WO2022212659A1

WO2022212659A1 - Multi-genic mrna vaccine compositions and methods of use

Info

Publication number: WO2022212659A1
Application number: PCT/US2022/022764
Authority: WO
Inventors: James Eberwine; Norbert PARDI; Hyunbum KIM; Tamas Bartfai; Jai-Yoon Sul
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2021-03-31
Filing date: 2022-03-31
Publication date: 2022-10-06

Abstract

The present invention relates to multi-genic mRNA immunogenic compositions and methods for inducing an immune response against one or more pathogenic microorganism in a subject. In some embodiments, the present invention provides a composition comprising at least one mRNA molecule encoding a plurality of pathogen antigens.

Description

TITLE OF THE INVENTION

Multi-genic mRNA Vaccine Compositions and Methods of Use

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AA028409 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/168,661, filed March 31, 2021, which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Enhancing host immune responses to vaccination can improve the efficacy and durability of vaccines. Current RNA-based vaccines rely on a single antigen of interest to be expressed by host B-cells to stimulate immune responses. While effective at eliciting host immune responses, historical experience with vaccines imply that the most successful vaccination approaches use multiple antigens from pathogenic agents to educate the immune system.

Thus, there is a need in the art for improved multi-genic immunogenic compositions (e.g., vaccines). The present invention addresses this need.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a composition for inducing an immune response against one or more pathogenic microorganisms in a subject. In one embodiment, the composition comprises at least one mRNA molecule encoding a combination of two or more pathogenic microorganism antigens or fragments thereof.

In one embodiment, the two or more pathogenic microorganism antigens are antigens from different viral strains of the same virus. Attorney Docket No. 046483-6221-00WO

In one embodiment, the two or more pathogenic microorganism antigens are antigens from different viruses.

In one embodiment, at least one virus is selected from the group consisting of influenza virus, HIV and a beta coronavirus.

In one embodiment, at least one influenza virus antigen is a hemagglutinin (HA) antigen or a fragment thereof, neuraminidase (NA) antigen or a fragment thereof, NP antigen or a fragment thereof, or an M2 ion channel antigen or a fragment thereof. In one embodiment, at least one influenza virus antigen comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or any combination thereof.

In one embodiment, at least one HIV antigen is an Env antigen or a fragment thereof, a Nef antigen or a fragment thereof, a Pol antigen or a fragment thereof, a Gag antigen or a fragment thereof, a Tat antigen or a fragment thereof, a Rev antigen or a fragment thereof, a Vif antigen or a fragment thereof, a Vpr antigen or a fragment thereof, or a Vpu antigen or a fragment thereof. In one embodiment, at least one HIV antigen comprises an amino acid sequence of SEQ ID NO: 5, or SEQ ID NO: 6, or any combination thereof.

In one embodiment, at least one coronavirus antigen is a spike antigen or a fragment thereof, an envelope protein or a fragment thereof, a nucleocapsid protein or a fragment thereof, a membrane glycoprotein or a fragment thereof, an ORFlab polyprotein or a fragment thereof, an ORF3a protein or a fragment thereof, an ORF6 protein or a fragment thereof, an ORF7a protein or a fragment thereof, an ORF8 protein or a fragment thereof, or an ORFIO protein or a fragment thereof. In one embodiment, at least one coronavirus antigen comprises an amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, or any combination thereof.

In one embodiment, the composition further comprises an adjuvant or at least one mRNA molecule encoding an adjuvant. In one embodiment, the adjuvant comprises a T cell stimulatory molecule. In one embodiment, the T cell stimulatory molecule is selected from the group consisting of CD80 and CD86. In one embodiment, Attorney Docket No. 046483-6221-00WO the composition comprises an mRNA molecule encoding SEQ ID NO: 18 or SEQ ID NO:19.

In one embodiment, the composition further comprises a vehicle for delivery of the mRNA molecule. In one embodiment, the delivery vehicle is a lipid nanoparticle (LNP), a peptide or a combination of a lipid and a peptide. In one embodiment, at least one mRNA molecule is encapsulated within the delivery vehicle.

In one embodiment, the composition is a vaccine.

In one embodiment, the invention relates to a method of inducing an immune response against at least one pathogenic microorganism in a subject comprising administering to the subject an effective amount of a composition for inducing an immune response against one or more pathogenic microorganisms in a subject. In one embodiment, the composition comprises at least one mRNA molecule encoding a combination of two or more pathogenic microorganism antigens or fragments thereof. In one embodiment, the two or more pathogenic microorganism antigens are antigens from the same organism. In one embodiment, the two or more pathogenic microorganism antigens are antigens from different organisms.

In one embodiment, the two or more pathogenic microorganism antigens are antigens from different viral strains of the same virus. In one embodiment, the two or more pathogenic microorganism antigens are antigens from different viruses.

In one embodiment, the method comprises inducing an immune response against two or more different pathogenic microorganisms. In one embodiment, the method comprises inducing an immune response against two or more different viruses.

In one embodiment, the at least two different viruses are selected from the group consisting of influenza virus, HIV and a beta coronavirus.

In one embodiment, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.

In one embodiment, the method comprises a single administration of the composition. In one embodiment, the method comprises multiple administrations of the composition.

In one embodiment, the invention relates to a method of treating or preventing a disease or disorder associated with at least one pathogenic microorganism in Attorney Docket No. 046483-6221-00WO a subject comprising administering to the subject an effective amount of a composition for inducing an immune response against one or more pathogenic microorganisms in a subject. In one embodiment, the composition comprises at least one mRNA molecule encoding a combination of two or more pathogenic microorganism antigens or fragments thereof. In one embodiment, the two or more pathogenic microorganism antigens are antigens from the same organism. In one embodiment, the two or more pathogenic microorganism antigens are antigens from different organisms.

In one embodiment, the method comprises treating or preventing a disease or disorder associated with at least one virus.

In one embodiment, the at least two different viruses are selected from the group consisting of influenza virus, HIV and a beta coronavirus. In one embodiment, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery.

BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of several embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently exemplified. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 depicts representative images demonstrating the co-expression of co-transfected mRNAs encoding eGFP, COVID-19 spike protein and CD86. Attorney Docket No. 046483-6221-00WO

Figure 2 depicts representative images demonstrating the co-expression of co-transfected mRNAs encoding eGFP, COVID-19 spike protein, COVID-19 nucleocapsid and CD80.

Figure 3 depicts representative images demonstrating the co-expression of co-transfected mRNAs encoding eGFP, COVID-19 spike protein, COVID-19 nucleocapsid and CD80.

Figure 4 depicts exemplary experimental data demonstrating the ratio of expression of nucleocapsid and eGFP protein amounts correlates with the amount of transfected mRNAs encoding COVID-19 nucleocapsid and eGFP.

DETAILED DESCRIPTION

The present invention relates to compositions and methods for inducing an immune response against a pathogen (e.g., a pathogenic microorganism) or target protein in a subject. In some embodiments, the invention provides a composition comprising at least one mRNA molecule encoding at least one pathogen antigen. For example, in one embodiment, the composition is a vaccine comprising at least one mRNA encoding at least one pathogen antigen, wherein the vaccine induces an immune response in the subject to a pathogenic microorganism. In some embodiments, the composition is a vaccine comprising at least one mRNA encoding two or more pathogen antigens, wherein the vaccine induces an immune response in the subject to at least one pathogenic microorganism. In some embodiments, the composition is a vaccine comprising at least two mRNA molecules, wherein each mRNA molecule encodes at least one antigen, wherein the vaccine induces an immune response in the subject to at least one pathogenic microorganism. In one embodiment, the two or more antigens are antigens from two or more different strains of a virus. In one embodiment, the two or more antigens are two or more different antigenic peptides from the same virus.

Exemplary pathogen antigens that can be included in the multi-genic immunogenic compositions (e.g., vaccines) of the invention include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, mycobacterial antigens and protozoan antigens. Exemplary viral antigens that can be included in the multi-genic immunogenic compositions (e.g., vaccines) of the invention include, but are not limited Attorney Docket No. 046483-6221-00WO to, influenza antigens, HIV antigens, and SARS-CoV-2 antigens. In some embodiments, the viral antigens are from disease-causing viruses. In some embodiments, the viral antigens are from viruses known to cause cancer, including, but not limited to, human papilloma virus (HPV) and the human T cell lympothropic virus HTLV-1.

In some embodiments, the invention relates to compositions and methods for inducing an immune response against a pathogen or target antigen in a subject, wherein the compositions comprise a combination of at least one pathogen antigen and at least one antigen to stimulate a lasting T cell response. Exemplary T cell stimulatory antigens that can be included in the immunogenic composition of the invention include, but are not limited to, CD80 and CD86.

In some embodiments, the invention provides a multi-genic immunogenic composition (e.g., vaccine) composition comprising at least one mRNA molecule encoding at least one pathogen antigen. In some embodiments, the invention provides a combination vaccine comprising at least one mRNA molecule encoding at least one pathogen antigen and at least one mRNA molecule encoding at least one T cell stimulatory antigen. In one embodiment, the combination vaccine comprises a single mRNA molecule encoding at least one pathogen antigen and at least one T cell stimulatory antigen. In one embodiment, the combination vaccine comprises a first mRNA molecule encoding at least one pathogen antigen and at least one additional mRNA molecule encoding at least one T cell stimulatory antigen.

In some embodiments, the invention provides methods of using the multi- genic immunogenic compositions (e.g., vaccines) of the invention for inducing an immune response against one or more target pathogen or protein. In some embodiments, the immune response is a cellular immune response, a humoral immune response, or a combination of a cellular and humoral immune response.

In some embodiments, the invention provides methods of treating or preventing a disease or disorder associated with a target pathogen or protein. In one Attorney Docket No. 046483-6221-00WO embodiment, the invention provides methods of treating or preventing a disease or disorder associated with a pathogenic microorganism.

Definitions

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

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immune-reactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et ak, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et ak, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et ak, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et ak, 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, Attorney Docket No. 046483-6221-00WO linear antibodies, scFv antibodies, and multi-specific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations k and l light chains refer to the two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibody, which is generated using recombinant DNA technology. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term should also be construed to mean an antibody, which has been generated by the synthesis of an RNA molecule encoding the antibody. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned), synthesizing the RNA, or other technology, which is available and well known in the art.

By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., Attorney Docket No. 046483-6221-00WO an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

The term “immunogen” as used herein, is intended to denote a substance of matter, which is capable of inducing an adaptive immune response in an individual, where said adaptive immune response is capable of inducing an immune response, which significantly engages pathogenic agents, which share immunological features with the immunogen. “Immunogen” refers to any substance introduced into the body in order to generate an immune response. That substance can a physical molecule, such as a protein, or can be encoded by a vector, such as DNA, mRNA, or a virus.

The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

“Immune response,” as the term is used herein, means a process involving the activation and/or induction of an effector function in, by way of non-limiting Attorney Docket No. 046483-6221-00WO examples, a T cell, B cell, natural killer (NK) cell, and/or an antigen-presenting cell (APC). Thus, an immune response, as would be understood by the skilled artisan, includes, but is not limited to, any detectable antigen-specific activation and/or induction of a helper T cell or cytotoxic T cell activity or response, production of antibodies, antigen presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, and the like.

As used herein, an “immunogenic composition” may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, a virus expressing or presenting an antigen or cellular component, or a combination thereof. In particular embodiments, the composition comprises or encodes all or part of any peptide antigen described herein, or an immunogenically functional equivalent thereof. In other embodiments, the composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell, lipid nanoparticle, or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination.

As used herein, the term “vaccine” refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a Attorney Docket No. 046483-6221-00WO gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology. Attorney Docket No. 046483-6221-00WO

As used herein, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the original nucleotide sequence at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

As used herein, an amino acid sequence is “substantially homologous” to any of the amino acid sequences described herein when its amino acid sequence has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%. The identity between two amino acid sequences can be determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

The term “variant” as used herein with respect to a nucleic acid refers (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. A variant may be a nucleic acid sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof.

The term “variant” as used with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also refer to a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino Attorney Docket No. 046483-6221-00WO acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

As used herein, the terms “fragment” or “functional fragment” refer to a fragment of an influenza virus antigen or a nucleic acid sequence encoding an influenza virus antigen that, when administered to a subject, provides an increased immune response. Fragments are generally 10 or more amino acids or nucleic acids in length. “Fragment” may mean a polypeptide fragment of an antigen that is capable of eliciting an Attorney Docket No. 046483-6221-00WO immune response in a subject. A fragment of an antigen may be 100% identical to the full length except missing at least one amino acid from the N and/or C terminal, in each case with or without signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length antigen, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.

A fragment of a nucleic acid sequence that encodes an antigen may be 100% identical to the full length except missing at least one nucleotide from the 5’ and/or 3’ end, in each case with or without sequences encoding signal peptides and/or a methionine at position 1. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Attorney Docket No. 046483-6221-00WO

In the context of the present invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell. Exemplary modified nucleosides are described elsewhere herein. For example, an mRNA where some or all of the uridines have been replaced with pseudouridine, 1 -methyl psuedouridine, or another modified nucleoside, such as those described elsewhere herein. In some embodiments, the nucleotide sequence may contain a sequence where some or all cytodines are replaced with methylated cytidine, or another modified nucleoside, such as those described elsewhere herein.

The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic Attorney Docket No. 046483-6221-00WO acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

In some instances, the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. By way of one non-limiting example, a promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response. Attorney Docket No. 046483-6221-00WO

In some embodiments, “pseudouridine” refers to nriacp³'!' (l-methyl-3- (3 -amino-3 -carboxypropyl) pseudouridine). In another embodiment, the term refers to m^l F (1-methylpseudouridine). In another embodiment, the term refers to Yih (2’-0- methylpseudouridine. In another embodiment, the term refers to m⁵D (5- methyldihydrouridine). In another embodiment, the term refers to m³ F (3- methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more lipids.

The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides. Attorney Docket No. 046483-6221-00WO

The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH.

The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid.

The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monom ethoxy-poly ethyleneglycol)-2, 3 -dimyristoylglycerol (PEG-s- DMG) and the like.

“Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et ah, 1991 Glycobiology 5: 505- 10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

The terms “subject,” “patient,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some non-limiting embodiments, the patient, subject or individual is a mammal, bird, poultry, cattle, pig, horse, sheep, ferret, primate, dog, cat, guinea pig, rabbit, bat, or human.

A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less Attorney Docket No. 046483-6221-00WO favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as a human.

To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, prevention, or eradication of at least one sign or symptom of a disease or disorder.

The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a Attorney Docket No. 046483-6221-00WO polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (1985, Genaro, ed.,

Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention relates to compositions and methods for inducing an immune response against one or more target pathogen antigens or proteins in a subject. In some embodiments, the invention provides a composition comprising at least one mRNA molecule encoding a combination of at least two antigens. For example, in one embodiment, the composition is a vaccine comprising at least one mRNA molecule Attorney Docket No. 046483-6221-00WO encoding at least two pathogen antigens, wherein the immunogenic composition induces an immune response in the subject to one or more pathogenic microorganisms, and therefore the immunogenic composition is a multi-genic immunogenic composition (e.g., vaccine).

Vaccine

In one embodiment, the present invention provides an immunogenic composition for inducing an immune response against a pathogen antigen in a subject. For example, in one embodiment, the immunogenic composition is a vaccine. For a composition to be useful as a vaccine, the composition must induce an immune response against the pathogen antigen in a cell, tissue or subject. In some embodiments, the composition induces an immune response against the pathogen antigen in a cell, tissue or subject. In some instances, the immunogenic composition induces a protective immune response in the subject.

An immunogenic composition of the present invention may vary in its composition of nucleic acid and/or cellular components. In one embodiment, the immunogenic composition comprises a nucleic acid encoding at least one pathogen antigen. In a non-limiting example, a nucleic acid encoding at least one pathogen antigen might also be formulated with an adjuvant. Of course, it will be understood that various compositions described herein may further comprise additional components. For example, one or more vaccine components may be comprised in a delivery vehicle, including, but not limited to a peptide, a lipid, a liposome, a lipid nanoparticle or any combination thereof. In another non-limiting example, a vaccine may comprise one or more adjuvants. An immunogenic composition of the present invention, and its various components, may be prepared and/or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

In various embodiments, the induction of immunity by the expression of the pathogen antigen can be detected by observing in vivo or in vitro the response of all or any part of the immune system in the host against the pathogen antigen.

In some embodiments, the induction of immunity by expression of one or more pathogen antigens can be confirmed by observing the induction of antibody Attorney Docket No. 046483-6221-00WO production against the pathogen antigen. For example, when antibodies against an antigen are induced in a subject immunized with the composition encoding the antigen, and when antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.

The specificity of the antibody response induced in a subject can include binding to many regions of the delivered antigen, as well as, the induction of neutralization capable antibodies that that prevent infection or reduce disease severity.

The induction of immunity by expression of the pathogen antigen can be further confirmed by observing the induction of T cells, such as CD4+ T cells, CD8+ T cells, or a combination thereof. For example, CD4+ T cells can also lyse target cells, but mainly supply help in the induction of other types of immune responses, including CTL and antibody generation. The type of CD4+ T cell help can be characterized, as Thl, Th2, Th9, Thl7, Tregulatory (Treg), or T follicular helper (Tfh) cells. Each subtype of CD4+

T cell supplies help to certain types of immune responses. In one embodiment, the composition selectively induces T follicular helper cells, which drive potent antibody responses.

The therapeutic compounds or compositions of the invention may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from, or at risk of (or susceptible to) developing a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity, which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Attorney Docket No. 046483-6221-00WO

Antigen

The present invention provides a composition for inducing an immune response in a subject. In one embodiment, the composition comprises at least one mRNA molecule encoding two or more antigenic polypeptides. In some embodiments, the two or more antigenic polypeptides are from a pathogenic microorganism. Pathogenic microorganisms, include, but are not limited to, bacteria, viruses, mycobacteria, fungi, and protozoa.

For example, in some embodiments, the composition comprises at least one mRNA molecule encoding two or more viral antigenic peptides, or a fragment or variant thereof. In some embodiments the two or more antigenic viral peptides are from, but are not limited to, viruses of families Adenoviridae, Adenoviridae, Alphaflexiviridae, Anelloviridae, Arenavirus, Arteriviridae, Asfarviridae, Astroviridae, Benyviridae, Betaflexiviridae, Birnaviridae, Bornaviridae, Bromoviridae, Caliciviridae, Caulimoviridae, Circoviridae, Closteroviridae, Coronaviridae, Filoviridae, Flaviviridae, Geminiviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Kitaviridae, Luteoviridae, Nairoviridae, Nanoviridae, Nimaviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Polyomaviridae, Pospiviridae, Potyviridae, Poxviridae, Reoviridae, Retroviridae, Retrovirus, Rhabdoviridae, Secoviridae, Togaviridae, Tombusviridae, Tospoviridae, Tymoviridae, and Virgaviridae. For example, exemplary viral antigens include, but are not limited to, antigens of African swine fever, Avian hepatitis E, Avian infectious laryngotracheitis, Avian nephritis virus, Bamboo mosaic virus, Banana bunchy top virus, Barley stripe mosaic virus, Barley yellow dwarf virus, Potato leafroll virus, Borna disease, Brome mosaic virus, wheat, Cauliflower mosaic virus, Chikungunya, Eastern equine encephalitis virus, Citrus leprosis, Citrus sudden death associated virus, Citrus tristeza virus, Coconut cadang- cadang viroid, Curly top virus, African cassava mosaic virus, Cytomegalovirus, Epstein- Barr virus, Dengue, Yellow fever, West Nile, Zika, Ebola virus, Marburg virus, Equine arteritis virus, Porcine reproductive and respiratory syndrome virus, Equine infectious anemia, Foot and mouth disease, Foot and mouth disease, Enteroviruses, Rhinoviruses, Hepatitis B virus, Hepatitis E virus, HIV, HIV-1, HIV-2, Infectious bursal disease virus (poultry), Infectious pancreatic necrosis (salmon), Infectious canine hepatitis, Attorney Docket No. 046483-6221-00WO aviadenoviruses of fowl, Influenza viruses, Lassa virus, Lymphocytic choriomeningitis virus, Monkeypox, Nairobi sheep disease, Newcastle disease virus (poultry), Norwalk virus, Numerous examples of crop damaging viruses, including Potato virus Y, Porcine circovirus 2, Beak and feather disease virus (poultry), Potato virus M, Rabies virus, Respiratory and enteric adenoviruses, Respiratory syncytial virus, Rice stripe necrosis virus, Rift Valley fever, rotaviruses, SARS, SARS-CoV-2, MERS, Sheeppox virus, Lumpy skin disease virus, Sin Nombre virus, Andes virus, SV40, Tobacco ringspot virus, Tomato bushy stunt virus, Tomato spotted wilt virus, Torque teno virus, Venezuelan equine encephalitis virus, Vesicular stomatitis Indiana virus, Viral hemorrhagic septicemia (trout), and White spot syndrome virus (shrimp), Primate T-lymphotropic virus 1, Primate T-lymphotropic virus 2, Primate T-lymphotropic virus 3, Human immunodeficiency virus 1, Human immunodeficiency virus 2, Simian foamy virus, Human picobimavirus, Colorado tick fever virus, Changuinola virus, Great Island virus, Lebombo virus, Orungo virus, Rotavirus A, Rotavirus B, Rotavirus C, Banna virus,

Borna disease virus, Lake Victoria Marburgvirus, Reston ebolavirus, Sudan ebolavirus, Tai forest ebolavirus, Zaire virus, Human parainfluenza virus 2, Human parainfluenza virus 4, Mumps virus, Newcastle disease virus, Human parainfluenza virus 1, Human parainfluenza virus 3, Hendra virus, Nipah virus, Measles virus, Human respiratory syncytial virus, Human metapneumovirus, Chandipura virus, Isfahan virus, Piry virus, Vesicular stomatitis Alagoas virus, Vesicular stomatitis Indiana virus, Vesicular stomatitis New Jersey virus, Australian bat lyssavirus, Duvenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Mokola virus, Rabies virus, Guanarito virus, Junin virus, Lassa virus, Lymphocytic choriomeningitis virus, Machupo virus, Pichinde virus, Sabia virus, Whitewater Arroyo virus, Bunyamwera virus, Bwamba virus, California encephalitis virus, Caraparu virus, Catu virus, Guama virus, Guaroa virus,

Kairi virus, Marituba virus, Oriboca virus, Oropouche virus, Shuni virus, Tacaiuma virus, Wyeomyia virus, Andes virus, Bayou virus, Black creek canal virus, Dobrava-Belgrade virus, Hantaan virus, Laguna Negra virus, New York virus, Puumala virus, Seoul virus, Sin Nombre virus, Crimean-Congo haemorrhagic fever virus, Dugbe virus, Candiru virus, Punta Toro virus, Rift Valley fever virus, Sandfly fever Naples virus, Influenza A virus, Influenza B virus, Influenza C virus, Dhori virus, Thogoto virus, Hepatitis delta virus, Attorney Docket No. 046483-6221-00WO

Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1,

Human coronavirus OC43, SARS coronavirus, Human torovirus, Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D, Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Encephalomyocarditis virus, Theilovirus, Equine rhinitis A virus, Foot and mouth disease virus, Hepatitis A virus, Human parechovirus, Ljungan virus, Aichi virus, Human astrovirus, Human astrovirus 2, Human astrovirus 3, Human astrovirus 4, Human astrovirus 5, Human astrovirus 6, Human astrovirus 7, Human astrovirus 8, Norwalk virus, Sapporo virus, Aroa virus, Banzi virus, Dengue virus, Ilheus virus, Japanese encephalitis virus, Kokobera virus, Kyasanur forest disease virus, Louping ill virus, Murray Valley encephalitis virus, Ntaya virus, Omsk haemorrhagic fever virus, Powassan virus, Rio Bravo virus, St Louis encephalitis virus, Tick-borne encephalitis virus, Usutu virus, Wesselsbron virus, West Nile virus, Yellow fever virus, Zika virus, Hepatitis C virus, Hepatitis E virus, Barmah Forest virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Getah virus, Mayaro virus, Mucambo virus, O'nyong-nyong virus, Pixuna virus, Ross River virus, Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Whataroa virus, and Rubella virus. In some embodiments, the viral antigens are from viruses known to cause cancer, including, but not limited to, human papilloma virus (HPV) and the human T cell lympothropic virus HTLV-1.

In some embodiments, the composition comprises a purified, mRNA encoding at least one antigenic pathogen polypeptide. In some embodiments, the mRNA encodes a fusion molecule wherein one or more peptide is linked or fused to the N- terminus or C-terminus of at least one antigenic polypeptide. In some embodiments, a signal peptide is fused to the N-terminus of at least one antigenic polypeptide. In some embodiments, a signal peptide is fused to the C-terminus of at least one antigenic polypeptide.

Influenza antigen

In some embodiments, the multi-genic mRNA immunogenic composition of the invention comprises at least one mRNA molecule having an open reading frame encoding at least one influenza virus antigenic polypeptide. Exemplary influenza antigens Attorney Docket No. 046483-6221-00WO that can be encoded by the mRNA molecule(s) of the multi-genic immunogenic composition (e.g., vaccine) of the invention include an HA antigen or a fragment thereof, an NA antigen or a fragment thereof, a NP antigen or a fragment thereof, an M2 ion channel antigen or a fragment thereof, or any combination thereof.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of influenza antigens from two or more different strains of influenza. The influenza virus antigen may be of any type or strain of influenza virus. For example, in one embodiment, the influenza virus antigen is a protein, or fragment thereof, of an influenza virus strain including, but not limited to, an influenza virus A strain, or a fragment or variant thereof, influenza virus B strain, or a fragment or variant thereof, influenza virus C strain, or a fragment or variant thereof, influenza virus D strain, or a fragment or variant thereof, or any combination thereof. In one embodiment, the influenza virus antigen is a protein, or fragment thereof, of an influenza virus strain including, but not limited to, H1N1 strain, or a fragment or variant thereof, H2N2 strain, or a fragment or variant thereof, H3N2 strain, or a fragment or variant thereof, H5N1 strain, or a fragment or variant thereof, H7N7 strain, or a fragment or variant thereof, H1N2 strain, or a fragment or variant thereof, H9N2 strain, or a fragment or variant thereof, H7N2 strain, or a fragment or variant thereof, H7N3 strain, or a fragment or variant thereof, H10N7 strain, or a fragment or variant thereof, H7N9 strain, or a fragment or variant thereof, H6N1 strain, or a fragment or variant thereof, and any combination thereof.

In one embodiment, the influenza virus antigen is a protein, or fragment thereof, of an influenza virus strain including, but not limited to, an influenza HA group 1 virus strain, or a fragment or variant thereof, influenza NA group 1 virus strain, or a fragment or variant thereof, and any combination thereof. In one embodiment, the influenza HA group 1 virus strain includes, but is not limited to, HI strain, or a fragment or variant thereof, H2 strain, or a fragment or variant thereof, H3 strain, or a fragment or variant thereof, H4 strain, or a fragment or variant thereof, H5 strain, or a fragment or variant thereof, H6 strain, or a fragment or variant thereof, H7 strain, or a fragment or variant thereof, H8 strain, or a fragment or variant thereof, H9 strain, or a fragment or variant thereof, H10 strain, or a fragment or variant thereof, HI 1 strain, or a fragment or Attorney Docket No. 046483-6221-00WO variant thereof, H12 strain, or a fragment or variant thereof, H13 strain, or a fragment or variant thereof, HI 5 strain, or a fragment or variant thereof, HI 5 strain, or a fragment or variant thereof, HI 6 strain, or a fragment or variant thereof, HI 7 strain, or a fragment or variant thereof, HI 8 strain, or a fragment or variant thereof, and any combination thereof. In one embodiment, the influenza NA group 1 virus strain includes, but is not limited to, N1 strain, or a fragment or variant thereof, N2 strain, or a fragment or variant thereof, N3 strain, or a fragment or variant thereof, N4 strain, or a fragment or variant thereof, N5 strain, or a fragment or variant thereof, N6 strain, or a fragment or variant thereof, N7 strain, or a fragment or variant thereof, N8 strain, or a fragment or variant thereof, N9 strain, or a fragment or variant thereof, N10 strain, or a fragment or variant thereof, N11 strain, or a fragment or variant thereof, and any combination thereof.

In one exemplary non-limiting embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of a HA from A/Puerto Rico/8/1934, comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a HA from HlNlpdm09-like A/Michigan/45/2015, comprising an amino acid sequence as set forth in SEQ ID NO:2. In another non-limiting exemplary embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of a NA from HlNlpdm09-like A/Michigan/45/2015, comprising an amino acid sequence as set forth in SEQ ID NO: 3 and aNA from A/Singapore/INFIMH- 16-0019/2016(H3N2), comprising an amino acid sequence as set forth in SEQ ID NO:4. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of at least two different influenza antigens. For example, in one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA encoding at least one HA antigen and at least one NA antigen. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecule encoding two or more of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

In some embodiments, the influenza virus antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an influenza virus antigen described herein and retains the immunogenic function of the Attorney Docket No. 046483-6221-00WO original amino acid sequence. For example, in some embodiments, the amino acid sequence of the influenza virus antigen has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

Human immunodeficiency virus (HIV) antigen

In some embodiments, the multi-genic mRNA immunogenic composition of the invention comprises at least one mRNA molecule having an open reading frame encoding at least one HIV antigenic polypeptide. Exemplary HIV antigens that can be encoded by the mRNA molecule(s) of the multi-genic immunogenic composition (e.g., vaccine) of the invention include, but are not limited to, an Env antigen or a fragment thereof, a Nef antigen or a fragment thereof, a Pol antigen or a fragment thereof, a Gag antigen or a fragment thereof, a Tat antigen or a fragment thereof, a Rev antigen or a fragment thereof, a Vif antigen or a fragment thereof, a Vpr antigen or a fragment thereof, and a Vpu antigen or a fragment thereof, or any combination thereof. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of HIV antigens from two or more different strains of HIV. In one exemplary non-limiting embodiment, the multi -genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of at least two different HIV antigens. For example, in one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA encoding a combination of a Gag antigen and an Env antigen. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecule encoding a combination of SEQ ID NO:5 and SEQ ID NO:6.

In some embodiments, the HIV antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an HIV antigen described herein and retains the immunogenic function of the original amino acid sequence. For example, in some embodiments, the amino acid sequence of the HIV antigen has a degree Attorney Docket No. 046483-6221-00WO of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

Coronavirus (CoV) Antigens

In some embodiments, the multi-genic mRNA immunogenic composition of the invention comprises at least one mRNA molecule having an open reading frame encoding at least one CoV antigenic polypeptide. In some embodiments, the CoV is SARS-CoV2. In some embodiments, the CoV is MERS-CoV. In some embodiments, the CoV is SARS-CoV. In some embodiments, the CoV is HCoV-OC43. In some embodiments, the CoV is HCoV-229E. In some embodiments, the CoV is HCoV-NL63. In some embodiments, the CoV is HCoV-HKUl. In some embodiments, at least one antigenic polypeptide is a coronavirus structural protein. For example, a coronavirus structural protein may be spike protein (S), envelope protein (E), nucleocapsid protein (N), membrane protein (M) or an immunogenic fragment thereof. In some embodiments, a coronavirus structural protein is a spike protein (S). In some embodiments, a coronavirus structural protein is a SI subunit or a S2 subunit of spike protein (S) or an immunogenic fragment thereof.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises one or more mRNA molecule encoding a combination of coronavirus antigens from two or more different strains of coronavirus. The coronavirus antigen may be of any type or strain of coronavirus. For example, in one embodiment, the coronavirus is a protein, or fragment thereof, of a SARS-CoV-2 coronavirus strain including, but not limited to, an Alpha (B.1.1.7 and Q lineages) strain or variant thereof, a Beta (B.1.351 and descendent lineages) strain or variant thereof, a Delta (B.1.617.2 and AY lineages) strain or variant thereof, an Epsilon (B.1.427 and B.1.429) strain or variant thereof, a Gamma (P.1 and descendent lineages) strain or variant thereof, an Eta (B.1.525) strain or variant thereof, an Iota (B.1.526) strain or variant thereof, a Kappa (B.1.617.1) strain or variant thereof, a Mu (B.1.621, B.1.621.1) strain or variant thereof, an Omicron Attorney Docket No. 046483-6221-00WO

(B.1.1.529 and BA lineages) strain or variant thereof or a Zeta (P.2) strain or variant thereof, or any combination thereof.

In some embodiments, the CoV mRNA polynucleotides of the multi-genic immunogenic compositions (e.g., vaccines) provided herein may encode one or more viral protein components of coronaviruses, for example, accessory proteins, replicase proteins and the like, or a fragment thereof. The present disclosure also encompasses multi-genic mRNA immunogenic compositions comprising one or more mRNA polynucleotides encoding two or more coronavirus proteins, for example a combination of at least one accessory protein or replicase protein and at least one structural protein.

For example, in one embodiment, the multi -genic immunogenic composition (e.g., vaccine) comprises one or more mRNA encoding a combination of two or more of a spike antigen (S), membrane glycoprotein (M) antigen, envelope protein (E), nucleocapsid protein (N), ORFlab polyprotein, ORF3a protein, ORF6 protein, ORF7a protein, ORF8 protein, and ORF10 protein or a fragment thereof. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecule encoding a combination of two or more of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:17.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises a combination of an mRNA molecule encoding a spike antigen (S), and an mRNA molecule encoding a nucleocapsid protein (N). In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecule encoding a combination of SEQ ID NO:7 and SEQ ID NO: 10.

In some embodiments, the CoV antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of a CoV antigen described herein and retains the immunogenic function of the original amino acid sequence. For example, in some embodiments, the amino acid sequence of the CoV antigen has a degree of identity with respect to the original amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least Attorney Docket No. 046483-6221-00WO

90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

Adjuvant

In one embodiment, the composition comprises an adjuvant. In one embodiment, the composition comprises a nucleic acid molecule encoding an adjuvant.

In one embodiment, the adjuvant-encoding nucleic acid molecule is an mRNA molecule.

Exemplary adjuvants include, but are not limited to, alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFa, ΈNEb, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86. Other genes which may be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P- selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95,

PEC AM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC 5 , TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and functional fragments thereof.

In one embodiment, the adjuvant molecule comprises a T cell stimulatory molecule. Exemplary T cell stimulatory molecules that can be included in the compositions of the invention as adjuvant molecules include, but are not limited to, CD80 and CD86.

In some embodiments, the composition comprises an mRNA molecule encoding a combination of least one pathogen antigen and at least one mRNA molecule encoding an adjuvant molecule. Attorney Docket No. 046483-6221-00WO

In some embodiments, the composition comprises a combination of at least one mRNA molecule encoding a pathogen antigen and at least one mRNA molecule encoding an adjuvant molecule.

In one embodiment, the adjuvant molecule comprises an mRNA molecule encoding CD80, wherein the CD80 comprises an amino acid sequence of SEQ ID NO: 18, or a variant thereof. For example, in some embodiments, the amino acid sequence of the CD80 adjuvant has a degree of identity with respect to the SEQ ID NO: 18 of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

In one embodiment, the adjuvant molecule comprises an mRNA molecule encoding CD86, wherein the CD86 comprises an amino acid sequence of SEQ ID NO: 19, or a variant thereof. For example, in some embodiments, the amino acid sequence of the CD86 adjuvant has a degree of identity with respect to the SEQ ID NO: 19 of at least 60%, of at least 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 91%, of at least 92%, of at least 93%, of at least 94%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, of at least 99%, or of at least 99.5%.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) comprises a combination of an mRNA molecule encoding a spike antigen (S), an mRNA molecule encoding a nucleocapsid protein (N) and at least one mRNA molecule encoding at least one of CD80 and CD86. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecule encoding a combination of SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 18. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecule encoding a combination of SEQ ID NO:7, SEQ ID NO: 10 and SEQ ID NO: 19. In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA Attorney Docket No. 046483-6221-00WO molecule encoding a combination of SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 18 and SEQ ID NO: 19.

Flagellin Adjuvants

Flagellin is an approximately 500 amino acid monomeric protein that polymerizes to form the flagella associated with bacterial motion. Flagellin is expressed by a variety of flagellated bacteria {Salmonella typhimurium for example) as well as non- flagellated bacteria (such as Escherichia coli). Sensing of flagellin by cells of the innate immune system (dendritic cells, macrophages, etc.) is mediated by the Toll-like receptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and NLRs have been identified as playing a role in the activation of innate immune response and adaptive immune response. As such, flagellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known flagellin polypeptides are publicly available in the NCBI GenBank database. The flagellin sequences from S.

Typhimurium, H. Pylori, V. Cholera, S. marcesens, S. flexneri, T Pallidum, L. pneumophila, B. burgdorferei, C. difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P. Mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa , and E. coli , among others are known.

A flagellin polypeptide, as used herein, refers to a full length flagellin protein, immunogenic fragments thereof, and peptides having at least 50% sequence identify to a flagellin protein or immunogenic fragments thereof. Exemplary flagellin proteins include flagellin from Salmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella enteritidis (AOAOC9BAB7), and Salmonella choleraesuis (Q6V2X8)). In some embodiments, the flagellin polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identify to a flagellin protein or immunogenic fragments thereof.

In some embodiments, the flagellin polypeptide is an immunogenic fragment. An immunogenic fragment is a portion of a flagellin protein that provokes an immune response. In some embodiments, the immune response is a TLR5 immune response. An example of an immunogenic fragment is a flagellin protein in which all or a Attorney Docket No. 046483-6221-00WO portion of a hinge region has been deleted or replaced with other amino acids. For example, an antigenic polypeptide may be inserted in the hinge region. Hinge regions are the hypervariable regions of a flagellin. Hinge regions of a flagellin are also referred to as “D3 domain or region, “propeller domain or region,” “hypervariable domain or region” and “variable domain or region.” “At least a portion of a hinge region,” as used herein, refers to any part of the hinge region of the flagellin, or the entirety of the hinge region.

In other embodiments an immunogenic fragment of flagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment of flagellin.

In some embodiments, the multi-genic mRNA immunogenic composition comprises an mRNA molecule that encodes a fusion protein of flagellin and one or more antigenic polypeptides. A “fusion protein” as used herein, refers to a linking of two components of the construct. In some embodiments, a carboxy-terminus of the antigenic polypeptide is fused or linked to an amino terminus of the flagellin polypeptide. In other embodiments, an amino-terminus of the antigenic polypeptide is fused or linked to a carboxy-terminus of the flagellin polypeptide. The fusion protein may include, for example, one, two, three, four, five, six or more flagellin polypeptides linked to one, two, three, four, five, six or more antigenic polypeptides.

Nucleic Acids

In one embodiment, the invention includes a nucleic acid molecule encoding at least one pathogen antigen, an adjuvant molecule, or a combination thereof.

In one embodiment, the invention includes one or more mRNA molecule encoding at least one pathogen antigen, an adjuvant molecule, or a combination thereof. In one embodiment, the mRNA molecule encodes a plurality of pathogen antigens. In some embodiments, the mRNA molecule encodes an plurality of pathogen antigens that induces an adaptive immune response against one or more pathogenic microorganisms. In some embodiments, the mRNA molecule encodes an plurality of pathogen antigens that induces an adaptive immune response against one or more antigens from different pathogenic microorganisms. In some embodiments, the mRNA molecule encodes an plurality of pathogen antigens that induces an adaptive immune response against one or more antigens from different viral strains. In some embodiments, the mRNA molecule Attorney Docket No. 046483-6221-00WO encodes an plurality of viral antigens that induces an adaptive immune response against one or more antigens from different viruses. In one embodiment, the invention includes an mRNA molecule encoding an adjuvant.

The nucleic acid molecule can be made using any methodology in the art, including, but not limited to, in vitro transcription, chemical synthesis, or the like.

The nucleotide sequences encoding a pathogen antigen or adjuvant, as described herein, can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a polypeptide according to the invention. Therefore, the scope of the present invention includes nucleotide sequences that are substantially identical to the nucleotide sequences recited herein and encode one or more pathogen antigen or adjuvant of interest.

A nucleotide sequence that is substantially identical to a nucleotide sequence encoding an antigen can typically be isolated from a producer organism of the antigen based on the information contained in the nucleotide sequence by means of introducing conservative or non-conservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

Further, the scope of the invention includes nucleotide sequences that encode amino acid sequences that are substantially identical to the amino acid sequences recited herein and preserve the immunogenic function of the original amino acid sequence.

In one embodiment, the invention relates to a construct, comprising a nucleotide sequence encoding at least one pathogen antigen. In one embodiment, the construct comprises a plurality of nucleotide sequences encoding a plurality of pathogen antigens. For example, in some embodiments, the construct encodes at least 1, at least 2, at least 3, or more than 3 pathogen antigens. In some embodiments, the construct encodes at least 1, at least 2, at least 3, or more than 3 viral antigens. In one embodiment, the Attorney Docket No. 046483-6221-00WO invention relates to a construct, comprising a nucleotide sequence encoding an adjuvant. In one embodiment, the construct comprises a first nucleotide sequence encoding one or more pathogen antigen and a second nucleotide sequence encoding an adjuvant.

In one embodiment, the composition comprises a plurality of constructs, each construct encoding one or more pathogen antigen. In some embodiments, the composition comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more,

7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more constructs. In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding a first virus antigen; and a second construct, comprising a nucleotide sequence encoding a second virus antigen. In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding a plurality of pathogen antigens; and a second construct, comprising a nucleotide sequence encoding an adjuvant.

In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding a first pathogen antigen; a second construct, comprising a nucleotide sequence encoding a second pathogen antigen; and a third construct, comprising a nucleotide sequence encoding an adjuvant.

In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding a first virus antigen; a second construct, comprising a nucleotide sequence encoding a second virus antigen; and a third construct, comprising a nucleotide sequence encoding an adjuvant.

In some embodiments, the construct of the invention is operatively bound to a translational control element. The construct can incorporate an operatively bound regulatory sequence for the expression of the nucleotide sequence of the invention, thus forming an expression cassette.

Vectors

The nucleic acid sequences coding for the influenza virus antigen or adjuvant can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues Attorney Docket No. 046483-6221-00WO containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically.

The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, a PCR-generated linear DNA sequence, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors and vectors optimized for in vitro transcription.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, carbohydrates, peptides, cationic polymers, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In one embodiment, the delivery vehicle comprises one or more cell penetrating peptide. Exemplary cell penetrating peptides that can be used for a delivery vehicle include, but are not limited to, self-associating cationic cell penetrating peptides.

In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/RNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be Attorney Docket No. 046483-6221-00WO naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long- chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as it is more readily evaporated than methanol.

In some embodiments, phototransfection can be used to introduce genetic matter into cells. Phototransfection uses femtosecond laser pulses for the targeted introduction of genetic matter into cells. Exemplary methods of phototransfection include, but are not limited to, those described by Barret et al., 2006, Nat Methods, 3(6):455-60 and Sul et al., Proc Natl Acad Sci U S A, 106(18):7624-9, each of which is incorporated herein by reference in its entirety.

In some embodiments, ultrasound-mediated gene transfection (sonotransfection) can be used to introduce genetic matter into cells. Sonotransfection uses repeated pules of ultrasound at different intensities, pulse repetition frequencies and exposure times for introduction of genetic matter into cells. Exemplary methods of sonotransfection include, but are not limited to, those described by Yoon et al., 2017, Scientific Reports volume 7, Article number: 5275, which is incorporated herein by reference in its entirety.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to a composition of the present invention, in order to confirm the presence of the mRNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Northern blotting and RT-PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunogenic Attorney Docket No. 046483-6221-00WO means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention. N-Linked Glvcosylation Site Mutants

N-linked glycans of viral proteins play important roles in modulating the immune response. Glycans can be important for maintaining the appropriate antigenic conformations, shielding potential neutralization epitopes, and may alter the proteolytic susceptibility of proteins. Some viruses have putative N-linked glycosylation sites. Deletion or modification of an N-linked glycosylation site may enhance the immune response. Thus, in some embodiments, the mRNA molecules of the invention encode antigenic polypeptides that comprise a deletion or modification at one or more N-linked glycosylation sites. In Vitro Transcription of mRNA

Multi-genic mRNAn immunogenic compositions of the present disclosure comprise at least one mRNA polynucleotide. In one embodiment, the mRNA is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

A “5' untranslated region” (5'UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.

A “3' untranslated region” (3'UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

A “5¹ cap” refers to a structure found on the 5' end of an mRNA molecule, and generally consists of a guanine nucleotide connected to mRNA via a 5' to 5' Attorney Docket No. 046483-6221-00WO triphosphate linkage. This guanosine is methylated on the 7 position directly after capping in vivo by a methyltransf erase. The 5’ cap provides stability to an mRNA.

An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTRthat contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

Modifications

In some embodiments, the present invention encompasses multi-genic mRNA molecules comprising a modified nucleoside, such as pseudouridine. In some embodiments, the composition comprises an isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside. In some embodiments, the composition comprises a vector, comprising an isolated nucleic acid encoding an antigen, adjuvant, or combination thereof, wherein the nucleic acid comprises at least one modified nucleoside.

In another embodiment, at least one nucleoside that is modified in the nucleoside-modified RNA the present invention is uridine (U). In another embodiment, Attorney Docket No. 046483-6221-00WO the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

In one embodiment, the modified nucleoside is m^lvP (1- methylpseudouridine). In another embodiment, the modified nucleoside is m¹acp^3vP (1- m ethyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine. In another embodiment, the modified nucleoside is I'm (2'-0-methylpseudouridine). In another embodiment, the modified nucleoside is m⁵D (5-methyldihydrouridine). In another embodiment, the modified nucleoside is m^3vP (3-methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

In another embodiment, the modified nucleoside of the present invention is m⁵C (5-methylcytidine). In another embodiment, the modified nucleoside is m⁵U (5- methyluridine). In another embodiment, the modified nucleoside is m⁶A (N⁶- methyladenosine). In another embodiment, the modified nucleoside is s²U (2- thiouridine). In another embodiment, the modified nucleoside is Y (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-0-methyluridine).

In other embodiments, the modified nucleoside is nriA (1- methyladenosine); m²A (2-methyladenosine); Am (2'-0-methyladenosine); ms²m⁶A (2- methylthio-N⁶-methyladenosine); i⁶A (N⁶-isopentenyladenosine); ms¾6A (2-methylthio- N⁶isopentenyladenosine); io⁶A (N⁶-(cis-hydroxyisopentenyl)adenosine); ms²io⁶A (2- methylthio-N⁶-(cis-hydroxyisopentenyl) adenosine); g⁶A (N⁶- glycinylcarbamoyladenosine); t⁶A (N⁶-threonylcarbamoyladenosine); ms²t⁶A (2- methylthio-N⁶-threonyl carbamoyladenosine); m⁶t⁶A (N⁶-methyl-N⁶- threonylcarbamoyladenosine); hn⁶A(N⁶-hydroxynorvalylcarbamoyladenosine); ms²hn⁶A (2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0-ribosyladenosine (phosphate)); I (inosine); nril (1-methylinosine); nrilm (l,2'-0-dimethylinosine); m³C (3- methylcytidine); Cm (2'-0-methylcytidine); s²C (2-thiocytidine); ac⁴C (N⁴- acetylcytidine); CC (5-formylcytidine); m⁵Cm (5,2'-0-dimethylcytidine); ac⁴Cm (N⁴- acetyl-2'-0-methylcytidine); k²C (lysidine); m'G ( 1-methylguanosine); m²G (N²- Attorney Docket No. 046483-6221-00WO methylguanosine); m⁷G (7-methylguanosine); Gm (2'-0-methylguanosine); nAG (N²,N²- dimethylguanosine); m²Gm (N²,2'-0-dimethylguanosine); m²2Gm (N²,N²,2'-0- trimethylguanosine); Gr(p) (2'-0-ribosylguanosine (phosphate)); yW (wybutosine); 02yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine);

D (dihydrouridine); m⁵Um (5,2'-0-dimethyluridine); s⁴U (4-thiouridine); m⁵s²U (5- methyl-2-thiouridine); s²Um (2-thio-2'-0-methyluridine); acp³U (3-(3-amino-3- carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U (5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo⁵U (uridine 5-oxyacetic acid methyl ester); chm⁵U (5- (carboxyhydroxymethyl)uridine)); mchm⁵U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U (5-methoxycarbonylmethyluridine); mcm⁵Um (5- methoxycarbonylmethyl-2'-0-methyluridine); mcm⁵s²U (5-methoxycarbonylmethyl-2- thiouridine); nm⁵s²U (5-aminomethyl-2-thiouridine); mnm⁵U (5- methylaminomethyluridine); mnm⁵s²U (5-methylaminomethyl-2-thiouridine); mnm⁵se²U (5-methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine); ncm⁵Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm⁵U (5- carboxymethylaminomethyluridine); cmnm⁵Um (5-carboxymethylaminomethyl-2'-0- methyluridine); cmnm⁵s²U (5-carboxymethylaminomethyl-2-thiouridine); m⁶2A (N⁶,N⁶- dimethyladenosine); Im (2'-0-methylinosine); m⁴C (N⁴-methylcytidine); m⁴Cm (N⁴,2'-0- dimethylcytidine); hm⁵C (5-hydroxymethylcytidine); m³U (3-methyluridine); cm⁵U (5- carboxymethyluridine); m⁶Am (N⁶,2'-0-dimethyladenosine); m⁶2Am (N⁶,N⁶,0-2'- trimethyladenosine); m²⁷G (N²,7-dimethylguanosine); nA⁷G (N²,N²,7- trimethylguanosine); m³Um (3,2'-0-dimethyluridine); m⁵D (5-methyldihydrouridine); f^Cm (5-formyl-2'-0-methylcytidine); nriGm (l,2'-0-dimethylguanosine); m⁴Am (1,2'- O-dimethyladenosine); rrrfU (5-taurinomethyluridine); xm⁵s²U (5-taurinomethyl-2- thiouridine)); imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A (N⁶- acetyladenosine).

In another embodiment, a nucleoside-modified mRNA of the present invention comprises a combination of 2 or more of the above modifications. In another Attorney Docket No. 046483-6221-00WO embodiment, the nucleoside-modified mRNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified mRNA comprises a combination of more than 3 of the above modifications.

In various embodiments, between 0.1% and 100% of the residues in the nucleoside-modified mRNA of the present invention are modified (e.g., either by the presence of pseudouridine, 1 -methyl-pseudouridine, or another modified nucleoside base). In one embodiment, the fraction of modified residues is 0.1%. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In Attorney Docket No. 046483-6221-00WO another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%.

In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%.

In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is

14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is

18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is

25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is

35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is

45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is Attorney Docket No. 046483-6221-00WO

55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%. In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

Linker

In some embodiments, one or more mRNA molecule of the invention encodes a fusion protein. Each of the components of a fusion protein may be directly linked to one another or they may be connected through a linker. For instance, the linker may be an amino acid linker. The amino acid linker encoded for by the RNA (e.g., mRNA) vaccine to link the components of the fusion protein may include, for instance, at least one member selected from the group consisting of a lysine residue, a glutamic acid residue, a serine residue and an arginine residue. In some embodiments the linker is 1-30, 1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

Pharmaceutical Compositions Attorney Docket No. 046483-6221-00WO

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to subjects of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various subjects is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, peptide wrapping, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be Attorney Docket No. 046483-6221-00WO administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable Attorney Docket No. 046483-6221-00WO sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. In some embodiments, the formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In some Attorney Docket No. 046483-6221-00WO embodiments, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. In one embodiment, the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (in some instances having a particle size of the same order as particles comprising the active ingredient).

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to Attorney Docket No. 046483-6221-00WO the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Liposomes. Lipoplexes. and Lipid Nanoparticles

The multi-genic mRNA immunogenic compositions of the disclosure can be formulated using one or more peptide wrappings, liposomes, lipoplexes, lipid nanoparticles, or a combination thereof. In some embodiments, pharmaceutical compositions of mRNAn immunogenic compositions include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are Attorney Docket No. 046483-6221-00WO dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

In some embodiments, the multi-genic mRNA immunogenic composition of the invention may be formulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle vaccine formulation comprising the polynucleotide is a nanoparticle which may comprise at least one lipid selected from, but not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C 12-200, DLin-MC3 -DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids.

In some embodiments, the multi-genic mRNA immunogenic composition of the invention may be formulated in a peptide particle or peptide wrap. In one emboidment, the multi-genic mRNA immunogenic composition of the invention may be formulated using a delivery vehicle comprising combination of peptides and lipids.

Multi-mRNA compositions

In some embodiments, the invention provides a combination of at least two mRNA molecules. In some embodiments, at least two mRNA molecules are combined in a single composition. In some embodiments, at least two mRNA molecules are formulated in separate compositions for administration at different times or at different sites. For example, in one embodiment, at least two mRNA molecules are formulated for use as a combination prime-boost vaccine.

In some embodiments, at least two mRNA molecules may be present in a composition, or combination of compositions, of the invention in any appropriate ratio such that each of the at least two mRNA molecules induces or enhances an immune response. In some embodiments, for a composition comprising two mRNA molecules, the two mRNA molecules may be present in a ratio of a:b, with “a” being any number from 1 to greater than 10,000 relative to the concentration of “b ” For example, in some embodiments, two mRNA molecules are present in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 1000:1, 5000:1, 10,000:1, greater Attorney Docket No. 046483-6221-00WO than 10,000:1 or any ratio there between. In some embodiments, for a composition comprising three mRNA molecules, the three mRNA molecules may be present in a ratio of a:b:c, with each of “a” and “b” being any number from 1 to greater than 10,000 relative to the concentration of “c ” For example, in some embodiments, three mRNA molecules are present in a ratio of 1:1:1, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 10:1:1, 20:1:1, 30:1:1, 40:1:1, 50:1:1, 60:1:1, 70:1:1, 80:1:1, 90:1:1, 100:1:1, 1000:1:1, 5000:1:1, 10,000:1:1, greater than 10,000:1:1, 2:2:1, 3:2:1, 4:2:1, 5:2:1, 10:2:1, 20:2:1, 30:2:1, 40:2:1, 50:2:1, 60:2:1, 70:2:1, 80:2:1, 90:2:1, 100:2:1, 1000:2:1, 5000:2:1, 10,000:2:1, greater than 10,000:2:1, 2:3:1, 3:3:1, 4:3:1, 5:3:1, 10:3:1, 20:3:1, 30:3:1, 40:3:1, 50:3:1, 60:3:1,

70:3:1, 80:3:1, 90:3:1, 100:3:1, 1000:3:1, 5000:3:1, 10,000:3:1, greater than 10,000:3:1, 2:4:1, 3:4:1, 4:4:1, 5:4:1, 10:4:1, 20:4:1, 30:4:1, 40:4:1, 50:4:1, 60:4:1, 70:4:1, 80:4:1, 90:4:1, 100:4:1, 1000:4:1, 5000:4:1, 10,000:4:1, greater than 10,000:4:1, 2:5:1, 3:5:1, 4:5:1, 5:5:1, 10:5:1, 20:5:1, 30:5:1, 40:5:1, 50:5:1, 60:5:1, 70:5:1, 80:5:1, 90:5:1, 100:5:1, 1000:5:1, 5000:5:1, 10,000:5:1, greater than 10,000:5:1, 2:10:1, 3:10:1, 4:10:1, 5:10:1, 10:10:1, 20:10:1, 30:10:1, 40:10:1, 50:10:1, 60:10:1, 70:10:1, 80:10:1, 90:10:1, 100:10:1, 1000:10:1, 5000:10:1, 10,000:10:1, greater than 10,000:10:1, 2:100:1, 3:100:1, 4:100:1, 5:100:1, 10:100:1, 20:100:1, 30:100:1, 40:100:1, 50:100:1, 60:100:1, 70:100:1, 80:100:1, 90:100:1, 100:100:1, 1000:100:1, 5000:100:1, 10,000:100:1, greater than 10,000:100:1, 2:1000:1, 3:1000:1, 4:1000:1, 5:1000:1, 10:1000:1, 20:1000:1, 30:1000:1, 40:1000:1, 50:1000:1, 60:1000:1, 70:1000:1, 80:1000:1, 90:1000:1, 100:1000:1, 1000:1000:1, 5000:1000:1, 10,000:1000:1, greater than 10,000:1000:1, 2:10,000:1, 3:10,000:1, 4:10,000:1, 5:10,000:1, 10:10,000:1, 20:10,000:1, 30:10,000:1, 40:10,000:1, 50:10,000:1, 60:10,000:1, 70:10,000:1, 80:10,000:1, 90:10,000:1, 100:10,000:1, 1000:10,000:1, 5000:10,000:1, 10,000:10,000:1, greater than 10,000:10,000:1, or any ratio there between. Similarly, for compositions comprising more than three mRNA molecule the mRNA molecules may be present at any appropriate ratio such that each of the more than three mRNA molecules each induce or enhance an immune response.

Methods

Some embodiments of the present disclosure provide methods of inducing an antigen specific immune response in a subject, comprising administering to the subject Attorney Docket No. 046483-6221-00WO any of the multi-genic mRNA immunogenic compositions as provided herein in an amount effective to produce an antigen-specific immune response. In some embodiments, an antigen-specific immune response comprises a T cell response or a B cell response.

In some embodiments, a method of producing an antigen-specific immune response comprises administering to a subject a single dose (no booster dose) of the multi-genic mRNA immunogenic composition of the present disclosure. In some embodiments, the method further comprises administering to the subject a second (booster) dose of the multi-genic mRNA immunogenic composition. Additional doses of the multi-genic mRNA immunogenic composition may be administered.

In some embodiments, the subjects exhibit a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) following the first dose or the second (booster) dose of the immunogenic composition. Seroconversion is the time period during which a specific antibody develops and becomes detectable in the blood. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but antibodies are considered absent. During seroconversion, antibodies are present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.

Some embodiments, of the present disclosure provide methods of inducing an antigen specific immune response in a subject, including administering to a subject a multi-genic mRNA immunogenic composition in an effective amount to produce an antigen specific immune response in a subject. Antigen-specific immune responses in a subject may be determined, in some embodiments, by assaying for antibody titer following administration to the subject of any of the multi -genic mRNA immunogenic compositions of the present disclosure. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by at least 1 log relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased by 1-3 log relative to a control,

In some embodiments, the anti-antigenic polypeptide antibody titer produced in a subject is increased at least 2 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is Attorney Docket No. 046483-6221-00WO increased at least 5 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased at least 10 times relative to a control. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is increased 2-10 times relative to a control.

In some embodiments, the control is an anti-antigenic polypeptide antibody titer produced in a subject who has not been administered a multi -genic mRNA immunogenic composition of the present disclosure.

In some embodiments, the multi-genic mRNA immunogenic composition of the present disclosure is administered to a subject in an effective amount (an amount effective to induce an immune response). In some embodiments, the effective amount is an amount to produce an antigen specific immune response in a subject. In some embodiments, the effective amount is a total dose of 25 pg to 1000 pg, or 50 pg to 1000 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a dose of 25 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 pg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 pg administered to the subject a total of two times.

In another embodiment, the multi-genic mRNA immunogenic composition of the present invention induces a significantly more robust adaptive immune response as compared with mRNAn immunogenic composition comprising a single antigen. In another embodiment, the multi-genic mRNA immunogenic composition of the present induces an adaptive immune response that is 2-fold greater than a mRNAn immunogenic composition comprising a single antigen. In another embodiment, the adaptive immune response is increased by a 3 -fold factor. In another embodiment, the adaptive immune response is increased by a 4-fold factor. In another embodiment, the adaptive immune response is increased by a 5-fold factor. In another embodiment, the adaptive immune response is increased by a 6-fold factor. In another embodiment, the adaptive immune response is increased by a 7-fold factor. In another embodiment, the adaptive immune response is increased by an 8-fold factor. In another embodiment, the adaptive immune Attorney Docket No. 046483-6221-00WO response is increased by a 9-fold factor. In another embodiment, the adaptive immune response is increased by a 10-fold factor. In another embodiment, the adaptive immune response is increased by a 15-fold factor. In another embodiment, the adaptive immune response is increased by a 20-fold factor. In another embodiment, the adaptive immune response is increased by a 50-fold factor. In another embodiment, the adaptive immune response is increased by a 100-fold factor. In another embodiment, the adaptive immune response is increased by a 200-fold factor. In another embodiment, the adaptive immune response is increased by a 500-fold factor. In another embodiment, the adaptive immune response is increased by a 1000-fold factor. In another embodiment, the adaptive immune response is increased by a 2000-fold factor. In another embodiment, the adaptive immune response is increased by another fold difference.

In another embodiment, “induces significantly more robust adaptive immune response” refers to a detectable increase in an adaptive immune response. In another embodiment, the term refers to a fold increase in the adaptive immune response (e.g., 1 of the fold increases enumerated above). In another embodiment, the term refers to an increase such that the multi-genic mRNA immunogenic composition can be administered at a lower dose or frequency than a single-antigen vaccine while still inducing a similarly effective adaptive immune response. In another embodiment, the increase is such that the multi-genic mRNA immunogenic composition can be administered using a single dose to induce an effective adaptive immune response.

Methods of Treatment or Prevention

The present invention provides methods of inducing an adaptive immune response against one or more antigens in a subject, comprising administering an effective amount of a composition comprising one or more isolated nucleic acids encoding one or more antigens. In some embodiments, the invention provides methods of inducing an adaptive immune response against one or more pathogenic microorganisms in a subject, comprising administering an effective amount of a composition comprising one or more isolated nucleic acids encoding one or more pathogen antigens.

In one embodiment, the method provides immunity in the subject to a disease or disorder associated with one or more encoded antigen. The present invention Attorney Docket No. 046483-6221-00WO thus provides a method of treating or preventing a disease, or disorder associated with the one or more encoded antigen. In some embodiments, the present invention provides a method of treating or preventing a disease, or disorder associated with the one or more pathogenic microorganism.

In one embodiment, the composition is administered to a subject having a viral infection, or a disease, or disorder associated with a viral infection. In one embodiment, the composition is administered to a subject at risk for developing a viral infection or a disease, or disorder associated with viral infection. For example, in one embodiment, a multi-genic mRNA immunogenic composition comprising at least one influenza antigen of the invention may be administered to a subject who is at risk for being exposed to, or in contact with, influenza virus. In one embodiment, a multi-genic mRNA immunogenic composition comprising at least one HIV antigen of the invention may be administered to a subject who is at risk for being exposed to, or in contact with, HIV. In one embodiment, a multi-genic mRNA immunogenic composition comprising at least one SARS-CoV2 antigen of the invention may be administered to a subject who is at risk for being exposed to, or in contact with, SARS-CoV2. In one embodiment, the composition is administered to a subject who lives in, traveled to, or is expected to travel to a geographic region in which a target virus is prevalent. In one embodiment, the composition is administered to a subject who is in contact with or expected to be in contact with another person who lives in, traveled to, or is expected to travel to a geographic region in which a target virus is prevalent. In one embodiment, the composition is administered to a subject who has knowingly been exposed to a target virus through their occupation, or other contact.

In one embodiment, the method comprises administering a composition comprising one or more mRNA molecules encoding two or more antigens. In one embodiment, the method comprises administering a composition comprising a first mRNA molecule encoding one or more pathogen antigens and a second mRNA molecule encoding molecule encoding one or more pathogen antigens. In one embodiment, the method comprises administering a composition comprising an mRNA molecule encoding a plurality of pathogen antigens. Attorney Docket No. 046483-6221-00WO

In one embodiment, the method comprises administering one or more composition, each composition comprising one or more mRNA encoding one or more pathogen antigens. In one embodiment, the pathogen antigens are different antigens of the same pathogenic microorganism. In one embodiment, the pathogen antigens are different antigens of the different pathogenic microorganisms. In one embodiment, the pathogen antigens are antigens from different strains of a virus. In one embodiment, the viral antigens are antigens from different viruses.

In one embodiment, the method comprises administering a first composition comprising one or more mRNA molecules encoding one or more pathogen antigens and administering a second composition comprising one or more mRNA molecules encoding one or more pathogen antigens. In one embodiment, the method comprises administering a plurality of compositions, each composition comprising one or more mRNA molecules encoding one or more pathogen antigens. In some embodiments, the method comprises a staggered administration of the plurality of compositions.

In some embodiments, the method of the invention allows for sustained expression of the pathogen antigen, or adjuvant, for at least several days following administration. In some embodiments, the method of the invention allows for sustained expression of the pathogen antigen for at least 2 weeks following administration. In some embodiments, the method of the invention allows for sustained expression of the pathogen antigen for at least 1 month following administration. In some embodiments, also provides for transient expression, as in some embodiments, the nucleic acid is not integrated into the subject genome.

In some embodiments, the method comprises administering the multi- genic mRNA immunogenic composition of the invention, which provides stable expression of multiple antigens. In some embodiments, administration of the multi-genic mRNA immunogenic composition of the invention results in little to no innate immune response, while inducing an effective adaptive immune response.

In some embodiments, the method provides sustained protection against at least one virus or a disease or disorder associated with at least one virus. In one embodiment, the virus is a human virus, a plant virus or an animal virus. Exemplary viruses include, but are not limited to, viruses of families Adenoviridae, Adenoviridae, Attorney Docket No. 046483-6221-00WO

Alphaflexiviridae, Anelloviridae, Arenavirus, Arteriviridae, Asfarviridae, Astroviridae, Benyviridae, Betaflexiviridae, Birnaviridae, Bomaviridae, Bromoviridae, Caliciviridae, Caulimoviridae, Circoviridae, Closteroviridae, Coronaviridae, Filoviridae, Flaviviridae, Geminiviridae, Hantaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Kitaviridae, Luteoviridae, Nairoviridae, Nanoviridae, Nimaviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Picornaviridae, Polyomaviridae, Pospiviridae, Potyviridae, Poxviridae, Reoviridae, Retroviridae, Retrovirus, Rhabdoviridae, Secoviridae, Togaviridae, Tombusviridae, Tospoviridae, Tymoviridae, and Virgaviridae. For example, exemplary viruses include, but are not limited to, African swine fever, Avian hepatitis E, Avian infectious laryngotracheitis, Avian nephritis virus, Bamboo mosaic virus, Banana bunchy top virus, Barley stripe mosaic virus, Barley yellow dwarf virus, Potato leafroll virus, Boma disease, Brome mosaic virus, wheat, Cauliflower mosaic virus, Chikungunya, Eastern equine encephalitis virus, Citrus leprosis, Citrus sudden death associated virus, Citrus tristeza virus, Coconut cadang-cadang viroid, Curly top virus, African cassava mosaic virus, Cytomegalovirus, Epstein-Barr virus, Dengue, Yellow fever, West Nile, Zika, Ebola virus, Marburg virus, Equine arteritis virus, Porcine reproductive and respiratory syndrome virus, Equine infectious anemia, Foot and mouth disease, Foot and mouth disease, Enteroviruses, Rhinoviruses, Hepatitis B virus,

Hepatitis E virus, HIV, HIV-1, HIV-2, Infectious bursal disease virus (poultry),

Infectious pancreatic necrosis (salmon), Infectious canine hepatitis, aviadenoviruses of fowl, Influenza viruses, Lassa virus, Lymphocytic choriomeningitis virus, Monkeypox, Nairobi sheep disease, Newcastle disease virus (poultry), Norwalk virus, Numerous examples of crop damaging viruses, including Potato virus Y, Porcine circovirus 2, Beak and feather disease virus (poultry), Potato virus M, Rabies virus, Respiratory and enteric adenoviruses, Respiratory syncytial virus, Rice stripe necrosis virus, Rift Valley fever, rotaviruses, SARS, SARS-CoV-2, MERS, Sheeppox virus, Lumpy skin disease virus, Sin Nombre virus, Andes virus, SV40, Tobacco ringspot virus, Tomato bushy stunt virus, Tomato spotted wilt virus, Torque teno virus, Venezuelan equine encephalitis virus, Vesicular stomatitis Indiana virus, Viral hemorrhagic septicemia (trout), and White spot syndrome virus (shrimp), Primate T-lymphotropic virus 1, Primate T-lymphotropic virus 2, Primate T-lymphotropic virus 3, Human immunodeficiency virus 1, Human Attorney Docket No. 046483-6221-00WO immunodeficiency virus 2, Simian foamy virus, Human picobirnavirus, Colorado tick fever virus, Changuinola virus, Great Island virus, Lebombo virus, Orungo virus, Rotavirus A, Rotavirus B, Rotavirus C, Banna virus, Borna disease virus, Lake Victoria Marburgvirus, Reston ebolavirus, Sudan ebolavirus, Tai forest ebolavirus, Zaire virus, Human parainfluenza virus 2, Human parainfluenza virus 4, Mumps virus, Newcastle disease virus, Human parainfluenza virus 1, Human parainfluenza virus 3, Hendra virus, Nipah virus, Measles virus, Human respiratory syncytial virus, Human metapneumovirus, Chandipura virus, Isfahan virus, Piry virus, Vesicular stomatitis Alagoas virus, Vesicular stomatitis Indiana virus, Vesicular stomatitis New Jersey virus, Australian bat lyssavirus, Duvenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Mokola virus, Rabies virus, Guanarito virus, Junin virus, Lassa virus, Lymphocytic choriomeningitis virus, Machupo virus, Pichinde virus, Sabia virus, Whitewater Arroyo virus,

Bunyamwera virus, Bwamba virus, California encephalitis virus, Caraparu virus, Catu virus, Guama virus, Guaroa virus, Kairi virus, Marituba virus, Oriboca virus, Oropouche virus, Shuni virus, Tacaiuma virus, Wyeomyia virus, Andes virus, Bayou virus, Black creek canal virus, Dobrava-Belgrade virus, Hantaan virus, Laguna Negra virus, New York virus, Puumala virus, Seoul virus, Sin Nombre virus, Crimean-Congo haemorrhagic fever virus, Dugbe virus, Candiru virus, Punta Toro virus, Rift Valley fever virus,

Sandfly fever Naples virus, Influenza A virus, Influenza B virus, Influenza C virus, Dhori virus, Thogoto virus, Hepatitis delta virus, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1, Human coronavirus OC43, SARS coronavirus,

Human torovirus, Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D, Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Encephalomyocarditis virus, Theilovirus, Equine rhinitis A virus, Foot and mouth disease virus, Hepatitis A virus, Human parechovirus, Ljungan virus, Aichi virus, Human astrovirus, Human astrovirus 2, Human astrovirus 3, Human astrovirus 4, Human astrovirus 5, Human astrovirus 6, Human astrovirus 7, Human astrovirus 8, Norwalk virus, Sapporo virus, Aroa virus, Banzi virus, Dengue virus, Ilheus virus, Japanese encephalitis virus, Kokobera virus, Kyasanur forest disease virus, Louping ill virus, Murray Valley encephalitis virus, Ntaya virus, Omsk haemorrhagic fever virus, Powassan virus, Rio Bravo virus, St Louis encephalitis virus, Tick-borne encephalitis virus, Usutu Attorney Docket No. 046483-6221-00WO virus, Wesselsbron virus, West Nile virus, Yellow fever virus, Zika virus, Hepatitis C virus, Hepatitis E virus, Barmah Forest virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Getah virus, Mayaro virus, Mucambo virus, O'nyong-nyong virus, Pixuna virus, Ross River virus, Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, Whataroa virus, and Rubella virus.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecules encoding one or more Influenza virus antigen, and thus the immunogenic composition of the invention provides protection against one or more strain of Influenza virus, or a disease or disorder associated therewith. In one embodiment, the disease or disorder associated with Influenza virus is influenza.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecules encoding one or more HIV antigen, and thus the immunogenic composition of the invention provides protection against one or more strain of HIV, or a disease or disorder associated therewith. In one embodiment, the disease or disorder associated with HIV is AIDS.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecules encoding one or more SARS-CoV-2 antigen, and thus the immunogenic composition of the invention provides protection against one or more strain of SARS-CoV-2, or a disease or disorder associated therewith. In one embodiment, the disease or disorder associated with SARS-CoV-2 is COVID-19.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecules encoding one or more HPV antigen, and thus the immunogenic composition of the invention provides protection against one or more strain of HPV, or a disease or disorder associated therewith. In one embodiment, the disease or disorder associated with HPV is cervical cancer, anal cancer, oral cancer, oropharyngeal cancer, or other cancers.

In one embodiment, the multi-genic immunogenic composition (e.g., vaccine) of the invention comprises one or more mRNA molecules encoding one or more Attorney Docket No. 046483-6221-00WO

HTLV-1 antigen, and thus the immunogenic composition of the invention provides protection against one or more strain of HTLV-1, or a disease or disorder associated therewith. In one embodiment, the disease or disorder associated with HTLV-1 is adult T-cell leukaemia/lymphoma (ATL).

In one embodiment, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for more than 2 weeks. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 1 month or more. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 2 months or more. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 3 months or more. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 4 months or more. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 5 months or more. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 6 months or more. In some embodiments, the method provides sustained protection against at least one disease or disorder associated with an infection by a pathogenic microorganism for 1 year or more.

In one embodiment, a single immunization of the composition induces a sustained protection against a disease or disorder associated with an infection by a pathogenic microorganism for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, or 1 year or more.

Administration of the compositions of the invention in a method of treatment can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration. In some embodiments, the method comprises intradermal delivery of the composition. In another Attorney Docket No. 046483-6221-00WO embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions comprising a multi-genic mRNA immunogenic composition as described herein to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 1 ng/kg/day and 100 mg/kg/day. In one embodiment, the invention envisions administration of a dose, which results in a concentration of the compound of the present invention from 10 nM and 10 mM in a mammal.

Typically, dosages which may be administered in a method of the invention to a mammal, such as a human, range in amount from 0.01 pg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In some embodiments, the dosage of the compound will vary from about 0.1 pg to about 10 mg per kilogram of body weight of the mammal. In some embodiments, the dosage will vary from about 1 pg to about 1 mg per kilogram of body weight of the mammal.

The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months, several years, or even less frequently, such as every 10-20 years, 15-30 years, or even less frequently, such as every 50-100 years. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc. Attorney Docket No. 046483-6221-00WO

In some embodiments, administration of an immunogenic composition or immunogenic composition of the present invention may be performed by single administration or boosted by multiple administrations.

In one embodiment, the invention includes a method comprising administering one or more compositions encoding multiple antigens or adjuvants described herein. In some embodiments, the method has an additive effect, wherein the overall effect of the administering the combination is approximately equal to the sum of the effects of administering each pathogen antigen or adjuvant. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each pathogen antigen or adjuvant.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

Example 1: Multi-genic mRNA Vaccines

Here the inventors propose a vaccination strategy based on co-transfecting multiple RNAs encoding multiple antigens, or alternatively a combination of both antigens and immune-enhancing factors. Without being bound by theory, the inventors propose that the combination of multiple distinct antigens from different genes of the target pathogen will make a more effective vaccine. Co-transfection of multiple RNAs, Attorney Docket No. 046483-6221-00WO each encoding a different antigen, produces a greater antigenic challenge to the vaccinated host, and consequently elicits a stronger immune response. A stronger immunological response is also elicited using different antigens from the same protein, where some regions might be more antigenic than others. Combining multiple RNA- encoded antigens in a single vaccine has the additional advantage of training the immune system to recognize multiple molecular targets on a potential pathogen, decreasing the possibility that mutant pathogen strains can escape immune recognition easily. Furthermore, administering multiple RNA-encoded antigens comprising different mutated components of a single viral protein facilitates greater protection against multiple mutated viral strains in a single vaccine dose.

An additional possible advantage of co-transfecting multiple RNAs simultaneously is the ability to combine antigen-encoding RNAs with immune-enhancing RNAs. One example involves co-transfection antigen encoding RNAs with RNA encoding one of the B7 proteins (CD80 or CD86). Expression of B7 proteins elicits greater immune responses by enhancing recruitment and activation of dendritic cells, macrophages, activated B-cells, and activated T cells. These additional immune stimulatory factors enhance host responses to vaccination by eliciting a more comprehensive immune response.

Finally, in one embodiment, the invention provides methods for co transfection of multiple antigens from the same pathogen target, which promotes the discovery of suitable antigen targets for vaccine development. Oftentimes the most optimal antigen for vaccine development is not known in advance. Co-transfection of multiple potential RNAs encoding different antigens simultaneously in a “shot-gun” approach facilitates a more rapid discrimination between good antigen targets for further development and those that do not elicit significant immune responses.

Example 2: Multi-genic mRNA Vaccine Compositions and Methods of Use

In an effort to improve upon the current SARS-CoV-2 (COVID-19) vaccine, the experiments described herein sought to co-express multiple COVID-19 antigens as well as T-cell activating proteins. Experimentally multiple COVID-19 genes as well as CD80 and/or CD86 have been able to be co-expressed. In Figure 1, mRNAs Attorney Docket No. 046483-6221-00WO encoding SARS-CoV2 spike protein and CD86 were phototransfected into mouse 3T3 cells and 3 days later fixed and immunostained. The immunocytochemical detection was performed using detection wavelengths consistent with the labeled secondary antibodies directed against each of the primary antibodies. eGFP was visualized from its’ endogenous fluorescence. The top 4 panels are two neighboring cells that have been transfected while the bottom 4 are a different set of cells. The nucleus is generally absent staining in these studies while there are clear overlaps in, as well as distinct localizations of each of the translated proteins.

Next the potential for co-expression of 4 antigens from simultaneous mRNA co-transfection was explored using COVID-19s’ nucleocapsid and spike proteins, eGFP and CD80. CD86 was not tested in this paradigm as the primary antibody was made in rabbit just as the nucleocapsid protein antibody was, precluding their simultaneous detection. Figure 2 shows the pseudo-colored cell image of translated protein in cells that were co-transfected with these four mRNAs. These cells were processed for immunocytochemistry 24 hrs post-transfection. The merged image shows an overlap of protein localization as well as some unique protein localizations. These data highlight how ex vivo mRNA when introduced into a cell gives rise to translated proteins that can be differentially localized within the transfected cell. The eGFP signal is lower than the nucleocapsid and spike proteins as less eGFP RNA was introduced into the cells. The CD80 signal is lower as well due to lower amounts of CD80 protein being translated or higher levels of CD80 protein degradation.

Figure 3 shows a detailed quantitative analysis of the fluorescent signals from the multiple translated antigens in the merged cell image (white box) shown in Figure 2. The height of the fluorescent peaks for each of the proteins shows differing amounts of assayed protein in subregions of this individual cell. An example of the specificity of expression is that in these cells under the conditions of this experiment, the spike protein is observed in the nucleus (red color in right hand image as well as the high quantitated peaks of fluorescence quantified in the CoV-2 Spike graph) of the cell as well as cytoplasm. There is also more nucleocapsid, eGFP and CD80 protein towards the near region of the cell (white arrow) than there is spike protein. Such localization differences may impact antigen presentation and the strength of subsequent immune responses. Attorney Docket No. 046483-6221-00WO

Experiments to examine the mRNA dose dependency upon the amount of detectable protein were also performed. This was assessed by transfection two different RNAs in differing amounts into the same cells followed by quantitative analysis of the fluorescent intensities of the resultant translated proteins. The y-axes of the bar graphs in Figure 4 show the absolute amounts of fluorescence for COVID-19 nucleocapsid protein (left graph) as well as eGFP (right graph). The concentrations of mRNA added into the cultures are on the x-axis. In graphing the principal components of the ratio of nucleocapsid fluorescence vs eGFP fluorescence in the same cell when using twice as much nucleocapsid mRNA relative to eGFP RNA there is a dose dependent increase in nucleocapsid/eGFP ratio (red dots). This is absent when both mRNAs are present in similar amounts (blue dots).

Immunocytochemistry Process:

Media was removed from the cultured cells followed by washing with Dulbecco’s phosphate buffered saline (DPBS) and fixed with 4% paraformaldehyde (PFA) in DPBS buffer at room temperature for 15 minutes followed by three washes in DPBS. The residual PFA was quenched by incubation the cells in 0.2M glycine in DPBS for 5 minutes. Cells were then washed with DBPS for three times and permeablized with 0.1% Triton X-100 in DPBS for 5 minutes. After three DPBS washes, cells were blocked with 5% BSA in PBST (0.1% Tween20 in DPBS) for 1 hour at room temperature. Cells were then washed with PBST for 3 times, each time for 5 minutes. Primary antibodies were diluted in 3% BSA in PBST, and incubated with cells at 4 degree overnight. After five washes with PBST, each time for 5 minutes, cells were then incubated with the diluted secondary antibodies in 3% BSA in PBST at 37 degree for 1 hour. The cells were washed with PBST for five times, each time for 5 minutes, followed by one wash in distilled water. After totally dried in the room temperature, cells were mounted with Fluoromount-G (catalog no. 0100-01, SouthemBiotech). The outcomes were evaluated by confocal microscopy.

Antibodies:

Primary antibodies used for the immunofluorescence analysis include Attorney Docket No. 046483-6221-00WO mouse anit-SARS-CoV-2 (COVID-19) spike antibody [1A9] (catalog no. GTX632604, GeneTex; 1:1000 dilution), rabbit anti-SARS-CoV-2 nucleocapsid protein antibody (catalog no. ab273167, abeam; 1:2500 dilution), rabbit anti-CD86 antibody [EPR21962] (catalog no. ab239075, abeam; 1:3000 dilution), and goat anti-CD80 polyclonal antibody (catalog no. PA5-19211, ThermoFisher; 1:1000 dilution). Secondary antibodies used include Alexa Fluor 405 Donkey anti-Goat IgG (catalog no. A48259, Invitrogen; 1:500 dilution), Alexa Fluor 546 Goat anti-mouse IgG (catalog no. A11030, Invitrogen; 1 : 1000 dilution), and Alexa Fluor 633 Goat anti -Rabbit IgG (catalog no. A21071, Invitrogen; 1:1000 dilution).

Example 3 : Example sequences for mRNA immunogenic composition constructs

Influenza sequences for mRNA immunogenic composition constructs:

SEQ ID NO: 1 - HA from A/Puerto Rico/8/1934 (H1N1 mouse-adapted)

SEQ ID NO:2 - HA from HlNlpdm09-like A/Michigan/45/2015(HlNl)

SEQ ID NO:3 - NA from HlNlpdm09-like A/Michigan/45/2015(HlNl) >H1N1_NA SEQ ID NO:4 - NA from A/Singapore/INFIMH- 16-0019/2016(H3N2) >H3N2_NA SEQ ID NO : 5 - HIV GAG Protein

SEQ ID NO:6 - HIV Env Protein (go 160}

SEQ ID NO: 7 - Spike Protein SEQ ID NO: 8 - E Protein SEQ ID NO: 9 - Membrane Glycoprotein SEQ ID NO: 10 - nucleocapsid SEQ ID NO: 11 - ORFlab SEQ ID NO: 12 - ORF3a SEQ ID NO: 13 ORF6 SEQ ID NO: 14 - QRF7a SEQ ID NO: 15 - ORE 7b SEQ ID NO: 16 - ORE 8 SEQ ID NO : 17 - ORF 10 SEQ ID NO: 18: - CD80 Attorney Docket No. 046483-6221-00WO SEQ ID NO: 19: - CD86

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A composition for inducing an immune response against one or more pathogenic microorganisms in a subject, the composition comprising at least one mRNA molecule encoding a combination of two or more pathogen antigens or fragments thereof.

2. The composition of claim 1, wherein the two or more pathogen antigens are antigens from different viral strains of the same virus.

3. The composition of claim 1, wherein the two or more pathogen antigens are antigens from different viruses.

4. The composition of claim 1, wherein at least one pathogen is selected from the group consisting of influenza virus, HIV and a beta coronavirus.

5. The composition of claim 4, wherein the at least one influenza virus antigen is selected from the group consisting of a hemagglutinin (HA) antigen or a fragment thereof, neuraminidase (NA) antigen or a fragment thereof, NP antigen or a fragment thereof, and M2 ion channel antigen or a fragment thereof.

6. The composition of claim 5, wherein the at least one influenza virus antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and any combination thereof.

7. The composition of claim 4, wherein the at least one HIV antigen is selected from the group consisting of an Env antigen or a fragment thereof, a Nef antigen or a fragment thereof, a Pol antigen or a fragment thereof, a Gag antigen or a fragment thereof, a Tat antigen or a fragment thereof, a Rev antigen or a fragment Attorney Docket No. 046483-6221-00WO thereof, a Vif antigen or a fragment thereof, a Vpr antigen or a fragment thereof, and a Vpu antigen or a fragment thereof.

8. The composition of claim 7, wherein the at least one HIV antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 6, and any combination thereof.

9. The composition of claim 4, wherein the at least one coronavirus antigen is selected from the group consisting of a spike antigen or a fragment thereof, an envelope protein or a fragment thereof, a nucleocapsid protein or a fragment thereof, a membrane glycoprotein or a fragment thereof, an ORFlab polyprotein or a fragment thereof, an ORF3a protein or a fragment thereof, an ORF6 protein or a fragment thereof, an ORF7a protein or a fragment thereof, an ORF8 protein or a fragment thereof, and an ORFIO protein or a fragment thereof.

10. The composition of claim 8, wherein the at least one coronavirus antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, and any combination thereof.

11. The composition of any one of claims 1-10, wherein the composition further comprises an adjuvant or at least one mRNA molecule encoding an adjuvant.

12. The composition of claim 11 wherein the adjuvant comprises a T cell stimulatory molecule.

13. The composition of claim 12, wherein the T cell stimulatory molecule is selected from the group consisting of CD80 and CD86. Attorney Docket No. 046483-6221-00WO

14. The composition of claim 13, wherein the composition comprises an mRNA molecule encoding at least one selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19.

15. The composition of claim 8, wherein the composition comprises a combination of at least 2 mRNA molecules encoding a combination of a SARS-CoV-2 nucleocapsid protein and a SARS-CoV-2 spike antigen.

16. The composition of claim 15, further comprising at least one additional mRNA molecule encoding a T cell stimulatory molecule selected from the group consisting of CD80 and CD86.

17. The composition of any one of claims 1-16, wherein the one or more mRNA molecule is encapsulated in one or more lipid nanoparticle (LNP).

18. The composition of any one of claims 1-17, wherein the composition is a vaccine.

19. A method of inducing an immune response against at least one virus in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-18.

20. The method of claim 19, wherein the method comprises inducing an immune response against two or more different viruses or two or more strains of a virus.

21. The method of claim 19, wherein at least two different viruses are selected from the group consisting of influenza virus, HIV and a beta coronavirus. Attorney Docket No. 046483-6221-00WO

22. The method of claim 19, wherein the composition is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

23. The method of claim 19, wherein the method comprises a single administration of the composition.

24. The method of claim 19, wherein the method comprises multiple administrations of the composition.

25. A method of treating or preventing a disease or disorder associated with at least one virus in a subject comprising administering to the subject an effective amount of a composition of any one of claims 1-18.

26. The method of claim 25, wherein the method comprises treating or preventing a disease or disorder associated with at least one virus.

27. The method of claim 26, wherein at least two different viruses are selected from the group consisting of influenza virus, HIV and a beta coronavirus.

28. The method of claim 25, wherein the composition is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

29. The method of claim 25, wherein the method comprises a single administration of the composition.

30. The method of claim 25, wherein the method comprises multiple administrations of the composition.