Split formulated trans-amplifying RNA FLU vaccine

Abstract

This disclosure relates to split-formulated influenza vaccines and methods of immunizing a subject against influenza using the same.

Description

[0001] SPLIT FORMULATED TRANS -AMPLIFYING RNA FLU VACCINE

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional Application No. 63 / 680,540, filed August 7, 2024, entitled “SPLIT FORMULATED TRANSAMPLIFYING RNA FLU VACCINE”, the content of which is hereby incorporated by reference herein in its entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (A141470008WG00-SEQ-KGC.xml; Size: 348,400 bytes; and Date of Creation: August 6, 2025 are herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Vaccination can protect vaccinated subjects from disease and slow the spread of the disease. RNA vaccines have advanced vaccine technology, in part by increasing vaccine production rate. Typically, RNA vaccines comprise mRNAs encoding an antigen from a pathogen (e.g., a virus). When the mRNA is introduced into a host, host cells translate the mRNA to produce the antigen, which triggers an immune response in the host, and in turn provides protection against the pathogen.

[0008] SUMMARY

[0009] Trans-amplifying RNA (taRNAs), an RNA technology based on alphaviral gene replication, may also be useful as a potential RNA vaccine platform. Unlike mRNAs, taRNAs comprise two separate components: a first RNA encoding a replicase (i.e., a replicase construct) and a second RNA encoding a pay load (i.e., a trans-replicating RNA (trRNA)). When introduced into a host cell, the replicase construct is translated to produce the replicase, which in turn amplifies (e.g., replicates) the trRNA. The trRNAs (e.g., trRNA replicates) can be translated by the host cell. Thus, host cell expression of the payload encoded by taRNAs is based on both the initial amount of trRNA introduced to the host cell and subsequent amplification of the trRNA by the replicase construct. taRNAs, as used in RNA vaccines, may provide higher expression of an antigen in a host cell than mRNAs because the replicase continuously replicates the trRNA over a period of time. This disclosure relates to the surprising discovery that a trRNA (comprising nucleic acids encoding an influenza virus antigen) and a corresponding replicase construct can be formulated in separate nanoparticles (i.e., split-formulated) and concomitantly administered to produce a desired effect in the subject, for example, an immune response (e.g., antibody production against a vaccine antigen). This disclosure further shows that altering the 5' UTR of trRNA of a split- formulated influenza vaccine (e.g., via an oeSTR modification) results in an immune response in a subject that meets or surpasses an immune response induced by an mRNA flu vaccine. This is true even when 100-fold less trRNA than mRNA is administered to the subject.

[0010] Together, these findings indicate that production of split-formulated trRNA influenza vaccine formulations requires far less manufacture of trRNA compared to replicase construct to achieve the same or better immune response compared to mRNA. For example, preparation of split-formulated taRNA influenza vaccine doses for 35,000 people may require production of only about 42mg of trRNA, compared to 4148mg of the replicase construct. Based on these findings, the inventors have identified methods for improving influenza RNA vaccine manufacture and distribution. In some aspects, generic replicase constructs are manufactured and stockpiled (e.g., stored long term) in nanoparticles, while flu antigen-encoding trRNA are designed and manufactured on an as-needed basis (e.g., in response to a pandemic, evolving variants of a virus, clinical trial data) and formulated in separate nanoparticles. These separate nanoparticles can then be concomitantly administered to subjects. Importantly, only relatively small amounts of trRNA (comprising nucleic acids encoding an antigen) would need to be synthesized to produce amounts required for population- scale vaccinations. These methods thus greatly enhance the rate at which an RNA vaccine can be produced, allowing for rapid turnover and mass vaccination, as well as significant reductions in manufacturing risk as the scale of material to be manufactured is lower. Moreover, these methods provide a strategy for rapid formulation and testing of multiple different candidate influenza antigens to identify those with improved immunogenic properties.

[0011] In some embodiments, this disclosure provides a trans-replicating ribonucleic acid (trRNA) nanoparticle (Tr-NP), comprising a first trRNA, wherein the first trRNA is an RNA polynucleotide comprising: (i) a nucleic acid encoding a first influenza virus antigen, operably linked to a conserved sequence element (CSE); and wherein the Tr-NP does not comprise a polynucleotide that comprises a nucleic acid encoding a replicase cognate to the CSE.

[0012] In some embodiments, the first trRNA further comprises: (ii) a nucleic acid encoding an immune modulating protein (IMP). In some embodiments, the IMP is an influenza virus nonstructural protein 1 (NS1), a vaccinia virus (VACV) RNA-binding protein 30 E3 (E3L) protein, or a Toscana virus (TOSV) NSs. In some embodiments, the first trRNA comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii). In some embodiments, the linker is an IRES element or a 2A peptide. In some embodiments, the IRES element is an IRES from CVB3, EMCV, PKV, MPV, or TraV.

[0013] In some embodiments, the influenza virus antigen comprises a fragment of an influenza virus Hemagglutinin (HA) protein and / or a fragment of an influenza virus Neuraminidase (NA) protein. In some embodiments, the influenza virus antigen comprises a fragment of a first influenza virus HA protein and a fragment of a second influenza virus HA protein, wherein the first influenza virus HA protein and the second influenza virus HA protein are from different influenza viruses. In some embodiments, the Tr-NP further comprises a linker flanked by the fragment of the first influenza virus HA protein and the fragment of the second influenza virus HA protein. In some embodiments, the influenza virus antigen comprises a fragment of an influenza virus HA protein and a fragment of an influenza virus NA protein. In some embodiments, the fragment of the influenza virus HA protein and the fragment of the influenza virus NA protein are from the same influenza virus.

[0014] In some embodiments, the first trRNA comprises a 5' untranslated region (UTR) and a 3' UTR flanking the nucleic acid of (i), and the nucleic acid of (ii) if present. In some embodiments, the 5' UTR comprises the CSE. In some embodiments, the 5' UTR is a SINV 5' UTR or an SFV 5' UTR. In some embodiments, the 3' UTR is a SINV 3' UTR or an SFV 3' UTR. In some embodiments, the trNP further comprises a second trRNA, wherein the second trRNA is an RNA polynucleotide comprising: (i) a nucleic acid encoding a second influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen and the second influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the second trRNA are cognate to a same replicase.

[0015] In some embodiments, the Tr-NP further comprises a third trRNA, wherein the third trRNA is an RNA polynucleotide comprising: (i) a nucleic acid encoding a third influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen, the second influenza virus antigen, and the third influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the third trRNA are cognate to a same replicase.

[0016] In some embodiments, the Tr-NP further comprises a fourth trRNA, wherein the fourth trRNA is an RNA polynucleotide comprising: (i) a nucleic acid encoding a fourth influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, and the fourth influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the fourth trRNA are cognate to a same replicase.

[0017] In some embodiments, the Tr-NP further comprises a fifth trRNA, wherein the fifth trRNA is an RNA polynucleotide comprising: (i) a nucleic acid encoding a fifth influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, the fourth influenza virus antigen, and the fifth influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the fifth trRNA are cognate to a same replicase

[0018] In some embodiments, one or more of the second, third, or fourth trRNA further comprise: (ii) a nucleic acid encoding an immune modulating protein (IMP). In some embodiments, the second, third, and / or fourth trRNA comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii). In some embodiments, at least one influenza virus antigen is an influenza virus B antigen. In some embodiments, at least one influenza virus antigen is an influenza virus A antigen. In some embodiments, this disclosure provides a composition, comprising the Tr-NP, and a replicase-nanoparticle (R-NP), wherein the R-NP comprises a replicase construct, wherein the replicase construct is an RNA polynucleotide comprising: (i) a nucleic acid encoding a replicase cognate to the CSE of the first trRNA; wherein the R-NP does not comprise a CSE that is cognate to the replicase.

[0019] In some embodiments, the RNA polynucleotide of the R-NP further comprises: (ii) a nucleic acid encoding an immune modulating protein (IMP). In some embodiments, the RNA polynucleotide of the R-NP comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii). In some embodiments, the linker is an IRES element, a % junction amino acid sequence, a sub-genomic promoter, or a nucleic acid encoding a 2A peptide. In some embodiments, the replicase is an alphavirus replicase. In some embodiments, the replicase is a Semliki Forest virus (SFV) replicase or a variant thereof. In some embodiments, the Tr-NP and the R-NP are present at a 1:1, 5:1, 10:1, 25:1, 50:1, or 100:1 molar ratio. In some embodiments, the Tr-NP is a lipid nanoparticle (Tr-LNP). In some embodiments, the Tr-NP comprises ALC- 0315, DSPC, cholesterol and PEG-lipid. In some embodiments, the R-NP is a lipid nanoparticle (R-LNP). In some embodiments, the R-NP comprises ALC-0315, DSPC, cholesterol and PEG- lipid. In some embodiments, the combined amount of replicase construct and total trRNA is less than 2 pg. In some embodiments, the composition is a vaccine. In some embodiments, this disclosure provides a composition comprising: a transreplicating RNA (trRNA) trRNA nanoparticle (Tr-NP), comprising a trRNA, wherein the trRNA is an RNA polynucleotide comprising a nucleic acid encoding an influenza virus antigen, operably linked to a conserved sequence element (CSE); and a replicase nanoparticle (R-NP) comprising a replicase construct, wherein the replicase construct is an RNA polynucleotide comprising a nucleic acid encoding a replicase cognate to the CSE of the trRNA; wherein the Tr- NP does not comprise a polynucleotide that comprises a nucleic acid encoding a replicase cognate to the CSE; wherein the R-NP does not comprise a CSE that is cognate to the replicase; and wherein the R-NP and Tr-NP are separate nanoparticles. In some embodiments, this disclosure provides a method of vaccinating a subject, the method comprising administering to the subject the composition.

[0020] In some embodiments, this disclosure provides a kit, comprising: a first vial comprising the Tr-NP; and a second vial comprising a replicase-nanoparticle (R-NP), wherein the R-NP comprises an RNA polynucleotide comprising a nucleic acid encoding a replicase cognate to the CSE of the first trRNA of the Tr-NP, and wherein the R-NP does not comprise a CSE that is cognate to the replicase.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows an example taRNA vaccine comprising greater than 99% LNPs comprising a replicase construct (and not a trRNA) and less than 1% LNPs comprising a tRNA encoding a vaccine antigen (and not a replicase construct).

[0023] FIGs. 2A-2B show immune response in mice after administration of an mRNA influenza vaccine or a taRNA influenza vaccine. FIG. 2A shows antibody production in response to mRNA influenza vaccine or a taRNA influenza vaccine administration. FIG. 2B shows stable mouse weight after administration of the mRNA influenza vaccine or a taRNA influenza vaccine.

[0024] FIG. 3 shows that a taRNA influenza vaccine comprising an oeSTR 5' UTR modification can induce the same level of antibody response as a mRNA vaccine in a mouse even though 100 times less antigen encoding RNA was administered to the mouse. FIG. 3 also shows a similar result with a taRNA encoding a NS1 immunomodulatory protein.

[0025] DETAILED DESCRIPTION

[0026] Described herein are split-formulated taRNA vaccines that are useful for decreasing the likelihood of infection by one or more influenza viruses. In some embodiments, a split- formulated taRNA vaccine comprises a trans replicating RNA (trRNA) nanoparticle (Tr-NP) comprising a trRNA, wherein the trRNA is an RNA polynucleotide comprising nucleic acids encoding one or more influenza virus antigens and a conserved sequence element (CSE), wherein the Tr-NP does not comprise a polynucleotide (e.g., RNA polynucleotide) comprising a nucleic acid encoding a replicase cognate to the CSE; and a replicase nanoparticle (R-NP) comprising a replicase construct, wherein the replicase construct is an RNA polynucleotide comprising nucleic acids encoding a replicase cognate to the CSE, wherein the R-NP does not comprise a polynucleotide (e.g., RNA polynucleotide) comprising a polynucleotide (e.g., a polynucleotide encoding an antigen) operably linked to a conserved sequence element (CSE) that is cognate to the replicase.

[0027] Influenza Virus

[0028] Influenza viruses are enveloped, negative- sense, single-stranded RNA viruses of the Orthomyxoviridae family, and are associated with mild to severe respiratory illness. Infections by an influenza virus (e.g., influenza A virus, influenza B virus) may result in the development of one or more symptoms associated with respiratory illness, including, but not limited to, difficulty breathing (e.g., dyspnea, tachypnea, hypopnea, hyperpnea, apnea), cough (e.g., productive cough, non-productive cough), shortness of breath, wheezing, stridor, sneezing, nasal congestion, fever, fatigue, malaise, muscle pain, nausea, vomiting, loss of taste and smell, and dizziness. Subjects infected by an influenza virus may develop mild respiratory illness, such that the subject exhibits symptoms which are not life-threatening (e.g., the subject exhibits “coldlike” symptoms, but does not have difficulty breathing) and which do not require medical intervention (e.g., do not require supplemental oxygen). Some subjects infected by influenza virus may be asymptomatic, such that the infected subject does not exhibit symptoms associated with respiratory disease. Asymptomatic infection and mild respiratory illness are typically selflimiting (e.g., resolves without requiring medical intervention) in healthy adults, but may progress into severe respiratory illness. Subjects infected by an influenza virus may develop severe respiratory illness, such that the subject exhibits symptoms which are life-threatening (e.g., require medical intervention to resolve) and / or fatal. Examples of severe respiratory illness associated with infection by influenza virus include, but are not limited to, pneumonia and bronchiolitis. Certain populations, such as children, elder adults, and immunocompromised adults, are at higher risk of developing severe respiratory illness. Furthermore, though vaccines for influenza viruses are highly desirable, global circulation and antigenic evolution pose a major challenge for the development of respiratory virus vaccines. Influenza viruses exhibit a spatiotemporal pattern of emergence and circulation. Influenza viruses may circulate in a seasonal pattern, such that activity of the respiratory virus peaks annually in a given geographic region (e.g., northern hemisphere) during a certain period (e.g., between September and December). Further, without being bound by theory, higher rates of viral circulation and / or geographic migration are believed to increase antigenic evolution, severity of infection, and spread of epidemics (Bedford, T., et al. Nature. 2015:523(7559):217- 220.).

[0029] Influenza viruses are categorized into four types- A, B, C, and D- on the basis of surface proteins and host species. Of these, influenza A virus (IAV) and influenza B virus (IBV) viruses contribute significantly to global respiratory disease burden.

[0030] Both IAV and IBV follow a similar nomenclature which unambiguously refers to a specific isolate. This nomenclature includes reference to the virus type (e.g., A or B), species (if non-human), region of isolation, strain / laboratory reference number, and year of isolation. For example, an IAV isolated in Wyoming in 2003, having the strain number “3” would be referred to as A / Wyoming / 3 / 2003. A subtype of A / Wyoming / 03 / 2003 having a type-3 hemagglutinin and type-2 neuraminidase in its viral envelope is referred to as “A / Wyoming / 03 / 2003 (H3N2)”. Similarly, an IBV isolated in Phuket in 2013, having the strain number “3073” would be referred to as B / Phuket / 3073 / 2013.

[0031] IAV and IBV both typically infiltrate host cells using similar mechanisms. Two influenza virus surface proteins, hemagglutinin (H, also referred to synonymously herein as “HA”) and neuraminidase (N, also referred to synonymously herein as “NA”) are critical for host cell infection and release of nascent virus, respectively. HA functions by binding to sialic acid-containing receptors in host cell membranes, causing host cells to endocytose the virion. During host cell-mediated digestion of the virion, changes in pH induce conformational changes in HA, which cause the viral envelope and host cell endosome to fuse, thereby releasing viral contents into the cell. NA is an enzyme capable of cleaving sialic acid groups from glycoproteins. Typically, NA participates in release of nascent virus from host cells via interactions with sialic acid residues on the host cell membranes. Some influenza viruses having certain types of NA (e.g., N1 with a G147R mutation) can also use NA to initiate entry into a host cell. Typically, neutralizing antibodies against influenza virus target HA, and current influenza vaccines are typically designed to induce an immune response against HA. IAVS can be further refined by the composition of HA and NA in the viral envelope. At the time of this filing, at least eighteen HA subtypes (e.g., HA type-1 (Hl), HA type-2 (H2), etc.) and eleven NA subtypes (e.g., NA type-1 (Nl), NA type-2 (N2), etc.) are known, differing primarily in their antigenicity. Of these, Hl, H2, H3, Nl, and N2 are commonly associated with lAV-mediated respiratory disease in humans. In addition to the names of specific isolates, IAVs are named according to a standard shorthand nomenclature indicating each HA and NA type (e.g., H1N1 refers to an IAV having HA type-1 and NA type-1). IAVs are capable of infecting several species, including various mammalian species and avian species; this diversity in host species is thought to result in vast genetic diversity and the rapid rate at which IAVs obtain mutations and reassortments. The frequent evolution of IAVs contributes significantly to difficulties in the design and preparation of traditional (e.g., protein-based) influenza virus vaccines.

[0032] Unlike IAVs, IBVs mutate at a slower rate compared to IAV and exhibit less variability in HA and NA, possibly due to its limited range of host species, which include certain mammals, such as humans and pigs. IBVs are not categorized into subtypes, but comprise two antigenically distinct lineages which currently circulate in humans: B / Victoria (e.g., B / Victoria-like) and B / Yamagata (e.g., B / Yamagata-like). In addition to the names of specific isolates, a given IBV may be further referred to by its parental lineage- for example, B / Phuket / 3073 / 2013 (B / Yamagata lineage).

[0033] Various circulating influenza virus subtypes are provided by several databases, e.g., on the worldwide web at gisaid.org / influenza-subtypes.

[0034] Accordingly, it is desirable that antigens encoded or otherwise provided by vaccines for influenza viruses having patterns of seasonal activity be selected according to the antigens which are in circulation or predicted to be circulating or prevalent in a forthcoming virus season. RNA- based vaccines can be quickly designed and produced with more rapid scaling than traditional (e.g., protein-based vaccines). Thus, RNA-based vaccines (e.g., taRNA vaccines) can be quickly adapted to encode antigens of influenza viruses found to be in circulation in a geographic region in a given season, and are a promising solution to the challenges influenza viruses pose.

[0035] Split Formulated taRNA

[0036] A “trans-amplifying RNA,” hereinafter referred to as “taRNA,” comprises a first and second RNA polynucleotide, wherein the first RNA polynucleotide encodes a replicase, the second RNA polynucleotide comprises a nucleic acid encoding a payload (e.g., an influenza virus antigen), and wherein the second RNA polynucleotide can be replicated by the encoded replicase in trans. A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A), uridine (U) / deoxythymidine (T), guanosine / deoxyguanosine (G), cytidine / deoxycytidine (C)) and joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications. In some sequences described herein, T / U is used to denote a particular nucleotide may be a T or U depending on whether the polynucleotide is an RNA polynucleotide (U) or a DNA polynucleotide (T). The first and second RNA polynucleotides of a taRNA are separate polynucleotides (i.e., separate molecules which are not a single continuous strand of RNA). The terms “replicase construct” and “trans replicon” (trRNA) construct are used synonymously herein to refer to the first and second RNA polynucleotides of a taRNA, respectively. As used herein, a “construct” refers to an artificial (i.e.., not naturally occurring) polynucleotide.

[0037] Unlike self-amplifying RNAs (e.g., as described in Comes JDG et al., Trends Biotechnol. 2023 Nov;41( 11): 1417- 1429), which comprise both trRNA elements and nucleic acids encoding the replicase capable of amplifying the trRNA (e.g., a replicase construct) in the same polynucleotide, the first and second RNA polynucleotides of a taRNA are separate polynucleotides (i.e., separate molecules which are not a single continuous strand of RNA). When using trRNAs to express a payload in a cell (e.g., when administering a taRNA to a subject), a cognate replicase must be provided by another source (e.g., by the same cell, or by a separate RNA polynucleotide encoding the replicase). When either the trRNA or replicase construct are absent in a cell, the payload (e.g., vaccine antigen) is unlikely to be produced in sufficient quantities to have a desired effect. Accordingly, taRNA are generally “co-formulated” such that they are formulated for delivery together (e.g., in the same nanoparticle). However, the inventors have identified a “split-formulation” for taRNA which may have several advantages over co-formulated taRNA, particularly for vaccines (e.g., influenza virus vaccines).

[0038] The term “split-formulated taRNA”, as used herein, refers to compositions in which a trRNA and a corresponding replicase construct are formulated in separate nanoparticles (e.g., Tr- NP and R-NP) but concomitantly administered to produce a desired effect in the subject, for example, an immune response (e.g., antibody production against a vaccine antigen). Split formulated taRNA comprise a trans-replicating RNA nanoparticle (Tr-NP) and a replicase nanoparticle (R-NP).

[0039] Trans Replicon (trRNA) Constructs

[0040] A “trans replicon” construct (trRNA) refers to a polynucleotide (e.g., RNA polynucleotide) comprising a nucleic acid encoding a pay load (e.g., an influenza virus antigen) operably linked to a conserved sequence element (CSE), wherein the polynucleotide does not encode a replicase cognate to the CSE. A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A), thymidine / deoxythymidine (T), guanosine / deoxyguanosine (G), cytidine / deoxycytidine (C) and uridine (U) joined by phosphodiester bonds. However, the term polynucleotide may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not these modifications are found in naturally occurring nucleic acids; indeed, such molecules may be preferred for certain applications.

[0041] A “gene” refers to a nucleic acid (e.g., DNA, RNA) encoding a polypeptide (e.g., a vaccine antigen or a replicase). In some embodiments, a gene is a naturally occurring gene. In some embodiments, a gene is a transgene (e.g., derived from a different organism). In some embodiments, a gene encodes an antigen (e.g., a vaccine antigen).

[0042] An “antigen” refers to a protein or protein fragment of a pathogen. In some embodiments, an antigen is a nucleic acid (e.g., aptamer), polysaccharide, polypeptide, oligosaccharide, lipid, particulate antigen, or cancer antigen (e.g., tumor antigen). Preferably, an antigen is a protein or a fragment thereof (e.g., a surface protein). In some embodiments, an antigen is a protein derived from a pathogen (e.g., a surface protein derived from an influenza virus). Preferably, an antigen is an influenza virus antigen.

[0043] In some embodiments, an antigen comprises a protein fragment of a pathogen (e.g., a fragment of an HA protein of an influenza virus, wherein the protein fragment is at least 5 amino acids (e.g., at least 10 amino acids or at least 20 amino acids) in length. In some embodiments, an antigen comprises a surface protein or fragment thereof of a pathogen (e.g., a viral envelope protein).

[0044] In some embodiments, an antigen is a protein or peptide that can be recognized by the immune system of a host organism. In some embodiments, an antigen, when introduced into a host, induces an immune response in the host (e.g., antibody production against the antigen). In some embodiments, an antigen is a vaccine antigen (e.g., an influenza virus vaccine antigen). A vaccine antigen induces an immunogenic response in a subject (e.g., as measured by antibody production against the vaccine antigen).

[0045] A “conserved sequence element” (CSE) refers to a recognition site for an alphavirus replicase. Typically, a CSE functions as a core promoter or enhancer for initiation of replication of a downstream sequence, such that a 5'-CSE may initiate synthesis of a plus-strand and a 3'- CSE may initiate synthesis of a minus-strand. An RNA polynucleotide may comprise one or more 5'-CSEs and / or 3'-CSEs. CSEs may be comprised in a UTR, for example a 5'-UTR and / or a 3' UTR. In some embodiments, a CSE forms one or more secondary structure(s), such as one or more stem-loops. Non-limiting examples of CSEs include CSE1, CSE2, CSE3, CSE4, and variants or derivatives thereof. In some embodiments, the CSE is a CSE derived from SFV, SINV, VEEV, or CHIKV alphavirus. CSEs are known in the art, e.g., as described in Hyde JL, Virus Res. 2015 Aug 3;206:99-107. trRNA constructs comprise one or more CSEs.

[0046] In some embodiments, a trRNA comprises one or more untranslated regions (UTRs).

[0047] UTRs may act as stabilizing elements and / or provide regulation of transcription of a gene (e.g., a nucleic acid encoding an antigen). Typically, UTRs are found upstream and / or downstream of a gene or transgene. A UTR located directly upstream of a start codon operably linked to a gene is referred to herein as a 5'-UTR. As a skilled artisan will understand, 5'-UTRs may comprise sequence elements which play roles in regulation of expression (e.g., Kozak sequences) or structural elements which alter stability of the molecule (e.g., 5' cap structures). A UTR located directly downstream of a stop codon operably linked to a gene or transgene is referred to herein as a 3'-UTR. 3'-UTRs may comprise structural elements which alter the stability of a construct and / or provide transcriptional control, including, but not limited to AU-rich elements and polyA tails. A variety of 5'-UTRs and a 3'-UTRs are known to those of ordinary skill in the art. UTRs may be naturally occurring or synthetic.

[0048] In some embodiments, a trRNA comprises a UTR derived from an alphavirus. In some embodiments, an alphavirus UTR comprises a CSE. In some embodiments, a trRNA comprises a 5'-UTR of an alphavirus (e.g., an alphavirus 5'-UTR). In some embodiments, a trRNA comprises a 3'-UTR of an alphavirus (e.g., an alphavirus 3'-UTR). Alphavirus 5'-UTR and alphavirus 3'-UTR sequences are described in the art, e.g., by Hyde JL et al., Virus Res. 2015 Aug 3;206:99-107. In some embodiments, a trRNA comprises a 5'-UTR and / or 3'-UTR derived from a SFV hereinafter referred to as a “SFV-UTR”. In some embodiments, the RNA polynucleotide comprises a 5'-UTR and / or 3'-UTR derived from a SINV, hereinafter referred to as a “SINV-UTR”. In some embodiments, an alphavirus 5'-UTR comprises one or more mutations relative to a wildtype alphavirus 5'-UTR. In some embodiments, an alphavirus 3'-UTR comprises one or more mutations relative to a wildtype alphavirus 3'-UTR. In some embodiments, the RNA polynucleotide comprises a 5'-UTR and 3'-UTR from the same virus. Non-limiting examples include an RNA polynucleotide comprising a 5'-UTR derived from a SINV (5'-SINV-UTR), and a 3'-UTR derived from a SINV (3'-SINV-UTR) or the RNA polynucleotide comprising a 5'-UTR derived from a SFV (5'-SFV-UTR), and a 3'-UTR derived from a SFV (3'-SFV-UTR). In some embodiments, the RNA polynucleotide comprises a 5'-UTR and 3'-UTR from different alphaviruses.

[0049] In some embodiments, a trRNA comprises one or more CSEs, wherein the CSEs are present in one or more UTRs. In some embodiments, a trRNA comprises a 5'-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 3'-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5'-UTR having one or more CSEs, a nucleic acid encoding a payload, and a 3'-UTR having one or more CSEs. In some embodiments, a trRNA comprises a 5'-UTR having one or more CSEs, a nucleic acid encoding a payload, and a 3'-UTR having one or more CSEs. In some embodiments, a trRNA comprises UTRs having one or more CSEs, wherein the UTRs are derived from one or more alphaviruses. A skilled artisan will appreciate that UTRs derived from alphaviruses comprise one or more CSEs unless stated otherwise. In some embodiments, a trRNA comprises a 5'-UTR derived from a first alphavirus and a 3'-UTR derived from a second alphavirus. In some embodiments, a trRNA comprises a 3'- UTR comprising one or more repeat sequence elements (RSE).

[0050] Influenza Virus Antigens

[0051] In some embodiments, a trRNA encodes a payload which comprises an influenza virus antigen. In some embodiments, an influenza virus antigen is a wildtype protein (or fragment thereof) of an influenza virus (e.g., a protein which is expressed by an influenza virus). Preferably, the wildtype protein is a surface protein of the virus (e.g., a viral envelope protein). A wildtype surface protein (e.g., a wildtype influenza virus HA protein, wildtype hMPV F protein) can be understood to comprise a transmembrane domain and an ectodomain (i.e., extracellular domain).

[0052] In some embodiments, a respiratory virus antigen is a full-length protein of a virus. In some embodiments, a respiratory virus antigen is a fragment of a protein of a virus, such that the respiratory virus antigen is not the full-length protein of a virus (i.e., is truncated relative to the corresponding wildtype protein of the virus). In some embodiments, a respiratory virus antigen is a fragment of a surface protein. In some embodiments, a respiratory virus antigen is a secretary fragment of a surface protein, wherein the fragment comprises at least a fragment of the ectodomain of the surface protein, but does not comprise the transmembrane domain of the surface protein.

[0053] In some embodiments, an influenza virus antigen (e.g., full length protein of a virus, fragment of a protein of a virus) is a mutant protein (or fragment thereof) of an influenza virus, such that it comprises one or more amino acid residue deletions, substitutions, or insertions relative to a corresponding wildtype protein. In some embodiments, a respiratory virus antigen is a fusion protein comprising a protein (or fragment thereof) of a virus and a different protein (or fragment thereof). In some embodiments, a fusion protein is a chimeric fusion protein, such that it comprises a protein (or fragment thereof) of a virus and a protein (or fragment thereof) of a different organism (e.g., a different virus, bacterium, mammal). In some embodiments, a fusion protein comprises a protein (or fragment thereof) a virus and a bacterial protein (or fragment thereof). In some embodiments, a fusion protein comprises a protein (or fragment thereof) a virus and a mammalian protein (or fragment thereof). In some embodiments, a fusion protein comprises a protein of a virus (or fragment thereof) and a transmembrane domain. In some embodiments, a fusion protein comprises an ectodomain of a protein of a virus and a ferritin protein (e.g., of a bacterium). For example, a fusion protein may comprise an ectodomain of an influenza virus HA protein fused to a ferritin protein of Helicobactero pylori, as described in Kanekiyo et al. Nature. 499(7456): 102- 106 (2013). A fusion protein may comprise one or more mutations.

[0054] In some embodiments, the mutant protein is a stabilized protein, such that the one or more amino acid residue deletions, substitutions, or insertions is stabilizes the mutant protein in a certain state or conformation. Methods of stabilizing proteins or identifying stabilizing mutations are known by those of ordinary skill in the art, e.g., see Magliery, T.J., Curr Opin Struct Biol. 2016:33:161-168; Wijma, Hein J., et al. Protein Engineering, Design & Selection, 27.2 (2014): 49-58. Several classes of stabilizing mutations are known (e.g., introduction of disulfide bonds, prolines, and / or hydrophobic residues). In some embodiments, one or more cysteines are introduced to the amino acid sequence of a stabilized protein, such that disulfide bonds are formed by at least one of the introduced cysteines, for example within or between monomers of the protein (Wang et al., J Virol. 89(20): 10602-10611). In some embodiments, one or more prolines are introduced to the amino acid sequence of a stabilized protein, such that a helix of the protein is stabilized or destabilized relative to the wildtype protein (Lyu et al. 1990. Science. 250(4981), 669-673). In some embodiments, one or more large cavity-lining residue is replaced with a hydrophobic residue, such that the protein is stabilized relative to the wildtype protein (Bueno et al. J Mol Bio. 2006. 358(3):701-712).

[0055] Some embodiments relate to amino acid sequences or nucleotide sequences having some percentage identity to a reference amino acid sequence or reference nucleotide sequence, respectively. As used herein, the term “identity” refers to the degree to which two or more sequences (e.g., amino acid sequences, nucleotide sequences) are related, as determined by the number of matches between each sequence. “Percent (%) identity” refers to the percentage of residues or nucleotides in a first amino acid or first nucleic acid sequence that are identical to the residues in a second amino acid sequence or second nucleic acid sequence. The skilled artisan will understand that determining percent identity may require a step of aligning a first and second sequence and / or introducing gaps. Several algorithms for aligning sequences are known in the art, for example, on the worldwide web at blast.ncbi.nlm.nih.gov / Blast.cgi and genome.jp / tools-bin / clustalw.

[0056] In some embodiments, the influenza virus antigen is a protein of an influenza virus (e.g., HA or NA of IAV, HA or NA of IBV). An “influenza virus antigen” includes a protein that is expressed on the surface of an influenza virus, or a fragment thereof (e.g., an immunogenic fragment). An “immunogenic fragment” includes a fragment of an antigen (e.g., an influenza virus antigen) that is at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids in length. In some embodiments, an immunogenic fragment includes a fragment of an antigen that is at least 10 amino acids in length. In some embodiments, an antigen is an influenza A virus (IAV) antigen. In some embodiments, an antigen is an influenza B virus (IBV) antigen.

[0057] In some embodiments, the influenza virus antigen comprises influenza virus hemagglutinin (HA) or fragment thereof (e.g., an immunogenic fragment thereof). In some embodiments, the influenza virus antigen comprises an IAV HA protein. In some embodiments, the influenza virus antigen is a full-length IAV HA protein. In some embodiments, the influenza virus antigen is not a full-length IAV HA protein. In some embodiments, the influenza virus antigen is an ectodomain of an IAV HA protein. In some embodiments, the influenza virus antigen is a fusion protein comprising an IAV HA protein or fragment thereof (e.g., at least 10 amino acids of an IAV HA protein), and a protein or fragment thereof of another organism. In some embodiments, the influenza virus antigen is a fusion protein comprising an ectodomain of an IAV HA protein and a ferritin protein. In some embodiments, the influenza virus antigen is a fusion protein comprising an ectodomain of an IAV HA protein and a ferritin protein of H. pylori. In some embodiments, the IAV HA protein comprises one or more mutations relative to a wildtype IAV HA protein. In some embodiments, the IAV HA protein is a stabilized IAV HA protein. In some embodiments, the IAV HA protein is an HA type-1 (Hl) protein or fragment thereof, a H2 protein or fragment thereof, a H3 protein or fragment thereof, a H4 protein or fragment thereof, a H5 protein or fragment thereof, a H6 protein or fragment thereof, a H7 protein or fragment thereof, a H8 protein or fragment thereof, a H9 protein or fragment thereof, a H10 protein or fragment thereof, a Hl 1 protein or fragment thereof, a H12 protein or fragment thereof, a H13 protein or fragment thereof, a H14 protein or fragment thereof, a H15 protein or fragment thereof, a H16 protein or fragment thereof, a H17 protein or fragment thereof, or a Hl 8 protein or fragment thereof. In some embodiments, the influenza virus antigen comprises an IBV HA protein. In some embodiments, the IBV HA protein comprises one or more mutations relative to a wildtype IBV HA protein. In some embodiments, the IBV HA protein is a stabilized IBV HA protein. In some embodiments, the IBV HA protein is an influenza B / Victoria lineage (e.g., influenza B / Victoria-like) HA protein. In some embodiments, the IBV HA protein is an influenza B / Yamagata lineage (e.g., influenza B / Yamagata-like) HA protein.

[0058] In some embodiments, the influenza virus antigen comprises influenza virus neuraminidase (NA) or a fragment thereof (e.g., an immunogenic fragment thereof). In some embodiments, the influenza virus antigen is a full-length IBV HA protein. In some embodiments, the influenza virus antigen is not a full-length IBV HA protein. In some embodiments, the influenza virus antigen is an ectodomain of an IBV HA protein. In some embodiments, the influenza virus antigen is a fusion protein comprising an IBV HA protein or fragment thereof (e.g., at least 10 amino acids of an IBV HA protein), and a protein or fragment thereof of another organism. In some embodiments, the influenza virus antigen is a fusion protein comprising an ectodomain of an IBV HA protein and a ferritin protein. In some embodiments, the influenza virus antigen is a fusion protein comprising an ectodomain of an IBV HA protein and a ferritin protein of H. pylori. In some embodiments, the influenza virus antigen comprises an IAV NA protein. In some embodiments, the IAV NA protein comprises one or more mutations relative to a wildtype IAV NA protein. In some embodiments, the IAV NA protein is a stabilized IAV NA protein. In some embodiments, the influenza virus antigen comprises neuraminidase type-1 (Nl) or a fragment thereof, N2 or a fragment thereof, N3 or a fragment thereof, N4 or a fragment thereof, N5 or a fragment thereof, N6 or a fragment thereof, N7 or a fragment thereof, N8 or a fragment thereof, N9 or a fragment thereof, N10 or a fragment thereof, or N11 or a fragment thereof. In some embodiments, the influenza virus antigen comprises an IBV NA protein. In some embodiments, the IBV NA protein is an influenza B / Victoria lineage (e.g., influenza B / Victoria-like) NA protein. In some embodiments, the IBV NA protein is an influenza B / Yamagata lineage (e.g., influenza B / Yamagata-like) NA protein.

[0059] Methods for selecting influenza virus antigens for use in a vaccine are known in the art e.g., as described in Gao C. et al., Nat. Commun. 15, 1128 (2024); Nguyen QT 2021 May 24;13(6):973 and on the worldwide web at cdc.gov / flu / prevent / vaccine-selection.htm (accessed July 2, 2024). Preferably, an influenza virus antigen is selected based on influenza viruses predicted to be circulating or prevalent in a forthcoming virus season, for example, an influenza virus recommended by the World Health Organization (WHO) for inclusion in a vaccine.

[0060] Replicase Constructs

[0061] A “replicase construct” (i.e., the first RNA polynucleotide) refers to mRNA which comprises a nucleic acid encoding a replicase and does not comprise nucleic acids encoding an antigen. In some embodiments, a replicase construct is a non-replicating mRNA. As used herein, the term “non-replicating mRNA” refers to an mRNA which is processed for translation into a gene product or else degraded, and which does not self-replicate. Once introduced to an environment comprising translational machinery (such as a cell), replicase constructs can be translated to generate the encoded replicase.

[0062] A “replicase” is an RNA-dependent RNA polymerase capable of transcribing (i.e.., reading) an RNA template to produce an RNA (e.g., trRNA). A replicase construct may encode a modified replicase from an RNA virus, for example, an alphavirus. The term “alphavirus” refers to an RNA virus belonging to the Togaviridae family. In some embodiments, a replicase of the replicase construct comprises one or more mutations. Replicase constructs suitable for use in a split-formulated taRNA do not encode an influenza virus antigen that is operably linked to a CSE cognate to the replicase encoded by the replicase construct. In some embodiments, replicase constructs suitable for use in a split-formulated taRNA do not encode an antigen that is operably linked to a CSE cognate to the replicase encoded by the replicase construct. In some embodiments, replicase constructs suitable for use in a split-formulated taRNA do not encode an antigen (e.g., an influenza virus antigen). In some embodiments, replicase constructs suitable for use in a split- formulated taRNA do not comprise a CSE cognate to the replicase encoded by the replicase construct. In some embodiments, the alphavirus comprise a single-stranded RNA genome encoding at least nsPl, nsP2, nsP3, nsP4, El, E2, E3, 6K / TF and capsid proteins. In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Semliki Forest virus (SFV) (SFV replicase). In some embodiments, a wildtype SFV replicase comprises an amino acid sequence of SEQ ID NO: 79. In some embodiments, the SFV replicase is a RLE replicase. A RLE replicase is an SFV replicase variant comprising (i) an arginine at a position corresponding to A1211 of SEQ ID NO: 79; (ii) a leucine at a position corresponding to D1212 of SEQ ID NO: 79; and (iii) a glutamic acid at a position corresponding to A1213 of SEQ ID NO: 79. In some embodiments, the RLE replicase comprises an amino acid sequence of SEQ ID NO: 80 (also referred to as an RLE replicase). In some embodiments, a replicase construct of a taRNA encodes a replicase derived from a Sindbis virus (SINV) (SINV replicase).

[0063] In some embodiments, a taRNA comprises a replicase construct encoding an alphavirus replicase. Typically, an alphaviral replicase comprises a complex formed by the non-structural proteins nsPl, nsP2, nsP3, and nsP4. Thus, a nucleic acid encoding an alphaviral replicase (also referred to herein as a “replicase-coding sequence”) will be understood to encode at least nsPl, nsP2, nsP3, nsP4, and variants thereof. Once expressed, alphaviral replicase may interact with a RNA polynucleotide comprising one or more CSEs which are cognate to the replicase and generate mirrored copies of the RNA polynucleotide, which can be subsequently translated (e.g., by a host cell). Replicase constructs of taRNA necessarily do not comprise a CSE; thus, once the replicase of a replicase construct is translated, the encoded replicase cannot replicate the replicase construct.

[0064] A replicase that is “cognate” to a CSE refers to a replicase that is the capable of transcribing a portion of a polynucleotide comprising the CSE (e.g., capable of transcribing a polynucleotide comprising or encoding a pay load). A replicase construct and trRNA construct are considered “compatible” or “cognate” when a trRNA comprises a CSE to which the replicase encoded by the replicase construct can bind, such that the trans replicon is replicated. In some embodiments, a compatible replicase construct and CSE are derived from the same alphavirus. In some embodiments, a replicase and CSE from the same species are cognate; for example, an SFV replicase is assumed to be cognate to a CSE derived from an SFV. In some embodiments, a replicase construct encodes a replicase derived from an SFV (SFV replicase). In some embodiments, a replicase derived from a SFV is capable of binding to a trRNA comprising a CSE from an SFV. In some embodiments, a replicase construct encodes a replicase derived from a SINV (SINV replicase). In some embodiments, a replicase derived from a SINV is capable of binding to a trRNA comprising a CSE from an SINV. In some embodiments, a compatible replicase construct and CSE are derived from different alphaviruses. In some embodiments, a replicase derived from a SFV is capable of binding to a trRNA comprising a CSE from a SINV. In some embodiments, a replicase derived from a SINV is capable of binding to a trRNA comprising a CSE from an SFV.

[0065] In some embodiments, a replicase construct comprises a 5'-UTR and / or a 3'-UTR. In some embodiments, a replicase construct comprises a 5'-UTR. In some embodiments, a replicase construct comprises a 3'-UTR. In some embodiments, a replicase construct comprises a 5'-UTR and a 3'-UTR. In some embodiments, a replicase construct comprises a 5'-UTR derived from human alpha- globin (5'-HAG-UTR). An illustrative 5'-HAG-UTR is provided in SEQ ID NO: 37. In some embodiments, a replicase construct comprises a 3'-UTR derived from human alphaglobin (3'-HAG-UTR). An illustrative 3'-HAG-UTR is provided in SEQ ID NO: 41. In some embodiments, a replicase construct comprises a 5'-HAG-UTR and a 3'-HAG-UTR. In some embodiments, a replicase construct comprises a 5'-HAG-UTR, a SFV replicase-encoding sequence, and a 3'-HAG-UTR (5'-HAG-UTR-SFV replicase-3'-HAG-UTR).

[0066] Split-Formulated taRNA Nanoparticles

[0067] In some embodiments, this disclosure provides a trRNA nanoparticle (Tr-NP). A “trRNA nanoparticle” (Tr-NP) refers to a nanoparticle which comprises a trRNA and which does not comprise a replicase construct cognate to a CSE of the trRNA. In some aspects, a Tr-NP comprises a trRNA, wherein the trRNA is an RNA polynucleotide comprising a nucleic acid encoding a first influenza virus antigen, operably linked to a CSE; and wherein the Tr-NP does not comprise a polynucleotide comprising a nucleic acid encoding a replicase cognate to the CSE (e.g., does not comprise a replicase construct encoding a replicase cognate to the CSE of the trRNA). In some aspects, a Tr-NP comprises a trRNA, wherein the trRNA is an RNA polynucleotide comprising a nucleic acid encoding a first influenza virus antigen, operably linked to a CSE; and wherein the Tr-NP does not include a polynucleotide comprising a nucleic acid encoding a replicase cognate to the CSE (e.g., does not include a replicase construct encoding a replicase cognate to the CSE of the trRNA). In some embodiments, the trRNA of a Tr-NP is encapsulated in the nanoparticle.

[0068] In some embodiments, this disclosure provides a replicase nanoparticle (R-NP). A “replicase nanoparticle” (R-NP) refers to a nanoparticle comprising a replicase construct and which does not comprise a trRNA comprising a CSE cognate to the replicase encoded by the replicase construct. In some embodiments, the replicase construct of an R-NP is encapsulated in the nanoparticle.

[0069] In some embodiments, a nanoparticle described herein (e.g., a Tr-NP, an R-NP) is a polymeric nanoparticle. In some embodiments, a Tr-NP is a polymeric nanoparticle. In some embodiments, an R-NP is a polymeric nanoparticle.

[0070] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP) (e.g., a liposome). In some embodiments, the Tr-NP is a lipid nanoparticle (i.e., Tr-LNP). In some embodiments, the R-NP is a lipid nanoparticle (i.e., R-LNP). In some embodiments, a lipid nanoparticle (e.g., Tr-NP, R-LNP) comprises an anionic lipid. In some embodiments, a lipid nanoparticle (e.g., Tr- NP, R-LNP) comprises a cationic lipid. In some embodiments, a lipid nanoparticle (e.g., Tr-NP, R-LNP) comprises a neutral lipid. In some embodiments, the lipid nanoparticle (e.g., Tr-NP, R- LNP) comprises an ionizable lipid. In some embodiments, the ionizable lipid is an unsaturated ionizable lipid, a multi-tail ionizable lipid, a polymeric ionizable lipid, a biodegradable ionizable lipid and / or a branched-tail ionizable lipid. In some embodiments, the ionizable lipid is selected from Fig. 1 or Fig. 2 of Han, X., et al., Nat Commun 12, 7233 (2021). Figs. 1 and 2 of Han, X., et al., Nat Commun 12, 7233 (2021) are herein incorporated by reference.

[0071] In some embodiments, the lipid nanoparticle (e.g., Tr-NP, R-LNP) comprises one or more of 3060iio, tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2- Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-l-yl)-l-(3-(pyrrolidin-l-yl)propyl)-2, 5-dihydro- lH-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl) bis(hexane-6,l-diyl)bis(2- hexyldecanoate); ALC-0159, 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; 0- sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13- dimethyl-2,3,4,7 -,8,9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro- lH-cyclopenta[a]phenanthren-3- ol; BAME-O16B, bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia- 3,6-diazahexacosyl)azanediyl) dipropionate; BHEM-Cholesterol, 2- (((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)- 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 -tetradecahydro- 1 Hcyclopenta[a]phenanthren-3- yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-Nmethylethan-l-aminium bromide; C12-200, 1 , 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin- l-yl)ethyl)azanediyl) bis(dodecan2-ol); cKK-E12, 3,6-bis(4-(bis(2 hydroxydodecyl)amino)butyl)piperazine-2, 5-dione; DC-Cholesterol, 30-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate; DOPE, 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]- N,N-dimethyl-l-propanaminium trifluoroacetate; DOTAP, l,2-dioleoyl-3-trimethylammonium- propane; DOTMA, l,2-di-O-octadecenyl-3-trimethylammonium-propane; DSPC, 1,2-distearoyl- snglycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl) 9,9', 9", 9"', 9'"', 9"'"- ((((benzene- 1, 3, 5-tricarbonyl)yris(azanediyl)) tris (propane-3, 1-diyl)) tris(azanetriyl))hexanonanoate; Lipid H (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo- 6- (undecyloxy )hexyl)amino) octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2, 5- diyl)bis(butane-4, l-diyl))bis(azanetriyl))tetrakis(ethane-2, 1-diyl) (9Z,9'Z,9"Z,9"'Z, 12Z,12'Z,12"Z,12"'Z)-tetrakis (octadeca-9,12-dienoate); PEG2000-DMG, 1,2-dimyristoylrac- glycero-3-methoxypolyethylene glycol-2000; TT3, Nl,N3,N5-tris(3-(didodecylamino) propy l)benzene- 1,3, 5 - trie arboxamide .

[0072] In some embodiments, an LNP (e.g., a Tr-NP, an R-NP) comprises an ionizable lipid, a phospholipid, a neutral sterol (e.g., cholesterol), and a pegylated lipid. For example, an LNP (e.g., a Tr-NP, an R-NP) may comprise ALC-0315, DSPC, cholesterol, and PEG-lipid. Components of an LNP may be present in a particular amount or ratio (e.g., molar ratio). For example, an LNP may comprise ALC-0315, DSPC, cholesterol and PEG-lipid in a molar ratio of 35-55% ALC-0315: 1-20% DSPC:30-55% cholesterol:0.01-8% PEG-lipid, 40-50% ALC-0315: 5-15% DSPC: 35-50% cholesterol:0.01-3% PEG-lipid, or about 45% ALC-0315:about 10% DSPC: about 42% cholesterol: about 1.5% PEG-lipid.

[0073] Lipid nanoparticles for RNA polynucleotide delivery are known in the art e.g., as described in Jung HN., Therano sties. 2022 Oct 24;12(17):7509-7531; Paunovska, K., Nat Rev Genet 23, 265-280 (2022); and by Han, X., et al., Nat Commun 12, 7233 (2021). Additional LNP components, production, formulations, and RNA delivery are also known in the art, e.g., as described by Jung. Other lipid nanoparticle and RNA compositions are known by those of skill in the art, e.g., as described in Hou et al., Nature Reviews Materials 6.12 (2021): 1078-1094.

[0074] In some embodiments, a Tr-NP and a R-NP comprise the same nanoparticle composition, such that the Tr-NP and R-NP comprise different RNAs formulated in nanoparticles sharing the same components. In some embodiments, a Tr-LNP and a R-LNP comprise the same polymeric nanoparticle composition, such that the Tr-LNP and R-LNP comprise different RNAs formulated in polymeric nanoparticles sharing the same components. In some embodiments, a Tr-LNP and a R-LNP comprise the same LNP composition, such that the Tr-LNP and R-LNP comprise different RNAs formulated in LNPs sharing the same components.

[0075] Immunomodulatory Proteins (IMP)

[0076] In some embodiments, an trRNA and / or a replicase construct encodes an immune modulating protein. As used herein, the term “immune modulating protein,” referred to hereinafter as IMP, refers to a protein, peptide or protein fragment, or fusion protein, which interferes with (e.g., reduces, inhibits, slows) a host immune response to a non-self danger molecule (e.g., a nucleic acid of a pathogen, a synthetic nucleic acid). Entry of taRNA into a host cell can induce a host immune response to the taRNA, which may result in decreased taRNA expression. Accordingly, host immune responses pose a potential obstacle to the efficacy of taRNA-based vaccines. Provided herein, in some aspects, are trRNAs and / or replicase constructs encoding an IMP. Without being bound by theory, IMPs encoded by trRNAs and / or replicase constructs are thought to reduce host immune responses to the trRNA and / or replicase construct, which may result in increased expression of influenza virus antigen encoded by the trRNA and / or vaccine response in the subject (e.g., subject antibody production against the antigen encoded by the trRNA and / or increased survivability or decreased symptoms upon infection with a pathogen vaccinated against compared to a subject who did not receive the vaccine).

[0077] Influenza Virus Non- structural Protein 1 (NS1)

[0078] In some embodiments, the IMP is an influenza virus non-structural protein 1 (NS1) protein. In some embodiments, a taRNA comprises a polynucleotide (e.g., trRNA, replicase construct) encoding NS1. NS1 blocks recognition of RIG-I mediated signaling through various means, such as antagonizing RIG-I, inhibiting oligomerization of TRIM25, inhibiting IRF-3, inhibiting NF-KB, and interfering with mRNA export machinery. In some embodiments, a replicase construct encodes influenza virus NSlprotein and a replicase. In some embodiments, a trRNA and / or replicase construct encodes an influenza virus antigen and influenza virus NSlprotein. In some embodiments, a trRNA and / or replicase construct encodes an A / Puerto Rico / 8 / 34 influenza virus (PR8) NS1 (PR8 NS1). In some embodiments, a trRNA and / or replicase construct comprises the nucleic acid sequence set forth SEQ ID NO: 71, which encodes an Influenza A / Puerto Rico / 8 / 34 influenza virus (PR8) (PR8 NS1). In some embodiments, the PR8 NS1 comprises an amino acid sequence of SEQ ID NO: 95. In some embodiments, a trRNA comprises, from 5' to 3': a nucleic acid encoding an influenza virus antigen; a linker; and a nucleic acid encoding influenza virus NSlprotein. In some embodiments, a trRNA comprises, from 5' to 3': a nucleic acid encoding influenza virus NSlprotein; a linker; and an influenza virus antigen.

[0079] In some embodiments, this disclosure provides a replicase construct encoding a replicase-NSl fusion protein comprising a replicase fused to influenza virus NSlprotein. In some embodiments, a replicase-NSl fusion protein comprises a replicase joined to influenza virus NSlprotein via a 3 / 4J. In some embodiments, a replicase-NSl fusion protein comprises a replicase joined to NS1 via a linker. In some embodiments, a replicase-NSl fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-NSl. In some embodiments, a replicase-NSl fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-NSl. In some embodiments, a replicase-NSl fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-T2A-NSl. In some embodiments, a replicase-NS 1 fusion protein is an RLE SFV WT nsPl-nsP2-nsP3-nsP4-T2A-NSl.

[0080] Vaccinia Virus (VACV) RNA-binding protein 30 E3 (E3L)

[0081] In some embodiments, the IMP is a vaccinia virus (VACV) RNA-binding protein 30 E3 (E3L). In some embodiments, the taRNA comprises a polynucleotide (e.g., the first RNA polynucleotide or second RNA polynucleotide) encoding VACV E3L protein. VACV E3L is a dsRNA binding protein which has been shown to interfere with PKR, OAS, ADAR, IRF3, IRF7, and NF-KB-mediated signaling, as well as the expression of IFNs and ISGs. VACV E3L is also capable of binding directly to PKR to prevent its activation of elFs. In some embodiments, the first RNA polynucleotide encodes VACV E3L and a replicase. In some embodiments, the second polynucleotide encodes VACV E3L and a payload (e.g., an influenza virus antigen or a pneumovirus antigen). In some embodiments, VACV E3L comprises the sequence set forth in SEQ ID NO: 88. In some embodiments, the taRNA comprises a polynucleotide of SEQ ID NO: 64, which encodes VACV E3L.

[0082] In some embodiments, this disclosure provides a taRNA vaccine comprising a polynucleotide encoding a replicase- VACV E3L fusion protein comprising VACV E3L inserted between two domains of a replicase. In some embodiments, a replicase- VACV E3L fusion protein comprises VACV E3L inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4). In some embodiments, a replicase- VACV E3L fusion protein comprises VACV E3L inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4) and a 3 / 4J duplicate. In some embodiments, a replicase- VACV E3L fusion protein is an SFV WT nsPl-nsP2-nsP3-3 / 4J- VACV E3L-3 / 4J-nsP4 replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J-VACV E3L-3 / 4J-nsP4 replicase-VACV E3L fusion protein further comprises an FCS between the VACV E3L and 3 / J duplicate, such that the replicase-VACV E3L fusion protein comprises SFV WT nsPl-nsP2- nsP3-3 / 4J-VACV E3L-FCS-3 / 4J-nsP4. In some embodiments, an SFV WT nsPl-nsP2-nsP3- 3 / 4J-VACV E3L-FCS-3 / 4J-nsP4 replicase-VACV E3L fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J- VACV E3L-FCS-3 / 4J-nsP4 replicase-VACV E3L fusion protein is encoded by the nucleic acid sequence set forth in SEQ ID NO: 66.

[0083] In some embodiments, a replicase-VACV E3L fusion protein comprises a replicase fused to VACV E3L. In some embodiments, a replicase-VACV E3L fusion protein comprises a replicase joined to VACV E3L via a 3 / 4J. In some embodiments, a replicase-VACV E3L fusion protein comprises a replicase joined to VACV E3L via a linker. In some embodiments, a replicase-VACV E3L fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-VACV E3L. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-VACV E3L comprises the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4- 3 / 4J-VACV E3L replicase-VACV E3L fusion protein is encoded by the sequence set forth in SEQ ID NO: 67. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl- nsP2-nsP3-nsP4-3 / 4J-VACV E3L. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4- 3 / 4J-VACV E3L comprises the amino acid sequence set forth in SEQ ID NO: 92. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-VACV E3L replicase-VACV E3L fusion protein is encoded by the sequence set forth in SEQ ID NO: 68. In some embodiments, a replicase-VACV E3L fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-T2A-VACV E3L. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4-T2A-VACV E3L comprises the amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, an RLE nsPl-nsP2-nsP3-nsP4- T2A-VACV E3L replicase-VACV E3L fusion protein is encoded by the sequence set forth in SEQ ID NO: 69. In some embodiments, a replicase-VACV E3L fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-VACV E3L. In some embodiments, an SFV WT nsPl-nsP2-nsP3- nsP4-T2A-VACV E3L comprises the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-VACV E3L replicase-VACV E3L fusion protein is encoded by the sequence set forth in SEQ ID NO: 70.

[0084] In some embodiments, a trRNA comprises, from 5' to 3': a nucleic acid encoding a pay load or a nucleic acid pay load; a linker; and a nucleic acid encoding VACV E3L protein. In some embodiments, a trRNA comprises, from 5' to 3': a nucleic acid encoding VACV E3L protein; a linker; a nucleic acid encoding a payload or a nucleic acid payload.

[0085] Toscana Virus (TOSV) Non-Structural Proteins (NSs)

[0086] In some embodiments, the IMP is a Toscana virus (TOSV) non-structural (NSs) protein. In some embodiments, the taRNA comprises a polynucleotide (e.g., the first RNA polynucleotide or second RNA polynucleotide) encoding TOSV NSs protein. Toscana virus an enveloped RNA arbovirus of the family Phenuiviridae . The NSs proteins of TOSV have been associated with inhibition of IIRs in cells (Gori-Savellini, 2013; Kalveram & I., 2013). In particular, TOSV NSs have been shown to downregulate protein kinase R (PKR), and inhibit the induction of IFN production by Rig-like receptor- mediated innate immune response pathways. In some embodiments, the first RNA polynucleotide encodes TOSV NSs and a replicase. In some embodiments, the second polynucleotide encodes TOSV NSs and a payload (e.g., an influenza virus antigen or a pneumovirus antigen).

[0087] In some embodiments, this disclosure provides a taRNA vaccine comprising a polynucleotide encoding a replicase-TOSV NSs fusion protein comprising TOSV NSs inserted between two domains of a replicase. In some embodiments, a replicase-TOSV NSs fusion protein comprises TOSV NSs inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4). In some embodiments, a replicase- TOSV NSs fusion protein comprises TOSV NSs inserted between a third and fourth domain of the replicase (i.e., between nsP3 and nsP4) and a 3 / 4J duplicate. In some embodiments, a replicase- TOSV NSs fusion protein is an SFV WT nsPl-nsP2-nsP3-3 / 4J- TOSV NSs-3 / 4J-nsP4 replicase-IMP fusion protein. In some embodiments, an SFV WT nsPl-nsP2-nsP3-3 / 4J-TOSV NSs-3 / 4J-nsP4 replicase-TOSV NSs fusion protein further comprises an FCS between the TOSV NSs and 3 / J duplicate, such that the replicase-TOSV NSs fusion protein comprises SFV WT nsPl-nsP2- nsP3-3 / 4J-TOSV NSs-FCS-3 / 4J-nsP4.

[0088] In some embodiments, a replicase-TOSV NSs fusion protein comprises a replicase fused to TOSV NSs. In some embodiments, a replicase-TOSV NSs fusion protein comprises a replicase joined to TOSV NSs via a 3 / 4J. In some embodiments, a replicase-TOSV NSs fusion protein comprises a replicase joined to TOSV NSs via a linker. In some embodiments, a replicase-TOSV NSs fusion protein is an RLE nsPl-nsP2-nsP3-nsP4-3 / 4J-TOSV NSs. In some embodiments, a replicase-IMP fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-3 / 4J-TOSV NSs. In some embodiments, a replicase-TOSV NSs fusion protein is an RLE nsPl-nsP2-nsP3- nsP4-T2A-TOSV NSs. In some embodiments, a replicase-TOSV NSs fusion protein is an SFV WT nsPl-nsP2-nsP3-nsP4-T2A-TOSV NSs.

[0089] In some embodiments, a trRNA comprises, from 5' to 3': a nucleic acid encoding a payload or a nucleic acid payload; a linker; and a nucleic acid encoding TOSV NSs protein. In some embodiments, a trRNA comprises, from 5' to 3': a nucleic acid encoding TOSV NSs protein; a linker; a nucleic acid encoding a payload or a nucleic acid payload.

[0090] Linkers

[0091] In some embodiments, the trRNA encodes a linker between the payload (e.g., the influenza virus antigen) and the IMP. In some embodiments, a replicase construct encodes a linker between the replicase and the IMP. As used herein, the term “linker” refers to sequences which allow for the concatenation of one or more nucleic acids encoding a gene product, for example, in a multicistronic vector. Linkers may be nucleic acid linkers, for example, nucleic acid elements which can induce cap-independent translation and internal promoters, or peptide linkers, such as ribosomal skip proteins. Proteins are considered to be “joined” by a linker if the nucleic acids encoding the proteins and the nucleic acid linkers or nucleic acids encoding a linker are comprised within a continuous polynucleotide.

[0092] In some embodiments, a linker is an internal ribosome entry site (IRES) element. As used herein, the term “IRES element” refers to a cis RNA sequence region which allows for the initiation of translation of a sequence in a cap-independent manner. The terms “IRES element,” “IRES,” “IRES sequence,” and “IRES region” may be used interchangeably. The term “capindependent” refers to a mechanism for initiation of translation of an RNA polynucleotide which does not require a 5' cap (e.g., Cap-0, Cap-1, Cap-2); cap-independent mechanisms for initiating translation are found in many viral RNA and in some eukaryotic mRNA. In contrast, “capdependent” mechanisms require a 5' cap (e.g., Cap-0) which recruits a series of initiation factors and catalyzes the formation of a functional ribosome to initiate translation by a ribosome; capdependent mechanisms for initiating translation are found in most eukaryotic mRNA. When used as linkers in an RNA polynucleotide, IRES elements can initiate the translation of downstream nucleic acids (i.e., coding sequence). Though rates of translation of a downstream nucleic acid can vary between different IRES elements and / or promoters, IRES elements may be useful for expressing multiple gene products off of a single polynucleotide, as translation can be reinitiated at each IRES element. IRES elements also allow for expression of multiple proteins without further alteration of their amino acid sequence (e.g., as with some peptide-based linkers). IRES elements are often observed in viruses, though virus-like endogenous IRES elements in eukaryotes have been observed. Non-limiting examples of viral and eukaryotic IRES sequences can be found on IRESbase (available on the worldwide web at reprod.njmu.edu.cn / cgi-bin / iresbase / index.php).

[0093] In some embodiments, a linker is a viral IRES element. Non-limiting examples of viral IRES elements include IRES elements of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Manhattan Parechovirus (MPV), Human rhinovirus (iHRV), Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 (EV71), Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Porcine kobuvirus (PKV), Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG- 1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF- A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), tortoise rafivirus (TraV), and Poliovirus (PV). In some embodiments, a linker is an IRES from CVB3. In some embodiments, an IRES from CVB3 comprises the sequence set forth in SEQ ID NO: 44. In some embodiments, a linker is an IRES from EMCV. In some embodiments, an IRES from EMCV comprises the sequence set forth in SEQ ID NO: 45. In some embodiments, a linker is an IRES from PKV. In some embodiments, an IRES from PKV comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, a linker is an IRES from MPV. In some embodiments, an IRES from MPV comprises the sequence set forth in SEQ ID NO: 47. In some embodiments, a linker is an IRES from TraV. In some embodiments, an IRES from TRAV comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, a linker is a ribosomal skip peptide. Ribosomal skip peptides, also known as “self-cleaving peptides,” are peptides that induce ribosomal skipping during translation of an RNA polynucleotide, such that the ribosome fails to covalently bond an amino acid to the nascent chain, then resumes translation downstream, such that multiple, separate proteins are produced. Sequences encoding ribosomal skip peptides tend to be significantly shorter than most IRES elements, and so may be particularly useful when constraints on polynucleotide length are important. Ribosomal skip peptides are typically viral peptides such as 2A peptides. 2A peptides are peptides of about 18-22 amino acids in length which can induce ribosomal skipping. Non-limiting examples of 2A peptides include 2A peptides from: thosea asigna virus 2A (T2A), porcine teschovirus-1 2A (P2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 18 (F2A). In some embodiments, a linker is a 2A peptide. In some embodiments, a linker is a T2A peptide. In some embodiments, a T2A peptide comprises the sequence set forth in SEQ ID NO: 73. In some embodiments, a T2A peptide is encoded by the sequence set forth in SEQ ID NO: 49. In some embodiments, a linker is a P2A peptide. In some embodiments, a P2A peptide comprises the sequence set forth in SEQ ID NO: 74. In some embodiments, a P2A peptide is encoded by the sequence set forth in SEQ ID NO: 50. In some embodiments, a linker is an E2A peptide. In some embodiments, an E2A peptide comprises the sequence set forth in SEQ ID NO: 75. In some embodiments, an E2A peptide is encoded by the sequence set forth in SEQ ID NO: 51.

[0094] Though expression of multiple proteins linked by 2A sequences is generally even, the addition of a 2A peptide alone adds additional amino acids onto each protein flanking it, which may be undesirable for certain proteins which do not tolerate terminal modifications. 2A sequences can also be combined with furin recognition sites to allow for removal of these 2A residues from a translated protein. Furin, also known as Paired basic Amino acid Cleaving Enzyme (PACE), is a protease capable of cleaving proteins at furin recognition sites. Furin-2A comprises a furin cleavage site (FCS) upstream of a given 2A peptide (e.g., T2A, P2A, E2A or F2A). In some embodiments, a linker is a Furin-2A peptide. In some embodiments, a linker is a Furin-T2A peptide. In some embodiments, a Furin-T2A peptide comprises the sequence set forth in SEQ ID NO: 76. In some embodiments, a Furin-T2A peptide is encoded by the sequence set forth in SEQ ID NO: 52. In some embodiments, a linker is a Furin-P2A peptide. In some embodiments, a Furin-P2A peptide comprises the sequence set forth in SEQ ID NO: 77. In some embodiments, a Furin-P2A peptide is encoded by the sequence set forth in SEQ ID NO: 53. In some embodiments, a linker is an alphaviral subgenomic promoter. An alphaviral subgenomic promoter is a nucleic acid upstream (5') of a nucleic acid encoding a payload, to which it is operably linked. Subgenomic promoters (SGP) comprise recognition and binding sites for RNA polymerase (e.g., RNA-dependent RNA polymerase), and may also comprise recognition sites or binding sites for other transcription factors. An alphaviral (SGP) is a promoter which is native to an alphavirus, and which can regulate transcription of nucleic acids to which it is operably linked. An alphaviral subgenomic promoter may be a promoter which, in wildtype alphaviruses, regulates transcription of a structural protein or a non-structural protein. SGPs of alphaviruses are typically found at the junction between nsP4 and structural (STR) coding regions of the alphaviral genome. These SGPs are described by the position spanned relative to the subgenomic start site; for example, an SGP spanning 100 nucleotides, starting 50nt upstream of the start site and ending 50nt downstream of the start site would be described as -50 / 50. In some embodiments, an SGP is a Chikungunya virus (CHIKV) SGP. In some embodiments, a CHIKV SGP is CHIKV (-75 / 69). In some embodiments, an SGP is an Eastern equine encephalitis virus (EEV) SGP. In some embodiments, a EEV SGP is EEV (-84 / 58). In some embodiments, an SGP is a Ross River virus (RRV) SGP. In some embodiments, an RRV SGP is RRV (-75 / 48). In some embodiments, an SGP is an SFV SGP. In some embodiments, a SFV SGP is SFV (-37 / 51); SFV (-50 / 0); SFV (-50 / 51), SFV (-100 / 0), SFV (-100 / 51), SFV (- 150 / 0), SFV (-150 / 51), SFV (-200 / 0), or SFV (-200 / 51). In some embodiments, an SGP is an SINV SGP. In some embodiments, an SINV SGP is SINV (SINV-75 / 49). In some embodiments, an SGP is a western equine encephalitis virus (WEEV) SGP. In some embodiments, an WEEV SGP is WEEV (-84 / 37).

[0095] In some embodiments, a linker is an alphaviral replicase junction region. The term “junction” refers to a region between the C-terminal amino acid of the first domain of a protein and the N-terminal amino acid of the second domain of a protein. A protein “domain” refers to a distinct functional or structural subunit of a protein or protein complex; for example, a replicase protein is a protein complex comprising nsPl, nsP2, nsP3, and nsP4, each of which may be referred to herein as a domain of a replicase. For example, a junction between nsP3 and nsP4 domains is referred to herein as “3 / 4J”. In some embodiments, nsPl and nsP2 are in a fusion protein that comprises an amino acid sequence having 95% identity to the sequence set forth in SEQ ID NO: 96. In some embodiments, nsPl and nsP2 are in a fusion protein that comprises the amino acid sequence set forth in SEQ ID NO: 96. In some embodiments, nsP3 comprises an amino acid sequence having 95% identity to the sequence set forth in SEQ ID NO: 97. In some embodiments, nsP3 comprises the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, nsP4 comprises an amino acid sequence having 95% identity to the sequence set forth in SEQ ID NO: 98. In some embodiments, nsP4 comprises the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, a 3 / 4J comprises the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, a 3 / 4J is encoded by the sequence set forth in SEQ ID NO: 54. In some embodiments, a 3 / 4J linker is joined to an FCS.

[0096] Illustrative taRNA Constructs

[0097] Illustrative split-formulated taRNA are provided herein.

[0098] In some embodiments, a split-formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a replicase; and (b) a Tr-NP comprising a trRNA comprising: (i) a nucleic acid encoding an influenza virus antigen (e.g., an IAV antigen, an IBV antigen); (ii) a nucleic acid encoding a linker, and (iii) a nucleic acid encoding an IMP. In some embodiments, a split-formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a SFV replicase; and (b) a Tr-NP comprising a trRNA comprising: (i) a nucleic acid encoding an influenza virus antigen (e.g., an IAV antigen, an IBV antigen); (ii) a nucleic acid encoding an IRES; (iii) a nucleic acid encoding NS1 influenza virus protein. In some embodiments, a split- formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a SFV replicase; and (b) a Tr-NP comprising a trRNA comprising from 5' to 3': (i) a nucleic acid encoding an influenza virus antigen (e.g., an IAV antigen, an IBV antigen); (ii) a nucleic acid encoding an IRES; (iii) a nucleic acid encoding NS1 influenza virus protein. In some embodiments, a split-formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a SFV replicase; and (b) a Tr-NP comprising a trRNA comprising from 5' to 3': (i) a nucleic acid encoding NS1 influenza virus protein; (ii) a nucleic acid encoding an IRES; (iii) a nucleic acid encoding an influenza virus antigen (e.g., one or more of an IAV antigen, one or more of an IBV antigen In some embodiments, the trRNA comprises a 5' SINV UTR. In some embodiments, the trRNA comprises a 5' oeSTR SINV UTR (e.g., an oeSTR extension). In some embodiments, a trRNA comprises a 5' UTR having an “oeSTR” extension. An oeSTR extension is a sequence of 9 nucleotides in length (AGAAGAUGG) inserted into the 5' terminal of a 5' UTR. The term “5' terminal of a 5' UTR” refers to nucleotides at the 5' end of the 5' SINV UTR. The 5' terminal of the wildtype 5' SINV UTR comprises six conserved nucleotides AUCGGC. The 5' extension described herein is inserted between the AU and CGGC (e.g., AUAGAAGAUGGCGGC (SEQ ID NO: 99), as shown in SEQ ID NO: 40). trRNA constructs may comprise any suitable combination of elements described herein. For example, a trRNA construct can comprise a sequence a 5' UTR, a coding sequence (e.g., a nucleic acid sequence encoding an influenza virus antigen, a nucleic acid sequence encoding an IMP), a stop codon, and a 3' UTR, as exemplified in REF ID NO: 72. REF ID NO: 72 includes “GeneOflnterest”. In some embodiments, the GeneOflnterest comprises a nucleic acid encoding a payload (e.g., an influenza virus antigen). In some embodiments, the GeneOflnterest comprises a nucleic acid encoding an IMP (e.g., NS1). In some embodiments, the GeneOflnterest comprises a nucleic acid encoding a pay load (e.g., an influenza virus antigen) and a nucleic acid encoding an IMP (e.g., NS1). In some embodiments, the GeneOflnterest comprises a nucleic acid encoding a payload (e.g., an influenza virus antigen), a nucleic acid encoding a linker (e.g., a 2A peptide or IRES) and a nucleic acid encoding an IMP (e.g., NS1). In some embodiments, the linker is encoded between the payload and the IMP. In some embodiments, proteins / peptides (e.g., the pay load, a 2A peptide and / or the IMP) are encoded as polyprotein. In some embodiments, a payload, a 2A peptide and an IMP are expressed as a polyprotein. In some embodiments, the GeneOflnterest comprises a nucleic acid encoding, from 3’ to 5’, a payload (e.g., an influenza virus antigen), a nucleic acid encoding a linker (e.g., a 2A peptide or IRES) and a nucleic acid encoding an IMP (e.g., NS1). In some embodiments, the GeneOflnterest comprises a nucleic acid encoding, from 3’ to 5’, a nucleic acid encoding an IMP (e.g., NS1), a nucleic acid encoding a linker (e.g., a 2A peptide or IRES), and a payload (e.g., an influenza virus antigen).

[0099] In some embodiments, a split-formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a replicase; and (b) a Tr-NP comprising a trRNA comprising: (i) a nucleic acid encoding a first influenza virus antigen (e.g., an IAV antigen, an IBV antigen); (ii) a nucleic acid encoding a first linker, (iii) a nucleic acid encoding a second influenza virus antigen (e.g., an IAV antigen, an IBV antigen), (iv) a nucleic acid encoding a second linker, and (v) a nucleic acid an encoding an IMP. In some embodiments, a split-formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a replicase; and (b) a Tr-NP comprising a trRNA comprising: (i) a nucleic acid encoding an influenza virus HA protein (ii) a nucleic acid encoding a first linker, (iii) a nucleic acid encoding an influenza virus NA protein (iv) a nucleic acid encoding a second linker, and (v) a nucleic acid encoding an IMP, wherein the influenza HA protein and the influenza NA protein are from the same influenza virus. In some embodiments, a split-formulated taRNA comprises: (a) an R-NP comprising a replicase construct comprising: (i) a nucleic acid encoding a replicase; and (b) a Tr-NP comprising a trRNA comprising: (i) a nucleic acid encoding an influenza virus HA protein (ii) a nucleic acid encoding a first linker, (iii) a nucleic acid encoding an influenza virus NA protein (iv) a nucleic acid encoding a second linker, and (v) a nucleic acid encoding an IMP, wherein the influenza HA protein and the influenza NA protein are from different influenza viruses.

[0100] Compositions and Formulations

[0101] Described herein are compositions comprising split-formulated taRNA encoding an influenza virus antigen. A composition may comprise any Tr-NP comprising any trRNA described herein, and any R-NP comprising any replicase construct described herein, for example, as described in the section entitled “Illustrative taRNA Constructs.”

[0102] In some embodiments, a composition (e.g., a split-formulated taRNA composition) described herein comprises a plurality of R-NPs and a plurality of Tr-NPs. A “plurality” refers to at least 2. In some embodiments, a plurality comprises at least 5 (e.g., at least 10, at least 100, at least 1,000, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10) R-NPs and Tr-NPs.

[0103] In some embodiments, this disclosure provides compositions comprising an R-NP and a Tr-NP, wherein the Tr-NP comprises a trRNA encoding an influenza virus antigen. In some embodiments, a composition comprises an R-NP and two or more Tr-NPs, wherein each Tr-NP comprises a nucleic acid encoding a different influenza virus antigen.

[0104] In some embodiments, a composition described herein comprises a 1:1 molar ratio of a replicase construct to a trRNA. In some embodiments, a composition described herein comprises a 5:1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises a 10:1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises a 25:1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises a 50: 1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises a 100:1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises a 500: 1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises a 1000:1 molar ratio of replicase construct to trRNA. In some embodiments, a composition described herein comprises greater than or equal to 99% R-NP and less than or equal to 1% Tr-NP. In some embodiments, a composition described herein comprises greater than 99% R-NP and less than 1% Tr-NP.

[0105] In some embodiments, the combined amount of replicase construct and trRNA in a composition described herein is less than 2pg. In some embodiments, the combined amount of replicase construct and trRNA in a composition described herein is less than 1.5pg. In some embodiments, the combined amount of replicase construct and trRNA in a composition described herein is about Ipg. “About” refers to ±5% of the numerical values cited. For example, “about 25% to about 75%” should be understood to include any value between 25% and 75% (inclusive), but also any value between 20-25% and 75-80%. About does not refer to percentages that are above 100% (e.g., above 100% of a composition).

[0106] Administration

[0107] “Administering” includes any suitable method of administration. For example, administration of a split-formulated taRNA composition (e.g., vaccine) may be oral, intravenous, sub-cutaneous, aerosol, or intramuscular administration. A composition (e.g., pharmaceutical composition, taRNA vaccine) may be administered one or more times. In some embodiments, a composition is administered once. In some embodiments, a composition is administered more than once.

[0108] Subjects

[0109] A “subject” as used herein refers to any suitable subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a mouse or rat. In some embodiments, the subject is a human.

[0110] Vaccines

[0111] Contemplated herein are split-formulated taRNA vaccines. A “vaccine” comprises a therapeutic or prophylactic material providing one or more antigens. In some embodiments, a vaccine elicits an immune response to the one or more antigens. In some embodiments, a vaccine comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, a vaccine comprises an adjuvant.

[0112] A “split-formulated taRNA vaccine” is a composition comprising a Tr-NP and an R-NP, wherein the Tr-NP comprises a trRNA comprising a nucleic acid encoding one or more influenza virus antigens and wherein the R-NP comprises a replicase construct encoding a replicase cognate to a CSE of the trRNA, which, when administered to a subject stimulates a host immune response (e.g. production of antibodies that bind to the antigen). Split-formulated taRNA vaccines are designed to stimulate immune responses protective against the virus. Introduction of a taRNA (e.g., a Tr-NP and an R-NP) to a subject results in expression of an antigen by the subject, which stimulates production of taRNA antigen-binding antibodies by the subject and / or protection of the subject against infection by the pathogen corresponding to the split-formulated taRNA vaccine. For example, a split-formulated taRNA vaccine for influenza virus is expected to provide protection against infection by influenza virus in a subject vaccinated with the split-formulated taRNA vaccine. Protection provided by a split-formulated taRNA vaccine may decrease (e.g., eliminate) symptoms of infection by the pathogen against which the subject is vaccinated, decrease the likelihood of mortality due to infection by the pathogen, and / or prevent infection by the pathogen. Furthermore, because taRNA constructs amplify the nucleic acids encoding payloads (e.g., antigens), taRNA vaccines may offer several advantages over both traditional (e.g., protein-based) vaccines and mRNA vaccines.

[0113] In some embodiments, a split-formulated taRNA vaccine described herein may be useful for preventing infection by one or more influenza viruses. In some embodiments, a split- formulated taRNA vaccine described herein may be useful for slowing the development of symptoms associated with infection by an influenza virus. In some embodiments, a split- formulated taRNA vaccine described herein may be useful for decreasing the severity of one or more symptoms associated with respiratory illness associated with an influenza virus. In some embodiments, a split-formulated taRNA vaccine described herein may be useful for preventing one or more symptoms associated with infection by an influenza virus. In some embodiments, a split- formulated taRNA vaccine described herein may be useful for reducing the severity of a respiratory illness caused by an influenza virus. In some embodiments, a split-formulated taRNA vaccine described herein may be useful for decreasing the likelihood of mortality due to infection by an influenza virus. In some embodiments, a split-formulated taRNA vaccine described herein may be useful for preventing development of mild respiratory illness caused by an influenza virus. In some embodiments, a split-formulated taRNA vaccine described herein may be useful for preventing development of severe respiratory illness caused by an influenza virus.

[0114] Multivalent Vaccines

[0115] In some aspects, this disclosure provides multivalent split-formulated taRNA vaccines. In some embodiments, multivalent vaccines comprise two or more different trRNAs (e.g., in a Tr-NP) wherein each trRNA encodes an antigen of a different strain, subtype, variant, or clade of an influenza virus (e.g., two or more different influenza virus antigens). In some embodiments, multivalent vaccines comprise a trRNA (e.g., in a Tr-NP) wherein the trRNA encodes a first influenza virus antigen and a second influenza virus antigen, wherein each virus antigen is of a different strain, subtype, variant, or clade of an influenza virus (e.g., two or more different influenza virus antigens). Multivalent vaccines are designed to protect a subject against a variety of strains, subtype, variant, or clades of a given virus (e.g., two or more influenza viruses). Multivalent vaccines may be bivalent (e.g., designed to protect against two different influenza viruses), trivalent (e.g., designed to protect against three different influenza viruses), quadrivalent (e.g., designed to protect against four different influenza viruses), pentavalent (e.g., designed to protect against five different influenza viruses), hexavalent (e.g., designed to protect against six different influenza viruses), heptavalent (e.g., designed to protect against seven different influenza viruses), octavalent (e.g., designed to protect against eight different influenza viruses), or more.

[0116] In some embodiments, split-formulated multivalent taRNA vaccines comprise an R-NP and one or more Tr-NPs comprising two or more different trRNAs, wherein each trRNA encodes an antigen of a different strain, subtype, variant, or clade of an influenza virus (e.g., two or more different influenza virus antigens).

[0117] In some embodiments, the two or more different trRNAs are comprised in the same nanoparticle (e.g., are part of the same Tr-NP). In some embodiments, a Tr-NP (e.g., of a split- formulated multivalent taRNA vaccine) comprises a first trRNA, wherein the first trRNA is an RNA polynucleotide comprising a nucleic acid encoding a first influenza virus antigen, operably linked to a CSE. In some embodiments, the Tr-NP (e.g., of a split-formulated multivalent taRNA vaccine) further comprises a second trRNA, wherein the second trRNA is an RNA polynucleotide comprising a nucleic acid encoding a second influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen and the second influenza virus antigen are different, and wherein the CSEs of the first trRNA and the second trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine). In some embodiments, the Tr-NP (e.g., of a split-formulated multivalent taRNA vaccine) further comprises a third trRNA, wherein the third trRNA is an RNA polynucleotide comprising a nucleic acid encoding a third influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen, the second influenza virus antigen, and the third influenza virus antigen are different, and wherein the CSEs of the first trRNA, second trRNA, and third trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split- formulated multivalent taRNA vaccine). In some embodiments, the Tr-NP (e.g., of a split- formulated multivalent taRNA vaccine) further comprises a fourth trRNA, wherein the fourth trRNA is an RNA polynucleotide comprising a nucleic acid encoding a fourth influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, and the fourth influenza virus antigen are different, and wherein the CSEs of the first trRNA, second trRNA, third trRNA, and fourth trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine). In some embodiments, the Tr-NP (e.g., of a split-formulated multivalent taRNA vaccine) further comprises a fifth trRNA, wherein the fifth trRNA is an RNA polynucleotide comprising a nucleic acid encoding a fifth influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, the fourth influenza virus antigen, and the fifth influenza virus antigen are different, and wherein the CSEs of the first trRNA, second trRNA, third trRNA, fourth trRNA, and fifth trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine).

[0118] In some embodiments, the two or more different trRNAs are comprised in separate nanoparticles (e.g., are part of different Tr-NPs). In some embodiments, a composition (e.g., a split-formulated multivalent taRNA influenza virus vaccine) comprises a first Tr-NP comprising a first trRNA, wherein the first trRNA is an RNA polynucleotide comprising a nucleic acid encoding a first influenza virus antigen, operably linked to a CSE. In some embodiments, the composition comprises a second Tr-NP comprising a second trRNA, wherein the second trRNA is an RNA polynucleotide comprising a nucleic acid encoding a second influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen and the second influenza virus antigen are different, and wherein the CSEs of the first trRNA and the second trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine). In some embodiments, the composition comprises a third Tr-NP comprising a third trRNA, wherein the third trRNA is an RNA polynucleotide comprising a nucleic acid encoding a third influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen, the second influenza virus antigen, and the third influenza virus antigen are different, and wherein the CSEs of the first trRNA, second trRNA, and third trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine). In some embodiments, the composition comprises a fourth Tr-NP comprising a fourth trRNA, wherein the fourth trRNA is an RNA polynucleotide comprising a nucleic acid encoding a fourth influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, and the fourth influenza virus antigen are different, and wherein the CSEs of the first trRNA, second trRNA, third trRNA, and fourth trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine). In some embodiments, the composition comprises a fifth Tr-NP comprising a fifth trRNA, wherein the fifth trRNA is an RNA polynucleotide comprising a nucleic acid encoding a fifth influenza virus antigen, operably linked to a CSE, wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, the fourth influenza virus antigen, and the fifth influenza virus antigen are different, and wherein the CSEs of the first trRNA, second trRNA, third trRNA, fourth trRNA, and fifth trRNA are cognate to a same replicase (e.g., a replicase of an R-NP of the same split-formulated multivalent taRNA vaccine).

[0119] Generally, each different trRNA of the same split-formulated multivalent taRNA vaccine can be understood to comprise a CSE cognate to the same replicase, such that each different trRNA can be amplified by the same replicase.

[0120] Influenza Virus taRNA Vaccines

[0121] In some embodiments, this disclosure provides split-formulated influenza virus taRNA vaccines. In some embodiments, a split-formulated influenza virus taRNA vaccine comprises a Tr-NP and an R-NP, wherein the Tr-NP encodes an influenza virus antigen. In some embodiments, a split-formulated influenza virus taRNA vaccine comprises a Tr-NP and an R- NP, wherein the trRNA of the Tr-NP encodes an influenza virus antigen and an IMP. In some embodiments, the Tr-NP and / or the R-NP comprise a polymeric nanoparticle. In some embodiments, the Tr-NP and / or the R-NP comprise a lipid nanoparticle.

[0122] In some embodiments, the split-formulated influenza virus taRNA vaccine is a multivalent split-formulated taRNA vaccine comprising one or more Tr-NPs comprising trRNAs encoding two or more antigens of different influenza viruses. In some embodiments, the multivalent split-formulated taRNA vaccine comprises two or more different trRNAs, each encoding a different influenza virus antigen, wherein the two or more different trRNAs are formulated in the same Tr-NP. In some embodiments, the multivalent split-formulated taRNA vaccine comprises two or more different trRNAs, each encoding a different influenza virus antigen, wherein the two or more different trRNAs are each formulated in a separate Tr-NP. In some embodiments, the two or more antigens of different influenza viruses are antigens of different lAVs. In some embodiments, the different lAVs share a subtype (e.g., are of the IAV H3N2 subtype), but differ in clade (e.g., 3C.2alb.2a.2 and 3C.2a.lb.la). In some embodiments, the different lAVs are of different types or subtypes (e.g., H3N2 and H1N1). In some embodiments, the two or more antigens of different influenza viruses are antigens of different IB Vs. In some embodiments, the different IBVs are of the same lineage (e.g., two or more different influenza B / Victoria lineage viruses). In some embodiments, the different IBVs are of different lineages (e.g., one or more influenza B / Victoria lineage virus and one or more influenza B / Yamagata lineage virus). In some embodiments, the two or more antigens of different influenza viruses comprise one or more antigens of an IAV and one or more antigens of an IBV. As a first example, a multivalent influenza virus taRNA vaccine may comprise a taRNA encoding an influenza A / H1N1 antigen and a taRNA encoding an influenza A / H3N2 antigen. As a second example, a multivalent influenza virus taRNA vaccine may comprise a taRNA encoding an influenza B / Victoria lineage antigen, and a taRNA encoding an influenza B / Yamagata lineage antigen. As a third example, a multivalent influenza virus taRNA vaccine may comprise a taRNA encoding an influenza A / H1N1 antigen, a taRNA encoding an influenza A / H3N2 antigen, and a taRNA encoding an influenza B / Victoria lineage antigen. As a fourth example, a multivalent influenza virus taRNA vaccine may comprise a taRNA encoding an influenza A / H1N1 antigen, a taRNA encoding an influenza A / H3N2 antigen, a taRNA encoding an influenza B / Victoria lineage antigen, and a taRNA encoding an influenza B / Yamagata lineage antigen.

[0123] Kits

[0124] In some embodiments, this disclosure provides a kit or components of kits suitable for administering to a subject (e.g., as a vaccine). Notably, embodiments relating to replicase constructs, trRNAs, R-NPs and Tr-NPs described herein may be understood as kits and / or components of kits suitable for administering to a subject.

[0125] In some embodiments, a kit comprises a first vial comprising an R-NP; and a second vial comprising Tr-NP, wherein the Tr-NP comprises a trRNA comprising a nucleic acid encoding an influenza virus antigen operably linked to a CSE cognate to a replicase of the R-NP.

[0126] In some embodiments, this disclosure provides a kit comprising a first vial comprising a plurality of R-NPs; and a second vial comprising a plurality of Tr-NPs, wherein a Tr-NP of the plurality of Tr-NPs comprises a trRNA comprising a nucleic acid encoding an influenza virus antigen operably linked to a CSE cognate to a replicase of an R-NP of the plurality of R-NPs.

[0127] In some embodiments, this disclosure provides a kit comprising a first vial comprising a plurality of R-NPs; and a second vial comprising a plurality of Tr-NPs, wherein most Tr-NPs of the plurality of Tr-NPs comprise a trRNA comprising a nucleic acid encoding an influenza virus antigen operably linked to a CSE cognate to a replicase of most R-NPs of the plurality of R-NPs In some embodiments, this disclosure provides a kit comprising a first vial comprising a plurality of R-NPs; and a second vial comprising a plurality of Tr-NPs, wherein each Tr-NP of the plurality of Tr-NPs comprises a trRNA comprising a nucleic acid encoding an influenza virus antigen operably linked to a CSE cognate to the replicase of each R-NP of the plurality of R-NPs.

[0128] In some embodiments, the first vial and the second vial of a kit comprise two components of a vaccine.

[0129] In some embodiments, a kit described herein comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, a kit described herein comprises a multidose container for administration of a composition described herein (e.g., a vaccine). In some embodiment, a kit comprises instructions for administering a vaccine.

[0130] Methods of Expressing a Payload

[0131] In some aspects, this disclosure provides methods of expressing a replicase and / or an influenza virus antigen in a cell. A cell may be an isolated cell or a cell of a subject. In some embodiments, the cell is a derived from a subject. In some embodiments, the cell is a mammalian cell. In some embodiments, a cell is a human cell.

[0132] In some embodiments, expressing a replicase and / or an influenza virus antigen in a cell comprises contacting the cell with an R-NP and / or Tr-NP of the disclosure. In some embodiments, contacting the cell with an R-NP and a Tr-NP comprises physical interaction between the R-NP, Tr-NP, and the cell. In some embodiments, the physical interaction is transitory. In some embodiments, the physical interaction results in the trRNA of the Tr-NP and / or replicase construct of the R-NP entering the cell. In some embodiments, the physical interaction results in the trRNA of the Tr-NP and replicase construct of the R-NP entering the cell.

[0133] In some aspects, this disclosure provides methods of expressing a replicase, the method comprising contacting a cell (e.g., of a subject) with an R-NP. In some aspects, this disclosure provides methods of expressing an influenza virus antigen in a cell (e.g., a cell of a subject), the method comprising contacting a cell (e.g., of a subject) with a Tr-NP.

[0134] In some embodiments, methods of expressing an influenza virus antigen in a subject comprise contacting a cell with a R-NP and a Tr-NP. In some embodiments, methods of expressing an influenza virus antigen in a cell comprise contacting the cell with a Tr-NP, wherein the cell comprises an RNA polynucleotide encoding a replicase (e.g., a replicase construct) cognate to the Tr-NP. In some embodiments, methods of expressing an influenza virus antigen in a cell comprise contacting the cell with an R-NP, wherein the cell comprises an RNA polynucleotide comprising nucleic acids encoding the influenza virus antigen, operably linked to a CSE that is cognate to the replicase of the R-NP.

[0135] In some embodiments, a cell is comprised in a subject, such that contacting a cell comprises administering R-NPs and / or Tr-NPs to the subject in which the cell is comprised. R- NPs and Tr-NPs of the present disclosure may be administered to a subject in any manner known in the art; for example, an R-NP and / or Tr-NP may be administered to a subject via a route selected from the group consisting of subcutaneous, intradermal, intramuscular, intranasal, intravenous, and sublingual administration. In some embodiments, an R-NP and / or Tr-NP are administered to a subject as a vaccine.

[0136] Contact of R-NPs and / or Tr-NPs with a cell (e.g., a cell in a subject) may be determined by measuring trRNA amplification by the replicase or pay load expression in a plurality of cells of the same type as the putatively contacted cell. Cells of the same “type” refer to a set of cells having similar characteristics (e.g., being of the same tissue). In some embodiments, cells of the same “type” are cells derived from the same tissue or organ of a subject. For example, in some embodiments, liver cells are the same cell type, regardless of whether they are derived from the same subject. In some embodiments, the cell type is a cell type of a specific organ (e.g., brain, eyes, skin, spinal cord, peripheral nervous system, lung, heart, spleen, kidney, liver, intestine, testis, and ovaries). In some embodiments, the cell type is a muscle cell type. In some embodiments, a cell type is a bodily fluid specific cell type (e.g., bone marrow cell or blood cell). In some embodiments, the cell type is specific cell type of specific organ or specific bodily fluid. For example, liver cells can be subdivided into different cell types (e.g., hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells).

[0137] Methods of Inducing an Immune Response In some aspects, this disclosure provides methods of inducing an immune response against an influenza virus antigen in a subject, the methods comprising administering a split- formulated taRNA (e.g., a Tr-NP and a R-NP), a split-formulated taRNA vaccine, a composition, and / or tr-NP and R-NP components of a kit described herein. In some embodiments, inducing an immune response comprises inducing an adaptive immune response to an influenza virus antigen (e.g., an influenza virus antigen encoded by an influenza virus taRNA vaccine). An adaptive immune response includes, but is not limited to, subject production of antibodies that bind to the antigen encoded by the split-formulated taRNA vaccine, activation of T cells, and / or activation of B cells. In some embodiments, inducing an immune response comprises inducing production of antibodies that bind to the respiratory virus antigen encoded in the split-formulated taRNA vaccine.

[0138] In some aspects, this disclosure provides methods of inducing an immune response against an influenza virus antigen (e.g., one or more of an IAV antigen, one or more of an IBV antigen) in a subject, the method comprising administering a composition described herein (e.g., a split-formulated taRNA (e.g., a Tr-NP and a R-NP), a split-formulated taRNA vaccine, a composition, a kit) to the subject.

[0139] Methods of Production

[0140] In some embodiments, this disclosure provides a method of producing an influenza virus vaccine, the method comprising: (i) obtaining a plurality of R-NPs; (ii) determining an influenza virus strain or subtype for which vaccination is desired; (iii) obtaining a plurality of Tr-NPs comprising a trRNA, the trRNA comprising a nucleic acid encoding an antigen related to the influenza virus strain or subtype, operably linked to a CSE that is cognate to the replicase of R- NPs of the plurality of R-NPs. In some embodiments, this disclosure provides a method of producing a multivalent influenza virus vaccine, the method comprising: (i) obtaining a plurality of R-NPs; (ii) determining two or more influenza virus strains or subtypes for which vaccination is desired; (iii) obtaining a plurality of different Tr-NPs, each Tr-NP comprising a different trRNA, each different trRNA comprising a nucleic acid encoding a different antigen related to a different influenza virus strain or subtype, operably linked to a CSE that is cognate to the replicase of R-NPs of the plurality of R-NPs. In some embodiments, this disclosure provides a method of producing a multivalent influenza virus vaccine, the method comprising: (i) obtaining a plurality of R-NPs; (ii) determining two or more influenza virus strains or subtypes for which vaccination is desired; (iii) obtaining a plurality of Tr-NPs, each Tr-NP comprising two or more different trRNA, each different trRNA comprising a nucleic acid encoding a different antigen related to each influenza virus strain or subtype, operably linked to a CSE that is cognate to the replicase of R-NPs of the plurality of R-NPs. Preferably, the influenza virus strain or subtype for which vaccination is desired is an influenza virus strain or subtype predicted to be circulating or prevalent in a forthcoming virus season.

[0141] R-NPs may be obtained in any suitable way from any suitable source. In some embodiments, obtaining R-NPs comprises producing the R-NPs (e.g., using the methods described in the Examples). In some embodiments, obtaining R-NPs comprises obtaining R-NPs from a third party.

[0142] Determining an influenza virus for which vaccination is desired may be performed using any suitable means. In some embodiments, determining comprises collecting a specimen from a subject and sequencing the specimen to determine an influenza virus (e.g., a strain or subtype of an influenza virus). In some embodiments, determining a disease or disorder for which vaccination is required comprises selecting a given influenza virus that has resulted in a pandemic or endemic or is expected to result in a pandemic or endemic. In some embodiments, determining a disease or disorder for which vaccination is required comprises obtaining a sample from a subject and identifying one or more mutated peptides against which an immune response is desired. In some embodiments, identifying am antigen comprises in silico identification of the antigen (e.g., vaccine antigen), for example, as described by Rawal, K. et al. Sci Rep 11, 17626 (2021).

[0143] Tr-NPs may be obtained in any suitable way from any suitable source. In some embodiments, obtaining Tr-NPs comprises produces the Tr-NPs (e.g., using the methods described in the Examples). In some embodiments, obtaining Tr-NPs comprises obtaining Tr- NPs from a third party.

[0144] In some embodiments, the method of producing a vaccine further comprises (iv) combining the plurality of R-NPs and the plurality of Tr-NPs. Combining may be performed prior to administration to a subject (e.g., by combining the contents of two vials of a kit described herein into a single syringe) or in a subject (e.g., by separately administering the contents of two vials of a kit described herein to the same subject). In some embodiments, steps (i)-(iii) are performed in sequential order. In some embodiments, steps (i)-(v) are performed in sequential order.

[0145] In some embodiments, after step (i) and before step (ii), the method of producing a vaccine comprises placing a plurality of R-NPs into long term storage. In some embodiments, long term storage comprises storage at least -20°C. In some embodiments, long term storage comprises storage at least -70 °C. In some embodiments, long term storage comprises storage at least -80 °C. In some embodiments, long term storage comprises storage between at about -20°C and -70°C. In some embodiments, long term storage comprises storage between at about -60°C and -80°C. In some embodiments, long term storage comprises storage for at least 3 months. In some embodiments, long term storage comprises storage for at least 6 months. In some embodiments, long term storage comprises storage for at least 1 year. In some embodiments, long term storage is at least 2 years. In some embodiments, long term storage is at least 3 years. In some embodiments, long term storage is at least 4 years. In some embodiments, long term storage is at least 5 years. In some embodiments, long term storage is 3 months to 1 year. In some embodiments, long term storage is 3 months to 2 years. In some embodiments, long term storage is 3 months to 3 years. In some embodiments, long term storage is 3 months to 4 years. In some embodiments, long term storage is 3 months to 5 years.

[0146] In some embodiments, this disclosure provides a method of formulating and testing multiple different influenza virus antigens to identify those with improved immunogenic properties. In some embodiments, the method comprises (i) producing two or more different compositions, wherein each composition comprises a plurality of the same R-NP but different pluralities of Tr-NPs, wherein each plurality of Tr-NPs comprises trRNA comprising nucleic acids encoding different influenza virus antigens, (ii) administering the two or more compositions to different subjects, and (iii) measuring immune responses (e.g., antibody production against the antigen) induced by administration of the two or more compositions.

[0147] In some embodiments, the disclosure describes a plurality of compositions, each composition of the plurality comprising the same R-NP but different Tr-NPs, wherein the Tr- NPs comprise nucleic acids encoding different influenza virus antigens.

[0148] SEQUENCES

[0149] Table 1: Illustrative 5’ UTR Sequences

[0150] Table 2: Illustrative 3’ UTR Sequences

[0151] Table 3: Illustrative Linkers

[0152] Table 4: Illustrative Replicase Sequences VVETPRSALKVTAQPNDVLLGNYVVLSPQTVLKSSKLAPVHPLAEQVKIITHNG RAGRYQVDGYDGRVLLPCGSAIPVPEFQALSESATMVYNEREFVNRKLYHIAVH GPSLNTDEENYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHE FAYEGLKIRPSAPYKTTVVGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIV NDVKKHRGLDIQAKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRS KVVLCGDPKQCGFFNMMQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYG GKMRTTNPCNKPIIIDTTGQTKPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAA SQGLTRKGVYAVRQKVNENPLYAPASEHVNVLLTRTEDRLVWKTLAGDPWIK VLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAAPVDAFQNKANVCWAKSLVPV LDTAGIRLTAEEWSTIITAFKEDRAYSPVVALNEICTKYYGVDLDSGLFSAPKVSL YYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQAVIAERKIQP LSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYNLALPR RRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHA MKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAVVSSLSRKFSSARVLRPDC VTSNTEVFLLFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGCAPSYRVKRA DIATCTEAAVVNAANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCG SYPVIHAVAPNFSATTEAEGDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSG GRDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDV ELTTDLVRVHPDSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRL QEANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRS

[0153] HQVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDH SDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAG IADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTA FRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGHLQQK SVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRK VENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIA ACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHH AYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFK RYACSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPM DRFTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNA VLRPNVHTLFDMSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILE DLGVDQYLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIAS RVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKP PYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSK WFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPR LVR EANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRSH

[0154] QVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDHS

[0155] DRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAGI

[0156] ADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTAF

[0157] RNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGHLQQKS

[0158] VRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRK

[0159] VENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIA

[0160] ACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHH

[0161] AYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFK

[0162] RYACSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPM

[0163] DRFTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNA

[0164] VLRPNVHTLFDMSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILE

[0165] DLGVDQYLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIAS

[0166] RVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKP

[0167] PYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSK WFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPR LVR NTNTMKNYLLPIVAVAFSKWAREYKADLDDEKPLGVRERSLTCCCLWAFKTRK MHTMYKKPDTQTIVKVPSEFNSFVIPSLWSTGLAIPVRSRIKMLLAKKTKRELIPV LDASSARDAEQEEKERLEAELTREALPPLVPIAPAETGVVDVDVEELEYHAGAG VVETPRSALKVTAQPNDVLLGNYVVLSPQTVLKSSKLAPVHPLAEQVKIITHNG RAGRYQVDGYDGRVLLPCGSAIPVPEFQALSESATMVYNEREFVNRKLYHIAVH GPSLNTDEENYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHE FAYEGLKIRPSAPYKTTVVGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIV NDVKKHRGLDIQAKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRS KVVLCGDPKQCGFFNMMQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYG GKMRTTNPCNKPIIIDTTGQTKPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAA SQGLTRKGVYAVRQKVNENPLYAPASEHVNVLLTRTEDRLVWKTLAGDPWIK VLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAAPVDAFQNKANVCWAKSLVPV LDTAGIRLTAEEWSTIITAFKEDRAYSPVVALNEICTKYYGVDLDSGLFSAPKVSL YYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQAVIAERKIQP LSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYNLALPR RRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHA MKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAVVSSLSRKFSSARVLRPDC VTSNTEVFLLFSNFDNGKRPSTLHRMNTKLSAVYAGEAMHTAGCAPSYRVKRA DIATCTEAAVVNAANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCG SYPVIHAVAPNFSATTEAEGDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSG GRDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDV

[0168] ELTTDLVRVHPDSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRL QEANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRS HQVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDH SDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAG IADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTA FRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGHLQQK SVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRK VENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIA ACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHH AYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFK RYACSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPM DRFTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNA VLRPNVHTLFDMSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILE DLGVDQYLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIAS RVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKP PYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSK WFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPR LVR RDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDVE

[0169] LTTDLVRVHPDSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRLQ

[0170] EANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRSH

[0171] QVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDHS

[0172] DRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAGI

[0173] ADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTAF

[0174] RNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGHLQQKS

[0175] VRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRK

[0176] VENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIA

[0177] ACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHH

[0178] AYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFK

[0179] RYACSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPM

[0180] DRFTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNA

[0181] VLRPNVHTLFDMSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILE

[0182] DLGVDQYLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIAS

[0183] RVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKP

[0184] PYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSK WFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPR LVR

[0185] Table 5: Illustrative IMP Sequences

[0186] Table 6: Illustrative Replicase Fusion Sequences NDVKKHRGLDIQAKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRS KVVLCGDPKQCGFFNMMQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYG GKMRTTNPCNKPIIIDTTGQTKPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAA SQGLTRKGVYAVRQKVNENPLYAPASEHVNVLLTRTEDRLVWKTLAGDPWIK VLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAAPVDAFQNKANVCWAKSLVPV LDTAGIRLTAEEWSTIITAFKEDRAYSPVVALNEICTKYYGVDLDSGLFSAPKVSL YYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQAVIAERKIQP LSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYNLALPR RRVTWLSPLNVTGADRCYDLSLGLPADAGRFDLVFVNIHTEFRIHHYQQCVDHA MKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAVVSSLSRKFSSARVLRPDC VTSNTEVFLLFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGCAPSYRVKRA DIATCTEAAVVNAANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCG SYPVIHAVAPNFSATTEAEGDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSG GRDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDV ELTTDLVRVHPDSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRL QEANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRS HQVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDH SDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAG IADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTA FRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAGIFSSDTGMSKIYID ERSNAEIVCEAIKTIGIEGATAAQLTRQLNMEKREVNKALYDLQRSAMVYSSDDI PPRWFMTTEADKPDADAMADVIIDDVSREKSMREDHKSFDDVIPAKKIIDWKGA NPVTVINEYCQITRRDWSFRIESVGPSNSPTFYACVDIDGRVFDKADGKSKRDAK

[0187] NNAAKLAVDKLLGYVIIRFRKRRGRAGAYIFSSDTGSGHLQQKSVRQHNLQCAQ LDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRKVENMKATVVD RLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIAACNEYLSRNY PTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHHAYHQPTVRSA VPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFKRYACSGEYWE EYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPMDRFTVDMKRD VKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNAVLRPNVHTLFD MSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILEDLGVDQYLLD LIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIASRVLEQRLTDS ACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKPPYFCGGFIVF DSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSKWFRTGLGAE LEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPRLVR AASLTEGRLGKLSILRKKQLKPCDTVMFSVGSTLYTESRKLLRSWHLPSVFHLK GKQSFTCRCDTIVSCEGYVVKKITMCPGLYGKTVGYAVTYHAEGFLVCKTTDT VKGERVSFPVCTYVPSTICDQMTGILATDVTPEDAQKLLVGLNQRIVVNGRTQR NTNTMKNYLLPIVAVAFSKWAREYKADLDDEKPLGVRERSLTCCCLWAFKTRK MHTMYKKPDTQTIVKVPSEFNSFVIPSLWSTGLAIPVRSRIKMLLAKKTKRELIPV LDASSARDAEQEEKERLEAELTREALPPLVPIAPAETGVVDVDVEELEYHAGAG VVETPRSALKVTAQPNDVLLGNYVVLSPQTVLKSSKLAPVHPLAEQVKIITHNG RAGRYQVDGYDGRVLLPCGSAIPVPEFQALSESATMVYNEREFVNRKLYHIAVH GPSLNTDEENYEKVRAERTDAEYVFDVDKKCCVKREEASGLVLVGELTNPPFHE FAYEGLKIRPSAPYKTTVVGVFGVPGSGKSAIIKSLVTKHDLVTSGKKENCQEIV NDVKKHRGLDIQAKTVDSILLNGCRRAVDILYVDEAFACHSGTLLALIALVKPRS KVVLCGDPKQCGFFNMMQLKVNFNHNICTEVCHKSISRRCTRPVTAIVSTLHYG GKMRTTNPCNKPIIIDTTGQTKPKPGDIVLTCFRGWVKQLQLDYRGHEVMTAAA SQGLTRKGVYAVRQKVNENPLYAPASEHVNVLLTRTEDRLVWKTLAGDPWIK VLSNIPQGNFTATLEEWQEEHDKIMKVIEGPAAPVDAFQNKANVCWAKSLVPV LDTAGIRLTAEEWSTIITAFKEDRAYSPVVALNEICTKYYGVDLDSGLFSAPKVSL YYENNHWDNRPGGRMYGFNAATAARLEARHTFLKGQWHTGKQAVIAERKIQP LSVLDNVIPINRRLPHALVAEYKTVKGSRVEWLVNKVRGYHVLLVSEYNLALPR RRVTWLSPLNVTGADRCYDLSLGLPRLEGRFDLVFVNIHTEFRIHHYQQCVDHA MKLQMLGGDALRLLKPGGSLLMRAYGYADKISEAVVSSLSRKFSSARVLRPDC VTSNTEVFLLFSNFDNGKRPSTLHQMNTKLSAVYAGEAMHTAGCAPSYRVKRA DIATCTEAAVVNAANARGTVGDGVCRAVAKKWPSAFKGEATPVGTIKTVMCG SYPVIHAVAPNFSATTEAEGDRELAAVYRAVAAEVNRLSLSSVAIPLLSTGVFSG GRDRLQQSLNHLFTAMDATDADVTIYCRDKSWEKKIQEAIDMRTAVELLNDDV ELTTDLVRVHPDSSLVGRKGYSTTDGSLYSYFEGTKFNQAAIDMAEILTLWPRL QEANEQICLYALGETMDNIRSKCPVNDSDSSTPPRTVPCLCRYAMTAERIARLRS HQVKSMVVCSSFPLPKYHVDGVQKVKCEKVLLFDPTVPSVVSPRKYAASTTDH

[0188] SDRSLRGFDLDWTTDSSSTASDTMSLPSLQSCDIDSIYEPMAPIVVTADVHPEPAG IADLAADVHPEPADHVDLENPIPPPRPKRAAYLASRAAERPVPAPRKPTPAPRTA FRNKLPLTFGDFDEHEVDALASGITFGDFDDVLRLGRAGAYIFSSDTGSGHLQQK SVRQHNLQCAQLDAVEEEKMYPPKLDTEREKLLLLKMQMHPSEANKSRYQSRK VENMKATVVDRLTSGARLYTGADVGRIPTYAVRYPRPVYSPTVIERFSSPDVAIA ACNEYLSRNYPTVASYQITDEYDAYLDMVDGSDSCLDRATFCPAKLRCYPKHH AYHQPTVRSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTMDSAVFNVECFK RYACSGEYWEEYAKQPIRITTENITTYVTKLKGPKAAALFAKTHNLVPLQEVPM DRFTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNA VLRPNVHTLFDMSAEDFDAIIASHFHPGDPVLETDIASFDKSQDDSLALTGLMILE DLGVDQYLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMFLTLFINTVLNITIAS RVLEQRLTDSACAAFIGDDNIVHGVISDKLMAERCASWVNMEVKIIDAVMGEKP PYFCGGFIVFDSVTQTACRVSDPLKRLFKLGKPLTAEDKQDEDRRRALSDEVSK WFRTGLGAELEVALTSRYEVEGCKSILIAMATLARDIKAFKKLRGPVIHLYGGPR LVRGRAGAGIFSSDTGMSKIYIDERSNAEIVCEAIKTIGIEGATAAQLTRQLNMEK REVNKALYDLQRSAMVYSSDDIPPRWFMTTEADKPDADAMADVIIDDVSREKS MREDHKSFDDVIPAKKIIDWKGANPVTVINEYCQITRRDWSFRIESVGPSNSPTFY AC VDIDGRVFDKADGKSKRD AKNNAAKLAVDKLLG YVIIRF* * *

[0189] Ill LVRGSGEGRGSLLTCGDVEENPGPMSKIYIDERSNAEIVCEAIKTIGIEGATAAQL

[0190] TRQLNMEKREVNKALYDLQRSAMVYSSDDIPPRWFMTTEADKPDADAMADVII

[0191] DDVSREKSMREDHKSFDDVIPAKKIIDWKGANPVTVINEYCQITRRDWSFRIESV

[0192] GPSNSPTFYACVDIDGRVFDKADGKSKRDAKNNAAKLAVDKLLGYVIIRF

[0193] Table 7: Illustrative Antigen Sequences

[0194] Table 8: Illustrative trRNA Construct

[0195] Table 9: Illustrative nSP Sequences

[0196] EXAMPLES

[0197] Experiments were performed to determine whether replicase constructs (e.g., as R-NPs) stockpiled in lipid nanoparticles and then later administered to a subject in combination with different lipid nanoparticles comprising trRNAs (e.g., Tr-NPs) (see FIG. 1) could induce the same or better immune response against influenza as an mRNA vaccine. This is also referred to as a split-formulated taRNA influenza vaccine. Results in mice show that an immune response to a split- formulated taRNA influenza vaccine can be elicited with a 250: 1 molar ratio of replicase construct to trRNA. Additionally, the split-formulated taRNA influenza vaccine can produce an equivalent or better antibody response than an mRNA vaccine, with delivery of much less RNA that encodes the influenza antigen.

[0198] Split-formulated taRNA influenza vaccines were compared with mRNA influenza vaccines in mice (FIGs. 2A-2B). Results showed the taRNA and mRNA induced similar levels of antibody response in a dose dependent manner (FIG. 2A), and that both taRNAs and mRNAs had a minimal effect on mouse weight, which suggest minimal and similar toxicity (FIG. 2B).

[0199] The taRNA influenza vaccine construct was further improved by modifying the 5' UTR of the taRNA. This is referred to as the oeSTR trRNA. An oeSTR trRNA comprises a sequence of 9 nucleotides in length (AGAAGAUGG) inserted into the 5' terminal of the 5' SINV UTR of the taRNA. Additionally, an NS1 was optionally added to the taRNA. NS1 is an immunomodulatory protein. Results showed that the oeSTR split-formulated taRNA influenza vaccine could produce the same immune response as the mRNA influenza vaccine, even when split-formulated taRNA influenza vaccine had 250-fold less RNA encoding the influenza viral antigen than the mRNA vaccine (FIG. 3). These results indicate that the split-formulated replicase construct can be stock piled and then a relatively small amount of the trRNA (encoding the antigen) can be prepared in response to a flu epidemic or pandemic. Results also indicated that the NS1 immunomodulatory protein did not have a significant effect on antibody production, however, it is believed that the NS1 immunomodulatory protein will be important for inducing an immune response in higher organisms (e.g., ferrets, non-human primates, and humans)

Claims

1. CLAIMSWhat is claimed is:

1. A trans-replicating ribonucleic acid (trRNA) nanoparticle (Tr-NP), comprising a first trRNA, wherein the first trRNA is an RNA polynucleotide comprising:(i) a nucleic acid encoding a first influenza virus antigen, operably linked to a conserved sequence element (CSE); and wherein the Tr-NP does not comprise a polynucleotide that comprises a nucleic acid encoding a replicase cognate to the CSE.

2. The Tr-NP of claim 1, wherein the first trRNA further comprises:(ii) a nucleic acid encoding an immune modulating protein (IMP).

3. The Tr-NP of claim 2, wherein the IMP is an influenza virus nonstructural protein 1 (NS1), a vaccinia virus (VACV) RNA-binding protein 30 E3 (E3L) protein, or a Toscana virus (TOSV) NSs.

4. The Tr-NP of claim 2 or 3, wherein the first trRNA comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii).

5. The Tr-NP of claim 4, wherein the linker is an IRES element or a 2A peptide.

6. The Tr-NP of claim 5, wherein the IRES element is an IRES from CVB3, EMCV, PKV, MPV, or TraV.

7. The Tr-NP of any one of claims 1 to 6, wherein the influenza virus antigen comprises a fragment of an influenza virus Hemagglutinin (HA) protein and / or a fragment of an influenza virus Neuraminidase (NA) protein.

8. The Tr-NP of any one of claims 1 to 7, wherein the influenza virus antigen comprises a fragment of a first influenza virus HA protein and a fragment of a second influenza virus HAprotein, wherein the first influenza virus HA protein and the second influenza virus HA protein are from different influenza viruses.

9. The Tr-NP of claim 8, further comprising a linker flanked by the fragment of the first influenza virus HA protein and the fragment of the second influenza virus HA protein.

10. The Tr-NP of claim 7, wherein the influenza virus antigen comprises a fragment of an influenza virus HA protein and a fragment of an influenza virus NA protein.

11. The Tr-NP of claim 10, wherein the fragment of the influenza virus HA protein and the fragment of the influenza virus NA protein are from the same influenza virus.

12. The Tr-NP of any one of claims 1 to 11, wherein the first trRNA comprises a 5' untranslated region (UTR) and a 3' UTR flanking the nucleic acid of (i), and the nucleic acid of (ii) if present.

13. The Tr-NP of claim 12, wherein the 5' UTR comprises the CSE.

14. The Tr-NP of claim 12 or 13, wherein the 5' UTR is a SINV 5' UTR or an SFV 5' UTR.

15. The Tr-NP of any one of claims 12 to 14, wherein the 3' UTR is a SINV 3' UTR or an SFV 3' UTR.

16. The Tr-NP of any one of claims 1 to 15, further comprising a second trRNA, wherein the second trRNA is an RNA polynucleotide comprising:(i) a nucleic acid encoding a second influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen and the second influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the second trRNA are cognate to a same replicase.

17. The Tr-NP of claim 12, further comprising a third trRNA, wherein the third trRNA is an RNA polynucleotide comprising:(i) a nucleic acid encoding a third influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen, the second influenza virus antigen, and the third influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the third trRNA are cognate to a same replicase.

18. The Tr-NP of claim 17, further comprising a fourth trRNA, wherein the fourth trRNA is an RNA polynucleotide comprising:(i) a nucleic acid encoding a fourth influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, and the fourth influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the fourth trRNA are cognate to a same replicase.

19. The Tr-NP of claim 18, further comprising a fifth trRNA, wherein the fifth trRNA is an RNA polynucleotide comprising:(i) a nucleic acid encoding a fifth influenza virus antigen, operably linked to a CSE; wherein the first influenza virus antigen, the second influenza virus antigen, the third influenza virus antigen, the fourth influenza virus antigen, and the fifth influenza virus antigen are different from one another, and wherein the CSEs of the first trRNA and the fifth trRNA are cognate to a same replicase20. The Tr-NP of any one of claims 16 to 19, wherein one or more of the second, third, or fourth trRNA further comprise:(ii) a nucleic acid encoding an immune modulating protein (IMP).

21. The Tr-NP of claim 20, wherein the second, third, and / or fourth trRNA comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii).

22. The Tr-NP of any one of claims 1 to 21, wherein at least one influenza virus antigen is an influenza virus B antigen.

23. The Tr-NP of any one of claims 1 to 22, wherein at least one influenza virus antigen is an influenza virus A antigen.

24. A composition, comprising the Tr-NP of any one of claims 1-23, and a replicase- nanoparticle (R-NP), wherein the R-NP comprises a replicase construct, wherein the replicase construct is an RNA polynucleotide comprising:(i) a nucleic acid encoding a replicase cognate to the CSE of the first trRNA; wherein the R-NP does not comprise a CSE that is cognate to the replicase.

25. The composition of claim 24, wherein the RNA polynucleotide of the R-NP further comprises:(ii) a nucleic acid encoding an immune modulating protein (IMP).

26. The composition of claim 24, wherein the RNA polynucleotide of the R-NP comprises a linker between the nucleic acid of (i) and the nucleic acid of (ii).

27. The composition of claim 26, wherein the linker is an IRES element, a % junction amino acid sequence, a sub-genomic promoter, or a nucleic acid encoding a 2A peptide.

28. The composition of any one of claims 24 to 27, wherein the replicase is an alphavirus replicase.

29. The composition of claim 26, wherein the replicase is a Semliki Forest virus (SFV) replicase or a variant thereof.

30. The composition of any one of claims 24 to 29, wherein the Tr-NP and the R-NP are present at a 1:1, 5:1, 10:1, 25:1, 50:1, or 100:1 molar ratio.

31. The Tr-NP of any one of claims 1 to 21 or the composition of any one of claims 22 to 28, wherein the Tr-NP is a lipid nanoparticle (Tr-LNP).

32. The Tr-NP of any one of claims 1 to 23, or the composition of any one of claims 24 to 30, wherein the Tr-NP comprises ALC-0315, DSPC, cholesterol and PEG-lipid .

33. The composition of any one of claims 24 to 32, wherein the R-NP is a lipid nanoparticle (R-LNP).

34. The composition of any one of claims 24 to 32, wherein the R-NP comprises ALC-0315, DSPC, cholesterol and PEG-lipid.

35. The composition of any one of claims 24 to 34, wherein the combined amount of replicase construct and total trRNA is less than 2 pg.

36. The composition of any one of claims 24 to 35, wherein the composition is a vaccine.

37. A composition comprising: a trans-replicating RNA (trRNA) trRNA nanoparticle (Tr-NP), comprising a trRNA, wherein the trRNA is an RNA polynucleotide comprising a nucleic acid encoding an influenza virus antigen, operably linked to a conserved sequence element (CSE); and a replicase nanoparticle (R-NP) comprising a replicase construct, wherein the replicase construct is an RNA polynucleotide comprising a nucleic acid encoding a replicase cognate to the CSE of the trRNA; wherein the Tr-NP does not comprise a polynucleotide that comprises a nucleic acid encoding a replicase cognate to the CSE; wherein the R-NP does not comprise a CSE that is cognate to the replicase; and wherein the R-NP and Tr-NP are separate nanoparticles.

38. A method of vaccinating a subject, the method comprising administering to the subject the composition of any one of claims 24 to 37.

39. A kit, comprising: a first vial comprising the Tr-NP of any one of claims 1 to 23; and a second vial comprising a replicase-nanoparticle (R-NP), wherein the R-NP comprises an RNA polynucleotide comprising a nucleic acid encoding a replicase cognate to the CSE of the first trRNA of the Tr-NP, and wherein the R-NP does not comprise a CSE that is cognate to the replicase.

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