EP4677097A2 - Vaccin pancoronavirus multi-antigène à large spectre - Google Patents

Vaccin pancoronavirus multi-antigène à large spectre

Info

Publication number
EP4677097A2
EP4677097A2 EP24771552.7A EP24771552A EP4677097A2 EP 4677097 A2 EP4677097 A2 EP 4677097A2 EP 24771552 A EP24771552 A EP 24771552A EP 4677097 A2 EP4677097 A2 EP 4677097A2
Authority
EP
European Patent Office
Prior art keywords
protein
composition
coronavirus
spike
nucleoprotein
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
EP24771552.7A
Other languages
German (de)
English (en)
Inventor
Lbachir Benmohamed
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.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
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 PCT/US2023/068080 external-priority patent/WO2023240148A2/fr
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP4677097A2 publication Critical patent/EP4677097A2/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20071—Demonstrated in vivo effect

Definitions

  • the present invention relates to vaccines, for example, viral vaccines, such as those directed to coronaviruses, e.g., pan-coronavirus vaccines.
  • Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in nearly a century. As of January 2024, the number of confirmed SARS-CoV-2 cases has reached over 770 million, and COVID-19 disease caused nearly 7 million deaths. Since early 2020, the world has continued to contend with successive waves of COVID-19, fueled by the emergence of over 20 variants of concern (VOCs) with continued enhanced transmissibility. While the Wuhan strain Hu1 is the ancestral variant of SARS-CoV-2 that emerged in late 2019 in China, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (B.1.1.28) VOCs subsequently emerged between 2020 to 2021 in the United Kingdom, South Africa, and Brazil, respectively.
  • the most pathogenic Delta variant (B. 1 .617. 2) was identified in India in mid-2021 , where it led to a deadly wave of infections.
  • the fast and heavily Spike-mutated Omicron variants and sub-variants i.e., B.1.1 .529, XBB1.5, EG.5, HV.1 , BA.2.86, and JN.1
  • B.1.1 .529, XBB1.5, EG.5, HV.1 , BA.2.86, and JN.1 are less pathogenic but are more immune-evasive.
  • breakthrough infections by these VOCs contributed to repetitive seasonal surges that often strain the world's healthcare systems, causing sustained hospitalizations, illnesses, and deaths.
  • the present invention features a universal pre-emptive pan-Coronavirus vaccine composition.
  • the composition may comprise two (or three) or more Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the composition comprises a sequence encoding two (or three) or more Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • compositions may further comprise Coronavirus antigen derived from at least a portion of a Spike protein.
  • the composition may comprise two (or three) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein.
  • the composition may comprises a sequence encoding two (or three) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein.
  • the present invention features a universal pre-emptive pan-Coronavirus vaccine composition.
  • the composition may comprise two (or three) or more Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the composition comprises a sequence encoding two (or three) or more Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the aforementioned composition may further comprise Coronavirus antigen derived from a Spike protein.
  • the composition may comprise two (or three) Coronavirus antigens derived from a)an NSP2 protein; b) an NSP14 protein; c) Nucleoprotein protein and d) a Spike protein.
  • the composition may comprise a sequence encoding two (or three) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein and a Spike protein.
  • the present invention may also feature a pan-Coronavirus recombinant vaccine composition.
  • the composition comprising a delivery system encoding two (or three) or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the aforementioned delivery system may further encode a Coronavirus antigen derived from at least a portion of a Spike protein.
  • the aforementioned delivery system may further comprise an additional delivery system encoding a Coronavirus antigens derived from at least a portion of a Spike protein.
  • the composition may comprise a delivery system encoding two (or three) or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein; and d) at least a portion of a Spike protein.
  • the delivery system may comprise a single delivery system or may comprise two or more delivery systems.
  • the present invention may feature a pan-Coronavirus recombinant vaccine composition.
  • the composition comprises a delivery system encoding two (or three) or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the aforementioned delivery system may further encode a Coronavirus antigen derived from a Spike protein.
  • the aforementioned delivery system may further comprise an additional delivery system encoding a Coronavirus antigens derived from a Spike protein.
  • the composition may comprise a delivery system encoding two (or three) or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • the delivery system may comprise a single delivery system or may comprise two or more delivery systems.
  • the aforementioned Coronavirus antigens may be the Spike protein or a portion thereof and the NSP2 protein or a portion thereof.
  • the Coronavirus antigens may be the Spike protein or a portion thereof and the NSP14 protein or a portion thereof.
  • the Coronavirus antigens may be the Spike protein or a portion thereof and the Nucleoprotein or a portion thereof.
  • the Coronavirus antigens may be the Spike protein or a portion thereof, the NSP2 protein or a portion thereof, and the NSP14 protein or a portion thereof.
  • the Coronavirus antigens may be the Spike protein or a portion thereof, the NSP2 protein or a portion thereof, and the Nucleoprotein or a portion thereof. In some embodiments, the Coronavirus antigens may be the Spike protein or a portion thereof, the NSP14 protein or a portion thereof, and the Nucleoprotein or a portion thereof. In some embodiments, the Coronavirus antigens may be the Spike protein; the NSP2 protein or a portion thereof; the NSP14 protein or a portion thereof; and the Nucleoprotein or a portion thereof.
  • the aforementioned composition may further comprise one or more Coronavirus antigens derived from an NSP3 protein or portion thereof, a NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the aforementioned composition may further comprise two or more Coronavirus antigens derived from an NSP3 protein or portion thereof, a NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the aforementioned composition may further comprise three or more Coronavirus antigens derived from an NSP3 protein or portion thereof, a NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the present invention is not limited to the aforestated Coronavirus antigens.
  • the aforementioned compositions may further comprise T cell attracting chemokine, e g., CCL5, CXCL9, CXCL10, CXCL11 , or a combination thereof.
  • the composition may also comprise a composition that promotes T cell proliferation and T-cell memory, e.g., IL-7, IL-2, IL-15, or a combination thereof.
  • the vaccine compositions described herein may protect against disease caused by one or more coronavirus variants or coronavirus subvariants.
  • the coronavirus variants or coronavirus subvariants may comprise past or currently circulating coronavirus variants (e.g., alpha, beta, gamma, delta, and omicron) or coronavirus subvariants. Additionally, the coronavirus variants or coronavirus subvariants may comprise future variants or future subvariants of human and animal coronavirus.
  • the vaccine compositions herein protect against infection and re-infection of coronavirus variants or coronavirus subvariants.
  • the vaccine composition may protect against infection or reinfection of one or more coronavirus variant or coronavirus subvariant.
  • the vaccine compositions may protect against infection or reinfection of multiple coronavirus variants or coronavirus subvariants.
  • the vaccine composition protects against infection or re-infection of one coronavirus variants or coronavirus subvariants.
  • the vaccine compositions described herein may induce strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Th1) cells, and/or CD8+ cytotoxic T-cells (CTL).
  • Abs antibodies
  • Th1 CD4+ T helper
  • CTL cytotoxic T-cells
  • the present invention may further feature a composition comprising two (or three) or more ribonucleic acids (mRNAs) comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein.
  • the two or three mRNAs may be formulated in a lipid nanoparticle.
  • the composition may comprise two (or three) or more mRNAs comprising an open reading frame encoding an entire Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein.
  • the composition may further comprise an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof formulated in a lipid nanoparticle.
  • the composition may comprise two (or three) or more mRNAs comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein.
  • the composition may comprise two (or three) or more mRNAs comprising an open reading frame encoding an entire Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein.
  • the two (or three) or more mRNAs are formulated in a lipid nanoparticle.
  • the present invention may feature a pharmaceutical composition.
  • the pharmaceutical composition may comprise a plurality of lipid nanoparticles; where a first lipid nanoparticle comprises three messenger ribonucleic acids (mRNAs) encapsulated therein, and each mRNA comprises an open reading frame encoding a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein, and a second lipid nanoparticle comprising an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof encapsulated therein.
  • mRNAs messenger ribonucleic acids
  • the pharmaceutical composition may comprise a plurality of lipid nanoparticles; where a first lipid nanoparticle comprises three messenger ribonucleic acids (mRNAs) encapsulated therein, and each mRNA comprises an open reading frame encoding an entire Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein, and a second lipid nanoparticle comprising an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof encapsulated therein.
  • mRNAs messenger ribonucleic acids
  • the mRNAs may further comprise a 5’ untranslated region (UTR) and a 3’ UTR.
  • the mRNAs further comprise a 3’ poly(A) tail and/or a 5’ cap or cap analog.
  • One of the unique and inventive technical features of the present invention is the use of both B cell antigens and T cell antigens within a single composition (e.g., a vaccine composition, a pharmaceutical composition, ect.).
  • a single composition e.g., a vaccine composition, a pharmaceutical composition, ect.
  • the technical feature of the present invention advantageously provides for a universal vaccine composition that will protect from future human outbreaks and deter future zoonosis. None of the presently known prior references or work has the unique inventive technical feature of the present invention.
  • the present invention may feature a universal pre-emptive pan-Coronavirus vaccine composition comprising a B cell antigen and two (or three) or more T-cell antigens.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprises a sequence encoding a B cell antigen and two (or three) or more T-cell antigens.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprises a B cell antigen and three T-cell antigens.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprises a sequence encoding a B cell antigen and three T-cell antigens.
  • the B cell antigens may be derived from a Coronavirus Spike protein or portion thereof.
  • the T cell antigens may be derived from an NSP2 protein or portion thereof, an NSP14 protein or portion thereof, a Nucleoprotein or portion thereof, or a combination thereof.
  • the T cell antigens may be derived from an NSP3 protein or portion thereof, an NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • FIG. 1A, 1 B, and 1 C shows highly conserved non-spike, structural, non-structural, and accessory protein antigens identified in the SARS-CoV-2 genome:
  • FIG. 1A shows bioinformatic analysis and alignment of the 29903 bp single strand RNA of 8.7 million genome sequences of SARS-CoV-2 strains that circulated worldwide over the last 4 years, including 20 VOCs; SARS-CoV; MERS-CoV; common cold Coronaviruses; and twenty-five animal’s SARS-like Coronaviruses (SL-CoVs) genome sequences isolated from bats (Rhinolophus affinis, Rhinolophus malayanus), pangolins (Manis javanica), civet cats (Paguma larvata), and camels (Camelus dromedaries).
  • FIG. 1B depicts 10 highly conserved non-Spike antigens that comprise 3 structural (Membrane, Envelope, and Nucleoprotein), 12 non-structural (NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, and NSP-14) and 1 accessory protein (ORF7a/b) as T cell antigens (top) and Spike as the B cell antigen (bottom) used to construct the individual and combined mRNA/LNP vaccines.
  • FIG. 10 highly conserved non-Spike antigens that comprise 3 structural (Membrane, Envelope, and Nucleoprotein), 12 non-structural (NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, and NSP-14) and 1 accessory protein (ORF7a/b) as T cell antigens (top) and Spike as the B cell antigen (bottom) used to construct the individual and combined mRNA/LNP vaccines.
  • FIG. 1C illustrates the individual and combined mRNA/LNP vaccines that consist of modified mRNAs expressing the B and T cell antigens encapsulated in lipid nanoparticles (LNPs), as detailed herein, and delivery intramuscularly in the outbreed golden Syrian hamsters.
  • LNPs lipid nanoparticles
  • FIG. 2 shows a comparison of cumulative mutation frequencies between Spike B cell antigen and ten conserved non-Spike T cell antigens among 12 SARS-CoV-2 variants and sub-variants of concern, including the recent highly mutated COVID variants ‘Pirola’ BA.2.86 and JN.1 that may cause more severe disease.
  • FIG. 3A and 3B shows experimental plan and gating strategy: FIG. 3A shows the experimental plan followed for the flow-cytometry experiments and the ELISpot experiments presented in FIG. 4A, 4B, 4C, and 4D, starting with the COVID-19 blood samples collection, patient genotyping, PBMCs extraction, and peptide stimulation.
  • FIG. 3B shows the gating strategy applied when analyzing the flow cytometry data. Fresh peripheral blood mononuclear cells (PBMCs) were used in this study as they generally have higher viability and functionality compared to frozen PBMCs. Freezing and thawing can lead to cell damage and loss of T-cell functionality, which may affect the accuracy and reliability of experimental results.
  • PBMCs peripheral blood mononuclear cells
  • Frozen PBMCs may exhibit altered activation status compared to fresh cells. Cryopreservation can induce stress responses in cells, leading to changes in their activation state and potentially affecting immune response assays.
  • using fresh PBMCs ensures the accuracy and reliability of experimental results. A side-by-side comparison of frozen and fresh PBMCs and pre-pandemic healthy control PBMCs yielded no significant difference.
  • FIG. 4A, 4B, 4C, and 4D shows IFN-y-producing CD4 + and CD8 + T cell responses to highly conserved antigens in unvaccinated COVID-19 patients with various degrees of disease severity:
  • FIG. 4A illustrates a positive correlation between the severity of COVID-19 and the magnitude of SARS-CoV-2 common antigens -specific CD4 + and CD8 + T cell responses in 71 COVID-19 patients.
  • COVID-19 patients 71
  • the magnitude of CD4 + and CD8 + T cell responses specific to CD4 + and CD8 + T cell epitopes from all the ten selected conserved antigens (FIG. 4B), the 13 individual cross-reactive CD4 + T cell epitope peptides (FIG.
  • FIG. 4C The number of IFN-y-producing CD8 + T cells was quantified in each of the 71 patients using ELISpot assay. Shown are the average/mean numbers (+ SD) of IFN-y-spot forming cells (SFCs) after CD4 + T cell peptide stimulation detected in each of the 71 COVID-19 patients divided into six groups based on disease severity scored 0 to 5.
  • a mean SFCs between 25 and 50 SFCs corresponds to a medium/intermediate response, whereas a strong response is defined for mean SFCs > 50 per 0.5 x 10 6 stimulated PBMCs.
  • PHA was used as a positive control of T-cell activation.
  • Unstimulated negative control SFCs (DMSO - no peptide stimulation) were subtracted from the SFC counts of peptides-stimulated cells. Shown is the correlation between the overall number of IFN-y-producing CD4 + T cells induced by each of the 14 cross-reactive CD4 + T cell epitope peptides (FIG.
  • FIG. 5A, 5B, 5C, and 5D shows the screening of 10 highly conserved T cell antigens for protection against the highly pathogenic Delta variant (B.1.617.2) in golden Syrian hamsters:
  • FIG. 5A shows Omicron sub-variant BA.2.75-based sequences of 10 highly conserved non-Spike T-cell antigens (i.e., NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, NSP-14, ORF7a/b, Membrane, Envelope, and Nucleoprotein) are used to construct methyl-pseudouridine-modified (ml ⁇ P) mRNA and capped using CleanCap technology.
  • 10 highly conserved non-Spike T-cell antigens i.e., NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, NSP-14, ORF7a/b, Membrane, Envelope, and Nucleoprotein
  • FIG. 5B shows the experimental plan to screen the vaccine efficacy of the 10 highly conserved T-cell Ags.
  • FIG. 5C shows percent weight change for 14 days post-challenge normalized to the initial body weight on the day of infection in hamsters immunized with mRNA/LNP expressing Spike 2P and Spike 6P.
  • 5D shows percent weight change for 14 days post-challenge normalized to the initial body weight on the day of infection in hamsters immunized with mRNA/LNP expressing individual NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, NSP-14, ORF7a/b, Membrane, Envelope, and Nucleoprotein at 1 pg/dose or 10 pg/dose.
  • the dashed line indicates the 100% starting body weight.
  • the arrows indicate the first-day post-challenge when the weight loss is reversed in T cell antigen (back arrow), Spike (grey arrow), and mock (red circle) vaccinated hamsters.
  • the data represent two independent experiments; the graphed values and bars represent the SD between the two experiments.
  • the Mann-Whitney test (two groups) or the Kruskal-Wallis test (more than two groups) were used for statistical analysis, ns P > 0.05, * P ⁇ 0.05, ** P ⁇ 0.01 , *** p ⁇ 0.001 , **** p ⁇ 0.0001.
  • FIG. 6A, 6B, 6C, 6D, and 6E shows protection against the highly pathogenic Delta variant (B.1.617.2) induced by individual NSP-2, NSP-14, and Nucleoprotein T cell antigen-based mRNA/LNP vaccines in golden Syrian hamsters:
  • FIG. 6A illustrates the three mRNA/LNP vaccines that consist of highly conserved T-cell Ags, NSP-2, NSP-14, and Nucleoprotein expressed as nucleoside-modified mRNA sequences derived from BA.2.75 Omicron sub-variant (BA2) and encapsulated in lipid nanoparticles (LNP).
  • BA2 Omicron sub-variant
  • LNP lipid nanoparticles
  • 6B shows the experimental plan to screen the vaccine efficacy of the 10 highly conserved T-cell Ags (i.e., NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, NSP-14, ORF7a/b, Membrane, Envelope, and Nucleoprotein).
  • FIG. 6C shows representative H & E staining images of lung pathology at day 14 p.i. of SARS-CoV-2 infected hamsters mock vaccinated or vaccinated with three protective NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccines at 4x magnifications.
  • FIG. 6D shows percent weight change for 14 days post-challenge normalized to the initial body weight on the day of infection.
  • FIG. 6E shows two- and 6 days post-infection (p.i.), viral loads were analyzed, to evaluate vaccine-induced protection against virus replication, by comparing viral RNA copies in the hamster's throats and lungs between mock and vaccine groups. Viral RNA copies were quantified by RT-PCR and expressed as log 10 copies per milligram of throat or lung tissue. The graphs show a comparison of viral titers in the hamster lungs between vaccinated vs. mock-vaccinated hamsters.
  • the data represent two independent experiments; the graphed values and bars represent the SD between the two experiments.
  • the Mann-Whitney test (two groups) or the Kruskal-Wallis test (more than two groups) were used for statistical analysis, ns P > 0.05, * P ⁇ 0.05, ** p ⁇ 0.01 , *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • FIG. 7 A, 7B, 7C, 7D, and 7E shows the protection against multiple SARS-CoV-2 variants and sub-variants of concern induced by combined NSP-2, NS P-14, and Nucleoprotein-based mRNA/LNP vaccine in the hamster model:
  • FIG. 7A illustrates the combination of three vaccines that consist of highly conserved protective T-cell Ags, NSP-2, NSP-14, and Nucleoprotein expressed as nucleoside-modified mRNA sequences derived from BA.2.75 Omicron sub-variant (BA2) and encapsulated in lipid nanoparticles (LNP).
  • BA2 Omicron sub-variant
  • LNP lipid nanoparticles
  • FIG. 7B shows the hamster experimental design and timeline to study the vaccine efficacy in golden Syrian hamsters of 10 individual T cell antigen-based mRNA/LNP vaccines on COVID-19-like symptoms detected.
  • FIG. 7C shows representative H & E staining images of lung pathology at day 14 p.i.
  • FIG. 7D shows percent weight change for 14 days post-challenge normalized to the initial body weight on the day of infection for each variant and sub-variant.
  • the dashed line indicates the 100% starting body weight.
  • the arrows indicate the first-day post-challenge when the weight loss is reversed in T cell antigen (back arrow), Spike (grey arrow), and mock (red circle) vaccinated hamsters.
  • FIGS. 7E show two- and 6-days post-infection (p.i.) with the wild-type Washington variant (WA1/2020), the highly pathogenic Delta variant (B.1.617.2), or the highly transmissible Omicron sub-variant (XBB1.5), viral loads were analyzed, to evaluate vaccine-induced protection against virus replication, by comparing viral RNA copies in the hamster's throats and lungs between mock and vaccine groups. Viral RNA copies were quantified by RT-PCR and expressed as log 10 copies per milligram of throat or lung tissue. The graphs show a comparison of viral titers in the hamster lungs between vaccinated vs. mock-vaccinated hamsters.
  • Viral titration data showing viral RNA copy number in the throats of vaccinated vs. mock-vaccinated hamsters detected at days 2 and 6 post-challenge.
  • the data represent two independent experiments; the graphed values and bars represent the SD between the two experiments.
  • the Mann-Whitney test (two groups) or the Kruskal-Wallis test (more than two groups) were used for statistical analysis, ns P > 0.05, * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001 , **** P ⁇ 0.0001 .
  • FIG. 8A, 8B, 8C, 8D, and 8E shows protection induced by combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine against the highly pathogenic Delta variant (B.1.617.2):
  • FIG. 8A illustrates combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine that consists of Spike mRNA/LNP vaccine combined to highly conserved protective T-cell Ags, NSP-2, NSP-14, and Nucleoprotein mRNA/LNP vaccines. All sequences are derived from BA.2.75 Omicron sub-variant (BA2).
  • FIG. 1 BA.2.75 Omicron sub-variant
  • FIG. 8B shows the transfection of Spike, NSP-2, NSP-14, and Nucleoprotein mRNA and protein expression in vitro in the human epithelial HEK293T cells.
  • FIG. 8C the hamster experimental design and timeline to study the beneficial effect in golden Syrian hamsters of adding the Spike mRNA/LNP vaccine to the combined NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine on the protection against the highly pathogenic Delta variant (B.1.617.2).
  • FIG. 8D shows percent weight change for 14 days post-challenge normalized to the initial body weight on the day of infection with the highly pathogenic Delta variant (B.1.617.2). The dashed line indicates the 100% starting body weight.
  • 8E shows six days post-infection (p.i.), with the highly pathogenic Delta variant (B.1.617.2), the viral loads were analyzed, to evaluate vaccine-induced protection against virus replication, by comparing viral RNA copies in the hamster's throats and lungs between mock and vaccine groups. Viral RNA copies were quantified by RT-PCR and expressed as log 10 copies per milligram of throat or lung tissue.
  • the graphs show a comparison of viral titers in the hamster lungs between vaccinated vs. mock-vaccinated hamsters. The data represent two independent experiments; the graphed values and bars represent the SD between the two experiments.
  • FIG. 9A, 9B, 9C, 9D, 9E, and 9F shows protection induced by the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine against the wild-type Washington variant (WA1/2020) and the highly transmissible Omicron sub-variant (XBB1.5).
  • FIG. 9A illustrates combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine.
  • FIG. 9A illustrates combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine.
  • FIG. 9B and 9C shows percent weight change for 14 days post-challenge normalized to the initial body weight on the day of challenge with the wild-type Washington variant (WA1/2020) at 2 x 10 5 pfu/hamster and the highly transmissible Omicron sub-variant (XBB1.5) at 2 x 10 5 pfu/hamster, respectively.
  • the dashed line indicates the 100% starting body weight.
  • the arrows indicate the first-day post-challenge when the weight loss is reversed in T cell antigen (back arrow), Spike (grey arrow), and mock (red circle) vaccinated hamsters.
  • FIG. 9D shows representative H & E staining images of lung pathology at day 14 p.i.
  • Lungs of hamsters immunized with Spike mRNA/LNP alone show peri bronchiolitis (arrow), perivasculitis (asterisk), and multifocal interstitial pneumonia.
  • Lungs of hamsters that received a combination Spike mRNA/LNP vaccine and combined T cell antigens mRNA/LNP vaccine demonstrate mostly normal bronchial, bronchiolar (arrows), and alveolar architecture. Scale bars, 1 mm.
  • FIG. 9E and 9F shows viral titration data showing viral RNA copy number in the throats of vaccinated vs.
  • FIG. 10 shows a graph of the IgG level among hamsters vaccinated with a combination of NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccines, spike alone vaccine, and mock vaccination (Top Panel). Neutralization assay data among the vaccinated and mock-vaccinated groups showing vaccine-induced serum-neutralizing activities (Bottom Panel).
  • FIG. 11 shows Patient selection based on HLA-A*02:01 and HLA-DRB1*01:01 alleles: All the 600 patients enrolled in our study for were genotyped class I HLA-A*02:01 and class II HLA-DRB1*01 :01 by PCR. Out of the 600 COVID-19 patients, 147 patients were positive for HLA-A*02:01 or/and HLA-DRB1*01 :01 and were considered in this study.
  • the 147 patients were from mixed ethnicities (Hispanic (28%), Hispanic Latino (22%), Asian (16%), Caucasian (13%), mixed Afro-American and Hispanic (8%), Afro-American (5%), mixed Afro-American and Caucasian (2%), Native Hawaiian and Other Pacific Islander descent (1 %). Six percent of the patients did not reveal their race/ethnicity. The detailed demographic and clinical data forthe 147 patients enrolled in this study are shown.
  • Symptomatic and Asymptomatic COVID-19 patient stratification based on disease severity Following patient discharge, they were divided into six groups depending on the severity of their symptoms and their intensive care unit (ICU) and intubation (mechanical ventilation) status by medical practitioners. The scoring criteria were as follows: Severity 5: patients who died from COVID-19 complications; Severity 4: infected COVID-19 patients with severe disease who were admitted to the intensive care unit (ICU) and required ventilation support; Severity 3: infected COVID-19 patients with severe disease that required enrollment in ICU, but without ventilation support; Severity 2: infected COVID-19 patients with moderate symptoms that involved a regular hospital admission; Severity 1 : infected COVID-19 patients with mild symptoms; and Severity 0: infected individuals with no symptoms. Among the 147 COVID-19 Patients, subjects with a Severity score of 0 were defined as Asymptomatic and subjects with a severity score of 1-5 were defined as Symptomatic.
  • class-l HLA For class-l HLA, the screening was first performed (two-digit level) by HLA-A*02 flow cytometry staining (data not shown, mAbs clone BB7.2, BioLegend, San Diego, CA). The four-digit class-l HLA-A*02:01 subtype was subsequently screened by PCR on blood samples.
  • FIG. 12A show pre-clinical superiority of the mRNA-LNP Encoding Spike + T Cell Antigens Combination Vaccine described herein Versus mRNA-LNP Encoding Spike Protein Alone against SARS-CoV-2 Delta (B.1.617.2) variant in hACE2 mice.
  • the pre-Clinical protective efficacy of the Techlmmune mRNA-LNP encoding Spike + T Cell antigens combination vaccine versus Spike alone was assessed by measuring body weight loss of mRNA-LNP immunized hACE2 mice and challenged with 1 x 10 5 pfu of SARS-CoV-2 Delta (B.1.617.2) variant.
  • mice were followed-up for fourteen days post intranasal infection and their body weight was measured daily.
  • the graph show the body weight of mice immunized with mRNA-LNP encoding for Spike 2P protein alone (Square), the body weight of mice immunized with mRNA-LNP encoding for T cell Ag (Nucleoprotein, NSP2 and NSP14) proteins (triangle); the body weight of mice immunized with mRNA-LNP encoding for Spike SP2 + T cell Ag (Nucleoprotein, NSP2 and NSP14) proteins (Black circle) and the Body weight of mock immunized (unvaccinated) mice (empty circle).
  • mice were followed-up for fourteen days for changes in body weight in each group of mice after challenge with SARS-CoV-2 Delta (B.1 .617.2) variant.
  • a two-tailed unpaired t-test was applied in the comparison of weight loss and survival rates between groups assuming Gaussian distribution.
  • P ⁇ 0.05 was statistically significant at a 95% confidence interval.
  • FIG. 12B shows the mRNA-LNP Encoding Spike + T Cell Antigens Combination Vaccine Protection is Mediated by CD4+ and CD8+ T-Cells.
  • the effects of CD4+ and CD8+ T cell depletion on body weight loss against intranasal infection with SARS-CoV-2 Delta (B.1.617.2) variant were analyzed in Techlmmune mRNA-LNP encoding Spike + T cell antigens vaccinated hACE2 mice, CD4+ and/or CD8+ T cell depleted and challenged with 1 x 10 5 pfu of SARS-CoV-2 Delta (B.1 .617.2) variant.
  • mice were followed-up for fourteen days post intranasal infection and their weight was measured daily.
  • the graph show the body weight of mice immunized with the mRNA-LNP encoding for Spike SP2 + T cell Ag (Nucleoprotein, NSP2 and NSP14) proteins without depleted CD4+ and CD8+ T-cells (Black circle), or with depletion of CD4+ T cells (Triangle), depletion of CD8+ T-cell (circle half with half black), with both depletion of CD4+ and CD8+ T cells (empty square) and the Body weight of mock immunized (unvaccinated) mice (empty circle).
  • CD4+ or CD8+ T cell depletion in mice immunized with mRNA-LNP was conducted by intraperitoneal injection (2 times) of 300 pg of anti-CD4 Ab (GK1.5) and/or anti-CD8a Ab (2.43). 2 days after the last depletion with anti-CD4 and/or anti-CD8 mAb injections, mice were followed-up for fourteen days for changes in body weight in each group of mice after challenge with SARS-CoV-2 Delta (B.1.617.2) variant. A two-tailed unpaired t-test was applied in the comparison of weight loss and survival rates between groups assuming Gaussian distribution. P ⁇ 0.05 was statistically significant at a 95% confidence interval.
  • FIG. 13A and 13B shows non-limiting examples of antigen combinations that may be used for the vaccine compositions described herein.
  • the proteins may be covalently or non-covalently linked together for administration of the vaccine composition.
  • Spike protein may refer to a portion of the spike protein or a spike protein with one or more mutations (e.g., a spike protein with two or six proline substitutions). Additionally, proteins denoted in the figures may include only a portion of said protein.
  • the antigen combinations may be synthesized and delivered in a single delivery system. In other embodiments, the antigen combinations may be synthesized and delivered in different delivery systems.
  • the terms "immunogenic protein, polypeptide, or peptide” or “antigen” refer to polypeptides or other molecules (or combinations of polypeptides and other molecules) that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein.
  • the protein fragment has substantially the same immunological activity as the total protein.
  • a protein fragment according to the disclosure can comprises or consists essentially of or consists of at least one epitope or antigenic determinant.
  • An "immunogenic" protein or polypeptide, as used herein, may include the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof.
  • Immunogenic fragment refers to a fragment of a protein that includes one or more epitopes and thus elicits the immunological response described above.
  • Immunogenic fragments for purposes of the disclosure may feature at least about 1 amino acid, at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.
  • epitope refers to the site on an antigen or hapten to which specific B cells and/or T cells respond.
  • the term is also used interchangeably with "antigenic determinant” or “antigenic determinant site.”
  • Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • the term "immunological response" to a composition or vaccine refers to the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest.
  • an "immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host may display either a therapeutic or protective immunological response, so resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
  • a variant refers to a substantially similar sequence.
  • a variant comprises a deletion and/or addition and/or change of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or an amino acid sequence, respectively.
  • Variants of a particular polynucleotide of the disclosure can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present disclosure are biologically active; that is, they have the ability to elicit an immune response.
  • the terms “treat” or “treatment” or “treating” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow the development of the disease, such as slow down the development of a disorder, or reducing at least one adverse effect or symptom of a condition, disease or disorder, e.g., any disorder characterized by insufficient or undesired organ or tissue function.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced as that term is defined herein.
  • a treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or decrease of markers of the disease, but also a cessation or slowing of progress or worsening of a symptom that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treatment also includes ameliorating a disease, lessening the severity of its complications, preventing it from manifesting, preventing it from recurring, merely preventing it from worsening, mitigating an inflammatory response included therein, or a therapeutic effort to affect any of the aforementioned, even if such therapeutic effort is ultimately unsuccessful.
  • carrier or “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable vehicle” refers to any appropriate or useful carrier or vehicle for introducing a composition to a subject.
  • Pharmaceutically acceptable carriers or vehicles may be conventional but are not limited to conventional vehicles.
  • E. W. Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 15th Edition (1975) and D. B. Troy, ed. Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore MD and Philadelphia, PA, 21st Edition (2006) describe compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds or molecules.
  • Carriers are materials generally known to deliver molecules, proteins, cells and/or drugs and/or other appropriate material into the body.
  • the nature of the carrier will depend on the nature of the composition being delivered as well as the particular mode of administration being employed.
  • pharmaceutical compositions administered may contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like.
  • Patents that describe pharmaceutical carriers include, but are not limited to: U.S. Patent No. 6,667,371 ; U.S. Patent No. 6,613,355; U.S. Patent No. 6,596,296; U.S. Patent No.
  • the carrier may, for example, be solid, liquid (e.g., a solution), foam, a gel, the like, or a combination thereof.
  • the carrier comprises a biological matrix (e.g., biological fibers, etc.).
  • the carrier comprises a synthetic matrix (e.g., synthetic fibers, etc.).
  • a portion of the carrier may comprise a biological matrix and a portion may comprise synthetic matrix.
  • coronavirus may refer to a group of related viruses such as but not limited to severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). All the coronaviruses cause respiratory tract infections that range from mild to lethal in mammals. Several non-limiting examples of Coronavirus strains are described herein. In some embodiments, the compositions may protect against any Sarbecoviruses, including but not limited to SARS-CoV1 or SARS-C0V2. As used herein, “severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)” is a betacoronavirus that causes Coronavirus Disease 19 (COVID-19).
  • SARS-CoV2 severe acute respiratory syndrome coronavirus 2
  • a “subject” is an individual and includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird, a reptile, or an amphibian.
  • a mammal e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included.
  • a “patient” is a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects
  • administering refers to methods of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, administering the compositions orally, parenterally (e.g., intravenously and subcutaneously), by intramuscular injection, by intraperitoneal injection, intrathecally, transdermally, extracorporeally, topically or the like.
  • parenterally e.g., intravenously and subcutaneously
  • intramuscular injection e.g., intraperitoneal injection
  • intrathecally e.g., transdermally, extracorporeally, topically or the like.
  • a composition can also be administered by topical intranasal administration (intranasally) or administration by inhalant.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism (device) or droplet mechanism (device), or through aerosolization of the composition.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism.
  • an inhaler can be a spraying device or a droplet device for delivering a composition comprising the vaccine composition, in a pharmaceutically acceptable carrier, to the nasal passages and the upper and/or lower respiratory tracts of a subject. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intratracheal intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular composition used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • a composition can also be administered by buccal delivery or by sublingual delivery.
  • buccal delivery may refer to a method of administration in which the compound is delivered through the mucosal membranes lining the cheeks.
  • the vaccine composition is placed between the gum and the cheek of a patient.
  • sublingual delivery may refer to a method of administration in which the compound is delivered through the mucosal membrane under the tongue.
  • the vaccine composition is administered under the tongue of a patient.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves the use of a slow-release or sustained-release system such that a constant dosage is maintained. See, for example, U.S. Pat. No. 3,610,795, which is incorporated by reference herein.
  • the present invention features pre-emptive pan-coronavirus vaccines, methods of use, methods of producing said vaccines, methods of preventing coronavirus infections, etc.
  • the present invention also provides methods of testing said vaccines, e.g., using particular animal models and clinical trials.
  • the vaccine compositions herein can induce efficient and powerful protection against the coronavirus disease or infection, e.g., by inducing the production of antibodies (Abs), CD4 + T helper (Th1) cells, and CD8 + cytotoxic T-cells (CTL).
  • the present invention features a universal pre-emptive pan-coronavirus vaccine composition.
  • the composition may comprise two (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the composition may comprise three (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the composition comprises a sequence encoding two (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the composition comprises a sequence encoding three (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the aforementioned composition may further comprise Coronavirus antigen derived from at least a portion of a Spike protein.
  • the composition may comprise two (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein.
  • the composition may comprise three (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein
  • the composition may comprise a sequence encoding two (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein.
  • the composition may comprise a sequence encoding three (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein.
  • the present invention features a universal pre-emptive pan-Coronavirus vaccine composition.
  • the composition may comprise two (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the composition may comprise three (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the composition comprises a sequence encoding two (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the composition comprises a sequence encoding three (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the aforementioned composition may further comprise Coronavirus antigen derived from a Spike protein.
  • the composition may comprise two (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; c) Nucleoprotein protein and d) a Spike protein.
  • the composition may comprise three (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; c) Nucleoprotein protein and d) a Spike protein.
  • the composition may comprise a sequence encoding two (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein and a Spike protein. In other embodiments, the composition may comprise a sequence encoding three (or more) Coronavirus antigens derived from a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein and a Spike protein.
  • the present invention may also feature a pan-Coronavirus recombinant vaccine composition.
  • the composition comprising a delivery system encoding two (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the composition comprising a delivery system encoding three (or more) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the aforementioned delivery system may further encode a Coronavirus antigen derived from at least a portion of a Spike protein.
  • the aforementioned delivery system may further comprise an additional delivery system encoding a Coronavirus antigens derived from at least a portion of a Spike protein.
  • the composition may comprise a delivery system encoding two (or more) Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein; and d) at least a portion of a Spike protein.
  • the composition may comprise a delivery system encoding three (or more) Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein; and d) at least a portion of a Spike protein.
  • the delivery system may comprise a single delivery system or may comprise two or more delivery systems.
  • the present invention may feature a pan-Coronavirus recombinant vaccine composition.
  • the composition comprises a delivery system encoding two (or more) Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the composition comprises a delivery system encoding three (or more) Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • the aforementioned delivery system may further encode a Coronavirus antigen derived from a Spike protein.
  • the aforementioned delivery system may further comprise an additional delivery system encoding a Coronavirus antigens derived from a Spike protein.
  • the composition may comprise a delivery system encoding two (or more) Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • the composition may comprise a delivery system encoding three (or more) Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • the delivery system may comprise a single delivery system or may comprise two or more delivery systems.
  • the present invention may feature a universal pre-emptive pan-Coronavirus vaccine composition comprising a B cell antigen and two (or three) or more T-cell antigens.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprises a sequence encoding a B cell antigen and two (or three) or more T-cell antigens.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprising a B cell antigen and three T-cell antigens.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprises a sequence encoding a B cell antigen and three T-cell antigens.
  • the B cell antigens may be derived from a Coronavirus Spike protein or portion thereof.
  • the T cell antigens may be derived from an NSP2 protein or portion thereof, an NSP14 protein or portion thereof, a Nucleoprotein or portion thereof, or a combination thereof.
  • the T cell antigens may be derived from an NSP3 protein or portion thereof, an NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the present invention features may further universal pre-emptive pan-Coronavirus vaccine composition comprising two or more large sequences derived from at least a portion of an NSP2 protein; at least a portion of an NSP14 protein; at least a portion of a Nucleoprotein protein; and at least a portion of a Spike protein.
  • the universal pre-emptive pan-Coronavirus vaccine composition comprises at least three or more large sequences derived from at least a portion of an NSP2 protein; at least a portion of an NSP14 protein; at least a portion of a Nucleoprotein protein; and at least a portion of a Spike protein.
  • the aforementioned Coronavirus antigens may be the Spike protein (or a portion thereof) and the NSP2 protein (or a portion thereof).
  • the Coronavirus antigens may be the Spike protein (or a portion thereof) and the NSP14 protein (or a portion thereof).
  • the Coronavirus antigens may be the Spike protein (or a portion thereof) and the Nucleoprotein (or a portion thereof).
  • the Coronavirus antigens may be the Spike protein (or a portion thereof), the NSP2 protein (or a portion thereof), and the NSP14 protein (or a portion thereof).
  • the Coronavirus antigens may be the Spike protein (or a portion thereof), the NSP2 protein (or a portion thereof), and the Nucleoprotein (or a portion thereof). In some embodiments, the Coronavirus antigens may be the Spike protein (or a portion thereof), the NSP14 protein (or a portion thereof), and the Nucleoprotein (or a portion thereof). In some embodiments, the Coronavirus antigens may be the Spike protein (or portion thereof); the NSP2 protein (or a portion thereof); the NSP14 protein (or a portion thereof); and the Nucleoprotein (or a portion thereof). In some embodiments, the Spike protein (or a portion thereof) comprises one or more proline substitutions. Portions of the aforementioned may comprise immunogenic fragments.
  • the aforementioned composition may further comprise one or more Coronavirus antigens derived from an NSP3 protein or portion thereof, a NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the aforementioned composition may further comprise two or more Coronavirus antigens derived from an NSP3 protein or portion thereof, a NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the aforementioned composition may further comprise three or more Coronavirus antigens derived from an NSP3 protein or portion thereof, a NSP12 protein or portion thereof, or a protein encoded by ORF7a/b or portion thereof.
  • the present invention is not limited to the aforestated Coronavirus antigens.
  • the vaccine compositions herein may feature multiple antigens, e.g., large sequences, which may comprise multiple conserved epitopes, that help provide multiple opportunities forthe body to develop an immune response for preventing infection. Further, the vaccines herein may be designed to be effective against past, current, and future coronavirus outbreaks.
  • the vaccine composition comprises multiple antigens.
  • the antigens are conserved antigens, e.g..antigens that are highly conserved among human coronaviruses and/or animal coronaviruses (e.g., coronaviruses isolated from animals susceptible to coronavirus infections).
  • the vaccine composition comprises multiple large sequences.
  • the large sequences are conserved large sequences, e.g., sequences that are highly conserved among human coronaviruses and/or animal coronaviruses (e.g., coronaviruses isolated from animals susceptible to coronavirus infections).
  • Corona viruses used for determining conserved antigens may include human SARS-CoVs as well as animal CoVs (e.g., bats, pangolins, civet cats, minks, camels, etc.) as described herein.
  • SARS-CoV-2 strains obtained from humans (Homo Sapiens), along with the animal’s SARS-like Coronaviruses genome sequence (SL-CoVs) sequences obtained from bats (Rhinolophus affinis, Rhinolophus malayanus), pangolins (Manis javanica), civet cats (Paguma larvata), and camels (Camelus dromedarius).
  • the included SARS-CoV/MERS-CoV strains are from previous outbreaks (obtained from humans (Urbani, MERS-CoV, OC43, NL63, 229E, HKU1-genotype-B), bats (WIV16, WIV1 , YNLF-31 C, Rs672, recombinant strains), camel (Camelus dromedarius, (KT368891.1 , MN514967.1 , KF917527.1 , NC_028752.1), and civet (Civet007, A022, B039)).
  • the human SARS-CoV-2 genome sequences are represented from six continents.
  • coronaviruses may be used for determining conserved antigens (including human SARS-CoVs as well as animal Co Vs (e.g., bats, pangolins, civet cats, minks, camels, etc.)) that meet the criteria to be classified as “variants of concern’’ or “variants of interest.” Coronavirus variants that appear to meet one or more of the undermentioned criteria may be labeled "variants of interest” or "variants under investigation” pending verification and validation of these properties.
  • the criteria may include increased transmissibility, increased morbidity, increased mortality, increased risk of “long COVID,” ability to evade detection by diagnostic tests, decreased susceptibility to antiviral drugs (if and when such drugs are available), decreased susceptibility to neutralizing antibodies, either therapeutic (e.g., convalescent plasma or monoclonal antibodies) or in laboratory experiments, ability to evade natural immunity (e.g., causing reinfections), ability to infect vaccinated individuals, increased risk of particular conditions such as multisystem inflammatory syndrome or long-haul COVID or Increased affinity for particular demographic or clinical groups, such as children or immunocompromised individuals.
  • variants of interest are renamed “variant of concern” by monitoring organizations, such as the CDC.
  • the antigens e.g., conserved antigens may be derived from structural (e.g., spike glycoprotein, or Nucleoprotein) or non-structural proteins of the coronaviruses (e g., any of the 16 NSPs, e.g., NSP2 and NSP14, encoded by ORF1a/b).
  • structural e.g., spike glycoprotein, or Nucleoprotein
  • non-structural proteins of the coronaviruses e.g., any of the 16 NSPs, e.g., NSP2 and NSP14, encoded by ORF1a/b.
  • the antigens e.g., large sequences are each highly conserved among one or a combination of: SARS-CoV-2 human strains, SL-CoVs isolated from bats, SL-CoVs isolated from pangolin, SL-CoVs isolated from civet cats, and MERS strains isolated from camels.
  • the antigens e.g., large sequences are each highly conserved among one or a combination of: at least 50,000 SARS-CoV-2 human strains, five SL-CoVs isolated from bats, five SL-CoVs isolated from pangolin, three SL-CoVs isolated from civet cats, and four MERS strains isolated from camels.
  • the antigens e.g., large sequences are each highly conserved among one or a combination of: at least 80,000 SARS-CoV-2 human strains, five SL-CoVs isolated from bats, five SL-CoVs isolated from pangolin, three SL-CoVs isolated from civet cats, and four MERS strains isolated from camels.
  • the antigens e.g., large sequences are each highly conserved among one or a combination of: at least 50,000 SARS-CoV-2 human strains in circulation during the COVI-19 pandemic, at least one CoV that caused a previous human outbreak, five SL-CoVs isolated from bats, five SL-CoVs isolated from pangolin, three SL-CoVs isolated from civet cats, and four MERS strains isolated from camels.
  • the antigens e.g., large sequences are each highly conserved among at least 1 SARS-CoV-2 human strain in current circulation, at least one CoV that has caused a previous human outbreak, at least one SL-CoV isolated from bats, at least one SL-CoV isolated from pangolin, at least one SL-CoV isolated from civet cats, and at least one MERS strain isolated from camels.
  • the antigens e.g., large sequences are each highly conserved among at least 1 ,000 SARS-CoV-2 human strains in current circulation, at least two CoVs that have caused a previous human outbreak, at least two SL-CoVs isolated from bats, at least two SL-CoVs isolated from pangolin, at least two SL-CoVs isolated from civet cats, and at least two MERS strains isolated from camels.
  • the antigens e.g., large sequences are each highly conserved among one or a combination of: at least one SARS-CoV-2 human strain in current circulation, at least one CoV that has caused a previous human outbreak, at least one SL-CoV isolated from bats, at least one SL-CoV isolated from pangolin, at least one SL-CoV isolated from civet cats, and at least one MERS strain isolated from camels.
  • the present invention is not limited to the aforementioned coronavirus strains that may be used to identify conserved antigens, e.g., large sequences.
  • one or more of the conserved antigens are derived from one or more SARS-CoV-2 human strains or variants in current circulation; one or more coronaviruses that have caused a previous human outbreak; one or more coronaviruses isolated from animals selected from a group consisting of bats, pangolins, civet cats, minks, camels, and other animal receptive to coronaviruses; and/or one or more coronaviruses that cause the common cold.
  • SARS-CoV-2 human strains and variants in current circulation may include the original SARS-CoV-2 strain (SARS-CoV-2 isolate Wuhan-Hu-1), and several variants of SARS-CoV-2 including but not limited to variant B.1.177 (Spain); variant B.1.160 (Australia), variant B.1.1.7 (UK), variant P.1 (Japan/Brazil), variant B.1.351 (South Africa), variant B.1.427 (California), variant B.1.429 (California), variant B.1.258 (Scotland); variant B.1.221 (Belgium/Netherlands); variant B.1.367 (Norway/France); variant B.1.1.277 (UK); variant B.1.1.302 (Sweden); variant B.1.525 (North America, Europe, Asia, Africa, and Australia); variant B.1.526 (New York), variant S:677H; variant S:677P; B.1 .617.2-Delta, variant B.1 .1 ,5
  • the present invention is not limited to the aforementioned variants of SARS-CoV-2 and encompasses variants identified in the future.
  • the one or more coronaviruses that cause the common cold may include but are not limited to strains 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus).
  • the term “conserved” refers to an antigen or large sequence that is among the most highly conserved antigen or large sequences identified in a sequence alignment and analysis.
  • the conserved antigen or large sequences may be the 2 most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 3 most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 4 most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 5 most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 6 most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 7 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 8 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 9 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 10 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 15 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 20 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 25 most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 30 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 40 most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 50 most highly conserved sequences identified. In some embodiments, the conserved sequences may be the 50% most highly conserved antigen or large sequences identified. In some embodiments, the conserved antigen or large sequences may be the 60% most highly conserved sequences identified. In some embodiments, the large conserved sequences may be the 70% most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 80% most highly conserved sequences identified.
  • the conserved antigen or large sequences may be the 90% most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 95% most highly conserved sequences identified. In some embodiments, the conserved antigen or large sequences may be the 99% most highly conserved sequences identified. The present invention is not limited to the aforementioned thresholds.
  • the composition comprises one or more antigens.
  • the one or more antigens comprise at least one of one or more conserved coronavirus B-cell target epitopes; one or more conserved coronavirus CD4+ T cell target epitopes; and one or more conserved coronavirus CD8+ T cell target epitopes.
  • the vaccine composition comprises two or more antigens.
  • the two or more antigens comprise at least one of one or more conserved coronavirus B-cell target epitopes; one or more conserved coronavirus CD4+ T cell target epitopes; and one or more conserved coronavirus CD8+ T cell target epitopes.
  • the composition comprises one or more large sequences.
  • the one or more large sequences comprise at least one of one or more conserved coronavirus B-cell target epitopes; one or more conserved coronavirus CD4+ T cell target epitopes; and one or more conserved coronavirus CD8+ T cell target epitopes.
  • the vaccine composition comprises two or more large sequences.
  • the two or more large sequences comprise at least one of one or more conserved coronavirus B-cell target epitopes; one or more conserved coronavirus CD4+ T cell target epitopes; and one or more conserved coronavirus CD8+ T cell target epitopes.
  • the antigens may be each separated by a linker.
  • the linker allows for an enzyme to cleave between the antigens.
  • the present invention is not limited to particular linkers or particular lengths of linkers.
  • one or more antigens may be separated by a linker 2 amino acids in length, or a linker 3 amino acids in length, or a linker 4 amino acids in length, or a linker 5 amino acids in length, or a linker 6 amino acids in length, or a linker 7 amino acids in length, or a linker 8 amino acids in length, or a linker 9 amino acids in length, or a linker 10 amino acids in length.
  • one or more antigens may be separated by a linker from 2 to 10 amino acids in length.
  • Linkers are well known to one of ordinary skill in the art. Non-limiting examples of linkers include AAY, KK, and GPGPG.
  • the antigens e.g., large sequences may be derived from structural proteins, non-structural proteins, or a combination thereof.
  • structural proteins may include spike proteins (S) or Nucleoproteins (N)
  • non-structural proteins may include NSP2 and NSP14, encoded by ORF1a/b.
  • the antigens are derived from at least one SARS-CoV-2 protein.
  • the SARS-CoV-2 proteins may include ORFI ab protein, Spike glycoprotein, ORF3a protein, Envelope protein, Membrane glycoprotein, ORF6 protein, ORF7a protein, ORF7b protein, ORF8 protein, Nucleoprotein protein, and ORF10 protein.
  • the ORFI ab protein provides nonstructural proteins (Nsp) such as Nsp1 , Nsp2, Nsp3 (Papain-like protease), Nsp4, Nsp5 (3C-like protease), Nsp6, Nsp7, Nsp8, Nsp9, Nsp10, Nsp11 , Nsp12 (RNA polymerase), Nsp13 (5’ RNA triphosphatase enzyme), Nsp14 (guanosineN7-methyltransferase), Nsp15 (endoribonuclease), and Nsp16 (2 -O-ribose-methyltransferase).
  • the SARS-CoV-2 proteins include Spike glycoprotein, Nucleoprotein protein, Nsp2, Nsp14, or a combination thereof.
  • the SARS-CoV-2 has a genome length of 29,903 base pairs (bps) ssRNA.
  • the region between 266-21555 bps codes for ORFI ab polypeptide; the region between 21563-25384 bps codes for one of the structural proteins (spike protein or surface glycoprotein); the region between 25393-26220 bps codes for the ORF3a gene; the region between 26245-26472 bps codes for the envelope protein; the region between 26523-27191 codes for the membrane glycoprotein (or membrane protein); the region between 27202-27387 bps codes for the ORF6 gene; the region between 27394-27759 bps codes for the ORF7a gene; the region between 27894-28259 bps codes for the ORF8 gene; the region between 28274-29533 bps codes for the Nucleoprotein phosphoprotein (or the Nucleoprotein protein); and the region between 29558-29674 bps codes for the ORF10 gene.
  • the antigens may comprise a T-cell epitope restricted to a large number of human class 1 and class 2 HLA haplotypes and not restricted to HLA-0201 for class 1 or HLA-DR for class 2.
  • the conserved antigens may be restricted to human HLA class 1 and 2 haplotypes.
  • the conserved epitopes are restricted to cat and dog MHC class 1 and 2 haplotypes.
  • the vaccine compositions described herein protects against disease caused by one or more coronavirus variants or coronavirus subvariants.
  • the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants, including, but not limited to, alpha, beta, gamma, delta, and omicron.
  • the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • the vaccine compositions described herein may also protect against infection and reinfection of coronavirus variants or coronavirus subvariants.
  • the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants, including but not limited to alpha, beta, gamma, delta, and omicron.
  • the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • the vaccine compositions described herein protects against infection or reinfection of one or more coronavirus variants or coronavirus subvariants. In some embodiments, the vaccine composition described herein against infection or reinfection of multiple coronavirus variants or coronavirus subvariants. In other embodiments, the vaccine composition described herein composition protects against infection or re-infection caused by one coronavirus variant or coronavirus subvariant.
  • the vaccine composition induces strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Th1) cells, and/or CD8+ cytotoxic T-cells (CTL).
  • Abs antibodies
  • Th1 CD4+ T helper
  • CTL CD8+ cytotoxic T-cells
  • the antigen may comprise large sequences, such as conserved large sequences that are highly conserved among human and animal coronaviruses.
  • large sequence refers to a sequence having at least 25 amino acids or at least 75 nucleotides.
  • the large sequences comprise epitopes, such as the conserved epitopes described herein.
  • the large sequence has at least 75 nt. In some embodiments, the large sequence has at least 150 nt. In some embodiments, the large sequence has at least 200 nt. In some embodiments, the large sequence has at least 250 nt. In some embodiments, the large sequence has at least 300 nt. In some embodiments, the large sequence has at least 400 nt. In some embodiments, the large sequence has at least 500 nt. In some embodiments, the large sequence has at least 600 nt. In some embodiments, the large sequence has at least 700 nt. In some embodiments, the large sequence has at least 800 nt. In some embodiments, the large sequence has at least 900 nt. In some embodiments, the large sequence has at least 1000 nt. In some embodiments, the large sequence has at least 1500 nt. In some embodiments, the large sequence has at least 2000 nt.
  • the vaccine composition comprises one antigen. In some embodiments, the vaccine composition comprises one or more antigens. In some embodiments, the vaccine composition comprises two antigens. In some embodiments, the vaccine composition comprises two or more antigens. In some embodiments, the vaccine composition comprises three antigens. In some embodiments, the vaccine composition comprises three or more antigens. In some embodiments, the vaccine composition comprises four antigens. In some embodiments, the vaccine composition comprises four or more antigens. In some embodiments, the vaccine composition comprises five or more antigens, e.g., 5, 6, 7, 8, etc. In some embodiments, the vaccine composition comprises one antigen.
  • the vaccine composition comprises one large sequence. In some embodiments, the vaccine composition comprises one or more large sequences. In some embodiments, the vaccine composition comprises two or more large sequences. In some embodiments, the vaccine composition comprises three or more large sequences. In some embodiments, the vaccine composition comprises four or more large sequences. In some embodiments, the vaccine composition comprises five or more large sequences, e.g., 5, 6, 7, 8, etc.
  • the antigens, e.g., large sequences are derived from a whole protein sequence expressed by SARS-CoV-2. In other embodiments, the antigens, e.g., large sequences are derived from a partial protein sequence expressed by SARS-CoV-2. In some embodiments, the antigens, e.g., large sequence of said proteins comprises B cell epitopes and T-cell epitopes that are restricted to a large number, e.g., from 3 to 10, different haplotypes that encompass 100% of the population regardless of race and ethnicity)of human class 1 and class 2 HLA haplotypes, so they are not restricted only to HLA-0201 for class 1 or HLA-DR1 for class 2.
  • the antigens may be highly conserved among human and animal coronaviruses.
  • the antigens, e.g., large sequences are derived from one or a combination of: one or more SARS-CoV-2 human strains or variants in current circulation; one or more coronaviruses that have caused a previous human outbreak; one or more coronaviruses isolated from animals selected from a group consisting of bats, pangolins, civet cats, minks, camels, and other animal receptive to coronaviruses; and/or one or more coronaviruses that cause the common cold.
  • the SARS-CoV-2 human strains or variants in current circulation may include variant B.1.177; variant B.1.160, variant B.1 .1.7 (UK), variant P.1 (Japan/Brazil), variant B.1 .351 (South Africa), variant B.1.427 (California), variant B.1 .429 (California), variant B.1.258; variant B.1.221 ; variant B.1.367; variant B.1.1.277; variant B.1.1.302; variant B.1.525; variant B.1.526, variant S:677H; variant S:677P; B.1 617.2-Delta, variant B.1 .1 529-Omicron (BA.1); sub-variant Omicron (BA.1); sub-variant Omicron (BA.2); sub-variant Omicron (BA.3); sub-variant Omicron (BA.4); sub-variant Omicron (BA.5).
  • BA.1 sub-variant O
  • the antigen(s), e.g., large sequence(s) may be derived from structural proteins, non-structural proteins, or a combination thereof.
  • the large sequence(s) may be selected from ORFIab protein, Spike glycoprotein (e.g., the RBD), ORF3a protein, Envelope protein, Membrane glycoprotein, ORF6 protein, ORF7a protein, ORF7b protein, ORF8 protein, Nucleoprotein protein, and/or an ORF10 protein.
  • ORFI ab protein comprises nonstructural protein (Nsp) 1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10, Nsp11 , Nsp12, Nsp13, Nsp14, Nsp15 and Nsp16.
  • the large sequence(s) may be selected from ORFI ab protein (encoding Nsp2 and Nsp14), Spike glycoprotein (e.g., the RBD), Nucleoprotein protein, or a combination thereof.
  • the present invention features a universal pre-emptive pan-coronavirus vaccine composition.
  • the composition may comprise a sequence encoded by or comprises two (or three) or more Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • the aforementioned composition may further comprise Coronavirus antigen derived from at least a portion of a Spike protein.
  • the composition may comprise a sequence encoded by or comprises two (or three) Coronavirus antigens derived from a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein and d) at least a portion of a Spike protein. See Table 1 .
  • the present invention features a universal pre-emptive pan-Coronavirus vaccine composition
  • a universal pre-emptive pan-Coronavirus vaccine composition comprising two or more large sequences derived from at least a portion of an NSP2 protein, at least a portion of an NSP14 protein, at least a portion of a Nucleoprotein protein, and at least a portion of a Spike protein (See Table 1).
  • Table 1 SARS-CoV-2 Omicron sub-variant BA.2
  • the portion of the NSP2 protein is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the portion of the NSP2 protein comprises a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 3.
  • the portion of the NSP14 protein is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 4 or SEQ ID NO: 5.
  • the portion of the NSP14 protein comprises a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 6.
  • the portion of the Nucleoprotein is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 7 or SEQ ID NO: 8.
  • the portion of the Nucleoprotein protein comprises a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 9.
  • one or more antigens are derived from a full-length spike glycoprotein. In some embodiments, one or more antigens is derived from a partial spike glycoprotein. In some embodiments, the spike (S) protein comprises at least one proline substitution, or at least two proline substitutions, or at least four proline substitutions, or at least six proline substitutions. The spike (S) protein may comprise two consecutive proline substitutions at amino acid positions 986 and 987. The proline substitutions may comprise K986P and V987P mutations. In further embodiments, the spike (S) protein is receptor-binding domain (RBD). In some embodiments, the RBD comprises a trimerized SARS-CoV-2 receptor-binding domain (RBD). See Table 2.
  • Table 2 Shows non-limiting examples of Coronavirus Spike (S) protein that may be used in accordance with the present invention.
  • the Spike protein is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 10.
  • the portion of the Spike protein comprises a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 11 or SEQ ID NO: 12.
  • the Coronavirus antigens may further comprise NSP 3, NSP12, ORF7a/b, or a combination thereof (see Table 3).
  • the portion of the NSP3 protein is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 13 or SEQ ID NO: 14.
  • the portion of the NSP12 protein is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 15 or SEQ ID NO: 16.
  • the portion of the ORF7a/b is encoded by a sequence that is 100%, 98%, 95%, 90%, 85%, 80%, 75%, or 50% identical to SEQ ID NO: 17 or SEQ ID NO: 18.
  • the vaccine composition comprises a molecular adjuvant and/or one or more T cell enhancement compositions.
  • the adjuvant and/or enhancement compositions may help improve the immunogenicity and/or long-term memory of the vaccine composition.
  • molecular adjuvants include CpG, such as a CpG polymer, and flagellin.
  • the vaccine composition comprises a T cell attracting chemokine.
  • the T cell attracting chemokine helps pull the T cells from the circulation to the appropriate tissues, e.g., the lungs, heart, kidney, and brain.
  • T cell attracting chemokines include CCL5, CXCL9, CXCL10, CXCL11 , CCL25, CCL28, CXCL14, CXCL17, or a combination thereof.
  • the vaccine composition comprises a composition that promotes T cell proliferation.
  • compositions that promote T cell proliferation include IL-7, IL-15, IL-2, or a combination thereof.
  • the vaccine composition comprises a composition that promotes T cell homing in the lungs.
  • compositions that promote T cell homing include CCL25, CCL28, CXCL14, CXCL17, or a combination thereof.
  • the molecular adjuvant and/or the T cell attracting chemokine and/or the composition that promotes T cell proliferation are delivered with a separate antigen delivery system from the large sequences.
  • Table 4 shows non-limiting examples of T-cell enhancements that may be used to create a vaccine composition described herein.
  • the T-cell enhancement compositions described herein may be integrated into a separate delivery system from the vaccine compositions.
  • the T-cell enhancement compositions described herein e.g. CXCL9, CXCL10, IL-7, IL-2 may be integrated into the same delivery system as the vaccine compositions.
  • the vaccine composition comprises a tag.
  • the vaccine composition comprises a His tag.
  • the present invention is not limited to a His tag and includes other tags such as those known to one of ordinary skill in the art, such as a fluorescent tag (e.g., GFP, YFP, etc.), etc.
  • the present invention also features vaccine compositions in the form of a delivery system (e.g., an antigen delivery system). Any appropriate antigen delivery system may be considered for delivery of the antigens described herein.
  • the present invention is not limited to the antigen delivery systems described herein.
  • the antigen delivery system is for targeted delivery of the vaccine composition, e.g., for targeting to the tissues of the body where the virus replicates.
  • the antigen delivery system comprises adenoviruses such as but not limited to Ad5, Ad26, Ad35, etc., as well as carriers such as lipid nanoparticles, polymers, peptides, etc.
  • the antigen delivery system comprises a vesicular stomatitis virus (VSV) vector.
  • VSV vesicular stomatitis virus
  • the present invention is not limited to adenovirus vector-based antigen delivery systems.
  • the antigen delivery system comprises an adeno-associated virus vector-based antigen delivery system, such as but not limited to the adeno-associated virus vector type 9 (AAV9 serotype), AAV type 8 (AAV8 serotype), etc.
  • the adeno-associated virus vectors used are tropic, e.g., tropic to lungs, brain, heart, and kidney, e.g., the tissues of the body that express ACE2 receptors.
  • AAV9 is known to be neurotropic, which would help the vaccine composition to be expressed in the brain.
  • the one or more antigens are operatively linked to a promoter.
  • the one or more large sequences are operatively linked to a generic promoter.
  • the one or more antigens are operatively linked to a CMV promoter.
  • the one or more antigens are operatively linked to a CAG, EFIA, EFS, CBh, SFFV, MSCV, mPGK, hPGK, SV40, UBC, or another appropriate promoter.
  • the one or more antigens are operatively linked to a tissue-specific promoter (e.g., a lung-specific promoter).
  • a tissue-specific promoter e.g., a lung-specific promoter
  • the antigen may be operatively linked to a SpB promoter or a CD144 promoter.
  • the vaccine composition comprises a molecular adjuvant.
  • the molecular adjuvant is operatively linked to a generic promoter, e.g., as described above.
  • the molecular adjuvant is operatively linked to a tissue-specific promoter, e.g., a lung-specific promoter, e.g., SpB or CD144.
  • the vaccine composition comprises a T cell attracting chemokine.
  • the T cell attracting chemokine is operatively linked to a generic promoter, e.g., as described above.
  • the T cell attracting chemokine is operatively linked to a tissue-specific promoter, e.g., a lung-specific promoter, e.g., SpB or CD144.
  • the vaccine composition comprises a composition for promoting T cell proliferation.
  • the composition for promoting T cell proliferation is operatively linked to a generic promoter, e.g., as described above.
  • the composition for promoting T cell proliferation is operatively linked to a tissue-specific promoter, e.g., a lung-specific promoter, e.g., SpB or CD144.
  • Table 5 shows non-limiting examples of promoters that may be used to create a vaccine composition described herein.
  • the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter (e.g., the T cell attracting chemokine and the composition that promotes T cell proliferation are synthesized as a peptide). In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by different promoters. In certain embodiments, the antigen, the T cell attracting chemokine, and the composition that promotes T cell proliferation are driven by the same promoter. In certain embodiments, the antigen, the T cell attracting chemokine, and the composition that promotes T cell proliferation are driven by the different promoters. In certain embodiments, the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter, and the one or more large sequences are driven by a different promoter.
  • the antigen delivery system comprises one or more linkers between the T cell attracting chemokine and the composition that promotes T cell proliferation.
  • linkers are used between one or more of the large sequences.
  • the linkers may allow for cleavage of the separate molecules (e.g., chemokine).
  • a linker is positioned between IL-7 (or IL-2) and CCL5, CXCL9, CXCL10, CXCL11 , CCL25, CCL28, CXCL14, CXCL17, etc.
  • a linker is positioned between IL-15 and CCL5, CXCL9, CXCL10, CXCL11 , CCL25, CCL28, CXCL14, CXCL17, etc.
  • a linker is positioned between the antigen or large sequence and another composition, e.g., IL-15, IL-7, CCL5, CXCL9, CXCL10, CXCL11 , CCL25, CCL28, CXCL14, CXCL17, etc.
  • a non-limiting example of a linker is T2A, E2A, P2A (see Table 6), or the like.
  • the composition may feature a different linker between each open reading frame.
  • Table 6 Shows non-limiting examples of linkers that may be used in accordance with the present invention.
  • the present invention includes mRNA sequences encoding any of the vaccine compositions or portions thereof herein, e.g., a molecular adjuvant, a T cell enhancement, etc.
  • the present invention also includes modified mRNA sequences encoding any of the vaccine compositions or portions thereof herein.
  • the present invention also includes DNA sequence encoding any of the vaccine compositions or portions thereof herein.
  • the mRNA sequence encodes at least two (preferably different) antigen. In certain embodiments, the mRNA sequence encodes at least three (preferably different) antigens.
  • the mRNA sequence may encode an NSP2 protein (or a portion thereof); an NSP3 protein (or a portion thereof); and a Nucleoprotein (Nucleoprotein) (or a portion thereof). Additionally, a separate mRNA sequence may encode a Spike protein (or portion thereof).
  • the mRNA sequence encodes at least four (preferably different) antigens; e.g., an NSP2 protein (or a portion thereof); an NSP3 protein (or a portion thereof); a Nucleoprotein (Nucleoprotein) (or a portion thereof); and a Spike protein (or a portion thereof).
  • an NSP2 protein or a portion thereof
  • an NSP3 protein or a portion thereof
  • a Nucleoprotein Nucleoprotein
  • Spike protein or a portion thereof
  • nucleic acids of a vaccine composition herein are chemically modified. In some embodiments, the nucleic acids of a vaccine composition therein are unmodified. In some embodiments, all or a portion of the uracil in the open reading frame has a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is an N1-methyl pseudouridine. In some embodiments, all or a portion of the uracil in the open reading frame has an N1 -methyl pseudouridine in the 5-position of the uracil.
  • an open reading frame of a vaccine composition herein encodes one antigen or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes two or more antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes three antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes five or more antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes ten or more antigens or epitopes. In some embodiments, an open reading frame of a vaccine composition herein encodes 50 or more antigens or epitopes.
  • the mRNAs may further comprise a 5’ untranslated region (UTR) and a 3’ UTR.
  • the mRNAs further comprise a 3’ poly(A) tail and/or a 5’ cap or cap analog.
  • the present invention may further feature a composition comprising two (or more) ribonucleic acids (mRNAs) comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group comprising, consisting essentially or consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein.
  • the composition comprising three (or more) ribonucleic acids (mRNAs) comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group comprising, consisting essentially or consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein.
  • the two or three mRNAs may be formulated in a lipid nanoparticle.
  • the composition may comprise two (or more) mRNAs comprising an open reading frame encoding an entire Coronavirus protein selected from a group comprising, consisting essentially, or consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein.
  • the composition may comprise three (or more) mRNAs comprising an open reading frame encoding an entire Coronavirus protein selected from a group comprising, consisting essentially, or consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein.
  • the composition may further comprise an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof formulated in a lipid nanoparticle.
  • the composition may comprise two (or more) mRNAs comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group comprising, consisting essentially or consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein.
  • the composition may comprise three (or more) mRNAs comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group comprising, consisting essentially or consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein.
  • the composition may comprise two (or more) mRNAs comprising an open reading frame encoding an entire Coronavirus protein selected from a group comprising, consisting essentially, or consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein.
  • the composition may comprise three (or more) mRNAs comprising an open reading frame encoding an entire Coronavirus protein selected from a group comprising, consisting essentially, or consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein.
  • the two or three (or more) mRNAs are formulated in a lipid nanoparticle.
  • compositions described herein e.g., the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc.
  • the compositions described herein, e.g., the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc. may be used to prevent a coronavirus infection prophylactically in a subject.
  • the compositions described herein may elicit an immune response in a subject.
  • the compositions described herein e.g., the antigens, the vaccine compositions, the antigen delivery systems, the chemokines, the adjuvants, etc., may prolong an immune response induced by the universal pre-emptive pan-coronavirus vaccine composition and increase T-cell migration to the lungs.
  • Methods for preventing a coronavirus disease in a subject may comprise administering to the subject a therapeutically effective amount of a pre-emptive pan-coronavirus vaccine composition according to the present invention.
  • the composition elicits an immune response in the subject.
  • the composition induces memory B and T cells.
  • the composition induces resident memory T cells (Trm).
  • the composition prevents virus replication, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • the composition prevents inflammation or an inflammatory response, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney. In some embodiments, the composition improves the homing and retention of T cells, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • Methods for preventing a coronavirus infection prophylactically in a subject may comprise administering to the subject a prophylactically effective amount of a pan-coronavirus vaccine composition according to the present invention.
  • the composition elicits an immune response in the subject.
  • the composition induces memory B and T cells.
  • the composition induces resident memory T cells (Trm).
  • the composition prevents virus replication, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • the composition prevents inflammation or an inflammatory response, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • the composition improves the homing and retention of T cells, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • Methods for eliciting an immune response in a subject may comprise administering to the subject a vaccine composition according to the present invention, wherein the composition elicits an immune response in the subject.
  • the composition induces memory B and T cells. In some embodiments, the composition induces resident memory T cells (Trm). In some embodiments, the composition prevents virus replication, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney. In some embodiments, the composition prevents a cytokine storm, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney. In some embodiments, the composition prevents inflammation or an inflammatory response, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney. In some embodiments, the composition improves the homing and retention of T cells, e.g., in the areas where the virus normally replicates, such as the lungs, brain, heart, and kidney.
  • Methods for prolonging an immune response induced by a vaccine composition of the present invention and increasing T cell migration to particular tissues may comprise co-expressing a T-cell attracting chemokine, a composition that promotes T cell proliferation, and a vaccine composition (e.g., antigen) according to the present invention.
  • tissue e.g., lung, brain, heart, kidney, etc.
  • a vaccine composition e.g., antigen
  • Methods for prolonging the retention of memory T cells into the lungs induced by a vaccine composition of the present invention and increasing virus-specific tissue-resident memory T-cells may comprise co-expressing a T-cell attracting chemokine, a composition that promotes T cell proliferation, and a vaccine composition (e.g., antigen) according to the present invention.
  • a vaccine composition e.g., antigen
  • the vaccine composition may be administered through standard means, e.g., through an intravenous route (i.v.), an intranasal route (i.n.), or a sublingual route (s. I.) route.
  • i.v. intravenous route
  • i.n. intranasal route
  • s. I. sublingual route
  • the method comprises administering to the subject a second (e.g., booster) dose.
  • the second dose may comprise the same vaccine composition or a different vaccine composition. Additional doses of one or more vaccine compositions may be administered.
  • the present invention features a method of delivering the vaccine to induce heterologous immunity in a subject (e.g., prime/boost).
  • the method comprises administering a first pan-coronavirus vaccine composition dose using a first delivery system.
  • the method comprises administering a second vaccine composition dose using a second delivery system.
  • the second composition is administered 8 days after administration of the first composition.
  • the second composition is administered 9 days after administration of the first composition.
  • the second composition is administered 10 days after administration of the first composition.
  • the second composition is administered 11 days after administration of the first composition.
  • the second composition is administered 12 days after administration of the first composition.
  • the second composition is administered 13 days after administration of the first composition. In some embodiments, the second composition is administered 14 days after administration of the first composition. In some embodiments, the second composition is administered from 14 to 30 days after administration of the first composition. In some embodiments, the second composition is administered from 30 to 60 days after administration of the first composition. In other embodiments, the first delivery system and the second delivery system are different. In some embodiments, the peptide vaccine composition is administered 14 days after the administration of the first vaccine composition dose. In some embodiments, the peptide vaccine composition is administered 30 or 60 days after the administration of the first vaccine composition dose.
  • the first delivery system or the second delivery system comprises an mRNA, a modified mRNA, or a peptide vector.
  • the peptide vector comprises an adenovirus or an adeno-associated virus vector.
  • the present invention features a method of delivering the vaccine to induce heterologous immunity in a subject (i.e., prime/pull).
  • the method comprises administering a pan-coronavirus vaccine composition.
  • the method comprises administering at least one T-cell attracting chemokine after administering the pan-coronavirus vaccine composition.
  • the T-cell attracting chemokine is administered 8 days after the vaccine composition is administered.
  • the T-cell attracting chemokine is administered 9 days after the vaccine composition is administered.
  • the T-cell attracting chemokine is administered 10 days after the vaccine composition is administered.
  • the T-cell attracting chemokine is administered 11 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 12 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 13 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 14 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered from 14 to 30 days after administration of the vaccine composition. In some embodiments, the T-cell attracting chemokine is administered from 30 to 60 days after administration of the vaccine composition. In some embodiments, the T cell-attracting chemokine composition is administered 8 to 14 days after the administration of the final vaccine composition dose. In some embodiments, the cell-attracting chemokine composition is administered 30 or 60 days after the administration of the final vaccine composition dose.
  • the present invention also features a novel “prime, pull, and boost” strategy.
  • the present invention features a method to increase the size and maintenance of lung-resident B-cells, CD4+ T cells, and CD8+ T cells to protect against SARS-CoV-2.
  • the method comprises administering a pan-coronavirus vaccine composition.
  • the method comprises administering at least one T-cell attracting chemokine after administering the pan-coronavirus vaccine composition.
  • the method comprises administering at least one cytokine after administering the T-cell attracting chemokine.
  • the T-cell attracting chemokine is administered 14 days after administering the pan-coronavirus composition.
  • the T-cell attracting chemokine is administered 14 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered from 14 to 30 days after administration of the vaccine composition. In some embodiments, the T-cell attracting chemokine is administered from 30 to 60 days after administration of the vaccine composition. In some embodiments, the cytokine is administered 8 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 9 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 10 days after administering the T-cell attracting chemokine.
  • the cytokine is administered 11 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 12 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 13 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered 14 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered from 14 to 30 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine is administered from 30 to 60 days after administering the T-cell attracting chemokine. In some embodiments, the cytokine composition is administered 8 to 14 days after the administration of the T cell-attracting chemokine. In some embodiments, the cytokine composition is administered 30 or 60 days after the administration of the T cell-attracting chemokine.
  • the present invention further features a novel “prime, pull, and keep” strategy.
  • the present invention features a method to increase the size and maintenance of lung-resident B-cells, CD4+ T cells, and CD8+ T cells to protect against SARS-CoV-2.
  • the method comprises administering a pan-coronavirus vaccine composition.
  • the method comprises administering at least one T-cell attracting chemokine after administering the pan-coronavirus vaccine composition.
  • the method comprises administering at least one mucosal chemokine after administering the T-cell attracting chemokine.
  • the T-cell attracting chemokine is administered 14 days after administering the pan-coronavirus composition. In other embodiments, the mucosal chemokines are administered 10 days after administering the T-cell attracting chemokine. In some embodiments, the T-cell attracting chemokine is administered 8 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 9 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 10 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 11 days after the vaccine composition is administered.
  • the T-cell attracting chemokine is administered 12 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 13 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered 14 days after the vaccine composition is administered. In some embodiments, the T-cell attracting chemokine is administered from 14 to 30 days after administration of the vaccine composition. In some embodiments, the T-cell attracting chemokine is administered from 30 to 60 days after administration of the vaccine composition. In some embodiments, the mucosal chemokine is administered 8 days after administering the T-cell attracting chemokine.
  • the mucosal chemokine is administered 9 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 10 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 11 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 12 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 13 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered 14 days after administering the T-cell attracting chemokine.
  • the mucosal chemokine is administered from 14 to 30 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine is administered from 30 to 60 days after administering the T-cell attracting chemokine. In some embodiments, the mucosal chemokine composition is administered 8 to 14 days after the administration of the T cell-attracting chemokine. In some embodiments, the mucosal cytokine composition is administered 30 or 60 days after the administration of the T cell-attracting chemokine.
  • the mucosal chemokines may comprise CCL25, CCL28, CXCL14, CXCL17, or a combination thereof.
  • the T-cell attracting chemokines may comprise CCL5, CXCL9, CXCL10, CXCL11 , or a combination thereof.
  • the cytokines may comprise IL-15, IL-2, IL-7, or a combination thereof.
  • the efficacy (or effectiveness) of a vaccine composition herein is greater than 60%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 70%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 80%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 90%. In some embodiments, the efficacy (or effectiveness) of a vaccine composition herein is greater than 95%.
  • Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1 ; 201 (11 ): 1607-10) .
  • vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials.
  • AR disease attack rate
  • RR relative risk
  • vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1 ; 201 (11 ) :1607-10).
  • Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial.
  • Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs.
  • a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared.
  • the vaccine immunizes the subject against a coronavirus for up to 1 year. In some embodiments, the vaccine immunizes the subject against a coronavirus for up to 2 years. In some embodiments, the vaccine immunizes the subject against a coronavirus for more than 1 year, more than 2 years, more than 3 years, more than 4 years, or for 5-10 years.
  • the subject is a young adult between the ages of about 20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45, or 50 years old).
  • the subject is an elderly subject about 60 years old, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85, or 90 years old).
  • the subject is about 5 years old or younger.
  • the subject may be between the ages of about 1 year and about 5 years (e.g., about 1 , 2, 3, 5, or 5 years) or between the ages of about 6 months and about 1 year (e.g., about 6, 7, 8, 9, 10, 11 or 12 months).
  • the subject is about 12 months or younger (e.g., 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 months or 1 month).
  • the subject is about 6 months or younger.
  • the subject was born full term (e.g., about 37-42 weeks). In some embodiments, the subject was born prematurely, for example, at about 36 weeks of gestation or earlier (e.g., about 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, or 25 weeks). For example, the subject may have been born at about 32 weeks of gestation or earlier. In some embodiments, the subject was born prematurely between about 32 weeks and about 36 weeks of gestation. In such subjects, a vaccine may be administered later in life, for example, at the age of about 6 months to about 5 years or older.
  • the subject is pregnant (e.g., in the first, second, or third trimester) when administered a vaccine.
  • the subject has a chronic pulmonary disease (e.g., chronic obstructive pulmonary disease (COPD) or asthma) or is at risk thereof.
  • COPD chronic obstructive pulmonary disease
  • Two forms of COPD include chronic bronchitis, which involves a long-term cough with mucus, and emphysema, which involves damage to the lungs over time.
  • a subject administered a vaccine may have chronic bronchitis or emphysema.
  • the subject has been exposed to a coronavirus.
  • the subject is infected with a coronavirus.
  • the subject is at risk of infection by a coronavirus.
  • the subject is immunocompromised (has an impaired immune system, e.g., has an immune disorder or autoimmune disorder).
  • the vaccine composition further comprises a pharmaceutical carrier.
  • Pharmaceutical carriers are well known to one of ordinary skill in the art.
  • the pharmaceutical carrier is selected from the group consisting of water, an alcohol, a natural or hardened oil, a natural or hardened wax, a calcium carbonate, a sodium carbonate, a calcium phosphate, kaolin, talc, lactose and combinations thereof.
  • the pharmaceutical carrier may comprise a lipid nanoparticle, an adenovirus vector, or an adeno-associated virus vector.
  • the vaccine composition is constructed using an adeno-associated virus vectors-based antigen delivery system.
  • a vaccine of any one of the foregoing paragraphs formulated in a nanoparticle (e.g., a lipid nanoparticle).
  • the nanoparticle has a mean diameter of 50-200 nm.
  • the nanoparticle is a lipid nanoparticle.
  • the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid.
  • the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid.
  • the cationic lipid is an ionizable cationic lipid
  • the non-cationic lipid is a neutral lipid
  • the sterol is a cholesterol.
  • the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • the present invention may feature a pharmaceutical composition.
  • the pharmaceutical composition may comprise a plurality of lipid nanoparticles; where a first lipid nanoparticle comprises three messenger ribonucleic acids (mRNAs) encapsulated therein, and each mRNA comprises an open reading frame encoding a Coronavirus protein selected from a group comprising, consisting essentially, or consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein, and a second lipid nanoparticle comprising an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof encapsulated therein.
  • mRNAs messenger ribonucleic acids
  • the pharmaceutical composition may comprise a plurality of lipid nanoparticles; where a first lipid nanoparticle comprises three messenger ribonucleic acids (mRNAs) encapsulated therein, and each mRNA comprises an open reading frame encoding an entire Coronavirus protein selected from a group comprising, consisting essentially, or consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein, and a second lipid nanoparticle comprising an mRNA comprising an open reading fream encoding a Coronavirus Spike protein or portion thereof encapsulated therein.
  • the first lipid nanoparticle comprises two mRNAs (preferably different) encapsulated therein.
  • the first lipid nanoparticle comprises two or more mRNAs (preferably different) encapsulated therein. In some embodiments, the first lipid nanoparticle comprises three mRNAs (preferably different) encapsulated therein. In some embodiments, the first lipid nanoparticle comprises three or more mRNAs (preferably different) encapsulated therein. In some embodiments, the first lipid nanoparticle comprises four mRNAs (preferably different) encapsulated therein. In some embodiments, the first lipid nanoparticle comprises four or more mRNAs (preferably different) encapsulated therein.
  • the second lipid nanoparticle comprises one mRNA encapsulated therein. In some embodiments, the second lipid nanoparticle comprises two or more mRNAs (preferably different) encapsulated therein. In some embodiments, the second lipid nanoparticle comprises three mRNAs (preferably different) encapsulated therein. In some embodiments, the second lipid nanoparticle comprises three or more mRNAs (preferably different) encapsulated therein. In some embodiments, the second lipid nanoparticle comprises four mRNAs (preferably different) encapsulated therein. In some embodiments, the second lipid nanoparticle comprises four or more mRNAs (preferably different) encapsulated therein.
  • the pharmaceutical composition described herein may comprise a single lipid nanoparticle (e.g., a first nanoparticle).
  • the SARS-CoV-2 single-stranded genome is comprised of 29903 bp that encodes 29 proteins, including 4 structural, 16 nonstructural, and 9 accessory regulatory proteins.
  • VBM Variants of Interest
  • SARS-CoV SARS-CoV
  • MERS-CoV Common Cold Coronaviruses
  • SL-CoVs SARS-like Coronaviruses
  • NSP-2 Size: 1914 bp, Nucleotide Range: 540 bp - 2454 bp
  • NSP-3 Size: 4485 bp, Nucleotide Range: 3804 bp - 8289 bp
  • NSP-4 Size: 1500 bp, Nucleotide Range: 8290 bp - 9790 bp
  • NSP-5-10 Size: 3378 bp, Nucleotide Range: 9791 bp - 13169 bp
  • NSP-12 Size: 2796 bp, Nucleotide Range: 13170 bp - 15966 bp
  • NSP-14 Size: 1581 bp, Nucleotide Range: 17766 bp - 19347 bp
  • ORF7a/b Size: 492
  • NSP-2 non-Spike antigens
  • NSP-14 non-Spike antigens
  • Nucleoprotein the sequences of the three non-Spike antigens (NSP-2, NSP-14, and Nucleoprotein) remain relatively conserved in these sub-variants BA.2.86 and JN.1 (21 , 0, 57 mutations respectively).
  • the sequence of NSP-12 and NSP-14 antigens are fully conserved (100%) in all variants and sub-variants, including the recent BA.2.86 and JN.1 , supporting the vital role of these two antigens in the life cycle of SARS-CoV-2.
  • NSP3 58 cumulative mutations
  • Nucleoprotein 57 cumulative mutations
  • the Nucleoprotein was considered in the combined vaccine since it is the most abundant viral protein and one of the most predominantly targeted antigens by T cells in individuals with less severe COVID-19 disease.
  • CD4+ and CD8+ T cells preferentially target seven of the ten highly conserved SARS-CoV-2 antigens and correlated with improved disease outcome in unvaccinated asymptomatic COVID-19 patients.
  • CD4 + and CD8 + T cell responses specific to highly conserved epitopes were compared in unvaccinated asymptomatic individuals (those individuals who never develop any COVID-19 symptoms despite being infected with SARS-CoV-2) versus unvaccinated symptomatic COVID-19 patients (those patients who developed severe to fatal COVID-19 symptoms) (FIG. 4A).
  • PBMCs were then stimulated in vitro for 72 hours using recently identified highly conserved 13 HLA-DR-restricted CD4 + or 16 HLA-A*0201 -restricted CD8 + T cell peptide epitopes derived from the non-structural proteins (NSPs), the ORF7a//b, Membrane, and Envelope, and Nucleoprotein, as detailed in Materials & Methods.
  • NSPs non-structural proteins
  • Methyl-pseudouridine-modified (ml ⁇ P) mRNA that encodes each of the ten highly conserved T cell antigens i.e., NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, NSP-14, ORF7a/b, Membrane, Envelope, and Nucleoprotein
  • NSP-2, NSP-3, NSP-4, NSP-5-10, NSP-12, NSP-14, ORF7a/b, Membrane, Envelope, and Nucleoprotein were constructed, based on the Omicron sub-variant BA.2.75, that are capped using CleanCap technology (i.e., ten T cell antigen mRNA vaccines).
  • LNPs lipid nanoparticles
  • the “plug-and-play” mRNA/LNP platform was selected as an antigen delivery technology over other platforms, as over one billion doses of the clinically proven Spike mRNA/LNP-based vaccines being already distributed around the world showed a high level of safety.
  • the mRNA/LNP platform responds to current goals of the next-generation pan-CoV vaccines: (/) the ability to safely confer durable, cross-protective T cell responses; and (//) the ability to be manufactured at a large scale to support a rapid and a global mass vaccination.
  • each T-cell antigen mRNA/LNP-based vaccine delivered individually by intramuscular route, was compared against the highly pathogenic Delta variant (B.1.617.2) in the outbred golden Syrian hamster model (FIG. 5B).
  • the Golden Syrian hamsters are naturally susceptible to SARS-CoV-2 infection, owing to the high degree of similarity between hamster ACE2 and human ACE2 (hACE2), and develop symptoms of COVID-19-like disease that closely mimic the COVID-19 pathogenesis in humans.
  • the initial 1 pg and 10 pg doses were selected based on previous similar mRNA-LNP vaccine studies in mice and hamsters.
  • NSP-2 Three out of ten highly conserved T-cell antigens mRNA/LNP-based vaccines, NSP-2, NSP-14, and Nucleoprotein prevented weight loss of the hamsters at a dose of as low as 1 pg/dose (P ⁇ 0.05, FIG. 5D).
  • the NSP-2 antigen was the most protective antigen with only 2% of body weight loss, followed by 4% of body weight loss for the Nucleoprotein and 6% of body weight loss for the NSP-14 (Black arrows).
  • the hamsters that were vaccinated with NSP-2, NSP-14, or Nucleoprotein mRNA/LNP vaccine gradually reversed their lost body weight as early as 4-5 days after challenge (Black arrows, FIG. 5D).
  • the mock-vaccinated hamsters gradually reversed their lost body weight late starting 6 to 9 days after being challenged (Red circles, FIG. 5D).
  • two conserved T-cell antigens mRNA/LNP-based vaccines i.e., NSP-3 and, ORF-7a/b
  • the remaining 5 T-cell antigens mRNA/LNP-based vaccines i.e., NSP-4, NSP-5-10, NSP-12, Membrane, and Envelope
  • NSP-4, NSP-5-10, NSP-12, Membrane, and Envelope did not produce any significant protection against weight loss (P > 0.05, FIG. 5D).
  • the mock-vaccinated hamsters were not protected and started losing weight as early as two days following a challenge with the highly pathogenic Delta variant B.1 .617.2.
  • Infectious virus titers are retrieved from the respiratory tract of infected hamsters and are approximately 1-2 logs higher in the nasal turbinate than in the lung, peaking at 2-4 days after infection.
  • FIG. 6A and 6B the protective efficacy of NSP-2, NSP-14, and Nucleoprotein mRNA/LNP-based vaccines (FIG. 6A and 6B) delivered at an intermediate dose of 5 pg/dose was tested against lung pathology (FIG. 6C) and weight loss (FIG. 6D), viral replication (FIG. 6E) caused by a highly pathogenic Delta variant (B.1 .617.2) in the golden Syrian hamster model.
  • Sars-CoV-2 infected hamsters developed lung pathologies, including alveolar destruction, proteinaceous exudation, hyaline membrane formation, marked mononuclear cell infiltration, cell debris-filled bronchiolar lumen, alveolar collapse, lung consolidation, and pulmonary hemorrhage. These lung pathologies are largely resolved by day 14 after infection, with air-exchange structures being restored to normal. In contrast, vaccination with individual NSP-2, NSP-14, and Nucleoprotein mRNA/LNP-based vaccines significantly reduced lung pathology (P ⁇ 0.05, FIG. 6C), following a challenge with the highly pathogenic Delta variant B.1.617.2.
  • the lungs of hamsters vaccinated with NSP-14 mRNA/LNP show peri bronchiolitis (arrow), perivasculitis (asterisk), and multifocal interstitial pneumonia (arrowhead).
  • Lungs of hamsters that received NSP-2 or Nucleoprotein mRNA/LNP vaccine demonstrate normal bronchial, bronchiolar (arrows), and alveolar architecture (FIG. 6C).
  • the lungs of mock-vaccinated hamsters demonstrated bronchi with bronchiolitis (arrows) and adjacent marked interstitial pneumonia (asterisks). No serious local or systemic unwanted side effects were noticed in the mRNA/LNP vaccinated hamsters confirming the safety mRNA/LNP delivery system.
  • the NSP-2, NSP-14, and Nucleoprotein mRNA/LNP-based vaccines prevented weight loss of the hamsters, gradually reversing the lost body weight as early as 4-5 days after the challenge (Black arrows, FIG. 6D).
  • the Nucleoprotein was the most protective antigen when it comes to prevention of body weight, followed by NSP-14 and NSP-2, respectively.
  • the Nucleoprotein-vaccinated hamsters progressively lose their body weight declining by only 2% within the first 4 days after infection, before gradually and reversing the lost body weight starting on day 4 after challenge (black arrow, FIG. 6D).
  • the NSP14-vaccinated hamsters progressively lose their body weight declining by only 6% within the first 5 days after infection, before reversing the lost body weight starting on day 6 after challenge black arrow, FIG. 6D).
  • the NSP2-vaccinated hamsters progressively lose their body weight declining by only 3% within the first 4 days after infection, before gradually and reversing the lost body weight starting on day 4 after challenge (black arrow, FIG. 6D).
  • animals following intranasal inoculation of mock-vaccinated hamsters with 1 x 10 5 pfu of the highly pathogenic Delta variant B.1.617.2, animals progressively lose their body weight declining by greater than 10% within the first week after infection, before gradually and spontaneously reversing the lost body weight starting on day 7 after challenge (red circles, FIG. 6D).
  • Infectious virus titers retrieved on days 2 and 6 post-challenge from the nasal turbinate of mock-vaccinated hamsters are approximately 20- to 40-fold logs higher compared to hamsters that received modified mRNA/LNP vaccine expressing T cell NSP-2, NSP-14, and Nucleoprotein, at the dose of 5 pg/dose, suggesting a fast and strong reduction in median nasal viral titer in the NSP-2, NSP-14, and Nucleoprotein mRNA/LNP vaccinated animals following challenge with the highly pathogenic Delta variant B.1.617.2 (P ⁇ 0.05, FIG. 6E).
  • a combined NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine confers robust and broad protection against multiple SARS-CoV-2 variants and sub-variants of concern.
  • WA1/2020 wild-type Washington variant
  • XBB1.5 Omicron sub-variant
  • Vaccination with the combined NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine at 5 pg/dose, significantly reduced lung pathology (FIG. 7C), fast prevented weight loss of the hamsters (P ⁇ 0.05) (FIG. 7D), and elicited a 20- to 40-fold reduction in median lung viral titer two- and six-days (FIG. 7E) following wild-type Washington variant (WA1/2020), Delta variant (B.1.617.2), and Omicron sub-variant (XBB1.5) in hamsters.
  • WA1/2020 Washington variant
  • Delta variant B.1.617.2
  • XBB1.5 Omicron sub-variant
  • the mock-vaccinated mice did not show a significant reduction in lung pathology, weight loss, and lung viral replication (FIG. 7C, 7D, and 7E).
  • the mock-vaccinated mice started losing weight as early as 1-2 days post-challenge and did not reverse the weight loss until late, 7-8-days post-challenge with Washington, Delta, and Omicron variants (red circles, FIG. 7C, 7D, and 7E).
  • a combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine confers a more potent and rapid protection against the highly pathogenic Delta SARS-Co V-2 variant (B. 1.617.2).
  • the latter was selected as it is safe with over one billion doses of the clinically proven Spike-alone mRNA/LNP-based vaccines that were already administered around the world. Given that most of the human population already received one to four doses of the first generation of Spike 2P-based COVID-19 vaccine, given the combined Spike 2P, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine as boosters in humans with pre-existing Spike 2P immunity may boost the protective efficacy 4S .
  • the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine induces stronger, faster, and broader protection against multiple variants and sub-variants compared to Spike-alone-based mRNA/LNP vaccine.
  • the hamsters that received the Spike-alone-based mRNA/LNP vaccine reversed the weight loss late 6 days post-challenge with the wild-type Washington variant (WA1/2020) (grey arrow, FIG. 9B).
  • the hamsters that received the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine significantly reversed the weight loss as early as the first-day post-challenge with the heavily Spike-mutated and most immune-evasive Omicron sub-variant (XBB.1.5) (black arrow, FIG. 9C).
  • the hamsters that received the Spike-alone-based mRNA/LNP vaccine reversed the weight loss late 6 days post-challenge with the Omicron sub-variant (XBB.1.5) (grey arrow, FIG. 9C).
  • the mock-vaccinated hamsters lost weight fast as early as the first day post-challenge and did not reverse the weight loss until late 7 to 8 days post-challenge with the wild-type Washington variant (WA1/2020) and the Omicron sub-variant (XBB.1 .5) (red circle, FIG. 9B and 9C).
  • the virus titers determined on days 2 and 6 post-challenge confirmed the significant reduction of the lung viral burden by up to 5 logs by the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine following a challenge by wild-type Washington variant (WA1/2020) or the Omicron sub-variant (XBB.1 .5) (FIG. 9E and 9F).
  • the protective NSP-2 and NSP-14 and Nucleoprotein T cell antigens in the combined vaccine all belong to the early-transcribed RTC region and are selectively targeted by human lung-resident enriched memory CD4 + and CD8 + T cells from “SARS-CoV-2 aborters” (i.e., those SARS-CoV-2 exposed seronegative healthcare workers and in household contacts who were able to rapidly abort the virus replication).
  • SARS-CoV-2 aborters i.e., those SARS-CoV-2 exposed seronegative healthcare workers and in household contacts who were able to rapidly abort the virus replication.
  • Lungs from vaccinated and mock-vaccinated hamsters were collected 2 weeks after the SARS-CoV-2 challenge, and cell suspensions were stimulated with pools of 15-mer overlapping NSP-2, NSP-14, or Nucleoprotein (FIG. 8C).
  • the frequency and function of lung-resident NSP-2-, NSP-14-, and Nucleoprotein-specific CD8 + and CD4 + T cells were compared in vaccinated protected hamsters versus mock-vaccinated unprotected hamsters.
  • IFN-y and TNF-a were highly expressed by NSP-2-, NSP-14-, and Nucleoprotein-specific CD4 + and CD8 + T cells.
  • the combined vaccine appeared to induce higher NSP-2- and Nucleoprotein-specific IFN-y + TNF-a + CD4 + and IFN-y + TNF-a + CD8 + T cell responses compared to NSP-14-specific IFN-y + TNFa + CD4 + and IFN-y + TNFa + CD8 + T cell responses (P ⁇ 0.001 for IFN-y).
  • T cell responses in the lungs of protected and non-protected hamsters indicate that the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine induced high frequencies of NSP-2, NSP-14, and Nucleoprotein-specific lung-resident CXCR5 + CD4 + T follicular helper cells (T FH cells), compared to Spike-alone-based mRNA/LNP vaccine.
  • CXCR5 + CD4 + T FH cells likely contribute to the augmentation in the Spike-specific neutralizing antibodies and protection observed in the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine group compared to Spike-alone-based mRNA/LNP vaccine.
  • the combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine elicited stronger serum neutralizing activity against the wild-type virus (P ⁇ 0.005) the Delta variant (P ⁇ 0.005) and the Omicron variants (P ⁇ 0.005) (FIG. 10, lower panel).
  • serum from the mRNA/LNP-Spike alone vaccinated hamsters manifested strong neutralizing activity against the wild-type Washington variant but markedly reduced neutralizing activity (a 5-fold reduction) against the heavily Spike-mutated Delta and Omicron variants (FIG. 10).
  • the high frequency of CXCR5 + CD4 + T FH cells in the lungs of hamsters that received the combined vaccine likely contributed to stronger Spike-specific neutralizing antibody activities that cleared the virus in the lungs.
  • the airway-resident B- and T cell immunity induced by combined Spike, NSP-2, NSP-14, and Nucleoprotein-based mRNA/LNP vaccine likely contribute collectively to the enhanced protection capable of conferring broad cross-strain protective immunity against infection and disease caused by multiple variants and sub-variants.
  • Severity 5 patients who died from COVID-19 complications
  • Severity 4 infected COVID-19 patients with severe disease who were admitted to the intensive care unit (ICU) and required ventilation support
  • Severity 3 infected COVID-19 patients with severe disease that required enrollment in ICU, but without ventilation support
  • Severity 2 infected COVID-19 patients with moderate symptoms that involved a regular hospital admission
  • Severity 1 infected COVID-19 patients with mild symptoms
  • Severity 0 infected individuals with no symptoms.
  • liquid-nitrogen frozen PBMCs samples (blood collected pre-COVID-19 in 2018) were used from HLA-A*02:017HLA-DRB1*01 :01 + unexposed pre-pandemic healthy individuals- 8 males, 7 females; median age: 54 (20-76) as controls.
  • Peptide synthesis Peptide-epitopes from twelve SARS-CoV-2 proteins, including 16 9-mer long CD8 T cell epitopes (ORF1 ab 8 492, ORF1abi675-i683, ORF1 ab22io-22is> ORF1 ab2363-237i> ORF1ab3oi3-302i> previously.
  • the Epitope conserveancy Analysis tool was used to compute the degree of identity of CD8 + T cell and CD4 + T cell epitopes within a given protein sequence of SARS-CoV-2 set at 100% identity level.
  • PBMCs Peripheral blood mononuclear cells
  • a fraction of the blood was kept separated to perform HLA genotyping of only the HLA-A*02:01 and DRB1 *01 :01 positive individuals. Subsequently, cells were stimulated with 10 pg/ml of each one of the 29 individual T cell peptide-epitopes (16 CD8 + T cell peptides and 13 CD4 + T cell peptides) and incubated in a humidified chamber with 5% CO 2 at 37°C. Post-incubation, cells were stained for flow cytometry, or transferred in IFN-y ELISpot plates (FIG. 3A). The same isolation protocol was followed for HD samples obtained in 2018. Ficoll was kept frozen in liquid nitrogen in FBS DMSO 10%; after thawing, HD PBMCs were stimulated similarly for the IFN-y ELISpot technique.
  • the plate was blocked with 200 pl of RPMI media plus 10% (v/v) FBS for two hours at room temperature to prevent nonspecific binding. Twenty-four hours following the blockade, the peptide-stimulated cells from the patient’s PBMCs (0.5 x 10 s cells/well) were transferred into the ELISpot-coated plates. PHA-stimulated or non-stimulated cells (DMSO) were used as positive or negative controls of T cell activation, respectively. Upon incubation in a humidified chamber with 5% CO 2 at 37°C for an additional 48 hours, cells were washed using PBS and PBS-Tween 0.02% solution.
  • DMSO PHA-stimulated or non-stimulated cells
  • Flow cytometry analysis Surface markers detection and flow cytometry analysis were performed on 147 patients after 72 hours of stimulation with each SARS-CoV-2 class-l or class-ll restricted peptide, and PBMCs (0.5 x 10 6 cells) were stained. First, the cells were stained with a live/dead fixable dye (Zombie Red dye, 1 /800 dilution - BioLegend, San Diego, CA) for 20 minutes at room temperature, to exclude dying/apoptotic cells.
  • a live/dead fixable dye Zaombie Red dye, 1 /800 dilution - BioLegend, San Diego, CA
  • HLA-A*02*01 restricted tetramers and/or five HLA-DRB1 *01 :01 restricted tetramers (PE-labelled) specific toward the SARS-CoV-2 CD8 + T cell epitopes Orf1 ab 2210.2218 , and Orf1 ab 428 3.429i and the CD4 + T cell epitopes ORF1 a 1350 .i3 6 5, E 26.40 , and M 176 .I 90 respectively (FIG. 3A).
  • Cells were alternatively stained with the EBV BMLF-1 28 o-288-specific tetramer for control of specificity.
  • HLA-A*02*01- HLA-DRB1 *01 :01 -negative patients were stained with our 10 tetramers as a negative control aiming to assess tetramers staining specificity. Subsequently, anti-human antibodies were used for surface-marker staining: anti-CD45 (BV785, clone HI30 - BioLegend), anti-CD3 (Alexa700, clone OKT3 - BioLegend), anti-CD4 (BUV395, clone SK3 - BD), anti-CD8 (BV510, clone SK1 - BioLegend), anti-TIGIT (PercP-Cy5.5, clone A15153G - BioLegend), anti-TIM-3 (BV 711 , clone F38-2E2 - BioLegend), anti-PD1 (PE-Cy7, clone EH12.1 - BD), anti-CTLA-4 (APC, clon
  • mAbs against these various cell markers were added to the cells in phosphate-buffered saline (PBS) containing 1 % FBS and 0.1% sodium azide (fluorescence-activated cell sorter [FACS] buffer) and incubated for 30 minutes at 4°C. Subsequently, cells were washed twice with FACS buffer and fixed with 4% paraformaldehyde (PFA, Affymetrix, Santa Clara, CA). A total of -200,000 lymphocyte-gated PBMCs (140,000 alive CD45 + ) were acquired by Fortessa X20 (Becton Dickinson, Mountain View, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR). The gating strategy is detailed in FIG. 3B.
  • Viruses SARS-CoV-2 viruses specific to six variants, namely (/) SARS-CoV-2-USA/WA/2020 (Batch Number: G2027B); (v) Delta (B.1.617.2) (isolate h-CoV-19/USA/MA29189; Batch number: G87167), and Omicron (XBB1.5) (isolate h-CoV-19/USA/FL17829; Batch number: G76172) were procured from Microbiologies (St. Cloud, MN). The initial batches of viral stocks were propagated to generate high-titer virus stocks. Vero E6 (ATCC-CRL1586) cells were used for this purpose. Procedures were completed after appropriate safety training was obtained using an aseptic technique under BSL-3 containment.
  • TaqMan quantitative polymerase reaction assay A laboratory-developed modification of the CDC SARS-CoV-2 RT-PCR assay was used for the screening of SARS-CoV-2 Variants in COVID-19 patients, which received Emergency Use Authorization by the FDA on April 17 th , 2020. (https://www.fda.gov/media/137424/download [accessed 24 March 2021]).
  • RT-PCR was carried out using the ABI StepOnePlus thermocycler (Life Technologies, Grand Island, NY).
  • S-N501Y, S-E484K, and S-L452R assays were carried out under the following running conditions: 25°C for 2 minutes, then 50°C for 15 minutes, followed by 10 minutes at 95°C and 45 cycles of 95°C for 15 seconds and 65°C for 1 minute.
  • the A 69-70 / A 242-244 assays were run under the following conditions: 25°C for 2 minutes, then 50°C for 15 minutes, followed by 10 minutes at 95°C and 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Samples displaying typical amplification curves above the threshold were considered positive. Samples that yielded a negative result or results in the S-A69-70/A242-244 assays or were positive for S-501Y P2, S-484K P2, and S-452R P2 were considered screen positive and assigned to VOCs.
  • ELISA Human Enzyme-linked immunosorbent assay
  • the bound serum antibodies were detected with HRP-conjugated goat anti-mouse IgG and chromogenic substrate TMB (ThermoFisher, Waltham, MA). The cut-off for seropositivity was set as the mean value plus three standard deviations (3SD) in HBc-S control sera.
  • the binding of the epitopes to the sera of SARS-CoV-2 infected samples was detected by ELISA using the same procedure; 96-well plates were coated with 0.5 pg peptides, and sera were diluted at 1 :50.
  • Genome sequences of previous strains of SARS-CoV for humans (B.1.177, B.1.160, B.1.1.7, B.1.351 , P.1 , B.1 .427/B.1 .429, B.1.258, B.1.221 , B.1.367, B.1.1.277, B.1.1.302, B.1.525, B.1.526, S:677H.Robin1 , S:677P.Pelican, B.1 .617.1 , B.1.617.2, B, 1 ,1 ,529) and common cold SARS-CoV strains (SARS-CoV-2-Wuhan-Hu-1 (MN908947.3), SARS-CoV-Urbani (AY278741 .1), HKU1-Genotype B (AY884001), CoV-OC43 (KF923903), CoV-NL63 (NC_005831), COV-229E (KY983587)) and MERS (NC_01
  • mRNA synthesis and LNP formulation Sequences of Spike and 10 T cell non-Spike antigens were derived from the SARS-CoV-2 Omicron sub-variant BA.2 (NCBI GenBank accession number OM617939) Nucleoside-modified mRNAs expressing SARS-CoV-2 full-length of prefusion-stabilized Spike protein with two or 6 proline mutations (mRNA-S-2P and mRNA-S-6P (Size: 3804 bp, Nucleotide Range: 21504 bp - 25308 bp)) and part or full-length ten highly conserved non-Spike T cell antigens (NSP-2 (Size: 1914 bp, Nucleotide Range: 540 bp - 2454 bp), NSP-3 (Size: 4485 bp, Nucleotide Range: 3804 bp - 8289 bp), NSP-4 (Size: 1500 bp,
  • Modified mRNA transcript with full substitution of Pseudo-U was synthesized by TriLink Biotechnologies using proprietary CleanCap® technology.
  • the synthesized polyadenylated (80A) mRNAs were subjected to DNase and phosphatase treatment, followed by Silica membrane purification. Finally, the synthesized mRNA was packaged as a 1 .00 ⁇ 6% mg/mL solution in 1 mM Sodium Citrate, pH 6.4. Purified mRNAs were analyzed by agarose gel electrophoresis and were kept frozen at -20°C.
  • HEK293T American Type Culture Collection (ATCC), CRL-3216] cells before testing in animal experiments and plated 10 A 6 cells in 500 pl culture medium in a 6-well plate on Day 0. Once the cells reached confluency, HEK293T cells in six-well plates were directly transfected with 2 pg of mRNA-LNP or only transfected with LNR A transfection mix for mRNA was prepared and cells were transfected as described by the LipofectamineTM MessengerMAXTM Transfection Reagent -specific protocol (Thermo Fisher Scientific, Catalog # LMRNA001).
  • mice vaccinated with SARS-CoV-2 Delta (B.1.617.2) Variant. Although all the mice challenged with SARS-Cov-2 start losing weight 2-3 days post infection, unvaccinated mice start gaining weight only by days 11 and 12. Similarly but very less pronounced body weight loss, Spike alone and T cell antigen encoding mRNA-LNP start regaining weight by day 5-6. Importantly mice vaccinated with the combination Spike2P + T-cell Ag show no weight loss and keep regaining weight throughout all the experiment (FIG. 12A).
  • Embodiment 1 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising a sequence encoding, or comprising: two or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein, b) at least a portion of an NSP14 protein, and c) at least a portion of a Nucleoprotein protein.
  • Embodiment 2 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising a sequence encoding, or comprising: three or more Coronavirus antigens derived from:a) at least a portion of an NSP2 protein, b) at least a portion of an NSP14 protein, and c) at least a portion of a Nucleoprotein protein.
  • Embodiment 3 The composition of embodiment 1 or embodiment 2 further comprising at least a portion of a Spike protein.
  • Embodiment 4 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising a sequence encoding, or comprising: two or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein b) at least a portion of an NSP14 protein c) at least a portion of a Nucleoprotein protein, and d) at least a portion of a Spike protein.
  • Embodiment 5 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising a sequence encoding, or comprising: three or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein, b) at least a portion of an NSP14 protein, c) at least a portion of a Nucleoprotein protein, and d) at least a portion of a Spike protein.
  • Embodiment 6 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are: the Spike protein or a portion thereof and the NSP2 protein or a portion thereof.
  • Embodiment 7 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are: the Spike protein or a portion thereof and the NSP14 protein or a portion thereof.
  • Embodiment 8 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are the Spike protein or a portion thereof and the Nucleoprotein or a portion thereof.
  • Embodiment 9 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP2 protein or a portion thereof, and the NSP14 protein or a portion thereof.
  • Embodiment 10 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP2 protein or a portion thereof, and the Nucleoprotein or a portion thereof.
  • Embodiment 11 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP14 protein or a portion thereof, and the Nucleoprotein or a portion thereof.
  • Embodiment 12 The composition of any one of embodiments 1-5, wherein the Coronavirus antigens are: the Spike protein; the NSP2 protein or a portion thereof; the NSP14 protein or a portion thereof; and the Nucleoprotein or a portion thereof.
  • Embodiment 13 The composition of any one of embodiments 1-12, wherein the portion of the NSP2 protein comprises at least a portion of SEQ ID NO: 3; or wherein the portion of the NSP2 protein is encoded by a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 14 The composition of any one of embodiments 1-12, wherein the portion of the NSP14 protein comprises at least a portion of SEQ ID NO: 6; or wherein the portion of the NSP14 protein is encoded by a sequence according to SEQ ID NO: 4 or SEQ ID NO: 5.
  • Embodiment 15 The composition of any one of embodiments 1-12, wherein the portion of the Nucleoprotein protein comprises at least a portion of SEQ ID NO: 9; ; or wherein the portion of the Nucleoprotein is encoded by a sequence according to SEQ ID NO: 7 or SEQ ID NO: 8. .
  • Embodiment 16 The composition of any one of embodiments 1-12, wherein the portion of the spike protein comprises at least a portion of either SEQ ID NO: 11 or SEQ ID NO: 12.
  • Embodiment 17 The composition of any one of embodiments 1-16, further comprising one or more Coronavirus antigens derived from: at least a portion of NSP3 protein, at least a portion of NSP12 protein, or at least a portion of a protein encoded by ORF7a/b.
  • Embodiment 18 The composition of embodiment 17, wherein the portion of NSP3 protein is encoded by a sequence according to SEQ ID NO: 13 or SEQ ID NO: 14.
  • Embodiment 19 The composition of embodiment 17, wherein the portion of NSP12 is encoded by a sequence according to SEQ ID NO: 15 or SEQ ID NO: 16.
  • Embodiment 20 The composition of embodiment 17, wherein the portion of the protein encoded by ORF7a/b is encoded by a sequence according to SEQ ID NO: 17 or SEQ ID NO: 18.
  • Embodiment 21 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising, or comprising a sequence encoding: two or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • Embodiment 22 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising, or comprising a sequence encoding: three or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • Embodiment 23 The composition of embodiment 21 or embodiment 22, further comprising a Spike protein.
  • Embodiment 24 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising, or comprising a sequence encoding: two or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • Embodiment 25 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising, or comprising a sequence encoding: three or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • Embodiment 26 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are: the Spike protein and the NSP2 protein.
  • Embodiment 27 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are: the Spike protein and the NSP14 protein.
  • Embodiment 28 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are the Spike protein and the Nucleoprotein.
  • Embodiment 29 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are the Spike protein, the NSP2 protein, and the NSP14 protein.
  • Embodiment 30 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are the Spike protein, the NSP2 protein, and the Nucleoprotein.
  • Embodiment 31 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are: the Spike protein, the NSP14 protein, and the Nucleoprotein.
  • Embodiment 32 The composition of any one of embodiments 20-25, wherein the Coronavirus antigens are the Spike protein, the NSP2 protein, the NSP14 protein, and the Nucleoprotein.
  • Embodiment 33 The composition of any one of embodiments 20-32, wherein the NSP2 protein comprises a sequences according to SEQ ID NO: 3, or wherein the NSP2 protein is encoded by a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 34 The composition of any one of embodiments 20-32, wherein the NSP14 protein comprises a sequences according to SEQ ID NO: 6, or wherein the NSP14 protein is encoded by a sequence according to SEQ ID NO: 4 or SEQ ID NO: 5.
  • Embodiment 35 The composition of any one of embodiments 20-32, wherein the Nucleoprotein protein comprises a sequences according to SEQ ID NO: 9, or wherein the Nucleoprotein is encoded by a sequence according to SEQ ID NO: 7 or SEQ ID NO: 8.
  • Embodiment 36 The composition of any one of embodiments 20-32, wherein the spike protein is encoded by a sequence according to either SEQ ID NO: 11 or SEQ ID NO: 12.
  • Embodiment 37 The composition of any one of embodiments 20-36, further comprising one or more Coronavirus antigens derived from: an NSP3 protein, an NSP12 protein, or a protein encoded by ORF7a/b.
  • Embodiment 38 The composition of embodiment 37, wherein the NSP3 protein is encoded by a sequence according to either SEQ ID NO: 13 or SEQ ID NO: 14.
  • Embodiment 39 The composition of embodiment 37, wherein the NSP12 protein is encoded by a sequence according to either SEQ ID NO: 15 or SEQ ID NO: 16.
  • Embodiment 40 The composition of embodiment 37, wherein the protein encoded by ORF7a/b is encoded by a sequence according to either SEQ ID NO: 17 or SEQ ID NO: 18.
  • Embodiment 41 The composition of any one of embodiments 1-40 further comprising a T cell attracting chemokine, wherein the T cell attracting chemokine is CCL5, CXCL9, CXCL10, CXCL11 , or a combination thereof.
  • Embodiment 42 The composition of any one of embodiments 1-40 further comprising a composition that promotes T cell proliferation and T-cell memory, wherein the composition that promotes T cell proliferation and memory is IL-7, IL-2, or IL-15.
  • Embodiment 43 The composition of any one of embodiments 1-42, wherein the vaccine composition protects against disease caused by one or more coronavirus variants or coronavirus subvariants.
  • Embodiment 44 The composition of embodiment 43, wherein the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants wherein the coronavirus variants comprise alpha, beta, gamma, delta, and omicron.
  • Embodiment 45 The composition of embodiment 43, wherein the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • Embodiment 46 The composition of any one of embodiments 1-45, wherein the vaccine composition protects against infection and re-infection of coronavirus variants or coronavirus subvariants.
  • Embodiment 47 The composition of embodiment 46, wherein the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants, wherein the coronavirus variants comprise alpha, beta, gamma, delta, and omicron.
  • Embodiment 48 The composition of embodiment 46, wherein the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • Embodiment 49 The composition of embodiment 46, wherein the vaccine composition protects against infection or reinfection of one or more coronavirus variant or coronavirus subvariant.
  • Embodiment 50 The composition of embodiment 49, wherein the vaccine composition protects against infection or reinfection of multiple coronavirus variants or coronavirus subvariants.
  • Embodiment 51 The composition of embodiment 49, wherein the vaccine composition protects against infection or re-infection of one coronavirus variants or coronavirus subvariants.
  • Embodiment 52 The composition of any one of embodiments 1-51 , wherein the vaccine composition induces strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Th1) cells, and/or CD8+ cytotoxic T-cells (CTL).
  • Abs antibodies
  • Th1 CD4+ T helper
  • CTL cytotoxic T-cells
  • Embodiment 53 The composition of any one of embodiments 1-52, wherein the composition protects against Sarbecoviruses, wherein sarbecoviruses comprise SARS-CoV1 or SARS-C0V2.
  • Embodiment 54 A pan-Coronavirus recombinant vaccine composition, the composition comprising a delivery system encoding two or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • Embodiment 55 A pan-Coronavirus recombinant vaccine composition, the composition comprising a delivery system encoding three or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; and c) at least a portion of a Nucleoprotein protein.
  • Embodiment 56 The composition of embodiments 54 or 55, wherein the delivery system further encodes a Coronavirus antigen derived from at least a portion of a Spike protein.
  • Embodiment 57 The composition of embodiments 54 or 55, further comprising a delivery system (e.g., a second delivery system) encoding a Coronavirus antigens derived from at least a portion of a Spike protein.
  • a delivery system e.g., a second delivery system
  • Embodiment 58 A pan-Coronavirus recombinant vaccine composition, the composition comprising an delivery system encoding two or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein; and d) at least a portion of a Spike protein.
  • Embodiment 59 A pan-Coronavirus recombinant vaccine composition, the composition comprising an delivery system encoding three or more Coronavirus antigens derived from: a) at least a portion of an NSP2 protein; b) at least a portion of an NSP14 protein; c) at least a portion of a Nucleoprotein protein; and d) at least a portion of a Spike protein.
  • Embodiment 60 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof and the NSP2 protein or a portion thereof.
  • Embodiment 61 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof and the NSP14 protein or a portion thereof.
  • Embodiment 62 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof and the Nucleoprotein or portion thereof.
  • Embodiment 63 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP2 protein or a portion thereof, and the NSP14 protein or portion thereof.
  • Embodiment 64 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP2 protein or portion thereof, and the Nucleoprotein or portion thereof.
  • Embodiment 65 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP14 protein or portion thereof; and the Nucleoprotein or portion thereof.
  • Embodiment 66 The composition of any one of embodiments 54-59, wherein the Coronavirus antigens are the Spike protein or a portion thereof, the NSP2 protein or portion thereof, the NSP14 protein or portion thereof; and the Nucleoprotein or portion thereof.
  • Embodiment 67 The composition of any one of embodiments 54-66, wherein the portion of the NSP2 protein comprises at least a portion of SEQ ID NO: 3; or wherein the portion of the NSP2 protein is encoded by a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 68 The composition of any one of embodiments 54-66, wherein the portion of the NSP14 protein comprises at least a portion of SEQ ID NO: 6; or wherein the portion of the NSP14 protein is encoded by a sequence according to SEQ ID NO: 4 or SEQ ID NO: 5.
  • Embodiment 69 The composition of any one of embodiments 54-66, wherein the portion of the Nucleoprotein protein comprises at least a portion of SEQ ID NO: 9; or wherein the portion of the Nucleoprotein is encoded by a sequence according to SEQ ID NO: 7 or SEQ ID NO: 8.
  • Embodiment 70 The composition of any one of embodiments 54-66, wherein the portion of the spike protein comprises at least a portion of either SEQ ID NO: 11 or SEQ ID NO: 12.
  • Embodiment 71 The composition of any one of embodiments 54-70, further comprising one or more Coronavirus antigens derived from: at least a portion of NSP3 protein, at least a portion of NSP12 protein, or at least a portion of a protein encoded by ORF7a/b.
  • Embodiment 72 The composition of embodiment 71 , wherein the portion of NSP3 protein is encoded by a sequence according to either SEQ ID NO: 13 or SEQ ID NO: 14.
  • Embodiment 73 The composition of embodiment 71 , wherein the portion of NSP12 protein is encoded by a sequence according to either SEQ ID NO: 15 or SEQ ID NO: 16.
  • Embodiment 74 The composition of embodiment 71 , wherein the portion of the protein encoded by ORF7a/b is encoded by a sequence according to either SEQ ID NO: 17 or SEQ ID NO: 18.
  • Embodiment 75 The composition of any of embodiments 54-74, wherein the delivery system in a single delivery system.
  • Embodiment 76 The composition of any of embodiments 54-74, wherein the delivery system comprises two or more delivery systems.
  • Embodiment 77 The composition of any of embodiments 54-76, wherein the delivery system is an adeno-associated viral vector-based antigen delivery system.
  • Embodiment 78 The composition of embodiment 77, wherein the adeno-associated viral vector is an adeno-associated virus vector type 8 (AAV8 serotype) or an adeno-associated virus vector type 9 (AAV9 serotype).
  • AAV8 serotype adeno-associated virus vector type 8
  • AAV9 serotype adeno-associated virus vector type 9
  • Embodiment 79 The composition of embodiment any of embodiments 54-78, wherein the delivery system is a vesicular stomatitis virus (VSV) vector.
  • VSV vesicular stomatitis virus
  • Embodiment 80 The composition of any of embodiments 54-79, wherein antigens are operatively linked to a generic promoter.
  • Embodiment 81 The composition of embodiment 54-80, wherein the generic promoter is a CMV or a CAG promoter.
  • Embodiment 82 The composition of any of embodiments 54-81 , wherein antigens and the at least a portion of the spike protein are operatively linked to a lung-specific promoter.
  • Embodiment 83 The composition of embodiment 82, wherein the lung-specific promoter is SpB or CD144.
  • Embodiment 84 The composition of any of embodiments 54-83, wherein the delivery system further encodes a T cell attracting chemokine.
  • Embodiment 85 The composition of embodiment 84, wherein the T cell attracting chemokine is CCL5, CXCL9, CXCL10, CXCL11 , or a combination thereof.
  • Embodiment 86 The composition of any of embodiments 54-85, wherein the T cell attracting chemokine is operatively linked to a lung-specific promoter.
  • Embodiment 87 The composition of any of embodiments 54-86, wherein the T cell attracting chemokine is operatively linked to a generic promoter.
  • Embodiment 88 The composition of any of embodiments 54-87, wherein the delivery system further encodes a composition that promotes T cell proliferation.
  • Embodiment 89 The composition of embodiment 88, wherein the composition that promotes T cell proliferation is IL-7, IL-2, or IL-15.
  • Embodiment 90 The composition of any of embodiments 54-89, wherein the composition that promotes T cell proliferation is operatively linked to a lung-specific promoter.
  • Embodiment 91 The composition of any of embodiments 54-90, wherein the composition that promotes T cell proliferation is operatively linked to a generic promoter.
  • Embodiment 92 The composition of any of embodiments 54-91 , wherein the T cell attracting chemokine and the composition that promotes T cell proliferation are driven by the same promoter.
  • Embodiment 93 The composition of any of embodiments 54-92, wherein the composition encodes a peptide comprising a T cell attracting chemokine and a composition that promotes T cell proliferation.
  • Embodiment 94 The composition of embodiment 93, wherein the peptide is operatively linked to a lung-specific promoter.
  • Embodiment 95 The composition of embodiment 93 wherein the peptide is operatively linked to a generic promoter.
  • Embodiment 96 A pan-Coronavirus recombinant vaccine composition, the composition comprising a delivery system encoding two or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • Embodiment 97 A pan-Coronavirus recombinant vaccine composition, the composition comprising a delivery system encoding three or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; and c) a Nucleoprotein protein.
  • Embodiment 98 The composition of embodiments 96 or 97, wherein the delivery system further encodes a Coronavirus antigens derived from a Spike protein.
  • Embodiment 99 The composition of embodiments 96 or 97, further comprising a delivery system (e.g., a second delivery system) encoding a Coronavirus antigens derived from a Spike protein.
  • a delivery system e.g., a second delivery system
  • Embodiment 100 A pan-Coronavirus recombinant vaccine composition, the composition comprising an delivery system encoding two or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • Embodiment 101 A pan-Coronavirus recombinant vaccine composition, the composition comprising a delivery system encoding three or more Coronavirus antigens derived from: a) an NSP2 protein; b) an NSP14 protein; c) a Nucleoprotein protein; and d) a Spike protein.
  • Embodiment 102 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein and the NSP2 protein
  • Embodiment 103 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein and the NSP14 protein.
  • Embodiment 104 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein and the Nucleoprotein.
  • Embodiment 105 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein, the NSP2 protein, and the NSP14 protein.
  • Embodiment 106 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein, the NSP2 protein, and the Nucleoprotein.
  • Embodiment 107 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein, the NSP14 protein, and the Nucleoprotein.
  • Embodiment 108 The composition of any one of embodiments 96-101 , wherein the Coronavirus antigens are the Spike protein, the NSP2 protein, the NSP14 protein, and the Nucleoprotein.
  • Embodiment 109 The composition of any one of embodiments 96-101 , wherein the NSP2 protein comprises according to SEQ ID NO: 3; or where the NSP2 protein is encoded by a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.
  • Embodiment 110 The composition of any one of embodiments 96-109, wherein the NSP14 protein comprises sequence according to SEQ ID NO: 6; or wherein the portion of the NSP14 protein is encoded by a sequence according to SEQ ID NO: 4 or SEQ ID NO: 5.
  • Embodiment 111 The composition of any one of embodiments 96-109, wherein the Nucleoprotein comprises a sequence according to SEQ ID NO: 9; or wherein the portion of the Nucleoprotein is encoded by a sequence according to SEQ ID NO: 7 or SEQ ID NO: 8.
  • Embodiment 112 The composition of any one of embodiments 96-109, wherein the spike protein comprises a sequence according to either SEQ ID NO: 11 or SEQ ID NO: 12.
  • Embodiment 113 The composition of any one of embodiments 96-112, further comprising one or more Coronavirus antigens derived from: an NSP3 protein, an NSP12 protein, or a protein encoded by ORF7a/b.
  • Embodiment 114 The composition of any one of embodiments 96-113, wherein the antigen delivery system in a single delivery system.
  • Embodiment 115 The composition of any one of embodiments 96-113, wherein the antigen delivery system comprises two or more delivery systems.
  • Embodiment 116 The composition of any one of embodiments 96-115, wherein the vaccine composition protects against disease caused by one or more coronavirus variants or coronavirus subvariants.
  • Embodiment 117 The composition of embodiment 116, wherein the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants wherein the coronavirus variants comprise alpha, beta, gamma, delta, and omicron.
  • Embodiment 118 The composition of embodiment 116, wherein the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • Embodiment 119 The composition of any one of embodiments 96-118, wherein the vaccine composition protects against infection and re-infection of coronavirus variants or coronavirus subvariants.
  • Embodiment 120 The composition of embodiment 119, wherein the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants, wherein the coronavirus variants comprise alpha, beta, gamma, delta, and omicron.
  • Embodiment 121 The composition of embodiment 120, wherein the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • Embodiment 122 The composition of embodiment 120, wherein the vaccine composition protects against infection or reinfection of one or more coronavirus variant or coronavirus subvariant.
  • Embodiment 123 The composition of embodiment 122, wherein the vaccine composition protects against infection or reinfection of multiple coronavirus variants or coronavirus subvariants.
  • Embodiment 124 The composition of embodiment 122, wherein the vaccine composition protects against infection or re-infection of one coronavirus variants or coronavirus subvariants.
  • Embodiment 125 The composition of any one of embodiments 96-124, wherein the vaccine composition induces strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Th1) cells, and/or CD8+ cytotoxic T-cells (CTL).
  • Abs antibodies
  • Th1 CD4+ T helper
  • CTL cytotoxic T-cells
  • Embodiment 126 The composition of any one of embodiments 96-125, wherein the composition protects against Sarbecoviruses, wherein sarbecoviruses comprise SARS-CoV1 or SARS-C0V2.
  • Embodiment 127 A composition, comprising: two or more messenger ribonucleic acids (mRNAs) comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein; wherein the two or more mRNAs are formulated in a lipid nanoparticle.
  • mRNAs messenger ribonucleic acids
  • Embodiment 128 A composition, comprising: two or more messenger ribonucleic acids (mRNAs) comprising an open reading frame encoding an entire Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, a Nucleoprotein protein; and a Spike protein; wherein the two or more mRNAs are formulated in a lipid nanoparticle.
  • mRNAs messenger ribonucleic acids
  • Embodiment 129 A composition, comprising: three messenger ribonucleic acids (mRNAs) comprising an open reading frame encoding at least a portion of a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein; wherein the three mRNAs are formulated in a lipid nanoparticle.
  • mRNAs messenger ribonucleic acids
  • Embodiment 130 A composition, comprising: three messenger ribonucleic acids (mRNAs) comprising an open reading frame encoding an entire Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein protein; wherein the three mRNAs are formulated in a lipid nanoparticle.
  • mRNAs messenger ribonucleic acids
  • Embodiment 131 The composition of embodiment 129 or embodiment 130, further comprising an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof formulated in a lipid nanoparticle.
  • Embodiment 132 The composition of any one of embodiments 127-131 , wherein the mRNA further comprises a 5’ untranslated region (UTR) and a 3’ UTR.
  • UTR untranslated region
  • Embodiment 133 The composition of any one of embodiments 127-132, wherein the mRNA further comprises a poly(A) tail.
  • Embodiment 134 The composition of any one of embodiments 127-133, wherein the mRNA further comprises a 5’ cap or a 5’ cap analog.
  • Embodiment 135 The composition of any one of embodiments 127-134, wherein the mRNAs comprise N1 -methylpseudouridine (m1 j) modified mRNAs.
  • Embodiment 136 The composition of any one of embodiments 127-135, wherein the lipid nanoparticles comprise ionizable catatonic lipid, neutral lipid, a sterol, a PEG modified lipid, or a combination thereof.
  • Embodiment 137 A pharmaceutical composition comprising: a plurality of lipid nanoparticles; wherein a first lipid nanoparticle comprises three messenger ribonucleic acids (mRNAs) encapsulated therein, wherein each mRNA comprises an open reading frame encoding a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein, and a second lipid nanoparticle comprising an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof encapsulated therein.
  • mRNAs messenger ribonucleic acids
  • Embodiment 138 A pharmaceutical composition comprising: a plurality of lipid nanoparticles; wherein a first lipid nanoparticle comprises three messenger ribonucleic acids (mRNAs) encapsulated therein, wherein each mRNA comprises an open reading frame encoding at least a portion a Coronavirus protein selected from a group consisting of: an NSP2 protein, an NSP14 protein, and a Nucleoprotein, and a second lipid nanoparticle comprising an mRNA comprising an open reading frame encoding a Coronavirus Spike protein or portion thereof encapsulated therein.
  • mRNAs messenger ribonucleic acids
  • Embodiment 139 The composition of embodiment 137 or embodiment 138, wherein the mRNA further comprises a 5’ untranslated region (UTR) and a 3’ UTR.
  • UTR untranslated region
  • Embodiment 140 The composition of any one of embodiments 137-139, wherein the mRNA further comprises a poly(A) tail.
  • Embodiment 140 The composition of any one of embodiments 136-139, wherein the mRNA further comprises a 5’ cap or 5’ cap analog.
  • Embodiment 141 The composition of any one of embodiments 137-140, wherein the mRNAs comprise N1 -methylpseudouridine (m1 qj) modified mRNAs.
  • Embodiment 142 The composition of any one of embodiments 137-141 , wherein the lipid nanoparticles comprise ionizable catatonic lipid, neutral lipid, a sterol, a PEG modified lipid, or a combination thereof.
  • Embodiment 143 The composition of any one of embodiments 127-142, wherein the vaccine composition protects against disease caused by one or more coronavirus variants or coronavirus subvariants.
  • Embodiment 144 The composition of embodiment 143, wherein the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants wherein the coronavirus variants comprise alpha, beta, gamma, delta, and omicron.
  • Embodiment 145 The composition of embodiment 143, wherein the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • Embodiment 146 The composition of any one of embodiments 127-145, wherein the vaccine composition protects against infection and re-infection of coronavirus variants or coronavirus subvariants.
  • Embodiment 147 The composition of embodiment 146, wherein the coronavirus variants or coronavirus subvariants comprise past or currently circulating coronavirus variants or coronavirus subvariants, wherein the coronavirus variants comprise alpha, beta, gamma, delta, and omicron.
  • Embodiment 148 The composition of embodiment 147, wherein the coronavirus variants or coronavirus subvariants comprise future variants or future subvariants of human and animal coronavirus.
  • Embodiment 149 The composition of embodiment 147, wherein the vaccine composition protects against infection or reinfection of one or more coronavirus variant or coronavirus subvariant.
  • Embodiment 150 The composition of embodiment 149, wherein the vaccine composition protects against infection or reinfection of multiple coronavirus variants or coronavirus subvariants.
  • Embodiment 151 The composition of embodiment 149, wherein the vaccine composition protects against infection or re-infection of one coronavirus variants or coronavirus subvariants.
  • Embodiment 152 The composition of any one of embodiments 127-151 , wherein the vaccine composition induces strong and long-lasting protection mediated by antibodies (Abs), CD4+ T helper (Th1) cells, and/or CD8+ cytotoxic T-cells (CTL).
  • Abs antibodies
  • Th1 CD4+ T helper
  • CTL CD8+ cytotoxic T-cells
  • Embodiment 153 The composition of any one of embodiments 127-152, wherein the composition protects against Sarbecoviruses, wherein sarbecoviruses comprise SARS-CoV1 or SARS-C0V2.
  • Embodiment 154 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising a sequence encoding or comprising: a B cell antigen or two or more T cell antigens.
  • Embodiment 155 A universal pre-emptive pan-Coronavirus vaccine composition, the composition comprising a sequence encoding or comprising: a B cell antigen and three T-cell antigens.
  • Embodiment 156 The composition of embodiment 154 and embodiment 155, wherein the B cell antigen is derived from a Coronavirus Spike protein or portion thereof.
  • Embodiment 157 The composition of embodiment 154 and embodiment 155, wherein the T cell antigens are derived from at least a portion of: an NSP2 protein, an NSP14 protein, a Nucleoprotein, or a combination thereof.
  • Embodiment 158 A method of inducing an immune response in a subject, the method comprising administering to the subject a composition (e.g., a vaccine composition, a pharmaceutical composition, ect.) according to any one of the preceding embodiments.
  • a composition e.g., a vaccine composition, a pharmaceutical composition, ect.
  • Embodiment 159 Use of a composition (e.g., a vaccine composition, a pharmaceutical composition, ect.) according to any one of embodiments 1-157 to induce an immune response in a subject.
  • a composition e.g., a vaccine composition, a pharmaceutical composition, ect.
  • Embodiment 160 A composition (e.g., a vaccine composition, a pharmaceutical composition, ect.) according to any one of embodiments 1-157 for use in a method of inducing an immune response in a subject.
  • a composition e.g., a vaccine composition, a pharmaceutical composition, ect.
  • descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

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Abstract

L'immunité décroissante induite par la COVID-19 reposant uniquement sur la spicule de première génération n'a pas permis d'empêcher un échappement immunitaire par de nombreux variants d'intérêt (VOC) apparus entre 2020 et 2024, entraînant une pandémie de COVID-19 prolongée. Ainsi, un vaccin contre le coronavirus (CoV) de nouvelle génération incorporant des antigènes de SARS-CoV-2 de non spicule hautement conservés. Des antigènes de lymphocytes T de non spicule conservés en combinaison à un antigène de spicule encapsulé dans des nanoparticules lipidiques permettent : (i) d'induire des fréquences élevées des lymphocytes T auxiliaires folliculaires CXCR5+CD4 spécifiques d'un antigène résidant dans le poumon, des lymphocytes T cytotoxiques GzmB+CD4+ et GzmB+CD8+, et des lymphocytes T effecteurs CD69+IFN-y+TNFa+CD4+ et CD69+IFN-y+TNFa+CD8+ ; et (ii) de réduire une charge virale et des symptômes de type COVID-19 provoqués par divers COV. Le vaccin panCoV reposant sur antigène/LNP combiné peut être rapidement adapté à une utilisation clinique pour conférer une immunité de protection croisée plus large contre des COV fortement mutés et des pathogènes émergents.
EP24771552.7A 2023-03-10 2024-03-11 Vaccin pancoronavirus multi-antigène à large spectre Pending EP4677097A2 (fr)

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PCT/US2023/068080 WO2023240148A2 (fr) 2022-06-07 2023-06-07 Vaccin hybride contre le coronavirus et la grippe
US202463626937P 2024-01-30 2024-01-30
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