WO2022197940A1 - Vaccine compositions and methods of use thereof - Google Patents

Vaccine compositions and methods of use thereof Download PDF

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Publication number
WO2022197940A1
WO2022197940A1 PCT/US2022/020774 US2022020774W WO2022197940A1 WO 2022197940 A1 WO2022197940 A1 WO 2022197940A1 US 2022020774 W US2022020774 W US 2022020774W WO 2022197940 A1 WO2022197940 A1 WO 2022197940A1
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WIPO (PCT)
Prior art keywords
protein
polynucleotide
acid sequence
polynucleotide encoding
amino acid
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.)
Ceased
Application number
PCT/US2022/020774
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English (en)
French (fr)
Inventor
Barbara MERTINS
Thomas FOLLIARD
Imre Mäger
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.)
ExcepGen Inc
Original Assignee
ExcepGen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to BR112023018838A priority Critical patent/BR112023018838A2/pt
Priority to CN202280028754.0A priority patent/CN117279648A/zh
Priority to CA3212387A priority patent/CA3212387A1/en
Priority to MX2023010807A priority patent/MX2023010807A/es
Priority to JP2023557020A priority patent/JP2024511356A/ja
Priority to EP22772215.4A priority patent/EP4308129A4/en
Priority to IL305819A priority patent/IL305819A/en
Priority to AU2022239561A priority patent/AU2022239561A1/en
Priority to KR1020237035159A priority patent/KR20230156945A/ko
Application filed by ExcepGen Inc filed Critical ExcepGen Inc
Publication of WO2022197940A1 publication Critical patent/WO2022197940A1/en
Priority to US18/466,251 priority patent/US20240156948A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
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    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
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    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
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    • 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
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    • A61P31/14—Antivirals for RNA viruses
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
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Definitions

  • Viral infectious diseases e.g, the flu
  • Viral diseases result in a wide variety of symptoms that vary in character and severity depending on the type of viral infection and other factors, including the person’ s age and overall health.
  • Viral infections can be treated with varying degrees of success, depending on the type of virus and other factors. Sometimes, the treatment may involve just management of symptoms.
  • FIGS. 1 A and IB show the plasmid maps of CoVEG2 (FIG. 1 A) and CoVEGl (FIG. IB). Also shown are the relative positions of cytomegalovirus (CMV) enhancer and promoter and Simian virus 40 Poly A (SV40PA).
  • CMV cytomegalovirus
  • SV40PA Simian virus 40 Poly A
  • FIG. 2 shows the expression of SARS-CoV-2 S protein, SARS-CoV-2 M protein, SARS-CoV-2 E protein and SARS-CoV-2 N protein from CoVEG2 in HEK293 cells.
  • FIG. 3 shows that SARS-CoV-2 S, N, M and E proteins expressed from CoVEG2 are able to assemble into VLPs and are secreted from cells. See Example 3.
  • FIG. 3 A shows results from an SDS PAGE experiment showing S, N, M, and E protein bands in the size exclusion chromatography void sample.
  • FIG. 3B shows the chromatogram of the size exclusion VLP isolation run, whereas the void volume peak, which contains the VLPs, is indicated with an arrow.
  • FIG. 3C shows results from a Western Blotting experiment showing S, N, M, and E protein bands in the size exclusion chromatography void sample.
  • E envelope protein
  • M membrane protein
  • N nucleocapsid protein
  • S spike protein
  • NT non-transfected.
  • FIG. 4 shows the study design to test the immunogenicity of CoVEGl and CoVEG2 plasmids in mice.
  • FIG. 5A-50 show the plasmid maps of each of CoVEG 3-17, respectively.
  • FIG. 5P shows the plasmid map of the control S only plasmid.
  • CMV cytomegalovirus
  • SV40PA Simian virus 40 Poly A.
  • FIG. 6 shows a schematic depiction of the expression cassettes in each of CoVEG 3- 17.
  • Each of the plasmids CoVEG 3-17 vary in the genes that are encoded, the order of the genetic elements and the presence or absence of regulatory elements, e.g ., the viral packaging signal.
  • the boxes marked “M”, “S”, “N” and “E” indicate the genes encoding the membrane protein, spike protein, nucleocapsid protein and the envelope protein, respectively.
  • the box marked “L” refers to the gene encoding the L enhancer protein.
  • S (Mut) denotes the prefusion conformation-stabilized spike protein mutant in which an internal endogenous furin cleavage site has been mutated to comprise the following amino acid substitutions: R682G, R683S, R685S; and which further has the two amino acid substitutions, K986P and V987P.
  • FIG. 7 shows the images from anti-spike (S) protein antibody immunostaining of HEK293T cells transfected with each of the indicated plasmids or a control plasmid that expresses only the S protein without the other viral proteins (M, N or E). The images confirm the expression of the S protein in these cells.
  • FIG. 8 shows the results from the Western Blot analysis of a preparation of viral-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs. “S” denotes transient transfection with a control plasmid that expresses only the S protein without the other viral proteins (M, N or E).
  • VLPs viral-like particles
  • Staining with anti- RBD antibody (left) and anti-N protein antibody (right) show that the tested plasmids are capable of expressing and secreting viral structural proteins as VLPs into the cell culture supernatants.
  • the red arrow indicates the full length protein.
  • FIG. 9A shows the total signal values obtained from an ELISA analysis using 1:500 diluted serum samples from BALB/c mice injected with each of the indicated CoVEG plasmids after 56 days.
  • FIG. 9B shows the endpoint titer values obtained from an ELISA analysis using serum samples from BALB/c mice injected with each of the indicated CoVEG plasmids after 56 days.
  • Endpoint titer refers to the reciprocal maximal antibody dilution at which the ELISA signal (absorbance at 450 nm) is above 3 standard deviations of background signal.
  • FIG. 10 shows the percent (%) inhibition of the in vitro binding of the Spike protein receptor binding domain (RBD) to the ACE2 receptor by serum samples obtained from mice injected with each of the indicated plasmids using the commercial cPassTM neutralization assay (GenScript).
  • the results show that the serum samples obtained from CoVEG5 and C0VEG8- injected mice have neutralizing antibodies.
  • the positive and negative controls are the assay controls of the cPass neutralization assay kit and contain a known amount of SARS-CoV-2 neutralizing antibodies or a seronegative sample, respectively, and are used according to the manufacturer’s protocol.
  • FIG. 11 shows the results from the Western Blot analysis of a preparation of viral-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs. Staining with anti-RBD antibody (left) and anti-N protein antibody (right) show that the tested plasmids are capable of expressing and secreting viral structural proteins as VLPs into the cell culture supernatants with varying efficiencies.
  • VLPs viral-like particles
  • FIG. 12 shows the results from the Western Blot analysis of a preparation of viral-like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constructs. Staining with anti-RBD antibody (left) and anti-N protein antibody (right) show that the tested plasmids are capable of expressing and secreting viral structural proteins as VLPs into the cell culture supernatants with varying efficiencies.
  • VLPs viral-like particles
  • FIG. 13 shows the total signal values obtained from an ELISA analysis from BALB/c mice injected either intradermally (ID) or intramuscularly (IM) with each of the indicated CoVEG plasmids after 15 days. “S-only” denotes administration of the Spike protein alone.
  • FIG. 14A shows the map of a plasmid encoding West Nile vims proteins, preM protein and envelope protein, along with the enhancer protein (EMCV LI protein).
  • FIG. 14B shows the map of a control plasmid encoding just the West Nile vims proteins, preM protein and envelope protein CMV: cytomegalovims; SV40PA: Simian vims 40 Poly A.
  • FIGS. 15A and 15B show the total signal values obtained from an ELISA analysis of VLP secretion in HEK293 cells.
  • FIG. 15A shows ELISA analysis of VLP secretion performed with anti-spike (S protein) antibodies.
  • FIG. 15B shows ELISA analysis of VLP secretion performed with anti-nucleocapsid (N protein) antibodies.
  • FIG. 16 shows transmission electron micrographs (TEM) of CoVEGlO and CoVEG20 protein expression.
  • FIG. 16 left shows TEM of CoVEGlO which contains the L regulatory protein.
  • FIG. 16 right shows TEM of CoVEG20 which lacks the L regulatory protein.
  • FIG. 17 shows images from anti-nucleocapsid (N) protein antibody immunostaining of HEK293T cells transfected with each of the indicated plasmids.
  • FIG. 18 shows the results from Western Blot analysis of a preparation of viral -like particles (VLPs) isolated from cell culture supernatants when transiently transfected with the indicated CoVEG constmcts. Staining with anti-RBD antibody (left) and anti-N protein antibody (right) show that the tested plasmids are capable of expressing and secreting viral stmctural proteins as VLPs into the cell culture supernatants with varying efficiencies.
  • VLPs viral -like particles
  • FIG. 19 shows the western blot results of co-immunoprecipitation experiments of the CoVEG 10 plasmid.
  • the receptor binding domain (RBD) pull-down signal of the N protein in the elution indicates the N protein was retained within the particles.
  • RBD receptor binding domain
  • a co-IP was performed without the anti-RBD antibody demonstrating that the N protein did not bind the precipitation resin non-specifically.
  • FIG. 20 shows antibody binding titers from CoVEG 5 and 9-14 plasmids, as well as a spike (S) protein only containing plasmid, after intramuscular (IM) or intradermal (ID) injection in C57BL/6 mice.
  • IM intramuscular
  • ID intradermal
  • FIG. 21 shows the ELISA analysis of the neutralizing antibodies from the samples shown in FIG. 20.
  • FIG. 22 shows the antibody binding titers from CoVEG 9, 10, and 18-20 plasmids, as well as a spike (S) protein only containing plasmid, after intramuscular (IM) or intradermal (ID) injection in C57BL/6 mice.
  • IM intramuscular
  • ID intradermal
  • FIG. 23 shows the ELISA analysis of neutralizing antibodies produced in response to CoVEG 9, 10 and 20 plasmids intramuscular (IM) or intradermal (ID) injection in C57BL/6 mice.
  • FIGS. 24A and 24B show the results of T-cell analysis of CoVEGlO and CoVEG20.
  • FIG. 24 A shows the results of T-cell analysis of CoVEGlO.
  • FIG. 24B shows the results of T- cell analysis of CoVEG20.
  • FIG. 25 shows ELISA analysis of VLPs that were purified from cell culture supernatants of HEK293T cells transfected with isolated and resuspended West-Nile Virus (WNV) constructs with the enhancer protein (WNV + EG, circles) and without the enhancer protein (WNC, squares).
  • WNV West-Nile Virus
  • the specificity of the ELISA was tested against a plasmid containing GFP that does not give a signal in the specific ELISA assay.
  • the ELISA analysis revealed that the isolation of VLPs by ultracentrifugation with the addition of the enhancer protein contained more active VLPs than in the absence of the enhancer protein. This was especially surprising because the total amount of produced protein was higher in the absence of the enhancer protein, further demonstrating that the addition of an enhancer proteins increased the quality of the expressed target protein.
  • FIGS. 26A and 26B show the time course analysis of cell culture supernatants obtained from HEK293T cells overexpressing the supernatant containing over expressed West-Nile Virus (WNV) constructs with the enhancer protein (WNV + L) and without the enhancer protein (WNV).
  • FIG. 26A shows ELISA analysis demonstrating that the VLP concentration in the WNV + L (left) construct peaked at 72 h after transfection and gradually decreased thereafter. This was evidence of VLP secretion from healthy cells, as the expression profile followed expected production of VLP from transient transfections and on the related VLP particle half-life.
  • FIG. 26 B confirmed ELISA analysis with cell images.
  • WNV +L cell images (left) revealed very little to no cell death whereas the WNV cell images revealed visible signs of cell death starting at 72 hours after transfection (right, cell death indicated by black stars).
  • FIGS. 27A and 27B show the analysis of neutralizing antibodies of CoVEG9, the Spike construct with the enhancer protein (Spike + L) and the Spike construct without the enhancer protein (Spike) on days 42 and 70 after immunization of mice.
  • the presence of neutralizing antibodies was detected using the commercial cPassTM SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT) Kit (GenScript).
  • sVNT Surrogate Virus Neutralization Test
  • FIG. 27A shows the individual animals by group on days 42 and 70.
  • FIG. 28 shows immunofluorescence images from cells overexpressing either West-Nile Virus (WNV) constructs with the enhancer protein (WNV + L, left) or without the enhancer protein (WNV, right). As observed in other examples, the total amount of protein decreased when an enhancer protein was added, as indicated by the weaker Alexa Fluor 488 Fluor signal of the secondary antibody used in the immunostaining (WNV + L, left) compared to the WNV (right).
  • WNV West-Nile Virus
  • compositions for use as a vaccine comprising an expression cassette comprising a polynucleotide encoding a viral protein and a polynucleotide encoding an enhancer protein.
  • the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.
  • the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO: 1, or at least 95% identity to SEQ ID NO: 2.
  • the amino acid sequence of the enhancer protein is SEQ ID NO: 1, or SEQ ID NO: 2.
  • the polynucleotide encoding the enhancer protein is operatively linked to a polynucleotide encoding an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • the polynucleotide encoding the IRES is SEQ ID NO: 24.
  • the viral protein is a viral antigen.
  • the viral protein is derived from a virus selected from the group consisting of coronavirus, influenza virus, Hepatitis B virus, Human Papilloma virus (HPV), West Nile virus, and Human Immunodeficiency Virus (HIV) virus.
  • the viral protein is derived from a coronavirus.
  • the coronavirus is a betacoronavirus.
  • the betacoronavirus is severe acute respiratory syndrome (SAR.S) virus.
  • the SAR.S virus is a SARS-CoV-2 virus.
  • the betacoronavirus is Middle East respiratory syndrome (MERS) virus.
  • the coronavirus protein is a coronavirus spike protein.
  • the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 13.
  • the spike protein is SEQ ID NO: 13.
  • the coronavirus protein is a coronavirus membrane (M) protein.
  • the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 33.
  • the M protein is SEQ ID NO: 33.
  • the coronavirus protein is a coronavirus envelope (E) protein.
  • the E protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 22.
  • the E protein is SEQ ID NO: 22.
  • the coronavirus protein is a coronavirus nucleocapsid (N) protein.
  • the N protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 20.
  • the N protein is SEQ ID NO: 20.
  • the coronavirus protein forms a virus-like particle (VLP).
  • VLP virus-like particle
  • the viral protein is derived from West Nile virus.
  • the viral protein is precursor membrane protein (preM), envelope glycoprotein (E), or a combination thereof.
  • the disclosure provides vectors for use as a vaccine, comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 30.
  • the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 30.
  • the disclosure provides vectors for use as a vaccine, comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 31.
  • the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 31.
  • the disclosure provides vectors for use as a vaccine, comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-49, 55 and 62.
  • the vector is a naked polynucleotide.
  • the vector is a deoxyribonucleic acid (DNA) polynucleotide.
  • the vector is a ribonucleic acid (RNA) polynucleotide.
  • the vector comprises a plasmid.
  • the vector comprises linear DNA. Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that in some cases can replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine- conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating.
  • the vector is an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication competent adenovirus vector, a replication deficient adenovirus vector, a herpes virus vector, a baculovirus vector, a nonviral plasmid, a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, or an adenovirus vector.
  • AAV adeno-associated virus
  • the vector is a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
  • BAC bacterial artificial chromosome
  • PAC bacteriophage PI -derived vector
  • YAC yeast artificial chromosome
  • MAC mammalian artificial chromosome
  • the expression cassette comprises a promoter operatively linked to each of the polynucleotide sequences of the expression cassette.
  • the vector comprises a DNA polynucleotide, said DNA polynucleotide encoding a viral packaging signal.
  • the viral packaging signal is a RNA polynucleotide.
  • the viral packaging signal is derived from a coronavirus.
  • the disclosure provides vaccine compositions, comprising any one of the vectors disclosed herein, and a pharmaceutically acceptable carrier.
  • the vaccine composition comprises an adjuvant.
  • the adjuvant is alum.
  • the adjuvant is monophosphoryl lipid A (MPL).
  • the disclosure provides methods of expressing a viral antigen in a eukaryotic cell, comprising contacting the cell with any one of the vectors disclosed herein.
  • contacting the cell with the vector results in: (i) expression of the antigen at a higher expression level; and/or (ii) expression of the antigen for a longer period of time; and/or (iii) expression of the antigen with better protein quality, than a vector lacking the enhancer protein.
  • contacting the cell with the vector results in: (i) expression of a virus like particle (VLP) comprising the antigen at a higher expression level; and/or (ii) expression of a VLP comprising the antigen for a longer period of time; and/or (iii) expression of a VLP comprising the antigen with better protein quality, than a vector lacking the enhancer protein.
  • VLP virus like particle
  • the vector comprises a polynucleotide encoding a viral packaging signal, wherein contacting the cell with the vector results in expression of the viral packaging signal, and wherein the VLPs encapsidate the viral packaging signal.
  • the vector results in the formation of a greater number of VLPs, as compared to a control vector lacking the polynucleotide encoding the viral packaging signal.
  • the disclosure provides methods of eliciting an immune response in a subject, comprising administering an effective amount of any one of the vaccine compositions disclosed herein to the subject.
  • tissue at an administration site of the subject expresses the antigen and/or a VLP comprising the antigen.
  • tissue at an administration site of the subject (i) expresses the antigen and/or a VLP comprising the antigen at a higher expression level; and/or (ii) expresses the antigen and/or a VLP comprising the antigen for a longer period of time; and/or (iii) expresses the antigen and/or a VLP comprising the antigen with better protein quality, than when a vector lacking the enhancer protein is administered.
  • the vector comprises a polynucleotide encoding a viral packaging signal, wherein tissue at an administration site of the subject expresses the viral packaging signal, and wherein the VLPs encapsidate the viral packaging signal.
  • the vector results in the expression of a greater number of VLPs, as compared to a control vector lacking the polynucleotide encoding the viral packaging signal.
  • the VLPs encapsidating the viral packaging signal are more immunogenic than control VLPs comprising the antigen but lacking the viral packaging signal.
  • the method elicits an antibody response in the subject.
  • the antibody response is a neutralizing antibody response.
  • the method elicits a cellular immune response.
  • the method elicits a prophylactic, protective and/or therapeutic immune response in the subject.
  • the administration is intradermal administration, intramuscular administration, subcutaneous administration, or intranasal administration.
  • the disclosure provides polynucleotides comprising an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picomavirus leader (L) protein or a functional variant thereof.
  • the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO: 1, or at least 95% identity to SEQ ID NO: 2.
  • the amino acid sequence of the enhancer protein is SEQ ID NO: 1, or SEQ ID NO: 2.
  • the polynucleotide encoding the enhancer protein is operatively linked to a polynucleotide encoding an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • the polynucleotide encoding the IRES is SEQ ID NO: 24.
  • the coronavirus protein forms a virus-like particle (VLP).
  • the disclosure provides polynucleotides comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 30.
  • the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 30.
  • the disclosure provides polynucleotides comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 31.
  • the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 31.
  • the polynucleotide is a naked polynucleotide. In some embodiments, the polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide. In some embodiments, the polynucleotide is a ribonucleic acid (RNA) polynucleotide. In some embodiments, the expression cassette comprises a promoter operatively linked to each of the polynucleotide sequences of the expression cassette.
  • kits comprising a vector, wherein the vector comprises an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.
  • the vector comprises an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picornavirus leader (L) protein or a functional variant thereof.
  • the disclosure provides vectors, comprising an expression cassette, said expression cassette comprising a promoter linked to a target gene, wherein the vector comprises a nucleic acid sequence encoding a viral packaging element.
  • the viral packaging element is a RNA polynucleotide.
  • the viral packaging element is derived from a coronavirus.
  • the viral packaging element is derived from SAR.S- CoV2.
  • the nucleic acid sequence encoding the viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 34.
  • the disclosure provides methods of expressing a target protein in a eukaryotic cell, comprising contacting the cell with any one of the vectors disclosed herein.
  • contacting the cell with the vector results in the formation of virus-like particles (VLPs) comprising the target protein.
  • contacting the cell with the vector results in the formation of a greater number of virus-like particles (VLPs) comprising the target protein, as compared to a control vector comprising the expression cassette but lacking the nucleic acid sequence encoding the viral packaging element.
  • the nucleic acid sequence encoding the viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 34.
  • the disclosure provides vectors for use as vaccines, comprising an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding an S protein wherein the S protein comprises SEQ ID NO: 13 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a third proteolytic cleavage site, a polynucleotide encoding an E protein, wherein the E protein
  • the disclosure also provides vectors for use as a vaccine, comprising an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding an M protein or an amino acid sequence at least 95% identical thereto, wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding an S protein wherein the S protein comprises SEQ ID NO: 13 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding an E protein or a polynucleotide sequence at least 95% identical thereto, wherein the E protein comprises SEQ ID NO: 22 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding an expression cassette,
  • the disclosure also provides vectors for use as vaccines, comprising an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding an M protein, wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the mutated S protein comprise SEQ ID NO: 51 or SEQ ID NO: 52 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleot
  • the disclosure provides vectors for use as vaccines, comprising an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal, wherein the viral packaging signal comprises SEQ ID NO: 34 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an M protein, wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a mutated S protein, wherein the S protein comprises SEQ ID NO: 51 or SEQ ID
  • VLPs Virus-like particles
  • VLPs are composed of viral structural proteins. Although VLPs are immunogenic, they are non-infectious. Therefore, VLPs have enormous potential for use in the development of vaccines. VLPs may be produced in vitro , and then administered to a subject in need of immunization. Alternatively, VLPs may be produced in vivo in the subject. [0061] The production of VLPs is challenging primarily because it often requires the expression of more than one structural protein from more than one plasmid. In some cases, several plasmids carrying different structural proteins may need to be introduced into the host cell at defined ratios to support the formation of VLPs. This process can be unreliable and often fails to produce sufficient levels of VLPs of required quality.
  • VLPs can be expressed from a single plasmid or a single RNA transcript, that will greatly simplify the process of making VLPs and thus, provide a much-needed boost for the development of vaccines comprising VLPs.
  • compositions and methods disclosed herein enable the reliable formation of high levels of VLPs in vivo and thus, enable a robust induction of immune response against the viral antigens on the VLPs. Furthermore, these compositions and methods may be used to induce immune response against different viruses, e.g ., coronaviruses (e.g. SARS CoV-2), influenza viruses, and West Nile virus.
  • coronaviruses e.g. SARS CoV-2
  • influenza viruses e.g. SARS CoV-2
  • West Nile virus e.g., West Nile virus.
  • polypeptide As used herein, the term “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. For example, “about 100” encompasses 90 and 110.
  • nucleotide sequences are listed in the 5 to 3' direction, and amino acid sequences are listed in the N-terminal to C-terminal direction, unless indicated otherwise.
  • polypeptide As used herein, polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • the term “subject” includes humans and other animals.
  • the subject is a human.
  • the subject may be an adult, a teenager, a child (2 years to 14 years of age), an infant (1 month to 24 months), or a neonate (up to 1 month).
  • the adults are seniors about 65 years or older, or about 60 years or older.
  • the subject is a pregnant woman or a woman intending to become pregnant.
  • subject is not a human; for example a non-human primate; for example, a baboon, a chimpanzee, a gorilla, or a macaque.
  • the subject may be a pet, e.g. , a dog or cat.
  • immunogen As used herein, the terms “immunogen,” “antigen,” and “epitope” refer to substances e.g. , proteins, including glycoproteins, and peptides that are capable of eliciting an immune response.
  • an “immunogenic response” in a subject results in the development in the subject of a humoral and/or a cellular immune response to an antigen.
  • an effective amount refers to the amount of an agent that is sufficient to achieve an outcome, for example, to affect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue into which it is administered, and the physical delivery system in which it is carried.
  • virus-like particle refers to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious.
  • Virus- like particles in accordance with the disclosure do not carry genetic information encoding for the proteins of the virus-like particles. In general, virus-like particles lack a viral genome and, therefore, are noninfectious. In addition, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.
  • an amino acid substitution interchangeably referred to as amino acid replacement, at a specific position on the protein sequence is denoted herein in the following manner: “one letter code of the WT amino acid residue - amino acid position - one letter code of the amino acid residue that replaces this WT residue”.
  • a Spike (S) protein which is a R682G mutant refers to an S protein in which the wild type residue at the 682 nd amino acid position (R or arginine) is replaced with G or glycine.
  • the disclosure provides vectors comprising an expression cassette comprising a polynucleotide encoding an antigen and a polynucleotide encoding an enhancer protein.
  • the vector is used as a vaccine, or as part of a vaccine composition.
  • vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • a vector for use according to the present disclosure may comprise any vector known in the art. Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating.
  • the vector is an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, a replication competent adenovirus vector, a replication deficient adenovirus vector, a herpes virus vector, a baculovirus vector, a nonviral plasmid, a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome, or an adenovirus vector.
  • AAV adeno-associated virus
  • the vector is a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
  • the vector is a naked polynucleotide.
  • the vector is a deoxyribonucleic acid (DNA) polynucleotide.
  • the vector is a ribonucleic acid (RNA) polynucleotide.
  • the vector comprises a first polynucleotide encoding an antigen and a second polynucleotide encoding an enhancer protein.
  • the vector has a design as shown in FIG. 1A or FIG. IB.
  • the vector is CoVEGl.
  • the vector is CoVEG2.
  • Table 1 shows the nucleic acid sequences of important regions of the CoVEGl and CoVEG2, and amino acid sequences encoded by these regions.
  • the vectors disclosed herein may comprise one or more expression cassettes.
  • expression cassette refers to a defined segment of a nucleic acid molecule that comprises the minimum elements needed for production of another nucleic acid or protein encoded by that nucleic acid molecule.
  • the expression cassette comprises a promoter.
  • the promoter is operatively linked to each of the polynucleotide sequences of the expression cassette.
  • a vector may comprise an expression cassette, the expression cassette comprising a first polynucleotide encoding an antigen, and a second polynucleotide encoding an enhancer protein.
  • the expression cassette comprises a first promoter, operatively linked to the first polynucleotide; and a second promoter, operatively linked to the second polynucleotide.
  • the expression cassette comprises a shared promoter operatively linked to both the first polynucleotide and the second polynucleotide.
  • the expression cassette comprises a coding polynucleotide comprising the first polynucleotide and the second polynucleotide linked by a polynucleotide encoding a separating element (e.g ., a ribosome skipping site or 2A element), the coding polynucleotide operatively linked to the shared promoter.
  • a separating element e.g ., a ribosome skipping site or 2A element
  • the expression cassette comprises a coding polynucleotide, the coding polynucleotide encoding an enhancer protein and an antigen linked to by a separating element (e.g., a ribosome skipping site or 2 A element), the coding polynucleotide operatively linked to the shared promoter.
  • a separating element e.g., a ribosome skipping site or 2 A element
  • the expression cassette is configured for transcription of a single messenger RNA encoding both the antigen and the enhancer protein, linked by a separating element (e.g, a ribosome skipping site or 2 A element); wherein translation of the messenger RNA results in expression of the antigen and the enhancer protein (e.g., the L protein) as distinct polypeptides.
  • the expression cassettes disclosed herein comprise one or more proteolytic cleavage sites, for example, 1, 2, 3, 4, or 5 proteolytic cleavage sites.
  • the proteolytic cleavage site is located between a polynucleotide encoding a first antigen, and another polynucleotide encoding a second antigen. In some embodiments, the proteolytic cleavage site is located between a polynucleotide encoding an antigen, and a polynucleotide encoding an enhancer protein. In some embodiments, the proteolytic cleavage site comprises the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, the proteolytic cleavage site is a furin cleavage site.
  • the expression cassettes disclosed herein comprise a nucleic acid sequence encoding a viral accessory protein, for example ORF3a protein.
  • the polynucleotide encoding the ORF3 protein has a nucleic acid sequence with at least 70% sequence identity - for instance, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the nucleic acid sequence of SEQ ID NO: 54.
  • the polynucleotide encoding the ORF3 protein has a nucleic acid sequence of SEQ ID NO: 54.
  • ORF3 protein has a amino acid sequence with at least 70% sequence identity - for instance, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the amino acid sequence of SEQ ID NO: 53.
  • the ORF3 protein has an amino acid sequence of SEQ ID NO: 53.
  • the vector is selected from the group consisting of CoVEG3-17.
  • the vector comprises a nucleic acid sequence having at least about 70% identity, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or about 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-49.
  • the vector comprises a nucleic acid sequence having at least 70% (for example, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%) identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-49.
  • the nucleic acid sequence of the expression cassette of CoVEG3-17 and the genetic elements therein are listed in Table 2.
  • the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the SARS CoV2 Spike protein. In some embodiments, the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the SARS CoV2 membrane protein. In some embodiments, the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the SARS CoV2 envelope protein. In some embodiments, the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the SARS CoV2 nucleocapsid protein.
  • the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the EMCV L protein. In some embodiments, the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the internal ribosome entry site (IRES). In some embodiments, the vector disclosed herein comprises the polynucleotide from any one of CoVEG 1-17 that encodes the viral packaging signal. ii) Polynucleotides
  • Polynucleotides of the present disclosure may include DNA, RNA, and DNA-RNA hybrid molecules.
  • polynucleotides are isolated from a natural source; prepared in vitro , using techniques e.g., PCR amplification or chemical synthesis; prepared in vivo , e.g. , via recombinant DNA technology; or prepared or obtained by any appropriate method.
  • polynucleotides are of any shape (linear, circular, etc.) or topology (single-stranded, double-stranded, linear, circular, supercoiled, torsional, nicked, etc.).
  • Polynucleotides may also comprise nucleic acid derivatives e.g.
  • PNAS peptide nucleic acids
  • polypeptide-nucleic acid conjugates nucleic acids having at least one chemically modified sugar residue, backbone, internucleotide linkage, base, nucleotide, nucleoside, or nucleotide analog or derivative; as well as nucleic acids having chemically modified 5' or 3' ends; and nucleic acids having two or more of such modifications. Not all linkages in a polynucleotide need to be identical.
  • the polynucleotides disclosed herein may encode one or more antigens; and/or one or more enhancer proteins. In some embodiments, the polynucleotide encodes one antigen. In some embodiments, the polynucleotide encodes one enhancer protein. In some embodiments, the polynucleotide encodes more than one antigen; more than one enhancer protein, and/or one or more separating elements.
  • the polynucleotide may encode a polypeptide that is not antigenic.
  • the polypeptide that is not antigenic may form a part of a VLP.
  • the present disclosure provides vectors that comprise polynucleotides that encode one or more antigens, and/or polynucleotides that encode one or more non-antigenic polypeptides, and/or polynucleotides that encode one or more enhancer proteins.
  • the one or more antigens and the one or more non-antigenic polypeptides are capable of forming a virus like particle (VLP).
  • the one or more antigens may be derived from one or more proteins of a first virus
  • the one or more non-antigenic polypeptides may be derived from one or more proteins of a second virus.
  • the first polynucleotide or the second polynucleotide, or both are operatively linked to a polynucleotide encoding an internal ribosome entry site (IRES).
  • the polynucleotide encoding the enhancer protein and/or the polynucleotide encoding the antigen are operatively linked to a polynucleotide encoding an IRES.
  • Vectors according to the present disclosure may comprise one or more promoters.
  • the term “promoter” refers to a region or sequence located upstream or downstream from the start of transcription which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • the polynucleotide(s) or vector(s) according to the present disclosure may comprise one or more promoters.
  • the promoters may be any promoter known in the art.
  • the promoter may be a forward promoter or a reverse promoter.
  • the promoter is a mammalian promoter.
  • one or more promoters are native promoters.
  • one or more promoters are non-native promoters.
  • one or more promoters are non-mammalian promoters.
  • RNA promoters for use in the disclosed compositions and methods include Ul, human elongation factor-1 alpha (EF-1 alpha), cytomegalovirus (CMV), human ubiquitin, spleen focus-forming virus (SFFV), U6, HI, tRNA Lys , tRNA Ser and tRNA Arg , CAG, PGK, TRE, UAS, UbC, SV40, T7, Sp6, lac, araBad, trp, and Ptac promoters.
  • operatively linked refers to elements or structures in a nucleic acid sequence that are linked by operative ability and not physical location.
  • the elements or structures are capable of, or characterized by, accomplishing a desired operation. It is recognized by one of ordinary skill in the art that it is not necessary for elements or structures in a nucleic acid sequence to be in a tandem or adjacent order to be operatively linked.
  • a promoter comprised by a vector according to the present disclosure is an inducible promoter.
  • vectors according to the present disclosure may further comprise a polynucleotide sequence encoding a polymerase.
  • the polymerase is a viral polymerase.
  • the vectors disclosed herein comprises a polynucleotide sequence encoding a T7 RNA polymerase.
  • a vector may comprise a T7 promoter configured for transcription of either or both of the polynucleotide encoding an antigen, and the second polynucleotide encoding the enhancer protein by a T7 RNA polymerase.
  • the expression or quality of the antigen is significantly improved by expression according to the disclosed methods, e.g ., in conjunction with one or more enhancer proteins.
  • the antigen is derived from a single protein.
  • the antigen is derived from multiple proteins.
  • the antigen is a chimeric antigen comprising amino acid sequences from one or more proteins.
  • the antigen is a viral antigen.
  • the viral antigen may comprise the whole or part of an amino acid sequence derived from any viral protein, without limitation.
  • the viral antigen is the viral protein.
  • the amino acid sequence of the viral protein is the whole or part of a structural protein or multiple structural proteins of a virus.
  • the antigen or antigens assemble into VLPs and are released from the expressing cells.
  • the viral antigen comprises the whole or an antigen fragment of any coronavirus protein, without limitation.
  • the coronavirus is a betacoronavirus.
  • the betacoronavirus is severe acute respiratory syndrome (SARS) virus.
  • the betacoronavirus is Middle East respiratory syndrome (MERS) virus, OC43, or HKU1.
  • the SARS virus is SARS- CoV-1.
  • the SARS virus is SARS-CoV-2.
  • the viral antigen comprises the whole or an antigen fragment of any one or more of the following proteins: coronavirus spike protein, coronavirus M protein, coronavirus N protein, and coronavirus E protein.
  • the coronavirus spike protein is selected from the group consisting of a SARS-Cov-2 spike protein, a Middle East respiratory syndrome (MERS) spike protein, and SARS-CoV spike protein.
  • the coronavirus M protein is selected from SARS-Cov-2 M protein, MERS M protein and SARS-CoV M protein.
  • the coronavirus N protein is selected from SARS-Cov-2 N protein, MERS N protein, and SARS-CoV N protein.
  • the coronavirus E protein is selected from SARS-Cov-2 E protein, MERS E protein, and SARS-CoV E protein.
  • the polynucleotide encoding the coronavirus spike protein has a nucleic acid sequence with at least 70% sequence identity - for instance, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the nucleic acid sequence of SEQ ID NO: 14 or 70.
  • the polynucleotide encoding the coronavirus spike protein has a nucleic acid sequence of SEQ ID NO: 14 or 70.
  • the amino acid sequence of the coronavirus spike protein has at least 70% sequence identity - for instance, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the amino acid sequence of SEQ ID NO: 13.
  • the amino acid sequence of the coronavirus spike protein is SEQ ID NO: 13.
  • the SARS-Cov-2 spike protein is a mutant S protein (also denoted as “S (Mut)”) that comprises one or more amino acid mutations, as compared to SEQ ID NO: 13.
  • the mutant S protein is expressed at a higher level, as compared to the wild type S protein. In some embodiments, the mutant S protein is prefusion conformation-stabilized spike protein. In some embodiments, the mutation in the S protein stabilizes the trimeric state of the S protein. In some embodiments, the mutant S protein comprises one or more mutations in the internal endogenous proteolytic cleavage site of the S protein. In some embodiments, the mutant S protein comprises a deletion of the internal endogenous proteolytic cleavage site of the S protein. In some embodiments, the one or more mutations in the proteolytic cleavage site of the S protein inhibit the cleavage of the S protein during the assembly process.
  • a VLP comprising any one or more of the mutant S proteins disclosed herein is more immunogenic than a VLP comprising a wild type S protein, e.g ., an S protein comprising an amino acid sequence of SEQ ID NO: 13.
  • the mutant S protein comprises a modification (e.g. a substitution) of at least one amino acid residue selected from the group consisting of R682, R683, A684, R685, K986, and V987 in SEQ ID NO: 13.
  • the mutant S protein comprises at least one amino acid substitution selected from the group consisting of R682G, R683S, R685S, K986P, and V987P in SEQ ID NO: 13.
  • the mutation S protein comprises the amino acid substitutions, R682G, R683S, R685S, K986P, and V987P in SEQ ID NO: 13.
  • the mutant S protein comprises the following amino acid substitutions in an internal endogenous furin cleavage site: R682G, R683S, R685S. That is, in some embodiments, the mutant S protein comprises the following amino acids at an internal endogenous furin cleavage site: G at amino acid residue 682, S at amino acid residue 683, A at amino acid residue 684, and S at amino acid residue 685.
  • the mutant S protein has at least 70% sequence identity - for instance, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the amino acid sequence of SEQ ID NO: 51.
  • the amino acid sequence of the mutant S protein is SEQ ID NO: 51.
  • the polynucleotide encoding the mutant S protein has a nucleic acid sequence with at least 70% sequence identity - for instance, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the nucleic acid sequence of SEQ ID NO: 52.
  • the polynucleotide encoding the coronavirus spike protein has a nucleic acid sequence of SEQ ID NO: 52.
  • the polynucleotide encoding the coronavirus M protein has a nucleic acid sequence with at least 80% sequence identity - for instance, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the nucleic acid sequence of SEQ ID NO: 19 or 66.
  • the polynucleotide encoding the coronavirus M protein has a nucleic acid sequence of SEQ ID NO: 19 or 66.
  • the amino acid sequence of the coronavirus M protein has at least 80% sequence identity - for instance, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the amino acid sequence of SEQ ID NO: 33.
  • the amino acid sequence of the coronavirus M protein is SEQ ID NO: 33.
  • the polynucleotide encoding the coronavirus N protein has a nucleic acid sequence with at least 80% sequence identity - for instance, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the nucleic acid sequence of SEQ ID NO: 21 or 71.
  • the polynucleotide encoding the coronavirus N protein has a nucleic acid sequence of SEQ ID NO: 21 or 71.
  • the amino acid sequence of the coronavirus N protein has at least 80% sequence identity - for instance, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the amino acid sequence of SEQ ID NO: 20.
  • the amino acid sequence of the coronavirus N protein is SEQ ID NO: 20.
  • the viral protein is derived from the any one of Groups I, II, III, IV, V, VI, or VII of viruses according to the Baltimore classification.
  • the viral protein is derived from an enveloped negative-sense, single stranded, segmented RNA virus (e.g . Influenza virus).
  • the viral protein is derived from an enveloped DNA virus (e.g. Hepatitis B virus).
  • the viral protein is derived from a non-enveloped DNA virus (e.g. Human Papillomavirus).
  • the viral protein is derived from a positive strand enveloped RNA virus (e.g. a coronavirus, e.g.
  • the viral antigen comprises the whole or an antigen fragment of any protein derived from West Nile virus.
  • the West Nile viral protein is the precursor membrane (prM), the envelope glycoprotein (E), or a combination thereof.
  • the vector encoding one or more West Nile virus proteins, e.g, prM and/or E protein is West Nile Virus Minimal plasmid (WNV minimal plasmid), as depicted in FIG. 14A or West Nile Virus Standard plasmid (WNV standard plasmid), as depicted in FIG. 14B.
  • the vector encoding one or more West Nile virus proteins e.g.
  • the polynucleotide encoding the West Nile vims prM protein has a nucleic acid sequence with at least 80% sequence identity - for instance, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, including any values and subranges that lie there between - to the nucleic acid sequence of SEQ ID NO: 59.
  • the polynucleotide encoding the West Nile vims prM protein has a nucleic acid sequence of SEQ ID NO: 59.
  • the viral antigen comprises the whole or an antigen fragment of any protein derived from the Influenza virus.
  • the strain of the Influenza virus is not limited, and may be any strain that is currently known or later discovered, e.g ., for example, H1N1, H3N2, or an Influenza B strain.
  • the Influenza viral protein is the HA protein, NA protein, Ml protein, M2 protein, or any combination thereof.
  • the viral antigen comprises the whole or an antigen fragment of any protein derived from the Hepatitis B virus.
  • the Hepatatis B viral protein is the sAg (S protein), sAg (M protein), sAg (L protein), preSl, preS2, cAg (core antigen), or any combination thereof.
  • the viral antigen comprises the whole or an antigen fragment of any protein derived from the Human Papilloma virus.
  • Human Papilloma viral protein is the LI protein of HPV 6, LI protein of HPV 11, LI protein of HPV 16, LI protein of HPV 18, or any combination thereof.
  • the viral antigen comprises the whole or an antigen fragment of any one or more of the proteins derived from each of the viruses listed below in Table 4.
  • the viral antigen may comprise the whole or an antigen fragment of any protein derived from the avian Influenza virus (H5N3). Table 4
  • the co-expression of the enhancer proteins with an antigen may improve one or more aspects of antigen expression, including but not limited to yield, quality, folding, posttranslational modification, activity, localization, and downstream activity, or may reduce one or more of misfolding, altered activity, incorrect posttranslational modifications, and/or toxicity.
  • the enhancer protein is a picomavirus leader (L) protein, or a functional variant thereof.
  • the picomavirus leader (L) protein is capable of blocking the nuclear pore, thereby inhibiting nucleocytoplasmic transport (“NCT”).
  • NCT nucleocytoplasmic transport
  • the term “functional variant” refers to a protein that is homologous to the picomavirus leader (L) protein and/or shares substantial sequence similarity to the picomavirus leader (L) protein (e.g., more than 30%, 40%, 50%, 60%, 70%, 80%, 85% 90%, 95%, or 99% sequence identity).
  • the functional variant shares one or more functional characteristics of the picomavirus leader (L) protein.
  • a functional variant of the picomavirus leader (L) protein retains the ability to inhibit NCT.
  • the picomavirus leader (L) protein is an L protein from the Cardiovirus, Hepatovirus, or Aphthovirus genera.
  • the enhancer protein may be from Bovine rhinitis A vims, Bovine rhinitis B vims, Equine rhinitis A vims, Foot-and-mouth disease vims, Hepatovims A, Hepatovims B, Marmota himalayana hepatovims, Phopivims, Cardiovims A, Cardiovims B, Theiler's Murine encephalomyelitis vims (TMEV), Vilyuisk human encephalomyelitis vims (VHEV), Theiler-like rat vims (TRV), or Saffold vims (SAF- V).
  • TMEV Murine encephalomyelitis vims
  • VHEV Vilyuisk human encephalomyelitis vims
  • TRV Theiler-like rat vims
  • SAF- V Saffold vims
  • the picornavims leader (L) protein is the L protein of Theiler’s vims or a functional variant thereof.
  • the L protein shares at least 90% identity to SEQ ID NO: 1.
  • the enhancer protein may comprise or consist of SEQ ID NO: 1.
  • the enhancer protein may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1.
  • the picornavims leader (L) protein is the L protein of Encephalomyocarditis vims (EMCV) or a functional variant thereof.
  • the L protein may share at least 90% identity to SEQ ID NO: 2.
  • the enhancer protein may comprise or consist of SEQ ID NO: 2.
  • the enhancer protein may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 2.
  • the nucleic acid sequence encoding the enhancer protein may comprise or consist of SEQ ID NO: 68. In some embodiments, the nucleic acid sequence encoding the enhancer protein may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 68.
  • the picornavirus leader (L) protein is selected from the group consisting of the L protein of poliovirus, the L protein of HRV16, the L protein of mengo virus, and the L protein of Saffold virus 2 or a functional variant thereof.
  • the picornavirus leader (L) protein is selected from the proteins listed in Table 5 or functional variants thereof.
  • the polynucleotide encoding the picornavirus leader (L) protein may encode an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence listed in Table 2.
  • the amino acid sequence of the picornavirus leader (L) protein may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to an amino acid sequence listed in Table 2.
  • amino acid sequence of the picornavirus leader (L) protein may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 12.
  • an enhancer protein may have an amino acid sequence comprising, or consisting of, one of the amino acid sequences listed in Table 2.
  • an enhancer protein may have an amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 12.
  • the antigens, enhancer proteins, and/or fusion proteins, or the polynucleotides encoding such may be modified to comprise one or more markers, labels, or tags.
  • a protein of the present disclosure may be labeled with any label that will allow its detection, e.g ., a radiolabel, a fluorescent agent, biotin, a peptide tag, an enzyme fragment, or the like.
  • the proteins may comprise an affinity tag, e.g. , a His-tag, a GST-tag, a Strep-tag, a biotin-tag, an immunoglobulin binding domain, e.g. , an IgG binding domain, a calmodulin binding peptide, and the like.
  • polynucleotides of the present disclosure comprise a selectable marker, e.g. , an antibiotic resistance marker.
  • the vectors disclosed herein comprise a polynucleotide sequence encoding a viral packaging signal (interchangeably referred to herein as “viral packaging sequence” or packaging signal” or “psi sequence”).
  • the polynucleotide sequence encoding a viral packaging signal is a DNA polynucleotide, an RNA polynucleotide, or a combination thereof.
  • the viral packaging signal is an RNA polynucleotide.
  • the vectors comprise more than one copy of the polynucleotide sequence encoding a viral packaging signal, for example, 2, 3, 4 or 5 copies of the polynucleotide sequence.
  • the viral packaging signal may be derived from any virus.
  • the viral packaging signal is derived from the same virus as the antigens that are expressed from the vector.
  • the viral packaging signal is derived from a different virus as the antigens that are expressed from the vector.
  • the viral packaging signal is derived from a virus selected from the group consisting of SARS-CoV-2, SARS-CoV- 1, MERS-CoV, chikungunya virus, African Swine Fever virus, Dengue virus, Zika virus, Influenza virus (e.g, A, B, C), Human Immunodeficiency Virus (HIV), Ebola virus, Hepatitis virus (e.g, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, and Hepatitis E), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), West Nile virus, and Human Papillomavirus.
  • a virus selected from the group consisting of SARS-CoV-2, SARS-CoV- 1, MERS-CoV, chikungunya virus, African Swine Fever virus, Dengue virus, Zika virus, Influenza virus (e.g, A, B, C), Human Immunodeficiency
  • the polynucleotide encoding the viral packaging element has at least about 70% identity (for example, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%), including all values and subranges that lie therebetween, to the polynucleotide of SEQ ID NO: 34.
  • the location of the polynucleotide encoding the viral packaging signal on the vector is not limited. In some embodiments, the location of the polynucleotide encoding the viral packaging signal on the vector may be 5' to all the nucleic acid sequences encoding the viral antigens.
  • the location of the polynucleotide encoding the viral packaging signal on the vector may be 3' to all the nucleic acid sequences encoding the viral antigens. In some embodiments, the location of one copy of the polynucleotide encoding the viral packaging signal on the vector is 5' to all the nucleic acid sequences encoding the viral antigens, and the location of the other copy of the polynucleotide encoding the viral antigen is 3' to all the nucleic acid sequences encoding the viral antigens.
  • the size of the viral packaging signal is not limited and may be in the range of about 50 bps to about 3 kb, for example, about 100 bps, about 200 bps, about 300 bps, about 400 bps, about 500 bps, about 550 bps, about 600 bps, about 650 bps, about 700 bps, about 800, bps, about 900 bps, about 1 kb, about 2 kb, or about 3 kb, including all values and subranges that lie therebetween.
  • the size of the viral packaging signal is about 600 to about 700 bps, for example, about 650 bps.
  • the size of the viral packaging signal is about 661 bps.
  • the disclosure further provides vectors, comprising an expression cassette, said expression cassette comprising a promoter linked to a target gene, wherein the vector comprises a polynucleotide encoding any one of the viral packaging elements disclosed herein.
  • the polynucleotides sequences encoding one or more viral antigens, and the polynucleotide sequence encoding the enhancer, and/or one or more regulatory elements may be ordered in any possible combination. For instance, the order of elements in the expression cassette may be as depicted for any one of the plasmids CoVEG 3-17 in FIG. 6.
  • the order of elements in the expression cassette might be related to the expression of antigens encoded by the vector, and/or formation of VLPs. Furthermore, it is thought that when the expression cassette comprises the genes in the following order from 5' to 3' - M, N, S, and E - it might result in higher protein expression and more stable VLP formation.
  • the vector comprises an expression cassette, comprising the elements in the same 5' to 3' order as CoVEG4.
  • the vector comprises an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding an M protein wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein wherein the S protein comprises SEQ ID NO: 13 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a E protein wherein the E protein comprises SEQ ID NO: 22 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding IRES wherein the IRES sequence comprises SEQ
  • the vector comprises an expression cassette, comprising the elements in the same 5' to 3' order as C0VEG8.
  • the vector comprises an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding an M protein wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an S protein wherein the S protein comprises SEQ ID NO: 13 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding a E protein wherein the E protein comprises SEQ ID NO: 22 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding IRES wherein the IRES sequence comprises
  • the vector comprises an expression cassette, comprising the elements in the same 5' to 3' order as CoVEG9.
  • the vector comprises an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding an M protein wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, wherein the S protein comprises SEQ ID NO: 51 or SEQ ID NO: 52 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleot
  • the vector comprises an expression cassette, comprising the elements in the same 5' to 3' order as CoVEGl 1.
  • the vector comprises an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging wherein the viral packaging signal comprises SEQ ID NO: 34 or a polynucleotide sequence at least 9% identical thereto, a polynucleotide encoding an M protein wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding
  • the vector comprises an expression cassette, comprising the elements in the same 5' to 3' order as CoVEG15.
  • the vector comprises an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal wherein the viral packaging signal comprises SEQ ID NO: 34 or a polynucleotide sequence at least 95% identical thereto, a polynucleotide encoding an M protein wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding encoding a polynucleotide
  • the vector comprises an expression cassette, comprising the elements in the same 5' to 3' order as CoVEG17.
  • the vector comprises an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a first polynucleotide encoding a viral packaging signal wherein the viral packaging signal comprises SEQ ID NO: 34 or a polynucleotide sequence at least 9% identical thereto, a polynucleotide encoding an M protein wherein the M protein comprises SEQ ID NO: 33 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding an N protein, wherein the N protein comprises SEQ ID NO: 20 or an amino acid sequence at least 95% identical thereto, a polynucleotide encoding a proteolytic cleavage site, a polynucleotide encoding
  • the VLP comprises three antigens. In some embodiments, the VLP comprises four antigens. In some embodiments, the structural proteins that form a VLP and the immunogenic viral antigens that are a part of the VLP are derived from the same virus (i.e., a native VLP). In some embodiments, the structural viral proteins that form a VLP are derived from one virus and the immunogenic viral antigens that get incorporated to that said VLP are derived from another virus (i.e., a chimeric VLP). In some embodiments, the viral proteins are mutated to enhance VLP assembly, VLP secretion and/or loading of the immunogenic antigen or antigens to the said VLP.
  • the first antigen is a coronavirus spike protein
  • the second antigen is a coronavirus membrane (M) protein
  • the third antigen is a coronavirus envelope (E) protein.
  • the first antigen is a coronavirus spike protein
  • the second antigen is a coronavirus membrane (M) protein
  • the third antigen is a coronavirus envelope (E) protein
  • the fourth antigen is a coronavirus nucleocapsid (N) protein.
  • protein quality might refer to without limitation, protein folding, posttranslational modification, functional activity, localization, and downstream activity.
  • the antigen which is co-expressed with an enhancer protein using any of the methods or vectors or compositions disclosed herein may have improved protein folding, improved posttranslational modification, improved functional activity, improved localization, and improved downstream activity, as compared to the antigen which is not co-expressed with an enhancer protein.
  • Non limiting examples of methods include viral transfection, direct uptake, projectile bombardment, direct injection with or without electroporation/sonoporation while using or not using cationic polymers, lipids, lipid formulations, and jet-gene devices.
  • Antigens and enhancer proteins can be stably or transiently expressed in cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 “Vector Therapy” & Chapter 13 “Delivery Systems for Gene Therapy”).
  • vectors can be introduced into a host cell by insertion into the genome using standard methods to produce stable cell lines, optionally through the use of lentiviral transfection, baculovirus gene transfer into mammalian cells (BacMam), retroviral transfection, CRISPR/Cas9, and/or transposons.
  • polynucleotides or vectors can be introduced into a host cell for transient transfection.
  • transient transfection may be effected through the use of viral vectors, helper lipids, e.g, PEI, Lipofectamine, and/or Fectamine 293.
  • the genetic elements can be encoded as DNA on e.g.
  • mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero and Hep-2 cells. Xenopus laevis oocytes, or other cells of amphibian origin, may also be used.
  • Prokaryotic host cells include bacterial cells, for example, E. coli, B. subtilis, and mycobacteria.
  • the pharmaceutically acceptable carrier, excipient, and/or vehicle may comprise saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • the vaccine composition comprises an adjuvant.
  • adjuvant refers to a compound that, when used in combination with an immunogen, augments or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may include intensification or broadening the specificity of either or both antibody and cellular immune responses.
  • the adjuvant is alum.
  • the adjuvant is monophosphoryl lipid A (MPL).
  • other adjuvants may be used in addition or as an alternative.
  • Other adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant, GMCSP, BCG, MDP compounds, e.g.
  • the adjuvant may be a paucilamellar lipid vesicle; for example, Novasomes®.
  • Novasomes® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They comprise Brij 72, cholesterol, oleic acid and squalene.
  • the disclosure further provides methods of eliciting an immune response in a subject, comprising administering an effective amount of any one of the vaccine compositions disclosed herein to the subject.
  • tissue at an administration site of the subject expresses the antigen and/or a VLP comprising the antigen.
  • the method elicits an antibody response in the subject.
  • the antibody response is a neutralizing antibody response.
  • the method elicits a cellular immune response.
  • the method elicits a prophylactic, protective and/or therapeutic immune response in the subject.
  • the vector comprises a DNA polynucleotide encoding a viral packaging signal, such that the tissue at an administration site of the subject expresses the viral packaging signal.
  • the VLPs encapsidate the viral packaging signal.
  • the VLPs encapsidate a polynucleotide comprising the viral packaging signal.
  • the VLPs encapsidate a polynucleotide consisting of the viral packaging signal.
  • the VLPs encapsidating the viral packaging signal are more immunogenic than control VLPs comprising the antigen but lacking the viral packaging signal. Without being bound by a theory, it is thought that a greater number of VLPs may be formed in the presence of a viral packaging signal, as compared to in the absence of the viral packaging signal.
  • the disclosed vectors encoding a viral packaging signal promote the formation of a greater number of VLPs, as compared to a control vector which does not encode the viral packaging signal.
  • compositions or vectors disclosed herein include, but are not limited to, parenteral administration (e.g ., intradermal, intramuscular, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppositories), subdermal, and intraperitoneal.
  • parenteral administration e.g ., intradermal, intramuscular, intravenous and subcutaneous
  • epidural e.g., epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppositories), subdermal, and intraperitoneal.
  • mucosal e.g., intranasal and oral or pulmonary routes or by suppositories
  • subdermal e.g., intranasal and oral or pulmonary routes or by suppositories
  • intraperitoneal e.g., intranasal and oral or pulmonary routes
  • compositions or vectors may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g, oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • the administration is intradermal administration.
  • the administration is intramuscular administration.
  • the administration is subcutaneous administration.
  • the administration is intranasal administration.
  • the compositions or vectors disclosed herein are administered by injection.
  • the injection is performed using a needle, a syringe, a microneedle, or a needle-less injection device.
  • the compositions or vectors disclosed herein are administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract or small particle aerosol (less than 10 microns) or spray into the lower respiratory tract.
  • the injection is followed by electroporation.
  • a follow-on boost dose is administered within a time period of about 1 hour to about several years (for example, about 12 hours, about 1 day, about 2 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month, about 6 months, about 1 year, about 2 years, including all values and subranges that lie there between) after the prior dose.
  • inclusion of the enhancer protein in a polynucleotide encoding one or more viral antigen proteins increases the duration or the amount of neutralizing antibodies in a subject relative to a vaccine composition without an enhancer protein.
  • inclusion of the enhancer protein increases the duration or the amount of neutralizing antibodies in a subject by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold relative to a vaccine composition without an enhancer protein.
  • inclusion of the enhancer protein increases Thl cellular response relative to a vaccine composition without an enhancer protein. In some embodiments, inclusion of the enhancer protein increases Thl response by about 25%, about 50%, about 100%, about 200%, about 300%, about 400%, about 500%, or about 1000% relative to a vaccine composition without an enhancer protein.
  • kits comprising any one or more of the vectors disclosed herein.
  • the disclosure further provides kits comprising any one or more of the polynucleotides disclosed herein.
  • the disclosure also provides kits comprising any one or more of the vaccine compositions disclosed herein.
  • the kits disclosed herein are useful for performing, or aiding in the performance of, the disclosed methods.
  • the kits comprise a pharmaceutically acceptable carrier.
  • the kits comprise instructions for proper use and safety information of the product or formulation.
  • the kits comprise dosage information based on the application and method of administration as determined by a doctor.
  • the present application also provides articles of manufacture comprising any one of the vaccine compositions or kits described herein. Examples of an article of manufacture include vials ( e.g . sealed sterile vials).
  • kits comprise one or more containers or vials filled with one or more of the ingredients of the vaccine compositions disclosed herein.
  • the kit comprises two containers, one containing the vector, or polynucleotide, or vaccine composition disclosed herein, and the other containing an adjuvant.
  • the kits further comprise a notice reflecting approval by a governmental agency for manufacture, use or sale for human administration.
  • Example 1 Construction of polynucleotides encoding SARS-CoV-2 and L protein
  • CoVEGl and CoVEG2 plasmids encode SARS-CoV-2 and the L enhancer protein.
  • Plasmid CoVEGl comprises polynucleotides encoding viral proteins of full-length S protein (SEQ ID NO: 14), M protein (SEQ ID NO: 19), and E protein (SEQ ID NO: 23) of SARS-CoV-2.
  • Plasmid CoVEG2 comprises polynucleotides encoding viral proteins of full- length S protein, M protein, N protein (SEQ ID NO: 21) and E protein of SARS-CoV-2.
  • the backbone of CoVEGl and CoVEG2 plasmids is shown in FIG. 1.
  • the CoVEGl and CoVEG2 plasmids also comprise a polynucleotide encoding the L protein from EMCV (SEQ ID NO: 16).
  • the nucleic acid sequence of the complete insert in CoVEG2 is represented by SEQ ID NO: 30. See Table 1.
  • the expression of this construct gives rise to three polypeptides: the SARS-CoV-2 Spike protein having amino acid sequence of SEQ ID NO: 13, CoVEG2 polypeptide 1 having amino acid sequence of SEQ ID NO: 25, and CoVEG2 polypeptide 2 having amino acid sequence of SEQ ID NO: 26.
  • the nucleic acid sequence of the insert in CoVEGl is represented by SEQ ID NO: 31.
  • the expression of this construct gives rise to two polypeptides: the SARS-CoV-2 Spike protein having amino acid sequence of SEQ ID NO: 13, and CoVEGl polypeptide having amino acid sequence of SEQ ID NO: 32. See Table 1.
  • the plasmid backbone (based on the design principles of the pVaxl plasmid) and insert for both the plasmids were generated using gene synthesis and do not contain any animal or human source material.
  • the plasmid backbone consists of a Kanamycin resistance gene, the ColEl origin of replication, the Human cytomegalovirus immediate-early promoter and Simian virus (SV40) Poly A signal. Polynucleotides encoding viral proteins were cloned in between the CMV promoter and the SV40 PolyA signal. After gene synthesis and plasmid preparation, the plasmid was transformed into E. coli for cloning and then screened using kanamycin. A representative colony was selected, and its plasmid sequence verified and used as source plasmid for further development. After transcription, the viral proteins were expressed from a single polycistronic mRNA.
  • Example 2 Expression of SARS-CoV-2 S, E, M, and N proteins in eukaryotic cells observed by immunofluorescence
  • HEK 293 eukaryotic cells were transfected with the pCoVEG2 plasmid. Twenty-four hours later, cells were fixed, permeabilized and analyzed by immunocytochemistry using commercial Alexa Fluor 568 fluorescently labelled secondary antibodies for detection.
  • FIG. 2 shows the expression of S, M, N and E proteins in HEK 293 cells, demonstrating that pCoVEG2 disclosed herein expresses the viral antigens in cells.
  • Example 3 Expression of SARS-CoV-2 S, E, M, and N proteins in eukaryotic cells observed by SDS-PAGE and western blot
  • HEK 293 cells were transfected with the pCoVEG2 plasmid and incubated for 96 hours. Thereafter, cell culture supernatant was harvested and concentrated. The concentrate was run over Superose 6 GL resin packed in the Tricorn 10/300 column using PBS as eluant. The void fraction, which contains secreted VLPs, was analyzed by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) and/or western blotting using monoclonal antibodies against S, N, M or E to demonstrate the presence of S, N, M and E proteins. See FIG. 3.
  • SDS-PAGE sodium dodecyl sulfate poly acrylamide gel electrophoresis
  • Anti-SARS-CoV-2 antibody analysis comprising anti-S protein antibody ELISA assay is performed based on commercially available materials. Alternatively, in-house developed cell-based and VLP -based ELISA assays is used. For ELISpot analysis, spleen is collected and T cells are isolated. ELISpot assessment is performed by priming the T cells with Miltenyi Biotec Peptivator SARS-CoV-2 peptide pools to activate SARS-CoV-2 reactive T cells. In addition, the toxicokinetic and pharmacodynamic characteristics of the plasmids are determined. See FIG. 4.
  • mice Female BALB/c mice (6-8 weeks of age) weighing 15 to 25 grams are randomly assigned to 4 groups with each group containing 10 animals. Mice are dosed intradermally with either the vehicle -PBS, a reference item EG-BB, which encodes the enhancer protein(s) under the control a CMV promoter, and two doses of CoVEGl and CoVEG2 at 1 and 25 pg. Mice are evaluated twice daily for mortality and moribundity. Clinical observations and body weights are collected weekly starting Week -1 and thereafter at least every 2 weeks during the study period. [0190] Dosed mice are bled at pre-defmed timepoints before dosing and serum are separated by centrifugation. The obtained serum samples are then analyzed for antibodies against the full length recombinant S protein (S1+S2) using a quantitative ELISA, as shown below in Table 3.
  • S1+S2 full length recombinant S protein
  • the resultant serum is split into 2 approximately equal aliquots; the first aliquot will be used for anti-vaccine antibody (AVA) analysis and the second aliquot kept for testing for neutralizing antibodies.
  • the aliquots are frozen immediately over dry ice or in a freezer set to maintain -80°C.
  • AZA anti-vaccine antibody
  • Spleens are collected using cell culture clean procedures for IFN-g and IL-4 evaluation by ELISpot.
  • Example 5 Immunogenicity of polynucleotide vaccine in humans
  • Immune memory indications are assessed by immune phenotyping PBMCs by flow cytometry and validated ELISpot assay focusing on CD49b+T-bet+ resting memory Th cell precursors and CXCR4+ S1P1+ memory plasma cell precursors cells on Day 15, Day 29, Day 57, Day 180, Day 270 and/or Day 394.
  • the measurements include: Geometric mean fold rise (GMFR) in IgG, IgM and/or IgA titer from baseline (Day 1 to Day 394); Geometric mean titer (GMT) of antibody (Day 15, Day 29, Day 57, Day 180, Day 270 and Day 394); and Percentage of subjects who seroconverted (Day 1 to Days 15, 29, 57, 180, 270 and 394). Seroconversion is defined as 4-fold change in antibody titer from baseline; and (c) IFN-g response as a measure of CD8 T cell response and phenotype of memory immune cells will be measured in PBMC isolated from subjects by a validated ELISpot assays. PBMC will be isolated on Days 15, 29, 57, 180, 270 and 394.
  • Plasmids CoVEG 3-17 comprise expression cassettes encoding different viral proteins in the order indicated in FIG. 6.
  • the plasmid backbone (based on the design principles of the pVaxl plasmid) and insert for the plasmids were generated using gene synthesis.
  • the plasmid backbone consists of a Kanamycin resistance gene, the ColEl origin of replication, the Human cytomegalovirus immediate-early promoter and Simian virus (SV40) Poly A signal. Polynucleotides encoding viral proteins were cloned in between the CMV promoter and the SV40 PolyA signal. After gene synthesis and plasmid preparation, the plasmid was transformed into E.
  • coli for cloning and then screened using kanamycin. A representative colony was selected, and its plasmid sequence verified and used as source plasmid for further development. After transcription, the viral proteins were expressed from a single polycistronic mRNA.
  • Stain with an anti-spike (S) protein antibody that binds to the receptor binding domain (RBD) - also referred to herein as “anti-RBD” - was added at a dilution of 1:500 and incubated for 1 hour at room temperature. The stain was removed, cells were washed and secondary antibody (Alexa Fluor 568 fluorescently labelled secondary anti- Rabbit, 1:1000 dilution) was added. The stain was incubated for 1 hour at room temperature before removal of the stain and washing. Cells were imaged using a EVOS cell imaging system.
  • FIG. 7 shows the expression of the S protein in HEK293 cells, demonstrating that all tested CoVEG plasmids were capable of expressing the spike protein.
  • HEK293T cells were seeded at 40,000 cells / well in a 24 well plate 24 hours prior to transfection.
  • Cells were transfected with the pCoVEG 5, 9-12, and 14-20 plasmids using PEI complexes following the manufacturers description.
  • the media was changed 12 hours after transfection and cells were incubated at 37°C, 5% CO2 for 48 hours.
  • the cell media was removed, and cells were fixed with 10% neutral buffered formalin for 10 minutes following permeabilization with 0.2% Triton X-100 in PBS for 10 minutes.
  • FIG. 17 shows the expression of the N protein in HEK293 cells, demonstrating that all tested CoVEG plasmids were capable of expressing the nucleocapsid protein.
  • Example 8 L protein required for detectable SARS-CoV-2 VLP formation
  • VLPs virus-like particles
  • 4 x 10 6 HEK293 cells were transfected with the pCoVEG 3-20 plasmids in a 150 mm dish using PEI complexes following manufacturers description. The media was changed 12 hours after transfection and cells were incubated at 37 °C, 5% CO2 for 72 hours.
  • VLP containing supernatants were harvested, spun down (1,500 x g, 15 min) and concentrated using an Amicon centrifugal filter unit (100 kDa cut off). Concentrate was spun down (4,500 x g, 15 minutes) to remove precipitate and VLPs were pelleted (100,000 x g, 1.5 hours) through a 20 % sucrose cushion.
  • FIG. 8 shows that CoVEG 3-8 plasmids were capable of expressing the S protein and CoVEG 3 and 5-8 plasmids were capable of expressing the N protein.
  • FIG. 12 shows the expression of S protein and N protein from CoVEG 5, 12, 14, 13, 10, 9 and 11 plasmids.
  • FIG 18 shows the expression of S protein and N protein from CoVEG 5, 8, 9, 10, 15, 16, 17 and 20 plasmids.
  • the presence of the enhancer L protein resulted in different S and N protein expression ratios, e.g. as shown by the CoVEG 10 (L protein) versus the CoVEG 20 (no L protein) S and N western blotting in FIG. 18.
  • anti-RBD Session Biological, mouse anti-RBD SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse Mab, 40592-MM57 SARS- CoV-2, 1:500 dilution in EZ Block
  • anti-N Novus Biologicals, Mouse anti-SARS-CoV-2 Nucleocapsid, Clone: B3449M, N2787B09, 1:1000 dilution in EZ Block
  • Antibodies were incubated for 1 hour at room temperature before washing three times with 0.05% Tween-20 in PBS and adding 75 m ⁇ of secondary antibody (Goat-Anti-mouse, HRP-conjugate, 1:2,000 dilution, Southern Biotech, Goat anti-Mouse IgG(FFHL), horseradish peroxidase (HRP) , Polyclonal, OB 103405) and incubating for 1 hour at room temperature.
  • Wells were thoroughly washed (5x with 0.05% Tween-20 in PBS), and binding was developed using 75 m ⁇ 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Surmodisc Inc TMB One Component HRP Microwell Substrate). The reaction was carried out for 30 minutes with 75 m ⁇ Stop Solution (Surmodisc Inc 450 NM LIQ STOP REAGENT) and Absorbance was measured at 450 nm.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • FIG 15 shows ELISA results from VLP secretion of CoVEG 5 and 9-14 plasmids compared with single protein S and N expressing vectors. Both Spike and Nucleocapsid proteins secreted from HEK293 cells. However, while the S protein demonstrated high ELISA VLP signal relative to single protein expression, the N protein demonstrated a notably lower VLP signal relative to single protein expression. It may be that N signal in VLPs is lower than the S signal in VLPs because the N protein is on the interior of the VLP and not accessible to the antibody.
  • VLP containing supernatants were harvested, spun down (1,500 x g, 15 minutes) and concentrated using an Amicon centrifugal filter unit (100 kDa cut off).
  • Resin was incubated for 120 minutes before eluting with 0.1M glycine pH 2. Eluates were immediately neutralized by adding 5 times volume of 1M Tris pH 8.0. Fractions were analyzed for the presence of N protein by western blot using anti-N antibody (Novus Biologicals, Mouse anti-SARS-CoV-2 Nucleocapsid, Clone: B3449M, N2787B09).
  • FIG. 19 shows the western blot result of the co-IP experiments of the CoVEG 10 plasmid.
  • the N-protein is packed in intact VLPs as demonstrated by the presence of an N- signal in the elution fraction after incubation with RBD (left side, arrow). This signal, compared with the absence of signal in the washing fraction, indicates the N protein is retained within the particles.
  • the co-IP was run in parallel without the anti-RBD antibody (right side). The N protein signal was not detectable in the elution fraction, demonstrating that the N protein did not bind the resin non-specifically.
  • Serum was collected from the mice after 56 days and added to the wells (1:500 dilution for binding antibody detection, 1:100 - 1:7812500 for Endpoint Titer measurement). Serum was incubated for 1 hour at room temperature before washing thrice with 0.05% Tween-20 in PBS and adding 75 m ⁇ of secondary antibody (Goat-Anti-rabbit, HRP-conjugate, 1:4,000 dilution) and incubating for 1 hour at room temperature.
  • secondary antibody Goat-Anti-rabbit, HRP-conjugate, 1:4,000 dilution
  • FIG. 9A shows the total binding antibody measured using ELISA
  • FIG. 9B shows the measured endpoint titers.
  • FIG. 13 shows ELISA results from injection of CoVEG 5, 9, 11, 10, 12, 13, 8 and 14 plasmids, either intramuscularly (IM) or intradermally (ID).
  • IM intramuscularly
  • ID intradermally
  • the intramuscular injection of CoVEGlO induced a much higher immune response as compared to the injection of the Spike protein alone.
  • Intramuscular injections of CoVEG5, CoVEG9, and CoVEGl 1 also induced a higher immune response as compared to the injection of the S protein alone.
  • FIGS. 20 and 22 show antibody binding titers from CoVEG 5 and 9-14, 18, 19, and 20 plasmids, as well as S only, on day 42 after IM or ID injection. Interestingly, all of the tested CoVEGs were capable of inducing an immune response, with CoVEG 9 the most efficacious. Notably, the VLP forming CoVEG 9 performed better than the spike protein alone.
  • the cellular immune response was measured using the presence of antigen reactive T cells using IFN-g and IL-4 enzyme-linked immune absorbent Spot (ELISpot) assays.
  • ELISpot enzyme-linked immune absorbent Spot
  • spleen was collected and T cells were isolated. ELISpot assessment was performed by priming the T cells with Miltenyi Biotec Peptivator SARS-CoV-2 peptide pools to activate SARS-CoV-2 reactive T cells.
  • Mouse IL-4 Single color ELISPOT and Mouse INF-g Single color ELISPOT (Immunospot, Cellular Technology Limited) were used according to manufacturer’s instructions.
  • FIG 21 shows the neutralizing antibodies of the samples shown in FIG. 20. These data indicate that not all generated antibodies possess neutralizing capacity. A lower signal in this assay confirmed neutralization as defined by a signal less than 70% of the negative control (dashed line). Signals lower than 30% of the negative control were confirmed to be strongly neutralizing.
  • the tested CoVEGs performed differently depending on the design and the injection site. Surprisingly, ID injection did not induce strong neutralizing antibodies, whereas IM injections did induce strong neutralizing antibodies. Additionally, CoVEG13 and 14 did not meet the criteria for neutralization.
  • VLP containing supernatants were harvested, spun down (1,500 x g, 15 minutes) and concentrated using an Amicon Ultra centrifugal filter unit (100 kDa cut off). Concentrate was spun down (4,500 x g, 15 minutes) to remove precipitate and VLPs were pelleted through a 20% sucrose cushion at 100,000 x g for 1.5 hours. VLPs were resuspended in PBS and analyzed by ELISA.
  • Anti-West Nile Virus Antibody clone E16
  • EZ block (1 : 5,000)
  • Wells were washed and goat anti-mouse HRP labeled detection antibody (Southern Biotech) was added for detection, followed by washes and the incubation with TMB substrate and the stop solution.
  • Signal was read out as absorbance at 450 nm using a plate reader.
  • Isolated RVPs are incubated with mouse serum samples at different serial dilutions and added to pre-plated PHK-21 cells and incubated for 2 days, after which the reporter gene activity is measured using a microplate reader. The reduction in the reporter gene activity reflects the level of WNV neutralizing antibodies in mouse sera.
  • Example 13 Expression and immunogenicity of additional polynucleotide constructs
  • plasmids encoding viral proteins derived from other viruses e.g. , Influenza viral proteins (e.g, HA, NA, Ml, M2, or any combination thereof), Hepatitis B viral proteins (e.g., sAg (S protein), sAg (M protein), sAg (L protein), preSl, preS2, cAg (core antigen), or any combination thereof), Human Papillomavirus (e.g, LI protein of HPV 6, LI protein of HPV 11, LI protein of HPV 16, LI protein of HPV 18, or any combination thereof) is performed using the methods described in Example 6. Expression of these proteins in different combinations in HEK293T cells and isolation of the VLPs is performed using methods described in Examples 7, 8, 10 and 11. Finally, the immunogenicity of the plasmids encoding these proteins is tested using the methods described in Example 9 and 12.
  • Influenza viral proteins e.g, HA, NA, Ml, M2, or any combination thereof
  • Hepatitis B viral proteins e
  • Embodiment 2 The vector of embodiment 1, wherein the amino acid sequence of the enhancer protein has at least 95% identity to SEQ ID NO: 1, or at least 95% identity to SEQ ID NO: 2.
  • Embodiment 3 The vector of embodiment 1 or embodiment 2, wherein the amino acid sequence of the enhancer protein is SEQ ID NO: 1, or SEQ ID NO: 2.
  • Embodiment 4 The vector of any one of embodiments 1-3, wherein the polynucleotide encoding the enhancer protein is operatively linked to a polynucleotide encoding an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • Embodiment 5 The vector of embodiment 4, wherein the polynucleotide encoding the IRES is SEQ ID NO: 24.
  • Embodiment 6 The vector of any one of embodiments 1-5, wherein the viral protein is a viral antigen.
  • Embodiment 6.2 The vector of embodiment 6.1, wherein the viral protein is derived from a coronavirus.
  • Embodiment 7 The vector of any one of embodiments 1-6.2, wherein the coronavirus is a betacoronavirus.
  • Embodiment 8 The vector of embodiment 7, wherein the betacoronavirus is severe acute respiratory syndrome (SAR.S) virus.
  • SAR.S severe acute respiratory syndrome
  • Embodiment 9 The vector of embodiment 8, wherein the SAR.S virus is a SARS-CoV- 2 virus.
  • Embodiment 10 The vector of embodiment 7, wherein the betacoronavirus is Middle East respiratory syndrome (MERS) virus.
  • MERS Middle East respiratory syndrome
  • Embodiment 11 The vector of any one of embodiments 1-10, wherein the coronavirus protein is a coronavirus spike protein.
  • Embodiment 12 The vector of embodiment 11, wherein the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 13.
  • Embodiment 13 The vector of embodiment 12, wherein the spike protein is SEQ ID NO: 13.
  • Embodiment 13.1 The vector of embodiment 11, wherein the spike protein is a mutant spike protein.
  • Embodiment 13.2 The vector of embodiment 13.1, wherein the mutant spike protein comprises the amino acid substitutions, R682G, R683S, R685S, K986P, and V987P, in SEQ ID NO: 13.
  • Embodiment 13.3 The vector of embodiment 13.1, wherein the mutant spike protein comprises an amino acid sequence of SEQ ID NO: 51.
  • Embodiment 14 The vector of any one of embodiments 1-13.3, wherein the coronavirus protein is a coronavirus membrane (M) protein.
  • M coronavirus membrane
  • Embodiment 15 The vector of embodiment 14, wherein the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 33.
  • Embodiment 16 The vector of embodiment 14 or embodiment 15, wherein the M protein is SEQ ID NO: 33.
  • Embodiment 17 The vector of any one of embodiments 1-16, wherein the coronavirus protein is a coronavirus envelope (E) protein.
  • E coronavirus envelope
  • Embodiment 18 The vector of embodiment 17, wherein the E protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 22.
  • Embodiment 19 The vector of embodiment 17 or embodiment 18, wherein the E protein is SEQ ID NO: 22.
  • Embodiment 20 The vector of any one of embodiments 1-19, wherein the coronavirus protein is a coronavirus nucleocapsid (N) protein.
  • the coronavirus protein is a coronavirus nucleocapsid (N) protein.
  • Embodiment 21 The vector of embodiment 20, wherein the N protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 20.
  • Embodiment 22 The vector of embodiment 20 or embodiment 21, wherein the N protein is SEQ ID NO: 20.
  • Embodiment 23 The vector of any one of embodiments 1-22, wherein the coronavirus protein forms a virus-like particle (VLP).
  • VLP virus-like particle
  • Embodiment 23.1 The vector of embodiment 6.1, wherein the viral protein is derived from West Nile virus.
  • Embodiment 23.2 The vector of embodiment 23.1, wherein the viral protein is precursor membrane protein (preM), envelope glycoprotein (E), or a combination thereof.
  • preM precursor membrane protein
  • E envelope glycoprotein
  • Embodiment 24 A vector for use as a vaccine, comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 30.
  • Embodiment 25 The vector of embodiment 24, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 30.
  • Embodiment 26 A vector for use as a vaccine, comprising an expression cassette comprising a polynucleotide, wherein the polynucleotide comprises a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 31.
  • Embodiment 27 The vector of embodiment 26, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 31.
  • Embodiment 27.1 A vector for use as a vaccine, comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35-49, and 55.
  • Embodiment 28 The vector of any one of embodiments 1-27.1, wherein the vector is a naked polynucleotide.
  • Embodiment 29 The vector of any one of embodiments 1-28, wherein the vector is a deoxyribonucleic acid (DNA) polynucleotide.
  • DNA deoxyribonucleic acid
  • Embodiment 30 The vector of any one of embodiments 1-28, wherein the vector is a ribonucleic acid (RNA) polynucleotide.
  • RNA ribonucleic acid
  • Embodiment 31 The vector of any one of embodiments 1-30, wherein the vector comprises a plasmid.
  • Embodiment 32 The vector of any one of embodiments 1-30, wherein the vector comprises linear DNA.
  • Embodiment 33 The vector of any one of embodiments 1-32, wherein the expression cassette comprises a promoter operatively linked to each of the polynucleotide sequences of the expression cassette.
  • Embodiment 33.3 The vector of embodiment 33.2, wherein the viral packaging signal is derived from a coronavirus.
  • Embodiment 35 The vaccine composition of embodiment 34, wherein the vaccine composition comprises an adjuvant.
  • Embodiment 36 The vaccine composition of embodiment 35, wherein the adjuvant is alum.
  • Embodiment 37 The vaccine composition of embodiment 35, wherein the adjuvant is monophosphoryl lipid A (MPL).
  • MPL monophosphoryl lipid A
  • Embodiment 38 A method of expressing a viral antigen in a eukaryotic cell, comprising contacting the cell with the vector of any one of embodiments 1 to 33.4.
  • Embodiment 39 The method of embodiment 38, wherein contacting the cell with the vector results in: (i) expression of the antigen at a higher expression level; and/or (ii) expression of the antigen for a longer period of time; and/or (iii) expression of the antigen with better protein quality, than a vector lacking the enhancer protein.
  • Embodiment 40.1 The method of embodiment 40, wherein the vector comprises a DNA polynucleotide encoding a viral packaging signal, wherein contacting the cell with the vector results in expression of the viral packaging signal, and wherein the VLPs encapsidate the viral packaging signal.
  • Embodiment 40.2 The method of embodiment 40.1, wherein the vector results in the formation of a greater number of VLPs, as compared to a control vector lacking the DNA polynucleotide encoding the viral packaging signal.
  • Embodiment 41 A method of eliciting an immune response in a subject, comprising administering an effective amount of the vaccine composition of any one of embodiments 34 to 37 to the subject.
  • Embodiment 42 The method of embodiment 41, wherein tissue at an administration site of the subject expresses the antigen and/or a VLP comprising the antigen.
  • Embodiment 43 The method of embodiment 42, wherein tissue at an administration site of the subject: (i) expresses the antigen and/or a VLP comprising the antigen at a higher expression level; and/or (ii) expresses the antigen and/or a VLP comprising the antigen for a longer period of time; and/or (iii) expresses the antigen and/or a VLP comprising the antigen with better protein quality, than when a vector lacking the enhancer protein is administered.
  • Embodiment 43.1 The method of any one of embodiments 41-43, wherein the vector comprises a DNA polynucleotide encoding a viral packaging signal, wherein tissue at an administration site of the subject expresses the viral packaging signal, and wherein the VLPs encapsidate the viral packaging signal.
  • Embodiment 43.3 The method of embodiment 43-43.2, wherein the VLPs encapsidating the viral packaging signal are more immunogenic than control VLPs comprising the antigen but lacking the viral packaging signal.
  • Embodiment 44 The method of any one of embodiments 41 to 43, wherein the method elicits an antibody response in the subject.
  • Embodiment 45 The method of embodiment 44, wherein the antibody response is a neutralizing antibody response.
  • Embodiment 46 The method of any one of embodiments 41 to 43, wherein the method elicits a cellular immune response.
  • Embodiment 47 The method of any one of embodiments 41 to 46, wherein the method elicits a prophylactic, protective and/or therapeutic immune response in the subject.
  • Embodiment 48 The method of any one of embodiments 41 to 47, wherein the administration is intradermal administration, intramuscular administration, subcutaneous administration, or intranasal administration.
  • Embodiment 49 A polynucleotide comprising an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picomavirus leader (L) protein or a functional variant thereof.
  • L picomavirus leader
  • Embodiment 51 The polynucleotide of embodiment 49 or embodiment 50, wherein the amino acid sequence of the enhancer protein is SEQ ID NO: 1, or SEQ ID NO: 2.
  • Embodiment 52 The polynucleotide of any one of embodiments 49-51, wherein the polynucleotide encoding the enhancer protein is operatively linked to a polynucleotide encoding an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • Embodiment 53 The polynucleotide of embodiment 52, wherein the polynucleotide encoding the IRES is SEQ ID NO: 24.
  • Embodiment 54 The polynucleotide of any one of embodiments 49-53, wherein the coronavirus protein is a coronavirus antigen.
  • Embodiment 55 The polynucleotide of any one of embodiments 49-54, wherein the coronavirus is a betacoronavirus.
  • Embodiment 56 The polynucleotide of embodiment 55, wherein the betacoronavirus is severe acute respiratory syndrome (SAR.S) virus.
  • SAR.S severe acute respiratory syndrome
  • Embodiment 57 The polynucleotide of embodiment 56, wherein the SAR.S virus is a SARS-CoV-2 virus.
  • Embodiment 58 The polynucleotide of embodiment 55, wherein the betacoronavirus is Middle East respiratory syndrome (MERS) virus.
  • MERS Middle East respiratory syndrome
  • Embodiment 59 The polynucleotide of any one of embodiments 49-58, wherein the coronavirus protein is a coronavirus spike protein.
  • Embodiment 60 The polynucleotide of embodiment 59, wherein the spike protein shares at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 13.
  • Embodiment 61 The polynucleotide of embodiment 59 or embodiment 60, wherein the spike protein is SEQ ID NO: 13.
  • Embodiment 62 The polynucleotide of any one of embodiments 49-61, wherein the coronavirus protein is a coronavirus membrane (M) protein.
  • Embodiment 63 The polynucleotide of embodiment 62, wherein the M protein shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to SEQ ID NO: 33.
  • Embodiment 64 The polynucleotide of embodiment 62 or embodiment 63, wherein the M protein is SEQ ID NO: 33.
  • Embodiment 65 The polynucleotide of any one of embodiments 49-64, wherein the coronavirus protein is a coronavirus envelope (E) protein.
  • E coronavirus envelope
  • Embodiment 67 The polynucleotide of embodiment 65 or embodiment 66, wherein the E protein is SEQ ID NO: 22.
  • Embodiment 68 The polynucleotide of any one of embodiments 49-67, wherein the coronavirus protein is a coronavirus nucleocapsid (N) protein.
  • Embodiment 72 A polynucleotide comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 30.
  • Embodiment 73 The polynucleotide of embodiment 72, wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 30.
  • Embodiment 77 The polynucleotide of any one of embodiments 49-76, wherein the polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide.
  • DNA deoxyribonucleic acid
  • Embodiment 80 A kit comprising a vector, wherein the vector comprises an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picomavirus leader (L) protein or a functional variant thereof.
  • the vector comprises an expression cassette comprising a polynucleotide encoding a coronavirus protein and a polynucleotide encoding an enhancer protein, wherein the enhancer protein is a picomavirus leader (L) protein or a functional variant thereof.
  • L picomavirus leader
  • Embodiment 82 The kit of embodiment 80 or embodiment 81, wherein the polynucleotide encoding the enhancer protein is operatively linked to a polynucleotide encoding an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • Embodiment 85 The kit of any one of embodiments 80-84, wherein the coronavirus is a betacoronavirus.
  • Embodiment 98 The kit of any one of embodiments 80-97, wherein the coronavirus protein is a coronavirus nucleocapsid (N) protein.
  • the coronavirus protein is a coronavirus nucleocapsid (N) protein.
  • Embodiment 101 The kit of embodiment 80, wherein the expression cassette comprises a polynucleotide, comprising a nucleic acid sequence having at least 70% identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identity to the nucleic acid sequence of SEQ ID NO: 30.
  • Embodiment 108 The vector of any one of embodiments 104-107, wherein the nucleic acid sequence encoding the viral packaging element has at least about 70% identity to the nucleic acid sequence of SEQ ID NO: 34.
  • Embodiment 110 The method of embodiment 109, wherein contacting the cell with the vector results in the formation of virus-like particles (VLPs) comprising the target protein.
  • Embodiment 111 The method of embodiment 110, wherein contacting the cell with the vector results in the formation of a greater number of virus-like particles (VLPs) comprising the target protein, as compared to a control vector comprising the expression cassette but lacking the nucleic acid sequence encoding the viral packaging element.
  • Embodiment 114 A vector for use as a vaccine, comprising an expression cassette, comprising the following elements in the 5' to 3' order: a promoter, a polynucleotide encoding SEQ ID NO: 33 (M protein), a polynucleotide encoding a first proteolytic cleavage site, a polynucleotide encoding SEQ ID NO: 13 (S protein), a polynucleotide encoding a second proteolytic cleavage site, a polynucleotide encoding SEQ ID NO: 22 (E protein), polynucleotide encoding SEQ ID NO: 24 (IRES), a polynucleotide encoding SEQ ID NO: 2 (enhancer L protein), a polynucleotide encoding SEQ ID NO: 20 (N protein), and a polynucleotide encoding SEQ ID NO: 34 (viral packaging signal).
  • M protein a polyn

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CA3212387A CA3212387A1 (en) 2021-03-17 2022-03-17 Vaccine compositions and methods of use thereof
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