Methods and compositions for generating an immune response against a picornavirus

Abstract

Vaccine compositions for protecting a subject against diseases caused by viral pathogens of the Picornaviridae family are disclosed. These vaccines comprise nucleic acids encoding the capsid polyprotein (P1 polyprotein) mixed with the viral 3C protease that processes the P1 polyprotein, and encapsulated within nanoparticle carriers. Methods of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a viral pathogen, or of inducing an immune response against a viral pathogen in a subject are provided. Methods of making the vaccines are also described.

Description

METHODS AND COMPOSITIONS FOR GENERATING AN IMMUNE RESPONSE AGAINST A PICORNAVIRUSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application No. 63 / 712,700, filed October 28, 2024, which is incorporated by reference as if fully set forth.

[0002] The sequence listing electronically filed with this application titled "Sequence Listing XML,” which was created on October 25, 2025 and had a size of 71 ,999 bytes is incorporated by reference herein as if fully set forth.TECHNICAL FIELD

[0003] The present disclosure generally relates to the field of virology. In particular, the present invention is directed to vaccine compositions and methods for generating an immune response against a Picornavirus.BACKGROUND OF THE INVENTION

[0004] Developing an effective vaccine against picornaviruses is challenging, due to their obligate intracellular virion assembly and methods of immune evasion. Picornavirus virions assemble within host cells, which shields them from the immune system during critical stages of their lifecycle, limiting exposure to antibodies and uptake by antigen presenting cells. Additionally, these viruses have high mutation rates, especially in capsid proteins that antibodies typically target, allowing them to evade immune recognition and reducing the effectiveness of long-term immunity. With this high rate of mutation, many species of picornavirus have numerous serotypes, each requiring a distinct immune response due to antigenic variation. Developing a vaccine that covers all or most serotypes is difficult and often impractical. Together, these factors make picornaviruses particularly challenging vaccine targets, requiring innovative solutions. Nucleic acid approaches, such as mRNA vaccines, are useful for rapidly evolving pathogens such as picornaviruses because they can be quickly designed and synthesized to match new viral strains, reducing the time required for development. Additionally, mRNA vaccines elicit robust immune responses, producing both antibodies and T-cell responses, which are critical in combating picornavirus infection. To date however, no nucleic acid vaccine against a picornavirus has been demonstrated to induce neutralizing antibody titers that are the primary correlate of protection. The generation of structurally intact capsid antigen, properly processed by virally-encoded proteolytic activity, is useful to inducing such immune responses. This has yet to be achieved, neither with traditional protein-based vaccines nor with a nucleic acid approach. Furthermore, the properly processed capsid antigen must be presented to the immune system in an immunologically relevant context, with innate immune activation occurring in concert with antigen presentation.19406520.1

[0005] Nanoparticle compositions are disclosed herein that incorporate a plurality of nucleic acid molecules and formulations that, when administered to a mammalian subject, lead to translation and appropriate post-transcriptional processing of picornavirus capsid, with the capacity to induce innate immune stimulation such that protective humoral immune responses are produced.

[0006] Additionally, nanoparticle compositions disclosed herein are also useful in treating diseases caused by a Poliovirus and Enterovirus 71 (EV-A71 )SUMMARY OF THE INVENTION

[0007] In an aspect, the invention relates to a method of generating an immune response to picornaviruses. The method includes encoding a capsid polyprotein and viral protease as nucleic acid molecules. The method includes mixing these nucleic acid molecules in an appropriate ratio to produce the optimal biological outcome of capsid protein translation and proteolytic processing. The method furthermore includes generating a nanoparticle colloidal solution incorporating the nucleic acid mixture into nanoparticles containing an ionizable delivery compound that complexes with the nucleic acid molecules. The method also includes delivering the nucleic acid molecules encapsulated in the nanoparticle product to the cytoplasm of cells of a host organism.

[0008] In an aspect, the invention relates to a method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a virus of the Picornaviridae family, or of inducing an immune response against the picornavirus in a subject. The method comprises administering to the subject a vaccine composition comprising an effective amount of a first nucleic acid encoding a P1 polyprotein mixed with a second nucleic acid encoding a 3C protease, and a nanoparticle carrier that encapsulates the first nucleic acid and the second nucleic acid.

[0009] In an aspect, the invention relates to a vaccine composition for protecting a subject against a viral disease caused by a viral pathogen of the Picornaviridae family. The vaccine composition comprises a first mRNA sequence encoding the capsid P1 polyprotein of picornavirus and a second mRNA sequence encoding the viral 3C protease that processes the P1 polyprotein. These molecules are mixed at preferred ratios, encapsulated in a nucleic acid nanoparticle carrier to be delivered to cells. The carrier includes an ionizable molecule that complexes with the mRNA by charge-based interaction, and optionally additional excipients to produce a stable nanoparticle.

[0010] In an aspect, the invention relates to a kit comprising any one of the vaccine compositions disclosed herein. The kit is used for single administration of the vaccine, or sequential administration of the vaccine in a prime / boost regime.

[0011] In an aspect, the invention relates to a method of preparing any one of the vaccine compositions disclosed herein for protecting a subject against a viral pathogen of the Picornaviridae family. The method comprises mixing a first nucleic acid encoding a P1 polyprotein with a second nucleic acid29406520.1encoding a 3C protease to obtain a nucleic acid mixture. The method further comprises combining the nucleic acid mixture with a delivery compound that complexes with the first nucleic acid and the second nucleic acid.

[0012] These and other aspects and features of non-limiting embodiments of the present invention will become apparent to those skilled in the art upon review of the following description of specific non-limiting embodiments of the invention in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, particular embodiments are shown in the drawings. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0014] FIG. 1 is a diagram showing the structure of the foot-and-mouth disease virus (FMDV), a prototypical picornavirus;

[0015] FIG. 2 is a picture of an immunoblot performed on lysates of cells transfected with different mRNA molecules;

[0016] FIG. 3 is a chart depicting the humoral immune response as percent of inhibition to different candidate nanoparticle formulations of P1-only, or P1 and 3C protease, N1-methylpseudouridine-modified mRNA payloads mixed at different ratios (shown as 16:1 , 32:1 , and 64:1) in two types of delivery compounds: Delivery A and Delivery B;

[0017] FIG. 4 is a chart depicting the humoral immune response in cattle to different candidate nanoparticle formulations of P1 and 3C protease payloads mixed at different P1 :3C mRNA mass ratios and delivered as nanoparticles;

[0018] FIG. 5 is a chart showing the viral neutralizing titer of the same serum samples analyzed in FIG. 4;

[0019] FIG. 6 is a chart depicting the percent weight change of wild-type CD-1 mice administered nanoparticles (NPs) formulated with Delivery compound B containing the indicated mRNAs: NP1 (P1 mRNA), NP2 (P1 + 3C protease mRNA), and Neg (Saline Control);

[0020] FIG. 7 is a set of charts showing the concentrations of proinflammatory cytokines IL-6, IFN- alpha, RANTES, and MCP-1 in the serum of the mice described in FIG. 6 at 24 hours post-injection;

[0021] FIGS. 8A and 8B are sets of charts showing the concentrations of (FIG. 8A) cardiac troponin- I and (FIG. 8B) alanine transaminase in the serum of the mice described in FIG. 6 at 24 hours post-injection with NP1 (P1 mRNA), NP2 (P1 + 3C protease mRNA) and Neg (Saline Control);

[0022] FIGS. 9A and 9B are a scheme (FIG. 9A) and a table summarizing a cattle immunogenicity and challenge infection study performed to test the efficacy of the P1+3C protease mRNA (32:1 mass ratio) vaccine composition characterized in FIGS. 6, 7, 8A and 8B; and39406520.1

[0023] FIG. 10 is a set of charts showing the results of RT-qPCR-based detection of viral shedding in oral swabs from each animal described in FIGS. 9A and 9B.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Certain terminology is used in the following description for convenience only and is not limiting. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0025] The singular terms “a," “an," and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or" is intended to include “and” unless the context clearly indicates otherwise.

[0026] The phrase “at least one” followed by a list of two or more items, such as “A, B, or C,” means any individual one of A, B or C as well as any combination thereof.

[0027] The words "right," "left," "top," and "bottom" designate directions in the drawings to which reference is made.

[0028] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0029] Picornaviruses are a group of related nonenveloped RNA viruses which infect vertebrates including fish, mammals and birds The viruses in this family can cause a range of diseases including the common cold, poliomyelitis, meningitis, hepatitis, and paralysis. Notable examples are genera Enterovirus (including Rhinovirus and Poliovirus), Aphthovirus, Cardiovirus, and Hepatovirus.

[0030] As used herein, Foot-and-Mouth Disease (FMD) refers to a contagious degenerative viral disease of cloven-hoofed animals including domestic animals such as, cattle, sheep, goats, and swine, and wild animals, such as buffalo, bison, deer, and antelope.

[0031] It is caused by a Picornavirus, and its symptoms include fever, loss of appetite and weight, blistering or boils on mucous membranes of the mouth, feet and udder, lameness and, it results in death. FIG. 1 is a diagram showing the structure of the Foot-and-Mouth Disease Virus (FMDV), a prototypical picornavirus. Referring to this figure, the virion (top) comprises the RNA genome, and a capsid composed of viral structural proteins VP1 , VP2, VP3, and VP4. The genome is a single-stranded RNA molecule organized into three main coding segments— P1 , P2, and P3— each playing distinct roles in viral structure and replication

[0032] The P1 protein encodes the VP1 , VP2, VP3, and VP4 structural proteins of the virus. These proteins create the outer shell of the virion, which protects the viral RNA, facilitates attachment and entry into host cells, and serves as the primary antigen target of the humoral immune response. The protein P1 , also49406520.1referred to herein as the capsid polyprotein of picornavirus, or the P1 polyprotein, is processed by the FMDV 3C protease, also referred to herein as 3Cpro, into the mature VP1 , VP2, VP3, and VP4 proteins. The P2 and P3 segments are responsible for encoding non-structural proteins that are critical for viral replication and pathogenesis within host cells. The P3 segment encodes proteins 3A, 3B, 3C, and 3D, which also have key roles in viral RNA replication. The 3C protein, or protease, referred to herein as 3C protease, 3Cpro or simply 3C, acts as a protease that cleaves the viral polyprotein into functional components, including the junctures between VP2 and VP3, and between VP3 and VP1 .

[0033] Poliomyelitis is an illness caused by a Poliovirus that mainly affects nerves in the spinal cord or brain stem, and in its most severe form, poliomyelitis can lead to a person being unable to move certain limbs, being paralyzed, or result in death. It is caused by Poliovirus, a virus of the Picornaviridae family classified under the Enterovirus genus.

[0034] Hand, foot and mouth disease (HFMD) refers to a severe neurological disease in children. It is caused by Enterovirus 71 (EV-A71 ), a virus of the genus Enterovirus in the Picornaviridae family.

[0035] The term “vaccine” as used herein refers to a preparation which, when administered to a subject, induces or stimulates a protective immune response. A vaccine can render an organism immune to a particular disease. The disease may be an FMD, poliomyelitis, or HFMD.

[0036] To “protect a subject against a viral infection” means aiding in preventing, ameliorating or curing a pathogenic infection, or aiding in preventing, ameliorating or curing a disorder arising from that infection, for example to prevent or reduce one or more clinical signs resulting from a post treatment (i.e., post vaccination) infection with a pathogen. The pathogen may be a virus of the Picornaviridae family, such as a Picornavirus, a Poliovirus or an Enterovirus, for example, Enterovirus 71 (EV-A71). The term “Picornavirus” as used herein refers to Picornaviruses, Polioviruses and Enteroviruses.

[0037] The term “prevention” or “preventing” is intended to refer to averting, delaying, impeding or hindering the viral infection by a prophylactic treatment. The vaccine may, for example, prevent or reduce the likelihood of an infectious virus entering a host cell.

[0038] The terms “subject” and “individual" are used interchangeably herein, and mean a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g ., trout, catfish and salmon. The animals may be cloven- hoofed animals including domestic animals such as, cattle, sheep, goats, and swine, and wild animals, such as buffalo, bison, deer, and antelope. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In an embodiment,59406520.1the subject may be a mammal, e.g., a primate, e.g., a human. The terms, "patient” and “subject” are used interchangeably herein.

[0039] Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, or other cloven-hoofed animals, but are not limited to these examples. In addition, the methods described herein may be used to treat domesticated animals and / or pets. A subject may be male or female A subject may be a child or adult.

[0040] An embodiment provides a vaccine composition for protecting a subject against a Picornavirus comprising an effective amount of a first nucleic acid encoding a P1 polyprotein. The vaccine composition may further comprise an effective amount of the second nucleic acid encoding a 30 protease. The vaccine composition comprising a P1 - and 3C protease-encoding nucleic acids may be formulated with a nanoparticle carrier encapsulating the nucleic acid molecules.

[0041] An embodiment provides a vaccine composition for protecting a subject against a Poliovirus, comprising an effective amount of a first nucleic acid encoding a P1 polyprotein and a second nucleic acid encoding a 3C protease. The Picornavirus may be a Foot and Mouth Disease Virus (FMDV). The FMDV may be of serotype O, A, C, Asia 1 , SAT 1 , SAT 2, or SAT 3, or any combination thereof.

[0042] An embodiment provides a vaccine composition for protecting a subject against Enterovirus 71 (EV-A71 ), comprising an effective amount of a first nucleic acid encoding a P1 polyprotein and a second nucleic acid encoding a 3C protease. The vaccine composition may comprise mRNA or self-amplifying RNA (saRNA) molecules formulated in a nanoparticle carrier that encapsulates the nucleic acids.

[0043] The vaccine composition may be a prime vaccine. The prime vaccine may be combined with a boost vaccine composition. The boost vaccine composition may be similar to the prime vaccine composition. The boost vaccine composition may differ from the composition of the prime vaccine.

[0044] The first nucleic acids or the second nucleic acids may be an RNA or DNA molecule. The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides).

[0045] The RNA molecule may be a messenger RNA (mRNA), self-replicating RNA (saRNA) or replicon RNA (repRNA). An mRNA may encode a protein, polypeptide, or peptide that acts as an antigen. The messenger RNAs (mRNAs) are single-stranded RNAs that define the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA. The saRNA is an mRNA engineered to replicate itself within host cells, enhancing protein expression and boosting the immune response. SaRNA can sustain protein expression for a longer periods compared to that of a conventional mRNAs. SaRNA may be used for creation of vaccines and other therapeutic applications. The first nucleic acid or the second nucleic acids may be an mRNA, or self-amplifying RNA (saRNA) based on modified alphavirus genomes. The RNA molecules may contain modified nucleotides. The modified69406520.1nucleotides may be 5-methyluridine, 5-methylcytidine, pseudouridine, or N6-methyladenosine. The modified nucleosides may include N1-methylpseudouridine.

[0046] An embodiment provides a vaccine composition comprising mRNAs encoding P1 and 3C polypeptides homologous in amino acid sequence to those encoded by the genomic polyproteins of foot-and- mouth disease virus (FMDV) of serotype 0, A, C, Asia 1 , SAT 1 , SAT 2, or SAT 3. Table 1 shows representative embodiments of FMDV P1 Polyprotein and 3C Protease sequences.

[0047] Table 1 . Representative Embodiments of FMDV P1 Polyprotein and 3C ProteaseSequences** Each embodiment includes nucleic acids encoding the P1 polyprotein and 3C protease of FMDV serotypes O, A, C, Asia 1 , and SAT 1-3

[0048] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype O. The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 1 .

[0049] The first nucleic acid may encode the P1 polyprotein of FMDV of serotype O. The P1 polyprotein of the FMDV of serotype O may have an amino acid sequence as set forth in SEQ ID NO: 19. The first nucleic acid may encode a fragment of the P1 polyprotein of the FMDV of serotype O protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 730, 731 , 732, 733, 734, 735, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype O comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 19. The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype O that is a fragment of a P1 polyprotein of the FMDV of serotype O of 731 amino acids or longer.79406520.1

[0050] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype 0. The second nucleic acid encoding the 3C protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 2.

[0051] The second nucleic acid may encode the 3C protease of the FMDV of serotype O. The 3C protease of the FMDV of serotype O may have an amino acid sequence as set forth in SEQ ID NO: 20. The second nucleic acid may encode a fragment of the 30 protease of the FMDV of serotype O. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 205, 210, 21 1 , 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive The second nucleic acid may encode the 3C protease of the FMDV of serotype O comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 20. The second nucleic acid may encode the 3C protease of the FMDV of serotype O that is a fragment of a 3C protease of the FMDV of serotype O of 200 amino acids or longer.

[0052] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype A. The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 3.

[0053] The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype A. The P1 polyprotein of the FMDV of serotype A may have an amino acid sequence as set forth in SEQ ID NO: 21. The first nucleic acid may encode a fragment of the P1 polyprotein of the FMDV of serotype A protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 720, 730, 731 , 732, 733, 734, 735, 736, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype A comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 21 . The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype A that is a fragment of a P1 polyprotein of the FMDV of serotype A of 731 amino acids or longer.

[0054] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype A. The second nucleic acid encoding the 30 protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 4.

[0055] The second nucleic acid may encode the 3C protease of the FMDV of serotype A. The 3C protease of the FMDV of serotype A may have an amino acid sequence as set forth in SEQ ID NO: 22. The second nucleic acid may encode a fragment of the 3C protease of the FMDV of serotype A. The fragment may89406520.1TIB-PT019WQ be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211 , 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the FMDV of serotype A comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 22. The second nucleic acid may encode the 3C protease of the FMDV of serotype A that is a fragment of a 3C protease of the FMDV of serotype A of 200 amino acids or longer.

[0056] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype C. The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 5.

[0057] The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype C. The P1 polyprotein of the FMDV of serotype C may have an amino acid sequence as set forth in SEQ ID NO: 23. The first nucleic acid may encode a fragmentof the P1 polyprotein ofthe FMDV of serotype C protein. Thefragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 710, 720, 730, 731 , or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype C comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 23. The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype C that is a fragment of a P1 polyprotein of the FMDV of serotype C of 730 amino acids or longer.

[0058] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype C. The second nucleic acid encoding the 30 protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 6.

[0059] The second nucleic acid may encode the 3C protease of the FMDV of serotype C. The 3C protease of the FMDV of serotype C may have an amino acid sequence as set forth in SEQ ID NO: 24. The second nucleic acid may encode a fragmentof the 3C protease of the FMDV of serotype C. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211, 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the FMDV of serotype C comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 24. The second nucleic acid may encode the 3C protease of the FMDV of serotype C that is a fragment of a 3C protease of the FMDV of serotype C of 200 amino acids or longer.99406520.1

[0060] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype Asia 1 . The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 7.

[0061] The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype Asia 1 . The P1 polyprotein of the FMDV of serotype Asia 1 may have an amino acid sequence as set forth in SEQ ID NO: 25. The first nucleic acid may encode a fragment of the P1 polyprotein of the FMDV of serotype Asia 1 protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 710, 720, 730, 731 , or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype Asia 1 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 25. The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype Asia 1 that is a fragment of a P1 polyprotein of the FMDV of serotype Asia 1 of 730 amino acids or longer.

[0062] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype Asia 1 . The second nucleic acid encoding the 3C protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 8.

[0063] The second nucleic acid may encode the 3C protease of the FMDV of serotype Asia 1. The 30 protease of the FMDV of serotype SAT 3 may have an amino acid sequence as set forth in SEQ ID NO: 26. The second nucleic acid may encode a fragment of the 30 protease of the FMDV of serotype Asia 1. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 21 1 , 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the FMDV of serotype Asia 1 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 26. The second nucleic acid may encode the 3C protease of the FMDV of serotype Asia 1 that is a fragment of a 3C protease of the FMDV of serotype Asia 1 of 200 amino acids or longer.

[0064] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype SAT 1 . The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 9.

[0065] The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 1 . The P1 polyprotein of the FMDV of serotype SAT 1 may have an amino acid sequence as set forth in SEQ ID NO: 27.109406520.1TIB-PT019WQThe first nucleic acid may encode a fragment of the P1 polyprotein of the FMDV of serotype SAT 1 protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 710, 720, 730, 735, 740, 741 , 742, 743, 744, 745, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 1 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 27. The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 1 that is a fragment of a P1 polyprotein of the FMDV of serotype SAT 1 of 730 amino acids or longer.

[0066] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype SAT 1 . The second nucleic acid encoding the 3C protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 10.

[0067] The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 1 . The 3C protease of the FMDV of serotype SAT 1 may have an amino acid sequence as set forth in SEQ ID NO: 28. The second nucleic acid may encode a fragment of the 3C protease of the FMDV of serotype SAT 1. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 21 1 , 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 1 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 28. The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 1 that is a fragment of a 3C protease of the FMDV of serotype SAT 1 of 200 amino acids or longer.

[0068] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype SAT 2. The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 1 1 .

[0069] The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 2. The P1 polyprotein of the FMDV of serotype SAT 2 may have an amino acid sequence as set forth in SEQ ID NO: 29. The first nucleic acid may encode a fragment of the P1 polyprotein of the FMDV of serotype SAT 2 protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 710, 720, 730, 730, 735, 740, 741 , or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 2 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a119406520.1TIB-PT019WQ reference sequence as set forth in SEQ ID NO: 29. The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 2 that is a fragment of a P1 polyprotein of the FMDV of serotype SAT 2 of 730 amino acids or longer.

[0070] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype SAT 2. The second nucleic acid encoding the 3C protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 12.

[0071] The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 2. The 30 protease of the FMDV of serotype SAT 2 may have an amino acid sequence as set forth in SEQ ID NO: 30. The second nucleic acid may encode a fragment of the 30 protease of the FMDV of serotype SAT 2. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 21 1 , 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 2 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 30. The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 2 that is a fragment of a 3C protease of the FMDV of serotype SAT 2 of 200 amino acids or longer.

[0072] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the FMDV of serotype SAT 3. The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 13.

[0073] The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 3. The P1 polyprotein of the FMDV of serotype SAT 3 may have an amino acid sequence as set forth in SEQ ID NO: 31. The first nucleic acid may encode a fragment of the P1 polyprotein of the FMDV of serotype SAT 3 protein. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 710, 720, 730, 730, 735, 740, 741 , or any number of amino acids in a range between any two of the foregoing (end points inclusive). The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 3 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 31. The first nucleic acid may encode the P1 polyprotein of the FMDV of serotype SAT 3 that is a fragment of a P1 polyprotein of the FMDV of serotype SAT 3 of 730 amino acids or longer.

[0074] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the FMDV of serotype SAT 3. The second nucleic acid encoding the 3C protease may be an RNA129406520.1sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 14.

[0075] The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 3. The 30 protease of the FMDV of serotype SAT 3 may have an amino acid sequence as set forth in SEQ ID NO: 32. The second nucleic acid may encode a fragment of the 30 protease of the FMDV of serotype SAT 3. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 211 , 212, 213, 214, 215, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 3 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 32. The second nucleic acid may encode the 3C protease of the FMDV of serotype SAT 3 that is a fragment of a 3C protease of the FMDV of serotype SAT 3 of 200 amino acids or longer.

[0076] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the Human Poliovirus type 2. The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 15.

[0077] The first nucleic acid may encode the P1 polyprotein of the Human Poliovirus type 2. The P1 polyprotein of the Human Poliovirus type 2 may have an amino acid sequence as set forth in SEQ ID NO: 33. The first nucleic acid may encode a fragment of the P1 polyprotein of the Human Poliovirus type 2. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 810, 820, 830, 840, 850, 860, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the Human Poliovirus type 2 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 33. The first nucleic acid may encode the P1 polyprotein of the Human Poliovirus type 2 that is a fragment of a P1 polyprotein of the Human Poliovirus type 2 of 860 amino acids or longer.

[0078] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the of the Human Poliovirus type 2. The second nucleic acid encoding the 3C protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 16.

[0079] The second nucleic acid may encode the 30 protease of the Human Poliovirus type 2. The 3C protease of the Human Poliovirus type 2 may have an amino acid sequence as set forth in SEQ ID NO: 34. The second nucleic acid may encode a fragment of the 30 protease of the Human Poliovirus type 2. The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80,139406520.1TIB-PT019WQ90. 100. 110. 120. 130. 140. 150. 160. 170. 175. 180. 181. 182. 183. 184, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The second nucleic acid may encode the 3C protease of the Human Poliovirus type 2 comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 34. The second nucleic acid may encode the 3C protease of the Human Poliovirus type 2 that is a fragment of a 3C protease of the Human Poliovirus type 2 of 175 amino acids or longer.

[0080] In an embodiment, the first nucleic acid may be an mRNA or saRNA encoding the P1 polyprotein of the Human Enterovirus 71 (strain 7423 / MS / 87). The first nucleic acid encoding the P1 polyprotein may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence set forth in SEQ ID NO: 17.

[0081] The first nucleic acid may encode the P1 polyprotein of the Human Enterovirus 71 (strain 7423 / MS / 87. The P1 polyprotein of the Human Enterovirus 71 (strain 7423 / MS / 87) may have an amino acid sequence as set forth in SEQ ID NO: 35. The first nucleic acid may encode a fragment of the P1 polyprotein of the Human Enterovirus 71 (strain 7423 / MS / 87). The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 810, 820, 830, 840, 850, 860, 861 , 862, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive). The first nucleic acid may encode the P1 polyprotein of the Human Enterovirus 71 (strain 7423 / M S / 87) comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 35. The first nucleic acid may encode the P1 polyprotein of the Human Enterovirus 71 (strain 7423 / MS / 87) that is a fragment of a P1 polyprotein of the Human Enterovirus 71 (strain 7423 / MS / 87) of 860 amino acids or longer.

[0082] In an embodiment, the second nucleic acid may be an mRNA or saRNA encoding the 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87). The second nucleic acid encoding the 3C protease may be an RNA sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 18.

[0083] The second nucleic acid may encode the 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87). The 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87) may have an amino acid sequence as set forth in SEQ ID NO: 36. The second nucleic acid may encode a fragment of the 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87). The fragment may be of 10 amino acids or longer. The fragment may comprise 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175,180. 181. 182. 183. 184, or any number of amino acids in a range between any two of the foregoing (endpoints inclusive) The second nucleic acid may encode the 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87) comprising an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identity to a reference sequence as set forth in SEQ ID NO: 36. The second nucleic acid149406520.1may encode the 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87) that is a fragment of a 3C protease of the Human Enterovirus 71 (strain 7423 / MS / 87) of 175 amino acids or longer.

[0084] Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity is measured by the Smith Waterman algorithm (Smith TF, Waterman MS 1981 “Identification of Common Molecular Subsequences,” J Mol Biol 147: 195 - 197, which is incorporated herein by reference as if fully set forth).

[0085] An embodiment provides a vaccine composition comprising the P1 polyprotein mRNA and 3C protease mRNA molecules mixed such that the mass ratio of the mRNAs is 32:1 (P1 to 3C) or greater. The amount of the P1 polyprotein mRNA mixed with the 3C protease mRNA may be an amount satisfying a ratio of the P1 polyprotein mRNA to 3C protease mRNA selected from the value of greater than or equal to one of 8:1 (w:w), 16:1 (w / w) 32:1 (w:w), 64:1 (w:w), or 96: 1 (w:w), or any ratio in a range between any two of the foregoing (endpoints inclusive). For example, the P1 polyprotein mRNA to 3C protease mRNA ratio may be a value less than any integer or non-integer number selected from 16:1 to 32:1. The P1 polyprotein mRNA to 3C protease mRNA ratio may be equal to 16:1 (w:w), 17: 1 (w:w), 18:1 (w:w), 19:1 (w:w): 20:1 (w:w), 21 :1 (w:w), 22: 1 (w:w), 23:1 (w:w), 24:1 (w:w), 25:1 (w:w), 26:1 (w:w), 27:1 (w:w), 28:1 (w:w), 29:1 (w:w), 30:1 (w:w), 31 :1 (w:w) or 32:1 (w:w) or any ratio in a range between any two of the foregoing (endpoints inclusive). For example, the P1 polyprotein mRNA to 3C protease mRNA ratio may be a value equal to any integer or non-integer number in the range from 16:1 to 32:1 . The P1 polyprotein mRNA to 3C protease mRNA ratio may be 32:1 (w / w). The embodiment provides the 3C protease mRNA at a ratio low enough to avoid unwanted toxic effects in a human or animal subject upon administration, while still retaining sufficient proteolytic activity to process the P1 polypeptide.

[0086] An embodiment provides a composition comprising these mRNA molecules and a nanoparticle carrier. The embodiment also provides the preferred ratios described herein in which these mRNA molecules are delivered to cells in a nanoparticle carrier.

[0087] The carrier may include an ionizable molecule that complexes with the mRNA by chargebased interaction.

[0088] The first nucleic acid and / or the second nucleic acid may be non-covalently bound or covalently bound to the nanoparticle carrier. The first and / or second nucleic acids may be electrostatically bound to the charged nanoparticle carrier through an ionic bond. In an embodiment, the first and / or second nucleic acids may be encapsulated by the nanoparticle carrier.

[0089] As used herein, “encapsulated" can refer to a vaccine composition that provides an active agent, such as the first and second nucleic acids (e.g., a messenger RNAs), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acids are fully encapsulated in the nanoparticle.159406520.1In the context of nucleic acid therapeutic or preventive agents, full encapsulation may be determined by a Ribogreen® assay. RiboGreen® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA or RNA in solution (available from Thermo Fisher Scientific - US).

[0090] The first and / or second nucleic acid may be encapsulated in a nanoparticle carrier system for in vivo administration. The nanoparticle carrier may be, but is not limited to a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle or a dendrimer.

[0091] As used herein, the term “aminolipid” refers to an amphiphilic compound comprising at least one amine group covalently attached to a hydrophobic moiety, such as an alkyl, alkenyl, or alkoxyalkyl chain, which confers both water- and lipid-soluble character. Aminolipids include compounds having primary, secondary, tertiary, or quaternary amines, and optionally one or more ionizable nitrogen atoms capable of becoming protonated under acidic or physiological conditions. Aminolipids are typically used as components in nanoparticles for the encapsulation and intracellular delivery of nucleic acids. In certain embodiments, the aminolipid comprises: (i) an amine head group (e.g., diethylamine, dimethylamino, piperidine, morpholine, or imidazole derivatives), and (ii) a lipophilic tail region consisting of one or more C6-C24 hydrocarbon chains, optionally branched or unsaturated, linked through ester, ether, amide, or carbamate bonds. The aminolipid may be biodegradable, incorporating ester, disulfide, or other such labile linkages that readily break in the intra- or extracellular milieu within an organism. Representative examples of aminolipids include, but are not limited to aminolipids described in WO2025096681 , published May 8, 2025, which is incorporated herein by reference as if fully set forth. For example, aminolipids may include DLin-MC3-DMA, SM-102, ALC-0315, or other synthetic amine-containing lipids used for mRNA delivery.

[0092] As used herein, the term “polymeric nanoparticle” refers to a colloidal particle comprising one or more natural or synthetic polymers, having a mean hydrodynamic diameter typically in the range of 10 nm to 1000 nm, preferably 20 nm to 300 nm, and capable of encapsulating, adsorbing, or complexing a therapeutic or prophylactic nucleic acid payload Polymeric nanoparticles may be formulated as solid spheres, nanocapsules, micelles, or hybrid polymer-lipid particles, and may include biodegradable or non- biodegradable polymers comprising labile covalent linkages such as ester or disulfide bonds. The polymeric matrix can associate with nucleic acids through electrostatic, hydrophobic, or hydrogen-bonding interactions. In certain embodiments, the polymeric nanoparticle comprises one or more polymers selected from poly(l actic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(P-amino esters) (PBAEs), polyethyleneimine (PEI), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), polycaprolactone (PCL), chitosan, dextran, or poly(ethy lene glycol) (PEG) derivatives. The polymeric nanoparticle may be cationic, ionizable, or PEGylated, and can optionally include excipients such as surfactants, stabilizers, or targeting ligands.

[0093] Unless otherwise indicated, the term encompasses both self-assembled polymeric systems and crosslinked or emulsified polymer matrices that are capable of stably incorporating nucleic acids and facilitating their cellular uptake and cytoplasmic delivery.169406520.1

[0094] The liposomes may be an anionic liposomes or cationic liposomes. The term "anionic liposomes” refers to liposomes that include lipids comprising an anionic group. Anionic liposomes may be formed by anionic phospholipids. The phospholipids may include but are not be limited to 1 ,2-Didecanoyl-sn- Glycero-3-phosphatidylcholine (DDPC); 1 ,2-Dierucoyl-sn-Glycero-3-Phosphate (DEPA); 1 ,2-Erucoyl-sn- Glycero-3-phosphatidylcholine (DEPC); 1 ,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine (DEPE); 1 ,2- Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1 -glycerol .) (DEPG); 1 ,2-Linoleoyl-sn-Glycero-3- phosphatidylcholine (DLOPC); 1 ,2-Dilauroyl-sn-Glycero-3-Phosphate (DLPA); 1 ,2-Dilauroyl-sn-Glycero-3- phosphatidylcholine (DLPC); 1 ,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine (DLPE); 1 ,2-Dilauroyl-sn- Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) (DLPG); 1 ,2-Dilauroyl-sn-Glycero-3-phosphatidylserine (DLPS);1 .2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMG); 1 ,2-Dimy ristoyl-sn-Glycero-3-Phosphate (DMPA); 1 ,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine (DMPC); 1 ,2-Dimyristoyl-sn-Glycero-3- phosphatidylethanolamine (DMPE); 1 ,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1 -glycerol . . .) (DMPG);1 .2-Dimyristoyl-sn-Glycero-3-phosphatidylserine (DMPS); 1 ,2-Dioleoyl-sn-Glycero-3-Phosphate (DOPA); 1 ,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine (DOPC); 1 ,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine (DOPE); 1 ,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1 -glycerol . . .) (DOPG); 1 ,2-Dioleoyl-sn-Glycero-3- phosphatidylserine (DOPS); 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphate DPPC 1 ,2-Dipalmitoyl-sn-Glycero-3- phosphatidylcholine (DPPA); 1 ,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine (DPPE), 1 ,2- Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) (DPPG); 1 ,2-Dipalmitoyl-sn-Glycero-3- phosphatidylserine (DPPS); 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE); 1 ,2-Distearoyl-sn- Glycero-3-Phosphate (DSPA); 1 ,2-Distearoyl-sn-Glycero-3-phosphatidylcholine (DSPC); 1 ,2-Distearpyl-sn- Glycero-3-phosphatidylethanolamine (DSPE); 1 ,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1 -glycerol . . .) (DSPG); 1 ,2-Distearoyl-sn-Glycero-3-phosphatidylserine (DSPS); Egg-PC HEPC Hydrogenated Egg PC (EPC); High purity Hydrogenated Soy PC HSPC Hydrogenated Soy PC (HSPC); 1-Myristoyl-sn-Glycero-3- phosphatidylcholine (LYSOPC MYRISTIC); 1 -Palmitoyl-sn-Glycero-3-phosphatidylcholine (LYSOPC PALM ITIC); 1 -Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin (LYSOPC STEARIC); 1- Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine (MPPC); 1 -Myristoyl, 2-stearoyl-sn-Glycero-3- phosphatidylcholine (MSPC); 1 -Palmitoyl, 2-myristoyl-sn-Glycero-3-phosphatidylcholine (PMPC); 1- Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine (POPC); 1 -Palmitoyl-2-oleoyl-sn-Glycero-3- phosphatidylethanolamine (POPE); 1 ,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1 -glycerol) . . .] (POPG); 1- Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine (PSPC); 1 -Stearoyl, 2-myristoyl-sn-Glycero-3- phosphatidylcholine (SMPC); 1 -Stearoyl, 2-oleoyl-sn-Glycero-3-phosphatidylcholine (SOPC); and 1- Stearoyl ,2-palmitoyl-sn-Glycero-3-phosphatidylcholine (SPPC). Useful phospholipids may include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and phosphatidylglycerols.

[0095] The term "cationic liposomes” refers to liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic179406520.1portion. The positively charged moieties of cationic lipids used in cationic liposomes provide advantageous structural features. For instance, the lipophilic portion of the cationic lipid is hydrophobic and thus may direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species, or conversely, the cationic moiety may associate with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. The positively charged liposomes may interact with the negatively charged nucleic acid molecules to form a stable complex. Cationic lipids may include but are not be limited to, dioleoyl trimethylammonium propane (DOTAP), 1 ,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 1 ,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA), 1 ,2-dilinoleyloxy-N,N- dimethyl-3-aminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.

[0096] Liposomes may include zwitterionic lipids. As used herein the term "zwitterionic" refers to a molecule that contains both positive and negative charges, but have a net neutral charge, Zwitterionic lipids, also referred to herein as neutral lipids, may include but not be limited to acyl zwitterionic lipids and ether zwitterionic lipids. Zwitterionic lipids may include DPPC, DOPC and dodecylphosphocholine.

[0097] Liposomes may be formed from a single lipid or from a mixture of lipids. The hydrophilic portion of a lipid can be PEGylated, i.e., modified by covalent attachment of a polyethylene glycol to increase stability and prevent non-specific adsorption of the liposomes (Heyes et al. (2005) J Controlled Release 107:276-87, which is incorporated herein by reference as if fully set forth).

[0098] The term "dendrimer” refers to a highly branched macromolecule with a spherical shape. The surface of the dendrimer molecule may be modified in many ways, and many of the properties of the resulting construct may be determined by its surface. The dendrimers may be modified to have a positive surface charge, i.e., to be cationic dendrimers. The cationic dendrimers may form temporary association with the nucleic acids. Upon reaching its destination the dendrimer-nucleic acid complex may be then taken into the cell via endocytosis.An exemplary size for a single dendrimer-nucleic acid complex, also referred to herein as modified dendrimer nanoparticles (MDNPs), may be in the range of 30 nm to 1 ,000 nm in the longest dimension. MDNPs may have an average size from 30 nm to 450 nm, inclusive, from 50 nm to 300 nm, inclusive, or more from 60 nm to 250 nm, inclusive. MDNPS may be alkyl-modified dendrimer nanoparticles. Nanoparticle size may be influenced by the length of the alkyl chain that substitutes the core dendrimer. Methods of making and formulating modified dendrimer nanoparticles are described in WO2021 207020, published October 14, 2021 ; which is incorporated herein by reference as if fully set forth.

[0099] The nanoparticle carrier may comprise excipients to produce a stable nanoparticle.

[0100] In an embodiment, the vaccine composition may include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically-acceptable carrier1' means a pharmaceutically-acceptable material, composition or vehicle, for example a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved189406520.1in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which may serve as pharmaceutically-acceptable carriers include: (1) sugars, for example lactose, glucose, mannose and / or sucrose; (2) starches, for example corn starch and / or potato starch; (3) cellulose, and its derivatives, for example sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and / or cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, for example magnesium stearate, sodium lauryl sulfate and / or talc; (S) excipients, for example cocoa butter and / or suppository waxes;(9) oils, for example peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and / or soybean oil;(10) glycols, for example propylene glycol; (1 1) polyols, for example glycerin, sorbitol, and / or mannitol; (12) esters, for example glycerides, ethyl oleate and / or ethyl laurate; (13) agar; (14) buffering agents, for example magnesium hydroxide and / or aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) diluents, for example isotonic saline, and / or PEG400; (I8) Ringer's solution; (19) C2-C12 alcohols, for example ethanol; (20) fatty acids; (21 ) pH buffered solutions; (22) bulking agents, for example polypeptides and / or amino acids (23) serum component, for example serum albumin, HDL and LDL; (24) surfactants, for example polysorbates (Tween 80) and / or poloxamers; and / or (25) other non-toxic compatible substances employed in pharmaceutical formulations: for example, fillers, binders, wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives and / or antioxidants. The terms “excipient,” “carrier,” “pharmaceutically acceptable carrier,” or the like are used interchangeably herein.

[0101] In an embodiment, any vaccine composition described herein may be used as a single dose vaccine.

[0102] In an embodiment, any prime vaccine described herein may be used as the boost vaccine. Any boost vaccine described herein may be used as the prime vaccine. The prime vaccine may be the same vaccine as used as the boost vaccine.

[0103] In an embodiment, the prime vaccine may be a multi-modal vaccine. The multi-modal vaccine may be a vaccine comprising two or more components. The first component may be an RNA component as described herein. The second component may be a protein component as known in the art. Each component of the multi-modal vaccine may play a dominant role at different periods of an immunization schedule.

[0104] Method of Immunization

[0105] An embodiment comprises a method for treating or preventing a disease or condition in a subject. The method may comprise generating an immune response to picornaviruses. The method may comprise providing any one of the vaccine compositions described herein. The method includes delivering the nucleic acid molecules encapsulated in the nanoparticle product to the cytoplasm of cells of a host organism.199406520.1The method may comprise administering a therapeutically effective amount of the nanoparticle composition to a subject.

[0106] An embodiment provides a method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a viral pathogen, or of inducing an immune response against a viral pathogen in a subject, such as a mammal, preferably a hoofed animal, is provided. The viral pathogens may be a picornavirus, a picornavirus serotype 0, A, C, Asia 1 , SAT 1 , SAT 2, or SAT 3 or mutants thereof. The method may comprise administering to the subject a vaccine composition comprising an effective amount of any one of first nucleic acid encoding a P1 polyprotein, a second nucleic acid encoding a 30 protease, and a carrier. The method may involve administering the vaccine composition as a single dose vaccine.

[0107] The method may involve administration of the vaccine compositions disclosed herein as a single dose vaccine.

[0108] The method may involve “priming” and “boosting” immunization regimes, in which the immune response induced by a prime vaccine may be boosted by a boost vaccine. For example, following priming (at least once) with any one of the first and second nucleic acid encoding a P1 polyproteins and a 3C protease, a boost vaccine of the same or different composition as the prime vaccine, may be administered to boost the immune response in the primed host.

[0109] The boost vaccine may be administered subsequently to the prime vaccine. The boost vaccine may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59 or 60, or any number of weeks in a range between any two of the foregoing (endpoints inclusive) after the prime vaccine is administered.

[0110] The subject may be primed and / or boosted more than once. For example, the immunization strategy may be prime, prime, boost; or prime, boost, boost. In an embodiment, the prime vaccine may be administered as least twice, at least 3 times, at least 4 times, at least 5 times, or at least 6 times. In an embodiment, the boost vaccine may be administered as least twice, at least 3 times, at least 4 times, at least 5 times, or at least 6 times.

[0111] In an embodiment, administration of the boosting may be performed weeks or months following administration of the prime vaccine. For example, the boost vaccine may be administered 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 55 weeks, 60 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years or any period of time in a range between any two of the foregoing (endpoints inclusive) after the priming composition is administered.209406520.1

[0112] As used herein, the terms "treat," treating," "treatment," refer to reducing or ameliorating a disorder and / or symptoms associated therewith. Treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0113] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0114] The vaccine may be administered in an effective amount. The efficacy of the therapeutic treatment may involve monitoring pathogen infection following administration of vaccine compositions disclosed herein. The efficacy of prophylactic treatment may involve monitoring immune responses systemically and / or mucosally against the antigen. Systemic monitoring of the immune responses may involve monitoring the levels of lgG1 and lgG2a production whereas mucosal monitoring may involve assessing the level of IgA production. Antigen-specific serum antibody responses may be determined post-immunization but pre-challenge. Antigen-specific mucosal antibody responses may be determined post-immunization and postchallenge.

[0115] Assessing the immunogenicity of the vaccine compositions disclosed herein where the first or second nucleic acid encodes P1 polyprotein or a 3C protease may involve expressing the nucleic acids recombinantly for screening patient sera or mucosal secretions by immunoblot and / or microarrays. A positive reaction between the protein and the host sample may indicate that the host has mounted an immune response to the assessed protein.

[0116] The efficacy of the vaccine compositions may also be determined in vivo by challenging appropriate animal models of the pathogen infection, for example, the FMDV, Poliovirus, or Enterovirus infection .

[0117] In an embodiment, the vaccine compositions may be administered according to an appropriate dosage schedule. Dosage may involve administration of a single dose schedule or multiple doses. Multiple doses may be used for administration of prime vaccine or boost vaccine. Multiple doses of the vaccines may be administered by the same or different routes. For example, the prime vaccine may be administered parenterally and the boost vaccine may be administered via mucosal route. Alternatively, the prime vaccine may be administered via mucosal route, and the boost vaccine may be administered parenterally. Multiple doses may be administered at least 1 week apart.

[0118] A satisfactory effect may be obtained by systemic administration, e.g., intramuscular administration, subcutaneous administration or intravenous administration 1 -4 times at the amount of 103-1010Infectious Unit (IU) or 0.01-500 pg per time, preferably 105-1010IU or 0.1-100 pg per time, for example 107- 109IU or 1 -50 pg per one time The replicon may preferably be formulated in a vaccine composition suitable for administration in a conventional manner.219406520.1

[0119] In an embodiment, routes of administration may include, but not be limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, and intraocular injection. Oral and transdermal administration, as well as administration by inhalation or suppository may also be used. The preferred routes of administration may include intramuscular, intradermal and subcutaneous injection.

[0120] Method of making

[0121] An embodiment provides a method of preparing a vaccine composition for protecting a subject against an FMD, poliomyelitis, or HFMD.

[0122] An embodiment provides a method of preparing a vaccine composition described in embodiments herein. The method includes encoding a capsid polyprotein and viral protease as nucleic acid molecules. The method includes mixing the first and the second nucleic acids in an appropriate ratio, for example, as described herein, to produce the optimal biological outcome of capsid protein translation and proteolytic processing. The method furthermore includes generating a colloidal solution incorporating the nucleic acid mixture into nanoparticles containing ionizable delivery compounds that complex with the nucleic acid molecules. The first and second nucleic acids may be combined with alkyl-modified dendrimer-based materials (modified dendrimer nanoparticle, MDNP), by self-assembly of nucleic acids and alkyl-modified dendrimers, by self-assembly of nucleic acids and cationic and neutral lipids, or by self-assembly of nucleic acids, alkyl-modified dendrimers and appropriately charged lipids.

[0123] In an embodiment, prior to its application the first and second nucleic acid containing formulation may be mixed with specific depot-forming adjuvants such as squalene / water and other nanoparticulate delivery systems.

[0124] An embodiment provides a kit comprising any one of the vaccine compositions disclosed herein. The kit may be used for single administration, or for sequential administration of the vaccine in a prime / boost regime. The kit may comprise an immunoregulatory agent as described in embodiments herein. The immunoregulatory agent may be an adjuvant. The vaccine composition may be a single dose vaccine. The vaccine composition may comprise the prime and boost vaccines. The single dose vaccine, the prime and boost vaccines may be included in a container. The prime and boost vaccine may be included in the same container, or separate containers. The single dose vaccine, the prime and boost vaccines may be in liquid form or solid form.

[0125] The following list includes particular embodiments of the present invention. But the list is not limiting and does not exclude alternate embodiments, or embodiments otherwise described herein. Percent identity described in the following embodiments list refers to the identity of the recited sequence along the entire length of the reference sequence.EMBODIMENTS1 . A vaccine composition for protecting a subject against a viral disease comprising an effective amount of a first nucleic acid encoding a P1 polyprotein mixed with a second nucleic acid encoding a 3C229406520.1protease, and a nanoparticle carrier, wherein the nanoparticle carrier encapsulates the first nucleic acid and the second nucleic acid, and the viral disease is caused by a viral pathogen of the Picornaviridae family.2. The vaccine composition of embodiment 1 , wherein the first nucleic acid or the second nucleic acid is selected from the group consisting of mRNA, saRNA, and repRNA.3. The vaccine composition of one or both embodiments 1 and 2, wherein the viral disease is caused by a viral pathogen selected from the group consisting an FMDV, a Poliovirus and an Enterovirus.4. The vaccine composition of any one or more of embodiments 1 - 3, wherein the viral pathogen is an FMDV of a serotype 0, serotype A, serotype C, serotype Asia 1 , serotype SAT 1 , serotype SAT 2, or serotype SAT 3, or any combination thereof.5. The vaccine composition of any one or more of embodiments 1 - 4, wherein the viral pathogen is an FMDV of a serotype 0, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1 , and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 19.6. The vaccine composition of any one or more of embodiments 1 - 5, wherein the viral pathogen is an FMDV of a serotype 0, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2, and encodes a 30 protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 20.7. The vaccine composition of any one or more of embodiments 1 - 4, wherein the viral pathogen is an FMDV of a serotype A, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 21 .8. The vaccine composition of any one or more of embodiments 1 - 4 and 7, wherein the viral pathogen is an FMDV of a serotype A, the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4, and encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 22.9. The vaccine composition of any one or more of embodiments 1 - 4, wherein the viral pathogen is an FMDV of a serotype C, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 23.10. The vaccine composition of any one or more of embodiments 1 - 4 and 9, wherein the viral pathogen is an FMDV of a serotype C, the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6, and encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 24.11 . The vaccine composition of any one or more of embodiments 1 -4, wherein the viral pathogen is an FMDV of a serotype Asia 1 , the first nucleic acid comprises an RNA sequence with at least 90% identity239406520.1to the sequence set forth in SEQ ID NO: 7, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 25.12. The vaccine composition of any one or more of embodiments 1 - 4 and 1 1 , wherein the viral pathogen is an FMDV of a serotype Asia 1 , the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8, and encodes a 30 protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2613. The vaccine composition of any one or more of embodiments 1 -4, wherein the viral pathogen is an FM DV of a serotype SAT 1 , the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 9, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 27.14. The vaccine composition of any one or more of embodiments 1 - 4 and 13, wherein the viral pathogen is an FMDV of a serotype SAT 1 , the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 10, and encodes a 30 protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 28.15. The vaccine composition of any one or more of embodiments 1 -4, wherein the viral pathogen is an FMDV of a serotype SAT 2, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 11 , and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 29.16. The vaccine composition of any one or more of embodiments 1 - 4 and 15, wherein the viral pathogen is an FMDV of a serotype SAT 2, the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 12, and encodes a 30 protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 30.17. The vaccine composition of any one or more of embodiments 1 -4, wherein the viral pathogen is an FMDV of a serotype SAT 3, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 13, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 31 .18. The vaccine composition of any one or more of embodiments 1 - 4 and 17, wherein the viral pathogen is an FMDV of a serotype SAT 3, the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 14, and encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 32.19. The vaccine composition of any one or more of embodiments 1 - 3, wherein the viral pathogen is a Poliovirus, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 15, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 33.249406520.1TIB-PT019WQ20. The vaccine composition of any one or more of embodiments 1 - 3 and 19, wherein the viral pathogen is a Poliovirus, the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 16aand encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 34.21 . The vaccine composition of any one or more of embodiments 1 - 3, wherein the viral pathogen is an Enterovirus, the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 17, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 35.22. The vaccine composition of any one or more of embodiments 1 - 3 and 21 , wherein the viral pathogen is an Enterovirus, the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 18, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 36.23. The vaccine composition of any one or more of embodiments 1 - 22, wherein the first nucleic acid is mixed with the second nucleic acid at a ratio greater or equal to one selected from the group consisting of 8:1 , 16:1 , 32:1 , 64:1 , and 96:1 of the first nucleic acid to the second nucleic acid (w / w)24. The vaccine composition any one or more of embodiments 1 - 23, wherein the nanoparticle carrier is selected from the group consisting of: a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle and a dendrimer.25. A kit comprising the vaccine composition of any one or more of embodiments 1 - 24, wherein the kit is used for single dose vaccine administration, or for sequential administration in a prime / boost regime.26. The kit of embodiment 25 further comprising an immunoregulatory agent.27. The kit of both of embodiments 25 - 26 wherein the immunoregulatory agent is an adjuvant.28. The kit of any one or more of embodiments 25 - 27, wherein the vaccine composition comprise the prime and boost vaccines29. The kit of any one or more of embodiments 25 - 28, wherein the single dose vaccine is included in a container, or wherein the prime and boost vaccine are included in the same container, or separate containers.30. The kit of any one or more of embodiments 25 - 29, wherein the single dose vaccine, the prime and boost vaccines are in liquid form or solid form.31 . A method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a virus of the Picornaviridae family, or of inducing an immune response against the virus in a subject, the method comprising: administering to the subject a vaccine composition comprising an effective amount of a first nucleic acid encoding a P1 polyprotein mixed with a second nucleic acid encoding a 3C protease, and a nanoparticle carrier that encapsulates the first nucleic acid and the second nucleic acid.259406520.132. The method of embodiment 31 , wherein the first nucleic acid or the second nucleic acid is selected from the group consisting of an mRNA, saRNA, and a repRNA.33. The method of both of embodiments 31- 32, wherein the virus is an FMDV, a Poliovirus or an Enterovirus.34. The method of any one or more of embodiments 31 - 33, wherein the virus is an FMDV of a serotype 0, serotype A, serotype C, serotype Asia 1 , serotype SAT 1 , serotype SAT 2, or serotype SAT 3, or any combination thereof.35. The method of any one or more of embodiments 31 - 34, wherein the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence selected from the group consisting of SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , and 13, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence selected from the group consisting of SEQ ID NOS: 19, 21 , 23, 25, 27,29, and 31.36. The method of any one or more of embodiments 31 - 35, wherein the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence selected form the group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, and encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence selected from the group consisting of SEQ ID NOS: 20, 22, 24, 26, 28,30, and 32.37. The method of any one or more of embodiments 31 - 33, wherein the virus is a Poliovirus and the first nucleic acid comprises an RNA sequence with at least 90% identity to the set forth in SEQ ID NO: 15, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 33.38. The method of any one or more of embodiments 31 - 33 and 37, wherein virus is a Poliovirus, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 16, and encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 34.39. The method of any one or more of embodiments 31 - 33, wherein the virus is an Enterovirus and the first nucleic acid comprises an RNA sequence with at least 90% identity to the set forth in SEQ ID NO: 17, and encodes a P1 polyprotein comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 35.40. The method of any one or more of embodiments 31 - 39, wherein virus is an Enterovirus, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 18, and encodes a 3C protease comprising an amino acid sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 19.269406520.141 . The method of any one or more of embodiments 31 - 40, wherein the nanoparticle carrier is selected from the group consisting of: a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle and a dendrimer.42. The method of any one or more of embodiments 31 - 41 , wherein the vaccine composition is administered as a single dose vaccine.43. The method of any one or more of embodiments 31 - 42, wherein the vaccine composition comprises a prime vaccine and a boost vaccine, and is administered in a prime / boost regime.44. The method of any one or more of embodiments 31 - 43, wherein the boost vaccine is administered in a period of time ranging from 1 week to 60 weeks after the prime vaccine is administered.45. The method of any one or more of embodiments 31 - 44, wherein the step of administering results in preventing, treating, reducing the severity or slowing the progression of the disease in the subject.46. The method of any one or more of embodiments 31 - 45, wherein the subject is a cloven- hooved animal or a human.47. The method of any one or more of embodiments 31 - 46, wherein the cloven-hoofed animal is selected from the group consisting of: cattle, water buffalo, sheep, goat, pig, antelope, deer, bison, hedgehog, elephant, llama and alpaca.48. A method of preparing a vaccine composition of any one or more of embodiments 1 - 24 for protecting a subject against a viral pathogen of the Picornaviridae family comprising; mixing a first nucleic acid encoding a P1 polyprotein with a second nucleic acid encoding a 3C protease to obtain a nucleic acid mixture; and combining the nucleic acid mixture with a delivery compound that complexes with the first nucleic acid and the second nucleic acid.49. The method of embodiment 48, wherein the first nucleic acid is mixed with the second nucleic acid at a ratio greater or equal to one selected from the group consisting of 8:1 , 16:1 , 32:1 , 64:1 , and 96:1 of the first nucleic acid to the second nucleic acid (w / w).50. The method of both of embodiments 48 - 49, wherein the delivery compound is selected from the group consisting of: a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle and a dendrimer.51 . The method of any one or more of embodiments 48 - 50 further comprising formulating the vaccine composition with a pharmaceutically acceptable carrier.52. The method of any one or more of embodiments 48 - 51 further comprising formulating the vaccine compositions with one or more pharmaceutically acceptable diluents, adjuvants and / or excipient.279406520.1

[0126] Further embodiments herein may be formed by supplementing an embodiment with one or more elements from any one or more other embodiments herein, and / or substituting one or more elements from one embodiment with one or more elements from one or more other embodiments.EXAMPLES

[0127] The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and / or one or more element from an embodiment may be substituted with one or more detail from one or more example below.

[0128] Example 1. Vaccine Design

[0129] FMDV

[0130] FIG. 1 is a diagram showing the structure of the foot-and-mouth disease virus (FMDV), a prototypical picornavirus. The top of the figure shows the virion that comprises the RNA genome, and a capsid composed of viral structural proteins VP1 , VP2, VP3, and VP4. The genome is a single-stranded RNA molecule organized into three main coding segments— P1 , P2, and P3— each playing distinct roles in viral structure and replication. The P1 segment encodes the VP1 , VP2, VP3, and VP4 structural proteins of the virus. These proteins create the outer shell of the virion, which protects the viral RNA, facilitates attachment and entry into host cells, and serves as the primary antigen target of the humoral immune response. The P2 and P3 segments are responsible for encoding non-structural proteins that are critical for viral replication and pathogenesis within host cells. The P3 segment encodes proteins 3A, 3B, 3C, and 3D, which also have key roles in viral RNA replication. Notably, 3C acts as a protease that cleaves the viral polyprotein into functional components, including the junctures between VP2 and VP3, and between VP3 and VP1 . The nucleic acid vaccine strategy disclosed herein works by encoding the highlighted VP4-VP2-VP3-VP1 portion of the P1 segment (referred to as the P1 polyprotein), and the 3C portion of the P3 segment (referred to as the 3C protease), each as a nucleic acid molecule.

[0131] Poliovirus Several studies on poliovirus and related enteroviruses have established that co-expression of the structural polyprotein P1 and the viral protease 3CD (or 3C) is necessary and sufficient for capsid cleavage and assembly of virus-like particles (VLPs). Early work demonstrated that recombinant expression of the P1 region together with 3CD in mammalian, yeast, and insect cells results in proper proteolytic processing and self-assembly of capsid precursors into immunogenic particles (Ansardi et al., 1996, *J. Virol * 70: 1613-1620; Mason et al., 2006, *J. Virol.* 80: 11009-1 1016). Subsequent research confirmed that the balance of P1 and 3CD expression matters —excessive 3CD expression leads to host-cell toxicity and degradation of capsid precursors, whereas insufficient expression reduces VLP formation (Jiang et al., 201 1 , *Vaccine* 29: 7182-7192). These studies generally optimize the relative promoter strength or infection multiplicity for P1 and 3CD but do not define a specific mass or molar ratio between P1 -encoding and 3C / 3CD-encoding nucleic acids. The present disclosure surprisingly define and empirically validate289406520.1TIB-PT019WQ specific nucleic-acid mass ratios between P1 and 3C protease sequences, such as approximately 32:1 , to achieve efficient antigen maturation and maximal immunogenicity in nucleic acid vaccine formulations targeting poliovirus.

[0132] Enterovirus 71 (EV-A71)

[0133] Similarly numerous studies of EV-A71 and related enteroviruses have demonstrated that co-expression of the P1 structural polyprotein and the 3C protease is sufficient to drive capsid cleavage and virus-like particle (VLP) assembly (Kim et al., 2019, *PLoS ONE* 14(1): e0210477; https: / / doi.org / 10.1371 / journal.pone.0210477). For example, Kim et al. reported that the P1 region was placed under the strong baculovirus polyhedrin promoter while the 3CD gene was placed under a variety of weaker promoters (CMV-IE, Ief3, gp41 , chitinase) to optimize VLP yield. More recent studies have similarly modulated 3CD expression strength— for example by introducing upstream sequence elements or untranslated region (UTR) insertions to weaken translation— while maintaining robust P1 expression, thereby improving antigen processing and VLP assembly (Jiang et al., 2024, *Viruses* 16(6): 834; https: / / doi.org / 10.3390 / v16060834). Notably, however, these publications do not report a defined quantitative or mass ratio of P1 -encoding to 3CD-encoding nucleic acids, such as 32:1 or 16:1 , in the context of vaccine design. Accordingly, there remains an unmet need in the art for defining optimized stoichiometric ratios between these components to achieve maximal antigen processing efficiency with minimal cytotoxicity.

[0134] The present disclosure specify and validate such ratios for use in nucleic acid vaccine formulations targeting picornaviruses, including EV-A71 , nucleic acid molecules and formulations that, when administered to a mammalian subject, lead to translation and appropriate post-transcriptional processing of picornavirus capsid, with the capacity to induce innate immune stimulation such that protective humoral immune responses are produced.

[0135] Example 2. Expression and Processing of Picornavirus Capsid Polyprotein In Vitro

[0136] Baby hamster kidney (BHK) cells were transfected with mRNA molecules encoding either (i) the picornavirus P1 polyprotein alone or (II) the P1 polyprotein and the viral 3C protease (3Cpro) at indicated P1 :3C mRNA mass ratios (16:1 , 32:1 , and 64:1). The P1 polyprotein mRNA comprises the sequence put forth in SEQ ID NO: 1 , encoding the polypeptide sequence put forth in SEQ ID NO: 19. The 30 protease mRNA comprises the sequence put forth in SEQ ID NO: 2, encoding the polypeptide sequence put forth in SEQ ID NO: 20. Transfections were performed using a commercial lipid-based reagent (Mirus Bio). Lysates were harvested 24 hours post-transfection, separated by SDS-PAGE, and probed with an FMDV capsid-specific monoclonal antibody. FIG 2 is a picture of an immunoblot performed on lysates of cells transfected with different mRNA molecules. Referring to this figure, baby hamster kidney cells (BHKs) were transfected using a commercial transfection agent (Mirus Bio) with either P1 mRNA alone (P1 only, left panel) or a mixture of P1 polyprotein and 3C protease (P1 :3C; right panel) mRNAs at the indicated P1 :3C RNA mass ratio. One day after transfection, lysates were harvested, proteins separated by SDS-PAGE, transferred to PVDF membrane,299406520.1probed with an FMDV capsid-specific monoclonal antibody. The full-length P1 polypeptide appears as a -100 kDa species (molecular weights are indicated to the left of the blot). In the presence of the 3C protease mRNA, this full length P1 is not detected due to proteolytic activity of the 3C protein, and a band ~40kDa, characteristic of accumulating processed capsid (specifically, the VP4-VP2 polypeptide), is apparent. The highest concentration of the processed capsid signature band was observed at 32:1 and 64:1 ratios (P1 :3C mRNA mass).

[0137] As shown in FIG. 2, cells transfected with P1 mRNA alone exhibited a -100 kDa band corresponding to full-length P1 , while co-expression with 3C protease eliminated this band and produced a -40 kDa species characteristic of processed VP4-VP2 capsid polypeptide. The strongest processing was observed at 32:1 and 64:1 P1 :3C mRNA ratios, confirming that 3C-mediated proteolysis occurs in mammalian cells at low 3C protease abundance.

[0138] Example 3. Immunogenicity of P1 and 3C mRNA Nanoparticle Vaccines in Mice

[0139] Modified mRNA molecules encoding P1 polyprotein and 3C protease were encapsulated in ionizable lipid nanoparticles (LNPs) containing Delivery Compound A or B (delivery formulations described in PCT Patent Application Publication No. W02021207020A1 , published October 14, 2021 , “Carriers For Efficient Nucleic Acid Delivery,” Poulami Talukder, Jasdave S. Chahal, Jin Huang, Karl Ruping; and PCT Patent Application Publication No. WO2025096681 , “Novel Gene Delivery Agents,” published May 8, 2025, Poulami Talukder, Maria Varghese, Heliang Song, Nathan Ivanowsky; both of which are incorporated by reference as if fully set forth). Mice (CD-1 strain) were immunized intramuscularly (IM) in a prime-boost regimen (5 pig total mRNA per dose, 4-week interval). Serum was collected 2 weeks post-boost and analyzed by blocking ELISA (PrioCHECK™ FMDV Type O Antibody Kit). FIG. 3 is a chart depicting the humoral immune response as percent of inhibition to different candidate nanoparticle formulations of P1 -only, or P1 and 3C protease, N1-methylpseudouridine-modified mRNA payloads mixed at different ratios (16:1 , 32:1 , and 64:1 ). Mice were vaccinated with RNABL candidates by intramuscular injection (IM) in a prime / boost regimen, of 5 pig doses (total mRNA mass) at 4-week inter-dose interval. Serum was sampled 2-weeks post-boost and assayed using the PrioCHECK™ FMDV Type O Antibody ELISA Kit (Thermo Fisher), a blocking ELISA assay that measures the concentration of neutralizing antibodies against FMDV type O strain. In this assay, a percent inhibition (PI) value > 50 is suggestive of immunity to FMDV serotype O. Referring to FIG. 3, two different types of ionizable delivery compounds were tested in this experiment (Delivery A and Delivery B), with different innate immune stimulatory properties. The P1 :3C protease mRNA mass ratios tested, from left to right for each delivery candidate, were 16:1 , 32:1 , and 64:1 . Delivery formula B (Delivery A), which is more immunostimulatory than Delivery formula A (Delivery B), led to greater titers. Crucially, inclusion of the 3C protease mRNA payload was necessary to generate average humoral titers above the 50% protective threshold, as P1 mRNA alone did not consistently produce titers above this threshold.309406520.1TIB-PT019WC

[0140] As shown in FIG. 3, inclusion of the 3C protease mRNA payload significantly enhanced humoral titers, with P1 :3C ratios of 32:1 and 64:1 exceeding the 50% inhibition threshold indicative of protective antibody levels. Delivery Compound B produced the most robust immune responses, highlighting the importance of delivery vehicle and 3C protease co-expression.

[0141] Example 4: Immunogenicity in Cattle

[0142] Holstein cattle were immunized intramuscularly with 50 pig of nanoparticle vaccine containing P1 and 3C protease mRNAs mixed at mass ratios of 16:1 , 32:1 , or 64:1 using Delivery Compound B or a chemically similar analog. Serum was sampled 2 weeks post-boost and tested for anti-FMDV antibodies (PrioCHECK™ FMDV Type O ELISA). FIG. 4 is a chart depicting the humoral immune response in cattle to different candidate nanoparticle formulations of P1 and 3C protease payloads mixed at different P1 :3C protease mRNA mass ratios and delivered as nanoparticles. Referring to this figure, cattle were vaccinated with candidates by IM administration in a prime and boost regimen, with 50 pig doses with a 4-week inter-dose interval. Serum was sampled 2-weeks post-boost and assayed using the PrioCHECK™ FMDV Type O Antibody ELISA Kit (Thermo Fisher). Candidates 1 -3 were formulated with Delivery compound B as in the FIG. 3, and candidates 4-6 were formulated with an analog of Delivery compound B, possessing similar immunogenic potency. The P1 :3C ratios of the vaccine compositions were 16:1 for candidates 1 and 4, 32:1 for candidates 2 and 5, and 64:1 for candidates 3 and 6. All the compositions induced nearly complete inhibition in this blocking ELISA assay, indicating robust humoral responses against FMDV type O strains. As shown in FIG. 4, all vaccine formulations elicited near-complete inhibition in this assay. FIG. 5 is a chart showing the viral neutralizing titer of the same serum samples analyzed in FIG. 4. Referring to this figure, the serum samples were subjected to a viral neutralization test, with the y-axis indicating the virus neutralizing titer (VNT), i.e., the endpoint dilution of the serum at which complete inhibition of viral infection is observed in an in vitro infection assay. Average VNT values greater than 16, which correlate to protection against FMDV infection, were observed for all candidates, indicating that these vaccines are immunogenic in cattle.

[0143] Thus, corresponding virus-neutralization titers shown on FIG. 5 exceeded 1 :16, the threshold correlated with protection. These data confirm that co-delivery of P1 and 3C protease mRNAs induces robust, neutralizing humoral immunity in a target livestock species.

[0144] Example 5. Evaluation of Safety and Systemic Inflammatory Response in Mice

[0145] CD-1 mice were administered intramuscularly with 5 pig of nanoparticle formulations containing either P1 mRNA only (NP1), P1 + 3C protease mRNAs (NP2; 32:1 ratio), or saline (negative control). Body weight and serum biomarkers were monitored for 7 days post-injection. FIG. 6 is a chart depicting the percent weight change of wild-type CD-1 mice administered nanoparticles (NPs) formulated with Delivery compound B containing the indicated mRNAs: NP1 (P1 mRNA), NP2 (P1 + 3C protease mRNA), and Neg (Saline Control). Referring to this figure, the P1 and 3C protease mRNAs co-formulated to produce NP2 were mixed at a 32:1 mass ratio. CD-1 mice were injected intramuscularly with a high dose (5 pig) to319406520.1demonstrate that, despite its inherent cytotoxicity, 3C protease at this low concentration does not induce adverse health effects. The mice receiving the nanoparticle composition containing 3C protease mRNA did not exhibit greater body weight loss compared to control animals receiving only P1 mRNA (NP1 ) or vehicle- only injection of phosphate buffered saline (PBS) control solution. FIG. 6 shows no significant weight loss in vaccinated animals relative to controls. Serum cytokines IL-6, IFN-a, RANTES, and MCP-1 were quantified 24 hours post-injection. FIG. 7 is a set of charts showing the concentrations of proinflammatory cytokines IL- 6, IFN-alpha, RANTES, and MCP-1 in the serum of the mice described in FIG. 6 at 24 hours post-injection. This figure shows that systemic inflammatory side effects are not elevated by the inclusion of 3C protease mRNA at a mass ratio of 32:1 (P1 to 3C protease mRNAs). The NP2 composition, containing 3C protease mRNA, induced lower levels of IFN-alpha, RANTES, and MCP-1 . This effect is ascribed to the innate immune response suppressing function of the 3C protease. Inclusion of 3C protease mRNA did not elevate systemic inflammation; IFN-a and RANTES were modestly reduced relative to P1-only vaccination, consistent with known 3C protease-mediated suppression of innate signaling. FIGS. 8A and 8B are sets of charts showing the concentrations of (FIG. 8A) cardiac troponin-l and (FIG. 8B) alanine transaminase in the serum of the mice described in FIG. 6 at 24 hours post-injection with NP1 (P1 mRNA), NP2 (P1 + 3C protease mRNA) and Neg (Saline Control). These biomarkers of, respectively, heart and liver toxicity were not significantly elevated compared to the negative control saline-injected group, indicating that no tissue damage was associated with either P1-only or P1 +3C protease mRNA vaccines in these organs. Referring to FIGS. 8A and 8B, cardiac troponin-l and alanine transaminase (ALT) levels remained within baseline, demonstrating absence of cardiotoxicity or hepatotoxicity.

[0146] Example 6. Protective Efficacy Against Foot-and-Mouth Disease Virus Challenge in Cattle

[0147] Holstein cattle were immunized intramuscularly with two doses (50 g each, weeks 0 and 4) of the nanoparticle formulation containing P1 + 3C protease mRNAs (32:1 ratio, Delivery Compound B). At week 6, animals were challenged intranasopharyngeally with 10A6.8 TCID50of FMDV G / FRA / 1 / 2001. FIGS. 9A and 9B are a scheme (FIG. 9A) and a table summarizing a cattle immunogenicity and challenge infection study performed to test the efficacy of the P1 +3C protease mRNA (32:1 mass ratio) vaccine composition characterized in FIGS. 6, 7, 8A and 8B. Holstein cattle were immunized with 50 pg of the vaccine at weeks 0 and 4, and infected at week 6 with 10A6.8 TCIDso of FMDV C / FRA / 1 / 2001 via intranasopharyngeal deposition. The median tissue culture infectious dose (TCID50) is defined as the dilution of a virus required to infect 50% of a given cell culture. Clinical signs were monitored after infection up to week 10. Disease manifestation (oral lesion or multiple oral lesions and associated fever defined as rectal body temperature of 40°C and above) is reported in the table to the right, along with the serum viral neutralizing titer measured at week 6 pre-infection (dilution factor; titers of 6 and below are under the sensitivity limit of the assay and considered serologically negative). The vaccine resulted in 100% seroconversion and full protection against disease signs. All329406520.1vaccinated animals seroconverted (neutralizing titers > 1 :96) and exhibited no clinical signs of disease or fever, while control animals developed characteristic oral lesions and pyrexia as shown in FIGS. 9A and 9B.

[0148] FIG. 10 is a set of charts showing the results of RT-qPCR-based detection of viral shedding in oral swabs from each animal described in FIGS. 9A and 9B. Referring to this figure, the quantification cycle (Cq) value is plotted for each indicated sample collection day post-infection. Values up to cycle 35 indicate robust detection of shed FMDV 0 viral genome. All control animals exhibited readily detected virus, while vaccinated animals exhibited no or very low values, indicating that viral shedding was effectively controlled by the P1 +3C protease vaccine. Referring to FIG. 10, RT-qPCR analysis of oral swabs showed complete suppression of viral shedding in vaccinated animals compared to controls. These results demonstrate that coexpression of picornavirus P1 and 3C protease mRNAs delivered in nanoparticles provides sterilizing immunity and full clinical protection against FMDV challenge in cattle.

[0149] The foregoing examples collectively demonstrate that: (I) co-delivery of P1 and 3C protease mRNAs enables correct antigen processing and assembly, (II) the inclusion of 3C protease mRN A is essential for achieving neutralizing antibody titers, (iii) mRNA vaccine formulations expressing 3C protease mRNA is well tolerated in mice and livestock, and (iv) immunized cattle are completely protected from clinical disease and viral shedding following FMDV challenge.

[0150] Example 7. Vaccine Against Serotype A FMDV

[0151] mRNA molecules encoding a serotype A FMDV (strain A / 22 / IRQ / 24 / 64) P1 and 30 protease comprising sequences provided as SEQ ID NOs: 3 and 4 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. When administered to a subject, the P1 and 30 protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 21 and 22, respectively.

[0152] Example 8. Vaccine Against Serotype C FMDV

[0153] mRNA molecules encoding a serotype C FMDV (strain C / UK / 149 / 34) P1 and 3C protease comprising sequences provided as SEQ ID NOs:: 5 and 6 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. . When administered to a subject, the P1 and 30 protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 23 and 24, respectively.

[0154] Example 9. Vaccine Against Serotype Asia 1 FMDV

[0155] mRNA molecules encoding a serotype Asia 1 FMDV (strain Asia1 / PAK / 1 / 54) P1 and 30 protease comprising sequences provided as SEQ ID NOs: 7 and 8 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. . When administered to a subject, the P1 and 3C protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 25 and 26, respectively.

[0156] Example 10. Vaccine Against Serotype SAT 1 FMDV339406520.1

[0157] mRNA molecules encoding a serotype SAT 1 FMDV (strain SAT1 / RV / 11 / 37) P1 and 3C protease comprising sequences provided as SEQ ID NOs: 9 and 10 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. . When administered to a subject, the P1 and 3C protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 27 and 28, respectively.

[0158] Example 11. Vaccine Against Serotype SAT 2 FMDV

[0159] mRNA molecules encoding a serotype SAT 2 FMDV (strain SAT2 / SA / 106 / 59) P1 and 3C protease comprising sequences provided as SEQ ID NOs: 11 and 12 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. . When administered to a subject, the P1 and 30 protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 29 and 30, respectively.

[0160] Example 12. Vaccine Against Serotype SAT 3 FMDV

[0161] mRNA molecules encoding a serotype SAT 3 FMDV (strain SAT3 / SA / 57 / 59) P1 and 3C protease comprising sequences provided as SEQ ID NOs: 13 and 14 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. . When administered to a subject, the P1 and 3C protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 31 and 32, respectively.

[0162] Example 13. Vaccine Against Human poliovirus type 2

[0163] mRNA molecules encoding the Human poliovirus type 2 P1 and 30 protease comprising sequences provided as SEQ ID NOs: 15 and 16 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. When administered to a subject, the P1 and 30 protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 33 and 34, respectively.

[0164] Example 14. Vaccine Against Human enterovirus 71 (EV-A71)

[0165] mRNA molecules encoding a Human enterovirus 71 (strain 7423 / MS / 87) P1 and 30 protease comprising sequences provided as SEQ ID NOs: 17 and 18 respectively are mixed at an effective ratio of P1 :3C protease, preferably 32:1 or greater, and formulated in an acceptable nanoparticle carrier formulation. When administered to a subject, the P1 and 30 protease mRNAs are intracellularly translated into the polypeptide sequences put forth in SEQ ID NOs: 35 and 36, respectively.

[0166] ReferencesAnsardi et al., 1996, *J. Virol * 70: 1613-1620.Heyes et al. (2005) J Controlled Release 107:276-87.Jiang et al., 201 1 , *Vaccine* 29: 7182-7192Jiang et al., 2024, *Viruses* 16(6): 834; https: / / doi.org / 10.3390 / v16060834Kim et al., 2019, *PLoS ONE* 14(1 ): e0210477;349406520.1https : / / doi .org / 10.1371 / journal .pone.0210477.Mason et al., 2006, *J. Virol * 80: 1 1009-11016.Smith TF, Waterman MS 1981 “Identification of Common Molecular Subsequences," J Mol Biol 147: 195 -197.W02021207020A1 , “Carriers For Efficient Nucleic Acid Delivery,” published October 14, 2021 , Poulami Talukder, Jasdave S. Chahal, Jin Huang, Karl Ruping.WO2025096681 , published May 8, 2025, “Novel Gene Delivery Agents,” Poulami Talukder, Maria Varghese, Heliang Song, and Nathan Ivanowsky.

[0167] The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein . A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated . However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0168] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and / or shown in the attached drawings.359406520.1

Claims

CLAIMSWhat is claimed is:1 A vaccine composition for protecting a subject against a viral disease comprising an effective amount of a first nucleic acid encoding a P1 polyprotein mixed with a second nucleic acid encoding a 3C protease, and a nanoparticle carrier, wherein the nanoparticle carrier encapsulates the first nucleic acid and the second nucleic acid, and the viral disease is caused by a viral pathogen of the Picornaviridae family.

2. The vaccine composition of claim 1 , wherein the first nucleic acid or the second nucleic acid is selected from the group consisting of mRNA, saRNA and repRNA.

3. The vaccine composition of claim 1 , wherein the viral disease is caused by a viral pathogen selected from the group consisting an FMDV, a Poliovirus and an Enterovirus.

4. The vaccine composition of claim 3, wherein the viral pathogen is an FMDV of a serotype 0, serotype A, serotype C, serotype Asia 1 , serotype SAT 1 , serotype SAT 2, or serotype SAT 3, or any combination thereof5 The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype 0, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 1.

6. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype 0, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 2.

7. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype A, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 3.8 The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype A, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 4.

9. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype C, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 5.

10. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype C, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 6.11 . The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype Asia 1 , and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 7.369406520.

112. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype Asia 1 , and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8.

13. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype SAT 1 , and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 9.

14. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype SAT 1 , and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 10.

15. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype SAT 2, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 11.

16. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype SAT 2, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 12.

17. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype SAT 3, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 13.

18. The vaccine composition of claim 4, wherein the viral pathogen is an FMDV of a serotype SAT 3, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 14.

19. The vaccine composition of claim 3, wherein the viral pathogen is a Poliovirus, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO:15.

20. The vaccine composition of claim 3, wherein the viral pathogen Is a Poliovirus, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO:16.21 . The vaccine composition of claim 3, wherein the viral pathogen is an Enterovirus, and the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO:17.

22. The vaccine composition of claim 3, wherein the viral pathogen is an Enterovirus, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 18.379406520.

123. The vaccine composition of claim 1 , wherein the first nucleic acid is mixed with the second nucleic acid at a ratio greater or equal to one selected from the group consisting of 8:1 , 16:1 , 32:1 , 64:1 , and 96:1 of the first nucleic acid to the second nucleic acid (w / w).

24. The vaccine composition of claim 1 , wherein the nanoparticle carrier is selected from the group consisting of: a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle and a dendrimer.

25. A kit comprising the vaccine composition of any one of claims 1 - 24, wherein the kit is used for single administration, or sequential administration of the vaccine in a prime / boost regime.

26. The kit of claim 25 further comprising an immunoregulatory agent.

27. The kit of claim 26, wherein the immunoregulatory agent is an adjuvant.

28. The kit of claim 25, wherein the vaccine composition comprises the single dose vaccine or the prime and boost vaccines.

29. The kit of claim 28, wherein the single dose vaccine is included in a container, or, wherein the prime and boost vaccines are included in the same container, or separate containers.

30. The kit of claim 25, wherein the single dose vaccine, the prime and boost vaccines are in liquid form or solid form.31 . A method of preventing, reducing, inhibiting, or delaying the symptoms of an infection caused by a virus of the Picornaviridae family, or of inducing an immune response against the virus in a subject, the method comprising: administering to the subject a vaccine composition comprising an effective amount of a first nucleic acid encoding a P1 polyprotein mixed with a second nucleic acid encoding a 30 protease, and a nanoparticle carrier encapsulating the first nucleic acid and the second nucleic acid.

32. The method of claim 31 , wherein the first nucleic acid or the second nucleic acid is selected from the group consisting of an mRNA, saRNA, and a repRNA.

33. The method of claim 31 , wherein the virus is an FMDV, a Poliovirus or an Enterovirus.

34. The method of claim 33, wherein the virus is an FMDV of a serotype 0, serotype A, serotypeC, serotype Asia 1 , serotype SAT 1 , serotype SAT 2, or serotype SAT 3, or any combination thereof.

35. The method of claim 34, wherein the first nucleic acid comprises an RNA sequence with at least 90% identity to the sequence selected from the group consisting of SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , and 13.

36. The method of claim 34, wherein the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence selected form the group consisting of: SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

37. The method of claim 33, wherein the virus is a Poliovirus and the first nucleic acid comprises an RNA sequence with at least 90% identity to the set forth in SEQ ID NO: 15.389406520.1TIB-PT019WQ38. The method of claim 33, wherein virus is a Poliovirus, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 16.

39. The method of claim 33, wherein the virus is an Enterovirus and the first nucleic acid comprises an RNA sequence with at least 90% identity to the set forth in SEQ ID NO: 17.

40. The method of claim 33, wherein virus is an Enterovirus, and the second nucleic acid comprises an RNA sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 18.41 . The method of claim 31 , wherein the nanoparticle carrier is selected from the group consisting of: a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle and a dendrimer.

42. The method of claim 31 , wherein the vaccine composition is administered as a single dose vaccine.

43. The method of claim 42, wherein the vaccine composition comprises a prime vaccine and a boost vaccine, and is administered in a prim / boost regime.

44. The method of claim 43, wherein the boost vaccine is administered in a period of time ranging from 1 week to 60 weeks after the prime vaccine is administered.

45. The method of claim 31 , wherein the step of administering results in preventing, treating, reducing the severity or slowing the progression of the disease in the subject.

46. The method of claim 31 , wherein the subject is a cloven-hooved animal or a human.

47. The method of claim 46, wherein the cloven-hoofed animal is selected from the group consisting of: cattle, water buffalo, sheep, goat, pig, antelope, deer, bison, hedgehog, elephant, llama and alpaca.

48. A method of preparing a vaccine composition of any one of claims 1 - 24 for protecting a subject against a viral pathogen of the Picornaviridae family comprising; mixing a first nucleic acid encoding a P1 polyprotein with a second nucleic acid encoding a 3C protease to obtain a nucleic acid mixture; and combining the nucleic acid mixture with a delivery compound that complexes with the first nucleic acid and the second nucleic acid.

49. The method of claim 48, wherein the first nucleic acid is mixed with the second nucleic acid at a ratio greater or equal to one selected from the group consisting of 8:1 , 16:1 , 32:1 , 64:1 , and 96:1 of the first nucleic acid to the second nucleic acid (w / w).

50. The method of claim 48, wherein the delivery compound is selected from the group consisting of: a liposome, a lipid nanoparticle, a solid lipid nanoparticle, an aminolipid, a polymeric nanoparticle and a dendrimer.51 . The method of claim 48 further comprising formulating the vaccine composition with a pharmaceutically acceptable carrier.399406520.

152. The method of claim 48 further comprising formulating the vaccine compositions with one or more pharmaceutically acceptable diluents, adjuvants and / or excipient.409406520.1

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