Particle delivery for self-amplifying vaccines

Abstract

A cassette for the delivery of one or more mRNA is provided, the cassette comprising a 5' UTR, a Nodamura replicon, a ribosomal skipping polynucleotide, the one or more mRNA, a plant virus assembly origin polynucleotide ("OAS"), and a 3' UTR. In one aspect, the cassette further comprises a 5'mRNA cap. In a yet further aspect, the cassette does not comprise a poly A tail.

Description

PARTICLE DELIVERY FOR SELF-AMPLIFYING VACCINESCROSS-REFERENCE TO RELATED PATENT APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 660,427, filed on June 14, 2024, the contents of which are incorporated herein by reference in their entireties.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Nos. R21 AI161306, R01 CA224605 and R01 CA253615, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND100031 Traditional vaccines are based on the direct introduction of antigens that stimulate an immune response, whereas mRNA vaccines package a translatable RNA molecule encoding the antigen, which is expressed once the mRNA has been taken up by cells. Although mRNA vaccines have a history stretching back more than 40 years, they have recently gained more attention due to their widespread use during the COVID-19 pandemic. However, they have been developed for a range of indications beyond infectious diseases, including immunotherapy, genetic disorders, regenerative medicine, and cancer. The main benefits of mRNA vaccines include the universal design principles, scalability for mass production, safe translation without the risk of genomic integration, and the ability to generate a robust immune response due to the capacity of single mRNA molecules to produce many protein antigens. Conversely, the reactogenicity of mRNA vaccines is no different to that of conventional, protein-based counterparts. One key challenge with the delivery of mRNA vaccines is that naked mRNA degrades rapidly in vivo. This disclosure addresses this technical problem and provides related advantages and applications as well.SUMMARY OF THE DISCLOSURE

[0004] This disclosure provides compositions and methods for mRNA delivery that have therapeutic application in mRNA vaccines for the treatment of diseases and underlying research applications.

[0005] For example, the Covidl9 pandemic highlights the power of genetic medicine.However, there are shortcomings with current approaches and the use of lipid nanoparticles, e.g. supply chain shortcomings (chemical synthesis), instability of the formulation (needing ultralow freezers), etc.|0006| Applicant’s disclosure overcame these shortcomings and technical problems by packaging the mRNA coding for the gene of interest in virus-like particles (VLP) from plant viruses. These VLPs - or nanoparticles - are inherently stable and can be produced using less complicated methods so supply chain shortcomings are less likely an issue. In addition, formulated VLP mRNA vaccine is stable at room temperature. These improvements allow the use of Applicant’s vaccines in in developed and underdeveloped countries.

[0007] Herein, Applicant describes vectors and expression cassettes comprising an mRNA, and plant virus like particles containing them as well as methods of use of the vectors, cassettes and VLPs. Also described herein are compositions and kits comprising the vectors.

[0008] In one aspect, the plant virus like particle is selected from tobacco mosaic virus, tobacco mild green mosaic virus, and potato virus X.[00091 In one aspect, the mRNA vaccine is selected from an Omicron (SARS-CoV-2) vaccine or a Human papilloma virus vaccine.

[0010] Applicant also demonstrates efficacy of the mRNA vaccine against two targets: RBD from SARS-CoV-2; andE7 from HPV. These vaccines can be used to protect against infection, and the E7 vaccine also can be used for cancer therapy to treat or prevent HPV+ cancers.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1A - IB: Characterization of TMV.Nod.HPVE7.OAS VLPs (FIG. 1A)Transmission electron microscopy images of negatively-stained VLPs and size exclusion elution profiles of VLPs (black and red lines indicate 260 and 280 nm, respectively). Top panel = native TMV; middle panel = coat proteins; bottom panel = reassembled TMV.Nod.HPVE7.OAS VLPs. (FIG. IB) Encapsulation of Nod.HPVE7.OAS mRNA into TMV confirmed by isolating RNA from assembled VLPs for RT-PCR analysis. The arrowindicates the HPV E7 amplicon (anticipated size -300 bp). The lower-molecular-weight band is likely to be a primer dimer.

[0012] FIGS. 2A - 2C: Expression of E7 mRNA and Protein using the Nod.HPVE7.OAS Construct Delivered Directly as mRNA or Packaged in VLPs. (FIG.2A) Transfection of BHK-21 cells with Nod.HPVE7.OAS (lane 1), HPV E7 mRNA (lane 2) (500 ng / well), or exposure to TMV.Nod.HPVE7.OAS mRNA encapsulated in VLPs (lanes 3-5, 10, 20, and 40 pg VLPs containing -500 ng, -1 pg, and 2 pg of Nod.HPVE7.OAS mRNA, respectively). Total RNA was isolated from cell lysates after 24 h and was analyzed by RT-PCR. The anticipated size of the E7 amplicon was -300 bp, as confirmed by 1% (w / v) agarose gel electrophoresis. (FIG. 2B) Analysis of transcript levels by real-time quantitative PCR and multifold change relative to HPV E7 mRNA based on RNA collected from BHK-21 cells transfected with Nod.HPVE7.OAS or HPV E7 mRNA (500 ng / well). HPV E7 expression was normalized to the reference gene GAPDH. Data are means ± standard errors (n = 3). The statistical significance of multifold and relative multifold changes was determined by two-way ANOVA using Tukey’s multiple comparisons test compared to control cells and the presence of the replicon. (FIG. 2C) Immunofluorescence imaging of BHK-21 cells (blue channel showing the nucleus staining and cell membrane staining by WGA) transfected with Nod.HPVE7.OAS mRNA to confirm the presence of E7 protein (anti -HPV 16 E7 conjugated to Alexa Fluor 647). Scale bar = 10 pm.

[0013] FIGS. 3A - 3C: Immunogenicity of the TMV.Nod.HPVE7.OAS mRNA Vaccine. (FIG. 3A) BALB / c mice were immunized subcutaneously following a prime (PD, Prime dose)-boost (BD, Booster dose) schedule with 100 pg VLPs on days 1 and 14. Antisera were collected by retro-orbital bleeding on days 0 (PI, Pre-immune sera), 14 (B-I, Bleed-I), and 28 (B-II, Bleed-II) (FIG. 3B) E7-specific IgG antibody titers antibodies analyzed by ELISA. Data are means ± standard errors (n = 5 per group). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (***p < 0.0001). (FIG. 3C) The isotype profile (IgGl, IgG2a, IgG2b and IgM) suggested a Th2 -biased immune response to the VLP-based mRNA vaccine candidate. Data are means ± standard errors (n = 5 per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test.

[0014] FIGS. 4A - 4B: Splenocyte Proliferation Response to HPV E7. (FIG. 4A)Proliferation of splenocytes isolated from TMV.Nod.HPVE7.OAS or PBS-immunized mice and stimulated with HP VI 6 E7 protein (20 pg / mL) or PBS (control) for 24 and 48 h. Splenocyte proliferation was measured using an XTT cell proliferation assay. The poststimulation culture supernatant collected at different time points was tested for the presence of IFN-y and IL-4 using cytokine-specific ELISA kits. Data are means ± standard errors (n = 3). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. (FIG. 4B) ELISpot responses in splenocytes isolated from nice vaccinated with TMV.Nod.HPVE7.OAS or PBS and stimulated (S) with 20 pg / mL HPV E7, 10 pg / mL native TMV, or 50 ng / mL phorbol 12-myristate 13-acetate + 1 pg / mL ionomycin (PMA / IO) as a positive control, along with unstimulated (US) or PBS-stimulated negative controls. Spot-forming units per 5x 106 cells (SFU) were quantified for cytokines IL-4 and IFN-y. Data are means ± standard errors (n = 3 HPV E7 immunized group, n = 1 control group). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test.[0015[ FIGS. 5A - 5D: Characterization of TMV.Nov.OmicronRBD.OAS VLPs. (FIG. 5A) Schematic representation of Nod.OmicronRBD.OAS mRNA: 5’UTR (21 nts), Nodamura replicon (3129 nts), T2A with linker (96 nts), OmicronRBD (675 nts), TMV OAS (234 nts from 5313 to 5546 in the TMV genome), 3’UTR (218 nts). (FIG. 5B) Transmission electron microscopy (TEM) imaging of negatively stained native TMV, TMV CPs and reassembled TMV.Nod. OmicronRBD. OAS VLPs (FIG. 5C) Encapsulation of Nod.OmicronRBD.OAS mRNA into TMV was confirmed by RT-PCR analysis from the RNA isolated from assembled TMV VLPs. The arrow indicates the OmicronRBD specific primer binding site in panel A, with the expected fragment size at 680 bp. (FIG. 5D) Size exclusion elution profiles of native TMV, TMV coat proteins (CP), and assembled TMV.Nod. OmicronRBD. OAS VLPs; dotted and lines indicate detectors set at 260 nm and 280 nm, respectively.

[0016] FIG. 6: Particle Sizes of TMV.Nov.OmicronRBD.OAS VLPs After 1-Week at Various Temperatures. VLP sizes were derived from TEM images using the software Imaged and evaluated by analysis of variance (ANOVA) using the software OriginPro with a post hoc Bonferroni test38and a significance level of a = 0.05. Lowercase letters indicatesignificance groups; conditions that share the same letter were not significant different (p > 0.05). IQR - inter quartile range.

[0017] FIGS. 7A-7C: Expression Analysis of Nod.OmicronRBD.OAS. (FIG. 7A) Transfection of mRNA (500 ng / well) Nod.OmicronRBD.OAS (lane 1) and OmicronRBD (lane 2), or mRNA encapsulated TMV.Nod. OmicronRBD. OAS VLPs (lane 3; 10 pg TMV VLP with -500 ng Nod.OmicronRBD.OAS mRNA) in BHK-21 cells was confirmed by RT- PCR. Total RNA was isolated from cell lysates after 24 h of transfection. The amplicons of RBD were observed at the expected size -680 bp on 1% (w / v) agarose gel. (FIG. 7B) Quantitative analysis of mRNA transcripts by real-time quantitative PCR and multifold change levels of OmicronRBD mRNA were analyzed from the cell lysate collected posttransfection of Nod.OmicronRBD.OAS and OmicronRBD (500 ng / well) at 24 h. OmicronRBD was normalized to the reference gene GAPDH. Also plotted (right panel) is the relative multifold change of Nod. OmicronRBD with respect to the OmicronRBD.Significance level at 24 h was determined by two-way ANOVA using Tukey’s multiple comparison; **** p < 0.0001 calculated with respect to the control cells and **p < 0.005 was calculated with respect to the presence of replicon. (FIG. 7C) Immunofluorescence imaging of Nod.OmicronRBD.OAS mRNA transfection efficiency in BHK-21 cells using an Omicron-specific anti-SARS-CoV-2 Spike RBD antibody. The scale bar is 10 pm.|0018| FIGS. 8A - 8F: Immunogenicity of TMV.Nod.OmicronRBD.OAS mRNAVaccine. (FIG. 8A) Immunization and blood collection schedule scheme using BALB / c mice (n=5). Scheme made in Biorender. (FIG. 8B) Specific anti -OmicronRBD antibodies were elicited post s.c. immunization following a prime-boost schedule39'41. Mice received two doses (2 weeks apart) of 100 pg assembled TMV VLPs-based mRNA vaccine and antibody titers against OmicronRBD was analyzed by ELISA, 14- and 28-days post-immunization. (FIG. 8C) Endpoint titers showed a -1.5-fold increase in antibodies eliciting post-second immunization. Two-way ANOVA was used to compare between groups using pairwise multiple comparison followed by Tukey’s multiple comparison test. (FIG. 8D) IgG subtypes profile (IgGl, IgG2a, or IgG2b) was screened and (FIG. 8E) suggested a Th2 -biased immune response for Applicant’s mRNA vaccine candidate. One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between groups. (FIG. 8F) Inhibition percentage (%) above cut-off values indicate the presence of neutralizing anti-OmicronRBDantibodies. Std 1-6 is the standard curve - 2-fold serial dilution of a monoclonal control antibody (300 U / mL-4.688 U / mL). One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between groups. Asterisks< 0.0001, ***p < 0.0005, **p < 0.001, *p < 0.05) indicate significant differences between groups.[00.19] FIGS. 9A-9C: Schematic Representation of Plasmid Construct pNod.OmicronRBD.OAS. (FIG. 9A). Subcloning strategy for plasmid pNod.OmicronRBD.OAS and pOmicronRBD (FIG. 9B). Cloning of OmicronRBD gene with replicon and without replicon into the respective vector backbone- pNodLucOAS and pET22b was confirmed by double restriction digestion with Ndel and Age! on 1% (w / v) agarose gel in 1 *TAE (Tris-acetate-EDTA) running buffer. Arrow indicates the release of insert at the expected size -675 bp. (FIG. 9C) is a schematic of an HPV E7 expression construct.

[0020] FIG. 10: In vitro Transcribed Nod.OmicronRBD.OAS and OmicronRBD mRNA.Analysis of linearized plasmid template on 1 % (w / v) agarose gel in 1 *TAE running buffer for in vitro transcription was prepared by single digestion using Xbal (Top left panel). Electrophoresis (top right panel) and electropherogram analysis (bottom panel) of in vitro transcribed Nod.OmicronRBD.OAS (lane 1, expected size: -4388 bp) and OmicronRBD mRNA (lane 2: expected size: -681 bp) on Agilent 2100 bioanalyzer.DETAILED DESCRIPTION

[0021] Definitions

[0022] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0023] It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of this disclosure will be limited only by the appended claims.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0025] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); and Plotkin et al., Plotkin’s Human Vaccines, 7thedition (Elsevier).

[0026] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0027] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consistingessentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0028] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0029] The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1 % of the specified amount.

[0030] As used herein, comparative terms as used herein, such as high, low, increase, decrease, reduce, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 folds, or about 3 folds, or about 4 folds, or about 5 folds, or about 6 folds, or about 7 folds, or about 8 folds, or about 9 folds, or about 10 folds, or about 20 folds, or about 30 folds, or about 40 folds, or about 50 folds, or about 60 folds, or about 70 folds, or about 80 folds, or about 90 folds, or about 100 folds or more higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 0%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0031] As will be understood by one skilled in the art, for any and all purposes, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.100321 “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0033] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0034] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0035] The terms or “acceptable,” “effective,” or “sufficient” when used to describe the selection of any components, ranges, dose forms, etc. disclosed herein intend that said component, range, dose form, etc. is suitable for the disclosed purpose.

[0036] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits (which are also referred to as residues) may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0037] It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a polypeptide, protein, polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof’ is intended to be synonymous with “equivalent thereof’ when referring to a reference protein, polypeptide or nucleic acid,intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity, or at least about 85 % homology or identity, or alternatively at least about 90 % homology or identity, or alternatively at least about 95 % homology or identity, or alternatively at least about 96 % homology or identity, or alternatively at least about 97 % homology or identity, or alternatively at least about 98 % homology or identity, or alternatively at least about 99 % homology or identity (in one aspect, as determined using the Clustal Omega alignment program) and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to polynucleotides, an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complementary sequence.

[0038] An equivalent of a reference polypeptide comprises, consists essentially of, or alternatively consists of an polypeptide having at least 80%, or at least 85 %, or at least 90%, or at least 95%, or at least about 96%, or at least 97%, or at least 98%, or at least 99% amino acid identity to the reference polypeptide (as determined, in one aspect using the Clustal Omega alignment program or using BLAST (accessible at blast.ncbi.nlm.nih.gov / Blast.cgi, last accessed on August 1, 2021)), or a polypeptide that is encoded by a polynucleotide that hybridizes under conditions of high stringency to the complementary sequence of a polynucleotide encoding the reference polypeptide, optionally wherein conditions of high stringency comprises incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O. lx SSC, or deionized water.

[0039] In some embodiments, a first sequence (nucleic acid sequence or amino acid) is compared to a second sequence, and the identity percentage between the two sequences can be calculated. In further embodiments, the first sequence can be referred to herein as an equivalent and the second sequence can be referred to herein as a reference sequence. In yet further embodiments, the identity percentage is calculated based on the full-length sequence of the first sequence. In other embodiments, the identity percentage is calculated based on the full-length sequence of the second sequence.

[0040] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0041] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0042] The term “RNA” as used herein refers to its generally accepted meaning in the art. Generally, the term RNA refers to a polynucleotide comprising at least one ribofuranoside moiety. The term can include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, for example at one or more nucleotides of the RNA. Nucleotides in the nucleic acid molecules can alsocomprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. In some embodiments, the RNA is a messenger RNA (mRNA).100431 “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. In some embodiments, an mRNA as disclosed herein comprises, or consists essentially of, or yet further consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail.

[0044] Vaccination is the most successful medical approach to disease prevention and control. The successful development and use of vaccines has saved thousands of lives and large amounts of money. A key advantage of RNA vaccines is that RNA can be produced in the laboratory from a DNA template using readily available materials, less expensively and faster than conventional vaccine production, which can require the use of chicken eggs or other mammalian cells. In addition, mRNA vaccines have the potential to streamline vaccine discovery and development, and facilitate a rapid response to emerging infectious diseases, see, for example, Maruggi et al., Mol Ther. 2019; 27(4):757-772.

[0045] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and / or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, or protein, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, or protein, does not require “isolation” to distinguish it from its naturally occurring counterpart.

[0046] In some embodiments, the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention. As used herein, the term “recombinant protein” refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.10047] As used herein, “complementary” sequences refer to two nucleotide sequences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Paring of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementary sequences. It is not necessary for every nucleotide base in two sequences to pair with each other for sequences to be considered “complementary”. Sequences may be considered complementary, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two sequences pair with each other. In some embodiments, the term complementary refers to 100% of the nucleotide bases in two sequences pair with each other. In addition, sequences may still be considered “complementary” when the total lengths of the two sequences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementary” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementary sequences, but not a pair, are referred to herein as a mismatch.

[0048] A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0049] The term “express” refers to the production of a gene product, such as mRNA, peptides, polypeptides or proteins. As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0050] A “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated. In some embodiments, the gene product may refer to an mRNA or other RNA, such as an interfering RNA, generated when a gene is transcribed.[00511 The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide or a fragment thereof and optionally translated to produce the polypeptide or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. Further, as used herein an amino acid sequence coding sequence refers to a nucleotide sequence encoding the amino acid sequence.

[0052] The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population. In some embodiments, the term refers to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties. In further embodiments, the chemical modification is selected from pseudouridine, Nl- methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, or 2'-O-methyl uridine. In some embodiments the extent of incorporation of chemically modified nucleotides has been optimized for improved immune responses to the vaccineformulation. In other embodiments, the term excludes the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moieties.

[0053] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.100541 In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of residues of the RNA is chemically modified by one or more of modifications as disclosed herein. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher percentage of uridine residues of the RNA is chemically modified by one or more of modifications as disclosed herein.

[0055] It will be appreciated that an RNA as disclosed herein can be made using any appropriate synthesis method. For example, in some embodiments, an RNA is made using IVT from a single bottom strand DNA as a template and complementary oligonucleotide that serves as promotor. The single bottom strand DNA may act as a DNA template for in vitro transcription of RNA, and may be obtained from, for example, a plasmid, a PCR product, or chemical synthesis. In some embodiments, the single bottom strand DNA is linearized from a circular template. The single bottom strand DNA template generally includes a promotersequence, e.g., a bacteriophage promoter sequence, to facilitate IVT. Methods of making RNA using a single bottom strand DNA and a top strand promoter complementary oligonucleotide are known in the art. An exemplary method includes, but is not limited to, annealing the DNA bottom strand template with the top strand promoter complementary oligonucleotide (e.g., T7 promoter complementary oligonucleotide, T3 promoter complementary oligonucleotide, or SP6 promoter complementary oligonucleotide), followed by IVT using an RNA polymerase corresponding to the promoter sequence, e.g., a T7 RNA polymerase, a T3 RNA polymerase, or an SP6 RNA polymerase.10056] The term “a regulatory sequence”, “an expression control element” or “promoter” as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide.

[0057] A promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. In some embodiments, a promoter as used herein is corresponding to the RNA polymerase. In further embodiments, a promoter as sued herein comprises, or consists essentially of, or yet further consists of a T7 promoter, or a SP6 promoter, or a T3 promoter. Non-limiting examples of suitable promoters are provided in W02001009377A1.

[0058] An "RNA polymerase" refers to an enzyme that produces a polyribonucleotide sequence, complementary to a pre-existing template polynucleotide (DNA or RNA). In some embodiments, the RNA polymerase is a bacteriophage RNA polymerase, optionally a T7 RNA polymerase, or a SP6 RNA polymerase, or a T3 RNA polymerase. Non-limiting examples of suitable polymerase are further detailed in US10526629B2.

[0059] In some embodiments, the term “vector” intends a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly-dividing cells and optionally integrate into the target cell’s genome. Non-limiting examples of vectors include a plasmid, a nanoparticle, a liposome, a virus, a cosmid, a phage, a BAC, a YAC, etc. In some embodiments, plasmid vectors may be prepared from commercially available vectors. In other embodiments, viral vectors may be produced from baculoviruses, retroviruses,adenoviruses, AAVs, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. In one embodiment, the viral vector is a retroviral vector. In one embodiment, the vector is a plasmid. In one embodiment, the vector is a nanoparticle, optionally a polymeric nanoparticle or a lipid nanoparticle.100601 In one embodiment the vector includes a plant viral like particle.[00611 Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or alter 5' and / or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.

[0062] A “plasmid” is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for.[00631 As used herein, the term “Virus like particle” “viral like particle,” or “VLP” refers to a non-replicating, viral shell, derived from one or more plant viruses (e.g, one or more plant viruses described herein). VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g, comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012). As used herein, VLP intends naturally occurring (wild-type or native) VLP and engineered VLP, unless explicitly stated otherwise.

[0064] Virus and Virus-like Particles (VLPs) are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012).[0065| In some embodiments, the virus or VLP is derived from Cowpea chlorotic mottle virus (CCMV). CCMV is a spherical plant virus that belongs to the Bromovirus genus. Several strains have been identified and include, but not limited to, Carl (Ali, et al., 2007. J. Virological Methods 141 :84-86), Car2 (Ali, et al., 2007. J. Virological Methods 141 :84-86, 2007), type T (Kuhn, 1964. Phytopathology 54: 1441-1442), soybean (S) (Kuhn, 1968. Phytopathology 58: 1441-1442), mild (M) (Kuhn, 1979. Phytopathology 69:621-624), Arkansas (A) (Fulton, et al., 1975. Phytopathology 65: 741-742), bean yellow stipple (BYS) (Fulton, et al., 1975. Phytopathology 65: 741-742), R (Sinclair, ed. 1982. Compendium of Soybean Diseases. 2nded. The American Phytopathological Society, St. Paul. 104 pp.), and PSM (Paguio, et al., 1988. Plant Diseases 72(9): 768-770).

[0066] In some instances, the virus or VLP from CCMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type CCMV capsid, optionally expressed by Carl, Car2, type T, soybean (S), mild (M), Arkansas (A), bean yellow stipple (BYS), R, or PSM strain. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the CCMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03601 :|0067| MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYS VSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSC VTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAALT EGDVIVHLEVEHVRPTFDDSFTPVY (SEQ ID NO: 1), or an equivalent thereof.

[0068] In some cases, the virus or VLP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141 : 84-86 (2007).[0069| In some embodiments, the virus or VLP is derived from Cowpea mosaic virus (CPMV). CPMV is a non-enveloped plant virus that belongs to the Comovirus genus. CPMV strains include, but are not limited to, SB (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1) and Vu (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1). Cowpea mosaic virus (CPMV) is a VLP and a plant-infecting member of the orderPicornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the 24 K viral proteinase which is encoded by RNA-1. Thus capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell. In some embodiments, the VLP particles have been treated, prepared and / or inactivated by methods known in the art. In some embodiments, the CPMV particle further comprises one or more TLR agonists, that may be the same or different. In some instances, the virus or VLP from CPMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, CPMV produces a large capsid protein and a small capsid protein precursor (which generates a mature small capsid protein). In some cases, CPMV capsid is formed from a plurality of large capsid proteins and mature small capsid proteins. In some cases, the large capsid protein is a wild-type large capsid protein, optionally expressed by SB or Vu strain. In other instances, the large capsid protein is a modified large capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the large capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833):

[0070] MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYDVV NGQDFRATVAFLRTHVITGKIKVTATTNISDNSGCCLMLAINSGVRGKYSTDVYTICS QDSMTWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVIA KLDWSIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGGGAG ATQAFLANMPNSWISMWRYFRGELHFEVTKMSSPYIKATVTFLIAFGNLSDAFGFYE SFPHRIVQFAEVEEKCTLVFSQQEFVTAWSTQVNPRTTLEADGCPYLYAIIHDSTTGTI SGDFNLGVKLVGIKDFCGIGSNPGIDGSRLLGAIAQ (SEQ ID NO: 2), or an equivalent thereof.

[0071] In some cases, the mature small capsid protein is a wild-type mature small capsid protein, optionally expressed by SB or Vu strain. In other instances, the mature small capsidprotein is a modified mature small capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the mature small capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 834- 1022):[0072| GP VCAEASD VYSPCMIASTPPAPF SD VT AVTFDLINGKITPVGDDNWNTHIYN PPIMNVLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDARTF VISQPGSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNMRFD PNFRVAGNILMPPFPLSTETPPL (SEQ ID NO: 3), or an equivalent thereof

[0073] In some embodiments, the virus or VLP is derived from Physalis mottle virus (PhMV). PhMV is a single stranded RNA virus that belongs to the genus Tymovirus. In some instances, the virus or VLP from PhMV comprises, or consists essentially of, or yet further consists of, a plurality of coat proteins. In some instances, the coat protein is a wild-type PhMV coat protein. In other instances, the coat protein is a modified coat protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the PhMV coat comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351 :

[0074] MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSL QTADPITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVY GGQSFVLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTA SIQISGRIRLSKPMLIAN (SEQ ID NO: 4), or an equivalent thereof.

[0075] In some embodiments, the virus or VLP is derived from Sesbania mosaic virus (SeMV). SeMV is a positive stranded RNA virus that belongs to the genus Sobemovirus. In some instances, the virus or VLP from SeMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type SeMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the SeMV capsid comprises, orconsists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06:

[0076] MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRSAVQQLQPTQAGISM APSAQGAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWLR GVAANWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGYV SGQVWSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVDV NIATPLVPARLVIALLDGSSSTAVAAGRIYCTYTIQMIEPTASALNN (SEQ ID NO: 5), or an equivalent thereof.

[0077] In some embodiments, the virus or VLP is derived from Potato virus X (PVX). PVX positive-sense single-stranded RNA virus that belongs to the Potexvirus genus. PVX is rod shaped. In some instances, the virus or VLP from PVX comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type PVX capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the PVX capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in Uniprot Q07626:

[0078] MTTPANTTQAVGSTTSTTTTTAGATPANSGLFTIPDGDFFSTAKVVVASNAVA TNEDLTKIQKIWKDMKIPSDTMAQAAWDLVRHCADVGSSAQTEMIGTGPYSNGVSR ARLAAAIKEVCKLRQFCRKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFF DGVTNPAAITPKEGLIRPPFEAEMNAAQTATFVKITKARAQSNDFASLDAAVTRGRIT GTTAAEAVISLPPP (SEQ ID NO: 6), or an equivalent thereof.

[0079] In some embodiments, the virus or VLP is derived from Tobacco mild green mosaic virus (TMGMV). TMGMV is a positive-sense single-stranded RNA virus belongs to the Tobamovirus genus. TMGMV is rod shaped. In some instances, the virus or VLP from TMGMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type TMGMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the TMGMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03579:[0080| MPYTINSPSQFVYLSSAYADPVQLINLCTNALGNQFQTQQARTTVQQQFADA WKPVPSMTVRFPASDFYVYRYNSTLDPLITALLNSFDTRNRIIEVDNQPAPNTTEIVN ATQRVDDATVAIRASINNLANELVRGTGMFNQAGFETASGLVWTTTPAT (SEQ ID NO: 7), or an equivalent thereof.100811 In some embodiments, the virus or VLP is derived from Tobacco mosaic virus (TMV). TMV is a positive-sense single-stranded RNA virus that belongs to the Tobamovirus genus. TMV is rod shaped. In some instances, the virus or VLP from TMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type TMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the TMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P69687:

[0082] MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVW KPSPQVTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLD ATRRVDDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO: 8), or an equivalent thereof.

[0083] The virus can be obtained according to various methods known to those skilled in the art. In embodiments where plant virus particles are used, the virus particles can be obtained from the extract of a plant infected by the plant virus. For example, cowpea mosaic virus can be grown in black eyed pea plants, which can be infected within 10 days of sowing seeds. Plants can be infected by, for example, coating the leaves with a liquid containing the virus, and then rubbing the leaves, preferably in the presence of an abrasive powder which wounds the leaf surface to allow penetration of the leaf and infection of the plant. Within a week or two after infection, leaves are harvested and viral nanoparticles are extracted. In the case of cowpea mosaic virus, 100 mg of virus can be obtained from as few as 50 plants. Procedures for obtaining plant picornavirus particles using extraction of an infected plant are known to those skilled in the art. See Wellink J., Meth Mol Biol, 8, 205- 209 (1998). Procedures are also available for obtaining virus-like particles. Saunders et al., Virology, 393(2):329-37 (2009). The disclosures of both of these references are incorporated herein by reference.

[0084] Gene delivery vehicles also include DNA / liposome complexes, micelles and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to a cell or cell population, direct introduction of the proteins described herein to the cell or cell population can be done by the non-limiting technique of protein transfection, alternatively culturing conditions that can enhance the expression and / or promote the activity of the proteins disclosed herein are other non-limiting techniques.

[0085] The term “a regulatory sequence” “an expression control element” or “promoter” as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed and / or replicated, and facilitates the expression and / or replication of the target polynucleotide. A promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5’ or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. Polymerase II and III are examples of promoters.|0086| A polymerase II or “pol II” promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shRNA and microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EFl -alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lenti viral vectors.

[0087] An enhancer is a regulatory element that increases the expression of a target sequence. A “promoter / enhancer” is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer / promoter may be “endogenous” or “exogenous” or “heterologous.” An “endogenous” enhancer / promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter.

[0088] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotideresidues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi -stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.(0089] Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength / temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg2+normally found in a cell.100901 Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O. lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.|0091] When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing” and those polynucleotides are described as “complementary.” A double-stranded polynucleotide can be “complementary” or “homologous” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second. “Complementarity” or “homology” (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.100921 “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure. In some embodiments, the identity is calculated between two peptides or polynucleotides over their full-length, or over the shorter sequence of the two, or over the longer sequence of the two.

[0093] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDB J + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: blast.ncbi.nlm.nih.gov / Blast.cgi, last accessed on August 1, 2021.

[0094] In some embodiments, the polynucleotide as disclosed herein is a RNA or an analog thereof. In some embodiments, the polynucleotide as disclosed herein is a DNA or an analogthereof. In some embodiments, the polynucleotide as disclosed herein is a hybrid of DNA and RNA or an analog thereof.

[0095] In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide encodes the same sequence encoded by the reference. In some embodiments, an equivalent to a reference nucleic acid, polynucleotide or oligonucleotide hybridizes to the reference, a complement reference, a reverse reference, or a reverse-complement reference, optionally under conditions of high stringency.

[0096] Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70% sequence identity, or at least 75% sequence identity, or at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complementary. In one aspect, the equivalent must encode the same protein or a functional equivalent of the protein that optionally can be identified through one or more assays described herein. In addition or alternatively, the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.

[0097] The term “transduce” or “transduction” refers to the process whereby a foreign nucleotide sequence is introduced into a cell. In some embodiments, this transduction is done via a vector, viral or non-viral.

[0098] “Detectable label”, “label”, “detectable marker” or “marker” are used interchangeably, including, but not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Detectable labels can also be attached to a polynucleotide, polypeptide, protein or composition described herein.

[0099] As used herein, the term “label” or a detectable label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., N-terminal histidine tags (N-His), magnetically activeisotopes, e.g.,115Sn,117Sn and119Sn, a non-radioactive isotopes such as13C and15N, polynucleotide or protein such as an antibody so as to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to magnetically active isotopes, non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, or other property. In luminescence or fluorescence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescent labels that produce signals include but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.[OHIO] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0101] In some embodiments, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as acell surface marker. Suitable functional groups, include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule. The choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.

[0102] As used herein, a purification label or maker refers to a label that may be used in purifying the molecule or component that the label is conjugated to, such as an epitope tag (including but not limited to a Myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag), an affinity tag (including but not limited to a glutathione-S transferase (GST), a poly- Histidine (His) tag, Calmodulin Binding Protein (CBP), or Maltose-binding protein (MBP)), or a fluorescent tag.

[0103] A “selection marker” refers to a protein or a gene encoding the protein necessary for survival or growth of a cell grown in a selective culture regimen. Typical selection markers include sequences that encode proteins, which confer resistance to selective agents, such as antibiotics, herbicides, or other toxins. Examples of selection markers include genes for conferring resistance to antibiotics, such as spectinomycin, streptomycin, tetracycline, ampicillin, kanamycin, G 418, neomycin, bleomycin, hygromycin, methotrexate, dicamba, glufosinate, or glyphosate.10104] The term “culturing” refers to the in vitro or ex vivo propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0105] In some embodiments, the cell as disclosed herein is a eukaryotic cell or a prokaryotic cell. In some embodiments, the cell is a plant cell or a human cell. In some embodiments, the cell is a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293T cell, or an a549 cell.

[0106] “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.The host cell can be a prokaryotic or a eukaryotic cell. In some embodiments, the host cell is a plant cell or a cell line, such as a human embryonic kidney 293 cell (HEK 293 cell or 293 cell), a 293 T cell, or an a549 cell.

[0107] “Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished through a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically recited, the term “host” includes a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simian, canine, bovine, porcine, murine, rat, avian, reptilian and human.

[0108] “Prokaryotic cells” that usually lack a nucleus or any other membrane-bound organelles and are divided into two domains, bacteria and archaea. Additionally, instead of having chromosomal DNA, these cells’ genetic information is in a circular loop called a plasmid. Bacterial cells are very small, roughly the size of an animal mitochondrion (about l-2pm in diameter and 10 pm long). Prokaryotic cells feature three major shapes: rod shaped, spherical, and spiral. Instead of going through elaborate replication processes like eukaryotes, bacterial cells divide by binary fission. Examples include but are not limited to bacillus bacteria, E. coli bacterium, and Salmonella bacterium.101.09 [ A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers.

[0110] Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, lipid nanoparticle and the like, HK polymers, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant humanalbumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0111] A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.101121 “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. Pharmaceutically acceptable carriers include lipid nanoparticles, HK polymers, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They may be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0113] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of longterm stabilization, bulking up solid formulations, or to confer a therapeutic enhancement onthe active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0114] The compositions used in accordance with the disclosure can be packaged in dosage unit form for ease of administration and uniformity of dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0115] A unit dose, dosage, or regimen can be determined from the IC50 of a given polynucleotide, vector, cell, composition, or kit, for neutralizing activity against a SARS- CoV-2 polypeptide or polynucleotide, preferably a SARS-CoV-2 spike protein. “IC50” means the concentration of polynucleotide, vector, cell, composition, or kit required for 50% inhibition. Alternatively, the effective amount can be determined from the EC50 of a given polynucleotide, vector, cell, composition, or kit. “EC50” means the plasma concentration required to obtain 50% of the maximum neutralizing effect in vivo.| 0l16| A combination as used herein intends that the individual active ingredients of the compositions are separately formulated for use in combination and can be separately packaged with or without specific dosages. The active ingredients of the combination can be administered concurrently or sequentially.

[0117] As used herein, the term “animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term “mammal” includes both human and non-human mammals such as non-human primates (e.g., apes, gibbons,chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, bat, rat, rabbit, guinea pig).

[0118] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, nonhuman primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, bat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a disease.

[0119] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (z.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, treatment excludes prophylaxis.{0120] In some embodiments, the terms “treating,” “treatment,” and the like, as used herein, mean ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject. Reference to “treating,” or “treatment” of a patient is intended to include prophylaxis. Treatment may also be preemptive in nature, i.e., it may include prevention of disease in a subject exposed to or at risk for the disease. Prevention of adisease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen or may involve prevention of disease progression. For example, prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level but instead may mean prevention of the symptoms of a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of progression of a disease to a later stage of the disease.

[0121] “Immune response” broadly refers to the antigen-specific responses of lymphocytes to foreign substances. The terms “immunogen” and “immunogenic” refer to molecules with the capacity to elicit an immune response. All immunogens are antigens; however, not all antigens are immunogenic. An immune response disclosed herein can be humoral (via antibody activity) or cell-mediated (via T cell activation). The response may occur in vivo or in vitro. The skilled artisan will understand that a variety of macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to be immunogenic. The skilled artisan will further understand that nucleic acids encoding a molecule capable of eliciting an immune response necessarily encode an immunogen. The artisan will further understand that immunogens are not limited to full- length molecules but may include partial molecules.

[0122] As used herein, "viral load", also known as "viral burden," "viral titer", "viral level" or "viral expression" in some embodiments, is a measure of the severity of a viral infection, and can be calculated by estimating the amount of virus in an infected organism, an involved body fluid, or a biological sample.

[0123] As used herein, a biological sample, or a sample, is obtained from a subject. Exemplary samples include, but are not limited to, cell sample, tissue sample, biopsy, liquid samples such as blood and other liquid samples of biological origin, including, but not limited to, anterior nasal swab, ocular fluids (aqueous and vitreous humor), peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper’s fluid or pre-ejaculatory fluid, female ejaculate, sweat, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, ascites, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions / flushing, synovial fluid,mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.

[0124] In some embodiments, the sample may be an upper respiratory specimen, such as a nasopharyngeal (NP) specimen, an oropharyngeal (OP) specimen, a nasal mid-turbinate swab, an anterior nares (nasal swab) specimen, or nasopharyngeal wash / aspirate or nasal wash / aspirate (NW) specimen.

[0125] In some embodiments, the samples include fluid from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), umbilical cord blood, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), washings of female reproductive tract, urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof. In some embodiments, a liquid biological sample is a blood plasma or serum sample. The term "blood" as used herein refers to a blood sample or preparation from a subject. The term encompasses whole blood, blood product or any fraction of blood, such as serum, plasma, buffy coat, or the like as conventionally defined. In some embodiments, the term “blood” refers to peripheral blood. Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g., between 3-40 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.

[0126] The term “adjuvant” refers to a substance or mixture that enhances the immune response to an antigen. As non-limiting example, the adjuvant can comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g., US 8,241,610). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant can be formulated by the methods described inWO201 1150240 and US20110293700, each of which is herein incorporated by reference in its entirety.

[0127] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0128] “Administration” or “delivery” of a polynucleotide, vector, cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include inhalation, intramuscular administration, nasal administration, oral administration, intraperitoneal, infusion, injection, and topical application. In preferred embodiments, the route of administration is inhalation or intramuscular administration. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0129] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracistemal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The disclosure is not limited by the route of administration, the formulation or dosing schedule.

[0130] In some embodiments, an RNA, polynucleotide, vector, cell or composition as disclosed herein is administered in an effective amount. An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific agent employed, bioavailability of the agent, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. In general, one will desire to administer an amount of the agent that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0131] Modes For Carrying Out the Disclosure10132] While mRNA vaccines offer rapid production and avoid the need for BSL-3 facilities, they are dependent on efficient nanocarriers for delivery. Applicant turned toward the virus-like particles from tobacco mosaic virus (TMV) to package a self-replicating mRNA encoding the SARS-CoV-2 receptor binding domain (RBD) protein (FIG. 1). The plant virus nanotechnology is a unique platform offering several distinct advantages: First, nanoparticles are stable to high temperatures and solventsn, and therefore do not require refrigeration during storage or distribution; therefore, the resulting vaccine would not rely on the cold chain. Second, while plant viruses and their VLPs are not infectious toward mammals (thus offering safety), they are highly visible to the immune system - therefore the proposed delivery platform, not only serves as a delivery vehicle but also an adjuvant.101331 In one aspect, Applicant designed an exemplary COVID-19 vaccine by making use of a replicon encoding the RBD domain from SARS-CoV-2 spike protein packaged into tobacco mosaic virus (TMV) VLPs. Transduction efficiency and vaccine efficacy were evaluated in in vitro and in vivo models demonstrating neutralizing activity against SARS-CoV-2.

[0134] The rapid development of mRNA vaccines in response to COVID-19 has generated interest in the application of this technology against HPV. The efficacy of an LNP- encapsulated mRNA vaccine encoding HPV E7 (HPV mRNA-LNP) was demonstrated in a mouse model of HPV+ oropharyngeal squamous cell carcinoma.43As an alternative to LNPs, Applicant tested VLPs derived from the plant virus TMV because plant viruses have inherent immunostimulatory properties, acting as an adjuvant as well as a carrier.59Applicant demonstrated the suitability of TMV as a platform for development of therapeutic HPV vaccines by using it to deliver a self-amplifying shuffled E7 mRNA, which elicited a potent humoral and cellular immune response in mice.[0135[ Cassettes, Vectors and Compositions

[0136] The disclosure herein provides a cassette for the delivery of one or more of an mRNA, the cassette comprising, or consisting essentially of, or consisting of an optional 5’ mRNA cap, a 5’ UTR, a Nodamura replicon, a ribosomal skipping polynucleotide, the one or more mRNA, a plant virus assembly origin polynucleotide, and a 3’ UTR, optionally wherein the cassette does not comprise a polyA tail. In one aspect, the cassette comprises a RNA polynucleotide. Also provided is a plant virus nanoparticle capsid protein (VLP) comprising, or consisting of, or consisting essentially of the cassette, e.g., the RNA cassette.

[0137] According to one aspect, the one or mRNA is selected from an mRNA vaccine or an mRNA encoding a therapeutic polypeptide. “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally- occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. In some embodiments, an mRNA as disclosed herein comprises, or consists essentially of, or yet further consists of at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail.

[0138] In some embodiments, the mRNA constructs comprise a 5’ UTR. An exemplary DNA sequence encoding the 5’UTR is: GTATTGAATCCAAAACTCAAA (SEQ ID NO: 17). An exemplary RNA sequence for the 5’UTR is: GUAUUGAAUCCAAAACUCAAA (SEQ ID NO: 25).

[0139] In some embodiments, the mRNA constructs comprise a 3’ UTR. An exemplary DNA sequence encoding the 3 ’UTR is: GCAAGTTTTAGTTAATATTAGAAATGTGAAGATGTCAGCGGGTTTCTGTCCGCTT TCTCTGGAGTTTGTGTCGGTGTGTATTGTTTATAGAAATAATATAAAATTAGGTTT GAGAGAGAAGATTACAAACGTGAGAGACGGAGGGCCCATGGAACTTACAGAAG AAGTCGTTGATGAGTTCATGGAAGATGTCCCTATGTCGATCAGGCTTGCAAAGTT TCGATCTCGAACCG (SEQ ID NO: 18). An exemplary RNA sequence for the 3 ’UTR is: GCAAGUUUUAGUUAAUAUUAGAAAUGUGAAGAUGUCAGCGGGUUUCUGUCCG CUUUCUCUGGAGUUUGUGUCGGUGUGUAUUGUUUAUAGAAAUAAUAUAAAAU UAGGUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACU UACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGG CUUGCAAAGUUUCGAUCUCGAACCG (SEQ ID NO: 26).

[0140] The Nodamura virus is a member of the nodaviridae family. Nodaviruses are small, non-enveloped zoonotic viruses. Their genomes comprise of two segmented, linear, positivesense, single-stranded RNA molecules. RNA1 is approximately 3.1-3.2 kilobases (kb) in length, whereas RNA2 is approximately 1.2-1.4 kb. RNA 1 encodes the RdRP. RNA2 encodes the viral capsid protein, which forms the core of nodaviruses. Nodamura virus (NV) is a species of the genus Alphanodavirus of the Nodaviridae, a family of small riboviruses, with genera comprising alphanodavirus and betanodaviruses, with bipartite, positive-strandRNA genomes that also includes Flock House virus (FHV). NV is unique among alphanodaviruses in its ability to infect both insects and mammals.

[0141] As used herein, the term “replicon” refers to an autonomously replicating DNA or RNA molecule. A viral replicon has the genes encoding some or all structural proteins of a wild-type virus deleted. Therefore, while the replicon can self-replicate, it is unable to subsequently form infectious viral particles, making replicons of interest in clinical applications See K. Van Der Meulen and P.L. J. Rudelsheim (2022) Final COGEM Report CGM 2022-06, cogem.net / app / uploads / 2022 / 12 / CGM-2022-06-Viral-replicon-systems-and- their-biosafety-aspects.pdf. Thus, the Nodamura replicon is self-replicating RNA (RNA1) from the Nodamura virus. The Nodamura replicon can be engineered and therefore can vary in length. See L. Gitlin et al. (2014) PLOS Pathogens 10(12): el004529. In some aspects, the Nodamura replicon is 3129 nucleotides. An exemplary Nodamura replicon sequence is provided in SEQ ID NO: 19.10142] The plant virus assembly origin polynucleotide is also referred to as the origin of assembly sequence (“OAS”). The OAS is an assembly signal that interacts with coat proteins, and have been identified in plant viruses including Tobacco Mosaic Virus, Beet Mosaic Virus, and Turnip Crinkle Virus, and Potato Virus X. The OAS plays a role in the assembly of RNA viruses and can improve the efficiency of RNA encapsidation. See M.V. Arkhipenko et al. (2011) Acta Naturae 3(3): 40-46, K. Saunders et al. (2022) 434(24): 67873, and V.R. Basnayake et al. (2009) Virology 384(1): 169-178. For example, the rod-shaped monopartite Tobacco Mosaic Virus OAS can is 51 nucleotides in length, a bulged stem loop with a G residue at every third nucleotide in the terminal loop. The icosahedral monopartite Turnip Crinkle Virus OAS can a 28 nucleotide bulged hairpin loop. V.R. Basnayake et al. (2009) Virology 384(1): 169-178. However, the OAS can vary in length. See D.J. Hawang et al. (1994) Proc Natl Acad Sci U S A. 91(19): 9067-9071. As described herein, in one embodiment the OAS of the gene cassette is the TMV OAS, spanning nucleotides 5313 to 5546 in the TMV genome.

[0143] As used herein, the term “ribosomal skipping polynucleotide” refers to peptides which induce ribosomal skipping, such as “F2A self-cleaving peptide” or “T2A self-cleaving peptide.” Ribosomal skipping is a translational effect which produces an apparent ‘cleavage’ of polyproteins — no actual cleavage occurs. See C.A.C. Cruz-Teran et al. (2017) ACS Synth.Biol. 6(11): 2096-2107. 2A self-cleaving peptides are a class of 18-22 aa-long peptides, which can induce ribosomal skipping during translation of a protein in a cell. The apparent cleavage is triggered by ribosomal skipping of the peptide bond between the Proline (P) and Glycine (G) in C-terminal of 2A peptide. An exemplary T2A polypeptide sequence is : EGRGSLLTCGDVEENPGP (SEQ ID NO: 23), encoded by the RNA sequence: GAAGGCCGCGGCAGCCUGCUGACCUGCGGCGAUGUGGAAGAAAACCCGGGCCC G (SEQ ID NO: 14).

[0144] In some embodiments, the mRNA constructs comprise a TMV origin associated sequence (OAS). An exemplary DNA sequence encoding the mRNA is provided in SEQ ID NO: 21 and having the nucleotide sequence:GCAAGTTTTAGTTAATATTAGAAATGTGAAGATGTCAGCGGGTTTCTGTCCGCTT TCTCTGGAGTTTGTGTCGGTGTGTATTGTTTATAGAAATAATATAAAATTAGGTTT GAGAGAGAAGATTACAAACGTGAGAGACGGAGGGCCCATGGAACTTACAGAAG AAGTCGTTGATGAGTTCATGGAAGATGTCCCTATGTCGATCAGGCTTGCAAAGTT TCGATCTCGAACCGG (SEQ ID NO: 21). An exemplary RNA sequence is provided in SEQ ID NO: 22, having the nucleotide sequence:GCAAGUUUUAGUUAAUAUUAGAAAUGUGAAGAUGUCAGCGGGUUUCUGUCCG CUUUCUCUGGAGUUUGUGUCGGUGUGUAUUGUUUAUAGAAAUAAUAUAAAAU UAGGUUUGAGAGAGAAGAUUACAAACGUGAGAGACGGAGGGCCCAUGGAACU UACAGAAGAAGUCGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCGAUCAGG CUUGCAAAGUUUCGAUCUCGAACCGG (SEQ ID NO: 22).

[0145] In some embodiments, the mRNA constructs comprise a mutated HPV E7 mRNA encoded by the DNA nucleotide sequence. An exemplary E7 DNA sequence encoding the mRNA is:

[0146] ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACC AGAGACAACTGGTCTCTACG'G'TTATG'G'G'CAATTAAATGACAGCTCAGAGGAGGA GGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAA TATTGTAACCTTTTGTTGCAA676767TGACTCTACGCTTCGGTTGTGCGTACAAAGC ACACACGTAGACATCCGTACGTTGGAAGACCTGTTAATGGGCACACTAGGAATT GTGTGCCCCATCGG'TT CTCAGAAACCATAA (SEQ ID NO: 15).

[0147] The above (SEQ ID NO: 15) is a shuffled E7-Nodamura sequence (303 bp). Mutant E, Human papillomavirus type 16ZE7 GenBank: AF536180.1, mutated to G at 21, 24, 26, 61 and 64; mutated codon indicated as bold and italics.

[0148] In some embodiments, the mRNA constructs comprise a mutated HPV E7 mRNA having the RNA nucleotide sequence:AUGCAUGGAGAUACACCUACAUUGCAUGAAUAUAUGUUAGAUUUGCAACCAG AGACAACUGGUCUCUACGGUUAUGGGCAAUUAAAUGACAGCUCAGAGGAGGA GGAUGAAAUAGAUGGUCCAGCUGGACAAGCAGAACCGGACAGAGCCCAUUAC AAUAUUGUAACCUUUUGUUGCAAGGGUGACUCUACGCUUCGGUUGUGCGUAC AAAGCACACACGUAGACAUCCGUACGUUGGAAGACCUGUUAAUGGGCACACUA GGAAUUGUGUGCCCCAUCGGUUCUCAGAAACCAUAA (SEQ ID NO: 16).

[0149] In some embodiments, the mRNA constructs comprise a polynucleotide sequence encoding the HPV E6 polypeptide sequence:MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDL CIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQ KPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO: 24).

[0150] An exemplary RNA sequence for HPV E6 is:AUGCAUCAGAAACGCACCGCGAUGUUUCAGGAUCCGCAGGAACGCCCGCGCAA ACUGCCGCAGCUGUGCACCGAACUGCAGACCACCAUUCAUGAUAUUAUUCUGG AAUGCGUGUAUUGCAAACAGCAGCUGCUGCGCCGCGAAGUGUAUGAUUUUGC GUUUCGCGAUCUGUGCAUUGUGUAUCGCGAUGGCAACCCGUAUGCGGUGUGC GAUAAAUGCCUGAAAUUUUAUAGCAAAAUUAGCGAAUAUCGCCAUUAUUGCU AUAGCGUGUAUGGCACCACCCUGGAACAGCAGUAUAACAAACCGCUGUGCGAU CUGCUGAUUCGCUGCAUUAACUGCCAGAAACCGCUGUGCCCGGAAGAAAAACA GCGCCAUCUGGAUAAAAAACAGCGCUUUCAUAACAUUCGCGGCCGCUGGACCG GCCGCUGCAUGAGCUGCUGCCGCAGCAGCCGCACCCGCCGCGAAACCCAGCUG (SEQ ID NO: 17).[0151 [ In some embodiments, the mRNA constructs comprise a polynucleotide sequence encoding the Omicron receptor binding domain (RBD) polypeptide sequence. An exemplary RBD polypeptide encoded by the DNA and RNA polynucleotides is:RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFT FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCV IAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYF PLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 27). An exemplary RNA sequence for the Omicron RBD is:CGCGUGCAGCCGACCGAAAGCAUUGUGCGCUUUCCGAACAUUACCAACCUGUG CCCGUUUGAUGAAGUGUUUAACGCGACCCGCUUUGCGAGCGUGUAUGCGUGG AACCGCAAACGCAUUAGCAACUGCGUGGCGGAUUAUAGCGUGCUGUAUAACCU GGCGCCGUUUUUUACCUUUAAAUGCUAUGGCGUGAGCCCGACCAAACUGAACG AUCUGUGCUUUACCAACGUGUAUGCGGAUAGCUUUGUGAUUCGCGGCGAUGA AGUGCGCCAGAUUGCGCCGGGCCAGACCGGCAACAUUGCGGAUUAUAACUAUA AACUGCCGGAUGAUUUUACCGGCUGCGUGAUUGCGUGGAACAGCAACAAACU GGAUAGCAAAGUGAGCGGCAACUAUAACUAUCUGUAUCGCCUGUUUCGCAAA AGCAACCUGAAACCGUUUGAACGCGAUAUUAGCACCGAAAUUUAUCAGGCGG GCAACAAACCGUGCAACGGCGUGGCGGGCUUUAACUGCUAUUUUCCGCUGCGC AGCUAUAGCUUUCGCCCGACCUAUGGCGUGGGCCAUCAGCCGUAUCGCGUGGU GGUGCUGAGCUUUGAACUGCUGCAUGCGCCGGCGACCGUGUGCGGCCCGAAAA AAAGCACCAACCUGGUGAAAAACAAAUGCGUGAACUUU (SEQ ID NO: 20).[01521 As used herein, the term “a polyadenylation site (PolyA tail)” can be represented as “AA(A)„,” wherein n is an integer that is 0 or about from 1 to about 50, or about 75, or about 100, or about 150, or about 175, or about 200, or about 225, or about 250, or about 275, or about 300, or more.

[0153] In one aspect, the cassette further comprises a sequence encoding a CAP protein. In a further aspect, the cassette that further comprises the CAP protein comprises the mRNA encoding an HPV mRNA.

[0154] In some respects, the mRNA is selected from an mRNA vaccine or an mRNA encoding a therapeutic peptide. In one embodiment, the mRNA vaccine is selected The mRNA vaccine encodes a viral protein, e.g. selected from one or more of an Omicron spike protein, or fragment, or an HPV polypeptide, an HPV E6 polypeptide, an HPVE7 polypeptide, an HPV mutated E7 polypeptide, or an Omicron RBD polypeptide.

[0155] As used herein, the terms “spike protein,” “spike glycoprotein” or “S protein” are used interchangeably, referring to a glycoprotein projecting from the lipid bilayer of the surface of an enveloped virus, such as SARS-CoV-2. In some embodiments, an S protein refers to an S protein of a SARS-CoV-2. In further embodiments, an S protein or an equivalent thereof as used herein also refers to an S protein variant (for example, an S protein of a naturally occurring SARS-CoV-2 variant, such as an Omicron variant), an S protein mutant (for example, a mutated S protein as disclosed herein), an S protein fragment (such as an immunogenic fragment), or any combination thereof (such as, a naturally occurring variant engineered with additional mutation(s), or a fragment thereof).

[0156] In some embodiments, an S protein as used herein comprises, or consists essentially of, or yet further consists of an SI polypeptide or an S2 polypeptide or both. In some embodiments, an S protein as used herein is a precursor protein comprising, or consisting essentially of, or yet further consisting of both SI and S2. Such precursor can be processed into SI and S2 by Cathepsin L (CTSL), Transmembrane Serine Protease (TMPRSS2) or furin to yield the mature SI and S2 protein. In some embodiments, the S protein as used herein refers to a protein complex comprising, or consisting essentially of, or yet further consisting of a mature SI protein and a mature S2 protein. In other embodiments, the S protein as used herein refers to an SI protein. In yet other embodiments, the S protein as used herein refers to an S2 protein. Non-limiting exemplary sequences of an S protein or the underlying gene may be found under Gene ID: 43740568 (retrieved from ncbi.nlm.nih.gov / gene / 43740568, last accessed on August 1, 2021), NCBI Reference Sequence: NC_045512.2 (retrieved from ncbi.nlm.nih.gov / nuccore / NC_045512.2 / , last accessed on August 1st, 2021) or UniProtKB / Swiss-Prot: P0DTC2 (retrieved from uniprot.org / uniprot / P0DTC2, last accessed on August 1, 2021), which are incorporated by reference herein.

[0157] In some embodiments, a fragment (such as an immunogenic fragment) of an S protein comprises, or consists essentially of, or yet further consists of a receptor binding domain (RBD) of the S protein. In some embodiments, a receptor-binding domain (RBD) refers to a short immunogenic fragment from a virus that binds to a specific endogenous receptor sequence to gain entry into target cells. In some embodiments, RBD refer to a part of the ‘spike’ glycoprotein (S-domain) which is needed to interact with endogenous receptors to facilitate membrane fusion and delivery to the cytoplasm.

[0158] In some embodiments, the fragment or immunogenic fragment is at least about 5 amino acids long, or at least about 8 amino acids long, or at least about 10 amino acids long, or at least about 15 amino acids long, or at least about 20 amino acids long, or at least about 25 amino acids long, or at least about 30 amino acids long, or at least about 40 amino acids long, or at least about 50 amino acids long, or at least about 60 amino acids long, or at least about 70 amino acids long, or at least about 80 amino acids long, or at least about 100 amino acids long, or at least about 125 amino acids long, or at least about 150 amino acids long, or at least about 160 amino acids long, or at least about 170 amino acids long, or at least about 180 amino acids long, or at least about 190 amino acids long, or at least about 200 amino acids long, or at least about 250 amino acids long, or at least about 300, or longer; and optionally comprises, consists essentially of, or yet further consists of a RBD of the S protein or an equivalent thereof. The immunogenic fragment is useful for inducing an immune response to the SARS-CoV-2 or reducing or inhibiting the binding of SARS-CoV-2 to its receptor, such as ACE2, or both and a fragment that is non-immunogenic is useful as a control in the assays as provided herein.

[0159] According to one embodiment, the mRNA vaccine is selected from an Omicron vaccine or a Human papillomavirus (HPV) vaccine.

[0160] The vaccine can be a protein from the Omicron strain of SARS-CoV-2, for example the S-protein. The S-Protein of Omicron SARS-CoV-2 mRNA be encoded by a sequence according to NCBI LC731729.1 (Synthetic construct pVEE-101c.l gene for spike protein)(SEQ ID NO: 9).

[0161] According to one embodiment, the Omicron SARS-CoV-2 vaccine is the Omicron RBD, which is a fragment of the S-Protein. In one embodiment the Omicron RBD comprises or consists of 675 nucleotides (SEQ ID NO: 13).

[0162] The mRNA vaccine can encode a protein from HPV. The HPV genome encodes intracellular oncogenes include HPV E5, E6, and E7 which disrupt critical cell processes which leads to cancer development. HPV E7 specifically binds and inhibits Rb resulting in the release of E2F and activation cell cycling and aberrant cell proliferation which is a hallmark of cancer. E7 (wild type) can have an RNA sequence encoded by a DNA polynucleotide from according to AF536180.1 (Human papillomavirus type 16 isolateAfrican-1 type):ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGA CAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATG AAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTG TAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACA CGTAGACATCCGTACGTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA (SEQ ID NO: 10).

[0163] In one aspect, the amino acid sequence of the HPV E 7 comprises the amino acid sequence:MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTF CCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO: 12). Also provided herein is a DNA polynucleotide encoding the amino acid sequence.

[0014] In some respects, the E7 protein is a synthetic construct mutant E7 which has a nucleotide sequence according to AF536180.1 (SEQ ID NO: 10), with mutation to G at nucleotides 21, 24, 26, 61 and 64. In some embodiments, the mRNA constructs comprise a mutated HPV E7 mRNA encoded by the DNA nucleotide sequence:ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGGTCTCTACGGTTATGGGCAATTAAATGACAGCTCAGAGGAGGAGGATG AAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTG TAACCTTTTGTTGCAAGGGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACA CGTAGACATCCGTACGTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCGGTT CTCAGAAACCATAA (SEQ ID NO: 11).

[0165] In some embodiments, the mRNA constructs comprise a mutated HPV E7 mRNA having the RNA nucleotide sequence:

[0166] AUGCAUGGAGAUACACCUACAUUGCAUGAAUAUAUGUUAGAUUUGCAA CCAGAGACAACUGGUCUCUACGGUUAUGGGCAAUUAAAUGACAGCUCAGAGG AGGAGGAUGAAAUAGAUGGUCCAGCUGGACAAGCAGAACCGGACAGAGCCCA UUACAAUAUUGUAACCUUUUGUUGCAAGGGUGACUCUACGCUUCGGUUGUGCGUACAAAGCACACACGUAGACAUCCGUACGUUGGAAGACCUGUUAAUGGGCAC ACUAGGAAUUGUGUGCCCCAUCGGUUCUCAGAAACCAUAA (SEQ ID NO: 18).Insome aspects, the one or more mRNA encodes one or more of the HPV E6, HPV E7 or the mutated HPV E7. In a further aspect, the mRNA encodes HPV E6 and mutated E7 as separate RNAs or as a single polycistronic RNA.

[0167] In some embodiments, the cassette is about 4 to 5 kilobases, or about 3.5 to 4.5 kilobases, or about 4 kilobases.

[0168] In one embodiment, the cassettes comprise the elements shown in FIG. 5A or FIG. 9C.

[0169] In a further aspect, the cassettes further comprise a detectable label or selection marker that in one aspect, is a RNA or DNA molecule encoding the detectable label or selection marker.10170 [ Further provides is a plurality of the cassettes as described herein, wherein the cassettes of the plurality are the same or different from each other.[01711 Also provided is a DNA polynucleotide encoding the cassettes or plurality as described herein.

[0172] Also provided are vector comprising the cassettes as described herein, or DNA polynucleotides encoding them, wherein the vector is a viral vector or a plasmid. The cassettes, DNA polynucleotides, vectors and plurality can be combined with a carrier, such as a pharmaceutically acceptable carrier. In some embodiments, the vector further includes a detectable or purification label.

[0173] In one embodiment, an isolated host cell is provided, wherein the cell comprises one or more of the cassettes as described herein, or DNA polynucleotides encoding them, or the vectors. The host cells can be a prokaryotic or a eukaryotic cell. The host cells can be combined with a carrier, such as a pharmaceutically acceptable carrier.

[0174] A system for the manufacture of a VLP encapsulated cassette as describe above is provided. The capsid is from a plant virus as described herein, e.g., a TMV, a TMGMV, or a PVX.

[0175] In some embodiments, the plant virus like particle (VLP) is derived from a plant virus. In some embodiments, the plant virus is selected from TMV, TMGMV, or PVX. TheVLP can be comprised of the plant virus coat proteins. The cassette can be encapsulated in the VLP coat protein.

[0176] In some embodiments, the plant virus nanoparticle, e.g. TMV, TMGMV, PVX, or another rod-shape virus particle has a length of about 1 nm to about 400 nm, or from about 100 nm to about 300 nm, or from about 200 nm to about 300 nm. In some embodiments the plant virus nanoparticle is 188 nm.

[0177] In some embodiments, the plant virus nanoparticle, e.g., an icosahedral shaped particle has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.

[0178] In come embodiments, the vector is included in a composition. The composition further includes a carrier, optionally a pharmaceutically acceptable carrier.

[0179] In some embodiments, the composition further comprises an additional prophylactic or therapeutic agent.

[0180] In some embodiments, the additional prophylactic or therapeutic agent is suitable for preventing or treating a SARS-CoV-2 or HPV related disease as disclosed herein. In further embodiments, the additional prophylactic or therapeutic agent comprises, or alternatively consists essentially of, or yet further consists of an anti-viral agent, optionally remdesivir, lopinavir, ritonavir, ivermectin, tamiflu, or favipiravir; an anti-inflammatory agent, optionally dexamethasone, tocilizumab, kevzara, colcrys, hydroxychloroquine, chloroquine, or a kinase inhibitor; a covalescent plasma from a subject recovered from a SARS-CoV-2 infection; an antibody binding to SARS-CoV-2, optionally bamlanivimab, etesevimab, casirivimab, or imdevimab; or an antibiotic agent, optionally azithromycin.

[0181] In some embodiments, the additional prophylactic agent is suitable for preventing a disease that is not related to SARS-CoV-2 or HPV. For example, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for another coronavirus, such as SARS-CoV or MERS-CoV. Additionally or alternatively,the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for another papillomavirus. Additionally or alternatively, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for another virus, such as an influenza (flu) vaccine, a Hepatitis A vaccine, a Hepatitis B vaccine, a Hepatitis c vaccine, a polio vaccine, a chickenpox varicella vaccine, a measles vaccine, a mumps vaccine, a rubella vaccine, a rotavirus vaccine. In some embodiments, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for a bacterium or other pathogen, such as a diphtheria vaccine, a Haemophilus influenzae type b vaccine, a Pertussis vaccine, a pneumococcus vaccine, a Tetanus vaccine, or a Meningococcal vaccine. In some embodiments, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for a non-infectious disease, such as a cancer.101821 In some embodiments, the composition further comprises an adjuvant.101831 Also provided is a method to prepare an encapsulated mRNA vaccine comprising, or consisting of, or consisting essentially of, admixing a VLP capsid protein with the cassette, in a ratio of about 0.5:aboutl5 of the cassette to the VLP capsid protein in a buffer for about 15 to about 20 hours, and then isolated the VLPs. In other aspects, the ration is from about 1 :about 20 of the cassette to the VLP capsid protein. Further provided are the isolated encapsulated mRNA vaccines produced by these methods.

[0184] An isolated VLP comprising a VLP capsid protein and an mRNA cassette as described herein is provide. The VLP capsid can be from or derived from any VLP described herein, e.g., a plant virus selected from a TMV, a tobacco mild green mosaic virus (TMGMV), or a potato virus X (PVX).

[0185] In one aspect, the mRNA of the cassette is selected from an mRNA vaccine or an mRNA therapeutic peptide as described herein.

[0186] Further provided are a plurality of isolated VLPs encapsulated cassettes wherein the, wherein the VLPs and / or the mRNAs are the same or different from each other.]0187[ The VLP encapsulated cassettes can be combined with a carrier, optionally a pharmaceutically acceptable carrier.[0188 Methods of Delivery and Treatment

[0189] In one aspect, provided herein is a method to deliver mRNA to a cell, comprising contacting the cell with one or more of: the cassette or encapsulated cassette or composition as disclosed herein. In one aspect, the contacting is in vitro. In another aspect, the contacting is in vivo.

[0190] In one aspect, provided herein is a method to deliver mRNA to a subject in need thereof, comprising administering to the subject the encapsulated cassette or composition comprising the encapsulated cassette or a composition containing such, as disclosed herein. According to one aspect, the subject is a mammal, optionally a human patient.10191] In one aspect, provided herein is a method for preventing or treating a disease, or inducing an immune response in a subject in need thereof. According to one embodiment, the immune response is induced against a CO VID infection. According to another embodiment, the immune response is induced against HPV infection. According to yet another embodiment, the method is used to treat an HPV-related cancer.

[0192] The method comprises, or alternatively consists essentially of, or yet further consists of administering to the subject, optionally an effective amount of, one or more of comprising administering to the subject the encapsulated cassette or composition comprising the encapsulated cassette or a composition containing such, as disclosed herein, thereby preventing or treating the disease. In one aspect, the subject is a mammal, optionally a human patient.

[0193] A vector or composition including mRNA from SARS-CoV-2 may be used against a COVID infection. A vector or composition including mRNA from HPV may be used against an HPV infection or related cancer.

[0194] In some embodiments, the method further comprises treating the subject in need thereof, such as administering to the subject, an additional prophylactic or therapeutic agent.

[0195] In some embodiments, the additional prophylactic or therapeutic agent is suitable for preventing or treating a SARS-CoV-2 or HPV related disease as disclosed herein.

[0196] In further embodiments, the additional prophylactic or therapeutic agent comprises, or alternatively consists essentially of, or yet further consists of an anti-viral agent, optionallyremdesivir, lopinavir, ritonavir, ivermectin, tamiflu, or favipiravir; an anti-inflammatory agent, optionally dexamethasone, tocilizumab, kevzara, colcrys, hydroxychloroquine, chloroquine, or a kinase inhibitor; a covalescent plasma from a subject recovered from a SARS-CoV-2 infection; an antibody binding to SARS-CoV-2, optionally bamlanivimab, etesevimab, casirivimab, or imdevimab; or an antibiotic agent, optionally azithromycin.

[0197] In some embodiments, the additional prophylactic agent is suitable for preventing a disease that is not related to SARS-CoV-2 or HPV. For example, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for another coronavirus, such as SARS-CoV or MERS-CoV. Additionally or alternatively, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for another papillomavirus. Additionally or alternatively, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for another virus, such as an influenza (flu) vaccine, a Hepatitis A vaccine, a Hepatitis B vaccine, a Hepatitis c vaccine, a polio vaccine, a chickenpox varicella vaccine, a measles vaccine, a mumps vaccine, a rubella vaccine, a rotavirus vaccine. In some embodiments, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for a bacterium or other pathogen, such as a diphtheria vaccine, a Haemophilus influenzae type b vaccine, a Pertussis vaccine, a pneumococcus vaccine, a Tetanus vaccine, or a Meningococcal vaccine. In some embodiments, the additional prophylactic agent comprises, or alternatively consists essentially of, or yet further consists of a vaccine for a non-infectious disease, such as a cancer.

[0198] In some embodiments, the subject does not have a SARS-CoV-2 infection when administrated with the vector or the composition.

[0199] In some embodiments, the subject does not have an HPV infection when administrated with the vector or the composition.

[0200] In some embodiments, the administrations is by inhalation. In further embodiments, the vector or the composition is atomized by a nebulizer inhalation system prior to or during administration. In yet further embodiments, the nebulizer system is a portable nebulizer for whole respiratory tract drug delivery.

[0201] In some embodiments, the administration is by subcutaneous injection. In some embodiments, the administration is by intramuscular injection. In some embodiments, the administration is by intraperitoneal injection (i.p).

[0202] In some embodiment, a composition as disclosed herein can be in the form of an aerosol, dispersion, solution, or suspension and can be formulated for inhalation, intramuscular, oral, sublingual, buccal, parenteral, nasal, subcutaneous, intradermal, or topical administration. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.

[0203] The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg / kg and 100 mg / kg body weight / day of active ingredients is administered dependent upon potency of the formulated composition. In some embodiments, an effective dose of an RNA, or polynucleotide, or vector, or cell or composition as disclosed herein is administered twice. In some embodiments, an effective dose of an RNA, or polynucleotide, or vector, or cell or composition as disclosed herein is administered twice at an interval of at least 21 days, at least 28 days, at least 35 days, at least 42 days, at least 49 days, at least 56 days, or at least 64 days.

[0204] Kit{0205] In one aspect, provided is a kit for use in a method as disclosed herein.

[0206] In some embodiments, the kit comprises, or alternatively consists essentially of, or yet further consist of instructions for use and one or more of: a cassette, DNA polynucleotide, vector, encapsulated cassette or composition disclosed herein. In further embodiments, the kit is suitable for use in a method of treatment as disclosed herein.

[0207] The following examples are included to demonstrate some embodiments of the disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosedand still obtain a like or similar result without departing from the spirit and scope of the invention.

[0208] Experimental No. 1

[0209] Introduction

[0210] Traditional vaccines are based on the direct introduction of antigens that stimulate an immune response, whereas mRNA vaccines package a translatable RNA molecule encoding the antigen, which is expressed once the mRNA has been taken up by cells.1Although mRNA vaccines have a history stretching back more than 40 years,1they have recently gained more attention due to their widespread use during the COVID-19 pandemic.2However, they have been developed for a range of indications beyond infectious diseases, including immunotherapy, genetic disorders, regenerative medicine, and cancer.3'5The main benefits of mRNA vaccines include the universal design principles, scalability for mass production, safe translation without the risk of genomic integration, and the ability to generate a robust immune response due to the capacity of single mRNA molecules to produce many protein antigens.3, 6Conversely, the reactogenicity of mRNA vaccines is no different to that of conventional, protein-based counterparts.7One key challenge with the delivery of mRNA vaccines is that naked mRNA degrades rapidly in vivo. This has been addressed by the development of lipid nanoparticles (LNPs) for mRNA protection and targeted delivery to immune cells.8More recently, virus-like particles (VLPs), which resemble the structure of a natural virion but lack the endogenous genome, have been developed as delivery vehicles for drugs and nucleic acids, particularly in the field of immunotherapy, and have also been considered for the delivery of mRNA vaccines.9'11

[0211] Tobacco mosaic virus (TMV) is a naturally occurring plant virus that has been repurposed to develop a range of VLPs as non-infectious nanomaterials for medical applications.12The rod-shaped virion of wild-type TMV is 300 nm in length and 18 nm in diameter, with a central channel of 4 nm.13The genomic RNA intercalates with coat proteins and self-assembles into a virion with helical symmetry. However, TMV coat proteins will assemble with any RNA molecules that contain the appropriate packaging sequence, known as the origin of assembly sequence (OAS)14, forming particles longer or shorter than the wildtype virion according to the size of the RNA. This provides a versatile tool for theconstruction and delivery of mRNA vaccines. For example, TMV has been shown to package the self-amplifying Nodamura replicon, allowing the expression of functional mRNAs encoding reporters such as luciferase and derivatives of green fluorescent protein in vitro and in vivo.w’15It can also package other viral genomes when endowed with the correct packaging signal, including Semliki Forest virus and ebolaviruses.10, 16-18

[0212] In the context of mRNA vaccine development, TMV is particularly useful because the capsid is extremely robust, conferring stability against extreme temperature and pH, as well as various harsh solvents, allowing distribution without a cold chain.19This may be an advantage compared to the LNP technology, because some LNP -based mRNA vaccines, require storage and distribution in ultralow freezers therefore restricting distribution in low- income countries.20Further, plant virus large-scale production is inexpensive and straightforward because the coat protein can be produced in massive quantities by molecular farming in plants before mixing with synthetic RNA or RNA produced by in vitro transcription.21While in vitro assembly offers a plug-and-play technology, opportunity exists to produce and self-assemble the vaccine candidate in plants, which may allow to streamline production in the region-for the region and could make a contribution to less resourced areas of the world.22As native TMV virions contain a 6-kb genomic RNA,13TMV VLPs can encapsulate higher molecular weight RNA cargoes with ease - in stark contrast, encapsulation of longer mRNAs (3-5 kb or above) has been proven challenging with the LNP technology.4Finally, TMV-based nanoparticles interact directly with antigen-presenting cells and are transported to the draining lymph nodes.23They also function as pathogen- associated molecular patterns (PAMPs), triggering the innate immune system via pattern recognition receptors (PRRs).24-26TMV VLPs thus exhibit potent adjuvant properties, comparable to the LNPs (the latter often are engineered with immunomodulatory agents).23

[0213] Human papillomavirus 16 (HPV16) is associated with a high risk of cancer, including benign and malignant lesions.27This non-enveloped virus contains a double-stranded DNA genome of ~8 kb and primarily shows tropism for mucosal and cutaneous epithelia of the genital and upper respiratory tracts, and skin.28HPV16 expresses six early genes (El, E2, E4, E5, E6 and E7) and two late genes (LI and L2).29, 30The E6 and E7 are key oncogenic drivers of cervical carcinogenesis, transforming normal cells into cancerous ones by forming complexes with, and thus inactivating, the tumor suppressor proteins p53 (targeted by E6)and pRb (targeted by E7), respectively.31'33Both oncogenes are constitutively expressed throughout the HPV life cycle, making them crucial targets for therapeutic vaccine development. Mutations in E6 or E7 suppress their transforming functions in a dominant manner, so the delivery of mRNA encoding the suppressor mutant forms of E6 or E7 can prevent oncogenic transformation.32Preclinical and clinical studies of HPV16 / 18 E6ZE7- based vaccines have demonstrated efficacy in treating advanced-stage cervical cancers.32

[0214] Applicant’s provide evidence of a vaccine focused on E7. However, dual E7 and E6 vaccine construct also is provided herein

[0215] Materials and Methods

[0216] Plasmid construct|02l7] To express a modified HPV16 E7 protein with limited oncogenic potential, Applicant constructed a self-amplifying Nodamura replicon (Nod) plasmid, pT7.Nod.E7OAS.Synthetic constructs corresponding to the HPV16 E7 protein (GenBank AF536180.1) were prepared by GenScript with mutations in the conserved pRb-binding domain and cysteine repeat region (D21G, C24G, E26G, C61G and C94G) to suppress the transforming function of E7.35-37The mutated E7 sequence was transferred to pNod.LucOAS by insertion between the 5' restriction site Ndel and the 3' site Agel38(FIG. 9).{0218] Preparation of mRNA

[0219] The pT7.Nod.E7OAS construct contained the T7 promoter and 5' untranslated region (UTR), Nodamura replicon, HPV E7 (mutant) coding sequence, TMV OAS, and 3' UTR (FIG. 10). The plasmid was linearized with Xbal and purified using the QiaQuick PCR purification kit (Qiagen). In vitro transcription and capping were carried out using the HiScribe T7 ARCA mRNA Kit (New England Biolabs). The mRNA was then purified by lithium chloride precipitation. The purity and quality of the transcribed mRNA were assessed by UV-Vis spectroscopy, agarose gel electrophoresis, and evaluation using an Agilent 2100 bioanalyzer.

[0220] Characterization of Nod.HPVE7 mRNA

[0221] Transfection

[0222] Baby hamster kidney fibroblasts (BHK-21 cells) were seeded on coverslips in six- well plates containing Dulbecco’s modified Eagle’s complete medium supplemented with 10% fetal bovine serum (both from Thermo Fisher Scientific). The plates were incubated for 24 h at 37 °C in a 5% CO2 incubator until they reached 70-90% confluency and were then transfected with 500 ng Nod.HPV-E7 mRNA using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cells were incubated at 37 °C for 24-72 h as above and gene expression was assessed by confocal imaging, RT-PCR, and quantitative RT-PCR.

[0223] Confocal microscopy

[0224] Cells were fixed 24 h post-transfection in 4% (v / v) paraformaldehyde in PBS for 10 min at room temperature (RT) along with untransfected controls. The outer membrane was stained with wheat germ agglutinin (WGA) conjugated to Alexa Fluor 555 (Invitrogen; W32464), diluted 1 : 1000. The cells were then permeabilized with 0.2% Triton X-100 (Sigma- Aldrich, XI 00) for 2 min and blocked with 10% (v / v) goat serum (Thermo Fisher Scientific, 16210064) in PBS for 1 h. HPV E7 protein was detected by staining the cells with an HPV 16 E7-specific polyclonal antibody (Invitrogen; PA5-117383), diluted 1 :50 in 5% (v / v) goat serum in PBS. Bound primary antibody was detected using a secondary goat antirabbit antibody conjugated to Alexa Fluor 647 (Invitrogen; A-21245), diluted 1 :500 in 5% (v / v) goat serum in PBS. The cells were washed 3 x 5 min in PBS between treatments.Finally, the cells were mounted using Fluoroshield containing DAPI (Sigma-Aldrich, F6057) and sealed under the coverslips with clear nail polish. Images were acquired using a Nikon AIR confocal microscope with a 60* oil immersion objective, and the image data were processed using Nikon NIS-Elements.

[0225] RT-PCR

[0226] Total RNA was extracted from cells 24-72 h post-transfection with Nod.HPVE7 using the RNEasy Mini Kit (Qiagen). The isolated RNA was amplified by one-step RT-PCR using SuperScript IV reverse transcriptase (Thermo Fisher Scientific) and an E7-specific forward primer (5'-CAT ATG CAT GGA GAT ACA CCT-3') and reverse primer (5'-CTA GAA CCG GTT TAT GGT TTC-3'). Reverse transcription was carried out for 10 min at 60 °C, then the cDNA was denatured at 98 °C for 2 min and amplified by applying 40 cycles ofdenaturation at 98 °C for 10 s / kb, annealing at 60 °C for 10 s / kb, and extension at 72 °C for 30 s / kb, with a final extension step at 72 °C for 5 min. The products were analyzed by 1 % agarose gel electrophoresis in Tris-acetate-EDTA (l x TAE) buffer, pH 8.0; Fisher Scientific).

[0227] Quantitative real-time PCR

[0228] RNA extracted 24-72 h post-transfection as described above was reverse transcribed using the RT2 first strand kit (Qiagen) and amplified using RT2SYBR Green qPCR Mastermix (Qiagen) and an E7-specific forward primer (5'-CGG ACA GAG CCC ATT AC A ATA-3') and reverse primer (5'-CTT CCA ACG TAC GGA TGT CT A C-3') in a CFX96 real-time thermal cycler system (Bio-Rad, Hercules, CA, USA). GAPDH cDNA was amplified for normalization, using a specific forward primer (5'-GAC TTC AAC AGT GAC TCC CAC-3') and reverse primer (5'-TCT GTT GCT GT A GCC AAA TTC-3'). Mean CT values calculated using the 2AACT method were used to determine the relative expression level of the E7 gene and the fold change in gene expression in the presence or absence of the Nodamura replicon. All experiments were carried out using triplicate samples.

[0229] Encapsulation of Nod.HPV.E7 mRNA in TMV-like particles

[0230] TMV coat protein preparation

[0021] TMV was purified from infected Nicotiana benthamiana leaves as previously described39and 10 mg of TMV particles was mixed with two volumes of glacial acetic acid (Sigma-Aldrich; A6283; 34.8 M) for 20 min on ice, followed by centrifugation at 20,000 g for 20 min at 4 °C. The supernatant was collected and dialyzed against MilliQ water for 48 h using a Spectra Por S / P 1 6-8 kDa dialysis membrane (Thermo Fisher Scientific). The precipitated coat protein was collected by centrifugation as above, and the pellet was resuspended in 75 mM sodium phosphate buffer (pH 7.2).

[0232] In vitro assembly of Nod.HPV.E7 mRNA with TMV coat protein

[0233] Nod.HPV.E7 mRNA was allowed to self-assemble with TMV coat protein by mixing the two components at a 1 :20 mRNA / protein mass ratio in sodium phosphate buffer (pH 7.2) and incubating at 30 °C for 16-18 h. The VLPs were treated with 50 pg RNase A (Thermo Fisher Scientific) at 37 °C for 30 min and purified on a 100-kDa Amicon spin column.

[0234] Validation of assembled TMV VLPs encapsulating Nod.HPV.E7 mRNA

[0235] UV-Vis spectroscopy

[0236] The concentration of TMV, coat protein, and assembled VLPs encapsulating Nod.HPV.E7 mRNA was measured by UV-Vis spectroscopy using a Nanodrop 2000 device (Thermo Fisher Scientific). Beer’s law was applied with the following extinction coefficients: TMV, s260nm = 3 pLTMV coat protein, s260 nm = 1.3 pL pg1cm The purity of TMV coat proteins confirmed by measuring the A260 / A280 and A280 / A250 absorbance ratios. The integrity of the VLPs was confirmed by comparing the A260 / A280 absorbance ratio to that of native TMV.

[0237] Size exclusion chromatography102381 The purity and integrity of assembled VLPs (0.2 mg / mL) were compared to native TMV and its coat protein using an AKTA pure fast protein liquid chromatography (FPLC) system equipped with a Superose 6 Increase column (GE Healthcare Life Sciences). The chromatography system was operated at a flow rate of 0.5 mL / min, with detectors fixed at 260 nm (nucleic acid) and 280 nm (protein).

[0239] Transmission electron microscopy

[0240] Assembled VLPs (0.5 mg / mL) were visualized alongside native TMV and coat protein using a Tecnai G2 TF20 high-resolution electron microscope (FEZ, Hillsboro). Formvar carbon-coated 300-mesh copper grids (Electron Microscopy Sciences) were rendered hydrophilic using the PELCO easiGlow operating system before negative staining with 2% (w / v) uranyl acetate (Agar Scientific), followed by imaging at 300 kV.

[0241] Immunization studies

[0242] Animal studies were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego. BALB / c female mice (n = 5, 6-7 weeks old) were obtained from the Jackson Laboratory, Bar Harbor, MA, USA (strain #000651). Mice were immunized subcutaneously (s.c.) with 100 pg of TMV VLPs in PBS (pH 7.4) (containing ~5 pg of Nod.HPVE7 mRNA encapsulated) following a prime (day 0) and boost (day 14) schedule. Dosing was selected based on previous reports using sa- mRNA vaccines for various antigens, as well as mRNA vaccines targeting HPV16 E6ZE7.40-44In future studies dose-range studies will be conducted. Blood was collected by retro-orbital bleeding before the prime (day 0) and then on days 14 and 28. Plasma was collected by centrifugation at 2000 g for 10 min at 4 °C and stored at -20 °C.

[0243] ELISA

[0244] The titers of E7-specific IgG in mouse serum on days 0, 14 and 28 were evaluated by enzyme-linked immunosorbent assay (ELISA). High-binding nickel -activated ELISA plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 pL / well of Hise-tagged E7 protein (Genscript) at a concentration of 2 mg / mL in PBS (pH 7.4). The capture antisera were diluted 1 : 100 to 1 :2800 in PBS containing 2% bovine serum albumin (BSA, Sigma- Aldrich, A7030) and were incubated on a shaking platform at RT for 1 h. For detection, Applicant added 100 pL / well of a goat anti-mouse secondary antibody (Thermo Fisher Scientific) conjugated to horseradish peroxidase (HRP), diluted 1 :5000 in PBS containing 0.05% Tween-20 (PBST), and incubated for at RT 1 h. After each incubation step, the plates were washed 3 x 5 min with 200 pL PBST (pH 7.4). The signal was developed using 100 pL / well 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific), active ingredient 3,3',5,5'-tetramethylbenzidine, for 2 min at room temperature. The reaction was stopped with 50 pL 1 M sulfuric acid (Spectrum Chemical). Absorbance was measured at 450 nm using an Infinite 200 Pro microplate reader. The reciprocal dilution at which the absorbance was twice that of the blank wells was calculated as the endpoint titer of the anti- E7 antisera.

[0245] Antibody isotyping]0246[ HPV16 E7-specific isotypes (IgGl, IgG2a, IgG2b and IgM) in the antisera were determined using an ELISA protocol similar to that described above. The ELISA plate was coated as above, and the antisera were prepared and incubated as above. However, the detection step involved secondary antibodies specific for IgGl (Invitrogen; PA174421), IgG2a (Thermo Fisher Scientific; A-10685), IgG2b (Abeam; ab97250) and IgM (Invitrogen; 31172), all diluted 1 : 1000. The type of immune response was estimated from the IgG2a / IgGl ratio.

[0247] In vitro splenocyte proliferation assay

[0248] XTT cell proliferation assay

[0249] Spleens were harvested from vaccinated and PBS-injected mice on day 35. Splenocytes were prepared according to the Spleen Dissociation Kit, Mouse (Miltenyi Biotec) using a gentleMACS™ Octo Dissociator with Heaters. The collected cells were incubated with 1 x RBC Lysis Buffer (Invitrogen) for 5 min at RT. Viable splenocytes were cultured in a 96-well plate (1 x 105 cells / well in 100 pL medium) and were stimulated with 20 pg / mL of the HP VI 6 E7 protein or PBS (control), then incubated at 37 °C for 24, 48 and 72 h. Splenocyte proliferation was measured using the XTT Cell Proliferation Kit (Thermo Fisher Scientific, X12223). The culture supernatant was collected at different time points after stimulation to quantify interferon-gamma (IFN-y) and interleukin-4 (IL-4) levels using cytokine-specific ELISA kits (BD Pharmingen).

[0250] ELISPOT assay

[0251] Splenocyte activation was also analyzed using a mouse double-color ELISPOT kit (Cellular Technology Limited). Briefly, the ELISPOT plate was coated overnight at 4 °C with capture antibody, anti-mouse IFN-y and anti-mouse IL-4 antibodies (1 : 166). Spleens were harvested from Nod.HPV.E7 immunized and control mice on days 14 and 28. Splenocyte suspensions (5x l06cells / well) were stimulated with 100 pL of medium alone (negative control), HPV E7 (20 pg / mL), wild-type TMV (10 pg / mL) or 50 ng / mL phorbol 12-myristate 13-acetate (PMA) and 1 pg / mL ionomycin (both from Sigma-Aldrich) as a positive control, at 37 °C in a 5% CO2 incubator for 24 h. After washing with PBST, the plates were incubated with FITC-labeled anti-murine IFN-y (1 : 1000 dilution) and biotin- labeled anti-murine IL-4 (1 :666) antibodies for at RT 2 h. The plates were washed, incubated with streptavidin-alkaline phosphatase (1 : 1000) and anti-FITC-HRP secondary antibodies (1 : 1000) at RT for 1 h, then washed again with PBST and distilled water, and incubated with alkaline phosphatase (AP) substrate for 15 min and then with HRP substrate for 10 min at RT. The plates were then rinsed with water and air-dried at RT overnight before analysis with an S6 Entry M2 ELISpot reader (Cellular Technology Limited).

[0252] Results and Discussion

[0253] Cloning, in vitro transcription, and encapsulation of the E7 mRNA

[0254] A mutated HPV16 E7 sequence was inserted into the self-replicating Nod.HPVE7.OAS construct and the linearized vector was used for in vitro transcription.Applicant confirmed the recovery of transcripts representing the Nod.HPVE7.OAS construct (4010 nt) and the E7 mRNA (303 nt) by electrophoresis.

[0255] TMV was obtained from the infected leaves of N. benthamiana plants and the coat protein was isolated by glacial acetic acid disassembly. The TMV particles were rod-shaped with the typical native morphology as shown by TEM, and the absorbance ratios of A260 / A280 = 1.2 and A280 / A250 ~ 1 were as anticipated (FIG. 1A top panel). Disassembled coat proteins were also visualized by TEM, and the absorbance ratios of A260 / A280 ~ 0.65 and A280 / A250 ~ 2 indicated the coat protein was pure and devoid of viral genomic RNA (FIG. 1A middle panel). Size exclusion chromatography (SEC) showed typical elution profiles for the native TMV (~8 mL) and its coat proteins (-18 mL), confirming their integrity (FIG. 1A top and middle panels). The encapsulation of Nod.HPVE7.OAS mRNA in vitro at a protein / mRNA ratio of 20: 1 allowed the assembly of VLPs -188 nm in length, as confirmed by the high aspect ratio observed in TEM images, as well as A260 / A280 and A280 / A250 absorbance ratios and SEC profiles similar to native TMV particles (FIG. 1A bottom panel). The length of the packaged mRNA defines the length of TMV-based VLPs, and the predicted length of VLPs packaging Nod.HPVE7.OAS (4010 nt) was 188 nm. This was confirmed by TEM, which revealed VLPs with a mean length of 178.65 ± 17.03 nm. Total RNA was isolated from the VLPs and amplified by RT-PCR using E7-specific primers, and the expected amplicon size of -300 bp was confirmed by agarose gel electrophoresis (FIG. IB).

[0256] Expression of replicons in vitro

[0257] BHK-21 cells were transfected with the Nod.HPVE7.OAS self-replicating construct, E7 mRNA, or with VLPs containing the Nod.HPVE7.OAS construct, and in all cases Applicant detected E7 mRNA and protein in the cells. One-step RT-PCR using E7-specific primers resulted in the amplification of a ~300-bp product from cell lysates, confirming that the VLP-mediated delivery of Nod.HPVE7.OAS into the cells was similar in efficiency to direct transfection with Nod.HPVE7.OAS RNA (FIG. 2A). Quantitative real-time PCR showed a significant ~7-fold change in the abundance of E7 mRNA when delivered as the replicon vs the non-replicon mRNA (***p<0.0001 at 24 and 48 h and **p=0.0032 at 72 h), and they both showed a significant (***p< 0.0001) increase compared to the non-transfected control (FIG. 2B).

[0258] Kinetics and magnitude of antigen expression from self-amplifying mRNA have been reported in several animal models,41, 42, 45and Applicant’s expression data aligns with these findings. Applicant had previously shown a comparison kinetics of antigen expression from self-amplifying mRNA (sa-mRNA) to non-sa-mRNA using reporter genes such as renilla luciferase and fluorescent proteins.46At equal mRNA doses, expression from the sa-mRNA construct was initially lower due to the time required for replication of longer mRNA construct; important at the 24 h time point expression levels of the sa-mRNA construct significantly increased compared to mRNA alone - and this enhanced expression was pertinent over a 7-day time frame46Overall this is consistent with the kinetic analysis performed over 3 days showing a significant fold change that decreases over time (***p<0.0001 at 24 and 48 h and **p=.0009 at 72 h) (FIG. 2B). Lastly, expression of HPVE7 protein was confirmed by confocal imaging (FIG. 2C).

[0259] Immune response to the VLP-based mRNA vaccine candidate

[0260] Mice were subcutaneously injected with VLPs using a prime-boost regimen (immunization on days 0 and 14), and blood was collected on days 0, 14, and 28 (FIG. 3A). The titer of E7-specific IgG increased significantly (***p<0.0001 for dilutions of 1 / 100, 1 / 200, and 1 / 400) after the prime and boost immunizations (FIG. 3B left panel), with an endpoint titer of ~1 :5120 (FIG. 3B right panel). Isotyping revealed high levels of E7-specific IgM and significant levels (***p< 0.0001) of IgG isotypes IgGl, IgG2a, and IgG2b (FIG. 3C left panel), confirming immunoglobulin class switching and differentiation. The high levels of IgM were expected because this is the first class of antibody expressed during B cell development in response to primary antigenic exposure.47The development of IgM antibodies against SARS-CoV-2 spike protein (S) following vaccination is associated with the development of a more effective humoral immune response, leading to long-lasting protection due to the production of anti-S IgG with higher affinity and neutralizing efficacy.48, 49The IgG2a / IgGl ratio was < 1, indicating a Th2 -biased immune response (FIG. 3C right panel).

[0261] Murine splenocyte proliferation in vitro and E7-specific T cell memory response

[0262] Vaccines should induce long-lived memory T cells that clonally expand and differentiate into effector cells when they encounter the previously administered antigen byproducing IFN-y, TNFa and IL-2.50’51Spleen cell populations cultivated in vitro mature following stimulation with the E7 protein, causing naive T cells to proliferate into E7-specific effector T cells. The elicitation of a T cell memory response following exposure to TMV.Nod.HPV-E7.OAS was evident from the increased proliferation of E7-stimulated splenocytes harvested from TMV.Nod.HPV-E7.OAS-immunized mice compared to PBS- immunized controls after 24 and 48 h (FIG. 4A). Following stimulation, the proliferation index increased to -1.76 after 24 h and -1.5 after 48 h, as determined from the A450 stimulated / unstimulated absorbance ratio (FIG. 4A).

[0263] The post-stimulation culture supernatant showed significantly higher levels of IFN-y (**p = 0.0073 at 24 h ***p = 0.0003 at 48 h. ***p = 0.0002 at 72 h) in the immunized mice (FIG. 4B). ELISA revealed the presence of -4303 pg / mL IFN-y at 24 h, -6058 pg / mL at 48 h, and -6354 pg / mL at 72 h. ELISpot assays confirmed the significantly higher number of IFN-y-secreting cells (***p < 0.001) among E7-stimulated splenocytes from the immunized mice (FIG. 4B). Splenocyte proliferation and the generation of the Thl signature cytokine IFN-y following the stimulation of splenocytes with E7, suggest priming of T cell (memory) response in the vaccinated mice.

[0264] Conclusions

[0265] The rapid development of mRNA vaccines in response to COVID-19 has generated interest in the application of this technology against HPV. The efficacy of an LNP- encapsulated mRNA vaccine encoding HPV E7 (HPV mRNA-LNP) was demonstrated in a mouse model of HPV+ oropharyngeal squamous cell carcinoma.43As an alternative to LNPs, Applicant tested VLPs derived from the plant virus TMV because plant viruses have inherent immunostimulatory properties, acting as an adjuvant as well as a carrier.59Applicant demonstrated the suitability of TMV as a platform for development of therapeutic HPV vaccines by using it to deliver a self-amplifying shuffled E7 mRNA, which elicited a potent humoral and cellular immune response in mice.

[0266] Applicant’s work provides the foundation for the development of TMV-based therapeutic vaccines against HPV and can be expanded to include combinations of E6 and E7 to trigger a more potent and durable immune response.10267] Experiment No. 2

[0268] Introduction

[0269] COVID-19 has taught us that rapid development, distribution, and administration of vaccines to the global population is the most effective approach to quell emerging pandemics1'3. Vaccine design itself is challenging, but also its manufacture and global distribution; cold chain requirements present logistical and fiscal barriers to the availability of important, life-saving vaccines in resource-poor areas of the world. When designing a vaccine, there are several strategies to choose from and nanotechnology platforms offer great utility in modern vaccine design. The rapid development of a COVID-19 vaccine was possible because the genome and structural information of SARS-CoV-2 was made available in record time4'6; and the repurposing of a mature lipid nanoparticle delivery systems enabled rapid clinical development of a COVID-19 vaccine7. For fast emerging viral infections and pandemics such as CO VID-19, rapid development, and large-scale deployment of vaccines is a critical need.

[0270] While mRNA vaccines offer rapid production and avoid the need for BSL-3 facilities, they are dependent on efficient nanocarriers for delivery. Applicant turned toward the viruslike particles (VLPs) from tobacco mosaic virus (TMV) to package a self-replicating mRNA (making use of the Nodamura virus replicon8'13) encoding the SARS-CoV-2 receptor binding domain (RBD) protein (FIG. 5). Nucleic acid delivery using the VLPs from plant viruses and bacteriophages has been demonstrated14; and plant virus nanotechnologies including TMV have been applied as mRNA vaccine delivery technology13, 15. The plant virus nanotechnology is a unique platform offering several distinct advantages: First, native TMV nanoparticles are stable to high temperatures and solvents16, and therefore do not require refrigeration during storage or distribution. Second, while plant viruses and their VLPs are not infectious toward mammals (thus offering safety), they are highly visible to the immune system - therefore the proposed delivery platform, not only serves as a delivery vehicle but also an adjuvant. Their highly organized and repetitive structures act as pathogen-associated molecular patterns (PAMPs), triggering the innate immune system through pattern recognition receptors (PRRs), most commonly toll-like receptors (TLRs)17While the proteinaceous capsids are recognized by TLR218(and likely other PRRs), RNA-containing VLPs also signal through TLR7 / 819’20. These immune-stimulatory properties in combinationwith their size make them ideal candidates for vaccine delivery to the draining lymph nodes and priming interactions with antigen-presenting cells (APCs)21.

[0271] In this work, Applicant designed a COVID-19 vaccine candidate by making use of a self-amplifying Nodamura virus replicon encoding the RBD domain from SARS-CoV-2 spike protein. The construct was obtained by in vitro transcription and then packaged into TMV VLPs through reconstitution using purified coat protein. Nanoparticle vaccines were characterized and transduction efficiency and vaccine efficacy was evaluated in in vitro and in vivo models demonstrating neutralizing activity against SARS-CoV-2.

[0272] Materials and Methods

[0273] Molecular cloning and plasmid construct

[0274] The plasmid constructs pT7.Nov.OmicronRBD.OAS and pT7.OmicronRBD expressing ribosome binding domain of the spike protein against the Omicron variant of the SARS-CoV-2 with or without self-amplifying Nodamura replicon (Nod) was cloned into the plasmid pNod.Luc.OAS (kindly provided by Dr. Gelbart’s lab, UCLA) as described earlier in Gitlin et al 2014. For no replicon construct, cloning was performed in plasmid pET22b(+) using restriction enzyme sites-5’ Ndel and 3’AgeI respectively. These synthetic constructs were obtained (GenScript Co., Piscataway, NJ) and the spike protein region encoding 325- 547 amino acids of the ribosome binding domain from the Omicron variant (B.1.1.529) of SARS-CoV-2 was extracted from GenBank with the accession no. LC731729.1. The selected amino acid sequence contains nine cysteine residues indicated to facilitate folding and biological activity of RBD6,63. Among these nine cysteine residues, eight form four disulfide bonds (Cys336-Cys361, Cys379-Cys432, Cys391-Cys525) that contribute to the stability and functionality of the RBD protein. The ninth cysteine (Cys480) likely is involved in inter- molecular dimerization but does not impair the stability, folding or functionality of the RBD. Cloning of the coding region encoding for OmicronRBD domain into the plasmids pT7.Nod.OmicronRBD.OAS and pT7. OmicronRBD was confirmed by restriction digestion analysis and sequencing.

[0275] mRNA transcript preparation

[0276] In vitro synthesis of capped mRNA was achieved by transcribing the linearized plasmid DNA template using HiScribe T7 ARCA mRNA Kit (New England Biolabs,Ipswich, MA). The plasmid constructs pT7.Nod.OmicronRBD.OAS and pT7.OmicronRBD was linearized using A7 / I-a unique restriction enzyme site present at the C-terminus of the plasmid. The linearized plasmids pT7.Nod.OmicronRBD.OAS and pT7.OmicronRBD were transcribed using HiScribe T7 ARCA mRNA Kit (New England Biolabs, Ipswich, MA) following manufacturer’s instructions. The transcribed mRNA coding for Nod.OmicronRBD.OAS and OmicronRBD were precipitated by using RNase free Lithium chloride solution. The purity and quality of transcribed mRNA were analysed by UV-Vis spectroscopy, and Agilent 2100 bioanalyzer.102771 Functional characterization of transcribed mRNA

[0278] Transfection. The transcribed Nod.OmicronRBD.OAS and OmicronRBD mRNA were transfected into the mammalian cells by using lipofectamine 2000 reagent (Thermo Fisher Scientific) following manufacturer’s instructions. The Baby Hamster Kidney Fibroblast cells (BHK-21) were seeded into 6-well plates in (80,000; 500 pl) Dulbecco’s Modified Eagle Medium complete medium (Thermo Fisher Scientific) with 10 % (v / v) fetal bovine serum (Thermo Fisher Scientific) and 1% (w / v) penicillin / streptomycin (Thermo Fisher Scientific). The mRNA (500 ng) and lipofectamine dilution (5 pl) prepared in Opti- MEM™ reduced serum medium (Thermo Fisher Scientific) in a ratio of 1 : 1 followed by incubation for 5 min at room temperature. Then the cationic mRNA- lipids complex formulations were added to the cells and allowed for further incubations at 37°C for 24-72 h. The transfected cells were analyzed by RT-PCR, quantitative RT-PCR, Confocal imaging.

[0279] Reverse Transcription PCR (RT-PCR). To confirm the transfection of Nod.OmicronRBD.OAS and OmicronRBD mRNA, the total RNA was purified from the transfected cells between 24- 72 h using RNEasy Mini Kit (Qiagen) and the collected cell lysates were homogenised using QIAshredder homogenizer kit (Qiagen, Valencia, CA) as per the manufacturer’s descriptions. SuperScript™ IV One-Step RT-PCR (ThermoFischer Scientific) was performed using specific OmicronRBD primers to detect the OmicronRBD from the transfected cells. The reaction mixture comprises the RNA samples isolated from the cells, 2X Platinum™ SuperFi™ master mix, forward primer (Sequence: CAT ATG CGA GTT CAG CCT AC; 10 pM), reverse primer (Sequence: CTA GAC ACC GGT TCA GAA AT; 10 pM), and SuperScript™ IV enzyme in a nuclease-free water. The reaction mixture was incubated for reverse transcription of isolated RNA at 60°C; 10 min. For the polymerasereaction, initial denaturation at 98°C; 2 min and then amplification was followed for 40 cycles at 98°C; 10 s, 60°C; 10 s and 72°C; 30 s / kb with the final extension at 72°C for 5 min. Agarose gel electrophoresis was performed in Tris-acetate-EDTA electrophoresis buffer (l x TAE, pH 8.0) to analyse the amplified PCR product.|0280[ Quantitative real time PCR (RT-qPCR) . For quantitative analysis of OmicronRBD mRNA with or without the Nodamura replicon in the transfected cells, the RNA collected post transfection at 24-72 h was reverse transcribed by synthesizing complementary DNA using RT2first strand kit (Qiagen). SYBR Green-based real time quantification was performed using RT2SYBR Green qPCR mastermix (Qiagen) with OmicronRBD and GAPDH-gene specific primers (RBD forward: 5’ - ACG CTT CGC TTC AGT CTA TG-3’, RBD reverse: 5 ’-GTG AAG AAG GGT GCC AGA TTA (GAPDH forward: 5’- GACTTCAACAGTGACTCCCAC-3’, reverse: 5’- TCTGTTGCTGTAGCCAAATTC-3’). Mean CT values and the relative expression of OmicronRBD gene or multifold change of OmicronRBD in the presence or absence of Nodamura replicon were calculated by 2-delta delta CT values. Quantitative real time PCR was performed in triplicate in a CFX96 real time thermal cycler system (Biorad).

[0281] Confocal Imaging

[0282] To visualize the cellular expression of Nod. OmicronRBD. OAS mRNA on the BHK- 21 cells, the immunofluorescence staining of the transfected cells and confocal imaging was performed. BHK-21 cells (80,000 cells / 500 pL) were seeded on the coverslips (13 mm) in 6 well cell culture plate prior to transfection. The cells were allowed to attain confluency before proceeding for lipofectamine mediated transfection of Nod. OmicronRBD. OAS mRNA (500 ng) as described above. Post 24 h of transfection, the cells were washed with phosphate buffered saline (PBS, pH 7.4) and fixed in 4 % paraformaldehyde in PBS for 10 min at RT. The cell membrane of the transfected cells were stained with wheat germ agglutinin (WGA) conjugated with Alexa Fluor 555, (1 : 1000; Invitrogen™W32464) and then permeabilized with 0.2 % Triton™ X-100 for 2 min and blocked with 2 % BSA solution prepared in PBS, pH 7.4 buffer for 1 h at RT. Primary antibody labelling with Omicron specific anti-SARS- CoV-2 Spike RBD antibody, mouse IgGl (1 :50; ACROBiosystems, SPD M305) followed secondary antibody labelling using goat anti-mouse IgG (H&L) - Alexa Fluor™ 488 (1 : 100; Invitrogen™A-l 1001) in blocking solution. After every step, the cells were washed withPBS, pH 7.4 and were mounted onto slides with histology mounting medium fluoroshield containing DAPI (Sigma-Aldrich, St. Louis, MO) The stained cells were visualized using Nikon AIR confocal microscopy and the acquired images were analysed using Nikon NIS- Elements software.|0283| TMV Coat protein preparation, large scale assembly of TMV and transcribed Nod.OmicronRBD.OAS

[0284] TMV coat protein was prepared by using glacial acetic acid degradation method as described earlier29. In brief, the native TMV (10 mg in 0.1 M KP buffer, pH 7.4) was treated with 2 volumes of glacial acetic acid on ice for 20 min. The precipitated TMV genomic RNA was removed by centrifugation at 20,000 g for 20 min at 4 °C. The supernatant containing TMV coat protein was collected for dialysis. The supernatant was transferred into dialysis tubing using a 6-8 kDa dialysis membrane (Spectra Por S / P 1 Dialysis Membrane; Thermo Fisher Scientific) against MilliQ H2O for 48 h - 96 h at 4 °C, changing water every 12 h. After dialysis, the white precipitate of TMV CP was collected by centrifugation at 20,000*g for 20 min at 4 °C. The CP were then resuspended in 75 mM sodium phosphate buffer, pH 7.2. In vitro self-assembly of transcribed Nod.OmicronRBD.OAS were prepared with a 20:1 (CP:mRNA) mass ratio of TMV CP and mRNA in 75 mM sodium phosphate buffer, pH 7.2 and were incubated for 16-18 h at 30 °C64

[0285] Characterization of assembled TMV VLPs|0286| UV-Vis. Native TMV, TMV CP and assembled TMV.Nod.OmicronRBD.OAS VLPs concentration was measured by UV-Vis spectroscopy (Nanodrop 2000, Thermo Fisher Scientific), using Beer’s Law (TMV, s260nm = 3 pl.pg-l.cm-1 and TMV CP,S26o nm = 1.3 pL.pg-l.cm-1). TMV CP was confirmed by absorbance ratio of A260 / A280 = 0.65 and A280 / A250 = 2, compared to native or assembled TMV that shows absorbance ratio A260 / A280 = 1.2.

[0287] Size exclusion chromatography (SEC). Purity and integrity of native TMV (1 mg / mL), TMV CP (1 mg / mL) and assembled TMV.Nod.OmicronRBD.OAS (0.2 mg / mL) was assessed using AKTA pure Fast Protein Liquid chromatography system equipped with a Superose6 Increase size exclusion column (GE Healthcare LifeSciences), using a flow rate of 0.5 mL / min, fixing the detectors at 260 nm (nucleic acid) and 280 nm (protein).[0288| Transmission Electron Microscopy. Native TMV, TMV CP and assembled TMV.Nod.OmicronRBD.OAS (0.1-0.5 mg / mL in deionized water (DI H2O)) were loaded onto Formvar-carbon coated copper grids (300 mesh; Electron Microscopy Sciences); PELCO easiGlow operating system was used to render more hydrophilic grids. Grids were negative-stained with 2% (w / v) uranyl acetate (Agar Scientific) and imaged using Tecnai G2 TF20 High-Resolution electron microscope (FEZ, USA). VLP particle sizes were derived from TEM grids using the software ImageJ (https: / / imagej.net / ij / ) and evaluated by analysis of variance (ANOVA) using OriginPro 2024 (OriginLab) with a post hoc Bonferroni test38and a significance level of a = 0.05. In statistical tests comparing two groups, N refers to the total number of samples from both groups, whereas n refers to the number of samples from a single group.

[0289] Mice immunization

[0290] Female BALB / c mice (n=5, six- to seven-weeks-old) was obtained from The Jackson Laboratory (Strain #:000651) and animal experiments were conducted in accordance with Institutional Animal Care and Use Committee (IACUC), University of California San Diego. TMV.Nod.OmicronRBD.OAS VLPs (100 pg of VLPs containing ~5 pg Nod.OmicronRBD.OAS mRNA in lx PBS pH 7.4) were injected in the mice, subcutaneously (s.c.) behind the neck, following a biweekly prime-boost immunization schedule. Blood was collected using lithium-heparin-treated tubes (Thomas Scientific) by retro-orbital bleeding on day 0 (prior immunization, PI), and then on days 14 and 21 post-immunizations (B-l and B- 2). Plasma was separated by centrifugation at 2,000xg for 10 min at 4°C and stored at -20 °C until analyzed.

[0291] Detection of anti-Omicron-RBD of SARS-CoV-2 (B.l.1.529) specific antibodies in mice plasma

[0292] To determine the presence of specific antibodies against Omicron-RBD of SARS- CoV-2 (B.l.1.529), direct enzyme linked immunosorbent assay (ELISA) was performed using 96-well Pierce high binding nickel-activated ELISA plates (Thermo Fisher Scientific). Plates were coated with polyhistidinelO tagged Omicron-RBD protein (GenScript Co., 200 ng / 100 pL PBS pH 7.4 / well) overnight at 4 °C. After every incubation, plates were washed three times with 200 pL / well of PBST (0.05 % (v / v) Tween-20 in PBS, pH 7.4). Mice plasmawas diluted in two-fold serial dilution (2 % BSA, PBST), added to the plates and incubated for 1 h at room temperature (RT) in a microplate shaker incubator (400 rpm). After washing, plates were incubated with 1 :5000 dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (BSA 1% (v / v) in PBST; 100 pL / well; Thermo Fisher Scientific) and incubated for Ih at room temperature (RT) in a microplate shaker incubator (400 rpm). After a final washing, plates were developed using 1-Step Ultra TMB-ELISA substrate solution (100 pL / well; 3,3',5,5'-tetramethylbenzidine, Thermo Fisher Scientific) for 2 min at RT, followed by quenching with 50 pL of 2N sulfuric acid (Spectrum Chemical). Absorbance at 450 nm was measured using an Infinite 200 Pro microplate reader and i- control software (Tecan, Mannedorf, Switzerland). Endpoint antibody titers were determined as the reciprocal dilution at which the absorbance value was twice higher than the blank wells without mice plasma (background). Levels of different IgGs subclasses (IgGl, IgG2a and IgG2b) in mice plasma was determined as described above. Plasma samples were diluted at 1 :200 (in PBS) followed by incubation for Ih at room temperature (RT) in a microplate shaker incubator (400 rpm). Secondary HRP -labeled goat anti-mouse antibodies specific for IgGl (Invitrogen PA174421), IgG2a (Thermo Scientific A-10685), IgG2b (Abeam ab97250) were diluted 1 : 1000 followed by incubation for Ih at room temperature (RT) in a microplate shaker incubator (400 rpm). To determine the type of immune responses, the ratio of IgG2a / IgGl ratio was reported and ratio higher than 1 was considered as a Thl response.

[0293] Omicron SARS-CoV-2 Neutralization assay

[0294] ePass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript Co.) was used to detect the presence of neutralizing antibodies to Omicron SARS-CoV-2 RBD in mice plasma using a blocking Enzyme Linked Immunosorbent Assay (ELISA). The assay was performed according to the manufacturer’s protocol. Briefly, mice plasma, positive and negative controls (provided in the kit) were diluted to 1 : 10 in sample dilution buffer (Cat No Sl-60, GenScript Co.). SARS-CoV-2 (Omicron) neutralizing antibody calibration curve (monoclonal antibodies with neutralization activity to SARS-CoV-2 (Omicron)) was prepared by a 2-fold serial dilution in sample dilution buffer (300 U / mL-4.688 U / mL). Then, calibration curve, diluted mice plasma and controls were mixed with 1 : 1000 diluted RBD- HRP solution at 37 °C for 30 min; the RBD-HRP neutralization reaction mixtures was then added to the angiotensin converting enzyme-2 (ACE2)- coated assay microtiter plate foranother 30 min at 37 °C, to evaluate the interaction of free RBD-HRP with ACE2. Plate was washed four times with wash solution (provided by the manufacturer). For signal development, plate was incubated in dark with TMB solution at 25 °C for 15 min. After adding the stop solution, the absorbance measurement in microtiter plate reader was performed immediately at 450 nm. The signal inhibition (%) was calculated for the neutralizing Omicron SARS-CoV-2 antibodies present in the antisera as mentioned in the formula:Signal inhibition (%) = (1 - OD value of sample / OD value of negative control)* 100%

[0295] The cut-off percentage of signal inhibition >30%, should be considered positive for neutralizing antibodies as claimed by the manufacturer.

[0296] Results

[0297] Replicon design and synthesis

[0298] For mRNA synthesis, the plasmid constructs pNod.OmicronRBD.OAS and pOmicronRBD were designed by molecular cloning (FIG. 1A and FIG. 9). The vector cassettes contained the following features:■ A T7 promoter for in vitro transcription of mRNA from the plasmid DNA.■ The vectors were designed with and without the Nodamura replicon (Nod), which was added to increase longevity of the mRNA in vivo8'13.■ The T2A self-cleaving peptide sequence, known for its high “cleavage” efficiency (resulting in a ribosomal skip22), was inserted between the Nodamura replicon and OmicronRBD to enable translation of the OmicronRBD protein.■ The antigen was RBD from Omicron strain of SARS-CoV-2 (OmicronRBD).■ Finally, the TMV’s origin of assembly sequence (OAS), a hairpin structure, specifically the 234-nt sequence spanning nucleotides 5313 to 5546 in the TMV genome, was appended to the 3 ’ end of the mRNA. The OAS facilitates coat protein binding and subsequent nucleation of the cylindrical capsid23-27

[0299] The mRNA cassette was cloned under the control of the T7 promoter, and linearized plasmids were generated using the Xbal restriction enzyme (FIG. 10). Hiscribe anti-reverse capping (ARCA)-T7 polymerase was employed for highly efficient transcription of a capped mRNA cassette. The transcription reaction was concluded by "polymerase running-off fromthe linearized plasmid template, resulting in the production of capped transcripts Nod.OmicronRBD.OAS and OmicronRBD at the 5' end. Given the high stability of the Nodamura replicon construct, Applicant chose not to include poly(A) tailing28.

[0300] Analysis of the mRNA transcript concentration, purity, and size was conducted using UV-Visible spectroscopy (not shown) and a bioanalyzer (FIG. 10). The absorbance ratio of A260 / 280 - 2 indicated the purity of the capped mRNA transcripts. This was consistent also with bioanalyzer results confirming pure transcripts with the expected size of ~ 3.7 kb for Nod.OmicronRBD.OAS and ~ 0.7 kb for the OmicronRBD mRNA (FIG. 10).

[0301] Packaging the replicons into VLPs

[0302] For in vitro encapsulation of the transcribed mRNA containing the 234-nts OAS (FIG. 1A) of TMV, Applicant produced TMV coat proteins (CPs) using the established glacial acetic acid disassembly method29. TMV CP was obtained from TMV virions produced in Nicotiana benthamiana and established molecular farming and purification methods30. Transmission electron microscopy (TEM) imaging shows the typical high aspect ratio rods of native TMV as well as monodispersed and disassembled CP arranged as stacked disks (Figure IB). The resulting CPs were then used for packaging the mRNA transcripts at a mass ratio of 20: 1 (CP:mRNA). The in vitro self-assembled TMV.Nod. OmicronRBD. OAS VLPs revealed typical high aspect ratio nanotubes (length of -200 nm), which matched the theoretical length of the VLPs which was determined at 206 nm (Figure IB). The RNA serves as a ruler defining the length of TMV; native TMV packages a 6.3 kb genome and measures -300 nm. The Nod.OmicronRBD.OAS replicon measures 4.3 kb and hence results in -200 nm rods. High resolution size exclusion chromatography (SEC) was used to assess VLPs purity and integrity based on the physicochemical properties. The chromatographic monodisperse peaks observed in Applicant’s study indicated the purity of native TMV, TMV coat proteins (CP), and reassembled TMV.Nod. OmicronRBD. OAS VLPs. Size exclusion chromatography (SEC) was in good agreement with TEM and showed the typical elution profile of native TMV (~ 8 mL), TMV CPs (- 18 mL) and reassembled TMV VLPs (~ 8 mL) using a Superose6 Increase column (Figure ID). The SEC profiles provide insights into the purity of the plant viral expression vector: This method separates macromolecules (VLPs, CPs, mRNA) based on size, allowing for the separation of intact TMV VLPs from free CPs or unpackaged mRNA as well as other impurities. Due to high molecular weightsassembled TMV.Nod.OmicronRBD.OAS VLPs or native TMV eluate at ~ 8 mL from the Superose6 Increase column. In stark contrast, free CP elutes at ~ 18 mL, therefore allowing clear differentiation between the states (Figure 6D). Analyses of the obtained chromatogram from the TMV.Nod.OmicronRBD.OAS VLP samples shows a major peak at 8.7 mL with an A260 / 280 ratio of ~1.1 indicative of VLPs packaging the mRNA cassette. This peak corresponds to - 90% of the sample; a 10% fraction eluted > 20 mL which may indicate free CP or mRNA impurities. Overall, the SEC trace is in good agreement with the TEM data and absorbance readings by UV-Vis. Purity of TMV CP free from genomic RNA contamination was determined using UV-Vis, showing an absorbance ratio of A260 / A280 = -0.65 and A280 / A250 = -2; in contrast intact TMV has an absorbance ratio of A260 / A280 = 1.2 and A280 / A250 = -1 (not shown). Lastly, to confirm that indeed the target replicon was packaged into the VLPs, total RNA from VLPs was isolated and analyzed by RT-PCR using OmicronRBD gene specific primers - analysis by agarose gel electrophoresis confirmed the expected size and hence packing of Nod. OmicronRBD. OAS mRNA into TMV VLPs (Figure 1C). Finally, the yield of the assembled TMV.Nod.OmicronRBD.OAS VLPs was determined based on BCA assay or UV absorbance at A280, and recovery yields were -50- 60 % of the TMV starting amount.

[0303] An optimized purification method was established, and Applicant compared the use of spin filtration using a 100-kDa molecular weight cut-off (MWCO) vs. ultracentrifugation over a 30% (w / v) sucrose cushion. Purified Nod. OmicronRBD. OAS VLP particles were then analyzed by TEM imaging (data not shown). When using the spin filters, the dominant structures, accounting for 66% of particles, were TMV disk aggregates (-18 nm, data not shown). Only -11% of all particles observed were the target Nod. OmicronRBD. OAS VLPs measuring -200 nm in length (data not shown). The remaining fraction were shorter assemblies or broken particles. The yield of -200 nm-sized Nod. OmicronRBD. OAS VLPs was significantly increased when using the ultracentrifugation method: the number of disc aggregates was drastically reduced and the frequency of -200 nm VLPs increased from 11% to 34% (data not shown). This observation was consistent with literature using ultracentrifugation to eliminate incomplete assemblies of virus particles31. Applicant concluded that ultracentrifugation was best suited for purification from assembledNod.OmicronRBD.OAS VLPs, whereas spin filtration could be used to purify and concentrate TMV disk aggregates before assembly.

[0304] Stability of TMV-Nod.OmicronRBD.OAS VLPs

[0305] Plant viruses are recognized as stable biologies and native TMV remains structurally sound at temperatures up to 50°C32. Therefore, Applicant investigated the stability of the in vitro reconstituted TMV-Nod.OmicronRBD.OAS VLPs. Stability was assayed by TEM imaging after 1-week incubation of TMV-Nod.OmicronRBD.OAS VLPs in 10 mM KPO4 buffer pH 7 at -20°C, 4°C, 22°C, 37°C and 50°C. Frozen TMV-Nod.OmicronRBD.OAS VLPs were also subjected to 5 freeze-thaw cycles (-20°C, 22°C) (FIG. 6). The key findings are: VLPs were stable at 4°C as well as -20°C and tolerated repeated freezing and thawing. Applicant note that some particle breakage occurred (slight increase in the faction -100 nm- sized VLPs, albeit not statistically significant, see FIG. 6) after repeated freeze-thaw cycles (data not shown), and this is consistent with literature reports31. At 4°C and 22°C, there is tendency for more TMV disc aggregates (-18 nm) (data not shown), while at higher temperatures (37°C and 50°C), there was a tendency of larger assemblies, formed by free CPs, discs or by end-to-end assembly of VLPs (data not shown). The tendency for selfassembly as a function of temperature is consistent with other reports33‘34

[0306] Importantly, data indicate that TMV.Nod.OmicronRBD.OAS VLPs are suitable for storage in a fridge (4°C) or freezer (-20°C). Significant changes in particle size was observed at elevated temperatures with a size decrease at 22°C (ANOVA, p = 0.0015, N = 276, a = 0.05), and increase after storage at 37°C (ANOVA, p < 0.0001, N = 126, a = 0.05) or 50°C (ANOVA, p < 0.0001, N = 175, a = 0.05) (FIG. 6). Therefore this vaccine candidate omits the need specialized ultralow freezers (-60°C to -80°C) that were required for lipid nanoparticle-based COVID-19 vaccines35, 36. The tolerance of TMV-Nod.OmicronRBD.OAS towards repeated freeze-thaw cycles is another advantage, considering that cold chain breaches are rather common37

[0307] Expression of the replicon in vitro

[0308] To test expression of the transcribed mRNA cassette in mammalian cells, Applicant transfected either free OmicronRBD, free Nod.OmicronRBD.OAS or assembled TMV.Nod.OmicronRBD.OAS VLPs into BHK-21 cells, and observed the expression ofRBD. Applicant tested transfection using ‘naked’ mRNA to validate the designed mRNA is functional. RT-PCR analysis was performed from the RNA isolated from the transfected cell lysates indicating the amplicon at the expected size of -680 bp (FIG. 7A); and this was observed for the ‘naked’ mRNA’ and the TMV-packaged mRNA constructs. Next, Applicant carried out quantitative gene analysis of RBD in either presence or absence of self-amplifying Nodamura replicon. Here Applicant focused on the free mRNA constructs (not VLPs) - to determine the level of amplification as a result of the Nodamura replicon. Indeed significant increase of the OmicronRBD transcript was observed, resulting in a -10-fold increase of Nod.OmicronRBD.OAS vs. OmicronRBD 24 h post transfection (FIG. 7B

[0309] Finally, expression of the RBD protein in BHK-21 cells was confirmed using Omicron-specific anti-SARS-CoV-2 Spike RBD antibody and confocal imaging. Taken together this data confirms the functionality of the mRNA transcripts (FIG. 7C).

[0310] Mice immunization using the TMV.Nod.OmicronRBD.OAS mRNA vaccine candidate[03111 Effectiveness of the TMV.Nod.OmicronRBD.OAS mRNA vaccine candidate was evaluated in BALB / c mice following a prime-boost immunization schedule (n=5, 100 pg of vaccine candidate, s.c., 14 days apart), as previously reported39-41; PBS was used as a placebo. Blood was collected by retro-orbital bleeding prior the first immunization (preimmunization or PI, FIG. 8A), and then 14- and 28-days post-immunizations (bleed or B-l and B-2, FIG. 8A). Plasma was separated by centrifugation and screened for anti- OmicronRBD-specific antibodies and IgG subclasses using ELISA. Following the first immunization (prime), the TMV.Nod.OmicronRBD.OAS mRNA vaccine candidate elicited antibodies against Omicron-RBD of SARS-CoV-2 (B.1.1.529) and titers slightly increased after the second immunization (boost; FIG. 8B) reaching an endpoint titer of -1 :4,200 (prime) and -1 :6,400 (boost), respectively (-1.5-fold increase, FIG. 8C). IgG subclasses (IgGl, IgG2a / b) were determined (FIG. 8D) and data suggest an overall balanced Thl / Th2 with a trend toward Th2-bias (IgG2a / IgGl<l ratio) (FIG. 8E).

[0312] Finally Applicant assayed whether the antibodies elicited by the TMV.Nod.OmicronRBD.OAS mRNA vaccine candidate were neutralizing. A GenScript ePass SARS-CoV-2 Neutralization Antibody Detection Kit was used - the assay principle isa blocking ELISA that detects IgG that neutralize the interaction between RBD and hACE2 receptor. Pooled plasma (n=5) pre- (PI) and post-immunizations (B-l and B-2) were used and their neutralizing effect on the OmicronRBD-hACE2 interaction probed. Signal inhibition of >30% is a positive result - and with -35% and -38% signal inhibition Applicant’s vaccine candidate indeed elicited neutralization antibodies. Based on the standard curve and 10-fold dilution of the plasma analyzed, the mouse plasma reached a concentration of -1000 U / mL RBD-specific antibodies (FIG. 8F).

[0313] Discussion

[0314] Applicant successfully developed an mRNA vaccine candidate by making use of the TMV coat proteins and in vitro packaging of a Nodamura virus replicon-based mRNA cassette enabling RBD expression in cells and more importantly in mice yielding SARS- CoV-2 neutralizing antibodies, existing technologies - however the data highlight utility of this platform technology.

[0315] The COVID19 pandemic highlighted the opportunity of platform nanotechnology and power of genetic medicine. Viral and non-viral vector technologies are being developed, but only a small number of platforms is under consideration. Viruses are the most abundant biological entities on the planet with an estimated 1031viruses on Earth - this biodiversity offers tools for biotechnology. Since the discovery of tobacco mosaic virus (TMV) in 189842and its use for guiding principles of structural biology16, it has been repurposed for many different applications16. A few isolated prior works demonstrated feasibility of plant viral gene delivery: for example, TMV was utilized to deliver mRNA encoding GFP15; similar marker gene expression has been demonstrated using cowpea chlorotic mottle virus (CCMV)10. CCMV was further engineered to deliver replicons43, 44enabling amplification and expression of model antigens for vaccine development13. .

[0316] Compared to the contemporary viral vectors or synthetic nanoparticle systems, plant virus offer advantages: Plant viruses can be considered safer for use in humans compared to their mammalian counterparts, because plant viruses do not replicate in or infect mammals45. The system is non-integrating and therefore does not bear the risk insertional mutagenesis46. Applicant has shown that the plant virus based-delivery system can be administered at doses of up to 100 mg (1016particles) per kg body weight without clinical toxicity47,48. Theplatform demonstrates excellent blood and tissue compatibility49,50. Plant viruses are immunogenic and naturally target immune cells - thus providing a means of targeted delivery system with adjuvant properties. Antibody opsonization plays a fundamental role in nanoparticle and plant virus clearance and it aids the targeting immune cells. For example, Applicant demonstrated that prevalence of antibodies against cowpea mosaic virus (CPMV) enhances anti -tumor efficacy of the immunomodulatory CPMV nanoparticle51.

[0317] Further advantages of plant virus-based vectors are the availability of scalable manufacturing processes. Plant viruses and their virus-like particles (VLPs = genome-free versions) can be produced in plants or heterologous expression systems in high yields: manufacture in plants produces yields of up 1-2 grams plant virus-based particles per leaf tissue52. This is in stark contrast to production of mammalian viral vectors, which are produced in tissue culture which yielding milligrams per 1 1 cell culture53. The plant virusbased approach also provides advantages compared to synthetic systems: while manufacture of synthetic system is scalable, some systems are not stable in aqueous suspensions54'57. Finally, TMV exhibits exceptional stability in various media and across a wide range of temperatures - therefore ultralow freezers are not required for distribution. Innovating vaccine delivery platforms to break cold chain limitations is an efficient solution to safeguard potent vaccination for both wealthy and lower-income countries. Finally, one could further engineer the system to produce and self-assemble TMV-based VLPs laden with the desired replicons through molecular farming involving relatively non-sophisticated technology - possible allowing the production in the region for the region for low-resource areas58.

[0318] Conclusion

[0319] In conclusion, Applicant demonstrated a platform nanotechnology making use of the tobacco mosaic virus coat proteins self-assembled to package a replicon-based mRNA cassette - in vitro assembly is primed by inclusion of an origin-of-assembly site to facilitate packaging yielding mRNA-laden TMV. Through a combination of in vitro and in vivo assays Applicant demonstrated effectiveness of mRNA delivery and target antigen expression in mammalian cells and mice. SARS-CoV-2 specific immunoglobulins (IgG) were produced; the immune response was Thl / 2 balanced and most important IgGs were neutralizing as demonstrated by blocking of RBD-hACE2 receptor interactions. Because TMV and its coat proteins can be scaled through molecular farming or fermentation and the assembly-n-technology is simple while offering stability, this system holds promise to serve a platform nanotechnology for nucleic acid delivery.

[0320] Equivalents

[0321] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0322] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0323] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0324] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

[0325] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0326] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is alsothereby described in terms of any individual member or subgroup of members of the Markush group.

[0327] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0328] Clauses

[0329] Clause 1. A cassette for the delivery of one or more mRNA, the cassette comprising an optionally 5' mRNA cap, a 5' UTR, a Nodamura replicon, a ribosomal skipping polynucleotide, the one or more mRNA, a plant virus assembly origin polynucleotide ("OAS"), and a 3' UTR, optionally wherein the cassette does not comprise a polyA tail.

[0330] Clause 2. The cassette of clause 1, wherein the mRNA is selected from an mRNA vaccine or an mRNA encoding a therapeutic peptide.

[0331] Clause 3. The cassette of clause 2, wherein the mRNA vaccine is selected from an Omicron vaccine or a Human papillomavirus (HPV) vaccine.

[0332] Clause 4. The cassette of any one of clauses 1-3, wherein the mRNA encodes a polypeptide selected from an HPV E6 polypeptide, an HPVE7 polypeptide, a mutated HPV E7 polypeptide, or an Omicron RBD polypeptide.

[0333] Clause 5. The cassette of any one of clauses 1-4, wherein the plant virus assembly origin polynucleotide comprises a tobacco mosaic virus (TMV) OAS, optionally comprising of nucleotides 5313 to 5546 of the TMV genome.

[0334] Clause 6. The vector of any of clauses 1-5, wherein the ribosomal skipping polynucleotide comprises a polynucleotide encoding a T2A peptide.

[0335] Clause 7. The cassette of any one of clauses 1-6, further comprising a detectable label or a selection marker.

[0336] Clause 8. The cassette of any one of clauses 1-7, wherein cassette comprises a cassette as shown FIG. 5 A or FIG. 9C.

[0337] Clause 9. An isolated DNA polynucleotide encoding the cassette of any one of clauses 1-8.

[0338] Clause 10. A plurality of cassettes of any one of clauses 1-9, where the cassettes of the plurality are the same or different from each other.

[0339] Clause 11. A vector comprising the cassette of any one of clauses 1-9.

[0340] Clause 12. The vector of clause 11, wherein the vector is a viral vector or a plasmid.

[0341] Clause 13. A composition comprising the cassette of any one of clauses 1-9 or the plurality of claim 10, and a carrier, optionally a pharmaceutically acceptable carrier.

[0342] Clause 14. An isolated host cell comprising one or more of the cassettes of any one of clauses 1-9, the plurality of claim 10, or the vector of claim 11 and 12.

[0343] Clause 15. The isolated host cell of clause 12, wherein the cell is a prokaryotic cell or a eukaryotic cell.

[0344] Clause 16. A system for the manufacture of a VLP encapsulated mRNA, comprising a VLP capsid protein and a cassette of any one of clauses 1-9 or the plurality of claim 10.

[0345] Clause 17. The system of clause 16, wherein the VLP is derived from a plant virus selected from a TMV, a tobacco mild green mosaic virus (TMGMV), or a potato virus X (PVX).

[0346] Clause 18. A method to prepare an encapsulated mRNA vaccine comprising admixing the system of clause 17 at a ratio of about 0.5: 15 of the cassette to the VLP capsid protein in a buffer for about 15 to about 20 hours, and then isolated the VLPs.

[0347] Clause 19. An isolated VLP prepare by the method of clause 18.

[0348] Clause 20. An isolated VLP comprising a VLP capsid protein and an mRNA.

[0349] Clause 21. The isolated VLP of clause 20, wherein the VLP is derived from a plant virus selected from a TMV, a tobacco mild green mosaic virus (TMGMV), or a potato virus X (PVX).

[0350] Clause 22. The isolated VLP of clause 20 or 21, the mRNA is selected from an mRNA vaccine or an mRNA therapeutic peptide.

[0351] Clause 23. A plurality of isolated VLPs of any one of clauses 20 to 22, wherein theVLPs or the mRNAs are the same or different from each other.

[0352] Clause 24. A composition comprising the isolated VLP of any one of clauses 20 to 22, or the plurality of claim 23, and a carrier, optionally a pharmaceutically acceptable carrier.

[0353] Clause 25. A method to deliver mRNA to a cell, comprising contacting the cell with one or more of: the isolated VLP of any one of clauses 20 to 22, the plurality of claim 23, or the composition of claim 24.

[0354] Clause 26. The method of clause 25, wherein the contacting is in vitro or in vivo.[0355[ Clause 27. A method to deliver mRNA to a subject in need thereof comprising administering to the subject one or more of: the isolated VLP of any one of clauses 20 to 22, the plurality of claim 23, or the composition of claim 24.

[0356] Clause 28. The method of clause 27, wherein the subject is a mammal, optionally a human patient.

[0357] Clause 29. A method to induce an immune response in a subject in need thereof, comprising administering to the subject one or more of: the isolated VLP of any one of clauses 20 to 22, the plurality of claim 23, or the composition of claim 24.

[0358] Clause 30. The method of clause 29, wherein the subject is a mammal, optionally a human patient.

[0359] Clause 31. A kit comprising the cassette of any of clauses 1-9 or the plurality of claim 10, and instructions for use.

[0360] Partial Sequence Listing

[0361] SEQ ID NO: 9: S-Protein of Omicron SARS-CoV-2 mRNA be encoded by a sequence according to NCBI LC731729.1 (Synthetic construct pVEE-101c.l gene for spike protein):ATGTTTCTGCTCACAACCAAACGCACTATGTTTGTTTTCCTCGTGCTGCTCCCTTT GGTAAGTTCTCAGTGTGTAAACCTGACAACACGAACCCAGTTGCCTCCAGCTTAT ACCAACTCATTTACTCGCGGAGTATATTATCCCGATAAGGTCTTTAGAAGTAGCG TGTTGCACTCTACACAGGATCTGTTCTTGCCCTTCTTTAGTAACGTTACCTGGTTT CATGTGATAAGCGGAACAAATGGAACAAAAAGATTTGACAATCCAGTGCTTCCATTTAATGATGGGGTTTACTTTGCCAGTATCGAAAAGTCAAACATAATCCGGGGGTGGATCTTTGGAACCACTTTGGACTCTAAGACACAGTCTCTCCTCATAGTAAACAACGCCACCAATGTTGTCATAAAAGTATGCGAATTTCAGTTTTGCAACGATCCCTTTCTCGACCATAAGAATAATAAATCCTGGATGGAGTCTGAGTTCCGGGTTTATAGTAGTGCTAATAATTGCACTTTCGAATACGTGTCCCAACCATTCCTCATGGACCTTGAGGGCAAACAGGGGAATTTTAAAAACTTGCGCGAATTTGTCTTTAAGAATATCGACGGATACTTTAAGATCTATAGTAAACACACTCCTATCATCGTTCGGGAGCCCGAGGATCTTCCCCAAGGCTTTTCTGCTCTCGAACCCCTCGTAGACTTGCCAATTGGGATAAATATCACTCGCTTTCAAACTTTGCTTGCCCTCCACAGGAGCTACCTGACACCCGGCGACTCTTCTTCTGGTTGGACCGCCGGCGCCGCTGCCTATTATGTTGGTTACCTTCAGCCACGAACATTCTTGCTCAAGTATAACGAGAATGGCACCATTACCGACGCCGTCGATTGTGCATTGGATCCCTTGTCTGAAACAAAATGTACCTTGAAGTCCTTTACCGTAGAGAAAGGCATATACCAGACTTCCAACTTCCGAGTTCAGCCTACAGAATCCATCGTACGATTTCCCAACATCACAAACCTCTGCCCTTTCGACGAAGTATTTAATGCTACACGCTTCGCTTCAGTCTATGCCTGGAATAGGAAGCGCATATCAAATTGCGTGGCCGATTATTCAGTCCTCTATAATCTGGCACCCTTCTTCACTTTCAAGTGCTACGGCGTTTCCCCCACCAAACTCAATGATCTTTGCTTCACCAACGTCTATGCTGACAGTTTTGTCATACGAGGCGACGAAGTACGCCAGATTGCCCCCGGGCAGACAGGTAACATTGCTGATTATAATTATAAACTCCCAGATGACTTTACTGGATGCGTCATAGCCTGGAATTCCAACAAGCTAGATTCCAAGGTTTCCGGGAATTATAATTACCTTTATCGACTGTTCAGAAAGAGTAACTTGAAACCATTTGAGAGAGACATATCCACCGAGATTTACCAGGCAGGCAACAAGCCTTGTAACGGCGTTGCCGGATTTAACTGCTATTTTCCTTTGAGATCCTATTCCTTTAGACCAACATACGGGGTTGGCCACCAACCCTATCGAGTGGTTGTCCTCAGCTTTGAACTTTTGCACGCTCCCGCCACAGTCTGCGGACCAAAAAAGAGTACAAATCTTGTCAAGAATAAGTGCGTAAATTTCAATTTCAATGGCCTTAAGGGAACAGGCGTGCTGACTGAGTCAAACAAGAAGTTCCTGCCATTTCAGCAGTTTGGGCGGGATATAGCAGACACAACTGACGCTGTACGCGATCCTCAGACTTTGGAGATCTTGGACATCACTCCCTGTTCTTTCGGAGGGGTATCTGTCATCACCCCCGGAACTAATACATCAAATCAGGTCGCTGTGTTGTACCAAGGTGTCAACTGCACAGAAGTCCCCGTTGCTATACACGCAGACCAGCTCACCCCCACATGGCGGGTGTACTCAACTGGCTCAAACGTATTCCAGACCAGAGCTGGGTGCTTGATCGGTGCTGAATACGTGAACAATAGCTATGAATGCGATATTCCCATCGGTGCCGGGATCTGCGCTAGCTATCAGACACAGACCAAGTCCCACAGTGGAGCAGGATCTGTAGCATCCCAGTCTATTATTGCCTACACTATGTCATTGGGCGCCGAGAATAGCGTCGCATATTCAAATAATTCTATTGCAATACCCACCAACTTCACAATCTCCGTAACTACAGAAATACTTCCAGTTTCCATGACAAAGACATCAGTGGATTGTACAATGTATATATGCGGAGATTCCACAGAATGTTCAAATTTGCTCTTGCAGTACGGCTCCTTCTGCACCCAGCTCAAGAGGGCACTTACAGGTATTGCTGTCGAACAGGACAAGAACACACAAGAAGTCTTCGCCCAAGTCAAACAGATATACAAAACTCCTCCCATAAAGTACTTTGGCGGCTTCAACTTTAGTCAGATCCTCCCAGACCCTTCAAAACCATCTAAACGATCATTTATTGAAGATCTGCTGTTCAACAAGGTCACTCTTGCCGATGCTGGATTCATTAAGCAATACGGTGACTGCCTTGGTGATATTGCTGCCCGAGATCTGATCTGTGCCCAGAAATTCAAGGGGCTCACTGTACTCCCTCCACTGCTCACAGACGAAATGATTGCACAGTACACAAGTGCCCTGTTGGCAGGCACAATCACTAGCGGCTGGACCTTTGGCGCAGGTGCAGCACTCCAAATACCTTTTGCCATGCAGATGGCCTATCGGTTTAATGGGATAGGCGTGACTCAAAATGTCCTCTACGAAAACCAAAAGTTGATAGCTAACCAATTCAATTCAGCAATCGGGAAGATACAGGATTCACTGTCTAGTACTGCTAGTGCCCTTGGTAAGCTGCAGGACGTTGTCAACCACAATGCTCAAGCTCTGAATACATTGGTTAAGCAGCTCTCTAGTAAGTTTGGGGCCATCTCTTCAGTACTTAATGATATTTTCAGCCGATTGGACCCACCTGAAGCTGAAGTACAGATCGACAGGCTGATAACAGGCCGGCTCCAATCCCTCCAAACATACGTGACACAACAACTCATACGCGCAGCCGAAATCCGAGCCAGCGCTAACCTGGCAGCTACCAAGATGTCAGAATGCGTTCTGGGCCAGAGTAAACGCGTAGATTTCTGCGGGAAAGGGTACCACCTGATGTCCTTTCCACAATCTGCACCTCACGGGGTCGTCTTTTTGCATGTAACATACGTACCCGCACAAGAGAAGAATTTTACTACCGCTCCTGCCATCTGTCATGACGGGAAAGCTCATTTTCCTCGCGAAGGTGTGTTTGTATCTAATGGTACACATTGGTTTGTCACACAGCGGAATTTCTATGAACCCCAGATCATTACAACTGACAACACTTTTGTTTCCGGGAATTGTGACGTGGTCATAGGAATCGTAAATAACACTGTATATGATCCCCTCCAACCAGAGCTGGACTCTTTTAAAGAAGAACTGGATAAATATTTCAAGAACCACACAAGTCCCGACGTGGACCTTGGGGACATAAGTGGTATTAACGCATCTGTGGTTAACATTCAAAAGGAAATCGACAGACTCAACGAGGTGGCCAAAAACCTGAACGAAAGCTTGATAGATCTCCAGGAGTTGGGCAAGTATGAACAGTACATTAAATGGCCATGGTACATATGGCTTGGCTTTATCGCTGGCCTTATCGCCATCGTAATGGTTACAATCATGCTGTGCTGCATGAC CTCCTGCTGTTCTTGTTTGAAAGGGTGTTGTTCTTGTGGTAGTTGTTGCAAGTTTG ACGAAGATGATTCCGAACCTGTTCTTAAGGGGGTAAAGCTTCACTATACATGA (SEQ ID NO: 9).

[0362] Human papillomavirus type 16 isolate Frican-1 type, complete genome reproduced from Genbank AF536180.1 (SEQ ID NO: 12), https: / / www.ncbi.nlm.nih.gOv / nuccore / AF536180.l, last accessed on June 11, 2025.

[0363] LOCUS AF536180 7906 bp DNA circular VRL 25 -JUL-2016

[0364] DEFINITION Human papillomavirus type 16 isolate African- 1 type, complete genome.]0365{ ACCESSION AF536180

[0366] VERSION AF536180.1

[0367] KEYWORDS .

[0368] SOURCE Human papillomavirus 16

[0369] ORGANISM Human papillomavirus 16

[0370] Viruses; Monodnaviria; Shotokuvirae; Cossaviricota;

[0371] Papovaviricetes; Zurhausenvirales; Papillomaviridae;

[0372] Firstpapillomavirinae; Alphapapillomavirus; Alphapapillomavirus 9.

[0373] REFERENCE 1 (bases 1 to 7906)

[0374] AUTHORS Terai,M„ Fu,L., Ma,Z. and Burk, R D.

[0375] TITLE Cloning and Sequencing of Non-European Human Papillomavirus (HPV)

[0376] Variant Complete Genomes from Cervicovaginal Cells by an

[0377] Overlapping PCR Method

[0378] JOURNAL Unpublished

[0379] REFERENCE 2 (bases 1 to 7906)

[0380] AUTHORS Fu,L., Terai,M. and Burk, R D.

[0381] TITLE Direct Submission

[0382] JOURNAL Submitted (07-AUG-2002) Microbiology & Immunology, Albert Einstein

[0383] College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

[0384] FEATURES Location / Qualifiers

[0385] source 1..7906

[0386] / organism- 'Human papillomavirus 16"

[0387] / moljype- 'genomic DNA"

[0388] / isolate- ' African- 1 type"

[0389] / db xref="taxon:333760"

[0390] gene 83..559

[0391] / gene="E6"

[0392] CDS 83..559]0393| / gene="E6"

[0394] / codon_start=l

[0395] / product- 'putative transforming protein E6"YDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRYYCYSLYGTTLEQQYNKPLCDLLIRCIN CQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL"

[0398] gene 562..858

[0399] / gene="E7"

[0400] CDS 562..858

[0401] / gene="E7"

[0402] / codon_start=l

[0403] / product- 'putative transforming protein E7"

[0406] gene 865..2814

[0407] / gene="El"

[0408] CDS 865..2814

[0409] / gene="El"

[0410] / codon_start=l

[0411] / product- 'putative replication protein El"

[0412] / protein d=" AAQ.1.0722.,1"

[0413] / translation- 'MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGED LVDFIVNDNDYLTQAETETAHALFTAQEAKEHRDAVQVLKRKYLGSPLSDISGCVDNNISPRLKAICIEKQSRAAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGGSSQY SSGSGGEGVSERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCC DWCIAAFGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMVVLLLVRYKCGKNRETIEKLLSKL LCVSPMCMMIEPPKLRSTAAALYWYKTGMSNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQ MVQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQ MSMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVDFMSFLTALKRFLQGIPKKNCILLYGAA NTGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNAL DGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLVVFTFPNEFPFDENGNPVY ELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTL"

[0414] gene 2756.3853

[0415] / gene="E2"

[0416] CDS 2756..3853

[0417] / gene="E2"

[0418] / codon_start=l]0419[ / product- 'putative regulatory protein E2"

[0420] / protein id=" A AQ 10723,1"

[0421] / translation="MGTLCQRLNVCQDKILTHYENDSTDLRDHIDYWKQMRLECAIYYKAREMGFKHINHQVVPTLTVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMRYTNWKYIYICEDISVTVVEGQVDYYGLYYVHEGIQTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSDEVSSAEIIRQHLANHSAATHPKAVALGTKETQTTIQRPRSEPDTGNPCHTNKLLHRDSVDSAPILTAVNSSHKGRINCNSNTTPIVHLKGDANTLKCLRYRFKKHCKLYTAVSSTWHWTGHNVKHKSAIVTLTYDSECQREQFLSQVKIPKTITVSTGFMSI"[0422[ gene <3333..3620

[0423] / gene="E4"

[0424] CDS <3333..3620

[0425] / gene="E4"

[0426] / codon_start=l

[0427] / product- 'putative E4 protein"

[0428] / protein id="AAQ10724.1"

[0429] / translation- 'YYVLHLCLAATKYPLLKLLGSTWPTTPPRPIPKPSPWAPKKHRRLSSDQDQSQTPETPATPISCCTETQWTVLQSSLQLTAHTKDGLTVIVTLHP"[0430| gene 3850..4101

[0431] / gene="E5"

[0432] CDS 3850..4101

[0433] / gene="E5"

[0434] / codon_start=l

[0435] / product- 'putative E5 protein"

[0436] / protein id="AAQ 10725,1"

[0437] / translation- 'MTNLDTTSTTLLACFLLCFCVLLCVCLLIRPLLLSVSTYTSLILLVLLLWITAASAFRCFIVYILFVYIPLFLIHTHARFLIT"

[0438] gene 4237..5658

[0439] / gene="L2"

[0440] CDS 4237..5658

[0441] / gene="L2"

[0442] / codon_start=l

[0443] / product- 'putative minor capsid protein L2"0446 ILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSD

[0447] PSIVSLVEETSFIDAGAPTPVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDTFIVSTNPNTVTSSTPIPGSRPVARLGLYSRTTQQVKVVDPAFVTTPTKLITYDNPAYEGIDVDNTLYFPSNDNSINIAPDPDFLDIVALHRPALTSRRTGIRYSRIGNKQTLRTRSGKSIGAKVHYYYDLSTINPAEEIELQTITPSTYTTASHAASPTSINNGLYDIYADDFITDTSTTPVPSIPSTSLSGYIPANTTIPFGGAYNIPLVSGPDIPINTTDQTPSLIPIVPGSPQYTIIADGGDFYLHPSYYMLRKRRKRLPYFFSDVSLAA"

[0448] gene 556E.7156

[0449] / gene="Ll"

[0450] CDS 556E.7156

[0451] / gene="Ll"

[0452] / codon_start=l

[0453] / product- 'putative major capsid protein LI"DEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQYRVFRIYLPDPNKFGLPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCNNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGFKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL"

[0456] ORIGIN

[0457] 1 actacaataa ttcatgtata aaactaaggg tgtaaccgaa atcggttgaa ccgaaaccgg

[0458] 61 ttagtataaa agcagacatt ttctgcacca aaagagaact gcaatgtttc aggacccaca

[0459] 121 ggagcgaccc acaaagttac cagatttatg cacagagctg caaacaacta tacatgatat

[0460] 181 aatattagaa tgtgtgtact gcaagcaaca gttactgcga cgtgaggtat atgactttgc

[0461] 241 ttttcgggat ttatgcatag tatatagaga tgggaatcca tatgcagtgt gtgataaatg[0462 [ 301 tttaaagttt tattctaaaa ttagtgagta tagatattat tgttatagtt tgtatggaac

[0463] 361 aacattagaa cagcaataca acaaaccgtt gtgtgatttg ttaattaggt gtattaactg

[0464] 421 tcaaaagcca ctgtgtcctg aagaaaagca aagacatctg gacaaaaagc aaagattcca]0465 [ 481 taatataagg ggtcggtgga ccggtcgatg tatgtcttgt tgcagatcat caagaacacg

[0466] 541 tagagaaacc cagctgtaat catgcatgga gatacaccta cattgcatga atatatgtta

[0467] 601 gatttgcaac cagagacaac tgatctctac tgttatgagc aattaaatga cagctcagag

[0468] 661 gaggaggatg aaatagatgg tccagctgga caagcagaac cggacagagc ccattacaat

[0469] 721 attgtaacct tttgttgcaa gtgtgactct acgcttcggt tgtgcgtaca aagcacacac

[0470] 781 gtagacatcc gtacgttgga agacctgtta atgggcacac taggaattgt gtgccccatc

[0471] 841 tgttctcaga aaccataatc taccatggct gatcctgcag gtaccaatgg ggaagagggt[04721 901 acgggatgta atggatggtt ttatgtagag gctgtagtgg aaaaaaaaac aggggatgct

[0473] 961 atatcagatg acgagaacga aaatgacagt gatacaggtg aagattggt agattttata

[0474] 1021 gtaaatgata atgattattt aacacaggca gaaacagaga cagcacatgc gttgtttact [0475 J 1081 gcacaggaag caaaagaaca tagagatgca gtacaggttc taaaacgaaa gtatttgggt

[0476] 1141 agtccactta gtgatattag tggatgtgta gacaataata ttagtcctag attaaaggct

[0477] 1201 atatgtatag aaaaacaaag tagagctgca aaaaggagat tatttgaaag cgaagacagc

[0478] 1261 gggtatggca atactgaagt ggaaactcag cagatgttac aggtagaagg gcgccatgag

[0479] 1321 actgaaacac catgtagtca gtatagtggt ggaagtgggg gtggtagcag tcagtatagt

[0480] 1381 agtggaagtg ggggagaggg tgttagtgaa agacacacta tatgccaaac accactaca [04 1 { 1441 aatattttaa atgtactaaa aactagtaat gcaaaggcag caatgctagc aaaatttaaa [0482 [ 1501 gagttatacg gggtgagttt ttcagaatta gtaagaccat ttaaaagtaa taaatcaacg

[0483] 1561 tgttgcgatt ggtgtattgc tgcatttgga ctacaccta gtatagctga cagtataaaa

[0484] 1621 acattattac aacaatatg tttatattta cacatcaaa gtttagcatg ttcatgggga

[0485] 1681 atggttgtgt tactattagt aagatataaa tgtggaaaaa atagagaaac aattgaaaaa ]0486] 1741 ttgtgtcta aactatatg tgtgtctcca atgtgtatga tgatagagcc tccaaaattg

[0487] 1801 cgtagtacag cagcagcatt atattggtat aaaacaggta tgtcaaatat tagtgaagtg

[0488] 1861 tatggagaca cgccagaatg gatacaaaga caaacagtat tacaacatag ttttaatgat [0489[ 1921 tgtacatttg aattatcaca gatggtacaa tgggcctacg ataatgacat agtagacgat

[0490] 1981 agtgaaattg catataaata tgcacaattg gcagacacta atagtaatgc aagtgccttt

[0491] 2041 ttaaaaagta attcacaggc aaaaattgta aaggattgtg caacaatgtg tagacattat

[0492] 2101 aaacgagcag aaaaaaaaca aatgagtatg agtcaatgga taaaatatag atgtgatagg

[0049] 2161 gtagatgatg gaggtgattg gaagcaaatt gttatgtttt taaggtatca aggtgtagac

[0494] 2221 tttatgtcat ttttaactgc gttaaaaaga tttttgcaag gcatacctaa aaaaaatgc

[0495] 2281 atattactat atggtgcagc taacacaggt aaatcattat ttggtatgag tttgatgaaa

[0496] 2341 tttttgcaag ggtctgtaat atgttttgta aattctaaaa gccatttttg gttacaacca

[0497] 2401 ttagcagatg ccaaaatagg tatgttagat gatgctacag tgccctgttg gaactacata

[0498] 2461 gatgacaatt taagaaatgc attggatgga aatttagttt ctatggatgt aaagcataga

[0499] 2521 ccattggtac aactaaaatg ccctccatta ttaattacat ctaacattaa tgctggtaca ]0500] 2581 gattccaggt ggccttattt acataataga ttggtggtgt ttacatttcc aaatgagttt 10501] 2641 ccatttgacg aaaacggaaa tccagtgtat gagcttaatg ataagaactg gaaatccttt

[0502] 2701 ttctcaagga cgtggtccag attaagtttg cacgaggacg aggacaagga aaacgatggg

[0503] 2761 gactctttgc caacgtttaa atgtgtgtca ggacaaaata ctaacacatt atgaaaatga

[0504] 2821 tagtacagac ctacgtgacc atatagacta ttggaaacaa atgcgcctag aatgtgctat

[0505] 2881 ttattacaag gccagagaaa tgggatttaa acatattaac caccaggtgg tgccaacact

[0506] 2941 gactgtatca aagaataaag cattacaagc aattgaactg caactaacgt tagaaacaat

[0507] 3001 atataactca caatatagta atgaaaagtg gacattacaa gacgttagcc ttgaagtgta

[0508] 3061 tttaactgca ccaacaggat gtataaaaaa acatggatat acagtggaag tgcagtttga

[0509] 3121 tggagacata tgcaatacaa tgcgttatac aaactggaaa tatatatata tttgtgaaga ]0510[ 3181 catatcagta actgtggtag agggtcaagt tgactatat ggtttatact atgtccatga

[0511] 3241 aggaatacaa acatattttg tgcagtttaa agatgatgca gaaaaatata gtaaaaataa

[0512] 3301 agtatgggaa gttcatgcgg gtggtcaggt aatattatgt cctacatctg tgtttagcag

[0513] 3361 cgacgaagta tcctctgctg aaatattag gcagcacttg gccaaccact ccgccgcgac

[0514] 3421 ccatcccaaa gccgtcgcct tgggcaccaa agaaacacag acgactatcc agcgaccaag

[0515] 3481 atcagagcca gacaccggaa acccctgcca caccaataag ttgttgcaca gagactcagt

[0516] 3541 ggacagtgct ccaatcctca ctgcagtaa cagctcacac aaaggacgga taactgtaa

[0517] 3601 tagtaacact acacccatag tacatttaaa aggtgatgct aatactttaa aatgtttaag

[0518] 3661 atacagattt aaaaagcat gtaaatgta tacagcagtg tcgtctacat ggcatggac

[0519] 3721 aggacataat gtaaaacata aaagtgcaat tgtacactt acatatgata gtgaatgtca

[0520] 3781 acgggaacaa tttttgtctc aggttaaaat accaaaaact attacagtgt ctactggatt

[0521] 3841 tatgtctata tgacaaacct tgatactaca tccacaacat tactggcgtg ctttttgctt

[0522] 3901 tgcttttgtg tgcttttgtg tgtctgccta taatacgtc cgctgctttt gtctgtgtct

[0523] 3961 acatacacat cataatact attggtatta ttattgtgga taacagcagc ctctgcgttt

[0524] 4021 aggtgtttta tgtatatat tttatttgtt tatataccat tatttttaat acatacacat

[0525] 4081 gcacgcttct taattacata atgtatatgt acataatgta attgttacat ataattgctg

[0526] 4141 tatatcataa cttactgatt tttttttttt atttttatat atatagtttt tttttatttg

[0527] 4201 tttgtttgtt tttcaataaa ctgttattac ttaacaatgc gacacaaacg ttctgcaaaa

[0528] 4261 cgcacaaaac gtgcatcggc cacccaactt tataaaacat gcaaacaagc aggtacatgt 10529] 4321 ccacctgaca ttatacctaa ggttgaaggc aaaactattg ctgatcaaat attacaatat

[0530] 4381 ggaagtatgg gtgtattttt tggtgggtta ggaattggaa cagggtcagg tacaggcgga [0531 { 4441 cgcactgggt atattccatt aggaacaagg cctcccacag ctacagatac acttgctcct

[0532] 4501 gtaagacccc ctttaacagt agatcctgtg ggcccttctg atccttctat agtttcttta

[0533] 4561 gtggaagaaa ctagttttat tgatgctggt gcaccaacac ctgtaccttc cattccccca

[0534] 4621 gatgtatcag gatttagtat tacaacttca actgatacca cacctgctat attagatatt

[0535] 4681 aataatactg ttactactgt tactacacat aataatccca cttttactga cccatctgta

[0536] 4741 ttgcagcctc caacacctgc agaaactgga gggcatttta cactttcatc atccactatt

[0537] 4801 agtacacata attatgaaga aattcctatg gatacattta ttgttagcac aaaccctaac

[0538] 4861 acagtaacta gtagcacacc cataccaggg tctcgcccag tggcacgcct tggattatat

[0539] 4921 agtcgcacaa cacaacaagt taaagttgta gaccctgctt ttgtaaccac tcccactaaa

[0540] 4981 cttattacat atgataatcc tgcatatgaa ggtatagatg tggataatac attatatttt

[0541] 5041 cctagtaatg ataatagtat taatatagct ccagatcctg actttttgga tatagttgct05421 5101 ttacataggc cagcataac ctctaggcgt actggcatta gatacagtag aatggtaat

[0543] 5161 aaacaaacac tacgtactcg tagtggaaaa tctataggtg ctaaggtaca ttattattat

[0544] 5221 gatttaagta ctataatcc tgcagaagaa atagaattac aaactataac acctctaca ]0545[ 5281 tatactaccg cttcacatgc agcctcaccc acttctata ataatggat atatgatatt

[0546] 5341 tatgcagatg actttattac agatactct acaaccccag taccatctat accctctaca

[0547] 5401 tcctatcag gttatattcc tgcaaataca acaatcct tggtggtgc atacaatatt

[0548] 5461 cctttagtat caggtcctga tatacccatt aatacaactg accaaactcc tcattaatt

[0549] 5521 cctatagtc cagggtctcc acaatataca attattgctg atggaggtga cttttattta

[0550] 5581 catcctagtt atacatgt acgaaaacga cgtaaacgtt taccatattt tttttcagat

[0551] 5641 gtctctttgg ctgcctagcg aggccactgt ctactgcct cctgtcccag tatctaaagt

[0552] 5701 tgtaagcacg gatgaatatg ttgcacgcac aaacatatat tatcatgcag gaacatccag

[0553] 5761 actacttgca gttggacatc cctattttcc tattaaaaaa cctaacaata acaaaatatt

[0554] 5821 agttcctaaa gtatcaggat tacaatacag ggtatttaga atatatttac ctgaccccaa

[0555] 5881 taagtttggt cttcctgaca cctcatttta caatccagat acacagcggc tggtttgggc

[0556] 5941 ctgtgtaggt gttgaggtag gtcgtggtca gccattaggt gtgggcatta gtggccatcc 10557] 6001 tttataaat aaattggatg acacagaaaa tgctagtgct tatgcagcaa atgcaggtgt

[0558] 6061 ggataatagg gaatgtatat ctatggatta caaacaaaca caattgtgtt taattggttg

[0559] 6121 caaaccacct ataggggaac actggggcaa aggatcccca tgtaacaatg ttgcagtaaa

[0560] 6181 tccaggtgat tgtccaccat tagagtaat aaacacagtt attcaggatg gtgatatggt

[0561] 6241 tgataccggc tttggtgcta tggactttac tacattacag gctaacaaaa gtgaagttcc

[0562] 6301 actggatat tgtacgtcta tttgcaaata tccagattat ataaaatgg tgtcagaacc

[0563] 6361 atatggcgac agcttatttt tttatttacg aagggaacaa atgtttgtta gacatttatt 10564] 6421 taatagggct ggtgctgttg gtgaaaatgt accagacgat ttatacatta aaggctctgg

[0565] 6481 gtctactgca aatttagcca gttcaaata ttttcctaca cctagtggtt ctatggttac

[0566] 6541 ctctgatgcc caaatattta ataaaccata ttggtgcaa cgagcacagg gccacaataa

[0567] 6601 tggcatttgt tggggtaacc aactatttgt tactgttgtt gatactacac gcagtacaaa

[0568] 6661 tatgtcatta tgtgctgcca tatctactc agaaactaca tataaaaata ctaactttaa

[0569] 6721 agagtaccta cgacatgggg aggaatatga tttacagttt atttttcaac tgtgcaaaat

[0570] 6781 aaccttaact gcagacgtta tgacatacat acattctatg aattccacta ttttggagga 10571] 6841 ctggaatttt ggtttacaac ctcccccagg aggcacacta gaagatact ataggtttgt

[0572] 6901 aacatcccag gcaatgctt gtcaaaaaca tacacctcca gcacctaaag aagatcccct

[0573] 6961 taaaaaatat actttttggg aagtaaattt aaaagaaaag ttttctgcag acctagatca

[0574] 7021 gtttccttta ggacgcaaat ttttactaca agcaggattt aaggctaaac caaaatttac

[0575] 7081 attagggaaa cgaaaagcta cacccaccac ctcatctacc tctacaactg ctaaacgcaa

[0576] 7141 aaaacgtaag ctgtaagtat tgtatgtatg ttgaattagt gttgtttgtt gtttatatgc

[0577] 7201 ttgtatgtgc ttgtatgtgc ttgtaaatat tgagttgtat gtgtgtttgt atgtatggta

[0578] 7261 taataaacac gtgtgtatgt gtttttaaat gcttgtgtaa ctattgtgtc atgcaacata

[0579] 7321 aataaactta ttgtttcaac acctactaat tgtgttgtgg ttattcattg tatataaact

[0580] 7381 atatttgcta catcctgttt ttgttttata tatactatat tttgtagcgc cagcggccat

[0581] 7441 tttgtagctt caaccgaatt cggttgcatg ctttttggca caaaatgtat ttttttaaat

[0582] 7501 agttctatgt cagcaactat agtttaaact tgtacgtttc ctgcttgcca tgcgtgccaa

[0583] 7561 atccctgttt tcctgacctg cactgcttgc caaccattcc attgtttttt acactgcact

[0584] 7621 atgtgcaact actgaatcac tatgtacatt gtgtcatata aaataaatca ctatgcgcca

[0585] 7681 acgccttaaa taccgctgtt aggcacatat ttttggcttg ttttaactaa cctaattgca

[0586] 7741 tatttggcat aaggtttaaa cttttaaggc caactaaatg tcaccttagt tcatacatga

[0587] 7801 actgtgtaaa ggttagtcat acattgttca tttttaaaac tgcacatggg tgtgtgcaaa

[0588] 7861 ccgttttggg ttacaaattt acaagcaact tatataataa tactaa

[0589] SEQ ID NO: 19: Exemplary DNA sequence encoding a Nodamura replicon having the nucleotide sequence:ATGCTGAACTACGAGACAATCATCAACGGCGCATCGAGCGCTCTGAACATCGTTTCGCGTGCGTTAGGATACCGCGTGCCACTAGCCAAATCGCTGGCGCTGGTCGCGGGGTCCTGCGTGGTGTACAAAATAATCGTGCATCGACGCACGCTCGTGGCGTTCCTGGTAATCGGACCATACGCCACGGTGGTGCAGCACCGTCTGCCGATGGCCCTTCAGAGGGCCATCATTGAATATACACGAGAAGACCGTGAGATCAGCCTGTTTCCGCAAAATTCCATCGTTTCCGCAGAACACGCGCGGAAAGCGGATAATGGGCATCCGATCTCCGGGGGAACGCGTGATGTCGCGAGGGAGACTATTTCCCTTGCCATAAGGGCCGCTGGTTTTCGTCATTACGAAATCAGCCCCGCGAGGCAATCACCAGCTGAGGCGGCAAGCCACCAACATTATGCCGCCGCTGACCTCGTGAGAGCGGCTACTGAAGATAAGATCCAAGACGGTGATGTGGTAGTTGCCATCGACATCGATTACTACCTGCGTGACATTGACCGCTACCTGGGTCGTGGTGTCCCGTTCATGGCTTACACCTTCAATCCTGTTGAAGTAGCTGGCCGTGACGGTGACTCCTTTTTCCGGATCACGAACAATCAAGTCACGTTTGATGTTAGTGGCGGTGGATCTTGGTCCCATGAAGTTTGGGACTGGTGCGCGTTTGGTGAGTTCATCGAGACCCGAGACGCGAGCTGGCTTGCTTGGTTCGCCCGGGCGGTTGGACTCACCAAGTCGCAGATCCACAAAGTTCACTACTGCCGTCCATGGCCGCAATCGCCCCATCGCGCTTTGGTGTGGTGTCTGCCTGTAGCAAGCTACTG GCGCTTCACTTTCATTCCGACGGACCTGCATACGCGCACGCTTCGGCGTGTGCGTTATCAGGACACGTCCCGGCCCGGTTGGAATTCCATCGTCTCGACCGGGTCCGAAGGCCTTAATATCAGCCTTGGTCGCGAAGGAGCTGATCATTGCGTGACGATTCCAAAGGTGCACTACGACATGCTTATGGGTTTGTCGAGTGCGCAGTCGTTGTCGTCCCGCATGATCGGGCTCAAGTACACTGATCCTAGTGTACTCGCGACGGTTGCCCAATACTATCAGGGCAAGAATGTTGAAGTTGCCGACGCTGACAGGATCGGCCGCGCCATAAATCCCAAGGTCCACTGGCCAGCGCACGTCGAAGTTGACGAGGCGGAGGTTAGTGCTCGGGTGTACGCCAGCCCGTTGGTATCTGACGAAAATATGATGCCTATGATCAAGCGCTGGGAGACGCTGTCGTTGTCGCTGGACCGCCGGGTTACATTCCAACGTAATCCGAAGGTTCCTGGAAAACGGCTCAGGGCTTATGCCATTGAGTTCGTTGACTTGGTTGTGCCTGAGCGTGGTGTCGGAGTCCCCTATTCATTGGAGGACACCGCCGCCATGCTGGACAAACCAAGCCAGACCCTCGCCATCCAACAGGTGTGGGAGACTGTCGACATGCCCCCAAGAAGGCTCATCGAAGCGTTCGTGAAGAACGAACCGACCATGAAGGCTGGCCGTATCATCTCGTCGTTCGCTGACATGCGGTTCCTACTGCGGTTTTCCAGCTATACGCTGGCATTCCGTGATCAGGTGCTGCATGCAGAGCACAACCGGCATTGGTTTTGCCCGGGTTTGACCCCCGAGCAGATCGCCACAAAAGTGGTTGATTACGTGTCCGGTGTTGAAGAACCATCGGAGGGAGACTTTTCCAACTTTGATGGCACGGTTAGTGAGTGGCTACAACGCCACGTCATGAACGCCGTCTACCTGCGTTATTTCAATCACCGAGCGCAGCGAGACCTCAGGTCGTATACCGACATGCTGGTCTCATGCCCCGCGAGGGCGAAGCGATTCGGTTTTGCTTATGACGCGGGTGTCGGCGTTAAGAGCGGGTCGCCAACAACTTGCGACCTGAATACCGTGTGCAATGGTTTCCTCCAATATTGCTCCATTCGAATGACACACCCAGAGCTGACACCAATCGATGCTTTCCGGCTCATCGGTCTCGCGTTTGGGGACGATTCCCTCTTCGAGCGACGTTTCGCTAAGAACTATGCGAAGGTTTCCGCCGAGGTGGGGATGGTCCTCAAAATCGAGCGATTCGACCCGGCACAAGGCATCACTTTCCTCGCCCGTGTTTATCCCGACCCCTACACGTCGACCACAAGTTTCCAGGACCCTTTGCGTACCTGGAGGAAGCTCCACTTGACGACGCGCGATCCAACAATACCATTGGCAACGGCTGCCATCGATCGCGTTGAGGGCTACCTCGTCACCGACGGCCTGAGCCCGCTTACTGGCGCGTATTGTCGCATGGTTAAGCGGGTTTACGAGGCCGGCGGAGCCGAGGATGCCGCCAAGAGGAGGTCGCGAAAATCCCATTCCCGCGAAAAGCCGTATTGGTTGACTGTTGGAGGCGCTTGGCCCCAAGATGTCAAGGACGTTGATCTTATGTTCCAGTGTGCGGCCGCACGTACCGGAGTAGACCTCGAGACACTTCGGTCTCTGGATCAGCGTCTAGGAGAAATCACTGACGTCTGGGCGGATATTACCATCAACCGGGATAATGAACCAAACCCCTACAAGGATACACTGGACTTGGAGGGCCCGGCTGATGGCCGGGTGGACGATCGTGTATTTCAGAATGACAAACATGTCATGAGGTTAAGAGCTAATCAAGTCACTTCCAGCCAAGCTGGAGCAGCTGGCTCAGGAGACGCAAGCAACGATCCAAACGCTCATGATCGCGGATCCCAACGTCAACAAGGATCTGCGAGCGTTCTGCGAGTTCCTGACCGTGCAGCACCAGCGGGCGTATCGAGCGACGAACAGCCTGCTCATCAAACCGCGAGTCGCAGCAGCGCTTCGCGGGGAGGAGCTGGACCTGGGCGAGGCGGACGTCGCCGCCCGGGTCCGCCAGCTAAAACAACAGCTGGCGGAGCTCGAGATGGAAATCAAGCCAGGGCACCAACAAGTGGCCCGTCAAAACGCCAAGCTGAGGGAAGGTCCAGATCTAGTCGAGGACCGGCGGGTA GCCGTGGACGCGGGAAAGACTATAAGGAGGGTGGCAGCGGAGCTAGC.

[0590] SEQ ID NO: 20: Exemplary RNA sequence encoding a Nodamura replicon having the nucleotide sequence:

[0051] AUGCUGAACUACGAGACAAUCAUCAACGGCGCAUCGAGCGCUCUGAACAUCGU UUCGCGUGCGUUAGGAUACCGCGUGCCACUAGCCAAAUCGCUGGCGCUGGUCGCGGGGUCCUGCGUGGUGUACAAAAUAAUCGUGCAUCGACGCACGCUCGUGGCGUUCCUGGUAAUCGGACCAUACGCCACGGUGGUGCAGCACCGUCUGCCGAUGGCCCUUCAGAGGGCCA UCAUUGAAUAUACACGAGAAGACCGUGAGAUCAGCCUGUUUCCGCAAAAUUCCAUCGUUUCCGCAGAACACGCGCGGAAAGCGGAUAAUGGGCAUCCGAUCUCCGGGGGAACGCGUGAUGUCGCGAGGGAGACUAUUUCCCUUGCCAUAAGGGCCGCUGGUUUUCGUCAUUA CGAAAUCAGCCCCGCGAGGCAAUCACCAGCUGAGGCGGCAAGCCACCAACAUUAUGCCGCCGCUGACCUCGUGAGAGCGGCUACUGAAGAUAAGAUCCAAGACGGUGAUGUGGUAGUUGCCAUCGACAUCGAUUACUACCUGCGUGACAUUGACCGCUACCUGGGUCGUGGUG UCCCGUUCAUGGCUUACACCUUCAAUCCUGUUGAAGUAGCUGGCCGUGACGGUGACUC CUUUUUCCGGAUCACGAACAAUCAAGUCACGUUUGAUGUUAGUGGCGGUGGAUCUUGGUCCCAUGAAGUUUGGGACUGGUGCGCGUUUGGUGAGUUCAUCGAGACCCGAGACGCGAGCUGGCUUGCUUGGUUCGCCCGGGCGGUUGGACUCACCAAGUCGCAGAUCCACAAAGUUCACUACUGCCGUCCAUGGCCGCAAUCGCCCCAUCGCGCUUUGGUGUGGUGUCUGC CUGUAGCAAGCUACUGGCGCUUCACUUUCAUUCCGACGGACCUGCAUACGCGCACGCUUCGGCGUGUGCGUUAUCAGGACACGUCCCGGCCCGGUUGGAAUUCCAUCGUCUCGACCGGGUCCGAAGGCCUUAAUAUCAGCCUUGGUCGCGAAGGAGCUGAUCAUUGCGUGACG AUUCCAAAGGUGCACUACGACAUGCUUAUGGGUUUGUCGAGUGCGCAGUCGUUGUCGUCCCGCAUGAUCGGGCUCAAGUACACUGAUCCUAGUGUACUCGCGACGGUUGCCCAAU ACUAUCAGGGCAAGAAUGUUGAAGUUGCCGACGCUGACAGGAUCGGCCGCGCCAUAAAUCCCAAGGUCCACUGGCCAGCGCACGUCGAAGUUGACGAGGCGGAGGUUAGUGCUCGGGUGUACGCCAGCCCGUUGGUAUCUGACGAAAAUAUGAUGCCUAUGAUCAAGCGCUGGGAGACGCUGUCGUUGUCGCUGGACCGCCGGGUUACAUUCCAACGUAAUCCGAAGGUUCCUGGAAAACGGCUCAGGGCUUAUGCCAUUGAGUUCGUUGACUUGGUUGUGCCUGAGCGUGGUGUCGGAGUCCCCUAUUCAUUGGAGGACACCGCCGCCAUGCUGGACAAACCAAGCCAGACCCUCGCCAUCCAACAGGUGUGGGAGACUGUCGACAUGCCCCCAAGAAGGCUCAUCGAAGCGUUCGUGAAGAACGAACCGACCAUGAAGGCUGGCCGUAUCAUCUCGUCGUUCGCUGACAUGCGGUUCCUACUGCGGUUUUCCAGCUAUACGCUGGCAUUCCGUGAUCAGGUGCUGCAUGCAGAGCACAACCGGCAUUGGUUUUGCCCGGGUUUGACCCCCGAGCAGAUCGCCACAAAAGUGGUUGAUUACGUGUCCGGUGUUGAAGAACCAUCGGAGGGAGACUUUUCCAACUUUGAUGGCACGGUUAGUGAGUGGCUACAACGCCACGUCAUGAACGCCGUCUACCUGCGUUAUUUCAAUCACCGAGCGCAGCGAGACCUCAGGUCGUAUACCGACAUGCUGGUCUCAUGCCCCGCGAGGGCGAAGCGAUUCGGUUUUGCUUAUGACGCGGGUGUCGGCGUUAAGAGCGGGUCGCCAACAACUUGCGACCUGAAUACCGUGUGCAAUGGUUUCCUCCAAUAUUGCUCCAUUCGAAUGACACACCCAGAGCUGACACCAAUCGAUGCUUUCCGGCUCAUCGGUCUCGCGUUUGGGGACGAUUCCCUCUUCGAGCGACGUUUCGCUAAGAACUAUGCGAAGGUUUCCGCCGAGGUGGGGAUGGUCCUCAAAAUCGAGCGAUUCGACCCGGCACAAGGCAUCACUUUCCUCGCCCGUGUUUAUCCCGACCCCUACACGUCGACCACAAGUUUCCAGGACCCUUUGCGUACCUGGAGGAAGCUCCACUUGACGACGCGCGAUCCAACAAUACCAUUGGCAACGGCUGCCAUCGAUCGCGUUGAGGGCUACCUCGUCACCGACGGCCUGAGCCCGCUUACUGGCGCGUAUUGUCGCAUGGUUAAGCGGGUUUACGAGGCCGGCGGAGCCGAGGAUGCCGCCAAGAGGAGGUCGCGAAAAUCCCAUUCCCGCGAAAAGCCGUAUUGGUUGACUGUUGGAGGCGCUUGGCCCCAAGAUGUCAAGGACGUUGAUCUUAUGUUCCAGUGUGCGGCCGCACGUACCGGAGUAGACCUCGAGACACUUCGGUCUCUGGAUCAGCGUCUAGGAGAAAUCACUGACGUCUGGGCGGAUAUUACCAUCAACCGGGAUAAUGAACCAAACCCCUACAAGGAUACACUGGACUUGGAGGGCCCGGCUGAUGGCCGGGUGGACGAUCGUGUAUUUCAGAAUGACAAACAUGUCAUGAGGUUAAGAGCUAAUCAAGUCACUUCCAGCCAAGCUGGAGCAGCUGGCUCAGGAGACGCAAGCAACGAUCCAAACGCUCAUGAUCGCGGAUCCCAACGUCAACAAGGAUCUGCGAGCGUUCUGCGAGUUCCUGACCGUGCAGCACCAGCGGGCGUAUCGAGCGACGAACAGCCUGCUCAUCAAACCGCGAGUCGCAGCAGCGCUUCGCGGGGAGGAGCUGGACCUGGGCGAGGCGGACGUCGCCGCCCGGGUCCGCCAGCUAAAACAACAGCUGGCGGAGCUCGAGAUGGAAAUCAAGCCAGGGCACCAACAAGUGGCCCGUCAAAACGCCAAGCUGAGGGAAGGUCCAGAUCUAGUCGAGGACCGGCGGGUAGCCGUGGACGCGGGAAAGACUAUAAGGAGGGUGGCAGCGGAGCUAGC.

[0592] SEQ ID NO: 29: The Nod.HPV.E7 mRNA construct includes all essential elements: the 5' untranslated region (5'UTR), Nodamura virus replicon, a mutated HPV E7 sequence, TMV-origin-associated sequence (OAS), and the 3' untranslated region (3'UTR).

[0593] 5’ UTRGTATTGAATCCAAAACTCAAARNA 1 OF NODAMURA VIRUSATGCTGAACTACGAGACAATCATCAACGGCGCATCGAGCGCTCTGAACATCGTTT CGCGTGCGTTAGGATACCGCGTGCCACTAGCCAAATCGCTGGCGCTGGTCGCGG GGTCCTGCGTGGTGTACAAAATAATCGTGCATCGACGCACGCTCGTGGCGTTCCT GGTAATCGGACCATACGCCACGGTGGTGCAGCACCGTCTGCCGATGGCCCTTCA GAGGGCCATCATTGAATATACACGAGAAGACCGTGAGATCAGCCTGTTTCCGCA AAATTCCATCGTTTCCGCAGAACACGCGCGGAAAGCGGATAATGGGCATCCGAT CTCCGGGGGAACGCGTGATGTCGCGAGGGAGACTATTTCCCTTGCCATAAGGGC CGCTGGTTTTCGTCATTACGAAATCAGCCCCGCGAGGCAATCACCAGCTGAGGCG GCAAGCCACCAACATTATGCCGCCGCTGACCTCGTGAGAGCGGCTACTGAAGAT AAGATCCAAGACGGTGATGTGGTAGTTGCCATCGACATCGATTACTACCTGCGTG ACATTGACCGCTACCTGGGTCGTGGTGTCCCGTTCATGGCTTACACCTTCAATCCT GTTGAAGTAGCTGGCCGTGACGGTGACTCCTTTTTCCGGATCACGAACAATCAAG TCACGTTTGATGTTAGTGGCGGTGGATCTTGGTCCCATGAAGTTTGGGACTGGTG CGCGTTTGGTGAGTTCATCGAGACCCGAGACGCGAGCTGGCTTGCTTGGTTCGCC CGGGCGGTTGGACTCACCAAGTCGCAGATCCACAAAGTTCACTACTGCCGTCCAT GGCCGCAATCGCCCCATCGCGCTTTGGTGTGGTGTCTGCCTGTAGCAAGCTACTG GCGCTTCACTTTCATTCCGACGGACCTGCATACGCGCACGCTTCGGCGTGTGCGT TATCAGGACACGTCCCGGCCCGGTTGGAATTCCATCGTCTCGACCGGGTCCGAAG GCCTTAATATCAGCCTTGGTCGCGAAGGAGCTGATCATTGCGTGACGATTCCAAA GGTGCACTACGACATGCTTATGGGTTTGTCGAGTGCGCAGTCGTTGTCGTCCCGC ATGATCGGGCTCAAGTACACTGATCCTAGTGTACTCGCGACGGTTGCCCAATACT ATCAGGGCAAGAATGTTGAAGTTGCCGACGCTGACAGGATCGGCCGCGCCATAA ATCCCAAGGTCCACTGGCCAGCGCACGTCGAAGTTGACGAGGCGGAGGTTAGTG CTCGGGTGTACGCCAGCCCGTTGGTATCTGACGAAAATATGATGCCTATGATCAA GCGCTGGGAGACGCTGTCGTTGTCGCTGGACCGCCGGGTTACATTCCAACGTAATCCGAAGGTTCCTGGAAAACGGCTCAGGGCTTATGCCATTGAGTTCGTTGACTTGGTTGTGCCTGAGCGTGGTGTCGGAGTCCCCTATTCATTGGAGGACACCGCCGCCATGCTGGACAAACCAAGCCAGACCCTCGCCATCCAACAGGTGTGGGAGACTGTCGACATGCCCCCAAGAAGGCTCATCGAAGCGTTCGTGAAGAACGAACCGACCATGAAGGCTGGCCGTATCATCTCGTCGTTCGCTGACATGCGGTTCCTACTGCGGTTTTCCAGCTATACGCTGGCATTCCGTGATCAGGTGCTGCATGCAGAGCACAACCGGCATTGGTTTTGCCCGGGTTTGACCCCCGAGCAGATCGCCACAAAAGTGGTTGATTACGTGTCCGGTGTTGAAGAACCATCGGAGGGAGACTTTTCCAACTTTGATGGCACGGTTAGTGAGTGGCTACAACGCCACGTCATGAACGCCGTCTACCTGCGTTATTTCAATCACCGAGCGCAGCGAGACCTCAGGTCGTATACCGACATGCTGGTCTCATGCCCCGCGAGGGCGAAGCGATTCGGTTTTGCTTATGACGCGGGTGTCGGCGTTAAGAGCGGGTCGCCAACAACTTGCGACCTGAATACCGTGTGCAATGGTTTCCTCCAATATTGCTCCATTCGAATGACACACCCAGAGCTGACACCAATCGATGCTTTCCGGCTCATCGGTCTCGCGTTTGGGGACGATTCCCTCTTCGAGCGACGTTTCGCTAAGAACTATGCGAAGGTTTCCGCCGAGGTGGGGATGGTCCTCAAAATCGAGCGATTCGACCCGGCACAAGGCATCACTTTCCTCGCCCGTGTTTATCCCGACCCCTACACGTCGACCACAAGTTTCCAGGACCCTTTGCGTACCTGGAGGAAGCTCCACTTGACGACGCGCGATCCAACAATACCATTGGCAACGGCTGCCATCGATCGCGTTGAGGGCTACCTCGTCACCGACGGCCTGAGCCCGCTTACTGGCGCGTATTGTCGCATGGTTAAGCGGGTTTACGAGGCCGGCGGAGCCGAGGATGCCGCCAAGAGGAGGTCGCGAAAATCCCATTCCCGCGAAAAGCCGTATTGGTTGACTGTTGGAGGCGCTTGGCCCCAAGATGTCAAGGACGTTGATCTTATGTTCCAGTGTGCGGCCGCACGTACCGGAGTAGACCTCGAGACACTTCGGTCTCTGGATCAGCGTCTAGGAGAAATCACTGACGTCTGGGCGGATATTACCATCAACCGGGATAATGAACCAAACCCCTACAAGGATACACTGGACTTGGAGGGCCCGGCTGATGGCCGGGTGGACGATCGTGTATTTCAGAATGACAAACATGTCATGAGGTTAAGAGCTAATCAAGTCACTTCCAGCCAAGCTGGAGCAGCTGGCTCAGGAGACGCAAGCAACGATCCAAACGCTCATGATCGCGGATCCCAACGTCAACAAGGATCTGCGAGCGTTCTGCGAGTTCCTGACCGTGCAGCACCAGCGGGCGTATCGAGCGACGAACAGCCTGCTCATCAAACCGCGAGTCGCAGCAGCGCTTCGCGGGGAGGAGCTGGACCTGGGCGAGGCGGACGTCGCCGCCCGGGTCCGCCAGCTAAAACAACAGCTGGCGGAGCTCGAGATGGAAATCAAGCCAGGGCACCAACAAGTGGCCCGTCAAAACGCCAAGCTGAGGGAAGGTCCAGATCTAGTCGAGGACCGGCGGGTAGCCGTGGACGCGGGAAAGACTATAAGGAGGGTGGCAGCGGAGCTAGCTMV OASGCAAGT TTTAGTTAAT ATTAGAAATG TGAAGATGTC AGCGGGTTTCTGTCCGCTTT CTCTGGAGTT TGTGTCGGTG TGTATTGTTT ATAGAAATAATATAAAATTA GGTTTGAGAG AGAAGATTAC AAACGTGAGA GACGGAGGGCCCATGGAACT TACAGAAGAA GTCGTTGATG AGTTCATGGA AGATGTCCCTATGTCGATCA GGCTTGCAAA GTTTCGATCT CGAACCGG3’ UTRGCAAGT TTTAGTTAAT ATTAGAAATG TGAAGATGTC AGCGGGTTTCTGTCCGCTTT CTCTGGAGTT TGTGTCGGTG TGTATTGTTT ATAGAAATAATATAAAATTA GGTTTGAGAG AGAAGATTAC AAACGTGAGA GACGGAGGGCCCATGGAACT TACAGAAGAA GTCGTTGATG AGTTCATGGA AGATGTCCCTATGTCGATCA GGCTTGCAAA GTTTCGATCT CGAACCGREFERENCES - FOR EXPERIMENT NO. 1:(1) Verbeke, R.; Lentacker, I.; De Smedt, S. 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Claims

WHAT IS CLAIMED IS:

1. A cassette for the delivery of one or more mRNA, the cassette comprising an optional 5’ mRNA cap, a 5’ UTR, a Nodamura replicon, a ribosomal skipping polynucleotide, the one or more mRNA, a plant virus assembly origin polynucleotide (“OAS”), and a 3’ UTR, optionally wherein the cassette does not comprise a polyA tail.

2. The cassette of claim 1, wherein the mRNA is selected from an mRNA vaccine or an mRNA encoding a therapeutic peptide.

3. The cassette of claim 2, wherein the mRNA vaccine is selected from an Omicron vaccine or a Human papillomavirus (HPV) vaccine.

4. The cassette of any one of claims 1-3, wherein the mRNA encodes a polypeptide selected from an HPV E6 polypeptide, an HPVE7 polypeptide, a mutated HPV E7 polypeptide, or an Omicron RBD polypeptide.

5. The cassette of any one of claims 1-4, wherein the plant virus assembly origin polynucleotide comprises a tobacco mosaic virus (TMV) OAS, optionally comprising of nucleotides 5313 to 5546 of the TMV genome.

6. The vector of any of claims 1-5, wherein the ribosomal skipping polynucleotide comprises a polynucleotide encoding a T2A peptide.

7. The cassette of any one of claims 1-6, further comprising a detectable label or a selection marker.

8. The cassette of any one of claims 1-7, wherein cassette comprises a cassette as shown FIG. 5A or FIG. 9C.

9. An isolated DNA polynucleotide encoding the cassette of any one of claims 1-8.

10. A plurality of cassettes of any one of claims 1-9, where the cassettes of the plurality are the same or different from each other.

11. A vector comprising the cassette of any one of claims 1-9.

12. The vector of claim 11, wherein the vector is a viral vector or a plasmid.

13. A composition comprising the cassette of any one of claims 1-9 or the plurality of claim 10, and a carrier, optionally a pharmaceutically acceptable carrier.

14. An isolated host cell comprising one or more of the cassettes of any one of claims 1-9, the plurality of claim 10, or the vector of claim 11 and 12.

15. The isolated host cell of claim 12, wherein the cell is a prokaryotic cell or a eukaryotic cell.

16. A system for the manufacture of a VLP encapsulated mRNA, comprising a VLP capsid protein and a cassette of any one of claims 1-9 or the plurality of claim 10.

17. The system of claim 16, wherein the VLP is derived from a plant virus selected from a TMV, a tobacco mild green mosaic virus (TMGMV), or a potato virus X (PVX).

18. A method to prepare an encapsulated mRNA vaccine comprising admixing the system of claim 17 at a ratio of about 0.5: 15 of the cassette to the VLP capsid protein in a buffer for about 15 to about 20 hours, and then isolated the VLPs.

19. An isolated VLP prepare by the method of claim 18.

20. An isolated VLP comprising a VLP capsid protein and an mRNA.

21. The isolated VLP of claim 20, wherein the VLP is derived from a plant virus selected from a TMV, a tobacco mild green mosaic virus (TMGMV), or a potato virus X (PVX).

22. The isolated VLP of claim 20 or 21, the mRNA is selected from an mRNA vaccine or an mRNA therapeutic peptide.

23. A plurality of isolated VLPs of any one of claims 20 to 22, wherein the VLPs or the mRNAs are the same or different from each other.

24. A composition comprising the isolated VLP of any one of claims 20 to 22, or the plurality of claim 23, and a carrier, optionally a pharmaceutically acceptable carrier.

25. A method to deliver mRNA to a cell, comprising contacting the cell with one or more of: the isolated VLP of any one of claims 20 to 22, the plurality of claim 23, or the composition of claim 24.

26. The method of claim 25, wherein the contacting is in vitro or in vivo.

27. A method to deliver mRNA to a subject in need thereof comprising administering to the subject one or more of: the isolated VLP of any one of claims 20 to 22, the plurality of claim 23, or the composition of claim 24.

28. The method of claim 27, wherein the subject is a mammal, optionally a human patient.

29. A method to induce an immune response in a subject in need thereof, comprising administering to the subject one or more of: the isolated VLP of any one of claims 20 to 22, the plurality of claim 23, or the composition of claim 24.

30. The method of claim 29, wherein the subject is a mammal, optionally a human patient.

31. A kit comprising the cassette of any of claims 1-9 or the plurality of claim 10, and instructions for use.

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