depicts anti-spike and anti-nucleocapsid antibody responses in sera from hAd5 S- Fusion + N-ETSD vaccinated mice. Based on absorbance, there was significant production of both (a) anti-S antibodies and (c) anti-nucleocapsid antibodies (b, d) The ng equivalents are shown. Sera diluted 1:30 for anti-spike and 1:90 for anti-nucleocapsid antibodies. Data graphed as the mean and SEM. Statistical analysis was performed using an unpaired two-tailed Student’s t-test where * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001, and **** ⁇ 0.0001.
  • FIG.40 depicts cPass and Vero E6 cell SARS-CoV-2 confirm neutralization by antibodies
  • cPass Assay, inhibition of S RBD interaction with ACE2 was significant at both 1 :20 and 1:60 dilutions of serum from hAd5 S-Fusion + N-ETSD vaccinated mice
  • FIG.41 depicts isotypes for anti-spike and anti-nucleocapsid antibodies.
  • Panels A and C show that IgG2a and IgG2b isotype anti-spike and anti-nucleocapsid antibodies were significantly increased for hAd5 S-Fusion + N- ETSD mice compared to hAd5 Null mice.
  • Panels B and D shows the ng equivalents for antibody isotypes. Data graphed as the mean and SEM. Statistical analysis was performed using an unpaired two-tailed Student’s t-test where * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001, and **** ⁇ 0.0001.
  • FIG.42 depicts ELISpot of secreted cytokines
  • IFN-g secretion by hAd5 S-Fusion + N- ETSD splenocytes was significantly higher than hAd5 Null in response to both S peptide pool 1 and the N peptide pool; but
  • IL-4 was only secreted with hAd5 S-Fusion + N-ETSD in response to the N peptide pool (one high outlier in hAd5 null removed).
  • N 5 mice per group. All data sets graphed as the mean with SEM and all statistics performed using the Mann-Whitney test where * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001, and **** ⁇ 0.0001.
  • FIG.43 depicts ratios for T-cell and humoral responses reveal Thl predominance
  • a The ratio of total Thl (IFN-g) to Th2 (IL-4) spot-forming units is shown for responses to the combined S pools and to the N pool
  • b The Thl/Th2 ratio for antibodies against S and N is shown.
  • the dashed line indicates a ratio of 1 or a balance of Thl and Th2 (no predominance).
  • FIG.44 schematically illustrates thehAd5 vector, SARS-CoV-2, spike, and constructs.
  • A The human adenovirus serotype 5 with El, E2b, and E3 regions deleted (hAd5 [E1-, E2b-, E3-]) is shown.
  • B The SARS-CoV-2 virus displays spike (S) protein as atrimer on the viral surface.
  • S protein comprises the N-terminal (NT), the SI region including the RBD, the S2 and TM regions, and the C-terminal (CT); other function regions not labeled.
  • C Spike wild type (SWT), (D) spike fusion (S-Fusion), (E) nucleocapsid without ETSD and predominantly cytoplasmic localization (N); (F) N with the Enhanced T-Cell Stimulation Domain (N-ETSD), and (G) the bivalent S- Fusion + N-ETSD constructs are shown.
  • FIG.45 depicts photomicrographs establishing that N-ETSD localizes to endosomes, lysosomes, and autophagosomes.
  • MoDCs were infected with Ad5 N-ETSD or N without ETSD and were co-labeled with anti -flag (N, N-ETSD here have a flag tag) and anti-CD71 (endosomal marker), anti-Lamp 1 (lysosomal marker), or anti-LC3a/b antibodies.
  • N-ETSD N-ETSD
  • B CD71
  • C overlay
  • D N,
  • E CD71, and
  • F overlay.
  • G NETSD,
  • H Lamp-1
  • I overlay.
  • N N
  • K Lamp-1
  • L overlay
  • M N-ETSD
  • N/N-ETSD is red, other markers green, co-localization indicated by yellow arrows, and white arrows indicate lymphocytes.
  • FIG.46 demonstrates that patient plasma antibodies recognize SARS-CoV-2 antigens expressed by MoDCs after hAd5 S-Fusion + N-ETSD infection.
  • A MoDCs from two normal individuals were infected with hAd5 vaccines overnight, then exposed to previously infected patient plasma from a single individual; antibody binding to the DC cell surface was detected by flow cytometry. The flow histograms for hAd5 S-WT, S-Fusion, S-Fusion + N-ETSD, and Null are shown for MoDCs from two sources, (B) MoDCl and (C) MoDC2.
  • FIG.47 depicts exemplary results for T cell responses (of previously SARS-CoV-2 infected patient and virus-naive T-cell) to MoDCs pulsed with SARS-CoV-2 peptides.
  • T cells from all four previously SARS-CoV-2 infected patients (Pt) show significant IFN-g responses to SI, S2, and N peptide pool-pulsed MoDCs as compared to ‘none’.
  • T cells from virus-naive (unexposed, UnEx) control individuals showed far lower responses.
  • FIG.48 establishes that peptide-pulsed MoDCs from patients previously infected with SARS-CoV-2 stimulate autologous patient T cells to secrete IFN-g.
  • A MoDCs derived from previously infected SARSCoV-2 patients Pt4 and Pt 3 were pulsed with SARS-CoV-2 peptide mixes (SI, S2 or N) overnight and then incubated with autologous CD4+ (A, B) or CD8+ (C, D) T cells. IFN-g levels were determined by ELISpot.
  • FIG.49 demonstrates that IFN-g secretion by T cells from previously SARS-CoV-2 infected patients is greater in response to MoDC expression of N-ETSD compared to N.
  • A Experimental design.
  • B-D Secretion of IFN-g by autologous CD3+ T cells in response to hAd5- N-ETSD- and hAd5 Nexpressing MoDCs is shown.
  • E-G Secretion of IL-4 by CD3+ cells in response to infected MoDCs is shown with the same scales as IFN-g for each.
  • FIG.50 shows that previously SARS-CoV-2 infected patient T-cell responses to the bivalent vaccine and its individual components reveal distinct antigen specificity of T-cell populations.
  • A-C CD3+ T cell IFN-g responses for three patients.
  • D-F CD3+ T cell IL-4 responses.
  • G-I CD4+ IFN-g responses.
  • J-L CD8+ IFN-g responses.
  • RNA-based vaccines include intramuscular, subcutaneous, oral, and mucosal routes (alone or in combination), and may even be used as oral boost after currently known RNA-based vaccines.
  • a recombinant construct comprises a modified nucleocapsid protein and/or a modified spike protein.
  • the modified nucleocapsid protein comprises a trafficking sequence to so route the modified nucleocapsid protein to the endosomal/lysosomal subcellular compartments, thereby taking advantage of a key antigen presentation pathway to stimulate CD4+ T cells which in turn license dendritic cells to activate naive CD8+ cytotoxic T cells.
  • the modified spike protein has a modification that enhances surface expression of the modified spike protein to thereby render an immune response more robust against the spike antigen.
  • any antigen can be profitably redirected to the endosomal/lysosomal subcellular compartment.
  • Exemplary antigens to be tagged with ETSD for use in this manner in adenoviral or yeast vaccine vectors include CEA, human epidermal growth factor receptor 1 (HER1), HER2/neu, HER3, HER4, prostate-specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE- A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyuiy (TIVS7-2, polymorphism), Brachyury (IVS7 T/
  • N-ETSD refers to a modified nucleocapsid protein of the SARS-CoV-2 virus that includes an endosomal targeting sequence.
  • An exemplary N-ETSD has an amino acid sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO:2.
  • S-HA or “Spike” or “S” refers to a spike protein of the SARS-CoV-2 virus that has an HA tag.
  • An exemplary S-HA has an amino acid sequence of SEQ ID NO:3 and a nucleotide sequence of SEQ ID NO:5.
  • S-Fusion or “spike-fusion” refers to a modified spike protein of the SARS-CoV-2 virus that has increased surface expression.
  • An exemplary S-Fusion has an amino acid sequence of SEQ ID NO:4 and a nucleotide sequence of SEQ ID NO:6.
  • N or “N-wt” or “nucleocapsid” refers to the nucleocapsid protein of the SARS-CoV-2 virus.
  • An exemplary N protein has an amino acid sequence of SEQ ID NO:7.
  • ETSD refers to an endosomal targeting sequence.
  • An exemplary ETSD has an amino acid sequence of SEQ ID NO: 8.
  • ACE2 refers to the Angiotensin-converting enzyme 2.
  • An exemplary (human) ACE2 has an amino acid sequence of SEQ ID NO:9.
  • Soluble ACE2 protein refers to a mutant and truncated form of ACEs that is soluble under physiological conditions.
  • An exemplary soluble ACEs has an amino acid sequence of SEQ ID NO: 10.
  • viruses and yeasts are recombinant viruses and yeasts.
  • the viruses and yeasts disclosed herein may be useful for a variety of purposes, such as treating and/or preventing a coronavirus disease.
  • a replication defective adenovirus wherein the adenovirus comprises an El gene region deletion; an E2b gene region deletion; an E3 gene region deletion, a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein, a CoV2 nucleocapsid protein fused to an endosomal targeting sequence (N-ETSD), and a nucleic acid encoding a CoV2 spike protein sequence optimized for cell surface expression (S- Fusion).
  • CoV2 coronavirus 2
  • N-ETSD endosomal targeting sequence
  • S- Fusion a nucleic acid encoding a CoV2 spike protein sequence optimized for cell surface expression
  • the N-ETSD polypeptide may comprises a sequence with at least 80% identity to SEQ ID NO:l.
  • the identity value is at least 85%.
  • the identity value is at least 90%.
  • the identity value is at least 95%.
  • the identity value is at least 99%.
  • the identity value is 100%.
  • the N-ETSD fusion protein contains a linker between the N-ETSD domain and the nucleocapsid protein.
  • this linker may be a 16 amino acid linker having the sequence ((3 ⁇ 4S)4.
  • methods for enhancing the immunogenicity of an intracellular antigen, the methods comprising tagging the antigen with ETSD and expressing the tagged antigen in an antigen-presenting cell (e.g., a dendritic cell).
  • an antigen-presenting cell e.g., a dendritic cell
  • the fusion protein comprising N-ETSD and CoV-2 nucleocapsid protein may be encoded by a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2.
  • the identity value is at least 85%.
  • the identity value is at least 90%.
  • the identity value is at least 95%.
  • the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the CoV-2 spike protein is contemplated to have at least 85% identity to SEQ ID NO:3. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the nucleic acid encoding the CoV-2 spike protein has at least 85% identity to SEQ ID NO:5. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the CoV-2 spike fusion protein is contemplated to have at least 85% identity to SEQ ID NO:4. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the nucleic acid encoding the CoV-2 spike fusion protein has at least 85% identity to SEQ ID NO:6. In some embodiments, the identity value is at least 85%. In some embodiments, the identity value is at least 90%. In some embodiments, the identity value is at least 95%. In some embodiments, the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • a recombinant yeast comprising a nucleic acid encoding a protein selected from the group consisting of a coronavirus 2 (CoV-2) nucleocapsid protein, a CoV2 N-ETSD protein, a CoV2 spike protein, a CoV2 spike-fusion protein, and a combination thereof.
  • a protein selected from the group consisting of a coronavirus 2 (CoV-2) nucleocapsid protein, a CoV2 N-ETSD protein, a CoV2 spike protein, a CoV2 spike-fusion protein, and a combination thereof.
  • each of these encoded proteins may be further modified as described in more detail below.
  • the recombinant yeast is Saccharomyces cerevisiae.
  • the CoV-2 nucleocapsid protein or variant thereof comprises a sequence with at least 80% identity to SEQ ID NO:l or SEQ ID NO:7.
  • the identity value is at least 85%.
  • the identity value is at least 90%.
  • the identity value is at least 95%.
  • the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the CoV-2 spike protein or spike fusion protein comprises a sequence with at least 80% identity to SEQ ID NO:3 or SEQ ID NO:4.
  • the identity value is at least 85%.
  • the identity value is at least 90%.
  • the identity value is at least 95%.
  • the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the nucleic acid encoding the CoV-2 spike protein or spike fusion protein comprises a sequence with at least 80% identity to SEQ ID NO:5 or SEQ ID NO:6.
  • the identity value is at least 85%.
  • the identity value is at least 90%.
  • the identity value is at least 95%.
  • the identity value is at least 99%. In some embodiments, the identity value is 100%.
  • the adenoviruses and yeasts disclosed herein may further comprise a nucleic acid encoding a tralfi eking sequence, a co-stimulatory molecule, and/or an immune stimulatory cytokine.
  • the co-stimulatory molecule is selected from the group consisting of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1 A, ICAM-1, and LFA3.
  • the immune stimulatory cytokine may be selected from the group consisting of IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, and LMP1. Additionally or alternatively, the vaccines disclosed herein may also encode SARS-CoV-2 M protein, with or without an ETSD tag. Additionally or alternatively, the adenovirus and/or yeast may be administered in combination with one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1).
  • immune stimulatory cytokines e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1.
  • the immune stimulatory cytokine(s) is/are administered within 24 hrs of the adenovirus and/or yeast. That is to say, the adenovirus and/or yeast may be administered to a patient (e.g., a patient over 50 years of age), and then within the following 24 hrs, one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1) may be administered to the same patient.
  • IL-2, IL-12, IL-15 e.g., IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1
  • one or more immune stimulatory cytokines may be administered to a patient (e.g., a patient over 50 years of age), and then within the following 24 hrs, the adenovirus and/or yeast may be administered to the same patient.
  • a patient e.g., a patient over 50 years of age
  • the adenovirus and/or yeast may be administered to the same patient.
  • adenovirus and/or yeast as described herein in combination with one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1) may be particularly useful in elderly patients.
  • immune stimulatory cytokines e.g., IL-2, IL-12, IL-15, nogapendekin alfa-imbakicept, IL-21, IPS1, & LMP1
  • yielderly coveys patients over 50 years of age, e.g., patients over 55 years of age, over 60 years of age, over 65 years of age, over 70 years of age, over 75 years of age, over 80 years of age, over 85 years of age, or over 90 years of age.
  • the recombinant virus is administered via subcutaneous or subdermal injection.
  • administration may also be intravenous injection.
  • antigen presenting cells may be isolated or grown from cells of the patient, infected in vitro, and then transfused to the patient.
  • the composition is formulated in a pharmaceutically acceptable excipient suitable for administration to a subject.
  • the recombinant viruses and yeasts contemplated herein may further comprises a sequence that encodes at least one of a co-stimulatory molecule, an immune stimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition.
  • suitable co-stimulatory molecules include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1 A, ICAM-1, and/or LFA3, while suitable immune stimulatory cytokine include IL-2, IL-12, IL-15, IL-15 super agonist (N803), IL-21, IPS1, and/or LMP1, and/or suitable proteins that interfere include antibodies against or antagonists of CTLA-4, PD-1, TIM1 receptor, 2B4, and/or CD160.
  • suitable immune stimulatory cytokine include IL-2, IL-12, IL-15, IL-15 super agonist (N803), IL-21, IPS1, and/or LMP1, and/or suitable proteins that interfere include antibodies against or antagonists of CTLA-4, PD-1, TIM1 receptor, 2B4, and/or
  • the immunotherapeutic compositions disclosed herein may be either “prophylactic” or “therapeutic”.
  • the compositions of the present disclosure are provided in advance of the development of, or the detection of the development of, a coronavirus disease, with the goal of preventing, inhibiting or delaying the development of the coronavirus disease; and/or generally preventing or inhibiting progression of the coronavirus disease in an individual. Therefore, prophylactic compositions can be administered to individuals that appear to be coronavirus disease free (healthy, or normal, individuals), or to individuals who has not yet been detected of coronavirus. Individuals who are at high risk for developing a coronavirus disease, may be treated prophylactically with a composition of the instant disclosure.
  • the immunotherapy compositions are provided to an individual who is diagnosed with a coronavirus disease, with the goal of ameliorating or curing the coronavirus disease; increasing survival of the individual; preventing, inhibiting, reversing or delaying development of coronavirus disease in the individual.
  • a vaccine composition comprising the adenovirus or yeast as disclosed above, and wherein the composition is formulated for injection.
  • the vaccine composition may be used for inducing immunity against CoV-2 in a patient in need thereof, by administering to the patient the vaccine composition.
  • the method includes using a viral or yeast vector that encodes the wild-type or modified form of a nucleocapsid protein and/or the wild-type or modified form of a spike protein of the coronavirus in an immunogenic composition that is administered to a subject individual.
  • the virus and/or yeast vaccine thus administered, would infect the individual with CoV-2 the wild-type or modified form of the nucleocapsid or spike protein. With that in place, the individual would have an immune response against it, and be vaccinated.
  • the nucleocapsid protein and the spike protein are relatively conserved polypeptides, immune responses can be elicited for a variety of members of the coronavirus family.
  • the adenoviral vector may be modified to encode the wild-type or modified form of the nucleocapsid protein, and/or spike protein.
  • the yeast vector may also be modified to encode the wild-type or modified form of the nucleocapsid protein, and/or the spike protein.
  • positive immune responses were obtained on cell mediated immunity upon administration of immunogenic compositions comprising the viral and/or yeast vectors in patients in need thereof.
  • the present disclosure contemplates creating the coronaviral spikes to be expressed on the yeast surface.
  • the yeast is acting as an avatar coronavirus to stimulate B cells, which then results in humoral immunity.
  • hAd5 next generation bivalent human adenovirus serotype 5
  • hAd5 next generation bivalent human adenovirus serotype 5
  • S- Fusion S sequence optimized for cell surface expression
  • N conserved nucleocapsid
  • the next generation bivalent hAd5 S-Fusion+N-ETSD vaccine provides robust, durable cell-mediated and humoral immunity against SARS-CoV-2 infection.
  • the vaccine construct may be administered orally, intranasally, or sublingually.
  • the instant disclosure also provides beyond injectable formulations ( e.g .
  • the COVID-19 vaccine disclosed herein generates long-term T and B cell memory.
  • Coronaviruses are found in avian and mammalian species. They resemble each other in morphology and chemical structure: for example, the coronaviruses of humans and cattle are antigenically related. There is no evidence, however, that human coronaviruses can be transmitted by animals. In animals, various coronaviruses invade many different tissues and cause a variety of diseases in humans. One such disease was Severe acute respiratory syndrome (SARS) coronavirus disease that spread to several countries in Asia, Europe and North America in late 2002/early 2003. Another such disease is the novel Coronvirus Disease of 2019 (COVID 19) that has spread to several countries in the world.
  • SARS Severe acute respiratory syndrome
  • COVID 19 novel Coronvirus Disease of 2019
  • SARS-CoV-2 SARS-coronavirus 2
  • COVID- 19 The disease it causes is referred to as COVID- 19 and has rapidly become a worldwide pandemic that has disrupted socioeconomic life and resulted in more than 32 million infections and more than 1,100,000 deaths worldwide as of late October 2020.
  • COVID 19 usually begins with a fever greater than 38° C. Initial symptoms can also include cough, sore throat, malaise and mild respiratory symptoms. Within two days to a week, patients may have trouble breathing. Patients in more advanced stages of COVID 19 develop either pneumonia or respiratory distress syndrome. Public health interventions, such as surveillance, travel restrictions and quarantines, are being used to contain the spread of COVID 19. It is unknown, however, whether these draconian containment measures can be sustained with each appearance of the COVID 19 in humans. Furthermore, the potential of this new and sometimes lethal CoV as a bio-terrorism threat is obvious.
  • Coronavirus virions are spherical to pleomorphic enveloped particles.
  • the envelope is studded with projecting glycoproteins, and surrounds a core consisting of matrix protein enclosed within which is a single strand of positive-sense RNA (Mr 6 c 10 6 ) associated with nucleocapsid protein.
  • Mr 6 c 10 6 positive-sense RNA
  • nucleocapsid protein a single strand of positive-sense RNA
  • N coronavirus nucleocapsid
  • the coronavirus nucleocapsid (N) is a structural protein found in all coronaviruses, including COVID 19.
  • the nucleocapsid protein forms complexes with genomic RNA, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly.
  • coronavirus virions Another protein found throughout all coronavirus virions is the viral spike (S) protein.
  • Coronaviruses are large positive-stranded RNA viruses typically with a broad host range. Like other enveloped viruses, CoV enter target cells by fusion between the viral and cellular membranes, and that process is mediated by the viral spike (S) protein.
  • SARS-CoV-2 is an enveloped positive sense, single-strand RNA b coronavirus primarily composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope, as well as the viral membrane and genomic RNA. Of these, S is the largest and N the most prevalent. The S glycoprotein is displayed as a trimer on the viral surface (FIG.33, Panel A), whereas N is located within the viral particle. A schematic of the S primary structure is shown in FIG.33, Panel B. The sequence of SARS-CoV-2 was published and compared to that of previous coronaviruses. This was soon followed by reports on the crystal structure of the S protein.
  • the virus uses the S protein to enter host cells by interaction of the S receptor binding domain (S RBD) with angiotensin- converting enzyme 2 (ACE2), an enzyme expressed on a variety of cell types in the nose, mouth, gut, and lungs, as well as other organs, and importantly on the alveolar epithelial cells of the lung where infection is predominantly manifested.
  • S RBD S receptor binding domain
  • ACE2 angiotensin- converting enzyme 2
  • the S RBD is found within the SI region of the spike polypeptide.
  • the present disclosure provides a vaccine formulation comprising a recombinant entity, wherein the recombinant entity comprises a nucleic acid that encodes a nucleocapsid protein of coronavirus 2 (CoV2) or modified form thereof and/or wherein the recombinant entity encodes a spike protein of CoV2 or modified form thereof.
  • the vaccine formulation may be useful for treating a disease, such as a coronavirus mediated disease or infection.
  • a method for treating a coronavirus disease is contemplated for a patient in need thereof.
  • Such method will preferably include a step of administering to the subject an immunotherapy composition comprising a recombinant entity, wherein the recombinant entity comprises a nucleic acid that encodes a nucleocapsid protein of coronavirus 2 (CoV2) or a modified form thereof and/or a nucleic acid that encodes a spike protein of coronavirus 2 (CoV2) or a modified form thereof.
  • the coronavirus contemplated herein may be coronavirus disease 2019 (COVID-19) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
  • the present disclosure provides a method for treating coronavirus disease 2019 (COVID-19) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in a patient in need thereof, comprising: administering to the subject a first immunotherapy composition comprising a recombinant virus, wherein the recombinant virus comprises a nucleic acid that encodes a nucleocapsid protein of coronavirus 2 (CoV2) or modified form thereof, administering to the subject a second immunotherapy composition comprising a recombinant yeast, wherein the recombinant yeast comprises a nucleic acid that encodes a spike protein of CoV2.
  • the first and second immunotherapy compositions may be administered concurrently or sequentially to the patient.
  • a viral vector e.g., recombinant adenovirus genome, optionally with a deleted or non-functional E2b gene
  • a nucleic acid that encodes (a) at least a nucleocapsid protein or modified form thereof; and (b) at least one spike protein or modified form thereof.
  • the viral vector may further encode one or more co-stimulatory molecules.
  • the nucleic acid will also include a trafficking signal to direct a peptide product encoded by the nucleic acid to the cytoplasm, the endosomal compartment, or the lysosomal compartment, and the peptide product may also comprise a sequence portion that enhances intracellular turnover of the peptide product.
  • RBD as a key antigen was recently confirmed, and it was reported that in 44 hospitalized COVID-19 patients, RBD-specific IgG responses and neutralizing antibody titers are detectable in all patients by 6 days post-PCR confirmation of infection, and that the two are correlated. In addition to humoral responses, S epitopes are also frequent targets of COVID- 19 recovered patient T cells, providing further justification for inclusion of S in prophylactic immunization strategies.
  • N protein is a highly conserved and antigenic SARS-CoV- 2-associated protein that has been studied previously as an antigen in coronavirus vaccine design for SARS-CoV. N associates with viral RNA within the virus and has a role in viral RNA replication, virus particle assembly, and release. SARS-CoV-2 N is a highly antigenic protein and nearly all patients infected with SARS-CoV-2 have antibody responses to N. Furthermore, another study reported that most, if not all, COVID-19 survivors tested were shown to have N-specific CD4+ T-cell responses.
  • T-cell responses were predominantly to N, and it has been reported that in all 36 convalescent COVID-19 patients in their study, the presence of CD4+ and CD8+ T cells recognizing multiple regions of the N protein could be demonstrated.
  • These findings emphasize the importance of designing a vaccine with the highly conserved nucleocapsid present in both SARS-CoV and SARS-CoV-2.
  • recovered patients exposed to SARS-CoV-2 have been found without seroconversion, but with evidence of T-cell responses.
  • the T-cell based responses become even more critical given the finding in at least one study that neutralizing antibody titers decline in some COVID-19 patients after about 3 months.
  • the vaccines disclosed herein result in the generation of T-cell in addition to humoral responses.
  • a bivalent vaccine comprising many antigens, S RBD as displayed by inclusion of full-length S including SD1, SI and S2 epitopes, along with N, was contemplated and shown to be more effective in eliciting both T-cell and antibody-based responses than a construct with either antigen alone by presenting both unique and conserved SARS-CoV-2 antigenic sites to the immune system.
  • the importance of both S and N was highlighted by identifying that both S and N antigens as a priori potential B and T-cell epitopes for the SARS- CoV virus show close similarity to SARS-CoV-2 that are predicted to induce both T and B cell responses.
  • an additional consideration for design of an effective vaccine is the likelihood of antigen presentation on the surface of a recombinant protein-expressing cell, and expression in a conformation that recapitulates natural virus infection.
  • wild type N does not have a signaling domain that directs it to endosomal processing and ultimately MHC class II complex presentation to CD4+ T cells
  • the wild type N sequence is not optimal for induction of a vigorous CD4+ T-cell responses, a necessity for both cell-mediated and B cell memory.
  • an Enhanced T-cell Stimulation Domain (ETSD) to N allows the necessary processing and presentation.
  • One preferred ETSD polypeptide has an amino acid sequence of SEQ ID NO: 8. Of course, it should be appreciated that the sequence can be modified while maintaining the desired activity.
  • an ETSD sequence may have at least 85%, or at least 90%, or at least 95, or at least 98% identity to SEQ ID NO: 8.
  • an optimized the wild type S protein into an ‘S-Fusion’ sequence increases the likelihood of native folding, increased stability, and proper cell surface expression of the RBD.
  • the vaccine construct comprises an S-Fusion sequence and an N-ETSD sequence.
  • the vaccine platform utilized here is a next-generation recombinant human adenovirus serotype 5 (hAd5) vector with deletions in the El, E2b, and E3 gene regions (hAd5 [E1-, E2b-, E3-]).
  • This hAd5 [E1-, E2b-, E3-] vector (FIG.33, Panel C) is primarily distinguished from other first- generation [E1-, E3-] recombinant Ad5 platforms by having additional deletions in the early gene 2b (E2b) region that remove the expression of the viral DNA polymerase (pol) and in pre terminal protein (pTP) genes, and its propagation in the E.C7 human cell line.
  • the vector has an expanded gene-carrying/cloning capacity compared to the first generation Ad5 [El- , E3-] vectors.
  • Ad5 Ad5
  • This next generation hAd5 [E1-, E2b-, E3-] vaccine platform in contrast to Ad5 [E1-, E3-] -based platforms, does not promote activities that suppress innate immune signaling, thereby allowing for improved vaccine efficacy and a superior safety profile independent of previous Ad immunity.
  • this platform enables relatively long- term antigen expression without significant induction of anti-vector immunity. It is therefore also possible to use the same vector/construct for homologous prime-boost therapeutic regimens unlike first-generation Ad platforms which face the limitations of pre-existing and vaccine-induced Ad immunity. Importantly, this next generation Ad vector has demonstrated safety in over 125 patients with solid tumors. In these Phase I/II studies, CD4+ and CD8+ antigen-specific T cells were successfully generated to multiple somatic antigens (CEA, MUC1, brachyury) even in the presence of pre existing Ad immunity.
  • CEA somatic antigens
  • the instant disclosure provides findings of confirmed enhanced cell-surface expression and physiologically relevant folding of the expressed S RBD from S-Fusion by ACE2-Fc binding.
  • the N-ETSD protein was successfully localized to the endosomal/lysosomal subcellular compartment for MHC presentation and consequently generated both CD4+ and CD8+ T-cell responses.
  • Immunization of CD-I mice with the hAd5 S Fusion + N-ETSD vaccine elicited both humoral and cell-mediated immune responses to vaccine antigens.
  • CD8+ and CD4+ T-cell responses were noted for both S and N.
  • Statistically significant IgG responses were seen for antibody generation against S and N.
  • recombinant viruses it is contemplated that all known manners of making recombinant viruses are deemed suitable for use herein, however, especially preferred viruses are those already established in therapy, including adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, etc. Among other appropriate choices, adenoviruses are particularly preferred.
  • the virus is a replication deficient and non-immunogenic virus.
  • suitable viruses include genetically modified alphaviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.
  • adenoviruses are particularly preferred.
  • genetically modified replication defective adenoviruses are preferred that are suitable not only for multiple vaccinations but also vaccinations in individuals with preexisting immunity to the adenovirus (see e.g., WO 2009/006479 and WO 2014/031178, which are incorporated by reference in its entirety).
  • the replication defective adenovirus vector comprises a replication defective adenovirus 5 vector.
  • the replication defective adenovirus vector comprises a deletion in the E2b region. In some embodiments, the replication defective adenovirus vector further comprises a deletion in the El region. In that regard, it should be noted that deletion of the E2b gene and other late proteins in the genetically modified replication defective adenovirus to reduce immunogenicity. Moreover, due to these specific deletions, such genetically modified viruses were replication deficient and allowed for relatively large recombinant cargo.
  • WO 2014/031178 describes the use of such genetically modified viruses to express CEA (colorectal embryonic antigen) to provide an immune reaction against colon cancer.
  • CEA colonal embryonic antigen
  • relatively high titers of recombinant viruses can be achieved using genetically modified human 293 cells as has been reported (e.g., J Virol. 1998 Feb; 72(2): 926-933).
  • Ad5 [El -] are constructed such that a trans gene replaces only the El region of genes. Typically, about 90% of the wild-type Ad5 genome is retained in the vector.
  • Ad5 [E1-] vectors have a decreased ability to replicate and cannot produce infectious virus after infection of cells not expressing the Ad5 El genes.
  • the recombinant Ad5 [E1-] vectors are propagated in human cells allowing for Ad5 [E1-] vector replication and packaging.
  • Ad5 [E1-] vectors have a number of positive attributes; one of the most important is their relative ease for scale up and cGMP production.
  • Ad5 [E1-] vectors with more than two thousand subjects given the virus sc, im, or iv. Additionally, Ad5 vectors do not integrate; their genomes remain episomal. Generally, for vectors that do not integrate into the host genome, the risk for insertional mutagenesis and/or germ-line transmission is extremely low if at all. Conventional Ad5 [E1-] vectors have a carrying capacity that approaches 7kb.
  • Ad5 -based vectors One obstacle to the use of first generation (El -deleted) Ad5 -based vectors is the high frequency of pre-existing anti-adeno virus type 5 neutralizing antibodies. Attempts to overcome this immunity is described in WO 2014/031178, which is incorporated by reference herein. Specifically, a novel recombinant Ad5 platform has been described with deletions in the early 1 (El) gene region and additional deletions in the early 2b (E2b) gene region (Ad5 [E1-, E2b-]). Deletion of the E2b region (that encodes DNA polymerase and the pre-terminal protein) results in decreased viral DNA replication and late phase viral protein expression. E2b deleted adenovirus vectors provide an improved Ad-based vector that is safer, more effective, and more versatile than First Generation adenovirus vectors.
  • the adenovirus vectors contemplated for use in the present disclosure include adenovirus vectors that have a deletion in the E2b region of the Ad genome and, optionally, deletions in the El, E3 and, also optionally, partial or complete removal of the E4 regions.
  • the adenovirus vectors for use herein have the El and/or the preterminal protein functions of the E2b region deleted. In some cases, such vectors have no other deletions.
  • the adenovirus vectors for use herein have the El, DNA polymerase and/or the preterminal protein functions deleted.
  • E2b deleted refers to a specific DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one E2b gene product.
  • E2b deleted is used in relation to a specific DNA sequence that is deleted (removed) from the Ad genome.
  • E2b deleted or "containing a deletion within the E2b region” refers to a deletion of at least one base pair within the E2b region of the Ad genome.
  • more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted.
  • the deletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within the E2b region of the Ad genome.
  • An E2b deletion may be a deletion that prevents expression and/or function of at least one E2b gene product and therefore, encompasses deletions within exons of encoding portions of E2b-specific proteins as well as deletions within promoter and leader sequences.
  • an E2b deletion is a deletion that prevents expression and/or function of one or both of the DNA polymerase and the preterminal protein of the E2b region.
  • E2b deleted refers to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non functional. Such mutations include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein.
  • compositions and methods presented are not only suitable for directing virally expressed antigens specifically to one or another (or both) MHC systems, but will also provide increased stimulatory effect on the CD8+ and/or CD4+ cells via inclusion of various co-stimulatory molecules (e.g., ICAM-1 (CD54), ICOS-L, LFA-3 (CD58), and at least one of B7.1 (CD80) and B7.2 (CD86)), and via secretion or membrane bound presentation of checkpoint inhibitors.
  • co-stimulatory molecules e.g., ICAM-1 (CD54), ICOS-L, LFA-3 (CD58), and at least one of B7.1 (CD80) and B7.2 (CD86)
  • the virus may be used to infect patient (or non-patient) cells ex vivo or in vivo.
  • the virus may be injected subcutaneously or intravenously, or may be administered intranasally or via inhalation to so infect the patient’s cells, and especially antigen presenting cells.
  • immune competent cells e.g., NK cells, T cells, macrophages, dendritic cells, etc.
  • the patient or from an allogeneic source
  • immune therapy need not rely on a virus but may be effected with nucleic acid transfection or vaccination using RNA or DNA, or other recombinant vector that leads to the expression of the neoepitopes (e.g., as single peptides, tandem mini-gene, etc.) in desired cells, and especially immune competent cells.
  • nucleic acids will typically be delivered in association with a lipid formulation to protect the nucleic acid from degradation and to facilitate uptake of the nucleic acid into a target cell.
  • suitable promoter elements include constitutive strong promoters (e.g., SV40, CMV, UBC, EF1 A, PGK, CAGG promoter), but inducible promoters are also deemed suitable for use herein, particularly where induction conditions are typical for a tumor microenvironment.
  • inducible promoters include those sensitive to hypoxia and promoters that are sensitive to TGF-b or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive elements promoter).
  • suitable inducible promoters include the tetracycline-inducible promoter, the myxovirus resistance 1 (Mxl) promoter, etc.
  • the replication defective adenovirus comprising an El gene region deletion, an E2b gene region deletion, and a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid protein and/or a CoV2 spike protein, as disclosed herein may be administered to a patient in need for inducing immunity against CoV2.
  • Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage may vary from individual to individual, and the severity of the disease, and may be readily established using standard techniques.
  • the administration comprises delivering 4.8-5.2 x 10 11 replication defective adenovirus particles, or 4.9-5.1 x 10 11 replication defective adenovirus particles, or 4.95-5.05 x 10 11 replication defective adenovirus particles, or 4.99-5.01 x 10 11 replication defective adenovirus particles.
  • the administration of the virus particles can be through a variety of suitable paths for delivery.
  • One preferred route contemplated herein is by injection, such as intracutaneous injection, intramuscular injection, intravenous injection or subcutaneous injection. In some embodiments, a subcutaneous delivery may be preferred.
  • yeast expression and vaccination systems it is contemplated that all known yeast strains are deemed suitable for use herein.
  • the yeast is a recombinant Saccharomyces strain that is genetically modified with a nucleic acid construct encoding a protein selected from the group consisting of coronavirus 2 (CoV2) nucleocapsid protein, CoV2 spike protein, and a combination thereof, to thereby initiate an immune response against the CoV2 viral disease.
  • the yeast vehicle is a whole yeast.
  • the whole yeast in one aspect is killed.
  • the whole yeast is heat inactivated.
  • the yeast is a whole, heat-inactivated yeast from Saccharomyces cerevisiae.
  • yeast compositions for treatment of chronic hepatitis B infections.
  • any yeast strain can be used to produce a yeast vehicle of the present disclosure.
  • Yeasts are unicellular microorganisms that belong to one of three classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti.
  • One consideration for the selection of a type of yeast for use as an immune modulator is the pathogenicity of the yeast.
  • the yeast is a non-pathogenic strain such as Saccharomyces cerevisiae as non-pathogenic yeast strains minimize any adverse effects to the individual to whom the yeast vehicle is administered.
  • pathogenic yeast may also be used if the pathogenicity of the yeast can be negated using pharmaceutical intervention.
  • yeast strains include Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia.
  • yeast genera are selected from Saccharomyces, Candida, Hansenula, Pichia or Schizosaccharomyces, and in a preferred aspect, Saccharomyces is used.
  • yeast strains that may be used include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, and Yarrowia lipolytica.
  • yeast species used in the instant disclosure include S. cerevisiae, C. albicans, H. polymorpha, P. pastoris and S. pombe.
  • S. cerevisiae is useful due to it being relatively easy to manipulate and being "Generally Recognized As Safe” or "GRAS" for use as food additives (GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997). Therefore, particularly contemplated herein is a yeast strain that is capable of replicating plasmids to a particularly high copy number, such as a S.
  • the S. cerevisiae strain is one such strain that is capable of supporting expression vectors that allow one or more target antigen(s) and/or antigen fusion protein(s) and/or other proteins to be expressed at high levels.
  • any mutant yeast strains can be used, including those that exhibit reduced post-translational modifications of expressed target antigens or other proteins, such as mutations in the enzymes that extend N-linked glycosylation.
  • nucleic acid molecule encoding at least one protein is inserted into an expression vector such manner that the nucleic acid molecule is operatively linked to a transcription control sequence to be capable of eflecting either constitutive or regulated expression of the nucleic acid molecule when transformed into a host yeast cell.
  • nucleic acid molecules encoding one or more proteins can be on one or more expression vectors operatively linked to one or more expression control sequences.
  • Particularly important expression control sequences are those which control transcription initiation, such as promoter and upstream activation sequences.
  • Promoters for expression in Saccharomyces cerevisiae include promoters of genes encoding the following yeast proteins: alcohol dehydrogenase I (ADHl) or II (ADH2), CUP1, phosphogly cerate kinase (PGK), triose phosphate isomerase (TPI), translational elongation factor EF-1 alpha (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1), galactose- 1 -phosphate uridyl-transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome cl (CYC1)
  • Upstream activation sequences also referred to as enhancers
  • Upstream activation sequences for expression in Saccharomyces cerevisiae include the UASs of genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7 and GAL 10, as well as other UASs activated by the GAL4 gene product, with the ADH2 UAS being used in one aspect. Since the ADH2 UAS is activated by the ADR1 gene product, it may be preferable to overexpress the ADR1 gene when a heterologous gene is operatively linked to the ADH2 UAS.
  • Transcription termination sequences for expression in Saccharomyces cerevisiae include the termination sequences of the alpha-factor, GAPDH, and CYC1 genes.
  • Transcription control sequences to express genes in methyltrophic yeast include the transcription control regions of the genes encoding alcohol oxidase and formate dehydrogenase.
  • transfection of a nucleic acid molecule into a yeast cell can be accomplished by any method by which a nucleic acid molecule administered into the cell and includes diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion.
  • Transfected nucleic acid molecules can be integrated into a yeast chromosome or maintained on extrachromosomal vectors using techniques known to those skilled in the art.
  • yeast cytoplast, yeast ghost, and yeast membrane particles or cell wall preparations can also be produced recombinantly by transfecting intact yeast microorganisms or yeast spheroplasts with desired nucleic acid molecules, producing the antigen therein, and then further manipulating the microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplast, ghost or subcellular yeast membrane extract or fractions thereof containing desired antigens or other proteins.
  • Further exemplary yeast expression systems, methods, and conditions suitable for use herein are described in US20100196411A1, US2017/0246276, or US 2017/0224794, and US 2012/0107347.
  • viruses and yeasts may then be individually or in combination used as a therapeutic vaccine in a pharmaceutical composition, typically formulated as a sterile injectable composition with a virus of between 10 4 -10 13 virus or yeast particles per dosage unit, or more preferably betweenl0 9 -10 12 virus or yeast particles per dosage unit.
  • virus or yeast may be employed to infect patient cells ex vivo and the so infected cells are then transfused to the patient.
  • alternative formulations are also deemed suitable for use herein, and all known routes and modes of administration are contemplated herein.
  • second generation hAd5 [E1-, E2b-, E3-] based vaccines disclosed herein overcome pre-existing Anti-Ad5 immunity.
  • an advanced 2nd generation human adenoviral (hAd5) vector was constructed having two (2) additional deletions in the E2b region, removing the DNA polymerase and the preterminal protein genes [E1-, E2b-, E3-] (Former names of the adenovirus vector were Ad5, ETBX in literature)
  • E2b-deleted hAd5 vectors have up to a 12-14 kb gene-carrying capacity as compared to the 7-kb capacity of first generation Ad5 [E1-] vectors, providing space for multiple genes if needed.
  • hAd5 [E1-, E2b-, E3-] based recombinant vectors are produced using the human E.C7 cell line. Deletion of the E2b region also confers advantageous immune properties on these novel Ad vectors, eliciting potent immune responses to specific, non-viral antigens while minimizing the immune responses to Ad viral proteins.
  • hAd5 [E1-, E2b-, E3-] vectors induce a potent cell mediated immune (CMI) response, as well as Abs against the vectored antigens even in the presence of Ad immunity.
  • hAd5 [E1-, E2b-, E3-] vectors also have reduced adverse reactions as compared to Ad5 [E1-] vectors, in particular the appearance of hepatotoxicity and tissue damage.
  • the reduced inflammatory response against hAd5 [E1-, E2b-, E3-] vector viral proteins and the resulting evasion of pre-existing Ad immunity increases the capability for the hAd5 [E1-, E2b-, E3-] vectors to infect dendritic cells (DC), resulting in greater immunization of the vaccine.
  • DC dendritic cells
  • increased infection of other cell types provides high levels of antigen presentation needed for a potent CD8+ and CD4+ T cell responses, leading to memory T cell development.
  • hAd5 [E1-, E2b-, E3-] vectors are superior to Ad5 [E1-] vectors in immunogenicity and safety and will be the best platform to develop a COVID-19 vaccine in a rapid and efficient manner.
  • a prophylactic vaccine is tested against COVID-19 by taking advantage of this new hAd5 vector system that overcomes barriers found with other Ad5 systems and permits the immunization of people who have previously been exposed to Ad5.
  • Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine.
  • a novel platform (hAd5 [E1-, E2b-, E3-]) has been utilized to induce immune responses to various antigenic targets.
  • This vector platform expressed hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced.
  • Vaccination induced H1N1 immune responses in mice which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes.
  • the hAd5 [E1-, E2b-, E3-] has thus demonstrated the capability for the rapid development of effective vaccines against infectious diseases.
  • contemplated vaccine compositions when based on an adenoviral vector will utilize a recombinant hAd5 [E1-, E2b-, E3- ] platform to generate recombinant nucleic acids for therapeutic use in human.
  • Example 1 Selected hAd5 vaccine constructs and results
  • hAd5-COVID- 19 vaccine construct E1-, E2b-, E3- hAd5 vector with SARS-CoV-2 (S/N) protein insert (FIG.26).
  • This construct has been tested in preclinical experiments, including in vitro expression (FIG.27) and small animal immunogenicity.
  • Multiple COVID-19 constructs include RBD-alone, SI -alone, SI -fusion proteins, and combinations of RBD, SI and SI fusions with N.
  • Preliminary in-vitro studies demonstrate that these constructs (FIG.28) recognize convalescent serum antibodies and could serve as alternative vaccines.
  • the present disclosure confirms that by combining S with N, long-term cell- mediated immunity with a Thl phenotype can be induced. Indeed, significant potential exists for this combination vaccine to serve as a long-term “universal” COVID-19 vaccine in light of mutations undergoing in S and the finding that the structural N protein is highly conserved in the coronavirus family.
  • mice Homologous prime-boost immunogenicity in BALB-c mice. Mice have been treated with 1, 2 or 3 doses of the hAd5 COVID-19 vaccine and serum and splenocyte samples are being tested for SARS-CoV-2 antigen-specific immune responses. Serum is tested for anti-spike and anti-nucleocapsid antibody responses by ELISA. Splenocytes is tested for spike- and nucleocapsid-specific cell mediated immune responses by ELISPOT and intracellular cytokine simulation assays.
  • hAd5 [El - ,E2b-, E3-] N-ETSD, a vaccine containing SARS-CoV-2 nucleocapsid plus an enhanced T cell stimulation domain (ETSD), alters T cell responses to nucleocapsid.
  • Mice were immunized subcutaneously (SC) with a dose of 1010 VP twice at 7-day intervals. Blood was collected at several time points and spleen was collected upon sacrifice in order to perform immunogenicity experiments. Splenocytes were isolated and tested for cell mediated immune (CMI) responses.
  • CMI cell mediated immune
  • SARS-CoV-2 Virus Neutralization Studies Serum from the mice immunized during the course of the immunogenicity studies described above is used will be sent to a testing lab for SARS-CoV-2 neutralization studies to be performed in their ABSL-3 facility. Serum will be tested for COVID 19 virus neutralizing activity by mixing various dilutions of serum with COVID 19 virus, incubating the mixture, and then exposing the mixture to Vero cells to detect cytopathic effect (CPE). The last dilution that prevents CPE will be considered the endpoint neutralizing titer.
  • CPE cytopathic effect
  • Example 5 Phase lb Clinical trial testing of hAd5 [E1-. E 2b-, E3-1 CoV-2 vaccine.
  • Study Design This is a Phase lb open-label study in adult healthy subjects. This clinical trial is designed to assess the safety, reactogenicity, and immunogenicity of the hAd5- COVID-19-S and hAd5-COVID-19-S/N vaccines.
  • the hAd5-COVID-19-S and hAd5-COVID- 19-S/N vaccines are hAd5 [E1-, E2b-, E3-] vector-based targeting vaccines encoding the SARS- CoV-2 Spike (S) protein alone or together with the SARS-CoV-2 nucleocapsid (N) protein.
  • the hAd5 [E1-, E2b-, E3-] vector is the platform technology for targeted vaccines that has demonstrated safety in over 125 patients with cancer to date at doses as high as 5 c 10 11 virus particles per dose.
  • Co-administration of three different hAd5 [E1-, E2b-, E3-] vector-based vaccines on the same day at 5 c 10 11 virus particles per dose each (1.5 c 10 12 total virus particles) has also been demonstrated to be safe.
  • COVID-19 infection causes significant morbidity and mortality in a worldwide population.
  • the hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines are designed to induce both a humoral and cellular response even in individuals with pre-existing adenoviral immunity.
  • Each subject will receive a subcutaneous (SC) injection of hAd5-COVID-19-S or hAd5-COVID-19-S/N on Day 1 and Day 22 (i.e., 2 doses).
  • SC subcutaneous
  • This dosing schedule is consistent with hAd5 [E1-, E2b-, E3-] vector-based vaccines currently in clinical trials.
  • Cohorts 1-2 will enroll in parallel and may be opened at the same time or in a staggered manner depending upon investigational product supply. Subjects in cohorts 1A and 2A will complete the low-dose vaccination regimen first.
  • the primary objectives of the initial safety phase lb are to evaluate preliminary safety and reactogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines.
  • the secondary objectives are to evaluate the extended safety and immunogenicity of the hAd5-COVID-19-S and hAd5-COVID-19-S/N vaccines.
  • Phase lb expansion will proceed if the SRC determines it is safe to do so based on a review of safety data from the phase lb safety assessment.
  • Each subject will receive a SC injection of hAd5-COVID-19-S or hAd5-COVID-19- S/N on Day 1 and Day 22 (i.e., 2 doses).
  • follow-up study visits will occur at days 8, 22, 29, 52, and at months 3, 6, and 12 following the final vaccination. Additional follow up for safety information will occur via telephone contact as noted in the Schedule of Events.
  • the primary objective of the expanded phase lb is to select the most immunogenic construct between hAd5- COVID-19-S and hAd5-COVID-19-S/N and dose level as determined by changes in humoral and cellular immunogenicity indexes.
  • the secondary objectives are to assess safety and reactogenicity of hAd5-COVID-19-S and hAd5-COVID-19-S/N.
  • Example 7 The hAd5 GE1-. E2b-, E3-1 platform and constructs
  • FIG. 33 Panel C
  • S WT S protein comprising 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2)
  • FIG. 33 Panel E: S RBD-ETSD: S Receptor Binding Domain with an Enhanced T-cell Stimulation Domain (ETSD);
  • FIG. 33 Panel D: S WT: S protein comprising 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2)
  • S RBD-ETSD S Receptor Binding Domain with an Enhanced T-cell Stimulation Domain (ETSD)
  • FIG.33 Panel C
  • Panel F S Fusion: S optimized to enhance surface expression and display of RBD
  • FIG. 33 Panel G: N- ETSD: The nucleocapsid (N) sequence with the ETSD
  • FIG. 33, Panel H Bivalent S-Fusion + N-ETSD; S-WT + N-ETSD and S RBD-ETSD + N-ETSD constructs were also produced but are not shown.
  • Example 8 Enhanced HEK 293T cell-surface expression of RBD following transfection with Ad5 S- Fusion + N-ETSD
  • anti-RBD-specific antibodies did not detect RBD on the surface of HEK 293T cells transfected with hAd5 S-WT (FIG.34, Panel A) or hAd5 S-WT + N-ETSD (Fig. 9b) constructs, while hAd5 S-Fusion alone was slightly higher (FIG.34, Panel E).
  • both constructs with RBD, hAd5 RBD-ETSD and RBD-ETSD + N-ETSD showed high binding of anti-RBD antibody (FIG.34, Panels C and D).
  • FIG.35 depicts immunoblot analysis of S expression.
  • Cell surface RBD expression with (a) hAd5 S-WT, S-Fusion, and (c) S-Fusion + N-ETSD in HEK 293T cells shows high correlation with (d) expression of S in immunoblots of HEK 293T cell lysates probed using anti -full length (S2) antibody.
  • Y-axis scale is normalized to mode (NM).
  • the ETSD design successfully translocated N to the endosomal subcellular compartment.
  • N co-localized with the endosomal marker 45 transferrin receptor (CD71), as shown in FIG.37, Panel C, and also co-localized with the lysosomal marker Lampl (FIG.37, Panel D), demonstrating that N-ETSD is translocated throughout the endosomal pathway to lysosomes, enabling processing for MHC II presentation.
  • N-wild type (N-WT) compared to N-ETSD, shows dilfuse cytoplasmic distribution and does not co-localize with the lysosomal marker (FIG.37, Panel E).
  • mice received an initial injection on Day 0 and a second injection on Day 21. Sera were collected on Day 0 and at the end of the study on Day 28 for antibody and neutralization analyses.
  • Example 13 hAd5 S-Fusion + N-ETSD generates both CD8B+ and CD4+ T-cell responses
  • CD8+ activation by both S and N CD8 + splenocytes from hAd5 S-Fusion + N-ETSD vaccinated mice exposed to S peptide pool 1 (containing RBD and SI) show IFN-g expression that is significantly higher compared to hAd5 null mice (FIG.38, Panel A); splenocytes from these mice also expressed intracellular IFN-g in response to the N peptide pool. Evaluation of simultaneous IFN-y/TNF- a expression from CD8 b + splenocytes (FIG.38, Panel C) mirrored those for IFN-g expression alone. These results indicate that both S and N activate CD8+ T cells.
  • CD4+ activation by N Although CD8+ cytotoxic T cells mediate killing of virus infected cells, CD4+ T cells are required for sustained cytotoxic T lymphocyte (CTL) activity. Thus, CD4+ T cells in the vaccinated animals was evaluated. In contrast to CD8 b + splenocytes, only the N peptide pool stimulated CD4+ splenocytes from hAd5 S-Fusion + N-ETSD-inoculated mice to express IFN-g (FIG.38, Panel B) or IFN-y/TNF-a (FIG.38, Panel D) at levels that were substantially higher than hAd5 Null control. The contribution by N of CD4+ T-cell responses is vital to an effective immune response to the candidate vaccine.
  • CTL cytotoxic T lymphocyte
  • Example 14 hAd5 S-Fusion + N-ETSD generates antibody responses to both S and N antigens
  • Example 15 hAd5 S-Fusion + N-ETSD vaccine generates potent neutralizing antibodies as assessed by both cPass and live virus neutralization assays
  • Example 16 hAd5 S-Fusion + N-ETSD generates Thl dominant responses both in humoral and T-cell immunity
  • Antibody Thl dominance in response to N and S IgG2a, IgG2b, and IgG3 represent Thl dominance; while IgGl represents Th2 dominance.
  • IgG2a and IgG2b isotypes were predominant and significantly higher compared to the hAd5 Null control.
  • T-cell Thl dominance in response to N and S IFN-g production correlates with CTL activity 47 (Thl dominance), whereas, IL-4 causes delayed viral clearance 48 (Th2 dominance).
  • a ratio of IFN-g to IL-4 of 1 is balanced and a ratio greater than 1 is demonstrative of Thl dominance.
  • Thl -type predominance is also seen when the ratio of IFN-g to IL-4 based on spot forming units in response to the combined S peptide pools and the N peptide pool, is considered (FIG.43, Panel A). Thl predominance was seen again in humoral responses, where the ratio based on ng equivalence of Thl related antibodies (IgG2a, IgG2b, and IgG3) to Th2 related antibodies (IgGl) for both anti-S and anti-N antibodies is greater than 1 in all mice (FIG.43, Panel B).
  • This Thl dominant profile of the hAd5 S-Fusion + N-ETSD vaccine candidate provides further justification for hAd5 S-Fusion + N-ETSD to be the lead candidate for clinical testing.
  • the hAd5 S-Fusion + N-ETSD vaccine was designed to overcome the risks of an S- only vaccine and elicit both T-cell immunity and neutralizing antibodies, leveraging the vital role T cells play in generating long-lasting antibody responses and in directly killing infected cells.
  • Both CD4+ and CD8+ T cells are multifunctional, and induction of such multifunctional T cells by vaccines correlated with better protection against infection.
  • Enhanced CD4+ T-cell responses and Thl predominance resulting from expression of an S antigen optimized for surface display and an N antigen optimized for endosomal/lysosomal subcellular compartment localization and thus MHC I and II presentation led to increased dendritic cell presentation, cross-presentation, B cell activation, and ultimately high neutralization capability.
  • Contemporaneous MHC I and MHC II presentation of an antigen by the antigen presenting cell activates CD4+ and CD8+ T cells simultaneously and is optimal for the generation of memory B and T cells.
  • a key finding of the construct is that N-ETSD is directed to the endosomal/lysosomal compartment. There N-ETSD elicits a CD4+ response, a necessity for induction of memory T cells and helper cells for B cell antibody production. Others have also reported on the importance of lysosomal localization for eliciting the strongest T-cell IFN-g and CTL responses, compared to natural N.50,51
  • the T-cell responses to the S and N antigens expressed by hAd5 S-Fusion + N-ETSD were poly cytokine, including IFN-g and TNF-a, consistent with successful antimicrobial immunity in bacterial and viral infections.
  • Post-vaccination poly cytokine T-cell responses have been shown to correlate with vaccine efficacy, including those with a viral vector.
  • poly cytokine T-cell responses to SARS-CoV-2 N protein are consistent with recovered COVID- 19 patients, suggesting that the bivalent hAd5 S-Fusion + N-ETSD vaccine will provide vaccine subjects with greater protection against SARS-CoV-2.
  • the S protein here expressed as S-Fusion with confirmed enhanced RBD cell-surface expression and conformational integrity as evidenced by high ACE2-Fc binding, generated predominantly CD8+ T cells.
  • Our results confirmed the vaccine design goal, showing that S-Fusion induced elevated levels of antigen-specific T-cell responses against S compared to S-WT.
  • MHC I for CD8+ T-cell activation
  • MHC II for CD4+ T-cell activation
  • the hAd5 S-Fusion + N-ETSD construct described above is delivered by a next generation hAd5 [E1-, E2b-, E3-] platform wherein the E2b deletion (pol) alone enables prolonged transgene production and allows homologous vaccination (prime and the boost formulation is the same) in the presence of pre-existing adenoviral immunity.38
  • the same vaccine in an oral or sublingual formulation may also induce IgA mucosal immunity.
  • hAd5 [E1-, E2b-, E3-] vector was used (FIG.44, Panel A) to create viral vaccine candidate constructs.
  • hAd5 [E1-, E2b-, E3-] backbones containing SARS-CoV-2 antigen expressing inserts and virus particles were produced as previously described.
  • high titer adenoviral stocks were generated by serial propagation in the El- and E2b-expressing E.C7 packaging cell line, followed by CsC12 purification, and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) by ViraQuest Inc. (North Liberty, IA).
  • Viral particle counts were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm and viral titers were determined using the Adeno-XTM Rapid Titer Kit (Takara Bio). The constructs created included:
  • S-WT S protein comprising 1273 amino acids and all S domains: extracellular (1- 1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2);
  • S RBD- ETSD S Receptor Binding Domain (S RBD) with an ETSD (SEQ ID NO: 11);
  • N-ETSD Nucleocapsid (N) with ETSD;
  • S-WT + N-ETSD S-WT with an Enhanced T-cell Stimulation Domain (ETSD);
  • S-Fusion S optimized to enhance surface expression and display of RBD; and Bivalent S-Fusion + N-ETSD; Transfection of HEK 293T cells with hAd5 constructs
  • HEK 293T cells 2.5 x 10 5 cells/well in 24 well plates
  • DMEM Gibco Cat# 11995-065
  • FBS penicillin, 100 pg/mL streptomycin, 0.25 ug/mL Amphotericin B
  • Cells were transfected with 0.5 pg of hAd5 plasmid DNA using a JetPrime transfection reagent (Polyplus Catalog # 89129-924) according to the manufacturer’s instructions.
  • Cells were harvested 1, 2, 3, and 7 days post transfection by gently pipetting cells into medium and labeled with an anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog # 40150-D003) and F(ab’)2-Goat anti-Human IgG-Fc secondary antibody conjugated with R- phycoerythrin (ThermoFisher Catalog # H10104). Labeled cells were acquired using a Thermo- Fisher Attune NxT flow cytometer and analyzed using Flowjo Software.
  • an anti-RBD monoclonal antibody clone D003 Sino Biological Catalog # 40150-D003
  • F(ab’)2-Goat anti-Human IgG-Fc secondary antibody conjugated with R- phycoerythrin ThermoFisher Catalog # H10104.
  • cells were also incubated overnight at 4oC with a sheep anti-Lampl Alexa Fluor 488- conjugated (lysosomal marker) antibody (R&D systems, Cat# IC7985G) at 1:10 or a rabbit anti- CD71 (transferrin receptor, endosomal marker) antibody (ThermoFisher Cat# PA5- 83022) at 1:200.
  • a sheep anti-Lampl Alexa Fluor 488- conjugated (lysosomal marker) antibody R&D systems, Cat# IC7985G
  • a rabbit anti- CD71 (transferrin receptor, endosomal marker) antibody ThermoFisher Cat# PA5- 83022
  • HEK 293T cells transfected with hAd5 S-WT, S-Fusion, or S-Fusion + N-ETSD constructs were cultured and transfected as described in the main manuscript and harvested 3 days after transfection in 150 mL RIPA lysis buffer with IX final Protease Inhibitor cocktail (Roche). After protein assay, equivalent amounts of total protein were loaded into and run on a 4 to 12% gradient polyacrylamide gel (type) and transferred to nitrocellulose membranes using semi-dry transfer apparatus.
  • Anti-Spike S2 (SinoBiological Cat #40590-T62) was used as the primary antibody and IRDye® 800CW Goat anti-Rabbit IgG (H + L) (Li-Cor, 925-32211) as the secondary antibody using the Ibind Flex platform. Antibody-specific signals were detected with an infrared Licor Odyssey instrument.
  • HEK 293T cells were cultured at 37°C under conditions described above for transfection with hAd5 S-WT, S-Fusion, S-Fusion + N-ETSD, S RBD-ETSD, or S RBD-ETSD + N-ETSD and were incubated for 2 days and harvested for ACE2-Fc binding analysis.
  • Recombinant AC E2-IgGlFc protein was produced using Maxcyte transfection in CHO-S cells that were cultured for 14 days.
  • ACE2-IgGlFc was then purified using a MabSelect SuRe affinity column on AKTA Explorer.
  • ACE2-IgGlFc Purified ACE2-IgGlFc was dialyzed into 10 mM HEPES, pH7.4, 150 mM NaCl and concentrated to 2.6 mg/mL.
  • the ACE2-IgGlFc was used at a concentration of 1 pg/mL for binding.
  • Cells were incubated with ACE2-Fc for 20 minutes and, after a washing step, were then labeled with a PE conjugated F(ab’)2-goat anti-human IgG Fc secondary antibody at a 1 : 100 dilution, incubated for 20 minutes, washed and acquired on flow cytometer. Histograms are based on normalized mode (NM) of cell count - count of cells positive for signal in PE channel.
  • NM normalized mode
  • mice (Charles River Laboratories) 7 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA). After an initial blood draw, mice were injected with either hAd5 Null (a negative control) or vaccine candidate hAd5 S- Fusion + N-ETSD on Day 0 at a dose of 1 xlO 10 viral particles (VP). There were 5 mice per group. Mice received a second vaccine dose on Day 21 and on Day 28, blood was collected via the submandibular vein from isoflurane-anesthetized mice for isolation of sera and then mice were euthanized for collection of spleen and other tissues.
  • hAd5 Null a negative control
  • VP xlO 10 viral particles
  • Spleens were removed from each mouse and placed in 5 mL of sterile medium of RPMI (Gibco Cat # 22400105), HEPES (Hyclone Cat# SH30237.01), IX Pen/Strep (Gibco Cat # 15140122), and 10% FBS (Gibco Cat # 16140-089). Splenocytes were isolated within 2 hours of collection. ICS for flow cytometric detection of CD8 + and CD4+ T-cell-associated IFN-g and IFN-y/TNFa+ production in response to stimulation by S and N peptide pools.
  • Stimulation assays were performed using 10 6 live splenocytes per well in 96-well U- bottom plates. Splenocytes in RPMI media supplemented with 10% FBS were stimulated by the addition of peptide pools at 2 pg/mL/peptide for 6 h at 37°C in 5% C02, with protein transport inhibitor, GolgiStop (BD) added two hours after initiation of incubation. Stimulated splenocytes were then stained for lymphocyte surface markers CD8 and CD4, fixed with CytoFix (BD), permeabilized, and stained for intracellular accumulation of IFN-g and TNF-a.
  • BD protein transport inhibitor
  • Fluorescent- conjugated antibodies against mouse 6 ⁇ 8b antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-g (clone XMG1.2, BD), and TNF-a (clone MP6-XT22, BD) and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2).
  • Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.
  • ELISpot assays were used to detect cytokines secreted by splenocytes from inoculated mice. Fresh splenocytes were used on the same day, as were cryopreserved splenocytes containing lymphocytes. The cells (2-4 x 10 5 cells per well of a 96-well plate) were added to the ELISpot plate containing an immobilized primary antibodies to either IFN-g or IL-4 (BD), and were exposed to various stimuli (e.g. control peptides, target peptide pools/proteins) comprising 2 pg/mL peptide pools or 10 pg/mL protein for 36-40 hours.
  • stimuli e.g. control peptides, target peptide pools/proteins
  • cytokine conjugated to biotin was detected by a secondary antibody to cytokine conjugated to biotin (BD).
  • BD biotin
  • streptavidin/horseradish peroxidase conjugate was used detect the biotin-conjugated secondary antibody. The number of spots per well, or per 2-4 x 10 5 cells, was counted using an ELISpot plate reader.
  • ELISAs specific for spike and nucleocapsid antibodies as well as for IgG subtype (IgGl, IgG2a, IgG2b, and IgG3) antibodies were used.
  • a microtiter plate was coated overnight with 100 ng of either purified recombinant SARS-CoV-2 S-FTD (full-length S with fibritin trimerization domain, constructed and purified by Immunity Bio, Inc., 9920 Jefferson Boulevard, Culver City, CA 90232), SARS-CoV-2 S RBD (Sino Biological, Beijing, China; Cat # 401591- V08B1-100) or purified recombinant SARS-CoV-2 nucleocapsid (N) protein (Sino Biological, Beijing, China; Cat # 40588-V08B) in 100 pL of coating buffer (0.05 M Carbonate Buffer, pH 9.6).
  • SARS-CoV-2 S-FTD full-length S with fibritin trimerization domain, constructed and purified by Immunity Bio, Inc., 9920 Jefferson Blvd, Culver City, CA 90232
  • SARS-CoV-2 S RBD Sino Biological, Beijing, China; Cat # 401591- V08B1-
  • the wells were washed three times with 250 pL PBS containing 1% Tween 20 (PBST) to remove unbound protein and the plate was blocked for 60 minutes at room temperature with 250 pL PBST. After blocking, the wells were washed with PBST, 100 pL of diluted serum samples were added to wells, and samples incubated for 60 minutes at room temperature.
  • PBST Tween 20
  • a 100 pL of a 1/5000 dilution of rabbit anti-N IgG Ab or 100 pL of a 1/25 dilution of mouse anti-S serum were added to appropriate wells. After incubation at room temperature for 1 hour, the wells were washed with PBS-T and incubated with 200 pL o-phenylenediamine-dihydrochloride (OPD substrate (Thermo Scientific Cat # A34006) until appropriate color development. The color reaction was stopped with addition of 50 pL 10% phosphoric acid solution (Fisher Cat # A260-500) in water and the absorbance at 490 nm was determined using a microplate reader (SoftMax® Pro, Molecular Devices).
  • GenScript cPassTM for detection of neutralizing antibodies was used according to the manufacturer’s instructions.44 The kit detects circulating neutralizing antibodies against SARS- CoV-2 that block the interaction between the S RBD with the ACE2 cell surface receptor. It is suitable for all antibody isotypes and appropriate for use with in animal models without modification.
  • Vero e6 kidney epithelial cells from Cercopithecus aethiops (ATCC CRL-1586) were plated at 20,000 cells/well in a 96-well format and 24 hours later, cells were incubated with antibodies or heat inactivated sera previously serially diluted in 3-fold steps in DMEM containing 2% FBS, 1% NEAAs, and 1% Pen-Strep; the diluted samples were mixed 1:1 with SARS-CoV-2 in DMEM containing 2% FBS, 1% NEAAs, and 1% Pen-Strep at 10,000 TCID 50/mL for 1 hr.
  • the samples for testing included sera from the four mice that showed > 20% inhibition of ACE2 binding in cPass, pooled sera from those four mice, sera from a COVID-19 convalescent patient, and media only.
  • 120 pL of the virus/sample mixture was transferred to the Vero E6 cells and incubated for 48 hours before fixation with 4% PFA.
  • Each well received 60 pL of virus or an infectious dose of 600 TCID50. Control wells including 6 wells on each plate for no virus and virus-only controls were used.
  • the percent neutralization was calculated as 100-((sample of interest- [average of “no virus”] )/[average of “virus only”])* 100) with a stain for CoV-2 Np imaged on a Celigo Imaging Cytometer (Nexcelom Bioscience).
  • the presently disclosed dual-antigen candidate vaccine more broadly activates the immune system to combat SARS-CoV-2, by the inclusion of a modified viral nucleocapsid (N) antigen, a potent CD4+ and CD8+ T cell target, along with an optimized S protein (S-Fusion) to stimulate humoral responses.
  • N modified viral nucleocapsid
  • S-Fusion optimized S protein
  • the human adenovirus serotype 5 (hAd5) El, E2b, and E3 region-deleted [E1-, E2b-, E3-] vaccine platform (FIG.44, Panel A) is superior to the adenovirus platforms used in other COVID-19 vaccines currently in clinical trials because it is effective in the presence of pre-existing adenovirus immunity and has a reduced likelihood of generating a vector-targeted host immune response, thus can be used as both the prime and boost.
  • the vaccine comprises the optimized S surface protein, S-Fusion, to increase cell-surface display and humoral responses; as well as the highly conserved and antigenic N protein found within the viral particle, here with subcellular compartment targeting sequences for enhanced antigen presentation.
  • This strategy will be safe and robust in eliciting humoral and T cell responses to SARS-CoV-2.
  • the addition of N addresses the risk of loss of vaccine efficacy for S-only monovalent vaccines due to the emergence of mutations in S in the population over time.
  • N is highly conserved with a lower risk of mutation, while also being highly immunogenic.
  • Enhanced T cell Stimulation Domain directs N protein to the endosomal-lysosomal subcellular compartment after translation to support MHC class II presentation for T helper cell activation and promotion of CD8+ T cell activation through dendritic cell licensing.
  • ESD Enhanced T cell Stimulation Domain
  • Spike displayed as a trimer on the viral surface (FIG.44, Panel B), has a Receptor Binding Domain (RBD) that interacts with host angiotensin-converting enzyme 2 (ACE2) to facilitate entry into the host cells and propagation, thus antibodies against S are key to neutralization of infection.
  • RBD Receptor Binding Domain
  • ACE2 host angiotensin-converting enzyme 2
  • Antibodies against S RBD are commonly found in patients recovered from COVID19 51 as are antibodies against other S epitopes.
  • S-Fusion is S optimized by addition of a fusion linker to display the S RBD in a physiologically relevant form on the cell surface with the goal of improving generation of anti-S RBD antibodies that will be virus neutralizing.
  • the presently disclosed bivalent hAd5 S-Fusion + N-ETSD vaccine generates excellent T-cell responses.
  • Vaccines currently in clinical trials focus on generating humoral responses as a means to neutralize infection. However, given that antibodies, even if successfully generated and sufficiently neutralizing, may wane over time, the T cell response becomes critical. If T cell responses are absent due to virus-induced lymphopenia, even in the presence of abundant neutralizing antibodies, an infected person is at risk for developing acute symptoms of the disease. While it cannot be excluded that S (and other viral proteins) can induce T-cell responses, the evidence in the literature support a key role for N.
  • N is expected to not only elicit a humoral response, but also a T-cell response that better recapitulates disease-limiting natural immunity.
  • hAd5 S-Fusion + N-ETSD vaccine provided enhanced cell-surface expression of S RBD that was readily recognized by ACE2, reflecting its conformational integrity.
  • N-ETSD with endosomal/lysosomal localization for enhanced antigen presentation generated both neutralizing antibody and CD4+/CD8+ T-cell-mediated responses with Thl predominance in inoculated mice.
  • the present disclosure extends those findings using plasma and monocyte- derived dendritic cells (MoDCs) from previously SARS-CoV-2 infected patients to confirm native S antigen expression.
  • MoDCs monocyte- derived dendritic cells
  • hAd5-infected MoDCs presenting S-Fusion and N-ETSD elicit a predominant Thl response from autologous memory T cells of previously SARS-CoV-2 infected patients.
  • N in particular drives the CD8+ T cell responses in in vitro recall studies. Recapitulation of natural infection and immunity, to the degree it can be achieved by vaccination, by the hAd5 S- Fusion + N-ETSD vaccine makes it a prime candidate for clinical testing of its ability to protect individuals from SARS-CoV-2 infection and COVID-19 and this second-generation vaccine construct has now entered into Phase I clinical trials.
  • Example 18 The hAd5 1E1-. E2b-, E3-1 platform and constructs
  • FIG.44 Panel A
  • S WT S protein comprising 1274 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2)
  • FIG.44, Panel D S-Fusion: S optimized to enhance surface expression and display of RBD
  • FIG.44, Panel E N (N without ETSD): Nucleocapsid (wild type) sequences with tags for immune detection, but without ETSD modification, and predominantly cytoplasmic localization.
  • FIG.44 Panel F. N with the Enhanced T cell Stimulation Domain (N-ETSD): Nucleocapsid (wild type) with ETSD to direct lysosomal/endosomal localization and tags for immune detection; and FIG.44, Panel G: The Bivalent hAd5 S-Fusion + N-ETSD vaccine.
  • N-ETSD Enhanced T cell Stimulation Domain
  • Nucleocapsid antigen engineered with an Enhanced T cell Stimulation Domain directs N to endosomes, lysosomes and autophagosomes in MoDCs, driving enhanced CD4+ T- cell activation:
  • the hAd5 bivalent vaccine construct includes sequences designed to target N to MHC class II antigen loading compartments.
  • MoDCs from healthy subjects were infected with hAd5 N-ETSD or hAd5 N and localization was determined by immunocytochemistry.
  • N-ETSD showed localization to discrete vesicles, some coincident with CD71, a marker of recycling endosomes (FIG.45, Panels A-C), and LAMP-1, a marker for late endosome/lysosomes (FIG.45, Panels G-I), whereas N was expressed diffusely and uniformly throughout the cytoplasm (FIG.45, Panels D-F and J-L).
  • Lysosomes fuse with autophagosomes to enhance peptide processing and MHC class II presentation.
  • N-ETSD also displayed some co-localization with the autophagosome marker (FIG.45, Panels M-O).
  • Protein processing in autophagosomes plays a key role in MHC-mediated antigen presentation in DCs, providing a potential mechanism of enhanced CD4+ T cells induced by N-ETSD in the vaccine construct.
  • This binding reflects the presence of antibodies in plasma that recognize antigens expressed by the hAd5 vectored vaccines. Quantification of histograms showed little or no binding of virus-naive plasma antibodies to cells expressing either construct, and the highest binding of plasma antibodies from a previously SARSCoV-2 infected patient to cells expressing the bivalent S-Fusion + N-ETSD construct. This could be due to either the enhanced cell surface expression of S found in hAd5 S-Fusion + N- ETSD infected HEK 293T cells as compared to hAd5 S-Fusion alone or expression of both S and N antigens.
  • Example 19 N-ETSD optimizes spike antigen expression: binding of plasma antibodies from previously infected SARS-CoV-2 patients is enhanced for hAd5 S-Fusion + N-ETSD infected MoDCs compared to hAd5 S-Fusion or hAd5 S-WT
  • DCs are powerful antigen presenting cells for processing and presenting complex antigens acquired through infection or phagocytosis to elicit a T-cell response.
  • MoDCs from two healthy individuals were infected overnight with hAd5 S-WT, hAd5 S-Fusion, hAd5 S-Fusion + N-ETSD, or hAd5 Null then evaluated gene expression using plasma from a previously SARS-CoV-2 infected patient (FIG.46, Panel A).
  • Example 20 SARS-CoV-2 peptide pool immune reaction:
  • T cells from previously infected SARS-CoV-2 patients secrete significant levels of interferon-g (IFN-g) in response to SI, S2, and N SARSCoV-2 peptide pools compared to T cells from virus-naive controls
  • IFN-g interferon-g
  • T cells from each group were cultured with autologous MoDCs pulsed with peptide mixes spanning the sequences of N and S proteins.
  • T cells from previously infected SARS-CoV-2 patients but not unexposed subjects secreted IFNg in response to SARS-CoV-2 antigens (FIG.47), validating selective reactivity of T cells from patients previously infected with SARS-CoV-2.
  • Example 21 SARS-CoV-2 peptide pool immune reaction: CD4+ T cells from previously infected SARSCoV-2 patients recognize S and N peptide pool antigens but CD8+ T cells display greater recognition of N peptide antigens.
  • CD4+ T cells of the two patient samples tested responded to both the S and N peptide pools, with a higher response to N by Pt3 (FIG.48, Panel B).
  • CD8+ T cells from both patients responded to N with high significance, but not to S 1 or S2 peptide pools (FIG.48, Panels C and D).
  • Example 22 Autologous MoDCs infected with endo/lvsosome-directed nucleocapsid-ETSD elicit higher levels of IFN-g secretion from CD4+ and CD8+ T cells from previously infected SARS- CoV-2 patients compared to cytoplasmic nucleocapsid protein (hAd5 NY
  • Example 23 Thl dominant SARS-CoV-2 specific CD4+ and CD8+ memory T-cell recall to nucleocapsid and spike antigens is induced by hAd5 S-Fusion + N-ETSD infection of autologous MoDCs from previously SARS-CoV- 2 infected patients.
  • N-ETSD is more effective than N in eliciting patient T-cell cytokine responses.
  • IFN-g responses were similar for S-Fusion + N-ETSD and N-ETSD with responses to S-Fusion being relatively low (FIG.50, Panels A-C).
  • the number of IL-4 secreting T cells was very low for all (FIG.50, Panels D-F).
  • the increased T-cell response could be explained by either T cells recognizing increased S or the presence of N.
  • these T-cell responses were characterized by a predominance of IFN-g (Thl) relative to IL-4 (Th2).
  • CD4+ T cells from all three patients showed significantly greater recognition of all three constructs compared to Null (FIG.50, Panels G-I). While there were greater responses to specific constructs in some individuals, overall the responses to S-Fusion, S-Fusion + N-ETSD and N-ETSD were similar.
  • CD8+ T cells from all three patients recognized the bivalent and N-ETSD vaccines at a significantly higher level than Null; in only two of three patients did CD8+T cells recognize S- Fusion to a significant degree above Null (FIG.50, Panels J-L). These data indicate that T cells from previously infected SARS-CoV-2 patients have reactivity and immune memory recall to both of the vaccine antigens (S and N) in the vaccine vector.
  • One feature of S-Fusion is the higher expression of S RBD compared to S-WT. This was a goal of the vaccine design based on findings from earlier studies of S cryo-electron micrograph structures that suggested RBD epitopes would be largely unavailable for immune detection.
  • a further advantage accrues from combining S with N in the bivalent vaccine, through the ability of N to enhance immune detection of S, a phenomenon that has been observed by others for gene expression in general.
  • the vaccine-expressed N protein traffics to the endosomal/lysosomal subcellular compartments, a key antigen presenting pathway to stimulate CD4+ T cells so that they can license dendritic cells to activate naive CD8+ CTL.
  • N-ETSD localizes to endosomes and lysosomes, as well as to autophagosomes, in MoDCs. Both endosomal and lysosomal targeting are desirable for enhanced antigen presentation and CD4+ T-cell activation. Lysosomes can fuse acidic autophagosomes, facilitating protein processing; this has important implications for effective immune stimulation by modulation of MHC class II presentation. T cells of previously infected SARS-CoV-2 patients more readily recognized N- ETSD than N. The data presented here strongly support the potential of enhanced efficacy of a vaccine construct specifically expressing the modified N-ETSD.
  • T cells are critical for elimination of SARS-CoV infection. 36,74-78 Here, hAd5 expressed S and N elicited strong antigen-specific IFN-g, but virtually no IL-4 secretion from T cells of previously infected SARS-CoV-2 patients, pointing to Thl dominance. Antiviral Thl cytokine responses eliminate a variety of viruses from infected hosts 79 including the virus closely related to SARSCoV-2, SARS-CoV. 80 These data are also consistent with the studies in preclinical models. Importantly, the data suggests that both S and N are targets of CD4+ T cells that help both antibody production from B cells and CD8+ T cell memory, which together function to kill virus infected targets.
  • the recognition of these vaccine antigens by the T-cell subsets are consistent with immune control of the pathogen.
  • the hAd5 S-Fusion + N-ETSD T- cell biased vaccine has the potential to not only provide protection for uninfected patients, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.
  • the T cell recall of N-ETSD was shown to be Thl dominant as shown by the vigorous interferon-g response and the low IL-4 response.
  • Example 25 The hAd5 1E1- E2b-. E3-1 platform and constructs
  • hAd5 [E1-, E2b-, E3-] vector was used (FIG.44, Panel A) to create viral vaccine candidate constructs.
  • hAd5 [E1-, E2b-, E3-] backbones containing SARSCoV-2 antigen expressing inserts and virus particles were produced as previously described.
  • high titer adenoviral stocks were generated by serial propagation in the El- and E2b expressing E.C7 packaging cell line, followed by CsC12 purification, and dialysis into storage buffer (2.5% glycerol, 20 mM Tris pH 8, 25 mM NaCl) by ViraQuest Inc. (North Liberty, IA).
  • Viral particle counts were determined by sodium dodecyl sulfate disruption and spectrophotometry at 260 and 280 nm. Viral titers were determined using the Adeno-XTM Rapid Titer Kit (Takara Bio). The constructs created included: i. S-WT: S protein comprising 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2); ii. S Fusion: S optimized to enhance surface expression and display of RBD; iii. N: Nucleocapsid (N) wild type sequence protein containing tags for immune detection; iv. N- ETSD: N with an Enhanced T-cell Stimulation Domain (ETSD) together with tags for immune detection; and v. Bivalent S-Fusion + N-ETSD;
  • Monocyte-derived dendritic cells were differentiated from PBMC using GM- CSF (200U/ml) and IL-4 (lOOU/ml) as previously described86. Briefly, monocytes were enriched by adherence on plastic, while the non-adherent cells were saved and frozen as a source of lymphocytes, specifically T cells. Adherent cells were differentiated into dendritic cells (3-5 d in RPMI containing 10% FBS), then frozen in liquid nitrogen for later use. T cells were enriched from the non-adherent fraction of PBMC using MojoSort (BioLegend CD3 enrichment). CD4+ and CD8+ T cells were enriched using analogous kits from the same manufacturer. Efficiency of the cell separations was evaluated by flow cytometry.
  • cells were also incubated overnight at 4 C with a rabbit anti-CD71 (transferrin receptor, recycling/sorting endosomal marker) antibody (ThermoFisher) at 1:200; sheep anti-Lampl Alexa Fluor 488-conjugated (lysosomal marker) antibody (R&D systems) at 1 : 10; or a rabbit monoclonal anti human LC3a/b (Light Chain 3, autophagy marker) antibody (Cell Signaling Tech #12741S) used at 1 : 100.
  • a rabbit anti-CD71 (transferrin receptor, recycling/sorting endosomal marker) antibody ThermoFisher
  • sheep anti-Lampl Alexa Fluor 488-conjugated (lysosomal marker) antibody R&D systems
  • a rabbit monoclonal anti human LC3a/b (Light Chain 3, autophagy marker) antibody Cell Signaling Tech #12741S
  • Binding of plasma antibodies from previously SARS-CoV-2 infected patients to antigens expressed by vaccine-infected MoDCs Binding of plasma antibodies from previously infected subjects to antigens expressed on the surface of MoDCs was determined by differentiation of MoDCs from peripheral blood mononuclear cells (PBMC) to DC, infection of the MoDCs, incubation with previously infected patient plasma, and detection of binding to infected and uninfected MoDCs by flow cytometry.
  • PBMC peripheral blood mononuclear cells
  • MoDCs were infected (0.5 x 106/well in 12 well plates) at MOI 5000 using hAd5 S- WT, SFusion, S-Fusion + N-ETSD or a ‘Null’ construct that expresses green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the MoDCs were detached using EDTA (0.5 mM), gently pipetted and transferred for incubation with previously infected patient plasma at 1:100 dilution from a single patient (Pt4) at (4°C) for 30 minutes, and plasma antibodies detected on the MoDC surface by goat anti-human IgG (phycoerythrin conjugated). Cells were acquired as described above for flow cytometric analyses.
  • IFN-g spot forming cells enumerated by ELISpot.
  • ELISpot detection after aspiration and washing to remove cells and media, IFN-g was detected by a secondary antibody to cytokine conjugated to biotin.
  • a streptavidin/horseradish peroxidase conjugate was used detect the biotin-conjugated secondary antibody. The number of spots per well (1 x 10 5 cells), was counted using an ELISpot plate reader.
  • IL-4 was measured by ELISpot using a kit (MabTech) with wells precoated with antiIL-4 antibody and following the manufacturer’s instructions. Remaining steps for IL-4 detection were identical to those for IFN-g, but with alkaline phosphatase detection rather than peroxidase.
  • N-ETSD and/or S-Fusion or proteins with similarity to N-ETSD and/or S-Fusion
  • the N-ETSD and/or S-Fusion can also be expressed in yeast, either on yeast cells that present the antigen(s) or within yeast cells that may be prepared as lysates to so further enhance immunogenicity as is discussed in more detail below.
  • FIG.12 depicts a conceptual illustration of an ideal vaccine that will elicit durable and elfective immunity across multiple pathways.
  • a vaccine composition is preferably (but not necessarily) composed of a yeast vaccine composition and a viral vaccine composition, optionally in concert with a macrophage polarizing agent such as RP182 and an immune stimulatory cytokine or cytokine analog (e.g., N-803).
  • a macrophage polarizing agent such as RP182
  • an immune stimulatory cytokine or cytokine analog e.g., N-803
  • the yeast is a recombinant yeast that expressed from a recombinant nucleic acid one or more antigens of interest, and possibly further DAMP or PAMP or STING pathway signals.
  • the so produced recombinant yeast is typically heat inactivated, and after heat-inactivation the yeast is lysed.
  • the yeast is lysed using pressure homogenization and the homogenate is then clarified, preferably via filtration.
  • other methods of lysing are also deemed suitable, including enzymatic or chemical lysing of the cells wall, sonication, flash- freezing/thawing, etc.
  • clarification of the lysate may also include centrifugation, settling, flocculation, etc.
  • FIG.13 depicts results of an exemplary expression experiment where the SARS CoV nucleocapsid protein (N) is overexpressed in Saccharomyces cerevisiae. As can be seen, significant quantities of the recombinant protein were expressed in the yeast.
  • lysed tarmogen (yeast with recombinantly expressed antigen) was up to 150-fold more immunogenic than intact tarmogen with regard to induction of T cell responses (based on numerous antigen specific T cell activation studies in hundreds of mice).
  • the lysed yeast has a favorable safety profile and 10,000 10 YU doses can be prepared in 2 hours one small-scale pressure homogenizer.
  • Intactyeast are Thl-Thl7 inducing and trigger IFNy, IL2, IL12, IL6, TNFa, GM-CSF, while yeast lysates clearly induced strong Thl eflects and diminished Thl 7.
  • compositions of and methods for producing a vaccine composition using aragonite to form a solid dosage form e.g ., powder, tablet, or capsule
  • a solid dosage form e.g ., powder, tablet, or capsule
  • dissolves e.g., releases the antigenic or pre-anti genic vaccine molecule(s) after passing through the stomach of the subject receiving the vaccine.
  • the present disclosure is directed to an aragonite composition made of a plurality of aragonite particles loaded with vaccine active ingredient(s) rendering a solid dosage vaccine in the form of a powder, tablet, or capsule.
  • the vaccine composition may include a powder form (e.g., lyophilized) recombinant expression construct for expressing a corresponding antigen to the relevant infection/disease that has been blended with and thereby loaded on, the surface of the plurality of aragonite particles.
  • the vaccine composition immunizes against a coronavirus.
  • the recombinant expression construct is an adenovirus construct expressing at least one antigenic coronavirus protein or protein fragment.
  • the use of aragonite in the presently contemplated solid dosage form allows for cost effective manufacturing and easy administration of a stable vaccine composition.
  • the presently contemplated vaccine tablet or capsule can be mass produced and easily transported.
  • the solid dosage form allows for oral administration which for most persons can be self-administered without the need for a healthcare professional.
  • the tablet forms may also be made with additional excipients and/or additives (e.g., flavors and gelatins) to form a lozenge.
  • Aragonite e.g., oolitic aragonite
  • Aragonite is one of the purest forms of naturally precipitated calcium carbonate.
  • aragonite has a crystalline morphology of orthorhombic, bipyramidal, characteristically needle-shaped crystals, and as such is distinct from calcite and vaterite.
  • Aragonite can be processed to recrystallize and/or reform in various shapes, such that it can be used for various purposes that take advantage of the mechanical and chemical properties of the calcium carbonate minerals.
  • Aragonite particles as disclosed herein are solid matter having a regular (e.g., spherical, or ovoid) or irregular shape.
  • aragonite particles have an average particle size of between 100 nm to 1 mm.
  • Methods for milling aragonite particles are described in US 2020/0308015, the entire contents of which are herein incorporated by reference.
  • methods for milling aragonite particles are disclosed of 2.0 to 3.5 micron size with a clean top size.
  • a clean top size means that very few particles are larger than the 3.5 micron size when produced using the disclosed milling method with a classifier set at 2.0 to 3.5 micron or 2.5 to 3.5 micron size range.
  • aragonite particles as disclosed herein using the methods of U.S. 2020/0308015 have a cleaner top size than conventional GCC.
  • Aragonite s adsorption capacity is a function of three parameters: (1) surface charge (also known as “z (zeta) potential”); (2) surface area/void ratio; and (3) particle solubility.
  • surface charge also known as “z (zeta) potential”
  • surface area/void ratio also known as “z (zeta) potential”
  • particle solubility By accurately measuring these three parameters, one can determine what materials will adsorb to aragonite particle surfaces under given conditions.
  • the zeta potential of aragonite increases the stability of surfactants such as glycerol and sorbitol.
  • aragonite has a naturally high number of measurable pores in particles with diameters less than 2 nm (i.e., a high “microporosity”). See, e.g., EP 2719373.
  • the aragonite platform grips active ingredient particles strongly together allowing for the loaded aragonite to be formulated in a solid dosage form — e.g., powder, tablets, or capsules.
  • untreated aragonite has a neutral pH (7.8 to 8.2), a natural hydrophilic nature, electron charge (zeta potential), and already created nitrogenous pairing with amino acids and proteins.
  • these advantageous properties of aragonite render aragonite metastable under ambient conditions.
  • aragonite particles naturally include approximately 2-3% amino acid content, the majority of which are aspartic acid (approximately 25 to 30%) and glutamic acid (approximately 8 to 10%) rendering the aragonite surface hydrophilic.
  • a vaccine composition e.g., recombinant adenovirus
  • a vaccine composition is coupled directly to the natural, untreated surface of aragonite particles.
  • GCC ground calcium carbonate
  • PCC precipitated calcium carbonate
  • limestone production is a commodity grade with different attributes.
  • GCC ground calcium carbonate
  • PCC precipitated calcium carbonate
  • SCD particle sized distribution
  • aragonite refers to naturally occurring aragonite having a crystalline morphology of orthorhombic, bipyramidal, and characteristically needle-shaped crystals that is distinct from GCC, PCC, and limestone. For example, ball milled aragonite using the system and methods disclosed in U.S.
  • a clean top size means that very few particles are larger than the 3.5 micron size when produced using this system and method with a classifier set at 2. to 3.5 micron size range or 2.0 to 3.5 micron size range.
  • aragonite produced in this set range using the disclosed system only ⁇ 0.0005% are retained on a 325 mesh and only slightly more ⁇ 0.0007% are retained on a 500 mesh, as compared to a GCC product having the same median (D50) particle size distribution (PSD). Accordingly, aragonite produced using the contemplated system and methods have a cleaner top size than conventional GCC.
  • the solid dosage form made of aragonite provides a solid vaccine form capable of being ingested by oral administration.
  • the presently disclosed solid form having an enteric coating is ingested and the antigenic molecules loaded in the inner core are not released until after passing through the stomach, thereby allowing for absorption of the antigenic or pre- antigenic molecules into the bloodstream and delivery to immune cells.
  • antigenic molecule refers to the desired vaccine active ingredient(s) blended with and loaded on the surface of aragonite particles.
  • antigenic molecules may be antigenic in the form loaded on the aragonite particles or they may be a molecule or molecules ( e.g ., an expression vector) that are capable of producing (e.g., expressing) at least one antigenic protein or fragment.
  • the active ingredient or vaccine active ingredient as disclosed herein is referred to as an antigenic molecule which includes both antigenic molecules and pre-antigenic molecules, unless specified otherwise.
  • the aragonite vaccine composition includes: i) an inner core made of aragonite and the specific antigenic molecules; ii) an outer core of aragonite that completely surrounds the inner core such that the entire outer surface of the inner core is in contact with only the outer core and no surface of the inner core is exposed; and iii) a coating covering all the outer surface of the outer core.
  • the inner core is made of aragonite particles having a diameter of at least 2 um or greater thereby providing a surface area for capturing and loading the specific antigenic molecules thereon.
  • the outer core does not include any antigenic molecules and is made of 90% to 100% aragonite. More preferably, the outer core comprises only 100% aragonite.
  • the outer coating of the solid composition may be any suitable coating (e.g., enteric coating) that is stable in the highly acidic, low pH environment of the stomach (e.g., at a pH of approximately 3) and dissolves in the higher pH of the small intestine (e.g., at a pH of approximately 7 to 9).
  • suitable examples of enteric coatings include biopolymer dispersions such as methacrylic acid, ethyl acrylate, and/or a plasticizer/stabilizer (e.g., triethyl citrate (TEC)).
  • an anti-tacking agent may also be combined with the enteric coating — e.g., glycerol monostearate.
  • the inner core is made of aragonite particles having a diameter of at least 2 um or greater (e.g., 2 to 3.5 um) that have been blended with a lyophilized powder of the antigenic molecules.
  • additional excipients are blended with the aragonite and antigenic molecules.
  • dimethyl glycine and/or methylsulfonylmethane (MSM) may be combined with the lyophilized powder of antigenic molecules and blended with the aragonite particles.
  • the lyophilized antigenic molecules comprise a lyophilized recombinant expression vector having nucleic acids corresponding to (i.e., encoding) at least one antigenic protein or optimized protein or fragment thereof of the SARS-coronavirus 2 (SARS-CoV-2 or CoV2).
  • SARS-CoV-2 or CoV2 SARS-coronavirus 2
  • the contemplated solid dosage form vaccine composition disclosed herein may encode an antigen of at least one of the nucleoprotein (N) protein and the spike (S) protein of the coronavirus 2 virus (CoV2), both of which are conserved in all types of coronaviruses.
  • the antigen encoding molecules are a lyophilized recombinant entity, wherein the recombinant entity comprises a nucleic acid that encodes the nucleocapsid protein of CoV2 or a fragment thereof, and/or wherein the recombinant entity encodes the spike protein of CoV2 or fragment thereof.
  • the vaccine formulation may be useful for treating a disease, such as a coronavirus mediated disease or infection.
  • a method for treating a coronavirus disease in a patient in need thereof, wherein the method includes: administering to the subject the solid dosage form vaccine composition comprising the recombinant entity comprising a nucleic acid that encodes at least the CoV2 N protein or fragment thereof, and preferably encodes both the CoV2 N protein or fragment thereof and the CoV2 S protein or fragment thereof.
  • the coronavirus contemplated herein may be coronavirus disease 2019 (COVID-19) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • the contemplated solid dosage form vaccine composition disclosed herein comprises aragonite blended with a lyophilized recombinant entity made of a bivalent human adenovirus serotype 5 (hAd5) expression vector.
  • the hAd5 is capable of inducing immunity in patients with pre-existing adenovirus immunity and expresses antigens for producing antibodies that target the coronavirus 2 spike (S) protein and/or nucleocapsid (N) protein.
  • the hAd5 CoV2 may encode a modified nucleocapsid protein having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.
  • the hAd5 includes both an S sequence optimized for cell surface expression (S- Fusion) and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal subcellular compartment, with the potential to generate durable immune protection against CoV2, as disclosed in U.S. Application No. 16/883,263, the entire contents of which are herein incorporated by reference.
  • the hAd5 CoV2 may encode a S-Fusion or S-HA protein having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:4 or SEQ ID NO:3, respectively.
  • the bivalent hAD5 vaccine provides: (i) optimized S-Fusion having improved S receptor binding domain (RBD) cell surface expression as compared to S-WT (wild type) where little surface expression was detected; (ii) the expressed RBD from S-Fusion retained conformational integrity and recognition by ACE2-Fc; (iii) the viral N protein modified with an enhanced T-cell stimulation domain (ETSD) localized to endosomal/lysosomal subcellular compartments for MHC I/II presentation; and (iv) these optimizations to S and N (S-Fusion and N-ETSD) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T-cell responses in antigen-naive pre-clinical models as is shown in more detail below.
  • RBD S receptor binding domain
  • the lyophilized bivalent hAd5 vaccine comprises a replication defective adenovirus having an El, an E2b, and an E3 gene region deletion along with a nucleic acid encoding a coronavirus 2 (CoV2) nucleocapsid (N) protein fused to an endosomal targeting sequence (N-ETSD), and a nucleic acid encoding a CoV2 spike (S) protein sequence optimized for cell surface expression (S-Fusion).
  • CoV2 coronavirus 2
  • S CoV2 spike
  • the nucleic acid encoding the CoV2 N-ETSD protein in the hAd5 adenovirus has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2. More typically, the CoV2 N-ETSD protein encoded in the hAd5 adenovirus has a least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.
  • the nucleic acid encoding the CoV2 S-HA or S-Fusion protein in the hAd5 adenovirus has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5 or SEQ ID NO: 6, respectively. More typically, the CoV2 S-HA or S-Fusion protein encoded in the hAd5 adenovirus has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3 or SEQ ID NO: 4.
  • the lyophilized bivalent hAd5 vaccine may also include a nucleic acid encoding a trafficking sequence, a co-stimulatory molecule, and/or an immune stimulatory cytokine.
  • the encoded co-stimulatory molecule include one or more of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3.
  • the immune stimulatory cytokine include one or more of IL-2, IL-12, IL-15, IL-15 super agonist (N803), IL- 21, IPS1, and LMP1.
  • a corona virus vaccine composition made of aragonite particles blended with a lyophilized form a viral vector (e.g ., a lyophilized powder composition comprising a recombinant adenovirus genome, optionally with a deleted or non-functional E2b gene) that comprises a nucleic acid that encodes (a) at least a wild-type or modified nucleocapsid protein; and/or (b) at least one wild-type or modified spike protein.
  • the viral vector may further comprise co-stimulatory molecule.
  • the nucleic acid encodes a trafficking signal to direct a peptide product encoded by the nucleic acid to the cytoplasm, the endosomal compartment, or the lysosomal compartment, and the peptide product will further comprise a sequence portion that enhances intracellular turnover of the peptide product.
  • contemplated methods for making the disclosed solid dosage form vaccine include forming the inner core of aragonite and the vaccine active ingredients by loading the vaccine active ingredients (e.g., by mixing) the milled aragonite particles as disclosed herein and in U.S. 2020/0308015, the entire contents of which are herein incorporated by reference. Any suitable method of blending the lyophilized vaccine active ingredients with the aragonite particles may be used.
  • Loading or mixing of milled aragonite particles (e.g., having a D50 PSD of 2 um to 3.5 um) with the lyophilized vaccine active ingredients may be carried out by any conventional methodology.
  • the mixing of the aragonite with the active ingredients includes mixing dry weights of both the aragonite and the lyophilized active ingredients into a closed container suitable for rotation/inversion.
  • inversion or rotation of the aragonite and active ingredients may be carried out for about 5 minutes up to 30 minutes.
  • the vaccine composition may be loaded onto the solid dosage form in a mixer (e.g., tumbling mixer) or a blender.
  • the amount of aragonite may vary depending on the amount of lyophilized active ingredients and a determined titer of the active ingredients for inducing an immune response.
  • an effective dose of lyophilized bivalent hAd5 vaccine as disclosed herein may be about 1 x 10 9 IU per capsule (or tablet).
  • a lyophilized bivalent hAd5 vaccine composition having a titer of 2.21 x 10 7 IU/mg requires about 45.25 mg of lyophilized bivalent hAd5 powder per capsule.
  • the weight of the solid single capsule may be of between about 300 mg to 600 mg.
  • lyophilized active ingredient e.g ., lyophilized bivalent hAD5 expression both N and S CoV2 proteins
  • 490 to 510 mg aragonite for an active ingredient having a titer of 2.21 x 10 7 IU/mg, 45.25 mg of the lyophilized active ingredient may be blended with about 504.75 mg aragonite.
  • the tablet or caplet formation of the mixture may be carried out using any suitable encapsulation method and/or kit known in the art. For example, the Capsule Machine Filler (Item # CPMlOOl/2081677).
  • the contemplated method for making a solid dose vaccine tablet or capsule also includes making an outer core of aragonite that encompasses (e.g., completely encloses) the inner core.
  • the outer core is made of mostly (e.g., at least 90%) aragonite and more typically, the outer core is made of at least 99% aragonite.
  • the antigenic molecules In order to deliver the antigenic molecules to the bloodstream in a tablet or capsule for oral administration, the antigenic molecules must remain in the inner core encompassed by the outer core until they have passed through the stomach and are available for absorption by in the intestines (e.g., the small intestines). Accordingly, the outer core is coated with an enteric coating that is stable in the low pH of the stomach (e.g., at a pH of approximately 3) and dissolves in the higher pH of the intestines (e.g., at a pH of approximately 7 to 9).
  • the outer coating of the solid composition may be any suitable enteric coating.
  • enteric coating examples include methacrylic acid and/or ethyl acrylate polymers in tri ethyl citrate (TEC).
  • TEC tri ethyl citrate
  • Methods for applying an enteric coating are well known in art.
  • a coating device or apparatus may be used.
  • Specific examples of a coating device include the ProCoater (manufactured by Torpac).
  • Coated (C) and non-coated (NC) capsules made of aragonite particles or lactose mixed with hAD5-COVID-S/N were exposed to acid (HC1) to determine the acid permeability of the various capsules.
  • FIG.2 shows three photographs (right to left: 1, 2, 3) of bivalent human adenovirus serotype 5 COVID-Spike and Nucleocapsid antigen vaccine (hAD5-COVID- S/N) in a non-coated aragonite capsule (Sample #6) in 0.1 M hydrochloric acid (HCL) with observed wrinkling, swelling, or a hole in capsule as indicated: 1): 2 minutes post HCL acid exposure; 2): 2 hours post HCL acid exposure; and 3) 2 hours post HCL acid exposure and dried.
  • HCL hydrochloric acid
  • FIG.3 shows three photographs (right to left: 1, 2, 3) of hAD5-COVID-S/N in a non-coated aragonite capsule (Samples #7 or #8) in 0.1 M HCL with observed swelling, twisting, or a hole in capsule as indicated: 1): Sample #7 at 2 hours post HCL acid exposure; 2): Sample #8 at 2 hours post HCL acid exposure; 3) At 2 hours post HCL acid exposure and dried.
  • FIG.4 shows two photographs (right to left: 1, 2) of hAD5-COVID-S/N in a non-coated lactose capsule (Samples #3 or #4) in 0.1 M HCL with observed swelling of capsule as indicated: 1): Sample #3 at 2 hours post HCL acid exposure; 2): Sample #4 at 2 hours post HCL acid exposure.
  • FIG.5 shows two photographs (right to left: 1, 2) of hAD5-COVID-S/N in a coated aragonite capsule (Samples #1 or #5) in 0.1 M HCL with observed swelling of capsule as indicated: 1): Sample #1 at 2 hours post HCL acid exposure; 2): Sample #5 at 2 hours post HCL acid exposure.
  • the coated capsule was stable in acid.
  • FIG.6 shows Infectious Units per Gram (IFU/Gram) (y-axis) for indicated hAD5-COVID-S/N Capsule Type, as indicated.
  • FIG.7 shows the percentage (%) of Virus Recovery (y-axis) for each hAD5-COVID-S/N Capsule Type as indicated.
  • FIG.8 shows IFU/Gram for each hAD5-COVID-S/N Capsule Type and corresponding pH as indicated.
  • FIG.9 shows the percentage (%) of Virus Recovery for each hAD5-COVID-S/N Capsule Type, as indicated.
  • FIG.10 shows Percent Virus Recovered for each hAD5-COVID-S/N Capsule Type as indicated, with acid treatment indicated for those with shading.
  • FIG.ll shows Infectious Units/gram for each hAD5-COVID-S/N Capsule Type as indicated, with acid treatment indicated for those with shading. The results are also summarized in Table 2 below.
  • the contemplated dosage form includes an inner core of aragonite impregnated with (i.e., coupled with) carbon dioxide (C02) prior to the addition of the vaccine composition. See, e.g., EP 2719373 and US 2020/0155458.
  • the contemplated dosage form includes aragonite with a biocompatible polymer and/or a disintegrating agent mixed and processed with the aragonite prior to the addition of the vaccine composition.
  • aragonite is impregnated with C02 and mixed with both a biocompatible polymer and a disintegrating agent prior to the addition of the vaccine composition.
  • aragonite is impregnated with C02, mixed with a biocompatible polymer and a disintegrating agent, and formed (e.g., compressed) into a solid form prior to the addition of the vaccine composition. See, e.g, EP 2719373 and US 2020/0155458.
  • the aragonite may be coupled with carbon dioxide (C02) and mixed with at least one biocompatible polymer.
  • C02 carbon dioxide
  • the weight ratio of C02-coupled aragonite to the biocompatible polymer is from about 95:5 to 5:95.
  • the biocompatible polymer is a hot melt extruded biocompatible polymer.
  • biocompatible polymers include polylactic acid (PLA), polyethylene, polystyrene, polyvinylchloride, polyamide 66 (nylon), polycaprolactame, polycaprolactone, acrylic polymers, acrylonitrile butadiene styrene, polybenzimidazole, polycarbonate, polyphenylene oxide/sulfide, polypopylene, teflon, polylactic acid, aliphatic polyester such as polyhydroxybutyrate, poly-3- hydroxybutyrate (P3HB), polyhydroxyvalerate, polyhydroxybutyrate-polyhydroxyvalerate copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyglyconate, poly(dioxanone) and mixtures thereof.
  • PLA polylactic acid
  • PEG polyethylene
  • polystyrene polyvinylchloride
  • polyamide 66 nylon
  • polycaprolactame polycaprolactone
  • acrylic polymers acrylonitrile buta
  • the biocompatible polymer resin is PLA.
  • Additional excipients may also be added to the inner core and/or the outer core of the solid dosage form as determined by the manufacturing and packaging needs. Additional excipients may include ion exchange resins, gums, chitin, chitosan, clays, gellan gum, crosslinked polacrillin copolymers, agar, gelatine, dextrines, acrylic acid polymers, carboxymethylcellulose sodium/calcium, hydroxpropyl methyl cellulose phthalate, shellac or mixtures thereof, lubricants, inner-phase lubricants, outer-phase lubricants, impact modifiers, plasticizers, waxes, stabilizers, pigments, coloring agents, scenting agents, taste masking agents, flavoring agents, sweeteners, mouth-feel improvers, binders, diluents, film forming agents, adhesives, buflers, adsorbents, odor- masking agents and mixtures thereof
  • the aragonite particle surface may be treated to modify the binding surface.
  • treatment with stearic acid i.e., octadecanoic acid
  • octadecanoic acid provides for a hydrophobic surface, as disclosed in U.S. 16/858,548 and PCT/US20/29949.
  • treatment of the aragonite with phosphoric acid forms lamellar structures. Additional conjugation techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art as disclosed, for example, in Bioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013.
  • the present disclosure also provides methods and compositions for administering, monitoring, and assaying a vaccine.
  • the contemplated methods include inducing immunity against a virus in a patient, administering a vaccine composition to the patient by administering a vaccine composition to the patient by delivery to the nasal mucosa, oral mucosa, and/or alimentary mucosa of the patient.
  • the vaccine targets SARS-like coronavirus (SARS-CoV-2).
  • SARS-CoV-2 SARS-like coronavirus
  • the oral vaccine compositions described herein can serve as a booster vaccination to any initial prime vaccination against SARS-CoV-2 S or N protein.
  • the oral vaccine compositions described herein can be used as a booster vaccine to any anti-SARS-CoV-2 vaccine directed against the SARS-CoV-2 spike (S) and/or nucleocapsid (N) proteins.
  • This booster can work even in patients who were immunized with an anti-S or anti-N vaccine other than those described herein.
  • the initial prime vaccine can be a lipid nanoparticle vaccine containing mRNA encoding the S protein, such as those vaccines currently being tested by Modema and by Pfizer.
  • the boost described herein is administered at least 7 days after the initial prime vaccination, for example at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 28 days, at least 35 days, or at least 42 days.
  • the boost as described herein can effectively improve both antibody production against SARS-CoV-2 and cell-mediate immunity against SARS-CoV-2.
  • the vaccine administered for inducing immunity in the mucosal tissue of a patient is a vaccine against SARS-CoV-2.
  • the vaccine a replication defective adenovirus construct, comprising an El gene region deletion and an E2b gene region deletion.
  • the adenovirus comprises a sequence (e.g. SEQ ID NO: 12) encoding a SARS-CoV-2 spike fusion protein antigen with at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) primary sequence identity to SEQ ID NO:4.
  • the adenovirus comprises a sequence (e.g.
  • SEQ ID NO: 13 encoding a SARS-CoV-2 modified spike protein antigen with at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) primary sequence identity to SEQ ID NO: 11.
  • the adenovirus includes a sequence encoding a soluble ACE2 protein coupled to an immunoglobulin Fc portion, forming an ACE2-Fc hybrid construct that may also include a J-chain portion, as disclosed in U.S. 16/880,804 and U.S. 63/016,048, the entire contents of both of which are herein incorporated by reference.
  • the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a mutant variant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10), wherein the mutant variant has at least one mutated amino acid residue (e.g., by substitution) that imparts an increased binding affinity of the ACE2 protein for the RBD protein domain of the SARS-CoV-2 spike protein as disclosed in U.S. 63/022,146, the entire content of which is herein incorporated by reference.
  • a mutant variant of a recombinant soluble ACE2 protein e.g., SEQ ID NO: 10
  • the mutant variant has at least one mutated amino acid residue (e.g., by substitution) that imparts an increased binding affinity of the ACE2 protein for the RBD protein domain of the SARS-CoV-2 spike protein as disclosed in U.S. 63/022,146, the entire content of which is herein incorporated by reference.
  • the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a CoV2 nucleocapsid protein or a CoV2 spike protein fused to an endosomal targeting sequence (N- ETSD), as disclosed in U.S. 16/883,263 and U.S. 63/009,960, the entire contents of both of which are herein incorporated by reference.
  • the SARS-CoV- 2 vaccine includes modified yeast cells (e.g., Saccharomyces cerevisiae) genetically engineered to express coronaviral spike proteins on the yeast cell surface thereby creating yeast presenting cells to stimulate B cells (e.g., humoral immunity) as disclosed in U.S. 63/010,010.
  • yeast cells e.g., Saccharomyces cerevisiae
  • B cells e.g., humoral immunity
  • Serum samples drawn at the indicated time points from these macaques was then assessed by ELISA for anti-spike protein IgG and IgM seroreactivity. Briefly, 96 well EIA/RIA plates (ThermoFisher, Cat#07-200-642) were coated with 50 pL/well of 1 pg/mL solution of purified recombinant SARS-CoV-2-derived Spike protein (S-Fusion. ImmunityBio, Inc.) suspended in coating buffer (0.05 M Carbonate-Bicarbonate, pH 9.6) and incubated overnight at 4°C.
  • coating buffer 0.05 M Carbonate-Bicarbonate, pH 9.6
  • FIG.16 Viral load over time in the nasopharynx is shown in FIG.16.
  • Panel A shows viral load (qPCR) in nasal swabs from Group 1 macaques following SC+SC+oral vaccination
  • Panel B shows viral load (qPCR) in nasal swabs from Group 1 macaques following SC+oral+oral vaccination.
  • Viral load over time in the lungs (BAL) is shown in FIG.17.
  • FIG.17 Panel A shows viral load (qPCR) in BAL from Group 1 macaques following SC+SC+oral vaccination
  • Panel B shows viral load (qPCR) in BAL from Group 1 macaques following SC+oral+oral vaccination.
  • Serum samples from various human volunteers who have received various experimental anti-S ARS-CoV -2 vaccines were collected and assayed by ELISA as described above for IgG and IgM seroreactivity against SARS-CoV-2 S protein. The results are shown in FIG.18 with ELISA results detecting IgG & IgM seroreactivity against SARS-CoV-2 spike in sera samples drawn from human patients immunized with various experimental anti-SARS-CoV-2 vaccines.
  • Cohort 1 (10 individuals) was immunized by subcutaneous injection with 5xl0 10 viral particles of a vaccine as described herein (El-/E2b- Ad5 containing SEQ ID NO: 12 or SEQ ID NO: 13).
  • Cohort 2 (10 individuals) was immunized by subcutaneous injection with 10 11 viral particles of a vaccine as described herein.
  • Cohort 3 (15 individuals) was immunized by subcutaneous injection with 10 11 viral particles of a vaccine as described herein (or 5xl0 10 viral particles if safety concerns indicated a lower dose). Blood was drawn from each volunteer on the same day as the initial prime vaccination was administered. Blood was drawn again on days 8, 15, & 22. A booster injection of the same vaccine was administered on day 22.
  • ELISpot tests were run on the blood collected on days 1 & 15 to assess cell-mediated immunity against SARS-CoV-2. 400,000 viable PBMCs from each blood draw per well (Cellometer K2 w/ AO/PI viability stain) were stimulated with empty medium, SARS-CoV-2 S, SARS-CoV-2 N, SARS-CoV-2 M, CD3/CD28/CD2, and CEFT. After 48 hrs of stimulation, supernatants were frozen (-80°C) for later testing.
  • FIG.19 shows the results of the ELISpot test from Thl N-responsive patients 3, 6, & 11.
  • FIG.20 shows results from patient 4 (N-unresponsive) and patient 10 (weakly Thl N-responsive). None of these patients showed a Th2 response to N.
  • the contemplated vaccine formulations and methods of use afforded protection of nasal and lung airways against SARS-CoV-2 challenge in a non-human primate.
  • a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike (S-Fusion) protein and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) increases MHC class I/II responses.
  • S-Fusion modified SARS-CoV-2 spike
  • N-ETSD Enhanced T-cell Stimulation Domain
  • the adenovirus serotype 5 platform used, hAd5 [E1-, E2b-, E3-] previously demonstrated to be effective in the presence of Ad immunity, can be delivered in an oral formulation that overcomes cold-chain limitations.
  • the hAd5 S-Fusion + N-ETSD vaccine was evaluated in rhesus macaques showing that a subcutaneous prime followed by oral boosts elicited both humoral and Thl dominant T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 10 6 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.
  • FIG.25 depicts the hAd5 platform and the hAd5 S-Fusion + N-ETSD construct.
  • the human adenovirus serotype 5 vaccine platform with El, E2b, and E3 regions deleted (*) is shown.
  • the vaccine construct is inserted in the El regions (red arrow).
  • the dual-antigen vaccine comprises both S-Fusion and N-ETSD under control of cytomegalovirus (CMV) promoters and with C-terminal SV40 poly-A sequences delivered by the hAd5 [E1-, E2b-, E3-] platform.
  • CMV cytomegalovirus
  • the dual-antigen hAd5 S-Fusion + N-ETSD vaccine of FIG.25 expresses a viral spike (S) protein (S-Fusion) fused to a signal sequence that, as predicted based on reports for similar sequences, in the in vitro studies enhances cell-surface expression of the spike receptor binding domain (S RBD) as compared to S wildtype, the antigen used in the majority of other vaccines being developed.
  • This vaccine also expresses the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) that directs N to the endo/lysosomal subcellular compartment which is predicted to enhance MHC class II responses.
  • the S ARS-CoV -2 vaccine antigens are delivered by an recombinant human adenovirus serotype 5 (hAd5) [E1-, E2b-, E3-] vector platform (FIG.25) can rapidly generate vaccines against multiple agents, allowing production of high numbers of doses in a minimal time frame.
  • the hAd5 platform has unique deletions in the early 1 (El), early 2 (E2b) and early 3 (E3) regions (hAd5 [E1-, E2b-, E3-]), which distinguishes it from other adenoviral vaccine platform technologies under development, and allows it to be effective in the presence of pre-existing adenovirus immunity.
  • This platform produces vaccines against viral antigens such as Influenza, HIV-1 and Lassa fever and have shown induction of both antibodies and cell mediated immunity.
  • the vaccination of mice with the hAd5 [E1-, E2b-, E3-] vector expressing H1N1 hemagglutinin and neuraminidase genes elicited both cell-mediated immunity and humoral responses that protected the animals from lethal virus challenge.
  • the overwhelming maj ority of other S ARS-CoV -2 vaccines in development target only the wildtype S antigen and are expected to elicit SARS-CoV-2 neutralizing antibody responses.
  • the development of the vaccine prioritized the activation T cells to enhance the breath and duration of protective immune responses; the addition of N in particular was predicted to afford a greater opportunity for T cell responses.
  • T cells may provide immune protection at least as important as the generation of antibodies.
  • virus-specific T cells were seen in most patients, including asymptomatic individuals, even those with undetectable antibody responses.
  • T cells from SARS-CoV-2 convalescent individuals ‘recall’ the S-Fusion and N-ETSD antigens presented by transduced MoDCs as if they were re- exposed to the virus itself.
  • This T-cell recall of vaccine antigens suggests that, conversely, hAd5 S-Fusion + N-ETSD vaccination will generate T cells that will recognize SARS-CoV-2 antigens upon viral infection and protect the vaccinated individual from disease.
  • T-cell responses may be a critical feature for a vaccine to be efficacious against the many variants whose emergence, at least in part, may be an escape response to antibodies generated by either first wave virus (28) or by antibody-based vaccines.
  • first wave virus 228 or by antibody-based vaccines.
  • neutralization by 14 of 17 of the most potent mRNA vaccine-elicited monoclonal antibodies (mAbs) was either decreased or abolished variants E484K, N501Y or the K417N:E484K:N501Y combination.
  • Compelling additional advantages of a thermally stable oral boost are that it would likely transform the global distribution of vaccines, especially in developing countries and potentially enable patients to self-administer the boost(s) at home. Because the hAd5 S-Fusion + N-ETSD construct induces both humoral and CMI responses to both antigens, it also has the potential to serve as a ‘universal’ heterologous booster vaccine to the multitude of SARS- CoV-2 vaccines under development.
  • the goals of the study were to assess the immunogenicity of a dual-antigen hAd5 vaccine in both SC and oral formulations, and the potential of oral dose to serve as a boost following a single SC prime.
  • Cell-mediated T-cell response and protection of nasal passages and lung from SARS-CoV-2 infection after challenge was assessed, as well as the rate of viral clearance.
  • Anti-S IgG production was similar for SC-SC-Oral (where the first boost was SC) hAd5 S Fusion + N-ETSD vaccinated NHP (Panels D and E) and sera from all five NHP in this group demonstrated inhibition in the surrogate assay for viral neutralization (Panel F).
  • SC prime, oral boost vaccination reduces viral load in nasal passages and lung after SARS CoV-2 challenge: RT-qPCR analysis of genomic RNA (gRNA) was performed on nasal swab and bronchoalveolar lavage (BAL) samples to determine the amount of virus present.
  • Viral gRNA in this group continued to diminish to levels that were very low or below the level of detection (LOD) in all vaccinated animals by Day 63, 7 days after challenge.
  • LOD level of detection
  • Placebo controls had moderate to high levels (range 2E+09 - 8.4E+03 gene copies/mL) of SARS-CoV-2 present in nasal swab samples for the duration of the study.
  • gRNA In the lungs (bronchoalveolar lavage, BAL) of SC-Oral-Oral NHP, gRNA also decreased rapidly, with the geometric mean showing a ⁇ 2 log decrease in vaccinated NHP compared to placebo NHP at Day 57, just one day after challenge (FIG.22, Panels C and D).
  • SARS-CoV-2 gRNA in nasal swab samples was also reduced similarly to that seen in SC-Oral-Oral vaccinated primates, with viral gRNA decreasing to levels that were very low or below the LOD in all vaccinated animals by Day 63 (FIG.22, Panels E and F).
  • gRNA In the lungs of SC-SC-Oral NHP, gRNA also showed a ⁇ 2 log decrease on Day 57 (FIG.22, Panels G and H).
  • SC prime, oral boost vaccination immediately inhibited viral replication 188 in nasal passages and lung after SARS-CoV-2 challenge: The presence of replicating virus in nasal swab samples was determined by RT qPCR of subgenomic RNA (sgRNA).
  • sgRNA subgenomic RNA
  • sgRNA was below the LOD for two SC-Oral-Oral primates and, starting on Day 61, below the LOD for all primates that received only oral boosts (FIG.23, Panels A and B).
  • sgRNA In the lungs of SC-Oral-Oral NHP, sgRNA also decreased as compared to placebo starting at Day 57 and was below the LOD in all by Day 63 (FIG.23, Panels C and D).
  • PBMC peripheral blood mononuclear cell
  • T cells from SC-Oral-Oral vaccinated primates secreted interferon-gamma (IFN-g) in response to both S and N peptides on Days 14 and 35 (FIG.24, Panel A).
  • Interleukin-4 (IL-4) secretion was very low (FIG.24, Panel B), indicating the T-cell responses were T helper cell 1 (Thl) dominant, as reflected by the IFN-y/IL-4 ratio (FIG.24, Panel C).
  • the potential of the hAd5 S-Fusion + N-ETSD SC prime, oral boost vaccine to generate cytotoxic T cells is a key feature, given the critical role of T cells play in protection from infection in COVID-19 convalescent patients where SARS-CoV-2 specific T cells were identified even in the absence of antibody responses.
  • the apparent nearly immediate reduction of viral replication by the hAd5 S-Fusion + N-ETSD vaccination is in contrast to the reported findings for other adenovirus-vectored S-only vaccine NHP studies, wherein there was evidence of continued viral replication in some animals for at least a day after challenge.
  • hAd5 S-Fusion + N-ETSD vaccination of NHPs by SC and oral boost administration particularly reveal the potential for this vaccine to be developed for worldwide distribution, especially in light of the escape variants resistant to antibodies and convalescent plasma, now rapidly spreading throughout the world.
  • the oral hAd5 S-Fusion + N- ETSD formulation would not require ultra-cold refrigeration like many COVID vaccines currently in development.
  • Dependence on the cold-chain for distribution to geographically remote or under developed areas causes shipping and storage challenges and will likely reduce the accessibility of the RNA-based COVID-19 vaccines.
  • thermally stable oral hAd5 S-Fusion + N-ETSD vaccine due its expression of S and N, also has the potential to act as a ‘universal’ boost to other previously administered vaccines that deliver only S antigens. This use would also be facilitated by cold-chain independence.
  • kits for administering, monitoring, and assaying a vaccine.
  • the contemplated methods include inducing immunity against a virus in a patient, administering a vaccine composition to the patient by administering a vaccine composition to the patient by delivery to the nasal mucosa, oral mucosa, and/or alimentary mucosa of the patient.
  • the vaccine targets SARS-like coronavirus (SARS-CoV-2).
  • the disclosed methods also include obtaining a sample of saliva from the patient at a period of time after administering the vaccine.
  • the sample of saliva is preserved in a stabilizing solution comprising glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, or any combination thereof.
  • the stabilizing solution comprises glutaraldehyde at 0.10 to 2.0% weight per volume (w/v), sodium benzoate at 0.10 to 1.0% w/v, and/or citric acid at 0.025 to 0.20% w/v.
  • Additional embodiments include analyzing the sample of saliva for at least one selected from antibodies targeting the virus or a protein specific to the virus, wherein in the absence of antibodies in the sample saliva, the method further comprises administering a booster of the vaccine to the patient.
  • the stabilizing solution further comprises aragonite particle beads having an average particle size of between 100 nm to 1 mm.
  • the aragonite particle beads are capable of binding to immunoglobulin (Ig) proteins, anti-SARS-CoV-2 antibodies, or a SARS-CoV-2 viral protein.
  • the aragonite particle beads are coupled to a recombinant ACE2 protein or a recombinant ACE2 alpha helix protein.
  • the contemplated subject matter also includes an aragonite composition formulated for binding an immunoglobulin (Ig) protein, an anti-SARS-CoV-2 antibody protein, or a SARS-CoV- 2 viral protein.
  • the aragonite composition includes a plurality of aragonite particle beads having an average particle size of between 100 nm to 1 mm, wherein the plurality of aragonite particle beads are functionalized with a moiety capable of binding to an immunoglobulin (Ig) protein, the anti-SARS-CoV-2 antibody protein and/or the SARS-CoV-2 viral protein.
  • the plurality of aragonite particle beads are functionalized with a moiety capable of binding to the anti-SARS-CoV-2 comprises a recombinant ACE2 protein.
  • the moiety capable of binding to the anti-SARS-CoV-2 may be selected from a recombinant ACE2 protein having at least 85% sequence identity to SEQ ID NO:9, a recombinant alpha-helix ACE2 protein of SEQ ID NO: 10, or the recombinant alpha-helix ACE2 protein having at least one mutation selected from T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and/ D355L.
  • the contemplated subject matter includes methods for administering a vaccine to a patient by more than one route of administration to induce both local and systemic immune responses to the vaccine.
  • the contemplated subject matter also includes compositions and methods for assaying the presence or absence of the relevant antibodies (e.g . , anti-SARS-CoV-2 antibodies) in a patient sample (e.g., saliva, nasal mucosa, alimentary mucosa, or serum).
  • a patient sample e.g., saliva, nasal mucosa, alimentary mucosa, or serum.
  • the antibody status in the patient’s sample may be used to assess the need for an additional vaccine dose (e.g. , a booster dose/shot).
  • the route of administration of the vaccine as well as the regimen for administering additional (i.e., booster) doses of the vaccine can also affect whether or not the patient’s immune response is robust enough to establish protection.
  • SARS-CoV-2 SARS-like coronavirus
  • the duration of immunity (both humoral and cell-mediated) in a patient recovered from a SARS-CoV-2 infection is not yet completely known, and furthermore, a vaccine protocol has not yet been tested across a varied population.
  • a vaccine protocol has not yet been tested across a varied population.
  • the current SARS-CoV-2 pandemic and the high rate of transmission for the SARS-CoV-2 virus there is a need for a robust vaccination protocol and effective testing for the virus or immunity to the virus (e.g., presence of anti-SARS-CoV-2 antibodies).
  • the presently disclosed contemplated methods for inducing immunity in a patient include administering a vaccine by at least oral administration, and preferably by oral administration and by injection to the blood supply.
  • Many vaccines are given via the intramuscular (IM) route to optimize immunogenicity with the direct delivery of the vaccine to the blood supply in the muscle to induce systemic immunity.
  • IM administration is typically preferred over subcutaneous (SC) injection which is more likely to have adverse reactions at the injection site than IM injections.
  • mucosal immunity In addition to IM injection, induction of mucosal immunity has been reported to be essential to stop person-to-person transmission of pathogenic microorganisms and to limit their multiplication within the mucosal tissue. Furthermore, for protective immunity against mucosal pathogens, (e.g., SARS coronaviruses) immune activation in mucosal tissues instead of the more common approach of tolerance to maintain mucosal homeostasis allows for enhanced mucosal immune responses and better local protection.
  • mucosal pathogens e.g., SARS coronaviruses
  • nasal vaccination delivery of a vaccine by nasal administration induces both mucosal immunity as well as systemic immunity (see, e.g., Fujkuyama et al., 2012, Expert Rev Vaccines, 11:367-379 and Birkhoff et ak, 2009, Indian J. Pharm. Sci., 71:729-731).
  • embodiments of the present disclosure include providing a vaccine to the patient by at least administration to the nasal mucosa, oral mucosa, and/or alimentary mucosa of the patient.
  • the routes of administration include administering the vaccine to the nasal mucosa, oral mucosa, and/or alimentary mucosa of the patient together with injection into the blood supply (e.g., intramuscular (IM), intravenous (IV), or subcutaneous (SC)).
  • IM intramuscular
  • IV intravenous
  • SC subcutaneous
  • oral administration of a vaccine composition includes nasal injection, nasal inhalation, ingestion by mouth, and administration (e.g., inhalation, ingestion, injection) to the alimentary mucosa.
  • the routes of administering the vaccine include oral administration selected from delivery to the alimentary mucosa, nasal injection, nasal inhalation, ingestion by mouth, or inhalation by mouth together with administration by intramuscular (IM) injection.
  • the vaccine administered for inducing immunity in the mucosal tissue of a patient is a SARS-CoV-2 vaccine.
  • the SARS-CoV-2 vaccine e.g., an adenovirus construct
  • the SARS-CoV-2 vaccine includes a soluble ACE2 protein coupled to an immunoglobulin Fc portion, forming an ACE2-Fc hybrid construct that may also include a J-chain portion, as disclosed in U.S. 16/880,804 and U.S. 63/016,048, the entire contents of both of which are herein incorporated by reference.
  • the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a mutant variant of a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10), wherein the mutant variant has at least one mutated amino acid residue (e.g., by substitution) that imparts an increased binding affinity of the ACE2 protein for the RBD protein domain of the SARS-CoV-2 spike protein as disclosed in U.S. 63/022,146, the entire content of which is herein incorporated by reference.
  • a mutant variant of a recombinant soluble ACE2 protein e.g., SEQ ID NO: 10
  • the mutant variant has at least one mutated amino acid residue (e.g., by substitution) that imparts an increased binding affinity of the ACE2 protein for the RBD protein domain of the SARS-CoV-2 spike protein as disclosed in U.S. 63/022,146, the entire content of which is herein incorporated by reference.
  • the SARS-CoV-2 vaccine (e.g., an adenovirus construct) includes a CoV2 nucleocapsid protein or a CoV2 spike protein fused to an endosomal targeting sequence (N-ETSD), as disclosed in U.S. 16/883,263 and U.S. 63/009,960, the entire contents of both of which are herein incorporated by reference.
  • the SARS-CoV-2 vaccine includes modified yeast cells (e.g., Saccharomyces cerevisiae) genetically engineered to express coronaviral spike proteins on the yeast cell surface thereby creating yeast presenting cells to stimulate B cells (e.g., humoral immunity) as disclosed in U.S.
  • more than one vaccine composition as disclosed herein may be administered to a patient to induce immunity to SARS-CoV-2.
  • a patient may be administered genetically modified yeast cells expressing corona viral spike proteins as a single type of vaccine, or the genetically modified yeast cells may be administered together or concurrently with one or more SARS-CoV-2 adenovirus constructs as disclosed herein.
  • a vaccine administered as disclosed herein e.g., by oral administration and injection into the blood supply
  • disclosed herein are compositions and methods for assessing the continued presence of antibodies in a patient’s respiratory and digestive mucosa following infection with SARS-CoV-2 or following inoculation against SARS-CoV-2 with administration of a SAR coronavirus vaccine.
  • the presence of antibodies against the pathogen may be carried out using any one of many diagnostic tests.
  • the diagnostic test is a cell viability assay that allows for the detection of antibodies in the presence of antigen. Diagnostic tests using a cell viability assay for anti-SARS-CoV-2 antibody detection are disclosed in U.S. 62/053,691, the entire contents of which are herein incorporated by reference.
  • the cellular diagnostic assay relies on the expression of the target receptor for a given pathogen (e.g, ACE2 for SARS-CoV-2 infection) on the surface of an immune effector cell line (e.g, killer T cells, natural killer cells, NK-92 cells and derivatives thereof, etc.) and the expression of the pathogen ligand (e.g, Spike proteins for SARS-CoV-2 infection) on the surface of a surrogate cell line (e.g, HEK293 cells or SUP-B15 cells).
  • a given pathogen e.g, ACE2 for SARS-CoV-2 infection
  • an immune effector cell line e.g, killer T cells, natural killer cells, NK-92 cells and derivatives thereof, etc.
  • the pathogen ligand e.g, Spike proteins for SARS-CoV-2 infection
  • a surrogate cell line e.g, HEK293 cells or SUP-B15 cells.
  • assaying a saliva sample from the patient allows for expedited sample collection, increased patient participation, and may allow for the patient to obtain the sample themselves and either mail or transport the sample to the lab for testing.
  • saliva for the presence of neutralizing antibodies against SARS-CoV-2
  • the saliva Upon collection of the saliva sample, the saliva is placed into a preservative solution to stabilize the components (e.g., anti-SARS CoV-2 antibody or viral spike protein) therein.
  • preservatives for biological samples are disclosed, for example, in Cunningham & al.
  • a stabilizing preservative solution for a patient’s saliva sample may include any one of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof.
  • saliva samples may be mixed with stabilizing preservative solutions of glutaraldehyde to achieve a final glutaraldehyde concentration between 0.1%(w/v) and 2.0%(w/v), for example about 0.2%(w/v), about 0.3%(w/v), about 0.4%(w/v), about
  • saliva samples may be mixed with a stabilizing preservative solution of about 0.10% to about 1.00% sodium benzoate (weight/volume of sample) and/or about 0.025% to about 0.20% citric acid (weight/volume of sample).
  • a stabilizing preservative solution of about 0.10% to about 1.00% sodium benzoate (weight/volume of sample) and/or about 0.025% to about 0.20% citric acid (weight/volume of sample).
  • the saliva sample may be mixed with 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, or 1.00% w/v sodium benzoate.
  • the saliva sample is mixed a stabilizing preservative solution of at least 0.5 mg/mL (for example, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, at least 1 mg/mL, at least 1.5 mg/mL, at least 2 mg/mL, at least 2.5 mg/mL, at least 3 mg/mL, at least 3.5 mg/mL, at least 4 mg/mL, at least 4.5 mg/mL, or even 5 mg/mL) of benzoic acid and/or at least 0.2 mg/mL (for example, at least 0.2 mg/mL, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.40 mg/mL, at least 0.50 mg/mL, at least 0.75 mg/mL, at least 1.0 mg/mL, at least 1.25 mg/mL, at least 1.5 mg/mL, at least 1.75 mg
  • the saliva samples with preservatives as described above are stable for storage at temperatures between 15°C and 40°C for at least one hour (e.g., at least 5 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 48 hours, or even 36 hours).
  • a method of preserving a saliva sample for neutralizing antibody testing including mixing the saliva sample with the stabilizing solution made of one or more of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and/or sodium azide and storing between 15°C and 25°C for at least one hour, and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours.
  • the stabilizing solution made of one or more of glutaraldehyde, sodium benzoate, citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and/or sodium azide and storing between 15°C and 25°C for at least one hour, and up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, or 48 hours.
  • the saliva sample is mixed with a glutaraldehyde concentration between 0.1% (w/v) and 2.0% (w/v), and the glutaraldehyde-saliva is stored between 15°C and 25°C.
  • the glutaraldehy de-saliva may further comprise citric acid and/or benzoic acid at a concentration of as disclosed herein.
  • any antibody proteins or any specific antibody protein may be captured from the saliva sample with oolitic aragonite particles.
  • the saliva preserving solution of glutaraldehyde, sodium benzoate and citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, sodium azide, and any combination thereof as disclosed herein may also include oolitic aragonite (calcium carbonate, CaCCb) particles.
  • aragonite particles for binding to proteins is disclosed, for example, in U.S. 16/858,548 and PCT/US20/29949, the entire contents of both of which are herein incorporated by reference.
  • aragonite particles may be added to that have been modified to capture ( e.g . , bind to) any antibodies present in the saliva sample or specifically capture an antibody against a specific antigen.
  • aragonite may be functionalized with moieties capable of binding to an immunoglobulin (Ig) protein.
  • the Ig protein is an immunoglobulin A (IgA), immunoglobulin G (IgG), or immunoglobulin E (IgE) protein.
  • the aragonite is functionalized to bind to an IgA protein.
  • the aragonite particles are functionalized with moieties capable of binding to specific antibodies.
  • the aragonite particles may be coupled with a moiety specific to anti-SARS-CoV-2 antibodies.
  • the aragonite particle is coupled with a recombinant ACE2 protein as disclosed, for example, in U.S. 16/880,804, supra.
  • the aragonite particle is coupled with a recombinant human ACE2 protein having at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 9.
  • the aragonite particle is functionalized (e.g., coupled to) a recombinant soluble ACE2 protein (e.g., SEQ ID NO: 10).
  • a recombinant soluble ACE2 protein e.g., SEQ ID NO: 10
  • the recombinant soluble ACE2 may be mutated to form ACE2 variants having higher binding affinities for SARS- CoV-2 spike protein (e.g., the RBD domain of the spike protein).
  • ACE2 variant mutants of the recombinant soluble ACE2 protein include T27F, T27W, T27Y, D30E, H34E, H34F, H34K, H34M, H34W, H34Y, D38E, D38M, D38W, Q24L, D30L, H34A, and/or D355L.
  • the term “functionalized” refers to coupling or binding of a moiety to the aragonite particle thereby imparting any function of the coupled moiety to the aragonite particle.
  • the aragonite particle may be functionalized with a protein moiety.
  • Methods for preparing and using aragonite particle beads are disclosed in U.S. 16/858,548 and PCT/US20/29949.
  • the aragonite composition includes a plurality of aragonite particle beads.
  • the plurality of aragonite particle beads have an average particle size of between 100 nm to 1 mm.
  • a protein moiety is coupled directly to the natural, untreated surface of aragonite particles.
  • Aragonite particles have approximately a 2-3% amino acid content, including aspartic acid and glutamic acid rendering the aragonite surface hydrophilic. Accordingly, in some embodiments, protein moieties may be directly coupled to the surface of the aragonite particles.
  • the aragonite particle surface may be treated to modify the binding surface.
  • treatment with stearic acid i.e., octadecanoic acid
  • octadecanoic acid provides for a hydrophobic surface, as disclosed in U.S. 16/858,548 and PCT/US20/29949.
  • treatment of the aragonite with phosphoric acid forms lamellar structures. Additional conjugation techniques for coupling reactive groups to the amino acid surface of aragonite are known in the art as disclosed, for example, in Bioconjugate Techniques, Third Edition, Greg T. Hermanson, Academic Press, 2013.
  • Patients who do not show sufficient titers of (e.g . , presence of) neutralizing antibody in their saliva may be sent oral dosages of the respective vaccine (e.g., a SARS-CoV-2 vaccine as disclosed herein).
  • the patients inhale or ingest these vaccine dosages, and then two weeks later send another saliva sample — prepared and stored in the same manner as above — to the test facility to confirm that the oral vaccine dose has restored their anti-SARS-CoV-2 antibody (e.g., IgA) titers.
  • the respective vaccine e.g., a SARS-CoV-2 vaccine as disclosed herein.
  • the patients inhale or ingest these vaccine dosages, and then two weeks later send another saliva sample — prepared and stored in the same manner as above — to the test facility to confirm that the oral vaccine dose has restored their anti-SARS-CoV-2 antibody (e.g., IgA) titers.
  • IgA anti-SARS-CoV-2 antibody
  • a kit for collecting a saliva sample from a patient includes a collection container with the saliva preservative solution as disclosed herein.
  • the kit includes a collection container with a solution of any of one or combination of glutaraldehyde, sodium benzoate and/or citric acid, propyl gallate, EDTA, zinc, actin, chitosan, parabens, and sodium azide.
  • the kit may also include adhesive packaging and/or mailing supplies in order to secure the collection container with the saliva sample for transport or mailing.
  • the kit may also include at least one dose of the vaccine for oral administration.
  • immune stimulation with one or more immune stimulatory cytokines may prevent or alleviate lymphopenia that is frequently associated with COVID-19.
  • immune stimulatory cytokines N-803 is particularly contemplated.
  • COVID-19 infection causes lymphopenia, specifically a suppression of NK and CD8 T cells, and severe cases and subsequent fatalities are associated with this significant decline in lymphocytes.
  • Evidence also suggests that COVID-19 infection results in macrophage killing and reduction of natural killer (NK) cells and CD8+ T cells.
  • NK natural killer
  • Another group of researchers showed that median lymphocyte counts were significantly lower for patients admitted to the ICU as compared to those who did not require ICU care.
  • N-803 has demonstrated significant ability to induce increases in broad T-cell reactivity both in preclinical (mice) and clinical (breast, lung, and pancreatic cancers) settings. It is possible that by activating T-cell diversity, cross-reactivity to past coronavirus infections may offer immunity to COVTD- 19 following N-803 enhancement of T-cell reactivity and overcome immune evasion.
  • N-803 significantly promotes NK cell and CD8- T-cell proliferation and activation in the peripheral circulation and lymphoid organs in healthy mice and cynomolgus monkeys, as well as in a variety of murine and rodent tumor models including bladder cancer, lung cancer, melanoma, lymphoma, multiple myeloma, colon cancer, breast cancer and glioblastoma. Additional preclinical evidence exists for N-803's antiviral effect, including in murine and nonhuman primate (NHP) models.
  • NEP nonhuman primate
  • N-803 increases the cytotoxic potential of these immune cells as indicated by upregulation of the expression of activation markers including perforin and granzyme B.
  • the phenotypic changes induced in NK and CD8+ T cells by N-803 resulted in enhanced anticancer activity (through antibody-dependent cellular cytotoxicity, direct cellular cytotoxicity, and enhanced tumor-specific cytotoxicity) and prolonged survival in vivo.
  • NK cell and CD8+ T-cell activation has been demonstrated.
  • N-803- treated subjects demonstrated an over 20-fold increase in Ki-67 stained NK and CD8+T cells.
  • Clinical efficacy of N-803 in stimulating lymphopenic response has been demonstrated in patients with acute myeloid leukemia (AML), breast, lung, and pancreatic cancers.
  • AML acute myeloid leukemia
  • N-803 binds with a higher affinity to IL-15 receptor presenting cells, has enhanced lymphoid distribution, prolonged half-life, and causes proliferation and activation of effector NK cells and CD8+ memory T cells resulting in antitumor activity
  • N-803 may be used as a treatment option to counter COVID-19 related lymphopenia in a manner similar to N-803 stimulating both NK and CD8 T cells and rescue lymphopenia in normal healthy subjects as well as patients with cancer.
  • N-803 may be used for reversal or alleviation of lymphopenia in patients infected with COVID-19 and to so improve disease outcomes.
  • an infected subject may be treated by receiving a subcutaneous (SC) injection of N-803 on day 1, and on day 15 (if needed) in the abdomen.
  • SC subcutaneous
  • the N-803 will be provided in liquid injectable form at a dosage of 10 mcg/kg.
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
  • administering refers to both direct and indirect administration of the pharmaceutical composition or drug, wherein direct administration of the pharmaceutical composition or drug is typically performed by a health care professional (e.g., physician, nurse, etc.), and wherein indirect administration includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.).
  • a health care professional e.g., physician, nurse, etc.
  • indirect administration includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.).
  • the terms “prognosing” or “predicting” a condition, a susceptibility for development of a disease, or a response to an intended treatment is meant to cover the act of predicting or the prediction (but not treatment or diagnosis ol) the condition, susceptibility and/or response, including the rate of progression, improvement, and/or duration of the condition in a subject.
  • the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
  • the term “coupled to” is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms “coupled to” and “coupled with” are used synonymously.

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Abstract

Composition vaccinale pour induire une immunité contre un coronavirus chez un sujet comprenant un acide nucléique recombinant qui code pour N-ETSD, une protéine de nucléocapside modifiée qui comprend une séquence de ciblage endosomal, et/ou qui code pour S-Fusion, une protéine de pointe modifiée qui a une expression de surface améliorée. Le vaccin peut être formulé sous la forme d'un acide nucléique recombinant, d'une levure recombinante et/ou d'un virus recombinant tel qu'un adénovirus et peut être administré par injection et/ou administration par voie muqueuse.
EP21768699.7A 2020-03-11 2021-03-10 Traitement du covid-19 et procédés correspondants Pending EP4118210A4 (fr)

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US202062988328P 2020-03-11 2020-03-11
US202063009960P 2020-04-14 2020-04-14
US202063010010P 2020-04-14 2020-04-14
US16/883,263 US11684668B2 (en) 2020-03-11 2020-05-26 Replication-defective adenoviruses comprising nucleic acids encoding SARS-CoV-2 s glycoprotein and modified N protein comprising an endosomal targeting sequence
US202063059975P 2020-08-01 2020-08-01
US202063064157P 2020-08-11 2020-08-11
US202063117460P 2020-11-24 2020-11-24
US202063117922P 2020-11-24 2020-11-24
US202063117847P 2020-11-24 2020-11-24
US202063118697P 2020-11-26 2020-11-26
US202163135380P 2021-01-08 2021-01-08
PCT/US2021/021737 WO2021183665A1 (fr) 2020-03-11 2021-03-10 Traitement du covid-19 et procédés correspondants

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WO2003066820A2 (fr) * 2002-02-05 2003-08-14 The Regents Of The University Of California Molecules d'acides nucleiques codant pour des proteines de ciblage endosomales derivees de cd1 et utilisations associees
WO2011129468A1 (fr) * 2010-04-14 2011-10-20 Mogam Biotechnology Research Institute Hexon isolé à partir d'un adénovirus simien de sérotype 19, région hypervariable dudit hexon et adénovirus chimérique l'utilisant
EP3045181B1 (fr) * 2015-01-19 2018-11-14 Ludwig-Maximilians-Universität München Nouveau vaccin contre le coronavirus du syndrome respiratoire du moyen orient (MERS-CoV)
KR20170140180A (ko) * 2015-02-24 2017-12-20 더 유나이티드 스테이츠 오브 어메리카, 애즈 리프리젠티드 바이 더 세크러테리, 디파트먼트 오브 헬쓰 앤드 휴먼 서비씨즈 중동 호흡기 증후군 코로나 바이러스 면역원, 항체 및 그 용도

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